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Enhancing the anti-cancer T cell response via a biomanufactured, acellular, pro-inflammatory, secretome-based… Yang, Xining 2018

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ENHANCING THE ANTI-CANCER T CELL RESPONSE VIA A BIOMANUFACTURED, ACELLULAR, PRO-INFLAMMATORY, SECRETOME-BASED IMMUNOTHERAPEUTIC by  Xining Yang  B.Sc., The Central South University, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2018  © Xining Yang, 2018     ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Enhancing the Anti-Cancer T Cell Response Via a Biomanufactured, Acellular, Pro-Inflammatory, Secretome-Based Immunotherapeutic  submitted by Xining Yang in partial fulfillment of the requirements for the degree of Doctor of Philosophy  in Pathology and Laboratory Medicine  Examining Committee: Mark Scott, Pathology and Laboratory Medicine Supervisor  Christian Steidl, Pathology and Laboratory Medicine Supervisory Committee Member Cedric Carter, Pathology and Laboratory Medicine University Examiner Edward M. Conway, Hematology/Medicine University Examiner  Additional Supervisory Committee Members: Hélène Côté, Pathology and Laboratory Medicine Supervisory Committee Member Jacqueline Quandt, Pathology and Laboratory Medicine Supervisory Committee Member Marc Horwitz, Microbiology & Immunology Supervisory Committee Member    iii Abstract  Pro-inflammatory responses play an important role in controlling the development and progression of cancer. Contrary to the radiotherapy/chemotherapy that cause off-target bystander injuries, immunotherapies activate the patient’s immune responses for cancer elimination. Consequent to the critical role T lymphocytes play in the anti-cancer response, substantial pharmacologic efforts have been made to activate the endogenous T cell response. Unfortunately, many of these approaches have shown noteworthy toxicity due to their pan T cell activation. In contrast, the less robust T cell alloresponse has demonstrated a synergistic effect on the anti-cancer response but poses an inherent risk of Graft versus Host Disease. To overcome this risk, an acellular, allorecognition-derived, secretome-based, inflammatory agent (IA1) has been developed. To assess IA1’s immunomodulatory activity, T cell proliferation and differentiation were determined in vitro. The acellular effectors of the secretome were soluble and exosome-encapsulated microRNA and, due to the conserved nature of microRNA, demonstrated cross-species efficacy. The proliferation induced by IA1 was approximately 50% that of the allogeneic response and dramatically less than that induced by mitogen/mitogen-like stimulations suggesting that bystander cell injury, relative to these agents, could be substantially reduced. IA1 exerted no direct leukocyte toxicity but induced a significant proliferation of resting CD3+ (CD4+ and CD8+) T cells and skewed the response towards a pro-inflammatory state as evidenced by an increased ratio of effector versus regulatory T cells. In assessing the in vitro efficacy of IA1-activated leukocytes on cancer cell proliferation, we showed that IA1-treatment of resting leukocytes resulted in an enhanced anti-proliferative effect on cancer cells relative to untreated or sham-treated donor-matched leukocytes. The inhibition of HeLa cell   iv proliferation by IA1-activated leukocytes was noted by ~12 hours versus 4-5 days for resting cells. Importantly, no toxicity of IA1-activated leukocytes to non-cancerous cells was noticed. A second biomanufactured therapeutic (IA2; produced using HeLa cells) surprisingly demonstrated broad direct toxicities to cancer cells but was less effective than IA1 in inducing a leukocyte-mediated response. Successful development of this secretome therapeutic approach may prove useful in enhancing the endogenous immune response to cancer, and consequent to enhanced immunosurveillance, in reducing the metastatic potential of existing cancers.     v Lay Summary  The goal of this study was to develop a ‘safer’ cancer treatment that activated the anti-cancer immune cell (called lymphocytes) response. One problem associated with current approaches that use activated foreign lymphocytes to kill cancer cells is that they can kill healthy cells as well. We developed a cell-free cocktail (denoted as IA1) that contained small molecules, called microRNA, to activate an individual’s own resting lymphocytes. By enhancing the production of resting lymphocytes and driving them towards a cancer-killing status, IA1 induced the type of immune response seen with foreign (e.g., donor) lymphocytes but without the same risks. We demonstrated that IA1-activated lymphocytes effectively inhibited cancer cell growth but had minimal side effects on normal cells. This cell-free approach may prove useful in treating cancer as an initial treatment or as an enhancement for existing adoptive cell transfer immunotherapies.    vi Preface  This dissertation is an original and unpublished work of the author, X. Yang. Studies using human samples in Chapters 2 to 5 were approved by The University of British Columbia’s Research Ethics Board (certificate #H02-70215, “Transfusion of Antigenically Modified Erythrocytes”). All murine experiments in Chapters 2 to 5 were covered by The University of British Columbia Animal Care Committee certificate #A17-0220, “2017 In vivo analysis of Immunocamouflaged (Stealth) Blood Cells”. Studies using human and murine cell lines in Chapters 2, 3 and 5 were covered by The University of British Columbia Biosafety Permit #B18-0008. The intravenous injection of splenocytes into mouse tail vein in Chapters 2 and 3 was conducted by Iryna Shanina. The manufacturing of tolerogeneic agent (TA1) from immunocamouflaged (PEGylated) mixed lymphocyte reactions (MLR) in Chapters 2 and 3 was performed with Dr. Ning Kang. The concentration of purified total RNA in Chapters 2 and 4 was done in conjunction with Wendy Toyofuku.    vii Table of Contents  Abstract ....................................................................................................................................iii Lay Summary ............................................................................................................................ v Preface ...................................................................................................................................... vi Table of Contents .................................................................................................................... vii List of Tables .......................................................................................................................... xiii List of Figures ........................................................................................................................ xiv List of Acronyms and Abbreviations ................................................................................... xvii Glossary ................................................................................................................................. xxii Acknowledgements .............................................................................................................. xxiii Dedication............................................................................................................................. xxiv Chapter 1: Introduction............................................................................................................ 1 1.1 Overview of Cancer .................................................................................................... 1 1.1.1 Cancer Facts ......................................................................................................... 1 1.1.2 Cancer Initiation, Progression, and Metastasis ...................................................... 2 1.1.3 Conventional Cancer Therapies ............................................................................ 5 1.2 Cancer Immunosurveillance and Immunotherapy ........................................................ 7 1.2.1 Cancer, Inflammation, and Immunity ................................................................... 7 1.2.2 Effector T (Teff) Cells in Immunosurveillance ..................................................... 9 1.2.3 Cancer Cytotoxicity via Cell-Cell Communication and Interaction ..................... 13 1.2.4 Measurement of Immune Cell-Mediated Cancer Cytotoxicity............................. 15 1.2.5 Cancer Immunotherapy ...................................................................................... 17   viii 1.2.5.1 Current Approaches ....................................................................................... 17 1.2.5.2 Strategies for T Cell Activation and Expansion in the Laboratory................... 20 1.3 Allorecognition and Pro-Inflammatory Anti-Cancer Responses ................................. 21 1.3.1 T Cell Allorecognition ........................................................................................ 21 1.3.2 Allostimulatory Anti-Cancer Approaches ........................................................... 23 1.4 Cell-Free Approaches and Acellular Therapeutics ..................................................... 24 1.4.1 Cell Secretome and Acellular Conditioned Media .............................................. 24 1.4.2 Mixed Lymphocyte Reactions (MLR) ................................................................ 26 1.4.3 The Scott Laboratory Allorecognition-Based Acellular Therapeutics .................. 27 1.5 MicroRNA (miRNA) ................................................................................................ 29 1.5.1 Discovery of miRNA.......................................................................................... 30 1.5.2 Biogenesis of miRNA ........................................................................................ 31 1.5.3 miRNA-Mediated Post-Transcriptional Gene Regulation ................................... 32 1.5.4 Intracellular and Extracellular miRNA ............................................................... 34 1.5.5 miRNA-Containing Exosomes ........................................................................... 36 1.5.6 miRNA Are Evolutionarily Conserved and Exhibit Cross-Species Efficacy........ 38 1.5.7 miRNA Are Biomarkers in Cancer ..................................................................... 38 1.5.8 miRNA-Based Cancer Therapeutics ................................................................... 39 1.6 Hypothesis and Specific Aims ................................................................................... 40 Chapter 2: Methods and Materials ........................................................................................ 46 2.1 General Methods ....................................................................................................... 46 2.1.1 Human Peripheral Blood Mononuclear Cells (PBMC) Isolation ......................... 46 2.1.2 Mouse Splenocyte Isolation ................................................................................ 47   ix 2.1.3 Mixed Lymphocyte Reactions (MLR) ................................................................ 48 2.1.4 Lymphocyte Proliferation and Immunophenotyping via Flow Cytometry ........... 49 2.1.5 Statistical Analysis: ............................................................................................ 51 2.2 Specific Aim 1: Biomanufacturing of Acellular Therapeutics .................................... 52 2.2.1 Biomanufacturing of the SYN, IA1, IA2, and TA1 Acellular Secretomes ........... 52 2.2.2 Optimization of the Biomanufacturing and Efficacy Assessment of Secretomes . 54 2.2.3 Molecular Weight (M.W.) Fractionation and Exosome Purification of IA1......... 54 2.2.4 Cross-Species Efficacy of IA1 ............................................................................ 56 2.2.5 In Vivo Biomanufacturing of IA1-Plasma from Immunocompetent Mice ............ 57 2.3 Specific Aim 2: Immunomodulatory Effects of Acellular Therapeutics on T Leukocyte Proliferation and Differentiation ................................................................................ 58 2.3.1 Comparative Effects of Acellular Therapeutics to Anti-CD3/Anti-CD28 and Mitogen T Cell Activation Approaches .............................................................. 58 2.3.2 Effects of IA1/IA2 Activation on Lymphocyte miRNA Expression .................... 59 2.3.3 Enhancement of IA1 on Allorecognition ............................................................ 61 2.4 Specific Aim 3: Anti-Proliferative Effects of Acellular Therapeutics-Activated Leukocytes on Cancer Cells In Vitro ......................................................................... 61 2.4.1 Cancer and Non-Cancerous Cell Lines ............................................................... 61 2.4.2 Effector Leukocyte Viability Assay .................................................................... 62 2.4.3 Real-Time Assessment of Cancer Cell Proliferation In Vitro .............................. 63 2.4.4 Inhibition of Acellular Therapeutics on Cancer Cell Proliferation ....................... 64 2.4.5 Toxicity of Acellular Therapeutics-Activated Leukocytes on Non-Cancerous Cells ........................................................................................................................... 65   x 2.4.6 Lymphocyte-Cancer Cell Conjugation Formation ............................................... 66 Chapter 3: Biomanufacturing of Acellular Therapeutics...................................................... 67 3.1 Rationale and Objectives ........................................................................................... 67 3.2 Results ...................................................................................................................... 68 3.2.1 Human Models: .................................................................................................. 68 3.2.1.1 Optimization of Secretome Biomanufacturing In Vitro ................................... 68 3.2.1.2 Optimization of Assessing the Acellular Therapeutics’ Immunomodulatory Activity In Vitro ............................................................................................ 69 3.2.1.3 Immunomodulatory Effects of IA1: Size Fractionation and Exosome Isolation ...................................................................................................................... 72 3.2.2 Mouse Models: ................................................................................................... 74 3.2.2.1 Biomanufacturing and Optimization of Murine-Sourced IA1 In Vitro ............ 74 3.2.2.2 Immunomodulatory Effect of Murine IA1: Size Fractionation ........................ 76 3.2.2.3 In Vivo Biomanufacturing of IA1-Plasma in Mice .......................................... 77 3.2.3 Cross-Species Efficacy of Human- and Murine-Sourced IA1.............................. 78 3.3 Summary .................................................................................................................. 81 3.4 Discussion................................................................................................................. 82 3.5 Limitations and Future Directions ............................................................................. 85 Chapter 4: Immunomodulatory Effects of Acellular Therapeutics on T Leukocyte Proliferation and Differentiation ............................................................................................ 88 4.1 Rationale and Objectives ........................................................................................... 88 4.2 Results ...................................................................................................................... 89 4.2.1 Human Models: .................................................................................................. 89   xi 4.2.1.1 Effects of IA1 and IA2 on Resting PBMC Proliferation ................................. 89 4.2.1.2 Effects of IA1 and IA2 on Resting T Cell Subset Differentiation .................... 92 4.2.1.3 Lymphocyte Activation: Differential Expression of Immune miRNA ............. 95 4.2.1.4 Can IA1 Enhance an Existing Pro-Inflammatory Response ............................ 97 4.2.2 Mouse Models: ................................................................................................. 100 4.2.2.1 Effects of Murine IA1 on Resting T Cell Proliferation and Subset Differentiation ............................................................................................. 100 4.2.2.2 Effects of Murine IA1 on Existing Pro-Inflammatory Alloresponse.............. 103 4.3 Summary ................................................................................................................ 105 4.4 Discussion............................................................................................................... 106 4.5 Limitations and Future Directions ........................................................................... 108 Chapter 5: Anti-Proliferative Effects of IA1- and IA2-Activated Leukocytes on Cancer Cells In Vitro.......................................................................................................................... 111 5.1 Rationale and Objectives ......................................................................................... 111 5.2 Results .................................................................................................................... 114 5.2.1 Direct Toxicity of IA1 and IA2 To Human and Murine Leukocytes ................. 114 5.2.2 Toxicity of IA1-Activated Splenocytes To Murine Non-Cancerous Cells ......... 115 5.2.3 Anti-Proliferative Effects on Cancer Cells: HeLa Epithelial Cancer Model....... 116 5.2.4 IA1 and IA2 Differentially Affected PBMC-HeLa Cell Interactions ................. 124 5.2.5 Anti-Proliferative Effects on Cancer Cells: SH-4 Melanoma Model ................. 126 5.3 Summary ................................................................................................................ 128 5.4 Discussion............................................................................................................... 129 5.5 Limitations and Future Directions ........................................................................... 131   xii Chapter 6: Closing Remarks ................................................................................................ 134 6.1 Research Significance and Future Investigations ..................................................... 134 6.2 Potential Application of Secretome-Based Acellular Therapeutics........................... 137 Bibliography .......................................................................................................................... 145 Appendices ............................................................................................................................ 168 Appendix A Yang, X., Kang, N., Toyofuku, W.M., and Scott, M.D. Enhancing the Pro-Inflammatory Anti-Cancer T Cell Response Via Biomanufactured, Secretome-Based, Immunotherapeutics. Immunobiology, (2019), In Press ....................... 168    xiii List of Tables  Table 2.1 Human Immunopathology miRNA PCR Array: Functional miRNA Grouping for Target miRNA Genes in Array Plate ......................................................................... 60 Table 4.1 Comparison of Pro-Inflammatory Responses ........................................................... 107 Table 5.1 Comparison of Anti-Proliferative Effects on Cancer Cells ....................................... 130    xiv List of Figures  Figure 1.1 Multistep Cancer Development. ................................................................................. 3 Figure 1.2 Cancer, Inflammation and Immunity. ......................................................................... 9 Figure 1.3 CD8+ and CD4+ T Cell-Mediated Cancer Cytotoxicity. ............................................ 10 Figure 1.4 Interaction of a Cytotoxic Lymphocyte with a Cancer Cell. ...................................... 15 Figure 1.5 The Process of Adoptive T Cell Immunotherapy. ..................................................... 19 Figure 1.6 Direct and Indirect Pathways of Allorecognition. ..................................................... 22 Figure 1.7 The Scott Laboratory Allorecognition-Based Acellular Therapeutics. ....................... 29 Figure 1.8 The Biogenesis of miRNA. ...................................................................................... 32 Figure 1.9 miRNA-Mediated Silencing of Gene Expression. ..................................................... 33 Figure 1.10 Intracellular and Extracellular miRNA. .................................................................. 35 Figure 1.11 Formation, Secretion and Transmission of Exosomes. ............................................ 37 Figure 1.12 Outline of Project. .................................................................................................. 45 Figure 2.1 Human PBMC Isolation. .......................................................................................... 47 Figure 2.2 Mixed Lymphocyte Reactions (MLR). ..................................................................... 49 Figure 2.3 CFSE Proliferation and Immunophenotyping via Flow Cytometry. .......................... 50 Figure 2.4 Manufacturing Scheme of Secretome Biotherapeutics. ............................................. 53 Figure 2.5 Size Fractionation Studies. ....................................................................................... 55 Figure 2.6 In Vivo Manufacturing of IA1-Plasma. ..................................................................... 57 Figure 2.7 Electrode Impedance-Based Target Cell Proliferation............................................... 63 Figure 2.8 In Vitro Cancer Cell Proliferation Assay................................................................... 64 Figure 3.1 Optimized IA1 Was Biomanufactured From MLR at Day 5. .................................... 69   xv Figure 3.2 Assessing IA1’s Immunomodulatory Activity at Day 10 in Resting PBMC and Day 7 in MLR. .................................................................................................................. 70 Figure 3.3 IA1 Promoted a Pro-Inflammatory T Lymphocyte Proliferation and Differentiation via the Secretome miRNA....................................................................................... 73 Figure 3.4 Assessing Murine IA1’s Immunomodulatory Activity at Day 7 in Resting PBMC and Day 3 in MLR. ........................................................................................................ 75 Figure 3.5 Murine IA1 Promoted a Pro-Inflammatory T Lymphocyte Proliferation and Differentiation via miRNA-Containing Fractions. ................................................... 77 Figure 3.6 Allo-Stimulation Enhanced an Immune Response in Immunocompetent Naïve Mice at Day 5. ..................................................................................................................... 78 Figure 3.7 IA1 and IA1-Derived Exosomes Demonstrate Cross-Species Efficacy on Resting Human and Murine CD3+ T Lymphocyte Proliferation and Subset Differentiation. . 80 Figure 3.8 Proposed Immunomodulatory Pathways of Allorecognition-Derived Secretome miRNA. .................................................................................................................. 85 Figure 4.1 IA1 and IA2 Promoted Differential Subset Proliferation of Resting PBMC and in a More Restrained Manner Than Pan T Cell Activators. ............................................ 91 Figure 4.2 IA1 and IA2 Both Promoted a Pro-Inflammatory T Cell Subset Differentiation in Resting PBMC but in Different Manners. ............................................................... 94 Figure 4.3 IA1 and IA2 Pretreatment Induced Differential Intracellular miRNA Expression Profiles in Resting PBMC. ...................................................................................... 97 Figure 4.4 IA1 Significantly Enhanced the Alloproliferative Response of the MLR. ................. 99 Figure 4.5 Murine IA1 Induced a Pro-Inflammatory Resting T Cell Proliferation and Subset Differentiation. ..................................................................................................... 101   xvi Figure 4.6 Murine IA1 Enhanced the Existing Pro-Inflammatory Proliferation and Differentiation in MLR. ........................................................................................ 104 Figure 5.1 IA1 and IA2 Exerted No Toxicity on Resting Leukocytes. ..................................... 115 Figure 5.2 Murine IA1-Activated Splenocytes Had Minimal Toxicity to Non-Cancerous Cells. ............................................................................................................................. 116 Figure 5.3 IA1 Enhanced PBMC-Mediated Inhibition of HeLa Cell Proliferation while IA2 Exerted Direct Toxicity to HeLa Cells. ................................................................. 120 Figure 5.4 miRNA-Containing Fraction of IA1 Mediated the Anti-Proliferative Effects on HeLa Cells. .................................................................................................................... 121 Figure 5.5 IA1 and IA2 Enhanced the Anti-Proliferative Effects of CD4+ and CD8+ T Cell Subsets on HeLa Cells. ......................................................................................... 123 Figure 5.6 IA1 and IA2 Differentially Affected PBMC-HeLa Cell Interactions as Shown by Photomicroscopy and Cell Conjugation Assays..................................................... 125 Figure 5.7 IA1 and IA2 Attenuated SH-4 Cell Proliferation via Both Direct SH-4 Toxicity as well as PBMC-Mediated Growth Inhibition. ......................................................... 127 Figure 6.1 Mechanism of Action for IA1 Secretome Therapeutic in ACT Therapy. ................. 139 Figure 6.2 Use of IA1 and IA2 As Different Acellular Secretome-Based Therapeutics. ........... 143    xvii List of Acronyms and Abbreviations  3’ UTR 3’ untranslated regions  7AAD  7-amino-actinomycin D  ACT Adoptive cell transfer  Ago Argonaute AICD Activation-induced cell death ANOVA Analysis of variance APC Antigen presenting cells BSA Bovine serum albumin ° C Degrees Celsius CAR Chimeric antigen receptor CCL Chemokine (C-C motif) ligand CD Cluster of differentiation CFSE  Carboxyfluorescein diacetate succinimidyl ester CLL Chronic lymphocytic leukemia cm Centimeter CO2 Carbon dioxide Ct  Threshold cycle CTL  Cytotoxic T lymphocytes CTLA-4 Cytotoxic T lymphocyte-associated antigen 4 DC  Dendritic cells   xviii DMEM  Dulbecco’s modified eagle’s medium DNA  Deoxyribonucleic acid DNase  Deoxyribonuclease DTH Delayed-type hypersensitivity ER Endoplasmic reticulum  FasL Fas ligand FBS  Fetal bovine serum FDA  Food and Drug Administration FOXP3 Forkhead box P3 g Gram ´ g Gravitational force GvHD  Graft versus Host Disease H-2 H-2 haplotype HBV/HCV  Hepatitis B/C virus HDL High density lipoprotein HEPES  4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  HLA  Human leukocyte antigen HPV Human papillomavirus IFN Interferon IL  Interleukin iNKT cell Invariant natural killer T cell IQR Interquartile range   xix IV Intravenous IVIg Intravenous immunoglobulin kDa  Kilodalton l Liter µg  Microgram µm  Micrometer µM  Micromolar mAb Monoclonal antibodies MAF Macrophage-activating factor mg  Milligram MHC  Major histocompatibility complex  mPEG Methoxypoly(-ethylene glycol) mRNA Messenger RNA miRNA microRNA ml  Milliliter MLR  Mixed lymphocyte reactions mM  Millimolar MMP Matrix metalloproteinases M.W.  Molecular weight NF-kB Nuclear factor-kB NKT cell  Natural killer T cell nm Nanometer   xx NOD Non-obese diabetic nt Nucleotide OH• Hydroxyl radical PBL Peripheral blood lymphocytes PBMC  Peripheral blood mononuclear cells  PBS  Phosphate buffered saline PD-1 Programmed death-1 PD-L1 Programmed death-ligand 1 % Percent PHA  Phytohemagglutinin qRT-PCR  Quantitative reverse transcription polymerase chain reaction RBC  Red blood cells RISC RNA-induced silencing complex RNA  Ribonucleic acid RNase  Ribonuclease  RNI Reactive nitrogen intermediates RORgt Retinoic acid receptor-related orphan receptor gamma-T ROS Reactive oxygen species SEM  Standard error mean STAT Signal transducer and activator of transcription SV40 Simian vacuolating virus 40 T1D Type 1 diabetes   xxi TAA Tumour-associated antigens T-ALL T-cell acute lymphoblastic leukemia T-bet T-box transcription factor Teff Effector T cells TGF Transforming growth factor  Th  T helper  TIL Tumour-infiltrating lymphocytes Treg  Regulatory T TLR Toll-like receptors TNF  Tumour necrosis factor  TRAIL TNF-related apoptosis inducing ligand U Unit WBC  White blood cells WHO World Health Organization    xxii Glossary IA1 Inflammatory Agent 1-Allo; IA1-Allo IA2 Inflammatory Agent 2-HeLa; IA2-HeLa SYN Syngeneic media TA1 Tolerogeneic Agent 1   xxiii Acknowledgements  I offer my gratitude to the Scott Laboratory for all the support and help during my PhD journey. Special thanks to my supervisor Dr. Mark Scott who has given me the chance to study abroad and guided me through the project; that first meeting with you 6 years ago is one of the most amazing things happened in my life. Thank you for always being professional, helpful and considerate for both my study and life. I would like to acknowledge my fellow Scott Laboratory members, Dr. Ning Kang, Dr. Meera Raj, Wendy Toyofuku, Luxi Wang, Dr. Dana Price and Dr. Li Li, for their helpful discussions and valuable friendship. I would also like to thank Iryna Shanina from the Horwitz Laboratory for her assistance on some murine experiments. A thank-you to the Centre for Blood Research colleagues who have made me feel at home in this scientific community thus could progress rapidly. I would like to thank the Department of Pathology and Laboratory Medicine which I always believe is the best program for graduate studies. Special thanks to Dr. Haydn Pitchard for your insights and caring, to Dr. Hélène Côté, Dr. Jacqueline Quandt, Dr. Christian Steidl and Dr. Marc Horwitz for your guidance and advice as amazing supervisory committee members. I also express my thankfulness to Graduate and Postdoctoral Studies of the University of British Columbia for being inclusive and helpful especially for international students and making my stay here so enjoyable and memorable. Importantly, I would like to thank my loving parents, fiancée, families and friends who have been supportive and encouraging throughout my years of education. Without your belief in me, I would not have achieved this much. Last but not least, a sincere thank-you to all my blood donors for your generous contribution of blood and kindness to help me to obtain this PhD.   xxiv Dedication  To my beloved fiancée Haoning (Howard) Cen.   1 Chapter 1: Introduction 1.1 Overview of Cancer  1.1.1 Cancer Facts  Cancer is the leading cause of death worldwide. In 2012, there were an estimated 14.1 million new cases of cancer and 8.2 million deaths attributed to cancer. [1] Current incidence and mortality rates indicate approximately 100 new cases and 50 deaths per 100,000 in both genders; males experience higher rates than females. [2] These numbers are increasing over time, as 22% more new cancer cases and 1.8 million additional deaths worldwide have been predicted for 2020. [1] The highest rates of cancer are generally in North America, Oceania, and Europe. [1, 2] In Canada, 1 out of 4 people (28% of males and 24% of females) are expected to die from cancer. [3] An estimated 565 new cases of cancer and 221 deaths from cancer occurred every day in Canada in 2017. [3] The most common types of cancer diagnosed worldwide are lung, female breast, bowel, and prostate, accounting for more than 42% of all new cases. [1] Interestingly, these cancers have been amongst the most common since 1975. [1, 4, 5] According to 2012 cancer statistics, lung cancer ranked first and prostate cancer second in males diagnosed with cancer, while breast cancer and bowel cancer were the most common types in females. [1] In both sexes, stomach, liver, cervix, esophagus, and bladder cancer, along with non-Hodgkin lymphoma were the other most commonly diagnosed cancers in 2012. [1] It is estimated that globally there was a combined loss of around 180 million years of healthy life in 2008, resulting from the above main contributing cancers. [6] Indeed, only an estimated 50-60% survival rate for all cancers was reported in Canada in the past decade. [3]   2 Cancer poses significant and increasing economic impacts. The global cost of cancer was estimated to be $1.16 (US) trillion in 2010. [7] According to a World Health Organization (WHO) report the economic burden of cancer care accounts for a 70% death rate, especially in low- to middle-income countries. [8] In Canada, costs of cancer care rose steadily from $2.9 billion in 2005 to $7.5 billion in 2012. Most of the increase came from the rising costs of hospital-based care. [9] In addition, the development of tools for cancer diagnosis, treatment and follow-up, cancer related research, and therapeutic trial expenses all account for cancer costs being estimated to reach $158 (US) billion in 2020 in the United States. [10] In conclusion, cancer is a significant global issue that causes death and economic burdens. Efforts have been made to reveal the biological basis of cancer and improve cancer treatment approaches.   1.1.2 Cancer Initiation, Progression, and Metastasis Beyond ‘biological bad luck’, cancer development is viewed as a multistep process involving mutations and selection for highly proliferative and metastatic cells (Figure 1.1). [11] Initiation of cancer begins when the cell experiences genomic instability. The key process is genetic alterations which lead to downregulation of the tumour suppressor protein p53 (known as the ‘guardian of the genome’ [12]), allowing uncontrolled proliferation of a single cell and the outgrowth of a population of clonally derived cancer cells. During the progression of cancer, additional mutations continue accumulating, presenting a selection of cells that are rapidly growing to form a new and dominant clone within the cancer population. This cancer cell clonal selection event continues throughout the development of cancer, leading to the evolution of more rapid-growing and invasive cancer cells. Indeed, pioneer cancer cells escape from the primary tumour site and establish a secondary site, in order to achieve metastasis and malignancy.    3  Figure 1.1 Multistep Cancer Development.  Cancer development initiates when mutation occurs to a single cell, leading to the abnormal proliferation and formation of the initial cancer cell population. Mutations continue occurring in this cancer cell population, followed by selection for a more rapidly-growing clone. The repeated mutation-and-selection events result in the progression of cancer to increasingly rapid-growth. Cells escape from the primary tumour site, invade the neighbouring tissues, and establish a secondary site to achieve metastasis and malignancy. During the process of cancer initiation, progression, and metastasis, cancer cells have gained six hallmarks that are shared by almost all types of cancer. These hallmarks breach the anti-cancer mechanisms enabling the unlimited proliferation and progression of cancer. Modified from Cooper 2000. [11]  During cancer development, six hallmarks are gained by cancer cells to successfully breach the anti-cancer mechanisms, thus enabling the cancer progression (Figure 1.1). [13] Initially, cancer cells acquire the capability of self-sufficiency in growth signals. They deregulate Mutation (e.g., p53)Cell ProliferationMutationInitial Cancer Cell PopulationSelection for Rapid-growing Clone Variant Cells with Increased Growth PotentialVariant Cancer Cell PopulationMutationSelectionMore Rapidly-growing Variant Cancer Cell PopulationInitiationProgressionCell Escape MetastasisEndothelial CellSecondary SiteSelf-sufficiency in Growth SignalsInsensitivity to Anti-growth SignalsEvading ApoptosisLimitless Replicative PotentialSustained AngiogenesisTissue Invasion & MetastasisHallmarks of Cancer  4 the growth-enabling signals and become masters of their own destinies. [14] At the same time, cancer cells gain insensitivity to anti-growth signals. By evading growth suppressors, cancer cells circumvent the anti-growth environment, allowing unlimited growth. [14] Moreover, acquired resistance to apoptosis is likely a hallmark of all types of cancer. Cancer cells develop a variety of strategies to limit apoptosis, most common of which is the loss of p53 function – leading to the corruption of apoptosis circuitry. [15] Cancer cells also carry an intrinsic potential to maintain telomere length, resulting in a capacity for limitlessly replication. This capability enables cellular immortality and the generation of macroscopic tumours. [14] To meet the needs of nutrients and oxygen, cancer cells acquire sustained angiogenesis – the tumour-associated neovasculature. By forcing the ‘angiogenic switch’ on, cancer cells trigger the normally quiescent vasculature to continuously generate new vessels to support the expanding neoplastic growths. [16] Finally, cancer cells travel from the primary mass to distant sites, establishing new colonies. The fugitive cancer cells invade adjacent tissues to form metastases, which cause 90% of human cancer deaths. [17] What causes cancer? Although many factors affect the likelihood of cancer, and one dominant factor for one type of cancer may not apply to other cancers, external agents including radiation, chemicals, and viruses have been found to trigger cancer in both experimental animals and humans. [11] Radiation and most chemicals generally cause mutations in normal cells. For example, ultraviolet solar radiation, which causes mutations in skin cells due to induced defective deoxyribonucleic acid (DNA) repair, is the major cause of malignant melanoma. [18] Smoking, as an example, is the undisputed leading cause (80-90%) of lung cancer cases, and it is also implicated in larynx, esophagus, and other types of cancer, contributing to one-third of all cancer deaths. [11] There are more than 60 cancer-inducing chemicals identified in tobacco   5 smoke, including aldehydes, N-nitrosamines, and nitro compounds, most of which cause damage to DNA and account for a higher rate of p53 mutation in smokers compared to nonsmokers. [19] Viral infections are estimated to be the cause of 15-20% of human cancers. [20] Viruses encode proteins to reprogram the host signaling pathways and have the ability to regulate cellular genetic stability, proliferation, and apoptosis, all of which can lead to mutations and malignancies. Indeed, human cancer viruses such as human papillomavirus (HPV), hepatitis B/C virus (HBV/HCV), and simian vacuolating virus 40 (SV40) are responsible for cervical cancer, hepatocellular carcinoma, and brain cancer, respectively. [21–24] In addition to these external carcinogens, internal substances such as hormones, particularly estrogens, and phorbol esters also contribute to cancer development, promoting cellular proliferation rather than causing mutations. [11] Nonetheless, the formation of cancer is a complex multistep process and it is overly simplistic to speak of single causes for most cancers.  1.1.3 Conventional Cancer Therapies In an attempt to shrink tumour mass directly and eliminate cancer cells, radiotherapy has been used as a cancer treatment for over 100 years – since the discovery of X-rays in 1895. [25] This therapy, together with surgery, provided advances in treating non- or low-metastatic cancer and has been a standard treatment for a wide range of malignancies. [26] Between 1991 and 1996, ~30% of prostate cancer and ~40% of lung cancer patients were treated with radiotherapy as an initial management option. More than 50% of patients, spanning all cancer types, require at least one radiotherapy treatment course during their care. [27, 28] Radiotherapy and surgery dominated the field of cancer therapy into the 1960s, up until the cure rates of ~30% of these therapies were found to reach plateaus after more radical local treatments. [29] This issue led to the application of cancer drugs, either in conjugation with   6 surgery and/or radiotherapy, or as a first-line therapy. The development of cytotoxic cancer drugs, or chemotherapy, grew exponentially during the past century; a variety of classes of chemotherapeutic agents have been established based on their mechanism of action. [30] For example, alkylating agents impair cancer cell function by binding covalently with biologically important molecules (i.e., amino and carboxyl moieties). The platinum agent cisplatin is an inorganic heavy metal complex that produces DNA crosslinks, thus inhibiting the synthesis of DNA, ribonucleic acid (RNA), and proteins. Anthracycline antibiotics intercalate between DNA base pairs and inhibit DNA topoisomerases. Taxanes block the cell cycle in mitosis by promoting microtube assembly and stability. Despite these distinct modes of action, the aim of all chemotherapies is cancer cell death and tumour shrinkage.  In combination with surgery, radiotherapy, or chemotherapy, adjuvant or supportive therapies have also been applied after the initial treatment to prevent cancer recurrence or to manage the side effects from first-line therapies. Indeed, bystander toxicities to normal tissue, and the cancer patient, are significant during surgery, radiotherapy, or chemotherapy. Surgical knives induce invasive trauma to the patient, increasing both economic and mental burdens due to the long period of hospital care before and after surgery. Radiotherapy induces long-term damage to healthy cells that are not directly irradiated; this has become increasingly evident since first reported in 1992. [31] Even worse, these late effects may become the root of repeated tumour development for the patient. Similarly, chemotherapy exerts off-target toxicity and problems of drug resistance. These problems further encouraged the use of high-dose chemotherapies. [26] However, severe multi-organ failures and the extremely low rate of therapeutic benefits over toxicity have limited their clinical utility. [32]   7 In summary, surgery, radiotherapy, and chemotherapy are standard cancer treatment approaches, yet they bear significant side effects. Because cancer is a complex microenvironment that forms and progresses upon continuous balancing interactions with the immune system, the understanding of this interrelationship has led to the emergence of a new concept, ‘immunotherapy’, which aims at strengthening the immune system’s ability to recognize and eliminate nonself (e.g., tumour cells), instead of targeting cancer cells directly.  1.2 Cancer Immunosurveillance and Immunotherapy 1.2.1 Cancer, Inflammation, and Immunity Central to the initiation of cancer are mutations that enable rapid-growth, metastatic features, and other hallmarks of cancer cells. However, these genetic alterations within the cancer cells themselves are not sufficient for cancer development. Cancer promotion and progression are dependent on auxiliary procedures associated with surrounding cells in the microenvironment; immune cells and inflammation have long been significant in this process. [33] Chronic inflammation can cause and promote cancer. Inflammation is a process of recruiting immune cells to the site of damaged tissues in order to eliminate pathogens and heal wounds. However, persistent infections and repeated tissue damage induce chronic inflammation. [34, 35] As previously described in Figure 1.1, cancer formation requires multiple oncogenic mutations before the initial tumour mass can establish. Long-term inflammation associated with infection and/or irritation increases the mutation rates, mostly via the production of toxic free radicals [i.e., reactive oxygen species (ROS), reactive nitrogen intermediates (RNI), and hydroxyl radicals (OH•)] inducing DNA damage and genomic instability. [33, 36]   8 Moreover, the cancer-promoting cytokines, chemokines, and growth factors released from immune cells support cancer progression, angiogenesis, and metastasis. [36] In return, cancer can also cause inflammation. Pre-malignant tumours are viewed as ‘wounds that never heal’ by the human body. Indeed, at various stages of cancer development, immune cells such as mast cells, pro-inflammatory factors such as matrix metalloproteinases (MMP), and/or activated platelets are attracted to the site, conducting wound-healing activities. [37–41] Unlike the normal course of wound healing, in which the inflammatory cells and agents appear transiently and disappear, persistent inflammation at the tumour site can lead to tissue pathologies (i.e., fibrosis, angiogenesis and neoplasia), further promoting cancer progression. [36, 42] However, pro-inflammation responses induced by effector immune cells can kill cancer. These immune cells infiltrate into the tumour microenvironment and demonstrate cancer immunosurveillance by recognizing tumour antigens and initiating cancer cell apoptosis/necrosis. This antigen-activated immune response involves cells from both the innate and adaptive immune system. As schematically shown in Figure 1.2, T cells, natural killer (NK) cells and the anti-cancer subsets of dendritic cells (DC) induce cytotoxicity to cancer cells. [43–46] Cytokines including interferon-g (IFN-g) and interleukin-17 (IL-17) promote the pro-inflammatory differentiation of immune cells and boost the cytotoxic immunity. [47, 48] Interestingly, as mentioned above, these immune cells can also participate in the cancer-promoting inflammation. For example, immunosuppressive subsets of T cells [e.g., regulatory T (Treg) cells), macrophages and DC support cancer development via the production of IL-1, IL-6, etc. (Figure 1.2). [49] Therefore, redirecting the balance between conflicting inflammatory responses of immune cells towards cancer destruction is centric in immunotherapeutic design.     9  Figure 1.2 Cancer, Inflammation and Immunity.  Inflammation and immunity can induce both immunosurveillance and wound-healing response-derived cancer-promoting events. The balance between anti- and pro-cancer inflammation depends on the immune cell subsets and activities. Effector anti-cancer immune cells inhibit cancer development via cytotoxic and/or cytokine-mediated mechanisms. In contrast, cancer-promoting immune cell subsets and cytokines dampen immunosurveillance and act on malignant cells to tilt the balance towards cancer progression. Modified from Grivennikov 2010. [36]    1.2.2 Effector T (Teff) Cells in Immunosurveillance As shown in Figure 1.2, effective immunosurveillance is mostly mediated by the effector T (Teff) cells that are capable of inducing a pro-inflammatory anti-cancer response. Cluster of differentiation 8 positive (CD8+) cytotoxic T lymphocytes (CTL) are typically believed to be the major mediators of cytotoxicity. These cytotoxic T cells directly kill foreign invaders or cancer cells by recognizing peptide-major histocompatibility complex (MHC) class I complexes (Figure 1.3 A). [50] Upon recognition, two effector mechanisms of CTL can be activated to destroy the target cells. One is the granule exocytosis pathway, via which CTLs elicit cytoplasmic granules perforin and granzymes. [51, 52] Perforin is a pore forming protein which Inflammation and ImmunityImmunosurveillanceImmunosurveillanceWound-healing ResponseCancer-promoting InflammationT cells, NK cells, DCT cellsMacrophagesDCCancerTNF, IL-6, IL-11, IL-1IL-1, IL-6, IL-17, IL-23IFN-후, IL-17, !GM-CSF, IL-12FasL, TNF, TRAIL, IFN-훂/훃  10 opens up the membrane of target cells for successful granzyme translocation. [53] Granzymes are a family of serine proteases that function as the dominant cytolytic granules in activating the cell-death pathway. [54] The other CTL killing pathway occurs via the expression or release (e.g., in the form of exosomes) of death ligands such as the Fas ligand (FasL) and tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL). [55–57] Upon interaction with death receptors on the target cells, these death ligands activate the caspase cascade, leading to cell rupture. Both killing pathways, in most cases, trigger programed intracellular events and apoptotic cell death. [58, 59]   Figure 1.3 CD8+ and CD4+ T Cell-Mediated Cancer Cytotoxicity.  Panel A. CD8+ cytotoxic T cells recognize MHC class I molecules expressed on cancer cells. The ligation of CD28 with CD80/CD86 helps activate T cell signaling. Activated CD8+ T cells secrete cytolytic granules or express cell death ligands (FasL and TRAIL) causing direct killing of cancer cells. Panel B. CD4+ T helper cells recognize MHC class II molecules, mostly expressed on the antigen presenting cells (APC), which present the cancer antigen to T cells. Activated CD4+ T cells kill cancer cells either directly via secreted granules or cytokines in a similar manner to CD8+ T cells, or indirectly by attracting and activating other immune cells, such BAssistClass II MHCCD3 CD4CD80!CD86CD28CD4+ T CellMacrophageTh17TregTh2Th1Attract & ActivateIL-2!IFN-후,!TNF-훂IL-4,!IL-5, IL-10IL-17A,!IL-21,!IL-22IL-10,!IL-35,!TGF-훃Cancer CellClass I MHCCD3 CD8CD80!CD86CD28CD8+ T CellFasL, !TRAILGranulesAIFN-후GranulesExosomeDampenAPCT-bet STAT1GATA3STAT6ROR후tSTAT3FOXP3  11 as macrophages. T cell subsets are differentiated from naïve CD4+ T cells upon activation. Th1 and Th17 assist CD8+ T cells in priming and functioning, while Treg cells dampen Teff cell responses. The activity of Th2 cells in cancer immunity remains controversial.  CD4+ T cells provide assistance in improving the anti-cancer efficacy of CD8+ T cells. [60–63] They play a vital role in the activation and expansion of reactive CD8+ T cells, as well as the generation and maintenance of long-lived memory CTL. [62, 64–67] In addition to helping CD8+ cells kill cancer, CD4+ T cells have been discovered to either directly or indirectly kill cancer cells. [68] CD4+ T cells that recognize antigens through an MHC class II-restricted manner directly kill cancer cells by releasing cytolytic granules or expressing death ligands (Figure 1.3 B), analogous to cytotoxic CD8+ T cells. [69–79] MHC class II molecules are expressed on DC, macrophages, and B cells, but few are expressed on cancer cells, explaining why CD8+, but not CD4+ T cells are predominant in direct cancer killing. [80, 81] For cancers lacking MHC class II molecules, CD4+ T cells are able to attract and activate inflammatory cells such as macrophages, to either process and present tumour antigens or to kill the cancer cells directly. [68, 82, 83] Secreted IFN-g from CD4+ T cells can also mediate the cytotoxicity [84–89] and/or upregulate the expression of MHC class II molecules on cancer cells. [70, 90]  More importantly, CD4+ and CD8+ T cells have synergistical roles within the tumour microenvironment and in cancer immunotherapies. [82, 91, 92] For example, both cancer-specific CD4+ and CD8+ T cells are enriched at the tumour site, impacting the course of cancer development. [93] Approaches co-transferring CD4+ and CD8+ T cells to mice revealed a dramatically elevated survival rate and complete tumour regression in animals synergistically treated with both populations, in comparison to the transfer of CTL alone. [94] The dynamic balance between CD4+ and CD8+ T cells can serve as a biomarker of Teff cell activities, tumour progression, and therapeutic efficacy. [95–97] Hence, CD4+ and CD8+ T cells play equally   12 important roles in anti-cancer inflammatory responses. Singular and synergistic efficacies of either population should be evaluated in cancer immunotherapeutic approaches.  Upon activation, naïve CD4+ T cells can differentiate into one of several subsets, as defined by their function and pattern of cytokine production and function (Figure 1.3 B). [98] Type 1 T helper (Th1) cell differentiation is mostly regulated by the T-box transcription factor (T-bet) and the signal transducer and activator of transcription 1 (STAT1). [99] T-bet significantly enhances the production of IFN-g which in turn activates STAT1. [47, 48, 99, 100] IFN-g is essential for the activation of immune cell phagocytosis and the release of IL-2, which is an important T cell growth factor. [99, 101] In contrast, Type 2 T helper (Th2) cell differentiation is suppressed by T-bet but upregulated by GATA3 (GATA-binding protein) and STAT6. [99, 102] Th2 cells are capable of secreting IL-4, IL-5, and IL-10, conveying controversial effects in cancer immunity. [103] Retinoic acid receptor-related orphan receptor gamma-T (RORgt) is the master regulator of Type 17 T helper (Th17) cell differentiation and induces the production of IL-17A. [47, 48, 99] STAT3 binds to IL-17A promotors and enhances RORgt expression. [99, 104] In our previous and current studies, Th17 cells are specifically used as a surrogate of Teff cells. Th17 cells are characterized by their capability to secrete IL-17A, IL-17F, IL-21, IL-22 and chemokine (C-C motif) ligand 20 (CCL20) which can break immune tolerance and facilitate the eradication of melanoma cells in mice. [105–108] Nonetheless, controversial effects of Th17 cells in tumourigenesis and immunosuppression have also been studied. [105, 109] The significance of analyzing the dynamics between Th17 and other immune cells have been underlined, as the cancer and immunity context governs the function of Th17 cells. [105, 109] One important dynamic is observed between Th17 and Treg cells. In contrast to Teff cells, Treg cells, which express transcription factor forkhead box P3 (FOXP3), suppress   13 inflammatory responses; thus, dampening the anti-cancer activities. [110, 111] Treg cells release immunosuppressive cytokines, such as IL-10 and transforming growth factor-b (TGF-b), and can compete for IL-2 with Teff cells, resulting in a skewed survival/response of Teff versus Treg cells. [112, 113] The Th17:Treg cell ratio was employed in our study to reflect the overall pro-inflammatory responses and anti-cancer T cell efficacy potentially induced by the acellular therapeutics.  Moreover, natural killer T (NKT) cells are another important Teff cell subset in cancer. These cells share physical and functional characteristics of both NK cells and T cells. [114] NKT cells recognize lipid antigens presented by CD1d, a nonclassical MHC molecule. [115] Similar to most immune cells in the tumour inflammatory environment, subsets of NKT cells play distinct and opposing roles. In cancer, type I or invariant NKT (iNKT) cells play a mostly protective role by releasing IFN-g to activate CTL, DC, and NK cells. [115, 116] Indeed, upregulation of the iNKT cell response in the mouse model leads to cancer rejection by up-regulating the costimulatory molecules for Teff cell priming. [117, 118] In contrast, type II NKT cells produce Th2-like cytokines, such as IL-4, and primarily inhibit tumour immunity. [116, 119] This thesis study focuses on the responses of Teff (CD4+, CD8+, Th1, Th17, and iNKT) cells and the Teff:Treg cell ratio.  1.2.3 Cancer Cytotoxicity via Cell-Cell Communication and Interaction The control of cancer cell proliferation and migration by Teff cells is achieved through cell-cell communication and interaction. [120] Two types of cell-cell crosstalk pathways have been discovered thus far: direct and indirect communication. [121] Direct pathways require cell-cell contact (e.g., death receptor ligation) and involve intercellular channels to allow the transfer of molecules (e.g., perforin and granzymes). One typical example is cytolytic granule-mediated   14 cancer cell death. [53] Cytotoxic lymphocytes (e.g., CTLs, NK cells, CD4+ T cells) recognize target cancer cells and conjugate with them, forming a biological synapse (i.e., gap junction) between the two. The cytotoxic secretory granules are then fused with the presynaptic membrane and released from the lymphocytes into the synaptic cleft. As mentioned above, perforin creates transmembrane pores at the postsynaptic membrane of target cells for the successful diffusion of granzymes into the target cell cytosol. [51–53] After granzymes have initiated the apoptosis of cancer cells, the cytotoxic lymphocytes detach from the dying cell, moving forward to interact with another target cell. A similar direct junction has also been described between cancer cells and macrophages, endothelial cells, or DC, as well as in the antigen cross-presentation between DC and Teff cells. [122] Immunotherapeutic adoptive T cells have been reported to simultaneously conjugate with multiple cancer cells, exerting multi-killing effects. [123] Importantly, the number of conjugations modulate the propensity and kinetics of T cell mediated apoptosis, thus allowing the magnitude of conjugation to be used as a parameter to evaluate the immunity-mediated cancer cell elimination.  Within indirect communication pathways, the cell-cell crosstalk occurs in a contact-independent manner. This allows immune cells to target distant cancer cells through the secretion of bioactive molecules, including cytokines, chemokines, and exosomes. [121] Cytokines and chemokines are accepted into the target cells by binding to cell surface receptors, where they regulate the cell fate in a process called paracrine signaling. [124] Exosomes have emerged as a newly discovered platform of cell-cell communication. [125] They function as a bi-directional mediator between the immune and cancer cells, underlining the complexity of cancer immunosurveillance. [126–128] Within the area of exosome research, the small non-coding microRNA (miRNA) molecules have attracted the most interest due to their transmittable post-  15 transcriptional regulation of gene expression. [129–131] Discoveries pertaining to exosomal miRNA-mediated immune activation and/or cancer inhibition will be discussed in Section 1.5. The pathways of intercellular communication between immune and cancer cells are summarized in Figure 1.4.   Figure 1.4 Interaction of a Cytotoxic Lymphocyte with a Cancer Cell.  The cytotoxic lymphocyte conjugates with the cancer cell resulting in death receptor ligations or the transmission of cytolytic granules (A. Direct Killing). Perforin and granzyme are released from the cytotoxic lymphocyte to the synaptic cleft. Perforin forms pores for the diffusion of granzyme into the cancer cell, resulting in target cell apoptosis. Cytokines, chemokines, and exosomes secreted from cytotoxic lymphocytes can also target cancer cells via paracrine signaling or miRNA-mediated gene expression in an Indirect Killing pathway (B). Modified from Trapani 2002. [54]  1.2.4 Measurement of Immune Cell-Mediated Cancer Cytotoxicity  The ultimate goal of Teff cells in cancer is to achieve death of cancer cells. The ‘gold standard’ of measuring immune cell-mediated cytotoxicity is the radioactive chromium (51Cr)-release assay. [132–134] This assay is based on the detection of a 51Cr probe, released from the lysed target cells, allowing the quantification of cytolytic activities of immune cells against cancer cells. [132] Despite still being widely used in laboratories due to its reproducibility, drawbacks of the 51Cr-release assay, including biohazard concerns about radioactive materials, the short half-life of the isotope, and a low level of sensitivity, have been noted. [135, 136] Therefore, alternative nonradioactive methods have been reported. The enzyme-linked immunospot (ELISpot) assay measures cell-mediated cytotoxicity from a different aspect – effector cell function – instead of target cell death. The ELISpot assay enumerates the cytokines Cancer CellCytotoxic LymphocytePerforin GranzymeExosomeExosome ExosomeCytokine/ChemokineA. Direct KillingB. Indirect KilingSynapseDeath ReceptorsDeath Ligands: FasL, TRAIL  16 derived from the immune-cancer cell interactions by means of antibody-bound plates or nitrocellulose membranes. [137] Several advantages of the ELISpot assay are the usage of a low number of effector cells, its high sensitivity, and its specificity in measuring immune proteins and lymphocytes frequency. [136] Indeed, results from clinical trials have indicated its suitability for monitoring T cell responses. [138] Another popular measurement of cancer specific T cell cytotoxicity is the flow cytometric assay. Flow cytometry rapidly measures the fluorescence of single cells which correlates with cell size, structure, and other physical aspects. [136] Owing to the advances in laser and fluorochrome technology, comprehensive measurements can be obtained by labeling cells with a combination of antibodies against surface or intracellular antigens. [139–141] Therefore, the cancer cell death and immune cell activity, which are singularly measured by the 51Cr-release and ELISpot assays, respectively, can be simultaneously monitored via flow cytometry. Nonetheless, none of these methods provides real time measurements of cell proliferation/killing kinetics. Accordingly, modern approaches have been developed to meet this need, especially in the context of drug discovery and immunotherapy. [142, 143] One approach has been the development of the ACEA iCELLigenceÒ system, which monitors the increase in electrode impedance induced by cell (e.g., cancer or non-cancerous cells) proliferation and attachment. [144, 145] Studies using this technology have shown good correlation with results determined by conventional methodologies. [146, 147] Moreover, this real-time assay has been increasingly accepted in evaluating therapeutic efficacies of cancer treatment approaches. [146, 148–152] In these studies, it was shown that the impedance curves correlated well with cell proliferation. Importantly, non-adherent cells such as lymphocytes, do not impact the impedance readings allowing for the measurement of lymphocyte-mediated   17 inhibition of cancer cell proliferation. A more detailed description of the ACEA technology can be found in Chapter 2.  1.2.5 Cancer Immunotherapy Based on an understanding of how immune cells function in controlling cancer development, immunotherapies have been designed to direct anti-cancer inflammation for efficient immunosurveillance performance. The theory behind immunotherapy originated from William Coley’s methods to treat cancer patients with bacteria in order to induce an inflammatory response that could exert a bystander effect on the tumour mass. [153–156] Starting in 1891, Coley injected more than 1,000 cancer patients with different types of bacteria or bacterial products known as Coley’s Toxins. [157, 158] Despite being named the ‘Father of Immunotherapy’, Coley’s approach garnered criticism from the medical community. Coley’s Toxins gradually disappeared and were supplanted by the newer, and ‘safer’, developments of radiation therapy and later chemotherapy – which, we now know, themselves pose both short- and long-term risks to the patient. Today, almost 130 years after Coley’s Toxins made their initial debut, modern immunotherapy has begun to revisit Coley’s core principles of inducing an endogenous inflammatory response.  1.2.5.1 Current Approaches Similar to Coley’s use of Streptococcus pyogenes and Serratia marcescens, bacterium-mediated tumour therapies using genetically modified strains of Salmonella sp. have recently been used to induce an anti-cancer inflammatory microenvironment at the tumour site. [159, 160] In addition, many other immunotherapeutic strategies, including cytokines, cancer vaccines, oncolytic viruses, immune checkpoint blockades and adoptive cell transfer (ACT) therapies, have been proved promising. [161, 162] Cytokines such as IL-2 and IFN-a stimulate the host’s   18 overall inflammatory response via Teff cell expansion and activation. [163] Recombinant poliovirus has recently been used as a therapeutic cancer vaccine to enhance host Teff cell responses in glioblastoma patients, leading to dramatically improved survival rates and reduced risk of metastatic spread. [164] Cancer cells from patients can also be isolated, irradiated, and modified for re-infusion into the patient, in an attempt to activate the host’s immune response and improve tumour killing. [165–169] Oncolytic virus therapy employs viruses that infect and lyse cancer cells, followed by an induction/initiation of systemic anti-cancer immunity subsequent to cancer antigen exposure. [170, 171] To overcome tumour associated immunosuppression, immunomodulatory checkpoint inhibitors have also been developed to release the immunological brakes on T cell activation as a means of treating cancer. [172–174] Accumulated evidence has confirmed the immune activation by using programmed death protein-1 (PD-1), programed death ligand-1 (PD-L1), or cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) inhibitors in treating cancer. [174] But, the most favoured approach has been the application of tumour-specific ACT immunotherapy and the development of chimeric antigen receptor (CAR)-T cell therapies. [175]  T cell-focused ACT immunotherapy was not established until the identification of tumour-associated antigens (TAA), demonstrating that T cells could distinguish cancerous from healthy cells. [176, 177] Contrary to other types of powerful immunotherapies (e.g., checkpoint inhibitors) described previously, ACT ‘creates’ rather than ‘unleashes’ a TAA-specific productive immune response. [178] Therefore, this approach is most applicable to non-responsive patients and non-immunogenic tumours.  ACT immunotherapy is most clearly illustrated by CAR-T therapy. CARs are hybrid receptors formed by a cancer-specific ligand-binding domain, a CD3-z activating moiety derived   19 from T cells, and a co-stimulatory signaling domain. [179–181] To date, two CAR-T cell drugs have been approved by the Food and Drug Administration (FDA) and have achieved success in treating B cell lymphoblastic leukemia and diffuse large cell lymphoma. [175, 182, 183] In addition to CAR-T cells, another two possible sources (Figure 1.5) of cancer-specific T cells for ACT immunotherapy are tumour-infiltrating lymphocytes (TIL) and antigen-specific peripheral blood lymphocytes (PBL). TIL are naturally enriched with cancer-specific T cells, [184] while the antigen-specific expansion of PBL can be achieved via genetic engineering [i.e., manually encoding the desired T cell receptor (TCR) specificity to T cells]. [185] All these ACT approaches are harnessing the patient’s immune system by infusion of the ex vivo expanded T cells that recognize, target, and destroy cancer cells.   Figure 1.5 The Process of Adoptive T Cell Immunotherapy.  Patient T cells are harvested from either tumour-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL). TIL are preferentially cancer-specific, so they can be expanded non-specifically. PBL can be engineered into either specific tumour-associated antigens (TAA) or CAR-T cells prior to culture. After weeks of ex vivo cell expansion in culture, cancer-specific T cells can be re-infused into the patient. Modified from Perica 2015. [178]  Unfortunately, most ACT approaches to enhance the T cell-mediated killing of cancer cells are expensive and require significant ex vivo processing. Costs ranging from $50,000 to $500,000 per course of treatment have been reported depending on cell culture protocols, labour, and patient preparation. [186, 187] Given the difficulties in identifying patient-specific TAA, the limited source of TAA-responsive T lymphocytes, and cell transportation both from and back to T Cell SourceCultureExpansionReinfusionAdoptive T Cell Immunotherapy- CAR-T !- TIL!- TAA-specific PBL  20 the patient, ex vivo preparation of T cells takes weeks; accordingly, patients are therefore hospitalized for weeks. [178] Side effects (e.g., cytokine release syndrome) and other issues, such as the immunosuppressive cancer microenvironment, also need to be investigated. [183, 188] To achieve a safer and more cost-effective outcome of cancer rejection with the adopted T lymphocytes, combination therapy has been recommended. [189, 190] New ACT adjuvant strategies that can accelerate the ex vivo cell expansion process would also be beneficial. 1.2.5.2 Strategies for T Cell Activation and Expansion in the Laboratory Normally, lymphocytes are activated upon ligation of their antigen receptors with specific cognate antigens. [191] However, because of the low frequency of antigen-specific lymphocytes, and difficulties in identification and isolation, agents that nonspecifically activate T cells in the absence of antigens have been used to systemically boost an anti-cancer pro-inflammatory response. [192] This approach is exemplified by the pan-activation of the entire T cell repertoire via mitogens, cytokines, or monoclonal antibodies (mAb). Mitogens have always been regarded promisingly, as they trigger massive division of T lymphocytes, including Teff cells, capable of killing cancer cells. For example, phytohaemagglutinin (PHA) is a lectin found in plants that can recognize and agglutinate the glycosylated proteins on the T cell surface to trigger mitosis. [193, 194] Cytokines, such as IL-2, a general T cell growth factor, has been used to stimulate a broad-based pro-inflammatory response. [195] Similarly, mAb therapies, such as anti-CD3 and anti-CD28, potently induce T cell proliferation by directly binding to the TCR-associated, activation-related chains to initiate signaling pathways. [196] However, consequent to the overly robust T cell response arising from these pan T cell agents, cytokine release syndrome is often induced resulting in multi-organ failures, severe morbidity, and increased mortality – leading to the   21 suspension or abrogation of multiple clinical trials. [195, 197–200] Thus, alternative approaches to activate endogenous T cells in a controlled manner, with less toxicity, are needed.  1.3 Allorecognition and Pro-Inflammatory Anti-Cancer Responses 1.3.1 T Cell Allorecognition Allorecognition of T cells is discriminative compared to pan T cell activation, as only 1-10% of T cells are alloreactive. [35, 201] Alloresponse studies were originally established in the 1940s and 1950s, when lymphocyte-dependent adaptive immune responses were found to mediate graft rejection. [35] The MHC was then discovered and named based on its role in murine graft rejection. [202] Now, the molecular basis – MHC polymorphism – underlying the allorecognition between MHC molecules, or human leukocyte antigen (HLA) complex molecules, is well understood. Every human being expresses six class I HLA alleles and more than six class II HLA alleles; each HLA allele can be expressed from hundreds of gene alleles. [35] Because these polymorphic HLA genes can be inherited and expressed in many different combinations, it is quite likely that one individual bears some HLA proteins that appear foreign to another individual (except for identical twins). [35] Consequently, TCR that evolve to recognize non-self MHC molecules trigger potent T cell activation and alloresponses.  There are two modes of TCR allorecognition: direct and indirect pathway modes (Figure 1.6). [203] The direct pathway is initiated by T cells recognizing intact allogeneic MHC molecules. In contrast, the indirect pathway involves presentation of processed allo-peptides bound to self-MHC molecules on recipient antigen presenting cells (APC). Interestingly, despite both CD4+ and CD8+ T cells participating in MHC allorecognition, CD4+ T cells exclusively recognize allo-antigens through the indirect pathway, while CD8+ T cells recognize the allo-MHC molecules in a direct fashion. [204] Moreover, murine studies have revealed that CD4+ T   22 cell-activated indirect allorecognition exhibits early proliferation by producing IL-2 cytokine, dominating the initial alloresponse. In contrast, CD8+ T cells exert later, but prolonged, responses via IFN-g mechanisms and usually require help from CD4+ alloreactive T cells to fully activate/differentiate. [203, 204] Importantly, CD4+ T cell-mediated indirect allorecognition is sufficient to reject allo-grafts, and thus constitutes an essential route of rejection. CD8+ T cells activated via the direct pathway, however, are not fully competent and cannot contribute to rejection alone. [204]  Figure 1.6 Direct and Indirect Pathways of Allorecognition.  Panel A. Direct pathway: Recipient CD8+ T cells directly recognize the donor APC through TCR-MHC binding. APC-presented antigen can be either generic or donor-derived. Panel B. Indirect pathway: Donor MHC peptides are processed by the recipient APC and are presented to the recipient CD4+ TCR via the self-MHC molecule. Modified from Boisgérault 2001. [204]   Despite a ‘low’ frequency of alloresponsive T cells, allorecognition is one of the strongest immune responses; a single allogeneic graft cell can express thousands of MHC molecules, every one of which may be recognized as foreign by recipient T cells. [35] In contrast, in the case of an infected cell, only a small fraction of self-MHC molecules on self-APC will present the foreign microbial peptide to the host T cells. [35] Not surprisingly, the alloresponse has been studied in the context of cancer immunity for decades. [205–208] A. Direct pathwayRecipient T CellsB. Indirect pathwayDonor APC Recipient APCDonor MHC PeptideRecipient T CellsGeneric PeptideCD8 CD4Allo-MHC Self-MHCTCR TCR+ +  23 1.3.2 Allostimulatory Anti-Cancer Approaches A strong alloresponse is believed to promote an effective systemic T cell response against cancer antigens through several mechanisms and encompasses both CD4+ and CD8+ T cells. [209, 210] For example Fabre et al. demonstrated that allo-generated CD4+ T cells significantly enhanced the responses to cancer peptides and bypassed the need for presentation of cancer peptides by self-MHC class II molecules. [211] Further enhancing the anti-cancer effect, the alloresponse gives rise to an environment rich in cytokines and soluble T cell costimulatory ligands that may aid in overcoming T cell anergy and cancer tolerance. [211] Multiple avenues of research have focused on the ability of alloresponses to suppress cancer. As mentioned above, Coley’s Toxins inspired the use of bacteria or viruses to infect cancer patients. These infections create an immunostimulatory milieu, either locally or systemically, leading to dramatically improved survival rates and reduced risk of metastatic spread. [159, 160, 164, 212, 213] Hematopoietic stem cell transplantation induces potent graft-versus-malignancy responses in the recipients. [187] Researchers have also used the fusions between allogeneic DC and syngeneic cancer cells to promote effective T cell responses to carcinomas. [206, 207] These fused cells express all of the DC’ allogeneic human HLA molecules that stimulate a T cell alloresponse used to provide a cross-activation of a patient’s naïve T cells, as well as all the patient’s HLA molecules that are responsible for self-MHC restricted cancer peptide presentation. In therapeutic vaccination studies, patients or animals treated with allogeneic, same-type, whole-cell cancers have demonstrated an induced rejection of malignant gliomas [208] or prostate cancer. [205] The allorecognition reactions caused by the allogeneic cancer cells overcome the immune tolerance to syngeneic cancer antigens, resulting in the inhibition of cancer growth or complete cancer elimination.    24 Despite anti-cancer efficacies, these allorecognition-based therapeutics demonstrate potential risks of Graft versus Host Disease (GvHD); the carry-over of allogeneic cells has limited their practical utility. Hence, the development of a cell-free approach mimicking the induction of MHC-dependent allorecognition could be of clinical significance and simultaneously dramatically reduce the risk of adverse events.  1.4 Cell-Free Approaches and Acellular Therapeutics  Cell-free approaches have long been of interest to researchers, as biomarkers or as a replacement for cell-based therapeutics in drug design. [214–216] Indeed, unlike cell-based therapeutics, acellular therapeutics are not dependent on cell behaviours and have a wider range of applications. Even though the cell-free synthetic biology has emerged as a powerful engineering technology to design proteins, metabolites, and even artificial cells, with unprecedented freedom, acellular products from bioreactor systems are irreplaceable in reflecting the complete and comprehensive biological responses of living cells. [216, 217] Cell secretomes, i.e. conditioned media, fall within this bioreactor acellular category.  1.4.1 Cell Secretome and Acellular Conditioned Media  The emergence of the cell secretome concept can be traced back to two decades ago, as a consequence of the stem cell theory. [218] Research discovered that cell-secreted paracrine factors, in the absence of donor cells, were beneficial in tissue regeneration and wound healing leading to the concept of cell-free therapies. [219, 220] Indeed, secretomes from endothelial progenitor cells can achieve a therapeutic angiogenic effect equivalent to cell therapy. [221] Similarly, adipose-derived stem cell secretome promotes better mouse liver regeneration than cell-based therapy does. [222] Despite the absence of cells, cell secretome contains a variety of biologically active components that include proteins, lipids, miRNA, and extracellular vesicles,   25 such as exosomes and microparticles. [223] With the exceedingly rapid development of cell culture techniques at the beginning of the twenty-first century, the concept of cell secretome has been cemented into a pre-clinically applicable format – acellular conditioned media. Not surprisingly, the soluble components released from the cultivation systems can characterize the origin and features of cultured cells or tissues. Indeed, conditioned media from cell lines are widely used to reflect genotypic and phenotypic characteristics of the investigated primary tissues. [224] Conditioned media derived from various cell types or different responses exert distinct biological functions. Consequently, the generation of conditioned media is well-designed for its intended purpose. For example, exposure of human macrophages to conditioned media from mitogen-stimulated lymphocytes protects the macrophages from cholesteryl ester accumulation. [225] Conditioned media from rat spleen cells activated with the same mitogen increase the neuritogenesis and survival of retinal cells. [226] Furthermore, diverse key active components have been identified between conditioned media generated from various culturing systems. For example, the activities of macrophage-activating factor (MAF) and IFN had been detected in the conditioned media from chicken lymphocytes. [227] Dissimilarly, in conditioned media from amniotic membrane cells, secreted prostaglandins were identified as key effectors of the immunomodulatory activity. [228] Moreover, bioactive exosomes have been detected in the apoptotic peripheral blood mononuclear cell (PBMC)-derived conditioned media that attenuate spinal cord injury in rats. [218, 229, 230] These promising preclinical data encouraged the initiation of a clinical trial using human PBMC secretomes. [231] When considered in combination, these studies have highlighted the enormous potential of cell secretomes and acellular conditioned media in both research fields and clinical practice.    26 1.4.2 Mixed Lymphocyte Reactions (MLR)   In the Scott Laboratory, mixed lymphocyte reaction (MLR)-induced allorecognition is the foundational response for the manufacturing and assessment of acellular therapeutics. MLR were originally used to study the potential donor-recipient incompatibility of graft transplants. [232] MLR generate an ex vivo cellular immune response between two allogeneic lymphocyte populations, designated as Stimulator and Responder cells, from HLA-disparate individuals. The severity of lymphocyte reactions, including cellular proliferation, DNA synthesis, and blast transformation, indicate, but not exclusively, how much HLA differs between the Stimulator and Responder cells. [233] MLR have been used in many other animal species, in fact, chicken leukocytes reacted in MLR, demonstrating that T cells mediate the major responding reactions. [234] In later studies, this T cell dominant response in MLR has also been proved in human and murine models. [235, 236]  For research purposes, the MLR is used as an in vitro cell-based assay to evaluate the recognition of allo-antigens by T cells. [237] The magnitude of the MLR response is proportional to the extent of the HLA differences between these individuals. There are two types of MLR assays, one-way and two-way MLR, both of which are conducted via co-culturing Responder cells with Stimulator cells. However, in a one-way MLR, only the Responder lymphocytes proliferate, while the Stimulator cells are pretreated with irradiation or mitomycin C (a DNA crosslinker), to prevent cell replication. [238] In contrast, both Responder and Stimulator cells can respond and proliferate in a two-way MLR. In this study, in order to achieve a maximal allorecognition-based immune response, two-way MLR were used.    27 1.4.3 The Scott Laboratory Allorecognition-Based Acellular Therapeutics The initial acknowledgement of allorecognition modulation through polymer grafting dates back two decades, when the Scott Laboratory extended the scope of ‘stealth cells’ from red blood cells (RBC) to white blood cells (WBC). [239–241] The chemical camouflage of RBC antigens precede a successful blood transfusion, [239, 240, 242–248] while the polymer grafting of WBC provide support for safer transplantation. [241, 249–253] Indeed, by covalently modifying the cell surface of lymphocytes using methoxypoly(-ethylene glycol) (mPEG) polymer, MHC class II-mediated T cell alloproliferation is dramatically inhibited due to TCR non-recognition of allogeneic antigens presented by MHC molecules. [241] Later on, similar immunotolerogeneic effects are also found to be exerted by the cell secretomes derived from these immunocamouflaged T cells and their responder cells in mPEG-MLR. [253] By applying polymer grafting to one population of lymphocytes in an initial MLR, tolerogeneic conditioned media can be generated to induce an inhibited allorecognition and thus allo-proliferation in a second, fresh, MLR via direct administration to the cell culture. [253] The conditioned plasma produced in a similar way from mice challenged with immunocamouflaged allogeneic cells is also able to shut down the alloresponse in an in vitro MLR in a dose-dependent manner. [254] More importantly, these tolerogeneic conditioned plasmas re-orientate the systemic Teff and Treg cell balance to arrest the progress of autoimmune type 1 Diabetes (T1D) in non-obese diabetic (NOD) mice. [254] Also of significance, a complex of miRNA species was found to account for the immunotolerogeneic role of the conditioned media or plasma and is denoted as Tolerogeneic Agent 1 (TA1). [253, 254] The ‘pattern of miRNA expression’, which encompasses increasingly-, decreasingly-, and steadily-expressed miRNA, reflects the anti-reductionist approach of TA1 manufacturing and the complex systemic regulation by TA1.   28 Even though the tolerogeneic TA1 therapeutic derived from mPEG-immunocamouflage has been well illustrated in treating autoimmune diseases, the pro-inflammatory therapeutic Inflammatory Agent 1 (IA1), which was used as an important control of TA1, has not been explored. IA1 was concurrently generated with TA1, but only from a fresh control MLR secretome or allo-challenged mice. Contrary to the immunosuppression induced by TA1, IA1 is hypothesized to promote a pro-inflammatory response of T cells, which is essential for the treatment of immunodeficiency or cancer (Figure 1.7). This study aims to systemically investigate immune activation by therapeutic IA1 for the goal of cancer cell attenuation. The performance of secretome miRNA-mediated immunomodulation will be tested. A deuterogenic preparation of Inflammatory Agent 2 (IA2), derived from the lymphocyte-to-cancer cell allorecognition, will also be discussed regarding potential cancer specificity. Allorecognition, which is modeled by MLR in vitro, is the foundational bioreactor response for producing and assessing IA1 or IA2 preparations.    29  Figure 1.7 The Scott Laboratory Allorecognition-Based Acellular Therapeutics.  A pair of acellular therapeutics have been developed in the Scott Laboratory. The secretome preparations are derived from allorecognition responses modeled by the MLR in vitro. The tolerogeneic TA1 therapeutic is generated from the polymer-camouflaged mPEG-MLR secretome, while the pro-inflammatory IA1 therapeutic is derived from the fresh MLR secretome and functions as an important control for TA1 in preliminary studies. A ‘pattern of miRNA expression’ (Panel A) has been identified in both therapeutics and accounted for the vastly different biological effects of TA1 and IA1 (Panel B). TA1 maximizes the function of tolerogeneic T cell populations (green shaded area) and potentially other immune cell types (blue shaded area) but reduces the effects of pro-inflammatory cells (red shaded area). TA1-induced immunosuppression and tolerance has successfully arrested the progression of autoimmune diseases (i.e., T1D) in the NOD mice model. In contrast, the IA1 secretome-based therapeutic enhances the pro-inflammatory populations but inhibits the tolerogeneic responses. The role of IA1 in activating the pro-inflammatory Teff cells for cancer treatment has been investigated in this study. Modified from Wang 2015 and Kang 2017. [250, 254]  1.5 MicroRNA (miRNA) miRNA are short [~22-nucleotide (nt)-long] RNA molecules that regulate messenger RNA (mRNA) expression at a posttranscriptional level in diverse eukaryotes. Currently, more than 2,000 miRNA have been identified in humans. [255] miRNA are synthesized within cells but can exist either intracellularly or extracellularly. Extracellular, especially exosome-associated, miRNA have gained increased attention during the past decade, their utility in cancer TA1Pro-inflammatory PopulationsSecretome-miRNA Therapeutic(Protective)Th2MemoryTh1Th17TregAnergyB CellAPCNaiveExhaustionHelperNKCTLTolerogeneic PopulationsIA1Tolerance?Secretome-miRNA TherapeuticEnhanced Immune ActivityThis Study} }2-Fold2-FoldLog2 Fold Regulation (vs naive miRNA)}hsa-let-7a-5phsa-let-7e-5phsa-miR-132-3phsa-miR-135b-5phsa-miR-147ahsa-miR-149-5phsa-miR-155-5phsa-miR-183-5phsa-miR-203ahsa-miR-206hsa-miR-21-5phsa-miR-214-3phsa-miR-9-5phsa-miR-363-3phsa-miR-34a-5phsa-miR-302a-3phsa-miR-298hsa-miR-27b-3phsa-miR-27a-3p-5 0 5MLR v mPEG-MLRMLRmPEG-MLR∆ 2-FoldabVolcano PlotClustergramIA1	TA1	hsa-miR-147a	has-miR-9-5p	has-miR-155- p	 hsa-miR-206	hsa-miR-302a-3p	hsa-miR 135b-5p	 hsa-miR-214-3p	hsa-miR-149-5p	hsa-miR-183-5p	 hsa-miR-203a	hsa-miR-36 - p	 hsa-miR-21-5p	hsa-miR-27a-3p	hsa-miR-27b-3p	 hsa-miR-298	hsa-miR-34a-5p	 hsa-let-7a-5p	hsa-let-7e-5p	hsa-miR-132-3p	"pattern of miRNA expression"AB  30 treatment as biomarkers or therapeutic targets will be described below. miRNA are highly conserved, and their high multiplicity underlies the investigation into networks of miRNA expression instead of single miRNA knockout or upregulation.  1.5.1 Discovery of miRNA The first miRNA, lin-4, was found in Caenorhabditis elegans in 1993. [256] The lin-4 gene does not code a protein, but negatively regulates gene lin-14 at a posttranscriptional level to control the development pattern of larval stages. [256–258] Moreover, the lin-4 transcripts are small. [256] In 2000, another miRNA, let-7, which also regulated the proper timing of C. elegans development, similar to lin-4, was discovered. [259] Both lin-4 and let-7 complementarily bind to the sequence in the 3’ untranslated regions (3’UTR) of the target gene, leading to a model of miRNA regulation through antisense interactions. [256, 258–261] However, unlike lin-4, let-7 is conserved across species and was recognized in humans and bilaterian animals soon after its discovery in C. elegans. [262] This finding triggered the research into this new class of non-coding RNA. These RNA were called ‘microRNA’ because by then, the only thing known about them was that they were small. [263, 264] Lin-4 and let-7 are named based on their mutant phenotypes, but subsequently discovered miRNA are named with numbers to indicate the order of their discovery. [265] Orthologs from different species are given the same name. Similar names are assigned to paralogs within a species, with letter suffixes (a, b, c, …) distinguishing genes producing similar mature miRNA, and number suffixes (-1, -2, -3, …) distinguishing genes producing identical mature miRNA. [266] miRNA are grouped into families based on their targeting properties. [267] Usually, miRNA of the same family are evolutionarily related and evolutionarily related miRNA are   31 members of the same family. [266] The conservative characteristics of miRNA will be described in Section 1.5.6.  1.5.2 Biogenesis of miRNA miRNA are initially transcribed in the genome by RNA polymerase II (Pol II) as long primary miRNA (pri-miRNA). [266, 268–270] Each pri-miRNA molecule forms a hairpin substrate and contains a cap structure at the 5’ end. [271, 272] Pri-miRNA are processed by the Drosha endonuclease which cuts the stem of the pri-miRNA hairpin, liberating a 60-110 nt stem-loop called precursor-miRNA (pre-miRNA). [272, 273] Pre-miRNA are exported from the nucleus to the cytoplasm through the action of Exportin-5. [274–276] In the cytoplasm, pre-miRNA are further processed by Dicer-1 endonuclease that cleaves the loop to generate a short imperfect double-stranded miRNA duplex with a 5’ phosphate (P) and 2nt 3’ overhang on each end. [273, 277] This duplex is then unwound by a helicase; the ‘guide’ strand becomes the mature miRNA, while the ‘passenger’ strand is usually discarded and degraded (Figure 1.8). Once the mature, single-stranded, ~22 nt miRNA are formed, they are loaded into an Argonaute (Ago) protein family member, forming the functional RNA-induced silencing complexes (RISC). [278, 279] Usually, the miRNA-loaded RISC are very stable, with a half-life of days. [280, 281] Human cells express four different Ago proteins (Ago1, Ago2, Ago3, and Ago4) that compose RISC and mediate gene silencing, but only Ago2 possess endonuclease activity, therefore bestowing it with mRNA cleavage capabilities. [282–284]   32  Figure 1.8 The Biogenesis of miRNA.  Once transcribed by Pol II, the long pri-miRNA fold back on themselves to form a double-stranded hairpin structure. The 5’ end of the pri-miRNA is capped. Dorsha cuts the stem of the hairpin and releases the 5’ phosphate (P) in the pre-miRNA. The pre-miRNA are exported from the nucleus to the cytoplasm by Exportin 5, followed by the cleavage of the hairpin loop by Dicer-1, to form the short miRNA duplex with a 5’ P and 2nt 3’ overhang on each end. One strand of the miRNA duplex, the mature miRNA, is incorporated into an Ago protein to form the RISC. The other strand is usually degraded. Modified from Bartel 2018. [266]  1.5.3 miRNA-Mediated Post-Transcriptional Gene Regulation The mature miRNA within the RISC can target mRNA using the respective nucleotide complementarity. [285] The pairing of nucleotides 2-7 at the 5’ end of the miRNA, known as the miRNA ‘seed’, is believed to dominate the regulatory function of miRNA. [267] Indeed, the 5’ region of miRNA and the 3’ untranslated regions (3’ UTR) of target mRNA are found to be particularly conserved, indicating the preferential interactions of the miRNA ‘seed’ with the 3’ UTR. [263, 286, 287] There are two consensus models of miRNA-mediated silencing of gene expression. Based on the degree of miRNA-mRNA complementarity, the target mRNA can be subject to cleavage or translational repression (Figure 1.9). In animals, when miRNA are miRNA GenePri-miRNAPre-miRNAPre-miRNAmiRNA DuplexPPCapRISCAgoNucleusCytoplasmPol IIDorshaExportin 5Dicer-1PPPP  33 completely complementary to the target mRNA, the mRNA are cleaved by Ago2 protein and further degraded by exonucleases. [288] When, in most cases, the complementarity between miRNA and mRNA is incomplete, translational repression occurs. [285] The Ago1-4 proteins recruit GW182 proteins, and the mRNA are decayed through the deadenylation, decapping, and 5’-to-3’ exonuclealytic decay process. [288–291]   Figure 1.9 miRNA-Mediated Silencing of Gene Expression.  The activity of miRNA on target mRNA bases on the base pair complementarity. When there is a complete miRNA-mRNA complementarity, Ago2 protein cleaves the target mRNA. When the miRNA-mRNA complementarity is incomplete, the Ago1-4 proteins recruit the GW812 proteins to induce translational repression. Modified from Bartel 2009. [267]  Multiplicity is a common feature in miRNA-mediated regulation. [292] Due to the short length (6-8 base pairs) and imperfect complementarity of mRNA 3’UTR, miRNA have the potential to target hundreds of different mRNA. [267, 293, 294] On the other hand, one mRNA 3’UTR often contains multiple binding sites for the same or various miRNA, allowing for this complex network of regulation. [285, 295] The ‘low fidelity’ of the miRNA system makes the GW182Ago1-4Ago2RISCAgoCap AAA Cap AAAmRNAComplete miRNA-mRNA ComplementarityIncomplete miRNA-mRNA ComplementaritymRNATranslational RepressionmRNA CleavageDeadenylationDecapping & 5'-to-3' decay  34 targeting of a single miRNA species of likely limited therapeutic value. Hence, the Scott Laboratory consciously chose an anti-reductionist approach to produce a relatively complex ‘pattern of miRNA expression’ that mimics normal biology in order to achieve maximal biological functionality. [253, 254] 1.5.4 Intracellular and Extracellular miRNA Little is known about the intracellular localization of miRNA and RISC until recently. A common assumption that miRNA-mediated post-transcriptional gene silencing occurs in the cytoplasm dominated the field for decades. [285] Recently, the detection of Ago proteins and RISC have also been confirmed in a wide range of cellular organelles, including endoplasmic reticulum (ER), Golgi apparatus, lysosomes, endosomes, and even the cell nucleus and mitochondria. [285, 296–299] Importantly, the localization of miRNA-associated Ago proteins is dynamic, and the distribution can depend on the translational status of the cell, [300] the cell cycle phase, [301] and post-transcriptional modifications such as phosphorylation or hydroxylation. [302, 303] Additionally, miRNA have been detected outside the cell in virtually all known body fluids and cell secretomes suggesting their role as signaling molecules (Figure 1.10).    35  Figure 1.10 Intracellular and Extracellular miRNA.  Intracellular miRNA can be widely located in all cellular compartments, including ER, Golgi apparatus, lysosomes, endosomes, nuclei, and mitochondria - where they exert various regulatory activities. The functional miRNA-RISC can also be secreted outside the cell, as part of the secretome, in the form of free-floating Ago2/miRNA complexes, microvesicles, exosomes, apoptotic bodies and HDL. It has been suggested that these extracellular miRNA act as signaling molecules. Modified from Makarova 2016. [285]  The presence of miRNA in blood plasma/serum was initially observed 10 years ago. [304, 305] Later cancer research revealed that cell-free miRNA can derive from both host and cancer cells, presenting in a nuclease-resistant form and demonstrating stability in blood plasma/serum. [306, 307] Soon after, miRNA were discovered in all other body fluids, including saliva, urine, breast milk, tears, etc. [308–311] Similarly, mammalian cells in culture have also been reported to release extracellular miRNA into the cell secretomes and culture media. [130, 312–314] Nonetheless, the packaging format and release mechanism of extracellular miRNA remains controversial.  Ago2/miRNAHDLMicrovesicleApoptotic BodiesNucleusERMitochondriaEndosomeExosomeGolgi ApparatusRISCLysosomeSecretome  36 To date, more than 90% of the extracellular miRNA are detected as solely free-floating complexes associated with Ago2 in both blood plasma/serum and cell-conditioned media. [315, 316] The stability of Ago2 perfectly protects the extracellular miRNA from denaturation. [316] Mature miRNA incorporated in RISC can also be enwrapped into membranous particles and transmitted intercellularly. Examples include 100-1000 nanometer (nm) microvesicles shed from the cell membrane, 40-100 nm endosome derived exosomes, 1-4 µm apoptotic bodies generated during cell apoptosis, and lipoproteins such as high-density lipoproteins (HDL). [317–320] Despite variable forms of extracellular miRNA, most are not selectively released or biologically functional. [321] Indeed, the Ago2/miRNA complexes are reported to be non-specific byproducts resulting from physiological activity and the death of cells. [316, 322, 323] Exosomes are currently the only form of extracellular miRNA proved to be selectively generated and released. [315, 323] 1.5.5 miRNA-Containing Exosomes Exosomes constitute an important component of the cellular secretomes, enwrapping proteins, DNA, RNA and most importantly, miRNA. [130, 324] Actually, the detection of miRNA in exosomes [> 100 kilodalton (kDa)] exported from cultured cells occurred even before the discovery of extracellular miRNA in biological fluids. [130] Factors including the post-transcriptional modification of miRNA and the levels of target mRNA have indicated the existence of specific mechanisms for miRNA sorting into exosomes. [325–327] Notably, this membranous vesicle provides perfect protection for extracellular miRNA, leading to their remarkable resistance to ubiquitous RNases. [328]  Moreover, the membranous endosome-derived exosomes were found to be formed by selective inward budding of the cell plasma membrane (Figure 1.11) in response to cellular   37 stimulations, suggesting that exosomes (in particular miRNA) function as intercellular message transmitters in addition to prognostic biomarkers. [329, 330] Indeed, accumulating evidence has demonstrated the exosome-mediated genetic exchange between host cells or host and cancer cells. [130, 331, 332] This idea is further reinforced by evidence of exosome intake pathways, including endocytosis, fusion with the plasma membrane, [333, 334] and the ligation of DC exosomes with signaling receptors on target cancer cells (Figure 1.11). [335] Upon engulfment by the recipient cells, miRNA are released from exosomes. These free miRNA are then able to target mRNAs in the recipient cells, causing the conventional degradation or transcriptional inhibition. [336] A novel function of cancer-secreted exosomal miRNA-binding to intracellular Toll-like receptors (TLR) in immune cells has also been identified during cancer progression. [337, 338] miRNA-containing exosomes have gained great attention regarding their potential regulatory effects and delivery capability.    Figure 1.11 Formation, Secretion and Transmission of Exosomes.  Exosomes are membranous vesicles ranging from 40-1,000 nm and can be released from all types of cells. Exosomes are formed by inward budding into early endosomes from the cell plasma membrane. More differentiated multivesicular endosomes either fuse with the plasma membrane to release the exosomes, or fuse with lysosomes for content degradation. Secreted exosomes can be engulfed by the recipient cells via endocytosis, membrane fusion or receptor ligation. Once intake occurs, miRNA will be released to target mRNA. Novel functions of cancer-secreted exosomal miRNA can be mediated by binding to intracellular TLR in the recipient immune cells. Modified from Raposo 2013. [329]  Early EndosomeInward BuddingExosomesMultivesicular EndosomesLysosomeCell PlasmaReleasing miRNATLRTargeting mRNAEndocytosis Fusion!Ligation Cell CompartmentsRecipient Cell   38 1.5.6 miRNA Are Evolutionarily Conserved and Exhibit Cross-Species Efficacy As indicated by let-7 and other miRNA families above, the gene regulation of miRNA has notably been highly conserved evolutionarily and has shown significantly less inter-species variability. [295] Indeed, 90 families of mammalian miRNA have been conserved since their progenitors and most of these miRNA have important biological functions. [266] Recent findings have suggested the conservation and transmission of miRNA between species, facilitating intercellular crosstalk in a cross-species, or even cross-kingdom manner, between mammals, plants, parasites, etc. [339–344] For example, mouse mast cell miRNA can be transferred to human mast cells via exosomes. [130] Mammals are able to intake plant exosomal miRNA through food; these exogenous miRNA serve functions similar to endogenous miRNA. [345] In turn, mammalian miRNA can also regulate protozoan gene expression, as shown by the translocation and translational repression of 100 human miRNA in the malaria parasite Plasmodium falciparum. [346] Notably, a small group of poorly conserved miRNA exist in mammals, but they tend to be lost due to a lack of retention-favoured targeting interactions. [266, 347, 348] Substantial variation in the editing efficiency of miRNA, depending on cell lineage, tissue, etc., has also been observed. [349] Nonetheless, the broad-spectrum conservation of miRNA has implied their evolutionary importance in gene regulation and provided a window into the benefits of using animal models when studying human diseases in preclinical research. [350] 1.5.7 miRNA Are Biomarkers in Cancer To date, the vast majority of research into the role of miRNA in cancer has been largely observational, with specific miRNA being used as biomarkers of disease. The first hint of miRNA’s involvement in cancer was reported in 2002 when miR-15 and miR-16 were found to   39 be dysregulated in chronic lymphocytic leukemia (CLL). [351] Since then, miRNA were discovered to have roles in all of the cancer hallmarks, including proliferation, migration, and metastasis (Figure 1.1). More recently, the discovery of circulating miRNA provokes the application of miRNA as non-invasive biomarkers. To date, miRNA detection and expression in serum and plasma has been described for several cancer types, including leukemia, lymphoma, breast, ovarian, prostate, and hepatocellular cancer. [352] Due to their rapid response and high sensitivity to cellular environment alterations, miRNA are also potential biomarkers for drug efficacy prediction and treatment therapy decisions. [353] Similarly, miRNA expression patterns in lymphocytes which monitor cancer development have been of interest to researchers. Primary activation of naïve T cells is shown to affect intracellular miRNA expression. [354–356] For example, upon TCR activation, miR-155 is upregulated upon T cell activation and its expression is proportional to the strength of  TCR signaling, [357, 358] while miR-31 is downregulated post TCR activation. [359] Moreover, the regulation of miRNA in response to external stimuli is dynamic during T cell activation and differentiation stages. [360–362] Anti-CD3/anti-CD28 activation results in an early expression of miR-18a and miR-155, whereas the expression of miR-451, miR-21, and miR-146a is identified in a later stage. [359] Hence, the intracellular miRNA expression profiles can be useful in imaging the epigenetic modulation networks during T cell activation. 1.5.8 miRNA-Based Cancer Therapeutics In addition to biomarkers, the regulatory function of miRNA in cancer triggers the design of miRNA-based therapeutics. For example, anti-miRNA using modified antisense oligonucleotides are used to target upregulated miRNA. [363] miRNA mimics that use synthetic RNA duplexes can be recognized by and incorporated into RISC, thus allowing the targeting of   40 mRNA, similar to endogenous miRNA. [364] Systemic delivery of exogenous miRNA has also been achieved; promising miRNA delivery systems include viruses, vectors, nanoparticles, and exosomes. [365, 366] Not only are miRNA involved in the fate of cancer cells, but they also play a key role in immune regulation, which significantly sculpt cancer development. miRNA control lymphocyte development, TCR signaling, cytokine production and secretion, and immunotolerance. [367–370] Interestingly, a miRNA pattern encompassing both upregulated and downregulated miRNA contributes to human T cell acute lymphoblastic leukemia (T-ALL); it is possible that a combination of knockout and upregulation of target miRNA will inhibit malignancy. [370–372] Actually, to date, no singular miRNA-based therapy has proved broadly effective. The ‘pattern of miRNA expression’ raised in the Scott Laboratory in contrast, would be of more therapeutic utility. [253, 254] Importantly, emerging evidence demonstrates that the miRNA-containing exosomes (Figure 1.11), as part of the secretome, released from both immune and cancer cells have systemic effects. [353] These biological functions are of great interest, especially in the field of host-cancer communication via exosomal miRNA (Figure 1.4). By using a secretome approach, we hypothesized that the miRNA enwrapped in exosomes could enhance an anti-cancer immune response in resting T cells.  1.6 Hypothesis and Specific Aims An individual’s immune system is a continuous balancing act between tolerance and inflammation. Cancers may occur when this balance is skewed towards a tolerogeneic state consequent to the loss of the inflammatory response to abnormal cells. [49] Despite the potential role of the immunity, most anti-cancer therapeutics have historically been cytotoxic drugs. These   41 cytotoxic drugs typically targeted all proliferating cells thus killing not only the cancer cell but also other proliferating cells (including immune cells further depressing the inflammatory response) leading to significant toxicity to normal cells and tissues. [31, 32] Consequent to this problem, research and clinical efforts have more recently become focused on enhancing the individual’s own immune response to cancer cells.  The T lymphocytes play a critical role in the anti-cancer responses. Even though immune checkpoint (e.g., PD-1/PD-L1 and CTLA-4) blockades have emerged as a promising approach to ‘release the brakes’ of the immune system to combat cancer, most strategies have been focusing on accelerating the inflammatory response of Teff cells which include: CD8+ CTL and CD4+ cells such as Th1 and Th17. Current nonspecific activators of T cells include mitogens (e.g., phytohemagglutinin, PHA) and mAb (e.g., anti-CD3 and anti-CD28). However, consequent to the overly robust T cell response arising from these pan T cell agents, a cytokine release syndrome was often induced leading to multi-organ failures, severe morbidity and increased mortality leading to the suspension or abrogation of multiple clinical trials. [195, 197–200] More recently, to improve antigen specificity, CAR-T therapy has been developed and shown to be a highly promising approach to enhancing the endogenous immunological response to cancers. [182, 183] While effective in early clinical studies, CAR-T therapy is expensive and has similarly shown adverse effects (e.g., cytokine release syndrome). [183, 188] Therefore, new ACT adjuvant strategies, which could activate endogenous T cell responses in a controlled manner with less toxicity, have been recommended to achieve a more cost-effective outcome. [189, 190] Allorecognition and proliferation exhibits several potentially desirable biologic characteristics with regards to Teff cell generation, function and cancer killing. Allorecognition   42 generates a more discriminatory T cell response as only 1-10% of an individual’s T lymphocytes are alloresponsive. [35, 201] Despite the ‘low’ number of potentially reactive cells, the alloresponse is still quite potent as exemplified by the severity of GvHD. Not surprisingly, the alloresponse has been studied in the context of cancer immunity for decades. [211] While the current cell-mediated allorecognition-based therapeutics demonstrated anti-cancer efficacies, the potential risk of GvHD has limited its practical utility. Hence, the development of a secretome approach mimicking the induction of MHC-dependent allorecognition could be of significant clinical utility and simultaneously dramatically reduce the risk of GvHD and other adverse events.   While the active components within the secretomes were traditionally viewed as paracrine factors (e.g., cytokines), the secretome contains a variety of biologically active components that include proteins, lipids, miRNA as well as extracellular vesicles (exosomes and microparticles). [223] Not surprisingly, the secretome components released are defined by their cell/tissue origin as well as the physiologic activation state of the cells. Consequently, secretomes will exert different biologic responses. [224, 226–228] Indeed, previous studies in the Scott Laboratory using human or murine MLR models demonstrated that we could generate either tolerogeneic or pro-inflammatory secretomes (i.e., conditioned media or plasma) by regulating the strength of the allorecognition response. [253, 254] Recently, the miRNA-containing exosomes, which constitute an important part of secretomes, are extensively investigated due to their biological functions and drug delivery capability. [130, 315, 323, 331, 332] Currently, miRNA are most commonly used as disease biomarkers versus therapeutic agents due to the inherent regulatory complexity of miRNA in which a single gene can be influenced by multiple miRNA and a single miRNA can interact with   43 multiple genes. [272, 292] The ‘low fidelity’ of the miRNA system makes the targeting of a single miRNA species of likely limited therapeutic value. In contrast, the Scott Laboratory therapeutics mimicking the ‘complex patterns of miRNA expression’ that are seen in vivo are capable of inducing either systemic tolerance or inflammation. [250, 253, 254] A previously studied tolerogeneic preparation TA1 inhibits Teff cell production and increases the Treg cell function thus prevent the autoimmune diabetes in NOD mice through a miRNA-induced regulatory pathway. [254] This study investigated a pro-inflammatory therapeutic IA1 produced using a lymphocyte-based secretome approach from ex vivo allorecognition reactions. A lymphocyte-cancer cell (HeLa) biotherapeutic IA2 was concurrently developed to explore the potential cancer type specificity. The pro-inflammatory effects of IA1 and IA2 on Teff cell enhancement and Treg cell inhibition in promoting an anti-proliferative response on cancer cells were the focus of this dissertation.  It is our hypothesis that the novel biomanufactured, acellular, allorecognition-derived, secretome-based immunotherapeutics (IA1 and IA2) can promote a pro-inflammatory Teff cell response capable of attenuating cancer cell proliferation. To test this hypothesis, the specific aims (Figure 1.12) were:  1. To biomanufacture the acellular therapeutics from in vitro and in vivo allorecognition systems. In vitro mixed lymphocyte reactions (MLR; human and mouse) and in vivo alloresponsive animals (mouse) were used for manufacturing of acellular secretome-based therapeutics. The biomanufacturing time was optimized based on the proliferation of resting human PBMC in vitro. The measuring time of efficacy was optimized in both human and murine Resting Lymphocyte and MLR models. Efficacy assessment and characterization of IA1 were conducted via size fractionation studies and purification of miRNA-containing exosomes. Due to   44 the conserved nature of miRNA, cross-species efficacy of IA1 and IA1 exosomes was also assessed in the human and mouse models.   2. To assess the pro-inflammatory effects of acellular therapeutics on leukocytes in vitro. Both in vitro human and murine Resting Lymphocyte models were tested for T cell proliferation and subset differentiation (i.e., CD4+ T, CD8+ T, Th1, Th17, Treg and iNKT cells) via flow cytometry. Effects of IA1 and IA2 were compared to potent T cell activators (i.e., allo-stimulation, anti-CD3/anti-CD28 and PHA) as well as control and tolerogeneic (TA1) preparations. Intracellular miRNA expression of activated human PBMC was also profiled via quantitative reverse transcription polymerase chain reaction (qRT-PCR). To examine if IA1 could enhance the existing pro-inflammatory response, the T cell responses to IA1 stimulation in MLR models were similarly assessed.  3. To examine the anti-proliferative effects of acellular therapeutics-activated leukocytes on cancer cells in vitro. The anti-proliferative effects of IA1 (complete and fractions)/IA2-activated leukocytes (total population and subsets; e.g., CD4+ and CD8+ T cells) on cancer cells were studied using the ACEA iCELLigenceÒ instrument in vitro. Human epithelial cervical cancer (HeLa) and melanoma (SH-4) cell lines were used. Direct toxicity of acellular therapeutics alone was similarly tested on both cancer models. The conjugation formation between PBMC and HeLa cells were assessed via time-lapse microscopy and flow cytometry. Toxicity of IA1 and IA2 on resting leukocytes (PBMC and splenocytes), and the effects of IA1/IA2-activated leukocytes on non-cancerous (mouse myoblast) cells were also examined.    45  Figure 1.12 Outline of Project.  To develop ‘safer’ cell-free allorecognition-based anti-cancer therapeutics, in vitro and in vivo secretome approaches were used in both human and mouse models. Optimization of manufacturing and efficacy characterization were conducted via size fractionation and miRNA-containing exosome purification. Cross-species efficacy between human and mouse models was assessed to confirm the role of miRNA. The effects of IA1 and IA2 on leukocyte proliferation, subset differentiation and miRNA expression were tested in comparison to allo-stimulation as well as potent T cell activators anti-CD3/anti-CD28 and PHA. Cancer-inhibitive effects of these activated leukocytes were further examined on cancer cell proliferation and immune-cancer cell conjugation. Toxicity of IA1/IA2 itself or IA1/IA2-activated leukocytes on non-cancerous cells was also investigated. Comparative studies of IA1 versus IA2 will be discussed accordingly in both Aim 2 and 3.   Altogether, in order to develop a ‘safer’ anti-cancer therapeutic obviating the risk of GvHD, allorecognition-derived, secretome-based acellular therapeutics were investigated. The acellular miRNA-mediated immunomodulatory effects of these therapeutics were characterized and compared to the potent T cell activators. Importantly, the activity of acellular therapeutics in enhancing the anti-cancer effects of resting leukocytes would suggest their role in adjuvating autologous ACT immunotherapy. The successful development of this secretome-based approach could prove useful in enhancing the endogenous immunosurveillance for cancer.  AIM 3AIM 2Biomanufacturing of the acellular therapeutics from in vitro and in vivo allorecognition systems. Pro-inflammatory effects of IA1 and IA2 on leukocytes in vitro . Anti-proliferative effects of IA1- and IA2-activated leukocytes on cancer cells in vitro.Biomanufacturing & OptimizationPBMC miRNA ExpressionSize Fractionation & Exosome PurificationCross-species EfficacyT Cell ProliferationT Subset DifferentiationToxicity to Non-cancerous CellsCancer Cell ProliferationPBMC-HeLa ConjugationDonor A LymphocytesDonor B LymphocytesAllo-MLRDonor A LymphocytesAllogenic Cancer CellsCancer-MLRIA1IA2 Pro-Inflammatpry Leukocyte ResponsesCancer CellsPrimary CellsIA1/IA2 !Direct ToxicityImmune ActivationAcellular PreparationAIM 1HYPOTHESIS:  The novel biomanufactured, acellular, allorecognition-derived, secretome-based immunotherapeutics (IA1 and IA2) can promote a pro-inflammatory  Teff cell response capable of attenuating cancer cell proliferation.IA1 versus IA2Anti-Proliferative Effects  46 Chapter 2: Methods and Materials 2.1 General Methods As described in the Chapter 1, numerous immunological methods were utilized to both biomanufacture, and to assess the immunomodulatory and cancer-inhibitive activity of our acellular therapeutics. Several methods were common to all three specific aims and are described in detail below. Experimental conditions unique to a single Specific Aim are described within the Specific Aim section. 2.1.1 Human Peripheral Blood Mononuclear Cells (PBMC) Isolation To manufacture human-sourced acellular therapeutics and assess their immunomodulatory effects, human peripheral blood mononuclear cells (PBMC) were used. All experiments using human blood cells were done following the protocol approved by the University of British Columbia Clinical Research Ethics Board and in accordance with the Declaration of Helsinki. Following informed written consent, donor whole blood was collected in heparinized Vacutainer® blood tubes (BD, Franklin Lakes, NJ). Then 2 parts of whole blood were gently overlaid over 1 part of Histopaque-1077 (Sigma-Aldrich, St. Louis, MO) creating two-phases of whole blood and Histopaque in a 50 milliliter (ml) conical tube (Figure 2.1). [252, 253] After a centrifugation for 30 minutes at 900 gravitational force (´ g) without break, the PBMC layer was sterilely transferred to a new tube and washed twice with RPMI 1640 (with L-glutamine; Invitrogen by Life Technologies, Carlsbad, CA) containing 25 millimolar (mM) 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.01% human albumin (Sigma-Aldrich, St. Louis, MO). Isolated human PBMC were cultured with AIM V media (research grade; ThermoFisher Scientific, Grand Island, NY).    47  Figure 2.1 Human PBMC Isolation.  Freshly collected human donor whole blood was overlaid over Histopaque at a 2:1 ratio. After a centrifugation for 30 minutes at 900 ´ g with no break, the PBMC layer was carefully isolated for downstream assays.   2.1.2 Mouse Splenocyte Isolation To manufacture murine-sourced acellular therapeutics and assess their immunomodulatory effects, mouse splenocytes were isolated from immunocompetent mice. All murine experiments were done in accordance with the Canadian Council of Animal Care and the University of British Columbia Animal Care Committee guidelines and were conducted within the Centre for Disease Modeling at the University of British Columbia. Two MHC (i.e., mouse H-2 haplotype complex) disparate mouse strains were used: C57BL/6, H-2b; and BALB/c, H-2d. Murine spleens were freshly harvested from carbon dioxide (CO2) euthanized mice and homogenized into a cell suspension in phosphate buffered saline (PBS; 137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, and 1.76 mM KH2PO4, pH 7.4) containing 0.2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO) using the frosted end of two microscope slides. [250, 254] RBC were removed from the cell suspension using BD Pharm Lyse buffer (BD Pharmingen, San Diego, CA). Purified mouse splenocytes were cultured in RPMI 1640 media supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products, West Sacramento, CA), 1% L-glutamine, 1% penicillin-streptomycin and 50 micromolar (µM) ß-mercaptoethaniol (all from Invitrogen by Life Technologies, Carlsbad, CA).    48 2.1.3 Mixed Lymphocyte Reactions (MLR) As described in the introduction, previous work within the Scott Laboratory demonstrated that both tolerogeneic and pro-inflammatory acellular therapeutics could be prepared from lymphocytes using the mixed lymphocyte reactions (MLR). [250, 253, 254] Hence, the two-way MLR were used both to biomanufacture and to assess the pro-inflammatory efficacy of the acellular therapeutics in vitro (Figure 2.2). In brief, PBMC from two MHC-disparate human donors or splenocytes from two mouse strains were resuspended in appropriate cell culture media (i.e., AIM V for PBMC, FBS supplemented RPMI 1640 for splenocytes) at a final cell density of 2 × 106 cells/ml. An equal number (1 × 106) of cells from each donor were plated in multiwell flat-bottom 24-well tissue culture plates (BD Biosciences, Discovery Labware, Bedford, MA). Negative control wells consisted of PBMC or splenocytes alone from one cell donor (‘resting cells’) and were used to assess any background cell activation during leukocyte isolation. Mixed or resting cells were incubated in a humidified, 5% CO2 incubator at 37 degrees Celsius (37° C) and harvested for downstream assays at various timepoints as indicated. In some studies, mPEG-MLR were conducted by grafting PBMC from one of the human donors with 20 kDa succinimidyl valerate activated (SVA) mPEG (Laysan Bio Inc. Arab, AL) at a concentration of 2.0 mM per 4 × 106 cells/ml as previously described. [253, 254] By using the ‘resting cell’ and MLR system, we can biomanufacture our acellular therapeutics. Moreover, these two models were also used to assess the efficacy of derived preparations. Resting Lymphocyte represents a patient model in which no pro-inflammatory T cell responses exist and MLR stand for a model bearing limited pre-existing T cell activation.   49  Figure 2.2 Mixed Lymphocyte Reactions (MLR).  Lymphocytes were isolated from human whole blood or mouse splenocytes. Resting cells were human PBMC or mouse splenocytes from the same donor/strain, generating no proliferation post cell culture. MLR were conducted by mixing lymphocytes from two MHC-disparate donors thus an alloproliferation was induced. In some studies, one population of lymphocytes were PEGylated. The polymer grafting immunocamouflaged the allorecognition and inhibited the alloproliferation.   2.1.4 Lymphocyte Proliferation and Immunophenotyping via Flow Cytometry T lymphocyte proliferation is indictive of immune activation and function. Hence, to assess the effects of acellular therapeutics on the activation of resting T cells or the enhancement of pro-inflammatory responses of T cells in an allorecognition (MLR), percent (%) lymphocyte proliferation was assessed in vitro via a dye-dilution assay using the CellTrace CFSE (carboxyfluorescein diacetate succinimidyl ester) Cell Proliferation Kit (Cat. No. C34554; CellTrace, Molecular Probes, Invitrogen, Eugene, OR). [250, 252–254] In brief, cell labeling was done according to the product insert at a final concentration of 2.5 µM CFSE per 2 ´ 106 cells total. CFSE-labeled cells were either incubated alone (in the Resting Lymphocyte model) or mixed with an allogeneic population of CFSE-labeled cells (in the MLR model) for appropriate days (determined in Specific Aim 1) in a humidified 5% CO2 incubator at 37° C. Cells were then harvested and washed before being assessed by flow cytometry, which measured the progressive halving of CFSE fluorescence following cell division (Figure 2.3). Usually, the proliferation assay was conducted in combination with phenotyping studies to reveal the responses of T cell subsets.  MLRDonor A+B PBMC!or Splenocytes +AlloproliferationRestingDonor A+A PBMC!or Splenocytes +No ProliferationmPEG-MLRDonor A+ mPEG-B !PBMC or Splenocytes +Inhibited Alloproliferation  50  Figure 2.3 CFSE Proliferation and Immunophenotyping via Flow Cytometry.  Freshly isolated human PBMC or murine splenocytes were counted and stained with CFSE. Stained cells were plated in a 24-well plate and cultured in a humidified 5% CO2 incubator at 37° C for desired days. Percent proliferation was assessed by the progressive halving of CFSE fluorescence following cell division by flow cytometry. CD3+ and CD4+ T cell subset and iNKT cell differentiation were measured by mAb staining using flow cytometry.   T lymphocytes are a diverse group of cells encompassing multiple subsets. To assess the effects of acellular therapeutics on subset differentiation, the T cell lymphocyte subpopulations (CD3+CD4+ and CD3+CD8+) were measured by flow cytometry using fluorescently labeled anti-CD3, CD4 and CD8 monoclonal antibodies (mAb; BD Pharmingen, San Jose, CA). Moreover, to assess the pro-inflammatory state of T cells, the ratio of Teff:Treg was examined. Th17 were used as the representative population of Teff based on an earlier study from the Scott Laboratory in NOD mice experiments. [254] Human and mouse Th17 and Treg subsets were measured using the BD Th17/Treg Phenotyping Kit (Cat. No. 560762 and 560767, respectively; BD Biosciences, San Jose, CA). The inflammatory Th17 lymphocytes were CD3+CD4+IL-17+ while Treg were CD3+CD4+FOXP3+. Other human inflammatory T cell subsets such as Th1 lymphocytes and type I or invariant NKT (iNKT) cells were stained as CD3+/CD4+/IFN-g+ and CD3+/6B11+ respectively. After the staining, the cells (1 ´ 106 total) were washed and resuspended in phosphate buffered saline (PBS with 0.5% BSA) prior to flow acquisition. Unstained controls   51 were used to determine background fluorescence. [250, 252–254] All samples were acquired using the FACSCalibur flow cytometer and CellQuest Pro software (BD Biosciences, San Jose, CA) for both acquisition and analysis (Figure 2.3). By assessing the T cell proliferation and subset differentiation in both Resting Lymphocyte and MLR models, we can determine the pro-inflammatory potential of our secretome-based acellular therapeutics. 2.1.5 Statistical Analysis: A minimum of three independent experiments were performed for all studies. Normally distributed data were expressed as mean ± standard error mean (SEM). Non-normally distributed data and cell proliferation curves (i.e., ACEA iCELLigenceÒ studies) were expressed as median ± interquartile range (IQR). In some instances, representative histograms, microscopy images or median cell proliferation curves were presented. Data analysis was conducted using GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA). For statistical comparisons, parametric tests were used for normally distributed data while nonparametric tests were used for non-normal distributions. For comparison between two independent samples, an independent two-tailed t-test [parametric, assuming same standard deviation (SD)] or Mann-Whitney test (nonparametric, comparing ranks) was performed. For comparison of three or more samples, a one-way analysis of variance (ANOVA; parametric) or Kruskal-Wallis test (nonparametric) was performed. When significant differences were found, a post-hoc Tukey (parametric, correcting for confidence intervals and significance) or Dunn’s (nonparametric, correcting for significance without confidence intervals) multiple comparisons test was conducted for pair-wise comparisons. For significance, a minimum p value of < 0.05 (labeled with ‘*’) was used. The symbols ‘**’, ‘***’ and ‘****’ were used for labeling p value of < 0.01, < 0.001 and < 0.0001, respectively.   52 2.2 Specific Aim 1: Biomanufacturing of Acellular Therapeutics  The biomanufacturing of acellular pro-inflammatory preparations using the secretome approach was foundational to the thesis. The allogeneic biomanufacturing methods account for multiple variables including determining the optimal time of acellular therapeutic collection and time of efficacy assessment. Using our previous findings [241, 250, 253, 254] on the immunomodulatory activity of the supernatants collected from control and PEGylated human and mouse MLR, in vitro and in vivo biomanufacturing processes were designed to reproducibly generate our pro-inflammatory acellular therapeutics. 2.2.1 Biomanufacturing of the SYN, IA1, IA2, and TA1 Acellular Secretomes    As described above, acellular therapeutics were manufactured using either resting cells or the MLR-based reactions. In brief, the allogeneic preparation IA1 (IA1-Allo) was manufactured using PBMC or splenocytes from two MHC-disparate human or mouse donors (cell ratio 1:1) suspended in AIM V media. A final of 1 ´	106 total PBMC from each donor were plated in multiwell flat-bottom 24-well tissue culture plates (BD Biosciences, Discovery Labware, Bedford, MA). [253] Production of the allogeneic cancer cell-stimulated biologics IA2 (IA2-HeLa) was done using the modified MLR in which one PBMC donor was replaced with cultured (allogeneic) HeLa cells (PBMC:HeLa reaction). In brief, freshly isolated human PBMC were co-cultured with HeLa cells at a ratio of PBMC:HeLa = 50:1 in 24-well tissue culture plates. The final total PBMC number was 1 × 106. Production of the negative control biologics Syngeneic (SYN) media was accomplished from untreated single donor PBMC or splenocytes seeded at 2 × 106 cells per 24-well plate well.   For some studies, the effects of the pro-inflammatory IA1 and IA2 were compared to our previous described TA1 preparation. [254] The TA1 preparation, previously shown to inhibit   53 alloproliferation and to increase Treg and reduce effector T cells (Teff), was derived from an allogeneic human MLR in which one HLA-disparate PBMC population was modified by mPEG (20 kDa, 2 mM per 4 × 106 cells). [253, 254] Post-production processing and utilization of TA1 was otherwise identical to that of IA1. Following extensive optimization studies, all conditioned media were collected at 5 days post plating (Figure 2.4).    Figure 2.4 Manufacturing Scheme of Secretome Biotherapeutics.  Human PBMC or mouse splenocytes were freshly isolated from whole blood or murine spleens. SYN was derived from the secretome of resting lymphocytes while IA1 was produced using an allogeneic MLR. IA2 was produced from PBMC-HeLa cell reactions at a cell ratio of 50:1. TA1 was manufactured from mPEG-MLR where one donor’s PBMC were PEGylated with 20 kDa mPEG at a grating concentration of 2 mM per 4 × 106 cells. Secretomes were collected at 5 days post and further processed via centrifugation and 0.2 µm filtration. For some experiments, additional processing steps included size fractionation, exosome purification and miRNA isolation.  Post collection, the media was processed via centrifugation (400 × g; 10 minutes) to remove cells and cellular debris followed by a 0.2 µm syringe filtration step (Pall Corporation, Port Washington, NY). The processed media were aliquoted and stored in the -80° C freezer. Studies (not shown) demonstrated that freezing and thawing had no significant impact on the immunomodulatory activity of the processed media. For cell culture studies, the processed media were mixed 1:1 with fresh media and then seeded with the indicated cells. In some studies, the secretome therapeutics were further processed via molecular weight fractionation, exosome purification or miRNA isolation (Section 2.2.3). Acellular(Components:(0.2(µm(Filter(((±#M.W.#Frac+ona+on;#exosome#purifica+on;#miRNA#isola+on)Gra8ed(mPEGDonor 2IA1 !(IA1-Allo)SYN !IA2 !(IA2-HeLa)TA1 !(Tolerogeneic)Processing0 5DaysProcessingDonor 1MLRmPEG AllogeneicSyngeneicHeLaAllogeneic (*Resting Syngeneic  or Autologous)*  54 2.2.2 Optimization of the Biomanufacturing and Efficacy Assessment of Secretomes Key to the biomanufacturing of pro-inflammatory preparation IA1 is the lymphocyte allorecognition pathway (i.e., secretome). To determine the optimal timing for collection of human-sourced IA1, acellular media were collected at day 3, 5 and 7 post MLR. The secretome containing media were then used to stimulate resting PBMC for 10 days to assess their pro-proliferative effects on lymphocytes. The collection day of the media which exerted the maximal pro-proliferative effects was determined to be the optimal time of biomanufacturing for IA1, as well as for other acellular therapeutics in both human and mouse systems.    To assess the efficacy of acellular therapeutics on T cell proliferation, both the Resting Lymphocyte and MLR models were treated with IA1 collected at the optimal time point over 14 days. The lymphocyte proliferation was assessed at various day points throughout the culture process, and the maximal proliferation determined the optimal time of efficacy assessment. The human and mouse systems were optimized individually using their own-derived IA1 preparation, but the determined time of IA1 assessment applied to all other acellular therapeutics manufactured from the same bioreactor system (i.e., human or mouse). By optimizing the biomanufacturing and efficacy assessment, the maximal effects of acellular therapeutics can be achieved in enhancing a pro-inflammatory response and attenuating cancer cell proliferation.  2.2.3 Molecular Weight (M.W.) Fractionation and Exosome Purification of IA1 To determine the active component(s) of acellular media, the pro-proliferative effects of complete and molecular weight (M.W.) fractionated IA1 as well as IA1-derived exosomes were assessed. The fractionation studies were done using the Amiconâ Ultra-0.5 ml 30/100 kDa fractionation tubes (EMD Millipore, Billerica, MA). [254] As shown in Figure 2.5, complete media was centrifuged at 14,000 ´ g for 15 minutes to collect the filtrate. Another 2 minutes of   55 1,000 ´ g spin was conducted to recover the concentrated solute. Both the filtrate and the concentrate were scaled up to the volume of initial complete media with appropriate cell culture media for downstream assays. The immunomodulatory activity of both the filtrate and concentrate were assessed on resting lymphocytes using a 1:1 dilution with fresh media. To assess the role of cytokines and chemokines in the pro-inflammatory and cancer-inhibitive effects of IA1, the proliferative effects of the cytokine-rich fraction (< 30 kDa) was compared to complete media and ≥ 30 kDa fraction.  Figure 2.5 Size Fractionation Studies.  Up to 0.5 ml acellular media was added to each device with desired M.W. cut-off (e.g., 30 kDa). The device was put into a centrifugation tube and spun at 14,000 ´ g for 15 minutes. The filtrate (e.g., < 30 kDa fraction) was collected in the tube, while the concentrate (e.g., ³ 30 kDa fraction) was left in the device. To recover the concentrate, the device was turned upside down in a new clean tube and spun for another 2 minutes at 1,000 ´ g. Both the filtrate and concentrate were scaled up to the volume of initial complete media (i.e., 0.5 ml) with appropriate cell culture media for downstream assays.   While significant amounts of stable, non-complex/encapsulated miRNA have been previously found within the TA1 media [254], additional miRNA are bound to higher molecular weight Ago2 (~97 kDa) and in very large exosomes in the IA1 preparation. However, as introduced above, only miRNA associated with exosomes are selectively generated and secreted, while Ago2 bound miRNA are mostly byproducts of cell death. Hence, to further assess the role   56 of exosome-encapsulated miRNA, exosomes from IA1 acellular media were purified using a Total Exosome Isolation (from cell culture media) Kit (Cat. No. 4478359; Invitrogen by Life Technologies, Carlsbad, CA). Briefly, acellular media and the Total Exosome Isolation reagent were mixed and incubated at 4° C overnight. After incubation, samples were centrifuged at 10,000 ´ g for 1 hour at 4° C. The pelleted exosomes were resuspended in a sufficient volume of fresh (cell specific) tissue culture media to the volume of initial acellular media. The immunomodulatory activity of exosomes was assessed on resting lymphocytes using a 1:1 dilution with fresh media. By characterizing the effects of IA1 via M.W. fractionation and exosome purification, we can determine the active component(s) of acellular therapeutics and indicate future steps of miRNA purification.  2.2.4 Cross-Species Efficacy of IA1  Because miRNA are evolutionarily conserved, the cross-species efficacy of human- and murine-sourced IA1 on mouse splenocytes and human PBMC was examined.  In vitro production of human and murine IA1 was done similarly except for different growth media. Human PBMC were cultured in serum-free AIM V media while the culture of mouse splenocytes required additional growth factors provided mostly by the supplemented FBS. Therefore, human IA1 was accordingly supplemented with 10% FBS before being administrated to mouse splenocytes to ensure the baseline cell proliferation. Both resting human PBMC and mouse splenocytes were treated with human- and murine-sourced IA1, respectively. Cell proliferation and the Th17:Treg cell ratio were determined as previously described. In addition, the effects of exosomes isolated from human IA1 on mouse splenocytes were similarly examined. By assessing the cross-species efficacy of IA1 and IA1 exosomes, we can confirm the role of miRNA in mediating the pro-inflammatory response in T lymphocytes.    57 2.2.5 In Vivo Biomanufacturing of IA1-Plasma from Immunocompetent Mice In addition to the in vitro preparations, an in vivo IA-Plasma therapeutic was also manufactured using immunocompetent mouse models (Figure 2.6). Naïve C57BL/6 mice aged ~8 weeks were used as the recipients, BALB/c mice at the same age were used as the donors of allogeneic splenocytes. A total of 20 × 106 cells were intravenously injected. 5 days post challenge, the cell-free conditioned plasma was collected as IA1-Plasma. [254] Control preparations including Fresh- and SYN-Plasma were simultaneously manufactured by injecting PBS or syngeneic C57BL/6 splenocytes respectively. Acellular components were processed through centrifugation steps to remove RBC, WBC and platelets. At day 5 of plasma collection, animal body weight, spleen size and splenocyte phenotype were also measured to indicate the pro-inflammatory reactions from which IA1-Plasma was manufactured.  Figure 2.6 In Vivo Manufacturing of IA1-Plasma. Schematic summary of the manufacturing of IA1-Plasma from naïve mice. 20 × 106 allogeneic splenocytes freshly isolated from BALB/c mice were intravenously injected into ~8-week naïve C57BL/6 mice. 5 days post challenge, cell-free conditioned plasma was collected as IA1-Plasma. Controls including Fresh- and SYN-Plasma were manufactured from mice injected with PBS or syngeneic cells respectively. Acellular components were processed via centrifugation steps. Allogeneic BALB/c Splenocytes!Naïve	C57BL/6	Mice	IA1-Plasma!Day 5!	Control Plasma: Fresh- and SYN-Plasma from PBS and syngeneic splenocyte injected miceAcellular	Components:	Centrifuga>onal	Processing		6	20 x 10cells!total!  58 2.3 Specific Aim 2: Immunomodulatory Effects of Acellular Therapeutics on T Leukocyte Proliferation and Differentiation One problem with the current pan T cell activation strategies is the overly robust response which accounts for the systemic bystander injury. To compare our acellular therapeutics with the current T cell activation approaches, resting lymphocytes were stimulated with acellular miRNA-enriched cocktails in comparison to potent T cell stimulations. To examine the effects of IA1 on an existing pro-inflammatory response, the MLR model was stimulated for the assessment of T cell proliferation and subset differentiation as described below.  2.3.1 Comparative Effects of Acellular Therapeutics to Anti-CD3/Anti-CD28 and Mitogen T Cell Activation Approaches As previously noted, past clinical studies have examined the utility of allo-based and direct T cell (anti-CD3/anti-CD28 and mitogen) activation therapies in cancer treatment. Hence, the pro-proliferation potential of IA1 on resting PBMC was compared to these stimulations. Resting PBMC were treated with IA1 for 10 days and the allorecognition-based proliferation assays (MLR) were also conducted for 10 days. For anti-CD3/anti-CD28 activation assays, freshly isolated human PBMC were stimulated with plate-bound (24-well plate precoated with antibody) anti-human CD3e [5 microgram (µg) /ml; BD Pharmingen, San Diego, CA], in the presence of soluble anti-human CD28 (1 µg/ml; BD Pharmingen) for 3 days. For mitogen stimulation studies, PBMC were treated with PHA (Sigma-Aldrich, St. Louis, MO) at an amount of 2 µg per 1 × 106 total cells for 4 days incubation. T cell percent proliferation and differentiation post treatments were assessed via flow cytometry as described above. The comparative study can illustrate the efficacy and potential superiority of our therapeutics over current pan T cell stimulations.    59 2.3.2 Effects of IA1/IA2 Activation on Lymphocyte miRNA Expression Moreover, intracellular miRNA expression profiles of PBMC were compared following stimulation. In brief, total RNA was extracted from resting PBMC ± treatment (SYN, IA1, IA2, TA1, anti-CD3/anti-CD28 and PHA) post 72 hours incubation using the mirVanaTM PARISTM kit (Cat. No. AM 1556; Ambion, Life Technologies, Grand Island, NY). Total cellular RNA of the samples was prepared using the Agilent RNA 6000 Nano Kit (Cat. No. 5067-1511; Agilent Technologies, Santa Clara, CA). Sample RNA concentration and quality (e.g., integrity) was assessed using the Agilent 2100 Bioanalyzer System (Agilent Technologies).  Samples were stored at -80° C until further use. To partially characterize and quantify the relative abundance the miRNA species present in the resting and differentially activated PBMC, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was done using the miScript miRNA PCR Array system (Qiagen, Frederick, MD) for the human immunopathology pathway. These miRNA microarrays plates were run using an Applied Biosystems StepOnePlusTM Real Time PCR System (ThermoFisher Scientific, Grand Island, NY).  This human immunopathology array plate is pre-configured with the appropriate RNA and quality controls and has been validated by Qiagen. This array profiles the expression of 84 miRNA differentially expressed during normal and pathological immune responses (Table 2.1). It is worth noting that the 84 miRNA examined are not all inclusive and that other miRNA are likely to be present and could be of immunoregulatory importance. Threshold and baseline were defined and the resultant Ct (threshold cycle) values were calculated using the StepOnePlus software (v.2.1).  Ct values were exported and analyzed using the Qiagen GeneGlobe Online Analysis Center using the Relative Quantification qRT-PCR method for analysis (DDCt). The data shown represent three independent experiments using PBMC from the same donor.   60 Table 2.1 Human Immunopathology miRNA PCR Array: Functional miRNA Grouping for Target miRNA Genes in Array Plate Functional miRNA Grouping miRNA Genes Leukemia & Lymphoma: Hodgkin's Lymphoma: miR-125b-5p, miR-126-3p, miR-128, miR-130a-3p, miR-132-3p, miR-134, miR-135a-5p, miR-135b-5p, miR-138-5p, miR-142-3p, miR-142-5p,  miR-145-5p, miR-147a, miR-15b-5p, miR-181a-5p, miR-183-5p, miR-185-5p,  miR-200a-3p, miR-205-5p, miR-20b-5p, miR-21-5p, miR-23b-3p, miR-26a-5p,  miR-26b-5p, miR-27a-3p, miR-29b-3p, miR-302a-3p, miR-30c-5p, miR-31-5p,  miR-325, miR-335-5p, miR-34a-5p, miR-9-5p. Other: miR-15a-5p. Primary Effusion Lymphoma: miR-103a-3p, miR-106a-5p, miR-142-3p, miR-148a-3p, miR-152, miR-16-5p, miR-182-5p, miR-186-5p, miR-191-5p, miR-194-5p, miR-19a-3p, miR-210, miR-23b-3p, miR-26a-5p, miR-29b-3p, miR-30e-5p, miR-34a-5p. T-cell Leukemia: miR-106a-5p, miR-132-3p, miR-18b-5p, miR-19b-3p, miR-20b-5p, miR-363-3p. Autoimmune Disorders: Idiopathic Thrombocytopenic Purpura: miR-196a-5p, miR-214-3p, miR-298,  miR-299-3p, miR-379-5p, miR-383, miR-409-3p. Systemic Lupus Erythematosus: miR-142-3p, miR-184, miR-196a-5p, miR-21-5p,  miR-298, miR-299-3p, miR-383, miR-409-3p. Inflammatory Responses: IL-1 Induced Inflammatory Response: let-7g-5p, miR-146a-5p, miR-195-5p,  miR-26b-5p. Inflammation Mediators: miR-155-5p, miR-203a. Macrophage Inflammatory Response: miR-132-3p, miR-155-5p. Vascular Inflammation: miR-126-3p, miR-155-5p, miR-21-5p. Others: miR-125a-5p. Innate Immune Response: miR-105-5p, miR-146a-5p, miR-19a-3p, miR-19b-3p. Induced in Monocytes By LPS: let-7e-5p, miR-132-3p, miR-146a-5p, miR-155-5p, miR-187-3p, miR-9-5p, miR-99b-5p. Immune Cells: B Cell Differentiation: miR-150-5p. Invariant Natural Killer Cell Development: miR-155-5p, miR-150-5p. Lymphocytes: let-7e-5p, miR-125a-5p, miR-142-5p, miR-143-3p, miR-145-5p,  miR-146a-5p, miR-148a-3p, miR-195-5p, miR-206, miR-223-3p, miR-451a,  miR-493-3p, miR-574-3p. Monocytopoiesis: miR-106a-5p, miR-20a-5p. Natural Regulatory T Cells: miR-103a-3p, miR-142-5p, miR-149-5p, miR-150-5p,  miR-15b-5p, miR-16-5p, miR-191-5p, miR-19a-3p, miR-21-5p, miR-214-3p,  miR-223-3p, miR-26a-5p, miR-27b-3p, miR-29c-3p, miR-30b-5p, miR-30c-5p,  miR-30e-5p. Th2 Cells: miR-126-3p. Regulates Relevant Genes: Cytokines: miR-98-5p. Interferons & Receptors: miR-146a-5p, miR-223-3p. Interleukins: miR-106a-5p, miR-146a-5p, miR-155-5p, miR-15a-5p, miR-181a-5p,  miR-223-3p, miR-223-3p. STAT3: miR-18a-5p, miR-19a-3p, miR-19b-3p, miR-20a-5p. TNF Alpha: miR-125b-5p, miR-155-5p. Signal Transduction: IL6 / STAT3 Signaling: let-7a-5p, let-7c, let-7d-5p, miR-21-5p. NFKB Signaling: miR-146a-5p, miR-155-5p.    61 2.3.3 Enhancement of IA1 on Allorecognition  To determine if IA1 could further enhance, or jumpstart, an existing pro-inflammatory response, MLR studies were conducted in the absence and presence of the SYN and IA1 preparations. Based on the optimization studies previously described, human MLR were treated with SYN and IA1 (both 1:1 with fresh media) for 7 days and murine MLR were treated for 3 days. The T cell (CD3+, CD4+ and CD8+) percent proliferation and subset differentiation (Th17:Treg) were then assessed as previously described and compared to the control MLR. By examining the effects of IA1 on the MLR model which represents patients bearing moderate pro-inflammatory responses, we can potentially expand the target-spectrum of our acellular therapeutics to enhance the existing anti-cancer inflammatory responses.  2.4 Specific Aim 3: Anti-Proliferative Effects of Acellular Therapeutics-Activated Leukocytes on Cancer Cells In Vitro The enhanced pro-inflammatory response of lymphocytes is central to inhibition of cancer cell proliferation. As discussed in Chapter 1, previous studies have shown that allorecognition events can also enhance the anti-cancer efficacy. To assess if the resting lymphocytes activated by our allorecognition-derived acellular therapeutics could induce an anti-proliferative effect on cancer cells, in vitro cancer cell proliferation assay was conducted.  2.4.1 Cancer and Non-Cancerous Cell Lines Human epithelial HeLa cell line (CCL-2TM) was purchased from ATCC and cultured under 5% CO2 in Dulbecco’s modified eagle’s medium (DMEM; high-glucose contains 4.5 gram per liter (g/L) D-glucose, without L-glutamine or sodium pyruvate; Invitrogen, Carlsbad, CA) supplemented with 10% FBS, 2 mM GlutaMAX, 10 mM HEPES, 100 unit (U) penicillin and 100 µg streptomycin (all from Invitrogen, Carlsbad, CA). Human melanoma SH-4 cell line   62 (CRL-7724TM) was purchased from ATCC and cultured under 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 4 mM L-glutamine, 1 mM Na pyruvate, 100 U penicillin and 100 µg streptomycin. Both cancer cell lines were used at ~80% confluency. To study the toxicity of acellular therapeutics-activated leukocytes, murine non-cancerous myoblast cell lines was used. Mouse (C3H, H-2k) immortalized, non-cancerous myoblast C2C12 cells (CRL-1772TM) was gifted by James Johnson Lab at the University of British Columbia. Myoblasts were cultured under 5% CO2 in DMEM supplemented with 10% heat-inactivated FBS, 100 U penicillin and 100 µg streptomycin. Myoblasts were used at ~80% confluency within 5 passages. 2.4.2 Effector Leukocyte Viability Assay To determine whether acellular therapeutics exerted toxicity to the effector leukocytes, 7-amino-actinomycin D (7AAD; BD Biosciences, San Jose, CA) viability studies were done. 7AAD is a fluorescent nucleic dye that is excluded from viable cells but can enter non-viable cells due to increased membrane permeability. Resting human PBMC or mouse splenocytes were treated with acellular therapeutics (diluted 1:1 with AIM V) for 24 hours and stained with 7AAD at a final concentration of 0.05 milligram (mg) /ml followed by a 15 minutes incubation at room temperature prior to flow cytometric analysis as previously described. [250, 252] For some experiments, the toxicity of these pretreated PBMC on autologous PBMC was also examined. The recipient PBMC from the same donor were labeled with CFSE as described above and co-cultured (1:1) with acellular therapeutics pretreated (24 hours) but non-CFSE-labeled PBMC at 37° C, 5% CO2 for 7 days. The viability of the recipient PBMC was assessed by 7AAD among the CFSE+ population. By using the viability assay, we can determine the effects of pre-activation on effector leukocytes before administrating them to cancer cells.    63 2.4.3 Real-Time Assessment of Cancer Cell Proliferation In Vitro Despite efficacy, conventional measurements of cytotoxicity via 51Cr-release assay or ELISpot assay exhibit safety issues and limitations. [142, 143] In this study, we used a label-free, real-time measurement of cell proliferation using the ACEA iCELLigenceÒ instrument (ACEA Biosciences, Inc., San Diego, CA). The ACEA system monitors the increase in the electrode impedance exerted by the viable cell attachment and records the target cell (cancer and non-cancerous cells) proliferation in real-time. [144, 145] Importantly, the leukocytes’ non-adherent property is useful in the cytolytic assays as only attached target cells elicit impedance. The impedance reflects the ‘fingerprint’ of the overall cell behaviour and is converted into cell index generating the kinetic cell proliferation curve (Figure 2.7).  Figure 2.7 Electrode Impedance-Based Target Cell Proliferation.  A side view of single well in the ACEA iCELLigenceÒ experiment is shown. The electron flow in culture medium is collected by electrode terminals and converted into impedance index-based cell proliferation curve. In the absence of cells, a complete circuit of electric current flows between the electrodes. No cell impedance index is generated (yellow curve). As cells attach and proliferation on the electrode, the current flow is impeded, generating increased cell impedance index and a real-time readout of cancer cell proliferation. Black curve represents generic impedance trace for target cell proliferation. Blue curve indicates the target cell growth inhibition by the addition of apoptosis inducer (e.g., lymphocytes). Each phase of the impedance trace is explained in the text. addition of cellsSingle Well (side view)impeded electron flowelectron flownegative terminal positive terminalwell bottomculture medium345210-1Target Cell Proliferation!(Cell Impedance Index)Hours24 48 72 96 120144168345210-1Target Cell Proliferation!(Cell Impedance Index)Hours24 48 72 96 120144168cell adhisioncell proliferationcellular confluencecell death/detachment  64 2.4.4 Inhibition of Acellular Therapeutics on Cancer Cell Proliferation In vitro cytotoxicity of leukocytes (human PBMC) against cancer cells was analyzed using the ACEA system. All studies were done with an initial seeding density of 5,000 HeLa, or 20,000 SH-4, cells per well of the ACEA E-8 electronic microtiter plate (ACEA Biosciences, Inc., San Diego, CA) in DMEM medium with an acclimation time for initial adherence for 1 hour. Sufficient PBMC were added to the wells to achieve Effector:Target ratios of 0:1, 10:1, 25:1 and 50:1. Importantly, following 24 hours of pretreatment with the acellular therapeutics, the leukocytes were extensively washed to remove the residual conditioned media, leaving only the activated leukocytes. Cells were incubated in the ACEA instrument maintained at 37° C in a humidified 5% CO2 incubator for 7 days (168 hours). The immune cell-induced alterations in cancer cell attachment were monitored via the current impedance index and presented in real-time cancer cell proliferation curves (Figure 2.8).   Figure 2.8 In Vitro Cancer Cell Proliferation Assay.  In E-8 plates, cancer cells had been seeded and accommodated for 1 hour. To examine the anti-proliferative effects of activated immune cells on cancer cells, freshly isolated human leukocytes were pretreated with acellular therapeutics for 24 hours. Pretreated leukocytes were then isolated and washed before overlaying on the cancer cells. Direct toxicity of acellular media (supplemented with 10% FBS) was also assessed on cancer cells without the presence of leukocytes using a 1:1 dilution with fresh cell culture media. The cell attachment induced impedance index was translated to cell proliferation curves and was monitored in real-time using the ACEA iCELLigenceÒ instrument for 7 days.   In some HeLa cell experiments, the inhibition of cancer cell proliferation by purified (resting and activated) CD4+ and CD8+ T cells was assessed. Human CD4+ and CD8+ T cells   65 were purified using a DynabeadsÒ UntouchedTM Human CD4 or CD8 T Cell Isolation Kit (Cat. No. 11352D and 11348D respectively; Invitrogen by Life Technologies, Carlsbad, CA) from resting human PBMC. Briefly, freshly isolated PBMC were incubated with a mixture of mouse IgG antibodies against the non-CD4+ or non-CD8+ T cells for 20 minutes at 4° C. DynabeadsÒ were added and bound to the antibody labeled cells during a 15 minutes incubation at 25° C with gentle tilting and rotation. After the wash steps, the bead-bound cells were separated on a magnet from the supernatant containing the untouched CD4+ or CD8+ T cells. Purified T cells (purity ³ 90%) were pretreated with the acellular secretome products (SYN, IA1 and IA2) for 24 hours and then overlaid on cancer cells with cell proliferation being measured continuously for 7 days.  We were also interested if acellular therapeutics might exert direct toxicity to cancer cells presumably via the induction of apoptosis. Direct toxicity of the secretome products (added 1:1 into DMEM) was assessed by measuring HeLa/SH-4 cells proliferation in the absence of PBMC. (Figure 2.8). Due to the high demand for nutrients during cancer cell proliferation, acellular therapeutics (5 days old) were supplemented with 10% FBS to support the fundamental growth. Similarly, cancer cell proliferation was monitored using the ACEA instrument for 7 days.  2.4.5 Toxicity of Acellular Therapeutics-Activated Leukocytes on Non-Cancerous Cells To examine the toxicity of acellular therapeutics-activated leukocytes on non-cancerous cells, myoblasts proliferation was examined using the ACEA system. Resting mouse splenocytes were pretreated (24 hours) with acellular therapeutics (SYN and IA1), washed and overlaid to the pre-seeded (1 hour, 5,000 cells total) myoblasts to achieve a ratio of 0:1, 10:1, 25:1 and 50:1. Co-cultured cells were incubated in the ACEA instrument maintained at 37° C in a humidified   66 5% CO2 incubator for 7 days. By studying the effects of activated leukocytes on non-cancerous cells, we can evaluate the bystander cell injury of using acellular therapeutics.  2.4.6 Lymphocyte-Cancer Cell Conjugation Formation An important mechanism in the leukocyte-mediated killing of cancer cells is the cell-cell interaction. To quantitatively assess this interaction, flow cytometric cell conjugation assays were done. [252] Briefly, PBMC or HeLa cells were stained with amine-reactive fluorescent probes at a final concentration of 0.5 µM CFSE or 0.2 µM Far Red-DDAO (CellTrace, Molecular Probes, Invitrogen, Eugene, OR) per 2 × 106 cells, respectively. Cells were washed 3 times in excess RPMI 1640 media to remove any unincorporated stain. Stained PBMC and HeLa cells were co-cultured in RPMI 1640 media at a ratio of PBMC:HeLa = 50:1 to a final concentration of 1 × 106 cells/ml. Co-cultures were centrifuged briefly at 100 × g, 4° C for 1 minute and incubated at 37° C for 20 minutes to allow conjugation of cells. Cells were fixed by addition of 2% methanol-free formaldehyde. The double-stained cell population (CFSE+Far Red-DDAO+) was examined to determine the percentage of cell conjugation using flow cytometry. In addition to the conjugation assay, time-lapse microscopic photographs of the PBMC-HeLa cell co-cultures were captured. PBMC and HeLa cells (seeded at 5,000 total) were co-cultured in RPMI 1640 media, at a ratio of PBMC:HeLa = 50:1, in a heated (37° C) humidity chamber (Becton Dickinson, Franklin Lakes, NJ). Photomicrographs were taken at 20X magnification every 10 seconds for 90 minutes using a Nikon Eclipse Ti microscope (Digital sight DS-U3) and analyzed using NIS-elements software. [252] Representative photos at specific timepoints were then extracted for presentation. By assessing the lymphocyte-cancer cell conjugation formation, we can illustrate the intercellular communications during the process of lymphocyte-mediated cancer cell growth attenuation.    67 Chapter 3: Biomanufacturing of Acellular Therapeutics 3.1 Rationale and Objectives The potential synergy between the allorecognition-induced immune activation and the anti-cancer inflammatory response has been suggested. [206, 208] However, current cell-mediated allostimulatory approaches to treat cancer bear the potential risks of MHC-mismatch and GvHD. [211] In order to avoid the use of problematic allogeneic cells, a secretome (acellular conditioned media) strategy has been used in our study. The pre-clinical practice of using conditioned media derived from lymphocytes has proved promising in the context of tissue repair. [218] This secretome approach (e.g., tolerogeneic TA1) has also been used by the Scott Laboratory to prevent autoimmune T1D in NOD mice. [253, 254] However, limited studies have investigated the biological potentials of the pro-inflammatory therapeutic IA1. In this study, in vitro MLR allorecognition systems were used to reproducibly manufacture the pro-inflammatory acellular conditioned media, including allorecognition-derived IA1 and cancer-derived IA2. Additionally, an IA1-comparable conditioned plasma preparation was similarly generated from in vivo allorecognition responses. To assess the immunomodulatory efficacy of these derived preparations, Resting Lymphocyte and Alloresponse (i.e., MLR) models were used, respectively.  The composition of functional acellular components in conditioned media is highly dependent on the bioreactor systems from which the media are generated. Functional miRNA have been reported to mediate the immunomodulatory effects of TA1, which was generated from the camouflaged allorecognition response. [253, 254] In order to characterize the efficacy of our IA1 therapeutic derived from an unmodified allorecognition response, we first determined the fractions in which the active components of IA1 reside, and then explored the activity of individual components (i.e., miRNA-containing exosomes). Indeed, among various packaging   68 formats of extracellular miRNA, only the exosomes have been identified to exert active biological modulation. [315] In addition, because miRNA are evolutionary conserved, [286] cross-species efficacy of IA1 was further evaluated in both human and murine resting lymphocytes.  In this chapter, I discussed the manufacturing of the acellular secretome-based therapeutics using the allorecognition-based bioreactor systems. The immunomodulatory effects of these acellular therapeutics on human PBMC and mouse splenocytes were also characterized. The objectives for this chapter were the following: Objective 1. To manufacture the optimal acellular therapeutics from in vitro (human and mouse) and in vivo (mouse) bioreactor secretome systems;  Objective 2. To characterize the effects of IA1 via size fractionation and exosome isolation studies;  Objective 3. To explore the cross-species (human⬌mouse) efficacy of IA1 on resting T lymphocyte proliferation.  3.2 Results  3.2.1 Human Models:  3.2.1.1 Optimization of Secretome Biomanufacturing In Vitro The Scott Laboratory has developed allorecognition-based biomanufacturing systems to produce immunomodulatory therapeutics. As demonstrated in Chapter 2 (Figure 2.4), acellular therapeutics were prepared from syngeneic resting cells (SYN), allogeneic (IA1), allogeneic-HeLa (IA2) and tolerogeneic (TA1) cell culture systems. To determine the optimal manufacturing time of these therapeutics, IA1 was tested as the representative preparation. As shown in Figure 3.1, resting PBMC were treated with IA1 preparations collected from various   69 timepoints (i.e., day 3, 5, and 7). Proliferation at 10 days post stimulation with the day 5 and 7 IA1 induced a significant increase in the percent proliferation of resting lymphocytes (p < 0.0001 and p < 0.001, respectively). The increase by day 5 IA1 was more pronounced. Interestingly, this finding agreed with previous studies which identified the optimal in vivo TA1 was generated at day 5. [254] In contrast, SYN from neither day points made obvious alterations. Therefore, day 5 was determined to be the optimal time point for the manufacturing of both IA1 and other secretome products. In conclusion, the manufacturing of acellular therapeutics was optimized and conducted at day 5 of MLR using bioreactor human PBMC. Of note, the data variability shown in Figure 3.1 reflects inter-individual donor variation. Intra-individual donor variance tended to be minimal (data not shown).   Figure 3.1 Optimized IA1 Was Biomanufactured From MLR at Day 5.  Resting PBMC were treated with IA1 preparations collected from various timepoints (day 3, 5 and 7). IA1 collected at day 5 and 7 significantly increased the percent proliferation of resting lymphocytes; IA1 from day 5 induced the maximum effects. Data variability reflects inter-individual variance rather than intra-replicate discrepancies. SYN made minimal alteration. SYN and IA1 were added 1:1 into fresh PBMC growth media AIM V. Lymphocyte proliferation was examined post 10 days reaction via flow cytometry. p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh, n ³ 5.  3.2.1.2 Optimization of Assessing the Acellular Therapeutics’ Immunomodulatory Activity In Vitro The optimization of IA1 biomanufacturing has been described above, but the optimal time of IA1 assessment also needed to be determined basing on the PBMC proliferation. Two PBMC models were used to assess the immunomodulatory effects of acellular therapeutics. Resting PBMC represents a unactivated situation of the immunity, while alloresponse (i.e., % Lymphocyte Proliferation !(Day 10) 2030100 Fresh SYN Day 3 Day 5 Day 7IA1 collected at**** ***  70 MLR) indicates the pre-existence of moderate pro-inflammatory responses. As shown in Figure 3.2 A, IA1 started to significantly (p < 0.0001) increase the percent proliferation of Resting PBMC at day 7 and reached its maximal efficacy at day 10. Interestingly, the proliferation rate slightly decreased at day 14 compared to day 10 with the treatment of IA1. In contrast, no matter how long the PBMC were cultured, Fresh and SYN induced minimal proliferation thus the data points (dots on bars) from all timepoints were integrated. Therefore, we determined that day 10 was the optimal time of IA1 assessment in the Resting PBMC model.   Figure 3.2 Assessing IA1’s Immunomodulatory Activity at Day 10 in Resting PBMC and Day 7 in MLR.  Human IA1 (day 5) was used to optimize the time of assessment basing on lymphocyte proliferation. Panel A. Starting from day 7, IA1 induced a significantly increased percent proliferation of resting PBMC relative to fresh media. The effects of IA1 at day 10 was most pronounced. Fresh and SYN had minimal effects throughout the whole process, thus the data points (dots on bars) from all timepoints were integrated. Panel B. Control MLR induced increasing percent proliferation overtime. IA1 enhanced the baseline proliferation at day 7 relative to the control MLR. SYN made minimal alterations at either time point. Data points were graphed according to different timepoints. Panel C. Schematic summary of the Resting PBMC and human MLR models with the optimized time of assessment. Human-sourced IA1 was manufactured at day 5; its immunomodulatory activity was assessed at day 10 in Resting PBMC and day 7 in human MLR model.  p < 0.05 (‘*’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh (Panel A) or control MLR (Panel B), n ³ 4. SYN Day 4 Day 7 Day 10IA10************% Lymphocyte Proliferation102030Day 14Resting PBMCAFreshBCMLRDonor A PBMCDonor B PBMCMLR Day 10IA1Day 7Day 5IA1:Fresh Media (1:1)Lymphocyte ProliferationResting PBMCMLRDay 4 Day 7 Day 10 Day 14% Lymphocyte Proliferation040506070302010*IA1SYNControl MLR  71 Similarly, effects of IA1 on PBMC proliferation in the MLR was measured at various day points including day 4, 7, 10 and 14. As shown in Figure 3.2 B, the control MLR conducted with fresh growth media itself induced a lymphocyte proliferation as a result of allorecognition. [373] The longer of the reaction, the greater magnitude of the proliferation. SYN did not increase the lymphocyte proliferation at any time point. In contrast, IA1 significantly (p < 0.05) increased the percent lymphocyte proliferation at day 7. A similar trend was also seen at day 10. Similar to what we saw in the Resting PBMC model, IA1 stopped further enhancing the proliferation at day 14 when the baseline of control MLR was already high. Therefore, day 7 was determined to be the optimal time of IA1 assessment in the MLR model.  Figure 3.2 C schematically summarizes the optimized manufacturing and time of assessment of IA1. A fresh MLR using human PBMC was conducted for the biomanufacturing of IA1 at day 5. Acellular IA1 was then added 1:1 into fresh media which supports the regular growth of responder cells. Two assessing models, Resting PBMC and MLR, were tested with IA1 to determine the optimal time of assessment. Based on the lymphocyte proliferation, IA1 exerted optimal enhancement at day 10 in the Resting PBMC model, and at day 7 in the MLR model. Of note, using the same PBMC donor(s) for the manufacturing and assessment was not necessary. Data points have been mixed from various healthy PBMC donors treated with different lots of IA1; the outcomes were considerably consistent. In conclusion, the optimal effects of IA1 (biomanufactured at day 5) were exerted at day 10 and 7 in Resting PBMC and MLR model respectively. These day points also applied to other acellular therapeutics (i.e., SYN, IA2 and TA1) in this study. The Resting PBMC and MLR models were continuously used in the human studies throughout the whole project.   72 3.2.1.3 Immunomodulatory Effects of IA1: Size Fractionation and Exosome Isolation Previous studies on TA1 have demonstrated that miRNA, but not cytokines/chemokines, mediated the tolerogeneic immune response. [254] To determine the immunomodulatory effects and the active components of the secretome-derived pro-inflammatory therapeutics, resting PBMC were treated with the indicated various acellular preparations. As demonstrated in Figure 3.3 A, the IA1, but not SYN, induced a significant (p < 0.0001) increase in resting CD3+ T cell proliferation at day 10 of culture. Previously we hypothesized that soluble cytokines contained in the IA1 or TA1 conditioned media mediated their respective immunomodulatory effects. [253, 254] However, size fractionation studies (Figure 3.3 A) found that the cytokine/chemokine rich fraction (< 30 kDa) exhibited no proliferative effect on CD3+ T cells (1.81 ± 0.14%). In contrast, higher molecular weight fractions containing the Ago2/miRNA complex (~97 kDa) and miRNA-rich exosomes (³ 100 kDa) retained significant proliferative activity (7.51 ± 0.37% and 7.38 ± 0.66% respectively, p < 0.0001) relative to the complete IA1 (12.2 ± 1.21%) supporting earlier findings of biological activity for exosomes [130, 315]. In contrast, the similarly sized fractions from the Fresh and SYN demonstrated no proliferative activity. Of note, some cytokines can exist as multimers and thus exceed 30 kDa and could partially contribute to the efficacy of 30-100 kDa fraction. However, the ³ 100 kDa fraction was equally effective suggesting that miRNA (Ago2 and exosome encapsulated) were likely to be the major mediators. Moreover, exosome isolation studies demonstrated that IA1 (2.55-fold, p < 0.01), but not the SYN (1.08-fold), exosomes induced resting PBMC proliferation (Figure 3.3 B) confirming the role of miRNA-containing exosomes of IA1 in promoting a pro-inflammatory T cell proliferation. Importantly, IA1 not only increased CD3+ T cell proliferation (Figure 3.3 A) but also increased the Teff:Treg (Th17:Treg) cell ratio (2.23-fold, p < 0.01) resulting in a pro-inflammatory environment; crucial   73 for cancer cell killing (Figure 3.3 C). The SYN had no effect on either proliferation or the Teff:Treg ratio relative to fresh media. In contrast, as previously reported, the tolerogeneic TA1 was characterized by decreased Teff and increased Treg cell population (data not shown). [253, 254] In conclusion, IA1 promoted a pro-inflammatory T cell proliferation and differentiation depending on the miRNA-enriched fractions and exosomes. The acellular therapeutics derived from the MLR can be used to induce a miRNA-mediated T cell activation obviating the risk of allogeneic cell-mediated GvHD.   Figure 3.3 IA1 Promoted a Pro-Inflammatory T Lymphocyte Proliferation and Differentiation via the Secretome miRNA.  Panel A. The T lymphocyte (CD3+) proliferative potential of IA1 was assessed for both complete IA1 and M.W. restricted sub-fractions (< 30, 30-100 and ³ 100 kDa). Shown is the CFSE percent proliferation of CD3+ PBMC measured at day 10 via flow cytometry. As shown, the proliferative potential of IA1 was not associated with the cytokine-rich fraction, but was solely observed in the miRNA-containing fractions. The table insert indicated the M.W. of representative cytokines and chemokines. Panel B: Treatment of PBMC with purified exosomes from Fresh, SYN and IA1 demonstrated that only IA1 exosomes induced CD3+ T cell proliferation. Exosomes from fresh PBMC were normalized to 100%. Panel C: Analysis of subset differentiation of resting PBMC treated with Fresh, SYN and IA1 demonstrated that only IA1 induced a pro-inflammatory state as defined by the increased Th17:Treg cell ratio. Th17 and Treg phenotyping of resting CD4+ PBMC was measured post 10 days treatment via flow cytometry. p < 0.01 (‘**’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh unless specified, n ³ 4.  AIA1SYNFresh****15201050Complete <)30 30,100 ≥)100Size)Frac6on)(kDa)**** ****ExosomesAgo2Cytokines)ChemokinesCytokines KDa IFN-γ 16.7 IL-1β 17.3 IL-4 14.9 IL-10 18.6 TNF-α 17.4 GM-CSF 14.6 Chemokines CCL2 11.0 CCL3 10.1 CCL5 10.0 CCL11 8.3 CXCL8 11.1 CXCL10 10.9 Day 10%)CD3))Prolifera6on+)CD3))Prolifera6on+ Ra6o)Th17:Treg0.40.30.20.00.1**CompleteFresh SYN IA1CExosomesFresh SYN IA1B****015010020050250300350  74 3.2.2 Mouse Models: 3.2.2.1 Biomanufacturing and Optimization of Murine-Sourced IA1 In Vitro  Based on the optimal manufacturing of IA1 determined by the human bioreactor system, murine-sourced IA1 was also manufactured at day 5 from the mouse MLR using splenocytes. The assessment time of IA1 on both Resting Splenocyte and murine MLR models were similarly optimized (Figure 3.4) based on lymphocyte proliferation. Resting splenocytes were treated with Fresh, SYN and IA1 for 1, 3, 5, 7, 10 and 14 days (Figure 3.4 A). IA1 significantly (p < 0.05) increased the percent proliferation of Resting Splenocyte starting from day 5. Unexpectedly, SYN also induced a moderate but statistically significant (p < 0.05) proliferation of resting cells at early timepoints (day 5 and 7). This SYN-induced proliferation rate grew linearly with time and eventually matched with that induced by IA1 (day 10 and 14). No clear evidence has been found, but the pro-proliferative characteristic of splenocytes in vitro, the splenocyte apoptosis/necrosis during handling and the contamination of mouse strains may all potentially contribute to the discrepancy. Fresh media showed no enhancement of proliferation throughout 14 days. In conclusion, Day 7 was determined to be the optimal time of assessment in the Resting Splenocyte model due to the maximized effects of IA1 compared to either Fresh or SYN.     75  Figure 3.4 Assessing Murine IA1’s Immunomodulatory Activity at Day 7 in Resting PBMC and Day 3 in MLR. IA1 from the mouse origin was manufactured from a two-way MLR in which splenocytes from C57BL/6 and BALB/c mice were mixed and cultured. Cell-free secretome was collected at day 5 followed by centrifugation and 0.2 µm filtration. SYN was collected from single strain splenocytes as the negative control. Panel A. Starting from day 5, murine IA1 induced a significant proliferation of resting PBMC relative to Fresh. Surprisingly, SYN increased the proliferation similarly; the effects were less than IA1 at day 5 and 7, but went equivalent or more significant at later time points. Day 7 was determined to be the optimal time of IA1 assessment in the Resting Splenocyte. Panel B. Control MLR induced increasing percent proliferation overtime. IA1 enhanced the baseline proliferation at day 3 relative to the control MLR and SYN treatment. Similar to the observations in Resting Splenocyte, SYN induced increasing proliferation of control MLR along with time and its effects were indistinguishable from IA1 at late time points (day 5, 7, 10 and 14). Day 3 was determined to be the optimal time of IA1 assessment in the MLR. Data points were graphed according to different timepoints in Panels A-B. Panel C. Schematic summary of the Resting Splenocyte and murine MLR models with the optimized time of assessment. Murine-sourced IA1 was manufactured at day 5; its immunomodulatory activity was assessed at day 7 in Resting Splenocyte and day 3 in murine MLR. p < 0.05 (‘*’) was calculated in comparison to Fresh (Panel A) or control MLR (Panel B), n ³ 3.  Effects of IA1 on splenocytes proliferation in the MLR was also measured at day 1, 3, 5, 7, 10 and 14. As shown in Figure 3.4 B, the rate of lymphocyte proliferation in the control MLR grew linearly with time. IA1 significantly (p < 0.05) further enhanced this lymphocyte proliferation at day 3 but not at later time points. Similar to the observations in the Resting Resting SplenocytesA% Lymphocyte Proliferation080100********Day 3 Day 5 Day 7 Day 10 Day 14Day 1604020IA1SYNFreshMLRBDay 3 Day 5 Day 7 Day 10 Day 14Day 1% Lymphocyte Proliferation080100*604020IA1SYNControl MLRCMLRC57BL/6 SplenocytesBALB/c SplenocytesMLR Day 7IA1Day 3Day 5IA1:Fresh Media (1:1)Lymphocyte ProliferationResting Splenocyte  76 Splenocyte model, SYN induced increasing proliferation of control MLR along with time and its effects were indistinguishable from IA1 at day 5, 7, 10 and 14. In conclusion, as schematically shown in Figure 3.4 C, fresh MLR using splenocytes from two mouse strains was conducted for 5 days to manufacture murine-sourced IA1. Day 7 and 3 were determined to be the optimal time of IA1 assessment in the Resting Splenocyte and MLR model respectively.  3.2.2.2 Immunomodulatory Effect of Murine IA1: Size Fractionation Similar to human studies, pro-inflammatory effects of murine-sourced IA1 on Resting Splenocyte was also characterized via size fractionation studies. IA1 and IA1 < 30, 30-100, ³ 100 kDa fractions were administrated to resting splenocytes for 7 days (Figure 3.5 A) as previously determined. IA1 significantly increased the CD3+ T cell percent proliferation (22.6 ± 1.90%, p < 0.0001) relative to resting cell (3.62 ± 0.55%). As anticipated by Figure 3.4, SYN also slightly but not significantly increased the proliferation (8.50 ± 0.98%). In consistent with human-sourced IA1, higher molecular weight fractions containing the Ago2/miRNA complex (~97 kDa) and miRNA-rich exosomes (³ 100 kDa) of murine IA1 retained the pro-proliferative effects (p < 0.0001). In contrast, the < 30 kDa fraction of IA1, which includes cytokines and chemokines, induced minimal CD3+ T cell proliferation. Interestingly, the pro-proliferative effects of SYN were also mediated by the miRNA-containing but cytokine/chemokine-free fractions (Figure 3.5 A). Not only could IA1 increase T cell proliferation, but IA1 also promoted a pro-inflammatory CD4+ subset differentiation of Resting Splenocyte by increasing the Th17:Treg cell ratio (Figure 3.5 B). Compared to the baseline ratio of 0.20 ± 0.02% in resting splenocytes, IA1 significantly (p < 0.01) increased the ratio to 0.53 ± 0.08% while SYN slightly decreased the ratio (0.10 ± 0.04%). In conclusion, murine-sourced IA1 promoted a pro-  77 inflammatory T cell proliferation and differentiation in Resting Splenocyte. The pro-proliferative effects were mediated by the miRNA-enriched fractions of IA1.   Figure 3.5 Murine IA1 Promoted a Pro-Inflammatory T Lymphocyte Proliferation and Differentiation via miRNA-Containing Fractions.  Panel A. The T lymphocyte (CD3+) proliferative potential of murine IA1 was assessed for both complete IA1 and M.W. restricted sub-fractions (< 30, 30-100 and ³ 100 kDa). Shown is the CFSE percent proliferation of CD3+ T cells measured at day 7 via flow cytometry. As shown, the proliferative potential of IA1 was not associated with the cytokine-rich fraction, but was solely observed in the miRNA-containing fractions. As anticipated from Figure 3.4, the miRNA-containing fractions of murine SYN also showed a pro-proliferative potential. Panel C: Analysis of subset differentiation of resting splenocytes treated with Fresh, SYN and IA1 demonstrated that only IA1 induced a pro-inflammatory state as defined by the increased Th17:Treg cell ratio. Th17 and Treg phenotyping of resting CD4+ splenocytes was measured post 7 days treatment via flow cytometry. p < 0.05 (‘*’), p < 0.01 (‘**’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh, n ³ 4.  3.2.2.3 In Vivo Biomanufacturing of IA1-Plasma in Mice To investigate whether acellular therapeutics can be produced from in vivo systems, an IA1 media counterpart IA1-Plasma was also manufactured at day 5 using the in vivo allorecognition response (Figure 2.6). To examine the immune responses in immunocompetent naïve mice challenged with allogeneic splenocytes, the mouse body weight, spleen size and splenocyte phenotypes were assessed at the day of IA1-Plasma manufacturing. As shown in Figure 3.6 A, the size of spleen in allo-stimulated mice was increased as demonstrated by the elevated Spleen Size (centimeter; cm) over Body Weight (gram; g) ratio. Significantly, this ratio was increased (1.18-fold; p < 0.001) at day 5 in mice that were injected with allogeneic cells BDay 70.60.81.0**Fresh SYN IA1CompleteRa#o%Th17:Treg0.40.20.0AIA1SYNFreshComplete <30 30-100 ≥100kDaCytokines%Chemokines0**************302010Ago2ExosomesDay 7%%CD3%%Prolifera#on+  78 relative to control PBS-injected mice. In contrast, injection of syngeneic cells had minimal effects on the Spleen Size:Body Weight ratio. Moreover, as shown in Figure 3.6 B, the challenge with allogeneic splenocytes significantly increased the in vivo Th17:Treg cell ratio (1.75-fold, p < 0.0001) compared to PBS injection (Figure 3.6 B). In contrast, the treatment with syngeneic cells had minimal effects. In conclusion, the findings from the murine bioreactor system confirmed an enhanced immune response in the in vivo allorecognition model from which the IA1-Plasma therapeutic was manufactured.   Figure 3.6 Allo-Stimulation Enhanced an Immune Response in Immunocompetent Naïve Mice at Day 5. Panel A. Injection with allogeneic splenocytes significantly increased the Spleen Size:Body Weight  ratio of naïve mice. A 1.18-fold increase was observed relative to PBS-injected mice at Day 5. Syngeneic cell injection had minimal effects. Panel B. Allo-stimulation significantly increased the in vivo Th17:Treg cell ratio of naïve mouse. A 1.75-fold increase was induced by allogeneic cells in comparison to PBS. Syngeneic cells made minimal alterations. Grey shaded areas represented the control (i.e., PBS, mean ± SEM) values for comparative purposes. The Th17 and Treg differentiation was measured using splenocytes via flow cytometry. p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to PBS group, n = 17.  3.2.3 Cross-Species Efficacy of Human- and Murine-Sourced IA1 In contrast to cytokines and chemokines that are widely used in protein homology studies, miRNA are highly conserved evolutionarily with well-established cross-species, and even cross-kingdom, efficacy. [344] To further confirm the role of miRNA in the pro-inflammatory effects of IA1, cross-species (Human ⬌ Mouse) studies were conducted using resting human PBMC and mouse splenocytes. As expected, human-sourced IA1 induced a 0.150.20Ratio Th17:Treg ****0.100.050.00PBS Syngeneic AllogeneicInjected withBA0.15Spleen Size (cm)!/Body Weight (g) ***0.100.050.00Day 51.75-fold1.18-fold  79 significant proliferation of resting human CD3+ T cells (Figure 3.7 A). Importantly, murine-sourced IA1 also induced proliferation (p < 0.05) of human CD3+ T cells. More dramatically, human IA1 stimulated a significant proliferation (p < 0.001) of mouse splenocytes at a level comparable to murine-sourced IA1 (Figure 3.7 B). It must be noted that the murine fresh and SYN did somewhat elevate human and murine CD3+ T cell proliferation over the expected baseline levels; perhaps due to the ongoing immune events within the donor mice and/or possibly the presence of FBS which could result in some immunostimulation (Figure 3.7 A-B).  Of note, human studies were done using bovine serum-free media. Importantly, in the context of pro-inflammatory responses, the proliferation induced by the IA1 preparations (human and murine) resulted in an increase in the Th17:Treg cell ratio both intra- and inter-species (Figure 3.7 C-D). Of interest, the murine IA1 induced a larger increase (p < 0.05) in the human Teff:Treg ratio than did the human IA1. In mouse splenocytes, both the murine and human IA1s were comparably effective at increasing the Teff:Treg ratio relative to the resting and SYN-treated samples. Moreover, human IA1-exosomes similarly showed cross-species efficacy on CD3+ T cell proliferation in mouse splenocytes. As shown in Figure 3.7 E, murine IA1-exosomes induced CD3+ T cell proliferation and human IA1-exosomes induced an even more potent increase in proliferation than did murine IA1-exosomes. Human SYN-exosomes exerted no proliferative effect while murine SYN-exosomes showed a modest increase in proliferation similar to that observed with murine SYN-media (Figure 3.7 B). In summary, both the human- and murine-sourced IA1 could promote a potent intra- and inter-species T cell activation. Human IA1 secretome-derived miRNA-containing exosomes exerted an even stronger pro-proliferative effect on resting splenocytes than murine IA1-miRNA. These data further supported the role of miRNAs in the derived acellular therapeutics.    80  Figure 3.7 IA1 and IA1-Derived Exosomes Demonstrate Cross-Species Efficacy on Resting Human and Murine CD3+ T Lymphocyte Proliferation and Subset Differentiation. Panels A-B. Human- and murine-sourced IA1 exhibited significant cross-species efficacy in increasing resting CD3+ T lymphocyte proliferation. Human PBMC and mouse splenocytes were treated (10 days and 7 days respectively) with either human- or murine-sourced Fresh, SYN and IA1. Panels C-D. Human- and murine-sourced IA1 increased the Th17:Treg cell ratio of human and mouse CD4+ T lymphocytes at day10 and 7, respectively. Of note, to control for the presence of FBS in the murine experiments, human-sourced secretomes were supplemented with 10% FBS before being administrated to murine splenocytes. Panels E-F. IA1-derived exosomes demonstrated significant cross-species efficacy in inducing CD3+ T lymphocyte proliferation. Mouse resting splenocytes were respectively treated with murine- (E) and human-sourced (F) exosomes for 7 days. In contrast, exosomes from Fresh or SYN media demonstrated no significant proliferative effects. CFSE percent proliferation and T subset differentiation of human and mouse lymphocytes were measured via flow cytometry. p < 0.05 (‘*’), p < 0.01 (‘**’), p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh unless specified, n ³ 3. FHUMAN EXOSOME SOURCECROSS-SPECIES EXOSOME EFFICACYCROSS-SPECIES IA1 EFFICACYHuman:MurineFresh SYN IA1Human IA1**** **************C DA B0.60.8Ratio Th17:Treg**0.40.20.00*********102030MOUSE/SPLENOCYTESHUMAN/PBMCHuman IA1 Murine IA1 Murine IA1****FreshSYNIA1EMurine:MurineFresh SYN IA1MURINE EXOSOME SOURCE15201050********** % CD3  Proliferation+%/CD3//Prolifera?on+  81 3.3 Summary  Cell-mediated allo-stimulation of immune cells can be effective but is beset by the risk of GvHD. Using a secretome approach, acellular therapeutics have been successfully biomanufactured from allorecognition reactions both in vitro and in vivo. These secretome-derived acellular therapeutics induced a pro-inflammatory T lymphocyte response, similar to allo-stimulation, by increasing proliferation and the Teff:Treg cell ratio, but they obviate the bystander injury caused by allogeneic cells. To optimize the manufacturing and efficacy assessment of secretome therapeutics, two lymphocyte models have been established in both human and mouse studies. The Resting Lymphocyte model (i.e., PBMC and Splenocyte) represents a situation in which no activated immune response exists. The Alloresponse model (i.e., MLR) represents an existing pro-inflammatory response which is moderate and likely not potent enough to eliminate abnormal cells (i.e., cancer cells) in the disease settings. By testing the effects of human IA1 in the Resting PBMC model, the optimal time for manufacturing was determined to be day 5, and the optimal time of assessment was day 10. Because of the pre-existence of pro-inflammatory responses in human MLR, the optimal effects of IA1 were achieved at day 7. This phenomenon may result from nutrient depletion in the culture system, the accumulation of toxic byproducts released from cells, or the potential activation-induced cell death (AICD) response that occurs when lymphocytes are repeatedly stimulated, resulting in cell apoptosis for maintenance of homeostasis. [374] Similarly, murine IA1 was biomanufactured from the mouse allorecognition reactions at day 5, and assessed at days 7 and 3 in the Resting Splenocyte and MLR model, respectively. These two efficacy-assessing models were extensively used for the downstream studies in this project. To characterize the efficacy of acellular therapeutics, size fractionation and exosome purification studies were conducted. Interestingly,   82 the miRNA-containing (but not cytokine/chemokine-rich) fractions of IA1 mediated the majority of its immunomodulatory effects on resting lymphocytes. Further purification of miRNA in the form of exosomes confirmed their pro-proliferative activity. Preliminary data in the lab also demonstrated the loss of IA1’s efficacy when the purified miRNA were degraded by RNase A (data not shown). These observations were supported by the cross-species pro-inflammatory effects of IA1 and IA1-derived exosomes on human and mouse resting lymphocytes due to the evolutionary conservation of miRNA. These findings enrich our understanding of miRNA-mediated immunomodulatory pathways and provide new insights into the development of bioreactor therapeutics.  3.4 Discussion Current cell-mediated allo-stimulatory approaches to treat cancer can be effective, but they are often highly toxic. [211] To eliminate problematic allogeneic cells and maintain an allorecognition-like response, we employed an acellular secretome approach to biomanufacture therapeutics (IA1 and IA2) that replicate the immunomodulatory efficacy observed with allogeneic cells. Previous studies have demonstrated that acellular conditioned media exerted equivalent or superior therapeutic effects when compared to cell-mediated therapy for tissue regeneration. [221, 222] In the Scott Laboratory, the previously studied secretome preparation, TA1, has also achieved success in preventing the onset of T1D in NOD mice. [254] In this study, we used the in vitro MLR allorecognition model to biomanufacture pro-inflammatory therapeutics that could be of clinical significance in treating immunodeficiency and cancer. Importantly, IA1 or IA2 production using human donor PBMC and the HeLa cell line can be scaled up as necessary. In fact, large numbers of PBMC can be sourced from the Canadian Blood Services leukoreduction filter system. Besides an ‘off-the-shelf’ manufacturing strategy,   83 personalized therapeutics can also be readily achievable. PBMC and/or cancer cell populations can be purified from the patient and used for the production of secretome-derived therapeutics in an attempt to equip the therapeutics with some specificity to either autologous lymphocyte activation or cancer type-specific cytotoxicity. Moreover, the cross-species efficacy of miRNA-enriched secretome indicates the applicability of treating human patients with murine (or other species) therapeutics, which can be generated in a large batch.  Interestingly, while the active components within lymphocyte-derived secretome are traditionally viewed as cytokines and chemokines, the essential acellular effector of the IA1 secretome therapeutic were soluble (i.e., Ago-associated) and exosome encapsulated miRNA. To date, selective modulatory effects have only been confirmed in exosomal miRNA and not in Ago2/miRNA complexes. [130, 315, 322, 323] Indeed, using the well-established exosome isolation method, [375] allorecognition-derived exosomes replicated the pro-proliferative effects of the IA1 secretome therapeutic, while exosomes from SYN media showed no difference from the control. Despite the fact that enzymes and cytokines are also enwrapped in exosomes, the presence and concentration of these proteins vary greatly according to the different cell origins of the exosomes or the generation pathways. [130, 324, 376] In contrast, miRNA are consistently detected in exosomes and their critical role in intercellular communication and cellular differentiation has been demonstrated. [130, 344] The cross-species efficacy of IA1 and miRNA-containing exosomes is also supportive of miRNA as the prime mediators of IA1 since cytokines and chemokines often exhibit species specificity, [377–384] while miRNA show significantly less inter-species variability. [295] Hence, a secretome miRNA-mediated immunomodulatory pathway has been suggested in T lymphocyte proliferation and differentiation (Figure 3.8). The secretome is derived from the   84 allorecognition reaction between T lymphocytes and allogeneic cells, which are mostly APC that display MHC molecules for TCR interaction. The alloresponse in T lymphocytes triggers the biogenesis of miRNA, followed by processing into exosomes or with Ago2 proteins. These extracellularly-secreted forms of miRNA can be taken up by recipient T lymphocytes, acting as cell-cell communicators. Engulfment of exosomes involves endocytosis, fusion with plasma membranes, and/or ligation with receptors, [333–335] while the Ago2/miRNA uptake is mostly facilitated by a receptor (e.g., neuropilin-1) mechanism. [385] Exosomal miRNA are released to the cytoplasm of the recipient T lymphocyte and target mRNA in a conventional manner. Novel pathways of miRNA regulation, by binding to intracellular TLR in lymphocytes, has also been indicated to modulate cancer processes and metastasis. [334, 337, 338] Ago2/miRNA complexes could similarly function in the recipient cells, but more studies, including the optimization of the purification process, are needed to confirm their potentials. Nonetheless, using either the mechanism of miRNA uptake or action, recipient T lymphocytes can be activated, as demonstrated by the increased proliferation and subset differentiation. Notably, the activation of recipient cells does not require TCR crosslinking, which is vastly different from the cell-mediated allo-stimulatory approaches. This novel secretome approach enriches our understanding of acellular therapeutics and indicates the immunomodulatory potential of allorecognition-derived miRNA.     85  Figure 3.8 Proposed Immunomodulatory Pathways of Allorecognition-Derived Secretome miRNA.  Alloreactive T lymphocytes are activated through TCR-MHC allorecognition. Upon activation, pro-inflammatory miRNA are synthesized in T lymphocytes (as well as other allorecognition-involved immune cells e.g., DC) and released extracellularly as part of the secretome, in the form of exosomes and/or Ago2/miRNA complexes. These miRNA can be transported in body fluids or cell culture media and taken up by the recipient T cells. Via endocytosis, fusion with the plasma membrane, and/or ligation with receptors, exosome miRNA are absorbed into the cell and released to target multiple mRNA. Ago2/miRNA complexes could also travel into recipient cells via a receptor mechanism. Consequently, without the crosslinking of TCR with allogeneic MHC molecules, these resting T lymphocytes are activated by extracellular miRNA via mRNA targeting and/or TLR ligation pathways. The secretome miRNA-mediated allorecognition-like proliferation and subset differentiation of recipient T lymphocytes can then be achieved.   3.5 Limitations and Future Directions In this chapter, human-sourced IA1 was manufactured from MLR using PBMC from any two HLA-mismatched individuals. Despite the fact that almost no two individuals in the world share identical HLA molecules – meaning that PBMC from every pair of donors will induce allorecognition – the severity of the response between different donor pairs does differ. To achieve data integrity, I used as many donor pairs as possible to manufacture IA1, remaining TCRAllogeneic MHCT LymphocyteNucleuspri-miRNApre-miRNAmiRNA genepre-miRNADuplexMature miRNAT LymphocyteNucleusEndocytosis, Fusion with plasma membrane and/or Ligation with receptorsReleasing miRNATargeting mRNATCRProliferation DifferentiationAllorecognitionAllogeneic APCNucleusTLRExosomeEndosomeAgo2/miRNATT TTTh1Th17Treg Th2  86 aware of the lot-to-lot differences and carefully recording the lot used in each experiment. In the future process of massive manufacturing and quality control, on the one hand, in order to achieve maximal biological activity and lot-to-lot consistency, it would be imperative to evaluate the donor-to-donor responses using MLR and pool the optimal candidate donors and/or donor pairs for IA1 manufacturing. On the other hand, as discussed above, personalized therapeutics could be realized by using the lymphocytes from the patients themselves or from designated donor pairs. Similarly, due to the human donor variance, the responses of PBMC to IA1 varied as well. Therefore, the data shown in this study were integrated with the results of different lots of IA1 and the results of various human responder PBMC.  When comparing the immunomodulatory effects of IA1 derived from various origins (i.e., human and mouse), and despite the fact that most of the conclusions are consistent between these two bioreactor systems, diverse behaviours of human PBMC and mouse splenocytes were observed. Mouse splenocytes were stronger responder cells to both IA1 and allo-stimulation (i.e., MLR) than was human PBMC. Consequently, the murine-sourced IA1 produced from the MLR might have greater pro-proliferative effects than human IA1. Indeed, in the cross-species studies, murine IA1 induced a higher rate of proliferation in resting human PBMC than human IA1. Moreover, dissimilar to human-sourced SYN which had minimal effects, murine-sourced SYN unexpectedly induced an inflammatory proliferation of resting cells similar to murine IA1. This may due to the potential run-away activation of resting splenocytes in the in vitro biomanufacturing system, possibly caused by the pro-proliferative cell content released after cell breakage; however, more experimental analyses are needed to address this issue. Downstream studies were conducted in both human and murine systems, but the focus was on human models due to their consistency and translational relevance to clinical settings.    87 In future studies, the composition of IA1 miRNA and their activity will be investigated. Our current study has determined the functioning cytokine/chemokine-free fractions and miRNA-containing exosomes of IA1; the ‘pattern of miRNA expression’ and the regulatory network yet need to be identified. We selectively examined the exosomes due to their acceptance as a stimulation-specific form of miRNA, but fractionation studies clearly showed that the fractions which excluded exosomes were also effective. By modifying the current Ago2/miRNA purification methodologies and employing additional characterization strategies of acellular media (e.g., mass spectrometry, proteomics, and DNA/RNA sequencing), neo-immunomodulatory ingredients of IA1 may also be identified.  Moreover, IA1-Plasma could be applied to both in vitro and in vivo assessing systems. Limited data from preliminary studies in the Scott Laboratory has demonstrated its role as a control for TA1 in NOD mice and autoimmune diseases, but the potential of IA1-Plasma in immune activation and cancer treatment has yet to be explored. This study focuses on in vitro IA1 media due to the availability of leukocytes and cell lines. In contrast, in vivo manufacturing of IA1-Plasma, though also achieved, required a substantial number of animals and cumbersome workload; this was also the case for the efficacy assessment. Indeed, the optimal conditions of manufacturing and assessing IA1-Plasma might be different from that of IA1 media, thus more optimization studies are needed. In the current study, therapeutics that were consistently manufactured (at day 5) serve better in comparative studies; yet we should also bear in mind that IA1-Plasma and other secretome therapeutics would achieve better efficacy if being manufactured differently. Based on what we have demonstrated in the in vitro models, we expect IA1-Plasma to promote a similar pro-inflammatory response in both isolated lymphocytes and naïve and/or cancer-bearing animals.    88 Chapter 4: Immunomodulatory Effects of Acellular Therapeutics on T Leukocyte Proliferation and Differentiation 4.1 Rationale and Objectives Effective anti-cancer immune responses are mediated by functional Teff lymphocytes. [111] Current immunotherapies have demonstrated efficacy in promoting the function of Teff cells and/or down-regulating Treg cells. [111, 386] In order to assess the immunomodulatory effects of our secretome-based acellular therapeutics, T lymphocyte (CD3+, CD4+ and CD8+) proliferation and Teff (Th17, Th1 and iNKT) cell differentiation were studied in both Resting PBMC and MLR models. Of clinical significance, Resting PBMC represents a patient model in which no pro-inflammatory T cell responses exist, while MLR represents a model bearing weak endogenous T cell activation. In both models, Treg cell levels have also been measured, allowing for quantification of the pro-inflammatory magnitude, expressed as the ratio of Th17:Treg cells.  Despite their efficacy, current pan T lymphocyte stimulation approaches cause severe systemic injuries, increasing the need for alternative activation strategies that bear fewer bystander toxicities. [197, 198] Therefore, comparative studies were conducted between our acellular secretome therapeutics and mAb/mitogen stimulations. Flow cytometric and qRT-PCR methods were used to examine cellular proliferation, differentiation, and immune activation-associated intracellular miRNA expression. Moreover, since two distinct pro-inflammatory preparations have been developed in Chapter 3, the different effects of IA1 and IA2 were examined in this chapter (lymphocyte activation) and in Chapter 5 (cancer-inhibitive efficacy). Effects of murine-sourced IA1 on mouse splenocyte proliferation and differentiation were similarly studied in Resting Splenocyte and MLR models.    89 In this chapter, I discussed the immunomodulatory effects of secretome-based acellular therapeutics on T lymphocytes proliferation and differentiation comparative to pan T cell stimulations. The objectives for this chapter were the following: Objective 1. To assess the effects of acellular therapeutics on T cell proliferation, subset differentiation and intracellular miRNA expression; Objective 2. To compare the immunomodulatory effects of IA1 and IA2 to mAb/mitogen stimulations; Objective 3. To study the enhancement of IA1 on an existing pro-inflammatory response within the MLR. 4.2 Results  4.2.1 Human Models: 4.2.1.1 Effects of IA1 and IA2 on Resting PBMC Proliferation One problem with potent T cell activation strategies (e.g., mAb and mitogens) is an overly robust response. [200] Hence, we examined the comparative proliferative efficacy and differentiation profiles of the secretome-derived IA1 relative to that of potent T cell activators such as anti-CD3/anti-CD28 and PHA. Moreover, we also tested the IA2 preparation generated from PBMC-cancer cell (HeLa; Figure 2.4) reactions to determine if a cancer cell specific agent would prove more efficacious and specific than IA1. As shown in Figure 4.1 A, the different effects of IA1 and IA2 on CD3+, CD4+ and CD8+ T lymphocytes were compared to that observed in a control MLR as well as direct mAb (anti-CD3/antiCD28) or mitogen (PHA) stimulation. Both IA1 and IA2 significantly increased the CD3+ T cell percent proliferation in resting PBMC (12.2 ± 1.21%, p < 0.0001 and 10.7 ± 0.47%, p < 0.01, respectively) relative to fresh media (1.54 ± 0.35%), and this increase encompassed both CD4+ and CD8+ T cells.   90 Interestingly, IA1 predominantly increased CD4+ T cell proliferation while IA2 predominantly increased CD8+ T cell proliferation. As anticipated, the acellular IA1 and IA2 induced proliferation of resting PBMC was slightly less than 50% of that observed in a control two-way MLR (30.9 ± 3.41%) where two HLA-disparate populations are both proliferating. In contrast, anti-CD3/anti-CD28 and PHA stimulation both resulted in very high levels of CD3+ T cell proliferation (78.1 ± 1.78% and 94.4±0.27%, respectively, p < 0.0001). This phenomenon was also noticed even when the mitogen stimulation was titrated by using the secretome (i.e., conditioned media) collected from PHA-stimulated PBMC (data not shown). Indeed, these overly robust responses are indicative of the adverse clinical events associated with mitogen/mitogen-like therapeutics. [200] In contrast to IA1, the SYN and tolerogeneic TA1 treatment had no pro-proliferative effects on CD3+, CD4+ or CD8+ T cells relative to the resting cells.    91  Figure 4.1 IA1 and IA2 Promoted Differential Subset Proliferation of Resting PBMC and in a More Restrained Manner Than Pan T Cell Activators.  Panel A. Shown is the percent proliferation of resting CD3+, CD4+ and CD8+ (top to bottom, respectively) human T lymphocytes at day 10. IA1 and IA2 significantly increased the percent proliferation of resting CD3+ T cells relative to fresh media; the magnitude was 50% of that seen in an allorecognition (MLR, black bar) and dramatically less than that induced by anti-CD3/anti-CD28 or PHA. IA1 induced a CD4+-centric proliferation while IA2 predominantly enhanced CD8+ T cells. The acellular secretome preparations were added 1:1 into AIM V growth media. Data for the MLR, anti-CD3/28 and PHA were collected at day 10, 3 and 4 days respectively. CD3, CD4 and CD8 T lymphocyte subset proliferation was determined via flow cytometry. Panel B. IA1 significantly shrank the CD8+ T cell subpopulation while IA2 mostly decreased the population of CD4+ T cells. MLR, anti-CD3/anti-CD28 and PHA all induced distinct alterations to CD4+ and CD8+ T cell percent populations to a larger magnitude than IA1 or IA2. Percent populations of each CD3+ T cells subpopulation (i.e., CD4-CD8-, CD4+CD8-, CD4+CD8+ and CD4-CD8+) were presented in pie charts. Values in the middle indicated total CD3+ T cell proliferation. Panel C. Consequent to the differential effects on CD4+ and CD8+ T cell subpopulation differentiation, the ratio of CD4:CD8 cells was decreased by IA1, MLR, anti-CD3/anti-CD28 and PHA, while increased by IA2, relative to fresh media. Ratios were calculated using the percent populations of CD4+ and CD8+ T cells from Panel B. p < 0.05 (‘*’), p < 0.01 (‘**’), p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh, n ³ 4.   Prolifera)onA CD4/CD8	Differen)a)onBCD4⁺	8.61±1.15% 35.1± 1.99% 54.6±2.74% 1.74±0.21% CD4⁺	1.38± 0.45%SYN4.08±0.27%39.6± 0.81% 55.6± 0.58% 0.73±0.03%CD4⁺	2.34± 0.07%TA117.6±3.45%40.5± 0.73%38.8± 3.45% ****3.02±0.47% 12.2± 1.21% ****IA119.2±0.43% *25.9± 3.26%53.7±3.00% 1.16±0.08%10.7± 0.47% **IA24.80±0.17%41.3± 0.74%48.0±1.16% * 5.96±0.70% ** 30.9± 3.41% ****MLR13.5±2.65%35.1±2.65%45.7± 0.54% 5.73±0.46% ** CD4⁺	 CD8⁺	78.1± 1.78% ****a-CD38.64±4.63%44.7± 3.56% ***39.7± 4.34% **** 6.97±2.30% **** CD4⁺	 CD8⁺	94.4± 0.27% ****PHACD4-CD8-CD4-CD8+CD4+CD8-CD4+CD8+C 	 Fresh SYN TA1 IA1 IA2 MLR PHAAnti-CD3/CD28CD4:CD8 1.70±0.08	 	 	 	 	 	 	1.82±0.31 1.41±0.04 0.97±0.10 2.22±0.38 1.17±0.05 1.34±0.11 0.94±0.11**** * ** * ****CD8⁺	8.47±1.18% 33.8± 0.74% 56.0±1.52% 1.76±0.16%1.54± 0.35%FreshCD4⁺	CD8⁺	 CD8⁺	 CD8⁺	CD8⁺	 CD8⁺	 CD8⁺	CD4⁺	 CD4⁺	CD4⁺	CD3080100604020 ******************CD4080100604020****************Fresh SYN IA1 IA2 TA1 MLR PHAAn>-CD3/28CD8**************080100604020Media%	CD8		Prolifera>on+%	CD4		Prolifera>on+%	CD3		Prolifera>on+  92 Consequent to the different magnitude of CD4+ and CD8+ T cell proliferation induced by the above stimulations, distinct CD4+ and CD8+ T cell differentiation patterns (i.e., CD4:CD8 cell ratio) were observed (Figure 4.1 B-C). The ratio of CD4:CD8 cells can be indicative of T cell priming and tumour immunity. [95, 97] IA1, similar to the MLR, anti-CD3/anti-CD28 and PHA, significantly (p < 0.0001) decreased the ratio of CD4:CD8 cells. Surprisingly, the predominant CD4+ T cell proliferation by IA1 (Figure 4.1 A) was accompanied with a decreased CD4+ T cell population (p < 0.0001) and an increase in CD8+ T cell population among total T cells (Figure 4.1 B), contrary to what we expected that the population would go hand-in-hand with proliferation. Clearly, the proliferation-induced cell death must have happened simultaneously, sculpting the expansion of CD4+ and CD8+ T cell subpopulations. In contrast, despite its similar overall (i.e., CD3) proliferation rate, IA2 significantly increased the proliferation of CD8+ T cells but shrank their percent population among total T cells resulting in an increased (p < 0.05) CD4:CD8 cell ratio (Figure 4.1 C). These findings demonstrated that while the MLR allorecognition, anti-CD3/anti-CD28, PHA and IA1 induced a primarily CD4+ T cell response, IA2 induced a strong CD8+ T cell response. SYN and TA1 had minimal effects on either total T cell proliferation or CD4/CD8 differentiation. In conclusion, similar to the allogeneic stimulation, the allorecognition-derived IA1 exerted a potent activation to resting T cells. IA1 induced a T cell proliferation in a more restrained manner than mAb and PHA, obviating the run-over inflammation. IA2 also promoted a pro-inflammatory T cell response but was different from IA1.  4.2.1.2 Effects of IA1 and IA2 on Resting T Cell Subset Differentiation Not only did IA1 and IA2 induce differential proliferation and phenotyping of CD4+ and CD8+ T cells, but they also distinctly altered the CD4+ T cell subset (Th17, Treg and Th1) and   93 iNKT cells differentiation pattern (Figure 4.2). IA1 significantly increased both Th17 (Figure 4.2 A) and Treg cell population (Figure 4.2 B), but the increase in Th17 cells was predominant resulting in an increased Th17:Treg cell ratio (2.21-fold, p < 0.01. Figure 4.2 C). In contrast, IA2 moderately expanded the population of Th17 cell but failed to expand the Treg cell population; the loss of Treg cell population by IA2 relative to IA1 (p < 0.001) further elevated the Th17:Treg cell ratio (4.43-fold, p < 0.0001). The allogeneic stimulation (i.e., MLR) significantly increased both populations of Th17 and Treg cells, leading to a slightly but not significantly increased Th17:Treg cell ratio. Therefore, while IA1 exhibited a pro-inflammatory Teff:Treg ratio, IA2 demonstrated a more significant (p < 0.01) increase in the Th17:Treg cell ratio (Figure 4.2 C). Moreover, as previously reported by Wang et al, TA1 shrank multiple pro-inflammatory subsets and dramatically decreased the Th17:Treg cell ratio rescuing NOD mice from the development of T1D. [254] As shown in Figure 4.2 D-E, different effects of IA1 and IA2 were also observed in Th1 (Figure 4.2 D) and iNKT (Figure 4.2 E) cell differentiation. IA1 significantly (p < 0.001) increased Th1 cell population but showed no effects on iNKT cell differentiation. In contrast, IA2 did not expand Th1 cells but significantly increased iNKT cell population relative to either fresh media (p < 0.0001) or IA1 (p < 0.01). Neither SYN nor TA1 induced obvious alterations to Th1 cells, though both resulted in a slight elevation of iNKT cell population. As demonstrated in Figure 4.1 A above and summarized in Figure 4.2 F, IA1 induced a CD4+-centric but IA2 induced a CD8+-centric resting PBMC proliferation. In conclusion, IA1 and IA2 exerted differential effects on T cell subset differentiation. All these differences in T cell proliferation and differentiation suggested distinct signaling patterns within the activated PBMC.    94  Figure 4.2 IA1 and IA2 Both Promoted a Pro-Inflammatory T Cell Subset Differentiation in Resting PBMC but in Different Manners.  Panels A-C. To characterize the pro-inflammatory state, the Th17 and Treg cell subsets of CD4+ T cells were determined for the Fresh, SYN, IA1, IA2 and MLR (black bar) at day 10. The pro-inflammatory state was determined by an increase in the Th17:Treg (i.e., Teff:Treg) cell ratio relative to PBMC incubated in fresh media. As shown, both IA1 and IA2 expanded the Th17 cell population while IA2 failed to increase Treg cell population, resulting in a higher magnitude of increase in the ratio of Th17:Treg cells relative to IA1. MLR increased the population of both Th17 and Treg cells but made minimal alteration to the Th17:Treg cell ratio. Grey areas represented the control (i.e., Fresh; mean ± SEM) values for comparative purposes. Panels D-E. Percent population of CD4+IFN-g+ Th1 and CD3+6B11+ iNKT cells. Resting PBMC were treated with Fresh, SYN, IA1, IA2 and TA1 for 10 days. IA1 significantly increased the Th1 cell population but had minimal effects on iNKT cells. In contrast, IA2 expanded iNKT cells relative to either fresh media or IA1. TA1 made minimal alterations to either Th1 or iNKT cells. Panel F. As described above, IA1 and IA2 induced differential T cell proliferation pattern of resting PBMC. Despite the broad similarity in CD3+ T cell proliferation, IA1 predominantly enhanced CD4+ T cells while IA2 induced a CD8+-centric response. Data derived from Figure 4.1. p < 0.05 (‘*’), p < 0.01 (‘**’), p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh unless specified, n ³ 4. Proliferation (IA1 vs IA2)Th1 ***2.01.51.00.50.0************FreshSYNIA1IA2FreshSYNIA1IA2FreshSYNIA1IA2CD3+ CD4+ CD8+0102030%4Prolifera;on **Th1721034*******Treg42068 ** **** iNKT ****0.00.51.01.5Fresh SYN IA1 IA2 TA1**ABCDEFRatio!Th17:Treg0.40.20.00.60.81.0*******Ra;o4Th17:TregFresh SYN IA1 IA2 MLRMedia%4CD444ILG17A44Cells++%4CD444IFNG!44Cells++%4CD3446B1144Cells++%4CD444FOXP344Cells++  95 4.2.1.3 Lymphocyte Activation: Differential Expression of Immune miRNA   Immune cell activation and function can depend on a number of factors including altered intracellular miRNA expression in response to immunomodulatory agents. [354, 387, 388] Indeed, miRNA are key regulators of cellular differentiation and proliferation and can serve as highly sensitive biomarkers of immune activation, tolerance, or quiescence. [389–391] To examine the differential miRNA expression profiles of resting PBMC treated with SYN, IA1, IA2, TA1, anti-CD3/anti-CD28 and PHA, qRT-PCR studies were conducted. As shown in Figure 4.3 A, clustergram expression profiles of resting PBMC incubated for 72 hours in fresh and SYN media were similar across the majority of the 84 miRNA examined. The 72-hour treatment time was determined by the potent stimulation of anti-CD3/anti-CD28 (Figure 4.1) which caused severe cell death at late time point (e.g., day 10, data not shown). Similar to SYN treatment, the tolerogeneic TA1 therapeutic was also very closely aligned to resting cells in the fresh media despite being generated in mPEG-MLR system biologically capable of allorecognition (blocked only by the membrane grafted polymer). In contrast, both IA1 and IA2 demonstrated significant variances from the profile of the control PBMC (Figure 4.3 B). Moreover, as highlighted by the yellow boxes in Figure 4.3 B, IA1 and IA2 were very dissimilar to each other over a broad range of miRNA demonstrating that the induced T cell activation pathways were not equivalent (IA1 ≠ IA2) as anticipated by the findings shown in Figures 4.1-4.2. The expression of numerous miRNA was vastly altered by IA1 treatment while IA2-treated PBMC more closely resembled the control sample. Interestingly, despite the disparities noted in miRNA expression of treated PBMC, IA1 and IA2 increased CD3+ T cell proliferation to a similar level; however, a significant (p < 0.05) discrepancy of CD4+ and CD8+ T cell proliferation (Figures 4.1-4.2 and 4.3 C) was noticed as IA1 was CD4+-centric while IA2 was   96 CD8+-centric. All these observations could contribute to the different functions (i.e., cancer-inhibitive efficacy) between IA1 and IA2. In contrast to the more restrained proliferation seen with IA1 and IA2, activation of resting PBMC using anti-CD3/anti-C28 or PHA resulted in distinctly divergent miRNA expression patterns to either IA1 or IA2; likely consequent to these agents’ near universal activation and proliferation of CD3+ T cells (Figure 4.1). In conclusion, the differential intracellular miRNA expression profiles as well as the differences in the activation of CD4+ and CD8+ T cells clearly suggested that IA1 and IA2 exerted immunologically distinct effects. The inequivalence of IA1 and IA2 in immunomodulating T cell responses suggested that they might exhibit differential effects on cancer cell proliferation (Chapter 5).     97  Figure 4.3 IA1 and IA2 Pretreatment Induced Differential Intracellular miRNA Expression Profiles in Resting PBMC.  To partially assess the effect of the secretome-derived products on PBMC, miRNA arrays were conducted on resting, SYN, IA1, IA2, anti-CD3/anti-CD28 and PHA stimulated PBMC at 72 post treatment. Total RNA were extracted from treated cells for the profiling of 84 miRNA differentially expressed during normal and pathological immune responses. Panel A. miRNA clustergram expression profiles of PBMC treated with SYN and TA1 in comparison to Control. Panel B. miRNA expression profiles of PBMC treated with IA1, IA2, anti-CD3/anti-CD28 or PHA in comparison to Control. Yellow boxed indicated differential miRNA patterns between IA1 and IA2 treatment. Yellow (*) represented pro-apoptotic miRNA differentially upregulated in IA2 relative to IA1. Clustergram data in A-B represented 3 independent experiments. Panel C. Percent proliferation rates of CD3+, CD4+ and CD8+ T cells in the indicated conditions (Derived from Figure 4.1). p < 0.05 (‘¢’), n ³ 4.  4.2.1.4 Can IA1 Enhance an Existing Pro-Inflammatory Response As demonstrated, IA1 significantly stimulated a pro-inflammatory proliferative response in resting PBMC (Figures 4.1-4.3). However, in cancers, the immune system is not in a ‘resting’ state (i.e., as modeled by Resting PBMC) and, while ineffective at the gross level, will exhibit ¢ = p < 0.05 IA1 vs IA2CONTA1SYNCONTA1SYNCONIA1IA2a-CD3PHAA BMagnitude of Gene ExpressionminavgmaxControl	 SYN	 TA1	CD3+	 1.54±0.35	 1.38±0.45	 2.34±0.07	CD4+	 1.30±0.30	 1.16±0.49	 1.99±0.12	CD8+	 1.35±0.45	 1.34±0.56	 2.16±0.08		Control	 IA1	 IA2	 a-CD3	 PHA	1.54±0.35	 12.2±1.21	 10.7±0.47	 78.1±1.78	 94.4±0.27	1.30±0.30	 12.3±2.64	 4.52±0.88	 74.6±1.92	 93.8±0.68	1.35±0.45	 4.53±1.12	 9.44±0.58	 79.1±1.16	 96.6±0.62	C ¢¢CONIA1IA2a-CD3PHAIA1 vs IA2***%	Prolifera=on  98 some degree of immune activation. To determine if IA1 could further enhance, or jumpstart, an existing pro-inflammatory response, MLR studies were conducted in the absence and presence of the SYN and IA1 preparations. As shown in Figure 4.4 A, IA1 greatly enhanced the alloresponse within the MLR as demonstrated by human CD3+ (histogram and bar graph), CD4+ and CD8+ (bar graph) T cell percent proliferation. In contrast, SYN-treated MLR exhibited no significant changes in CD3+ or CD4+ T cell proliferation relative to the control MLR. However, a slight, but statistically (p < 0.05) significant, increase in CD8+ T cell proliferation relative to the control MLR was observed. Interestingly, in contrast to a CD4+-centric proliferative response in resting PBMC, IA1 had a greater pro-proliferative effect on CD8+ T cells (p < 0.0001 relative to CD4+ T cells) in the MLR allorecognition model. This CD8+-centric proliferation was accompanied with a significant shrinkage of CD4+ T cell percent population among total T cells, leading to a significantly decreased CD4:CD8 cell ratio (Figure 4.4 B). SYN had minimal effects to CD4/CD8 differentiation pattern. As shown in Figure 4.4 C, within the CD4+ T cells, IA1 significantly increased Th17 cells relative to the control MLR while Treg cell population remained statistically unchanged. Interestingly, the SYN actually decreased Th17 cells (p < 0.05) but had no effect on Treg cell population. Consequently, as demonstrated in Figure 4.4 D, the Th17:Treg cell ratio of the control MLR was significantly increased (1.55-fold; p < 0.01) by IA1 further supporting its role as a potential pro-inflammatory agent. In contrast, the SYN preparation significantly decreased (0.59-fold; p < 0.05) the Teff:Treg cell ratio. In conclusion, IA1 could jumpstart the allorecognition pathway and enhance an existing pro-inflammatory T cell response within MLR. Both the baseline T cell proliferation rate and the Teff:Treg cell ratio were further increased by the stimulation of IA1. Moreover, in situations when immune activation has already occurred, IA1 may increase the response of effector CD8+ T cells.    99  Figure 4.4 IA1 Significantly Enhanced the Alloproliferative Response of the MLR.  Human two-way MLR were conducted following treatment with Fresh, SYN or IA1 for 7 days. Each acellular preparation was added 1:1 into AIM V media. CFSE percent proliferation and T cell subset differentiation were measured via flow cytometry. Panel A. IA1 significantly enhanced the proliferation of CD3+, CD4+ and CD8+ human T cells within an MLR alloproliferation model. SYN had minimal pro-proliferation effects. Shown are representative histograms from a minimum of 5 independent experiments. M1 demonstrated the CFSE dilution upon cell proliferation while M2 indicated the non-proliferative population. Panel B. Consequent to the differential proliferation rate of CD4+ and CD8+ T cells, IA1 shrank the CD4+ T cell population resulting in a decreased CD4:CD8 cell ratio relative to the control MLR. Values in the middle of pie charts indicated total CD3+ T cell proliferation. Ratio of CD4:CD8 cells was calculated using percent populations and summarized in the table. Panel C. Of Interest, IA1 significantly upregulated Th17 CD4+ cells while having minimal effects on Treg cells. Panel D. Consequent to the IA1-mediated increase in Th17 cells, the pro-inflammatory state was significantly enhanced relative to the control MLR. In contrast, SYN significantly decreased the Teff:Treg cell ratio. Grey areas represented the control MLR (mean ± SEM) values for comparative purposes. p < 0.05 (‘*’), p < 0.01 (‘**’) and p < 0.0001 (‘****’) were calculated in comparison to Control MLR, n ³ 4.  IA1 MLRM1 M2M2M2M1M1101 102 103 10410003000300030010.4%Control MLR!SYN MLR22.4%12.1%CFSE	Prolifera4on6810***420Th17 TregCD4 DifferentiationCD4  Subset+ABCControl MLRSYNIA1040*************%	Prolifera4on302010ProliferationT	LymphocytesCD3  + CD4  + CD8  +CD4-CD8-CD4-CD8+CD4+CD8-CD4+CD8+5.25±0.22%36.8±1.30% 55.4±1.57%  2.05±0.57%CD8⁺	CD4⁺	11.6± 0.47%SYN6.92±0.39% **39.4±1.02%50.7± 0.68% * 2.92±0.06% CD8⁺	CD4⁺	21.8± 0.70% ****IA14.79±0.23%36.5± 0.79%56.3±1.19%2.48±0.22%CD4⁺	CD8⁺	10.1± 0.75%CON	MLRCON MLRSYNIA1CD4:CD81.55!±0.07*	1.29!±0.051.59!±0.14D0.60.81.0***0.40.20.0Fresh SYN IA1MediaRa4o	Th17:TregRatio Th17:Treg%	CD3		Prolifera4on	(Day	7)+% CD4  Cells+  100 4.2.2 Mouse Models:  4.2.2.1 Effects of Murine IA1 on Resting T Cell Proliferation and Subset Differentiation Similar to human studies, to compare the effects of murine-sourced therapeutics to that of allogeneic T cell activator (i.e., MLR), we examined the proliferative efficacy and differentiation profiles of murine-sourced SYN and IA1.  As shown in Figure 4.5 A, murine-sourced IA1 significantly increased the CD3+ T cell percent proliferation in resting splenocytes (22.6 ± 1.90%, p < 0.0001) relative to fresh media (3.62 ± 0.55%), and this increase encompassed both CD4+ and CD8+ T cells. Interestingly, in contrast to human-sourced IA1 that predominantly increased CD4+ T cell proliferation (Figure 4.1 A), murine-sourced IA1 predominantly increased the proliferation of CD8+ (38.7 ± 4.62%, p < 0.0001) than that of CD4+ (6.70 ± 0.94%, p < 0.0001) T cells. As mentioned in Figure 3.4, murine-sourced SYN also slightly increased resting CD3+ T cell proliferation (8.50 ± 0.98%) compared to Fresh; this increase mostly resulted from the elevation of CD4+ T cell proliferation (8.38 ± 1.34%, p < 0.05; Figure 4.5 A). As anticipated and similar to the human PBMC responses, the IA1 induced proliferation of resting CD3+ T cells in splenocytes was slightly less than 50% of that induced by allo- (i.e., MLR) stimulation (52.5 ± 5.38%). In the fresh MLR, the proliferation of CD8+ T cells (75.8 ± 6.18%) was more predominant than that of CD4+ T cells (8.56 ± 0.48%).     101  Figure 4.5 Murine IA1 Induced a Pro-Inflammatory Resting T Cell Proliferation and Subset Differentiation.  Panel A. Murine-sourced IA1 significantly increased the percent proliferation of CD3+, CD4+ and CD8+ T cells in resting splenocytes post 7 days’ treatment relative to fresh media. The CD8+ T cell response was more pronounced. SYN had minimal effects on total CD3+ T cell proliferation but induced a significant enhancement on CD4+ T cell proliferation. SYN and IA1 acellular preparations were diluted 1:1 with RPMI 1640 growth media (supplemented with 10% FBS). In comparisons, allogeneic (e.g., MLR, black bar) stimulation resulted in a more significant increase of T cell proliferation, the rate of which roughly doubled that observed in IA1 treatment. Panel B. Consequent to the CD8+-centric proliferation induced by IA1, the percent population of CD8+ T cells were increased resulting in a decreased CD4:CD8 ratio relative to control. MLR increased the CD8+ T cell population more significantly and simultaneously decreased the CD4+ T cell population, making the CD4:CD8 cell ratio even more reduced. Values in the middle of pie charts indicated total resting CD3+ T cell proliferation. The ratio of CD4:CD8 cells was calculated using percent populations and summarized in the table. Panel C. To investigate the effects of murine IA1 on T cell subset differentiation, Th17 and Treg cell population were measured. Neither SYN nor IA1 had any effects on Th17 cells. However significantly, IA1 decreased while SYN increased the Treg cell population, resulting in an elevated Th17:Treg cell ratio by IA1 relative to control. This effect was similar to that observed in MLR (black bar). The ratio of Th17:Treg cells was calculated using percent populations. p < 0.05 (‘*’), p < 0.01 (‘**’), p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to Fresh, n ³ 4.  Due to the differential proliferation of CD4+ and CD8+ T cells by IA1, an alteration in the CD4+ and CD8+ T cell differentiation pattern was also observed (Figure 4.5 B). Interestingly, the T cell subpopulation composition was different between resting human PBMC and mouse splenocytes. Relative to a 56.0 ± 1.52% CD4+ and 33.8 ± 0.74% CD8+ T cell population in CD3 Th17TregRatio!Th17:TregProlifera)on CD4	Subset	Differen)a)onCD4CD8A CCD4	and	CD8	Differen)a)onB080********60402015*******1050080100********604020Fresh SYN IA1MediaMLRCD4-CD8-CD4-CD8+CD4+CD8-CD4+CD8+1.52.01.00.501520**1050Ra&o	Th17:Treg0.60.81.0***0.40.20.0 Fresh SYN IA1MediaMLR	 Fresh SYN IA1 MLRCD4:CD8 2.98±0.83 	 	 	4.96±0.27 1.49±0.30 0.75±0.08*¢ = p<0.001 vs SYN¢ **¢Fresh SYNIA1 MLR12.2±2.50%  13.5±1.54%  64.9±3.64%  9.46±1.50%  3.62± 0.55%CD8⁺	CD4⁺	3.01± 0.56%  12.1±0.99%  73.0±1.53%  12.0±0.76%  8.50± 0.98%CD8⁺	CD4⁺	10.1± 1.19%  24.5± 2.56% *  57.6±3.38%  7.78± 0.45%  22.6± 1.90%CD8⁺	CD4⁺	8.77± 0.75%  47.6± 2.69% ****  35.1± 2.37% ***  8.54±1.07%  52.5± 5.38% CD8⁺	CD4⁺	%	CD4		IL-17A		Cells++%	CD4		FOXP3		Cells++%	CD3		Prolifera&on+%	CD4		Prolifera&on+%	CD8		Prolifera&on+  102 human PBMC (Figure 4.1 B), mouse splenocytes demonstrated a higher CD4+ T cell percentage (64.9 ± 4.44%) but a greatly lower CD8+ T cell percentage (13.5 ± 1.54%) at the resting status. Moreover, dissimilar to the predominant efficacy of human IA1 in decreasing CD4+ T cell population (Figure 4.1 B), murine IA1 had minimal effects on CD4+ T cell population but significantly (p < 0.05) increased the percent population of CD8+ T cells (24.5 ± 2.56%). Not only did allo-stimulation (i.e., MLR) enhance a more massive gross proliferation than IA1, but it also decreased the CD4+ T cell population (35.1 ± 2.37%, p < 0.001) and induced a more significant increase in CD8+ T cell population (47.6 ± 2.69%, p < 0.0001), resulting in a significant (p < 0.01) decrease in the CD4:CD8 cell ratio (0.75 ± 0.08%, p < 0.01) relative to Fresh (2.98 ± 0.83%). In contrast, SYN conversely expanded the CD4+ population, leading to an increase (p < 0.05) in the CD4:CD8 cell ratio (4.96 ± 0.27%) relative to control. This ratio was also significantly (p < 0.001) higher than that induced by IA1 (1.49 ± 0.30%) or MLR (0.75 ± 0.08%).  As anticipated from the effects of human IA1 on CD4+ T cell subset differentiation (Figure 4.2), murine IA1 also promoted a pro-inflammatory (i.e., increased Th17:Treg cell ratio) response of resting mouse splenocytes. However, dissimilar to human IA1’s predominant effects on increasing Th17 cells, murine IA1 exerted minimal effects on Th17 but significantly shrank Treg cells, resulting in an increased Th17:Treg cell ratio (6.93-fold, p < 0.01. Figure 4.5 C). In contrast, SYN significantly expanded the Treg population leading to a slight decrease in the Th17:Treg cell ratio. Of interest, IA1 exhibited a pro-inflammatory Teff:Treg ratio that was similar to, even slightly higher than that of allo-stimulation (i.e., MLR; 6.38-fold, p < 0.05), maximizing the Teff response by decreasing the Treg cell population. In conclusion, murine-  103 sourced IA1 promoted a pro-inflammatory proliferation and differentiation of resting splenocytes in a similar manner to allogeneic stimulation but diminished the risks of cell-mediated MHC-mismatch.  4.2.2.2 Effects of Murine IA1 on Existing Pro-Inflammatory Alloresponse  As demonstrated, human IA1 enhanced the existing alloresponse in human MLR (Figure 4.4). To investigate if murine-sourced IA1 could also jumpstart the pro-inflammatory response in a murine splenocyte allorecognition, MLR studies were conducted. As shown in Figure 4.6 A, IA1 further enhanced the alloresponse within the MLR by increasing the mouse CD3+, CD4+ and CD8+ T cell percent proliferation. In contrast, SYN did no change the CD3+ or CD4+ T cell proliferation relative to the control MLR. However, a slight, but statistically (p < 0.05) significant, increase in CD8+ T cell proliferation was observed. This finding is consistent with what we saw in the human MLR model (Figure 4.4). Similar to the CD8+-centric proliferative response in Resting Splenocyte, IA1 also had a greater pro-proliferative effect on CD8+ (12.4 ± 0.77%, p < 0.001) than CD4+ (4.26 ± 0.65%, p < 0.001) T cells in the MLR. Interestingly, this CD8+-predominant proliferation by both SYN and IA1 did not further expand the CD8+ T cell population in MLR but only IA1 slightly shrank the percent population of CD4+ T cells among the total CD3+ T cells (Figure 4.6 B). Consequently, a decrease of the CD4:CD8 cell ratio was observed with the IA1 treatment (2.15 ± 0.29%) relative to control MLR (2.86 ± 0.30%). In contrast, SYN slightly increased the CD4:CD8 cell ratio (3.00 ± 0.77%). Nonetheless, none of these alterations were statistically significant nor as dramatic as the effects of human IA1 on human MLR (Figure 4.4). Within the CD4+ T cell population, Th17 cells remained unchanged relative to the control MLR but the Treg cell population was significantly (p < 0.001) decreased by IA1 (Figure 4.6 C). The loss of Treg, which was also observed in the Resting Splenocyte   104 model (Figure 4.5), contributed to a further increased Th17:Treg cell ratio of the control MLR by IA1(1.90-fold; p < 0.001). SYN surprisingly also showed a trend of decrease in Treg and increase in the Th17:Treg cell ratio, though neither was statistically significant. In conclusion, murine-sourced IA1 enhanced the existing pro-inflammatory responses in MLR using a similar stimulatory mechanism observed in the Resting Splenocyte system.  Figure 4.6 Murine IA1 Enhanced the Existing Pro-Inflammatory Proliferation and Differentiation in MLR.  Mouse two-way MLR were conducted with the treatment of Fresh, SYN or IA1 for 3 days. Each acellular preparation was added 1:1 into RPMI 1640 media supplemented with 10% FBS. CFSE percent proliferation and T cell subset differentiation were measured via flow cytometry. Panel A. Murine IA1 significantly enhanced the T cell proliferation (CD3+, CD4+ and CD8+, top to bottom respectively) relative to control MLR. SYN also slightly but significantly induced an increase in CD8+ T cell proliferation. Panel B. IA1 slightly shrank the CD4+ T cell population and expanded the CD8+ T cell population relative to the control MLR resulting in a decreased CD4:CD8 cell ratio. SYN had minimal effects on either population or the ratio. Values in the middle of pie charts indicated total CD3+ T cell proliferation. The ratio of CD4:CD8 cells was calculated using percent populations and summarized in the table. Panel C. Similar to its effects on resting splenocytes, IA1 had minimal impact on Th17 cells but significantly decreased the Treg cell population, leading to a further increased Th17:Treg cell ratio relative to the control MLR. The ratio of Th17:Treg cells was calculated using percent populations. Grey areas represented the control MLR (mean ± SEM) values for comparative purposes. p < 0.05 (‘*’), p < 0.01 (‘**’), p < 0.001 (‘***’) and p < 0.0001 (‘****’) were calculated in comparison to control MLR, n ³ 8. CD3 Th17TregRatio!Th17:TregProlifera)on CD4	Subset	Differen)a)onRa#o	Th17:TregCD4CD8A CControl MLR SYN IA1152025**10506810****4201520*****1050321015***10500.60.8***0.40.20.0Control MLR SYN IA1CD4	and	CD8	Differen)a)onB9.22±3.82%  21.7±1.14%  64.9±4.44%  4.17±0.40%  5.88± 1.09%CD8⁺	CD4⁺	9.21±1.65%  23.5±3.49%  63.2±3.63%  4.16±0.18%  7.24± 0.52%CD8⁺	CD4⁺	12.9±1.33%  27.1± 2.64%  55.9±2.43%  4.13±0.35%  11.8± 1.36%CD8⁺	CD4⁺	SYNIA1Control	MLRCD4-CD8-CD4-CD8+CD4+CD8-CD4+CD8+CON MLRSYNIA1CD4:CD82.86!±0.30	2.15!±0.293.00!±0.77%	CD4		IL-17A		Cells++%	CD4		FOXP3		Cells++%	CD3		Prolifera#on+%	CD4		Prolifera#on+%	CD8		Prolifera#on+  105 4.3 Summary A significant problem with the current potent pan T cell activators is the runaway inflammation and bystander injuries inflicted on normal tissues. To examine if allorecognition-derived, secretome-based acellular therapeutics could activate T cells in a more controlled manner, leukocyte proliferation and T cell subset differentiation were assessed. As described in Chapter 3, two lymphocyte models in both human and mouse studies were used for efficacy assessment. Resting Lymphocyte was used to model a patient who had no existing immune response to fight diseases (e.g., cancer) whereas MLR were used as a patient model that had baseline pro-inflammatory responses. As shown, both human- and murine-sourced IA1 preparations were capable of initiating the pro-inflammatory response of resting (i.e., unactivated) lymphocytes, as well as enhancing the existing alloresponse in the MLR, possibly overcoming systemic T cell anergy to fight cancer cells. The pattern of pro-inflammatory responses of resting T cells activated by IA1 and IA2 was more comparable to that of allogeneic stimulation (i.e., MLR) from which the secretome therapeutics were biomanufactured, but much more restrained than that induced by the anti-CD3/anti-CD28 or PHA stimulation. This finding suggests that the systemic toxicity relative to mitogen or mAb T cell activators should be greatly reduced. Interestingly, despite the broad similarity in the biomanufacturing of IA1 and IA2, they exerted distinct effects on resting T cell subset proliferation and differentiation. IA1 induced a CD4+-centric proliferation and significantly promoted Th1 cell differentiation, while IA2 predominantly increased CD8+ proliferation and expanded the iNKT cell population. IA2 induced a higher magnitude of increase in the Th17:Treg cell ratio than IA1, suggesting a more significant cancer-inhibitive efficacy. Moreover, these proliferation and differentiation discrepancies between IA1 and IA2 were accompanied by diverse PBMC intracellular miRNA   106 expression profiles. Of note, several miRNA (e.g., miR-29b-3p, miR-186-5p, and miR-16-5p) associated with apoptosis were upregulated in IA2-, but not IA1-, treated PBMC. These differences between IA1 and IA2 in T cell activation indicated differential leukocyte-mediated anti-cancer responses (Chapter 5).  4.4 Discussion In an allorecognition response from which the secretome-bases therapeutics were derived, only 1-10% of the total T cells responded. [35, 201] Despite the ‘low’ number of alloresponsive T cells in the biomanufacturing system, the secretomes derived from allorecognition were potent, as demonstrated by their pro-proliferative and pro-inflammatory (Th17:Treg cell ratio) effects on both resting lymphocytes and control MLR. Nonetheless, differential patterns of IA1 activation in these two models were observed (Table 4.1). IA1 induced a CD4+-centric proliferation in human Resting PBMC, but predominantly enhanced the proliferation of CD8+ T cells in the MLR, in which a baseline of CD8+-predominantly proliferation had already existed. However, the CD4/CD8 cell differentiation pattern (decreased CD4:CD8 cell ratio) was similar between the Resting PBMC and MLR. Relative to the human PBMC system, the discrepancies between the mouse Resting Splenocyte and MLR models were less obvious. However, diverse responses between human and mouse bioreactor systems were noticed (see below).    107 Table 4.1 Comparison of Pro-Inflammatory Responses Assessment Model Characteristic Human-Sourced Murine-Sourced SYN IA1 IA2 SYN IA1 Resting Lymphocyte Predominant Proliferation None CD4+ T Cells CD8+ T Cells CD4+ T Cells CD8+ T Cells CD4:CD8  Cell Ratio Unchanged Decreased Increased Unchanged Decreased Th17:Treg  Cell Ratio Unchanged 2.21-fold Increase (­ Th17) 4.43-fold Increase (­ Th17 & ¯ Treg) Unchanged 6.93-fold Increase (¯ Treg) Enhancement of Th1 or iNKT Cell Differentiation None Th1 iNKT - - Cross-species T Cell Activation None Yes - Yes Yes  MLR Predominant Proliferation CD8+ T Cells CD8+ T Cells - CD8+ T Cells CD8+ T Cells CD4:CD8  Cell Ratio Unchanged Decreased - Unchanged Unchanged Th17:Treg  Cell Ratio 0.59-fold Decrease (¯ Th17) 1.55-fold Increase (­ Th17) - Trend of Increase (¯ Treg) 1.90-fold Increase (¯ Treg)  IA1 and IA2 were both biomanufactured from allorecognition-based human PBMC reactions; one population of allogeneic PBMC was replaced with HeLa cells for the production of IA2, to determine if a cancer-specific agent would prove more efficacious than IA1. Upon stimulation of resting CD3+ PBMC, IA1 predominantly increased CD4+ proliferation, while IA2 predominantly increased CD8+ proliferation (Table 4.1). Among CD4+ subsets, both IA1 and IA2 expanded the Th17 cell subset but IA2 did not expand Treg cell population, resulting in a higher magnitude of increase in the Th17:Treg cell ratio of PBMC relative to IA1. IA1 promoted Th1 cell differentiation while IA2 activated iNKT cells. These findings were supported by the upregulated expression of pro-apoptotic miRNA in IA2-, but not IA1-, treated PBMC. Hence, IA1 ≠ IA2, suggesting that the cell types [lymphocyte:lymphocyte versus lymphocyte:epithelial (i.e., HeLa)] utilized in the bioproduction alters the composition of secretome as the end product. This would not be surprising since all cells produce and export secretome, the composition of   108 which will vary based on cell type and function. Future directions will include investigating the differential effects of cell types on bioreactor produced secretome or miRNA. As mentioned above, diverse responses between human and mouse lymphocytes have been noticed. As discussed in Chapter 3, a stronger proliferative response was observed in resting mouse splenocytes than in resting human PBMC by IA1 stimulation, possibly due to the higher magnitude of splenocyte activity. However, a less significant enhancement of IA1 on alloproliferation within murine MLR was observed relative to human MLR, reflecting the differences between the Resting Lymphocyte and MLR models. Moreover, contrary to the predominant enhancement of human IA1 on resting CD4+ T cell proliferation, murine IA1 induced a CD8+-centric proliferation and differentiation in resting splenocytes. Human IA1 increased the Th17:Treg cell ratio in human PBMC (Resting and MLR) by predominantly increasing the Th17 cells, while murine IA1 induced an even higher magnitude of increase in both models by dramatically decreasing the mouse Treg cells. Surprisingly, murine SYN also increased cell proliferation and/or the Th17:Treg cell ratio in a similar manner to murine IA1 (Table 4.1). Confounding factors may include the ongoing immune events within the donor mice and the fragility of splenocytes in vitro. Similar effects of murine SYN was also noticed in the cross-species studies, as demonstrated in Chapter 3. Nonetheless, this study is focused on human PBMC models and human-sourced secretome therapeutics (i.e., IA1 and IA2).  4.5 Limitations and Future Directions In this chapter, the effects between acellular therapeutics and mAb/mitogen stimulations have been compared. According to the optimization studies in Chapter 3, IA1 and IA2 exerted maximal proliferation in resting PBMC at day 10. However, the mAb and mitogen stimulations were so potent and caused an apparent proliferation of T cells by 4-24 hours, resulting in   109 minimum cell viabilities at a late-assessment timepoint (i.e., day 10, data not shown). [392, 393] Alternatively, maximal T cell proliferation induced by anti-CD3/anti-CD28 and PHA stimulation was achieved at day 3 and 4, respectively. Hence, in the comparative studies, the effects of IA1/IA2 and current T cell activators were compared at their characterized optimal conditions (day 10 versus day 3 or day 4). Moreover, only one conventional dosage of mAb and mitogen were used to activate T cells. Our findings demonstrated a more controlled in vitro activation by IA1 and IA2 relative to these current T cell activators. Nonetheless, more extensive dosing and timing experiments need to be conducted for a more conclusive comparison in in vivo or clinical trial studies.  T cell proliferation and differentiation were examined using flow cytometric gating strategies conducted on the total lymphocyte population. Indeed, IA1, IA2, and other T cell activators were administrated to the entire population of human PBMC or mouse splenocytes, both of which consisted of multiple lymphocyte subpopulations (e.g., monocytes, B cells, and DC). [394, 395] The differing cell concentrations between human PBMC and murine splenocytes [382] could partially explain their diverse responses to IA1 activation observed in Chapters 3 and 4. While some argument could be made that the effects of IA1 on purified leukocyte subsets (e.g., CD3+CD4+ versus CD3+CD8+) needed to be assessed, this would perhaps be of less value than expected due to the extensive cross communications between not only T cell subsets but monocytes and other leukocyte populations. The enhancement of IA1 on the cytotoxicity of leukocytes (total population and purified CD4+/CD8+ T cells) against cancer cells will be discussed in Chapter 5.  While clear evidence of the pro-inflammatory activity of IA1 and IA2 have been achieved in T cell proliferation and differentiation, a more extensive analysis of the differential   110 signaling pathways, including nuclear factor-kB (NF-kB), mitogen-activated protein kinase (MAPK), and STAT, would be of significant benefit. Moreover, the PBMC miRNA expression profiles did not differentiate the miRNA transmitted from IA1/IA2 within the exosomes, from those subsequently biosynthesized in response to the IA1/IA2 activation. Therefore, miRNA tracking could be conducted using a molecular imaging-based strategy, which is also applicable to the in vivo studies due to its noninvasiveness to the experimental animals. [396] These mechanistic studies would also help in explaining the differential effects of IA1 and IA2 on resting T cell activation and/or persistent immune cells memories. Nonetheless, the current study illustrates the phenomenological effects of IA1 and IA2 on T cell activation and the subsequent cell-mediated anti-cancer responses (Chapter 5).     Despite that isolated lymphocytes rested in vitro represented a scenario of an unactivated immunity in vivo, the immune microenvironment in an organism containing a wide spectrum of different biological responses is never as simple as a tissue culture flask. Therefore, the immunomodulatory effects of acellular therapeutics (i.e., conditioned media and plasma) will be assessed in vivo in future studies. In parallel to the Resting Lymphocyte model in vitro, immunocompetent wildtype mice would be used as the in vivo model. As to the MLR model which demonstrate an existing moderate pro-inflammatory response, an infection or cancer mouse model would be employed accordingly. The degree of inflammation can be indicated by T cell proliferation and differentiation; the potential immunotoxicity induced by acellular therapeutics can be tested via the delayed-type hypersensitivity (DTH) assay in vivo. [397–401] The additional confirmation of IA1’s efficacy using animal models would underline the translational potential of our allorecognition-derived acellular therapeutics.    111 Chapter 5: Anti-Proliferative Effects of IA1- and IA2-Activated Leukocytes on Cancer Cells In Vitro 5.1 Rationale and Objectives Adoptive cell transfer (ACT) immunotherapies are gaining increasing interest due to their potent cancer cytotoxicity. Despite the efficacies, most current approaches, including CAR-T cell therapy, are still expensive, time consuming, and require extensive ex vivo processing. [178, 186] Moreover, as previously described, current direct activators (e.g., mAb and mitogens) used for endogenous T cell expansion pose significant risk to the patients, thus limiting their clinical utility. Hence, a safer, faster and lower-cost ACT-immunomodulatory approach would be advantageous.  By using a secretome approach, acellular therapeutics were developed that could be used to effectively promoted a pro-inflammatory response in resting autologous leukocytes of a patient thus obviating the risks of allogeneic GvHD (Chapter 4). To assess the anti-proliferative efficacy of the IA1- and IA2-activated leukocytes on cancer cells, in vitro cancer models were tested. The HeLa cervical cancer cell line was used as our first human cancer model due to the abundance of literature on their proven suitability as a cancer model. Indeed, it is probable that this cell line has been used in cancer studies more frequently than any other single cell line. [402] HeLa cells were derived from Henrietta Lacks who died of cervical cancer in 1951. [403] These cells are immortal, highly durable, and prolific cancerous epithelial cells.  In addition, melanoma cell lines have been used. Melanoma has been a widely-employed model for T cell-based immunotherapies and vaccine development. Indeed, the identification of melanosomal antigens enabled the engineering of cancer-specific T lymphocytes. [185, 404, 405]   112 Current successful immunotherapies in treating malignant melanoma include checkpoint inhibitors and CAR-T cell therapy. [406] In this study, an SH-4 melanoma cell line, which bears high metastatic potential in vivo, was used as another in vitro cancer model. SH-4 cells are immortal pleural effusion cells derived from a patient with metastatic melanoma in 1975. [407] To examine the potential impacts of acellular therapeutics on non-cancerous cells, direct leukocyte toxicity and effects of activated leukocytes on mouse myoblasts were tested.  Cytotoxic CD8+ T cells are believed to be the major effector leukocytes involved in killing cancer cells. Therefore, they have been widely used in adoptive therapies. [93, 94, 150, 179] More recently, CD4+ T cells have also been shown to exert cancer killing effects. [68] But perhaps most importantly, the synergistic function of both T cell subpopulations has been emphasized. [94] Hence, the activation of both resting and allo-stimulated CD4+ and CD8+ T cells by IA1 and IA2 were measured via flow cytometry in Chapter 4. In this chapter, the anti-proliferative effects of both single CD4+/CD8+ T cell subpopulations and total leukocytes (i.e., PBMC) on cancer cells are examined.  The cell-cell interactions between immune and cancer cells are a mechanistically important part of the anti-cancer response. Contact-dependent cell-cell crosstalk is also critical for the release of cytolytic granules onto target cells. [53] Hence, immune-cancer cell conjugation is an important parameter of T cell-mediated cytotoxicity. In this study, the interactions between PBMC and HeLa cells have been examined via microscopic and flow cytometric assays. In addition, indirect attenuation and/or killing of cancer cells can also be achieved via secreted biological molecules, including exosomal pro-apoptotic miRNA, obviating the necessity for direct leukocyte interaction. [121, 126] In this chapter, both direct and indirect cell-cell communication pathways were accordingly discussed.   113 Unregulated proliferation is perhaps the most defining hallmark of cancer cells. In this study, we employed an ACEA iCELLigenceÒ system to assess the real-time proliferation of cancer cells in response to either the IA1/IA2-activated leukocytes or the acellular therapeutic itself. The ACEA system registers the increase in the electrode impedance exerted by the viable cell attachment and monitors the cancer cell proliferation as presented by cell index proliferation curves. [144, 145] Leukocytes are non-adherent, thus do not elicit impedance; however, their immune responses against cancer cells are reflected by the kinetic cell impedance index. The proliferation of attached non-cancerous cells treated with IA1/IA2-activated leukocytes were also examined using this method.  In this chapter, I have focused on human secretome-derived therapeutics (i.e., IA1 and IA2) and human cancer models. This decision was made in part due to the more consistent observations with human preparations in immunomodulatory assays as described in Chapters 3 and 4. Mouse non-cancerous myoblasts were tested for toxicology studies. In this section, I discussed the anti-proliferative effects of IA1 or IA2 (direct toxicity or via activated resting leukocytes) on cancer cells and potential IA1/IA2 toxicity to non-cancerous cells. The objectives for this chapter were the following: Objective 1. To study the IA1/IA2 direct toxicity on resting leukocytes and the effects of IA1/IA2-activated leukocytes on non-cancerous cell proliferation; Objective 2. To investigate the anti-proliferative effects of IA1 and IA2 via direct toxicity or resting leukocytes (total PBMC and CD4+/CD8+ T cell subpopulations) activation on cancer cells;  Objective 3. To assess the impact of IA1 and IA2 on cell-cell interactions between leukocytes and cancer cells.   114 5.2 Results  5.2.1 Direct Toxicity of IA1 and IA2 To Human and Murine Leukocytes As previously described, the primary mode of use envisioned for the IA1 and IA2 biotherapeutics is the ex vivo activation of autologous donor lymphocytes for subsequent reinfusion into the patient. Critical to this mode of use is the lack of direct toxicity of IA1 and IA2 to the donor leukocytes themselves. To examine if IA1 and IA2 secretomes exerted direct toxicity to the donor leukocytes, the viability of resting PBMC were examined. Of biologic significance, neither the IA1 or IA2 preparations exerted direct toxicity to resting PBMC following 24 hours of exposure for pretreatment (Figure 5.1 A). Indeed, the viability of the resting PBMC was slightly, but statistically significantly (p < 0.0001) increased, with both therapeutics. Moreover, to examine if these IA1 pretreated PBMC induced toxicity to resting autologous leukocytes, resting PBMC (labeled with CFSE) from the same donor were co-cultured (1:1) with IA1 pretreated (24 hours) PBMC (non-CFSE-labeled) for 7 days and assessed for viability (Figure 5.1 B). As shown, SYN and IA1 pretreated PBMC exerted minimal toxicity to the autologous resting PBMC, demonstrating that IA1-activated autologous PBMC would not adversely affect the patient’s endogenous PBMC during the ACT therapy. Similar to human studies, murine-sourced IA1 itself exerted minimal cytotoxicity on resting splenocytes for effector cell pre-activation; actually, IA1 increased (p < 0.0001) the viability of resting splenocytes following 24 hours pretreatment (Figure 5.1 C). Surprisingly, SYN treatment also resulted in an increased splenocyte viability similar to that induced by IA1. The similar phenomenon with murine SYN was previously observed in splenocyte proliferation and T subset differentiation (Chapter 4). This partially explains why the focus of this chapter is human-sourced acellular therapeutics and human cancer cell models.    115  Figure 5.1 IA1 and IA2 Exerted No Toxicity on Resting Leukocytes.  Panel A. Direct toxicity on resting PBMC. Neither IA1 or IA2 (diluted 1:1 with AIM V) exerted any direct PBMC toxicity over the 24 hours used for PBMC pretreatment. Actually, both IA1 and IA2 improved PBMC viability over the 24 hour incubation in comparison to the fresh media and SYN product. n = 7. Panel B. Moreover, PBMC pretreated (24 hours) with SYN or IA1 had no toxicity to autologous resting PBMC. Pretreated PBMC were washed and overlaid 1:1 on resting, CFSE-labeled, autologous PBMC. The viability of the recipient CFSE+ PBMC was assessed at day 7. n = 6. Panel C. Murine IA1 [diluted 1:1 with RPMI 1640 (supplemented with 10% FBS)] exerted no direct splenocyte toxicity over 24 hours used for splenocyte pretreatment. Actually, IA1 improved splenocyte viability in comparison to the fresh media. Surprisingly, SYN also increased splenocyte viability to a similar level as IA1. n = 8. Viability was assessed using 7AAD.  p < 0.0001 (‘****’) was calculated in comparison to Fresh.  5.2.2 Toxicity of IA1-Activated Splenocytes To Murine Non-Cancerous Cells While IA1 (and IA2) exerted no direct toxicity to leukocytes (human PBMC and mouse splenocytes), we also wanted to examine the effects of the activated leukocytes on non-cancerous cells. Using the ACEA system, the proliferation of murine myoblasts was examined upon exposure to IA1-activated splenocytes. Of note, C2C12 myoblasts were derived from C3H (H-2k) mice thus allogeneic from the splenocytes isolated from C57BL/6 (H-2b) mice. However, as shown in Figure 5.2 A-C, at either splenocyte-to-myoblast ratio (i.e., 10:1, 25:1 and 50:1), resting, but allogeneic splenocytes did not impact the growth of non-cancerous cells. The pretreatment of resting splenocytes with SYN or IA1 exerted no inhibition on myoblast proliferation either. In fact, a slight enhancement of control myoblast proliferation was observed with the overlay of resting or activated splenocytes. In summary, neither human nor murine IA1 Direct Splenocyte Toxicity C%	Splenocyte	Viability080604020Fresh SYN IA1**** ****Direct PBMC Toxicity A B9005%	PBMC	ViabilityFresh SYN IA1 IA295100**** ****Human-sourcedResting SYN IA1PBMCPBMC:PBMC (1:1) Murine-sourced  116 had direct toxicity to resting effector leukocytes (Figure 5.1). These activated leukocytes exerted no genitive effects on non-cancerous human PBMC or mouse myoblasts (Figures 5.1-5.2).    Figure 5.2 Murine IA1-Activated Splenocytes Had Minimal Toxicity to Non-Cancerous Cells. Addition of resting splenocytes at neither splenocyte:myoblast ratio of 10:1 (Panel A), 25:1 (Panel B) or 50:1 (Panel C) had any effect on myoblast proliferation in comparison to control cells. Moreover, neither SYN nor IA1 pretreated (24 hours) splenocytes inhibited myoblast proliferation relative to resting splenocytes. 5,000 total myoblasts were pre-seeded for 1 hour before the overlay of splenocytes. Co-cultures were incubated for 7 days. Proliferation curves shown represent median ± IQR from at least 3 independent experiments.  5.2.3 Anti-Proliferative Effects on Cancer Cells: HeLa Epithelial Cancer Model As demonstrated, our secretome-based acellular therapeutics had no direct toxicity to human or murine leukocytes; nor did the activated murine leukocytes adversely impact non-cancerous cell proliferation. To examine the cancer-inhibitive effects of acellular therapeutics, initial studies assessed direct toxicity of IA1 and IA2 on HeLa cells over 7 days. In the absence of PBMC, HeLa cells showed continuous growth over 5-6 days (Figure 5.3 A-D). Similar to their effects on resting PBMC or non-cancerous cells, IA1, as well as the SYN and TAI, had no direct toxicity to HeLa cells as shown by proliferation curves and the quantification of Area Under Curve (AUC; Figure 5.3 A). Somewhat surprisingly, direct treatment of HeLa cells with IA2 demonstrated an almost total inhibition of growth. By approximately 30 hours, no cell proliferation was noted in HeLa cell cultures treated directly with IA2. The AUC of IA2 C Splenocyte:Myoblast!50:1 Hours24 48 72 96 120 144 168B Splenocyte:Myoblast!25:1 Hours24 48 72 96 120 144 168A Splenocyte:Myoblast!10:1 Control MyoblastResting SplenocyteSYNIA168420Myoblast Proliferation(Cell Impedance Index)Hours24 48 72 96 120 144 168  117 treatment was also significantly decreased relative to all other groups (i.e., Fresh, SYN, IA1, IA2 and TA1). The direct toxicity of IA2 to HeLa cells, but not to PBMC was suggestive of a pro-apoptotic or necrotic effect on the HeLa cells that was not induced by IA1. Indeed, as noted in Figure 4.3, IA2-treated PBMC exhibited several miRNA associated with the apoptosis of cancer cells. To examine the effect of IA1 and IA2 (as well as the SYN and TA1) on the ability of resting PBMC to attenuate cancer cell growth, resting human PBMC were treated with the therapeutics for 24 hours, washed, and overlaid on the HeLa cells at PBMC:HeLa ratios of 0:1, 10:1, 25:1 and 50:1 (Figure 5.3 B-D). The extensive washing removed any residual extracellular therapeutics leaving only the PBMC to exert the anti-cancer efficacy. Addition of untreated, but allogeneic, resting PBMC resulted in some growth retardation and the eventual killing of HeLa cell by day 4 or 5 possibly consequent to a combination of allorecognition and anti-cancer immune responses (Figure 5.3 B-D). This effect was most apparent at a PBMC:HeLa ratio of 50:1 as shown by the significant AUC decrease relative to control HeLa cells (Figure 5.3 D). The SYN treated PBMC behaved similarly and the reduction of HeLa cell proliferation/AUC was dependent on the PBMC number. As shown in Figure 5.3 B, low concentration of PBMC (10:1) pretreated with either fresh or acellular therapeutics had minimal effects on HeLa cell proliferation. This was particularly true when comparing the effects of pretreated PBMC to the direct toxicity of IA2 as shown by the * dotted line and the black bar. In contrast, at higher PBMC concentrations (25:1 and 50:1), IA1- and IA2-pretreated PBMC demonstrated an enhanced anti-proliferative effect. As shown in Figure 5.3 C, IA1-activated PBMC rapidly killed HeLa cells by day 4-5; IA2-activated PBMC induced an even more dramatic inhibition on cancer cell proliferation. This conclusion was confirmed by the AUC graph which demonstrated that IA1 treatment only significantly decreased the AUC relative to control HeLa cells, while   118 IA2-activated PBMC also induced a significant AUC decrease relative to resting cells. Interestingly, the cancer-inhibitive effects of neither IA1 nor IA2 pretreated PBMC were stronger than the IA2’s direct toxicity to HeLa cells (* dotted line; black bar). At a PBMC:HeLa ratio of 50:1 (Figure 5.3 D), the effects of both IA1- and IA2-actiavted PBMC were most significant. Relative to the apparent inhibition of HeLa cell proliferation by 4-5 days for resting or SYN-treated PBMC, the cancer-inhibitive effects of IA1-activated PBMC were noted by ~12 hours. Indeed, the IA1 pretreatment significantly decreased the AUC relative to both control HeLa cells, and resting PBMC, which had already significantly reduced the proliferation of control HeLa cells. Similar to the observations at the 25:1 ratio, IA2-activated PBMC exerted an even more potent (relative to IA1) anti-HeLa effect at the 50:1 ratio, resulting in an almost equivalent cancer-inhibitive effect to the direct toxicity of IA2 (* dotted line; black bar). IA2 significantly reduced the AUC relative to resting and SYN-treated PBMC. Intriguingly, Treatment of allogeneic PBMC with the tolerogeneic TA1 demonstrated no anti-HeLa effect and actually enhanced HeLa cell proliferation (Figure 5.3 C-D). This was particularly apparent at the 25:1 ratio (Figure 5.3 C) as shown by a significant increase in the AUC relative to fresh media, SYN/IA1/IA2 pretreatment or IA2’s direct toxicity. At the 50:1 ratio (Figure 5.3 D), a significant AUC increase with TA1 treatment was also noted in comparison to IA1/IA2-activated PBMC or IA2 media alone. This findings agreed with previous studies which have demonstrated that TA1 reduced the inherent alloresponse resulting in a tolerogeneic microenvironment. [248, 253, 254]   119  **345210-1HeLa Proliferation(Cell Impedance Index)Control HeLa Fresh MediaSYNIA1IA2TA1Direct HeLa Toxicity ACDBHeLa Proliferation(Cell Impedance Index)50:1 (PBMC:HeLa)25:1 (PBMC:HeLa)Control HeLa Resting PBMCSYNIA1IA2TA110:1 (PBMC:HeLa)Hours24 48 72 96 120 144 168*##**345210-1#4005003002001000*** ****4005003002001000** * *CON HeLaRest-!ingSYN IA1 IA2 TA1 IA2!MediaPBMCHeLa Proliferation(Cell Impedance Index)HeLa Proliferation(Cell Impedance Index)4005003002001000*** *CON HeLaFresh SYN IA1 IA2 TA1MediaArea Under CurveArea Under CurveArea Under CurveArea Under Curve******4005003002001000*** *** * *345210-1345210-1  120 Figure 5.3 IA1 Enhanced PBMC-Mediated Inhibition of HeLa Cell Proliferation while IA2 Exerted Direct Toxicity to HeLa Cells. Panels A. The direct HeLa cell toxicity of the SYN, IA1, IA2 and TA1 secretome products (ratio of 1:1 with HeLa growth media) was assessed. As shown, SYN, IA1 and TA1 had no effect on HeLa cell proliferation relative to the control sample. In contrast, IA2 exerted a potent and direct toxicity to HeLa cells. The quantification of Area Under Curve (AUC) also demonstrated that IA2 significantly reduced the AUC relative to all other conditions. Panels B-D. Shown are HeLa cell growth curves when overlaid at a 10:1, 25:1 or 50:1 ratio (Panel B, C and D, respectively) with resting PBMC, or the same PBMC pretreated (24 hours) with SYN, IA1, IA2 or TA1 prior to overlay. Resting and SYN-treated allogeneic PBMC exerted generic inhibition of HeLa cell proliferation likely consequent to a combination of allorecognition and anti-cancer immune responses. This inhibitive effect was PBMC number dependent. Low concentration (10:1) of IA1- or IA2-activated PBMC had minimal effects. In contrast, IA1 pretreated PBMC demonstrated a dramatically enhanced anti-cancer effect at both the 25:1 and, most significantly, 50:1 ratios. Moreover, IA2-activated PBMC exerted an even more potent (relative to IA1) anti-HeLa effect at both ratios; though it was not as strong as the direct toxicity of IA2. Interestingly, treatment of allogeneic PBMC with TA1 demonstrated no anti-cancer effect. In fact, TA1 appeared to reduce the inherent alloresponse and actually enhanced HeLa cell proliferation. This was particularly apparent at the 25:1 ratio. The dotted line (indicated by *) and the black bar represent direct IA2 HeLa cell toxicity shown in Panel A. Cell proliferation was continuously monitored in real-time over 7 days as a function of the cell impedance index using an ACEA iCELLigenceâ instrument. Proliferation curves shown represent median ± IQR from at least 3 independent experiments. HeLa cells were seeded at an initial density of 5,000 cells per well. PBMC were pretreated with the indicated agent and then extensively washed prior to being overlaid onto the HeLa cells. AUC graphs were derived from the proliferation curves. p < 0.05 (‘*’) was calculated in comparison to control HeLa unless specified. n ≥ 3.  Based on data observed in Chapter 3, the active components of IA1 appeared to reside in the ³ 30 kDa fraction. To determine if the cancer-inhibitive efficacy also resided in the miRNA-enriched (≥ 30 kDa) IA1 fraction, the anti-proliferative effects of resting PBMC pretreated with either the < 30 or ≥ 30 kDa fractions of IA1 were examined (Figure 5.4). As anticipated by Figure 3.3, the cytokine-rich fraction (< 30 kDa) pretreated PBMC showed no significant anti-proliferative activity on cancer cells and were virtually identical to the sham (i.e., SYN)-treated PBMC (dotted blue line) at both 25:1 (Figure 5.4 A) and 50:1 (Figure 5.4 B) ratios. In contrast, the PBMC pretreated with miRNA-containing ³ 30 kDa fraction of IA1 mediated the anti-HeLa effects and was almost indistinguishable from the complete IA1 preparation (dotted red line) at both ratios.     121  Figure 5.4 miRNA-Containing Fraction of IA1 Mediated the Anti-Proliferative Effects on HeLa Cells.  PBMC pretreatment with the cytokine-poor, miRNA-enriched, ³ 30 kDa fraction of IA1 resulted in a potent anti-HeLa effect. In contrast, pretreatment with the cytokine rich < 30 kDa fraction of IA1 was indistinguishable from the SYN preparation. PBMC:HeLa ratio equaled to 25:1 (Panel A) or 50:1 (Panel B). Cell proliferation was continuously monitored in real-time over 7 days as a function of the cell impedance index using an ACEA iCELLigenceâ instrument. Proliferation curves shown represent median (± IQR) from at least 3 independent experiments. HeLa cells were seeded at an initial density of 5,000 cells per well. PBMC were pretreated with the indicated agent and then extensively washed prior to being overlaid onto the HeLa cells.  To address whether IA1 primarily enhanced the cancer-inhibitive activity of CD4+ or CD8+ Teff cell subsets, T cell subset purification studies were done (Figure 5.5). This was of interest as studies of IA1 on resting PBMC (Figure 4.1) suggested a critical role for CD4+ T cells while in an allorecognition (MLR; Figure 4.4) model CD8+ T cells were implicated. As shown in Figure 5.5 A, the anti-HeLa effects of resting PBMC resided with the CD8+ T lymphocytes. Indeed, resting CD4+ T cells demonstrated no anti-proliferative effect and failed to inhibit HeLa cell proliferation. This was also true for the AUC quantification study which demonstrated that the resting PBMC and CD8+ T cells significantly decreased the AUC relative to the control HeLa group. In contrast, resting CD4+ T cells made minimal alterations. As shown in Figure 5.3 D and Figure 5.5 B, SYN-treated PBMC, similar to resting cells, induced a significant inhibition of HeLa cell proliferation likely due to a combination of allorecognition and anti-cancer immune responses. IA1, and most significantly IA2, enhanced the HeLa-inhibitive response of PBMC and decreased the AUC relative to control HeLa cells. IA2-50:1 (PBMC:HeLa)HeLa Proliferation(Cell Impedance Index)345210-1A BHours24 48 72 96 120 144 168Control HeLaIA1< 30 kDa≥ 30 kDaSYN-PBMC25:1 (PBMC:HeLa)Hours24 48 72 96 120 144 168  122 activated PBMC also significantly decreased the AUC relative to sham (SYN)-treated cells. In contrast to resting CD4+ T cells, IA1 and IA2 pre-activated (24 hours) purified CD4+ T cells demonstrated significant anti-HeLa effects (Figure 5.5 C). However, the effect of IA1 on CD4+ T cells activation were more pronounced than that of IA2 (as was anticipated by Figure 4.1). Indeed, only IA1 pretreatment significantly decreased the AUC relative to the sham SYN treatment. Similarly, IA1 significantly enhanced the anti-HeLa efficacy of CD8+ T cells relative to both control HeLa cells and SYN treatment (Figure 5.5 D). IA2 was less effective than IA1 in the activation of CD8+ T cells to inhibit HeLa cell proliferation. This finding was somewhat surprising as IA2 treatment of resting PBMC (in the absence of HeLa cells) increased CD8+ T cell proliferation relative to CD4+ T cell proliferation (Figure 4.1). Of note, neither IA1 or IA2 pretreated purified CD4+ or CD8+ T cells alone inhibited HeLa cell proliferation as effectively as total PBMC (Figure 5.5 B); likely due to the well described synergistic interaction of CD4+ and CD8+ T cells in the anti-cancer response. [68, 93] What was also noticeable was that IA2-activated total PBMC seemed to exert a stronger anti-HeLa effect than IA1-activated PBMC (though still less significant than IA2 direct toxicity, Figure 5.3), but IA2 was less effective than IA1 in activating CD4+ and CD8+ T cells subpopulations.    123  Figure 5.5 IA1 and IA2 Enhanced the Anti-Proliferative Effects of CD4+ and CD8+ T Cell Subsets on HeLa Cells.  To assess the relative roles of CD8+ and CD4+ T subsets on HeLa cell proliferation, CD4+ and CD8+ T cells were purified from resting human PBMC and pretreated with Fresh, SYN, IA1 or IA2 for 24 hours. As shown in Panel A, CD8+ T cells solely conferred the anti-Hela effect observed with resting PBMC. Indeed, resting PBMC and CD8+ T cells significantly decreased the AUC relative to the control HeLa cells. In stark contrast, resting CD4+ T cells showed no anti-HeLa effects as shown by the proliferation curves and AUC graph. As shown in Panel B, SYN-PBMC induced a significant inhibition of HeLa cell proliferation. IA1-, and more significantly, IA2-activated PBMC attenuated the control HeLa cell proliferation. IA1-activated PBMC also significantly reduced the AUC relative to SYN treatment. Panel C demonstrated that pretreatment of purified CD4+ T cells with IA1, and to a lesser extent IA2, resulted in a potent anti-HeLa effect relative to the control HeLa cells or SYN pretreatment. Additionally, IA1 (in particular) and IA2 enhanced the HeLa-inhibitive effects of CD8+ T cells relative to control HeLa cells (Panel D). Pretreatment of IA1, but not IA2, significantly decreased the AUC relative to SYN. Cell proliferation was continuously monitored in real-time over 7 days as a function of the cell impedance index using an ACEA iCELLigenceâ instrument. Proliferation curves shown represent median ± IQR from at least 3 independent experiments. HeLa cells were seeded at an initial density of 5,000 cells per well. PBMC were pretreated with the indicated agent and then extensively washed prior to being overlaid onto the HeLa cells. p < 0.05 (‘*’) and p < 0.001 (‘***’) were calculated in comparison to control HeLa unless specified. n ≥ 3.  In summary, both IA1- and IA2-activated resting PBMC exerted a HeLa-inhibitive efficacy. The enhancement of PBMC-induced anti-HeLa response by IA1 was solely mediated 345210-1ABHeLa Proliferation(Cell Impedance Index)Hours24 48 72 96 120 144 168CDHeLa Proliferation(Cell Impedance Index)Control HeLa Resting CD4Resting CD8Total PBMCActivated PBMC (50:1)Activated CD4 T Cells (50:1)Activated CD8 T Cells (50:1)Control HeLa IA1 IA2SYNResting Subsets (50:1)HeLa Proliferation(Cell Impedance Index)HeLa Proliferation(Cell Impedance Index)345210-1345210-1345210-1Area Under CurveArea Under CurveArea Under CurveArea Under CurveCON HeLaIA2IA1SYN****0400500300200100PBMC0400500300200100CON HeLaIA2IA1SYNCD8CON HeLaCD8CD4PBMC**0400500300200100*0400500300200100CON HeLaIA2IA1SYNCD4*********  124 by the miRNA-containing but not cytokine-enriched fraction. T cell subset purification studies demonstrated that IA1 was more effective than IA2 on activating the CD4+ and CD8+ T cells. However, IA2, but not IA1, inhibited HeLa cell proliferation via direct toxicity. The distinct inhibitory effects of IA2 on HeLa cell proliferation indicated that IA2 was not equivalent to IA1.  5.2.4 IA1 and IA2 Differentially Affected PBMC-HeLa Cell Interactions A key component of T cell mediated inhibition of cancer cell proliferation is the crosstalk between immune and target cells. To investigate the interactions between control and IA1/IA2-activated PBMC with HeLa cells, time-lapse video microscopy and cell conjugation studies were conducted. As illustrated in Figure 5.6 A, IA1 and IA2 treated PBMC exhibited significantly greater interactions with HeLa cells than did resting PBMC. Microscopically, this was most pronounced with IA1, versus IA2, as noted by the greater degree of clustering of PBMC (white arrows) and individual HeLa cells (asterisk). Interestingly, the IA2-PBMC treated HeLa cells (black open arrows) demonstrated significant cellular vesiculating/blebbing and morphology alterations not observed in the IA1-PBMC sample. These morphological changes may be characteristic of IA2 associated apoptosis/necrosis. These microscopic cell:cell observations were further confirmed using a cell:cell conjugation assay (Figure 5.6 B). As noted, both IA1 and IA2 significantly increased PBMC:HeLa conjugation while the SYN media had no effect. As a result of the enhanced PBMC:HeLa cell interactions, HeLa cell proliferation was significantly decreased in the IA1 and IA2, but not SYN, treatment groups (Figure 5.6 C).  In conclusion, IA1 promoted the direct PBMC-HeLa cell conjugation and enhanced the anti-HeLa efficacy of resting PBMC. IA2 also increased the PBMC-HeLa cell conjugation formation but induced apoptosis/necrosis-like responses in HeLa cell with limited participation of PBMC.   125  Figure 5.6 IA1 and IA2 Differentially Affected PBMC-HeLa Cell Interactions as Shown by Photomicroscopy and Cell Conjugation Assays. Conjugation formation between resting, IA1 or IA2 pretreated (24 hours) PBMC and HeLa cells was measured through both light microscopy and flow cytometry. PBMC:HeLa ratio equaled to 50:1. Time-lapse video was acquired for 90 minutes after 72 hours of PBMC and HeLa cell co-culture. Panel A. Representative images of PBMC-HeLa conjugation shot at 0, 30, 60 and 90 minutes during the time-lapse video. The black asterisks indicated representative HeLa cells, the white arrows pointed at PBMC. Black open arrows demonstrated blebbing of HeLa cells. Images shown are representatives frames at the indicated times from 3 independent experiments. Size bar = 10 micrometer (µm). Panel B. IA1 and IA2 significantly enhanced conjugation between CFSE labeled PBMC and Far-Red labeled HeLa cells after 20 minutes’ co-culture as measured by flow cytometry. Panel C. HeLa cell proliferation index at 72 hours (derived from Figure 5.3 D) post overlay with IA1 or IA2 pretreated PBMC correlated with the microscopic and flow cytometric findings. p < 0.05 (‘*’) and p < 0.01 (‘**’) were calculated in comparison to Fresh, n ³ 4.  34210Fresh SYN IA1 IA2Resting PBMC Treated With0102030Fresh SYN IA1 IA2Resting PBMC Treated With***PBMCA MinutesB C0 30 60 9072h* * * ** * * ** * * *Percent		Conjuga7on	(PBMC-HeLa)HeLa	Prolifera7on	(72h)	(Cell Impedance Index)Res7ngIA1IA2**  126 5.2.5 Anti-Proliferative Effects on Cancer Cells: SH-4 Melanoma Model The differential effects of IA1 and IA2 on HeLa cell proliferation and PBMC-HeLa cell interactions were observed above. To further assess the anti-cancer utility of IA1 and IA2, and the potential specificity of IA2 to a certain type of cancer (i.e., HeLa) cells, the SH-4 melanoma cell line was examined. Interestingly, as shown in Figure 5.7 A, in the absence of any PBMC, both IA1 and IA2 directly inhibited SH-4 proliferation. The direct anti-proliferative effect was more pronounced for IA2 relative to IA1 (p < 0.01), and similar to that seen with HeLa cells, suggesting that IA2’s direct mechanism of action could be broad spectrum. However, despite IA1’s lack of direct toxicity on HeLa cells, IA1 significantly reduced SH-4 proliferation and the AUC relative to control SH-4 cells or sham (Fresh and SYN) treatment; though the SH-4 cells did demonstrate a slow but consistent proliferation over time which was not seen with IA2 (Figure 5.7 A). As shown in Figure 5.7 B-C, SYN-treated PBMC at a 50:1 ratio exhibited an allo-/anti-cancer response resulting in a slight loss of SH-4 proliferation and cell death starting after approximately 72 hours. More importantly, while both IA1 and IA2 pretreatment of same donor resting PBMC (Figure 5.7 B and C, respectively) enhanced their anti-proliferative effects on SH-4 cells, IA1 was clearly superior to IA2. IA1-activated PBMC significantly decreased the AUC relative to control SH-4 cells even at a very low 10:1 ratio; this effect was more dramatic (p < 0.05 relative to SYN as well) at increased PBMC numbers (Figure 5.7 B). Indeed, with IA1 activation at both the 25:1 and 50:1 ratios, minimal SH-4 proliferation was noted over the first 48 hours and the SH-4 cell death (i.e., decreased impedance) became readily apparent by 48 hours. IA2-activation also enhanced PBMC-mediated SH-4 inhibition but to a much lesser degree; they required a longer time (> 72 hours) and a larger PBMC number (50:1) to induce a significant cancer attenuation (i.e., decreased impedance and AUC).    127  Figure 5.7 IA1 and IA2 Attenuated SH-4 Cell Proliferation via Both Direct SH-4 Toxicity as well as PBMC-Mediated Growth Inhibition.  Panel A. In contrast to HeLa cells, both IA1, and more significantly IA2, demonstrated direct toxicity/growth arrest to the control SH-4 cells or sham (Fresh and SYN) treatment. IA2 also significantly decreased the AUC relative to IA1. The SYN product had minimal inhibitory effect on SH-4 proliferation relative to fresh media. Panel B: Shown are the effects of IA1-activated PBMC (red lines) at ratios to SH-4 cells of 10:1, 25:1 and 50:1 relative to SYN-treated PBMC at a ratio of 50:1 (shaded area). SH-4 proliferation was significantly inhibited by IA1-activated PBMC even at a low PBMC concentration (10:1). The AUC of IA1 pretreatment was significantly decreased especially at higher ratios (25:1 and 50:1) relative to both control SH-4 cells and the SYN treatment. Panel C: IA2-activated PBMC (10:1, 25:1 and 50:1 ratios) also inhibited SH-4 cell proliferation though to a much lesser extent than IA1. A larger PBMC number (50:1) was needed for a significant decrease in the AUC. Proliferation curves shown represent median ± IQR from at least 3 independent experiments. p < 0.05 (‘*’) was calculated in comparison to control SH-4 unless specified. n ≥ 3.  In conclusion, IA1 exerted both direct and PBMC-mediated inhibition on SH-4 cell proliferation. The cytotoxicity of IA1-activated PBMC were more pronounced and lymphocyte number-dependent. IA2 showed less enhancement on anti-melanoma effect of PBMC but demonstrated a broad-spectrum direct cytotoxicity to both HeLa cells (Figure 5.3) and SH-4 melanoma cells (Figure 5.7). The inhibitory effects of IA1 and IA2 (both via direct toxicity and SH-4 Proliferation(Cell Impedance Index)A Direct SH-4 Toxicity 1.52.02.51.00.50.0IA1Control SH-4FreshIA2SYNHours24 48 72 96 120 144 168010:125:150:150:1Control SH-4IA2 (ratios) SYN-PBMC 50:1Control SH-4IA1 (ratios) SYN-PBMC 50:1B C50:1Hours24 48 72 96 120 144 168010:125:150:1Hours24 48 72 96 120 144 1680Area Under CurveCON SH-4IA2IA1FreshMediaSYN0300200100 *** ***CON SH-450:125:1SYN (50:1) IA1-PBMC10:1 CON SH-450:125:1IA2-PBMC10:1IA1-Activated PBMC IA2-Activated PBMC SYN (50:1)****** *  128 PBMC activation) on cancer cell proliferation suggested that IA1 and IA2 were not equivalent and functioned, at least partially, via different mechanisms. 5.3 Summary  A synergy between an alloresponse and an anti-cancer response has long been suggested, but the risks of GvHD precluded significant research in this area. [211] Using a secretome approach, our acellular therapeutics derived from allorecognition reactions can effectively enhance lymphocyte activation and induce an anti-proliferative effect on cancer cells. Of biological importance, pretreatment with IA1 or IA2 had no negative effect on resting donor leukocytes. But, these secretome-activated leukocytes exerted a significant inhibition of cancer cell [human cervical cancer (HeLa) and melanoma (SH-4)] proliferation. Importantly, although the secretome-based therapeutics are biomanufactured from an allorecognition response, the anti-cancer T cell responses mediated by IA1 or IA2 were not MHC-restricted. IA1 had a broad-spectrum anti-proliferative effect on both HeLa and SH-4 cell lines; IA2, which was derived from HeLa-specific allorecognition and enhanced a more significant anti-HeLa response than IA1, showed additional toxicity to SH-4 melanoma cells. Not surprisingly, cancer cell lines show varied responsiveness, even to the same drug or immunotherapy. [408, 409] Similarly, HeLa and SH-4 cells showed varied responsiveness to the differential effects of IA1 and IA2. Contrary to the minimal toxicity of IA1 itself to HeLa cells, a direct inhibition of SH-4 cells by IA1 was observed. IA2-activated PBMC exerted a stronger anti-HeLa effect than IA1-activated PBMC, but SH-4 cells were clearly more sensitive to the IA1 treatment. Moreover, IA1 and IA2 seemed to inhibit cancer cell proliferation using distinct preferential mechanisms. IA1 significantly enhanced the anti-HeLa effects of both total PBMC and purified CD4+/CD8+ T cell subsets. IA2 activated total PBMC more significantly (relative to IA1) but had less potency on purified T cell   129 subsets. In addition, IA1 induced cancer cell death through a PBMC-HeLa cell conjugation-associated pathway, while IA2 triggered HeLa cell apoptosis/necrosis with limited direct contact with PBMC. Importantly, IA1-activated leukocytes had no toxicity to non-cancerous cells, indicating a reduction in off-target adverse events.  5.4 Discussion As biomanufactured potential therapeutics, IA1 or IA2 had few direct toxicities to resting lymphocytes (i.e., PBMC; Table 5.1). Moreover, the IA1- or IA2-activated PBMC exerted anti-proliferative effects solely on cancer cells, but showed no negative effects on normal tissues, including autologous resting PBMC and non-cancerous cells. As described previously and summarized in Table 5.1, IA1 and IA2 induced differential patterns or magnitudes of cancer-inhibitive activity. IA1 had minimal direct impact on cancer cell proliferation, except for moderate inhibition of SH-4 cell growth, but significantly enhanced the cancer-inhibitive effects of resting PBMC. This PBMC number-dependent anti-proliferative response on cancer cells was more pronounced in the SH-4 model relative to the IA2-activated PBMC. In contrast, IA2 predominantly attenuated cancer cell proliferation via direct toxicity, the magnitude of which was equal to or greater than the PBMC-mediated anti-HeLa/SH-4 responses. This is especially true for HeLa cells, where IA2-activated PBMC were more effective at inhibiting HeLa cell proliferation relative to IA1-activated PBMC, suggesting a HeLa cell specificity of IA2. However, the purification of T cell subpopulations diminished the enhancement of IA2 on CD4+ and CD8+ T cells. This result was somewhat unexpected, as both CD8+-centric proliferation and a higher Th17:Treg cell ratio (relative to IA1) were induced by IA2 in Chapter 4; alternative pathways of cancer elimination other than T cell-dominant cytotoxicity might have been pursued by IA2. Moreover, as summarized in Chapter 4, IA1 increased the Th17:Treg cell ratio by   130 expanding the population of Th17 cells, while IA2 might adopt a mechanism of passively releasing the immunosuppressive stress (i.e., Treg cell response) rather than actively enhancing the direct cytotoxicity of Teff cells during cancer cell growth inhibition.  Table 5.1 Comparison of Anti-Proliferative Effects on Cancer Cells Effects Assessment IA1 IA2 Toxicity to Non-Cancerous Cells Direct toxicity on resting leukocytes Human PBMC: - Mouse Splenocytes: - Human PBMC: -  PBMC-mediated toxicity to  autologous resting PBMC - Not applicable Anti-proliferative effects of activated splenocytes on non-cancerous myoblasts - Not applicable Anti-Proliferative Effects on Cancer Cells Direct toxicity on cancer cells HeLa: -  SH-4: ++ (delayed growth) HeLa: +++++ SH-4: +++++ Enhancement of PBMC-mediated anti-proliferative effects on cancer cells HeLa: ++++  SH-4: +++ HeLa: +++++  SH-4: ++ Enhancement of CD4+/CD8+ T cells-mediated anti-proliferative effects  on HeLa cells CD4+ T cells: ++++  CD8+ T cells: ++ CD4+ T cells: ++  CD8+ T cells: + Enhancement of PBMC-HeLa cell conjugation Cytometric: ++ Microscopic: cell death Cytometric: +++ Microscopic: cell blebbing  Pro-apoptotic PBMC miRNA + +++++  Note: ‘-’ and ‘+’ indicated the absence or presence of response, respectively. The larger the numbers of ‘+’, the higher the magnitude of response.   In addition, the direct HeLa cell toxicity and the morphological changes noted in HeLa cells were suggestive of a pro-apoptotic or necrotic effect of IA2 that was not induced by IA1. Cytoplasmic/nuclear condensation, formation of apoptotic bodies and maintenance of an intact plasma membrane are morphological characteristics of programmed cell death; apoptotic inducers indeed can cause cancer cell blebbing. [410, 411] Although both IA1 and IA2 increased the percent conjugation between PBMC and HeLa cells in the flow cytometric assay, in microscopic images, IA1 caused a morphological presentation of HeLa cell death depending on the direct PBMC-HeLa cell conjugation, while the IA2-induced HeLa cell blebbing and vesiculating did not seem to be associated with direct contact with PBMC (Table 5.1). As introduced in Chapter 1, immune cell-mediated cancer cytotoxicity can be achieved through either direct (e.g., conjugation) or indirect (e.g., miRNA-containing exosomes) pathways.   131 Indeed, as anticipated from work discussed in Chapter 4, several apoptotic miRNA have been identified as upregulated specifically in IA2 but not IA1, may accounting for the direct toxicity of IA2 and the conjugation-independent apoptosis/necrosis of cancer cells. In summary, all these findings indicated that IA2 was not equivalent to IA1 in either immunomodulation or cancer cell growth inhibition. 5.5 Limitations and Future Directions In this study, human allogeneic cancer cell line models were used to assess the anti-proliferative effects of IA1 and IA2-activated PBMC on cancer cells. These studies demonstrated that the anti-proliferative effects of PBMC could be greatly enhanced. However, due to the nature of cancer cell lines, the PBMC used were obviously allogeneic. Hence, the inhibition of cancer cell proliferation was a result of both allorecognition and, after ~4 days of incubation, an anti-cancer Teff cell response. However, based on my results presented in Chapter 4, it would not be surprising if secretomes also enhanced the alloresponse (as IA1 jumpstarted the MLR allorecognition pathway) as well as the anti-cancer immune response itself. Evidence for a specific anti-HeLa response can be observed by the finding that resting, allogeneic CD4+ T cells exerted no anti-proliferative effect on HeLa cells over the 7 days of incubation, while the IA1-activated donor-matched CD4+ T cells exerted a potent anti-HeLa effect within the first 24-48 hours (Figure 5.5). Interestingly, as shown in Chapter 4, IA1’s primary proliferative effect was on CD4+ T cells. In contrast, in resting PBMC, only the CD8+ T cells exerted an anti-HeLa effect; but only after 72-96 hours. While IA1 enhanced the CD8+ T cell efficacy against HeLa cells, its stimulative effect was not as pronounced as that seen with CD4+ T cells and did not as dramatically change the time course of recognition. Moreover, the MHC-agnostic miRNA-containing exosomes also induced an anti-HeLa response which began much earlier than the,   132 likely, MHC-dependent allogeneic response occurring at 72-96 hours. In aggregate, despite the limitations of the models used, substantial evidence exists for an enhancement of a cancer-inhibitive response independent of MHC allorecognition. To better identify the underlying mechanism(s), future murine studies should investigate the anti-cancer effects of syngeneic splenocytes on in vitro and in vivo cancer models. Multiple direct and indirect pathways of cell-mediated cancer cytotoxicity were discussed in Chapter 1. Despite the desirability of real-time monitoring, the ACEA system, which was used for cell proliferation assessment in this study, does not directly examine the mechanisms of cancer cell killing. The impedance index collected from cell attachment reflected the ‘fingerprint’ of cell proliferation; including cell number, viability, and cell size. We interpreted this overall impedance index as ‘cell proliferation’. Future studies examining the secretion of cytolytic enzymes, including perforin and granzymes, and the expression of death ligands such as FasL and TRAIL on the cell surface should be pursued. Moreover, direct cancer inhibition by IA2 was hypothesized to be mediated by apoptotic/necrotic miRNA in an indirect pathway; hence miRNA tracking strategies and functional assays can provide mechanistic evidence in differentiating apoptosis and necrosis, or IA1 and IA2. For future in vivo studies, the measurement of tumour mass size and metastatic locations would also be necessary. In our study, IA2 was biomanufactured as a potential cancer type-specific therapeutic from the PBMC-HeLa cell response. Indeed, IA2-activated PBMC exerted a stronger anti-HeLa effect, but a weaker anti-melanoma effect than IA1 pretreatment. However, IA2 exerted direct toxicity to both HeLa and melanoma cells; this direct toxicity was usually more significant than PBMC-mediated cancer cell growth inhibition. Therefore, more rigid specificity to target a certain type of cancer can still be incorporated into our acellular therapeutics. The method of   133 producing IA2 secretome using patient-derived cancer cells can be applied; this therapeutic would also be personalized and capable of enhancing the T cell cytotoxicity towards the type of cancer that the patient has. IA2 may also synergistically enhance the CAR-T cell activity in targeted cancer cell killing. Moreover, strategies such as immunizing IA2 (or even IA1)-producing animals with cancer antigens and producing the therapeutics from cancer-responsive Teff cells (e.g., TIL) can be applied. By obtaining their own cancer type-specificity, IA1 and IA2 can be used as first-line therapeutics without the necessity to combine them with other treatments. Finally, despite a cancer cell line bearing high metastatic potential has been used in our in vitro assays, the effects of IA1 and IA2 on cancer metastasis are still mysterious. As discussed repeatedly above, future in vivo studies should be conducted to manufacture acellular therapeutics, test their immunomodulatory effects, and examine the consequent anti-cancer activities. The DTH assay has been described previously for in vivo toxicology assessment; other animal health indicators such as body weight and observable abnormalities are also important in determining the toxicity of IA1/IA2 or IA1-/IA2-activated leukocytes to naïve animal recipients. Preliminary studies in the Scott Laboratory have tested the effects of immunosuppressive TA1 in preventing the onset of T1D in NOD mice. [254] In contrast, murine cancer models will be used to monitor how pro-inflammatory IA1 and IA2 could enhance the infiltration and persistence of Teff cells in the primary cancer site, thus controlling cancer development. Moreover, the invasion of metastatic cancer cells and the ways in which IA1/IA2 promote Teff cell transportation to these neoplastic deposits can also be readily examined. By exploring the role of IA1 and IA2 in vivo, the clinical utility of secretome-based therapeutics can be further confirmed.   134 Chapter 6: Closing Remarks 6.1 Research Significance and Future Investigations Cancer is initiated from genetic mutations, but its development and progression are extensively shaped by immunity. [33, 128] In many instances, the inflammatory immune response to mutated abnormal cells is weak, or even inhibited, allowing for cancer cell proliferation and malignancy. While differentially cytotoxic pharmacologic agents have been critical in the decades of successful advances in cancer therapy, these agents do exhibit significant systemic toxicity toward normal proliferating cells. More recently, research and clinical efforts have focused on the practice of pro-inflammatory (i.e., increased Teff:Treg cell ratio) immunotherapy, arguably originating from William Coley’s treatments in 1891. [153–156] Today, perhaps the most favoured approach to inducing an endogenous immune response has been the application of ACT immunotherapy, especially CAR-T cell therapy. [175, 182, 183] While CAR-T cells will prove to be a crucial tool in cancer immunotherapy, they are accompanied by significant issues, including cost ($50,000 per patient), manufacturing time (weeks/months), and safety (e.g., cytokine release syndrome). [178, 183, 186, 188] Yet, are there other safer, faster, and lower-cost ACT adjuvant approaches that could be used to stimulate a patient’s autologous immune response? Multiple studies suggest that a strong alloresponse can promote an effective systemic CD4+ and CD8+ T cell response against cancer antigens. [203, 204] In contrast to pan T cell activation (e.g., mitogens; anti-CD3/CD28), the allorecognition response is more limited – as only 1-10% of T cells are alloresponsive. [35, 201] However, despite the ‘low’ number of potentially reactive cells, the infusion of significant numbers of allogeneic donor cells is beset by the significant risk of morbidity and mortality due to GvHD, thus limiting its practical   135 applicability. However, by using a ‘secretome approach’, acellular conditioned media can be prepared that have tissue specific biologic activity. As evidenced in this study, and our previous publications, the secretome of immunological cells can be used to exert potent immunomodulatory effects, both in vitro and in vivo. [250, 253, 254] Importantly, the biomanufacturing conditions dictate the secretome generated, allowing for the reproducible production of either tolerogeneic or pro-inflammatory agents. Indeed, previous work from the Scott laboratory demonstrated that a tolerogeneic miRNA-based secretome (TA1; Chapters 2 and 3) could enhance the production of Treg cells (and decrease Teff cells) and effectively inhibit the onset of T1D in NOD mice. [250, 253, 254] Moreover, as demonstrated in the current report, the allorecognition-based acellular IA1 secretome preparation can be used as a pro-inflammatory adjuvant for autologous ACT therapy by enhancing the proliferative response of resting lymphocytes and increasing the Teff:Treg cell ratio, thereby increasing immunity-mediated anti-cancer activity.  Notably, despite IA1 being biomanufactured from the secretome of an allorecognition response, IA1 activated lymphocytes are not MHC-restricted and the acellular IA1 therapeutic poses minimal risk of GvHD. Efficacy of IA1 is mediated by a complex mixture of soluble and exosome-encapsulated miRNA arising from the MLR allorecognition microenvironment, not residual cytokines within the IA1 preparation (Chapter 3). Due to the complexity and low fidelity of miRNA bioregulatory pathways, the use of a bioreactor system to produce the therapeutic miRNA was critical. Based on the complicated regulatory action of miRNA, we consciously chose an anti-reductionist approach to produce a complex ‘pattern of miRNA expression’ that mimics normal biology in order to achieve maximal biological functionality. In biology, it is important to note that activation of some, and inhibition of other, pathways interplay to produce   136 a biological response to stimuli. Thus, a ‘pattern of miRNA expression’, comprising both increased and decreased miRNA species, is essential for effective immunomodulation of the recipient – making the miRNA cocktail similar to therapeutic intravenous immunoglobulin (IVIg) which consists of hundreds of thousands of unique IgGs from thousands of donors. [412, 413] Due to the evolutionary conservation of miRNA, and the miRNA bioregulatory process, significant cross-species (human ⬌ mouse) efficacy was noted with the IA1 biotherapeutic (Chapter 3).  To further explore the role of miRNA in the IA1 and IA2 secretome therapeutics, future studies should include the assessment of miRNA composition and biological function. miRNA profiling approaches (e.g., qRT-PCR, miRNA microarray, and RNA-sequencing) could be more applicable in identifying the composition as a ‘pattern of miRNA expression’ than gene knockout or miRNA degradation studies that target single miRNA. [254, 414, 415] To better understand the immunomodulatory activity of these secretome miRNA, mechanistic studies have also been proposed. Current findings are phenomenologically supportive of the differential activity of secretome therapeutics to pan T cell stimulations; investigations on signaling pathways during T cell activation and anti-cancer activity would further illustrate the underling mechanism. The pro-apoptotic/necrotic potential of IA1 and especially IA2 miRNA in cancer treatment is also under exploration. Moreover, a transition from in vitro to in vivo analysis would be imperative to demonstrate the translational significance of our acellular secretome therapeutics. Previous TA1 therapeutics have achieved success in treating autoimmune diseases by arresting the progress of T1D in NOD mice; [254] the pro-inflammatory efficacy of IA1 and IA2 miRNA-enriched conditioned media or plasma would be accordingly investigated in the settings of immune deficiency and cancer development using in vivo animal models.   137 6.2 Potential Application of Secretome-Based Acellular Therapeutics Importantly, the secretome-produced IA1 exerted no ex vivo toxicity to leukocytes. However, IA1 did induce a significant pro-inflammatory response within resting T cells (Chapters 3 and 4), increasing the Teff:Treg cell ratio and enhancing an existing pro-inflammatory response – effects that would be beneficial in individuals with absent or weak endogenous anti-cancer immune responses. Importantly, the proliferation induced by IA1 was dramatically less than that induced by mitogens (PHA) or mAb (anti-CD3/anti-CD28), suggesting that systemic toxicity, relative to these agents, should be significantly reduced.  Indeed, the clustergram analysis of IA1 (or IA2) on miRNA expression of treated cells was dramatically different than, and often inverse to, that of the anti-CD3/anti-CD28 or PHA stimulated cells (Chapter 4). The enhancement of an endogenous response can also be observed in our cancer cell line models which employed allogeneic PBMC (Chapter 5). As noted in the anti-HeLa responses, resting PBMC would, after ~4-5 days of co-culturing, inhibit and eventually kill the HeLa cell; this was likely due to both allogeneic and anti-cancer immune responses. However, the anti-proliferative response was vastly enhanced, and pre-primed, by treating the resting PBMC for 24 hours with IA1 prior to overlaying onto the cancer cells. In anti-melanoma responses, IA1-activated PBMC exerted a lymphocyte number-dependent inhibition on SH-4 cell proliferation. Importantly, the pretreatment of resting leukocytes with IA1 had no negative impact on autologous immune cells or non-cancerous cell lines relative to untreated or sham-secretome treated donor matched leukocytes, making IA1 suitable for autologous ACT therapy.  How could IA1 be utilized in ACT therapy? As diagrammatically illustrated in Figure 6.1 A, the bioproduction of IA1 (and IA2) from the secretome is both inexpensive and rapid (5   138 days) and can be stored for long periods (frozen in the laboratory for several months; data not shown). Moreover, neither IA1 (nor IA2) production actually requires tissues (PBMC or, for IA2, cancer cells) derived from the patient, making it an ‘off-the-shelf’ immune adjuvant. Due to the miRNA-mediated cross-species efficacy, human patients can benefit from therapeutics derived from other species (e.g., mouse). Most importantly for patient care, ex vivo activation of lymphocytes is rapid (24 hours); in stark contrast to the weeks to months necessary for production and expansion of CAR-T cells. The IA1-activated cells exhibited dramatically enhanced immune recognition of cancer cells over resting PBMC, as evidence in photomicrographs and proliferation assays (Figure 6.1 B-C). Hence, IA1 activation of autologous PBMC could be employed as a first-line therapy or, potentially, be used in an immunotherapeutic bridge while CAR-T cells are produced. Moreover, IA1 may also be useful in enhancing the function of CAR-T cells that are also patient autologous. Advanced CAR-T cells with the capability of blocking PD-1 have just made their debut, exerting improved anti-cancer activity in solid tumours in vivo. [190] Similarly, by activating CAR-T cells with our acellular therapeutics, the increased Teff:Treg cell ratio could help in releasing the stress of immunosuppression leading to better cancer-inhibitive activity of CAR-T cells. Due to the simplicity and low cost of the approach, multiple rounds could be used, as necessary, with large numbers of autologous PBMC or CAR-T cells employed. Indeed, due to the ability to infuse large numbers of IA1 treated autologous cells, enhanced recognition of not only the primary tumour but metastatic sites as well could be achieved, thus improving long-term survival.     139  Figure 6.1 Mechanism of Action for IA1 Secretome Therapeutic in ACT Therapy.   Panel A. Bioreactor production of IA1 secretome is readily accomplished using an allogeneic MLR.  Source materials include PBMC donors (A and B), autologous cells (a; as one donor), lymphocytic cell lines, or leukoreduction filters from blood collection bags. Acellular supernatant is collected at day 5 for processing into IA1. IA1 is stable for months when aliquoted and frozen. Panels B-C. Weak to absent immune responses to both the primary tumour and metastatic sites allows for cancer progression. PBMC or CAR-T cells (D) from the patient are treated ex vivo for 24 hours with IA1 and then reinfused into the individual, where they show enhanced recognition and killing of the primary tumour (b) and, potentially, improved immune surveillance at metastatic sites (c). This is supported by photomicrographs of allogeneic PBMC responding to HeLa cells. As shown, after 72 hours of incubation, resting (weak responders; left) PBMC show limited interaction when overlaid on HeLa cells. In contrast, the same PBMC, when treated for 24 hours with IA1, show a robust enhanced interaction (right) with the HeLa cell monolayer.    In an attempt to determine if the alloresponse-generated IA1’s cancer-inhibitive response could be improved upon, the anti-HeLa acellular secretome product (IA2) was similarly studied. Somewhat surprisingly, despite the broad similarity in the bioproduction of IA1 and IA2 (Chapters 2 and 3), the resultant miRNA-enriched agents exhibited significant immunological and anti-cancer differences. At the T cell level (Chapter 4), upon stimulation of resting CD3+ T lymphocytes, IA1 predominantly increased CD4+ proliferation, while IA2 predominantly 24 HoursPatient PBMC, CAR-TA BIA1 BiomanufacturingIA1Stable in Freeze-Thaw & Storage StudiesPBMC%Donors%Autologous%%Cell%Lines%Leukoreduc6on%Filters%DA BAllogeneic 'Derived' aResting PBMCIA1-PBMCPrimary!MassMetastatic!SiteWEAK endogenous !immune responseENHANCED endogenous immune response5 DaysIA1miRNA!ProcessingbcHeLaAutologous ACT ImmunotherapyAcellular SecretomeAB CDIn vivoLYMMDS © 2018Patient PBMC, CAR-T  140 increased CD8+ T cell proliferation. Among CD4+ subsets, both IA1 and IA2 expanded the Th17 cell subset, but IA2 simultaneously shrank the Treg cell subset – resulting in a larger magnitude of increase in the Th17:Treg cell ratio in the IA2 treated PBMC. Evidence of these immunologic disparities may be seen in the inverse expression patterns of several miRNA noted in resting PBMC treated with IA1 versus IA2, suggesting differential activation pathways and downstream activities (Chapter 4). As noted in Chapter 5, IA2-treated PBMC were more effective at inhibiting HeLa cell proliferation than were the same PBMC pretreated with IA1 – suggesting a HeLa cell specificity. Indeed, this observation was supported by the finding that IA2-treated PBMC were less effective than IA1-treated PBMC in inhibiting SH-4 proliferation. Intriguingly, despite the enhanced killing of HeLa cells by the IA2-treated PBMC, purified CD4+ or CD8+ T cell subpopulations treated with IA2 were not as effective as the same subpopulations pre-treated with IA1 (Chapter 5).  More interestingly, the IA2 biotherapeutic demonstrated significant direct toxicity to not only HeLa cells (from which it was derived), but also SH-4 cells (Chapter 5). This direct toxicity was morphologically suggestive of IA2-activated PBMC-induced apoptosis, with significant blebbing observed in the HeLa cells (Chapter 5). This was supported by the observation that in IA2-, but not IA1-, treated PBMC, upregulation of several miRNA (Chapter 4; e.g., miR-29b-3p, miR-186-5p, and miR-16-5p) associated with apoptosis was apparent. [416–418] Hence, IA1 ≠ IA2, suggesting that the cell types [lymphocyte:lymphocyte versus lymphocyte:epithelial (i.e., HeLa)] utilized in the bioproduction alters the composition of the secretome and the derived miRNA-enriched product. This would not be surprising since all cells produce and export free and exosome-encapsulated miRNA, the composition of which will vary based on cell type and function. Indeed, some miRNA (e.g., miR-451 and miR-150) are preferentially sorted into   141 exosomes derived from normal cells (e.g., leukocytes), while other miRNA such as miR-214 and miR-155 are enriched in secretomes derived from cancer cell lines or PBMC from cancer patients. [419, 420] Ongoing studies are investigating the differential effects of cell types on bioreactor produced secretomes and miRNA.  As illustrated in Figures 6.1-6.2, the IA1 secretome-based therapeutic is biomanufactured from allogeneic stimulations, while IA2 is produced from an PBMC-cancer cell response which can be either allogeneic or syngeneic. Indeed, either allogeneic donor or autologous patient leukocytes can be used; as can cancer cell lines or patient-derived tumour cells. Moreover, by using either patient-derived leukocytes or cancer cells, the produced ‘personalized’ IA2 therapeutic would specifically equip the patient’s immunity with killing capability towards the type of cancer that the patient bears. Similar to our previous study on tolerogeneic TA1 inhibition of T1D in NOD mice, [254] IA1 or IA2 could be intravenously injected directly into the recipient, yielding a systemic reset of the immune system. Moreover, IA1 and IA2 by themselves induce a broad-spectrum direct toxicity to cancer cells, making them even more efficacious without causing GvHD. Indeed, previous murine studies in the lab demonstrated no evidence of GvHD or other adverse responses caused by direct IA1 or TA1 injection over 300 days (data not shown). Although, perhaps a more intriguing method of use of IA1 and IA2 is to activate the patient’s autologous Teff cells ex vivo, as partially explained in ACT therapy (Figure 6.1). Indeed, our data demonstrated that IA1 and IA2 significantly, but differentially, enhanced the anti-proliferative response of resting PBMC on cancer cells. Perhaps surprisingly, IA1 seemed to induce potent cancer-inhibitive CD4+ T cell response – completely absent in resting T cells. In contrast, IA2 exhibit significant direct toxicity to cancer cells and, when used to activate PBMC, operated primarily via CD8+ T cells. For IA1, the pro-  142 inflammatory (i.e., T cell proliferation) and cancer-inhibitive response was driven by miRNA, not cytokines, and was not MHC-based. The endogenous Teff cells activated by IA1 and IA2 would exert an enhanced anti-cancer efficacy at the primary tumour site and/or metastatic location post-reinfusion (Figures 6.1-6.2). Both IA1 and IA2 could also be used in combination with cancer cell-targeted CAR-T cells to achieve a faster and more cost-effective outcome. Taken together, IA1 and IA2 are both promising acellular therapeutics but different in terms of biomanufacturing, cancer-inhibitive activities and the potential method of use (IA1 ¹ IA2).  Metasta&c(CellsTumor(MassEndogenous)Immune)ResponseD Pa&ent(CellStrongWeak(Allogeneic((A)(Cells((Autologous((D)(Cells(Cancer((Pa&ent/Cell(Line)(CDACancer 'Derived'DDDDDD24 HoursIA2Autologous Teff or CAR-T ActivationPBMC(Donors((Cell(Lines(Leukoreduc&on(Filters(24 HoursAutologous Teff or CAR-T ActivationDDDA BDDIA1Allogeneic 'Derived' IA1 ≠ IA2IA1IA1A B5 DaysMDS © 2018Stable in Freeze-Thaw & Storage StudiesIA25 DaysIA2Ex Vivo Treatment of Autologous LeukocytesDirect In Vivo  (IV) Administration12  143 Figure 6.2 Use of IA1 and IA2 As Different Acellular Secretome-Based Therapeutics.  Generic or personalized IA1 can be biomanufactured using PBMC donors, patient lymphocytes, cell lines, or leukoreduction filter systems (A and B). The biomanufacturing of secretome approach is rapid (5 days). Cancer-derived IA2, alternatively, is biomanufactured using cancer cells (C) either from the patient or the cell lines. Two major methods of use of IA1 and IA2 acellular therapeutics are direct injection for systemic immune-orientation and ex vivo autologous leukocyte (D) activation. Importantly, in addition to activating patient-derived Teff cells, both IA1 and IA2 can potentially enhance the activity of CAR-T cells, which are also patient-autologous and, cancer cell-targeted. The rapid (24 hours) activation of Teff and CAR-T cells with IA1 or IA2 can enhance the immunosurveillance against not only the primary tumour but metastatic cells as well, thus improving long-term survival. Meanwhile, no toxicity of IA1/IA2-activated leukocytes to non-cancerous cells/tissues has been noticed. IA1 and IA2 are both stable therapeutics and can be easily stored. Nonetheless, IA1 and IA2 are not equivalent in terms of either their biomanufacturing, mechanism of action, or preferential method of use.  While Figure 6.2 envisions the use of IA1 and IA2 in human medicine, extensive animal studies will be needed. Potential investigations could utilize the well-established murine Lewis lung carcinoma and melanoma models. These in vivo (and in vitro) models are similar to the human epithelial (HeLa) and melanoma (SH-4) cancer models used in this thesis. The murine epithelial (Lewis lung carcinoma, LL/2) and melanoma (B16-F10) cell lines are readily obtained from ATCC and have been used extensively in cancer models. [421, 422] Importantly, both murine cancer cell lines are derived from C57BL/6 (H-2b) mice, allowing for the assessment of the anti-cancer effects of syngeneic splenocytes which can also be isolated from normal C57BL/6 donor mice. Similar to human studies, cancer type-specific IA2 could also be biomanufactured using the murine cancer cells. This animal ‘patient’ derived IA2 therapeutic should represent a personalized approach which uses autologous leukocytes and/or cancer cells as discussed above (Figure 6.2). Moreover, both the LL/2 and B16-F10 cancer cell lines are highly metastatic in vivo, making them desirable models to investigate if IA1 or IA2 ACT therapy could prevent metastatic spread consequent to improved immunosurveillance. [423, 424] The B16-F10 murine model also has implications for human melanoma (i.e., the SH-4 cell line) due to their homologous cancer-associated antigens including gp100, MART-1, tyrosinase, etc. [425–428] By examining the efficacy of IA1 and IA2 (administrated via two methods of use   144 described in Figure 6.2) on cancer development and/or progression using the in vivo animal models, the anti-proliferative and potential anti-metastasis significance of our acellular therapeutics would be further illustrated. In summary, bioreactor production of the lymphocyte allorecognition secretome yields a miRNA-based, MHC-independent therapeutic (IA1) that can be reproducibly manufactured and exhibits potent immunomodulatory activity. The IA1 biotherapeutic is both inexpensive and easy to produce and can be used ex vivo to induce a rapid (24 hours of incubation) pro-inflammatory response in resting, patient-sourced, autologous lymphocytes that may dramatically enhance their cancer-inhibitive efficacy upon reinfusion into the donor. Furthermore, by altering the type of tissue (e.g., cancer cell versus lymphocyte) to which the lymphocyte population is responding, other secretome-biotherapeutics (e.g., IA2) can be derived that may be capable of inducing apoptosis in targeted tissues. Successful development of this novel secretome biotherapeutic/manufacturing approach may prove useful in enhancing the endogenous immune response to cancer, and consequent to enhanced immunosurveillance, in preventing/reducing the metastatic potential of existing cancers.    145 Bibliography  1. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C et al. 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Overwijk WW, Tsung A, Irvine KR, Parkhurst MR, Goletz TJ et al. gp100/pmel 17 is a murine tumor rejection antigen: induction of “self”-reactive, tumoricidal T cells using high-affinity, altered peptide ligand. J Exp Med. 1998;188:277-286.    168 Appendices  Appendix A   Yang, X., Kang, N., Toyofuku, W.M., and Scott, M.D. Enhancing the Pro-Inflammatory Anti-Cancer T Cell Response Via Biomanufactured, Secretome-Based, Immunotherapeutics. Immunobiology, (2019), In Press     Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 1   Enhancing the Pro-Inflammatory Anti-Cancer T Cell Response Via Biomanufactured, Secretome-Based, Immunotherapeutics   Xining Yang, a,b Ning Kang, b.c Wendy M. Toyofuku ,b,c and Mark Scott a,b,c  a Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 1Z3 Canada; b University of British Columbia Centre for Blood Research; and c Canadian Blood Services  *CORRESPONDENCE AUTHOR: Mark D. Scott, Ph.D., Canadian Blood Services and the Centre for Blood Research, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3 Canada. Phone: 1-604-822-4976; Fax: 604-822-7635; E-Mail: mdscott@mail.ubc.ca      Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 2  RUNNING HEAD Pro-Inflammatory Secretome Immunotherapy  Non-Standard Abbreviations:  SYN Syngenic or autologous secretome derived therapeutic IA1 Inflammatory Agent 1 IA2 Inflammatory Agent 2 TA1 Tolerogenic Agent 1    Graphical Abstract    Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 3  CONFLICT OF INTERESTS All authors have read the journal's policy on disclosure of potential conflicts of interest. Canadian	Blood	Services	is	pursuing	patents	related	to	the	production	and	utilization	of	the	described	acellular	immunomodulatory	agents.	 	 Canadian Blood Services, a not-for-profit organization responsible for collecting, manufacturing and distributing blood and blood products to all Canadians (except Quebec), is the assignee for relevant patents. MDS	and	WMT	are	inventors	on	these	patents.  XY and NK have no conflicts of interest beyond bring paid by Canadian Blood Services.   Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 4  Abstract T lymphocytes play a critical role in the pro-inflammatory anti-cancer response; hence, significant pharmacologic efforts have been made to enhance the endogenous T cell response. Unfortunately, significant toxicity arises consequent to pan T cell activation. In contrast, the less robust T cell alloresponse has also demonstrated an anti-cancer effect, but poses an inherent risk of GvHD. To overcome the GvHD risk, an acellular pro-inflammatory agent (IA1) has been biomanufactured from the secretome of the allorecognition response. To assess IA1’s immunomodulatory activity, T cell proliferation and differentiation were determined in vitro. The pro-inflammatory properties of the IA1 therapeutic were mediated by the miRNA-enriched fractions.  Moreover, cross-species efficacy was observed consequent to the evolutionary conservation of miRNA. IA1 exerted no toxicity to resting PBMC but induced significant proliferation of resting CD3+ (CD4+ and CD8+) T cells and skewed the response towards a pro-inflammatory state (i.e., increased Teff:Treg ratio). Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC. The anti-proliferation effect of IA1-activated PBMC was noted within ~12 hours versus 4-5 days for resting cells. A second biomanufactured therapeutic (IA2; produced using HeLa cells) surprisingly demonstrated direct toxicity to cancer cells but was less effective than IA1 in inducing a cell-mediated response. This study demonstrates that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent pro-inflammatory, anti-cancer, effect on resting lymphocytes.  Keywords:  T lymphocyte; secretome; pro-inflammatory; cancer; miRNA  	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 5  Introduction		 An	 individual’s	 immune	 system	 is	 a	 continuous	 balancing	 act	 between	 tolerance	 and	inflammation.[1]	Cancers	may	occur	when	this	balance	is	skewed	towards	a	tolerogenic	state	consequent	 to	 the	 loss	 of	 the	 inflammatory	 response	 to	 abnormal	 cells.[2]	 This	immunological	 tenet	 is	 clearly	 supported	 by	 the	 finding	 that	 immunocompromised	individual	(either	inherent	or	drug	induced)	have	a	higher	incidence	of	cancers.[3,	4]	Despite	the	potential	role	of	a	nonresponsive	immune	environment,	most	anti-cancer	therapeutics	have	 historically	 been	 cytotoxic	 drugs.[5]	 These	 cytotoxic	 drugs	 typically	 targeted	 all	proliferating	 cells;	 thus	 killing	 not	 only	 the	 cancer	 cell	 but	 also	 other	 proliferating	 cells	(including	 immune	 cells	 further	 depressing	 the	 immune	 response)	 leading	 to	 significant	toxicity	 to	 normal	 cells	 and	 tissues.[6]	 Consequent	 to	 this	problem,	 research	 and	 clinical	efforts	have	more	recently	focused	on	enhancing	the	individuals	own	immune	response	to	cancer	cells.		 The	T	lymphocyte	(T	cell)	plays	a	critical	role	in	the	anti-cancer	inflammatory	responses.	An	effective	anti-cancer	pro-inflammatory	T	cell	response	is	dependent	upon	the	activation	of	 effector	 T	 cells	 (Teff)	which	 include:	 CD8+	 cytotoxic	 T	 lymphocytes	 (CTL)	 and	 CD4+	 T	helper	cells	(Th)	such	as	Th1	and	Th17.	Normally,	lymphocytes	are	activated	upon	ligation	of	their	 antigen	 receptors	 with	 specific	 cognate	 antigens.[7]	 However,	 because	 of	 the	 low	frequency	of	antigen-specific	lymphocytes,	as	well	as	difficulty	in	identification	and	isolation,	research	initially	focused	on	the	use	of	agents	that	directly	activated	T	cells	in	the	absence	of	antigens.	This	approach	is	exemplified	by	the	nonspecific	activation	of	T	cells	via	mitogens	(e.g.,	phytohemagglutinin;	PHA),	cytokines	(e.g.,	IL-2),	or	monoclonal	antibodies	(e.g.,	anti-CD3	and	anti-CD28).	However,	consequent	to	the	overly	robust	T	cell	response	arising	from	these	pan-T	cell	 agents,	 a	 cytokine	 release	 syndrome	was	often	 induced	 leading	 to	multi-organ	 failures,	 severe	 morbidity	 and	 increased	 mortality	 leading	 to	 the	 suspension	 or	abrogation	 of	multiple	 clinical	 trials.[8–13]	More	 recently,	 to	 improve	 antigen	 specificity,	chimeric	 antigen	 receptor	 T	 cell	 (CAR-T)	 therapy	 has	 been	 developed	 and	shown	 to	 be	 a	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 6  highly	promising	approach	to	enhancing	the	endogenous	immunological	response	to	cancers.	However,	 while	 highly	 effective,	 clinical	 studies	 have	 similarly	 shown	 significant	 adverse	effects	(e.g.,	cytokine	release	syndrome)	leading	to	enhanced	morbidity	and	mortality.[14]	Additionally,	 CAR-T	 therapy	 is	 expensive	 with	 costs	 approaching	 $500,000	 (US)	 per	patient.[15]	Thus,	 alternative	approaches	 to	activate	 the	endogenous	T	 cell	 response	 in	a	controlled	manner,	with	less	toxicity,	and	improved	affordability	are	needed.		 Multiple	studies	suggest	that	a	strong	T	cell-mediated	(CD4+	and	CD8+)	alloresponse	can	promote	an	effective	anticancer	response.[16–21]	In	contrast	to	pan-T	cell	activators	(e.g.,	mitogens;	anti-CD3/CD28),	the	allorecognition	response	is	more	limited	as	only	1-10%	of	T	cells	are	alloresponsive.[22,	23]	However,	despite	 the	 ‘low’	number	of	potentially	reactive	cells,	the	infusion	of	significant	numbers	of	allogeneic	donor	cells	is	beset	by	a	significant	risk	of	morbidity	and	mortality	due	to	Graft	versus	Host	Disease	(GvHD)	thus	limiting	its	practical	applicability.	However,	by	using	a	‘secretome	approach’,	acellular	conditioned	media	can	be	prepared	 that	 have	 tissue	 specific	 biologic	 activity.[24–28]	While	 the	 active	 components	within	 the	 secretome	were	 traditionally	 viewed	 as	 paracrine	 factors	 (e.g.,	 cytokines),	 the	secretome	contains	a	variety	of	biologically	active	components	that	include	proteins,	lipids,	microRNA	(miRNA)	as	well	as	extracellular	vesicles	(exosomes	and	microparticles).[24,	29–32]	Not	 surprisingly,	 the	 secretome	 components	 released	 are	 defined	 by	 their	 cell/tissue	origin	as	well	as	the	physiologic	activation	state	of	the	cells.[27,	28,	33,	34]	Consequently,	secretome	conditioned	media	can	be	designed	to	exert	differential	biologic	responses.[35–37]	Indeed,	previous	studies	from	our	laboratory	using	human	or	murine	mixed	lymphocyte	reaction	 (MLR)	 models	 demonstrated	 that	 we	 could	 generate	 either	 tolerogenic	 or	 pro-inflammatory	 secretomes	 (i.e.,	 conditioned	 media)	 by	 regulating	 the	 strength	 of	 the	allorecognition	response.[35–41]	 		 Using	 a	 pro-inflammatory,	 lymphocyte-allorecognition,	 secretome	 approach,	 we	hypothesized	 that	 the	 anti-cancer	 efficacy	 of	 resting	 peripheral	 blood	mononuclear	 cells	(PBMC)	 could	 be	 significantly	 enhanced.	 As	 demonstrated	 in	 this	 study,	 secretome	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 7  biotherapeutics	 can	 be	 reproducibly	 biomanufactured.	 The	 allorecognition-based	Inflammatory	 Agent	 1	 (IA1)	was	 found	 to	 be	 a	 potent	 activator	 of	 resting	 human	 PBMC	promoting	 proliferation	 of	 both	 CD8+	 CTL	 and	 CD4+	 Teff	 cell	 proliferation.	 The	 essential	acellular	effectors	of	the	secretome	were	soluble	and	exosome	encapsulated	miRNA	and,	due	to	 the	 conserved	 nature	 of	miRNA,	 demonstrated	 cross-species	 efficacy.	 Significantly,	 the	proliferation	 induced	 by	 IA1	was	 approximately	 50%	 that	 of	 the	 allogenic	 response	 and	dramatically	 less	 than	 that	 induced	 by	 mitogens	 (PHA)	 or	 monoclonal	 antibodies	 (anti-CD3/anti-CD28)	 suggesting	 that	 systemic	 toxicity,	 relative	 to	 these	 agents,	 should	 be	significantly	reduced.	Importantly,	IA1	exerted	no	direct	toxicity	to	PBMC.	However,	as	will	be	demonstrated,	IA1-treatment	of	resting	PBMC	resulted	in	a	significantly	enhanced	anti-cancer	effect	relative	to	untreated	or	sham-miRNA	treated	donor	matched	PBMC.	Concurrent	studies	were	similarly	done	using	a	lymphocyte-cancer	cell	(HeLa)	secretome	biotherapeutic	(IA2).	As	shown	in	this	study,	IA1	and	IA2	demonstrated	significant	biologic	and	anti-cancer	differences.	Successful	development	of	this	novel	secretome	therapeutic	approach	may	prove	useful	 in	 enhancing	 the	 endogenous	 immune	 response	 to	 cancer	 and	 in	 reducing	 the	metastatic	potential	of	existing	cancers	consequent	to	enhanced	immunosurveillance.	 		Methods	and	Materials	 	General	Methods	Human	PBMC:	All	human	experiments	were	done	in	accordance	with	the	University	of	British	Columbia	 Clinical	 Research	 Ethics	 Board	 and	 the	 Code	 of	 Ethics	 of	 the	 World	 Medical	Association	 (Declaration	 of	 Helsinki).	 Following	 informed	 written	 consent,	 donor	 whole	blood	was	collected	in	heparinized	Vacutainer®	blood	tubes	(BD,	Franklin	Lakes,	NJ).	PBMC	were	 prepared	 using	Histopaque-1077	 (Sigma-Aldrich,	 St.	 Louis,	MO)	 as	 per	 the	 product	instructions.	The	PBMC	layer	was	washed	twice	with	25	mM	HEPES/RPMI	1640	(with	L-glutamine;	Invitrogen	by	Life	Technologies,	Carlsbad,	CA)	containing	0.01%	human	albumin	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 8  (Sigma-Aldrich,	St.	Louis,	MO).	Human	PBMC	were	suspended	in	the	appropriate	media	as	needed	for	biomanufacturing	of	IA1,	cell	phenotyping	and	cell	proliferation	assays.	 	Statistical	 analysis:	 All	 data	 were	 expressed	 as	 mean	 ±	 standard	 error	 mean	 (SEM).	 A	minimum	of	 three	 independent	experiments	were	performed	in	duplicates	 for	all	studies.	Data	analysis	was	conducted	using	GraphPad	Prism	6.0	(GraphPad	Software,	Inc.,	San	Diego,	CA).	For	significance,	a	minimum	p	value	of	<0.05	was	used.	For	comparison	of	two	means,	an	independent	t-test	was	performed.	For	comparison	of	three	or	more	means,	a	one-way	analysis	of	 variance	 (ANOVA)	was	performed.	When	significant	differences	were	 found,	 a	post-hoc	 Tukey	 test	 was	 conducted	 for	 pair-wise	 comparison	 of	 means.	 For	 all	 studies	significance	was	denoted	as:	*	p<0.05;	**	p<0.01;	***	p<0.001;	and	****	p<0.0001.	 	 	Biomanufacturing	of	Acellular	Biotherapeutics	Production	of	the	alloresponse-based	acellular	Inflammatory	Agent	1	(IA1;	Figure	1A)	was	done	 using	 the	 secretome	 obtained	 from	 in	 vitro	 human	 two-way	 MLR	 as	 previously	described.[35–37]	In	brief,	the	allogenic	preparation	IA1-Allo	(IA1)	was	manufactured	using	PBMC	from	two	MHC-disparate	human	donors	suspended	in	AIM	V	media	(research	grade;	ThermoFisher	Scientific,	Grand	Island,	NY).	A	final	of	1´106	total	PBMC	from	each	donor	were	plated	 in	 multiwell	 flat-bottom	 24-well	 tissue	 culture	 plates	 (BD	 Biosciences,	 Discovery	Labware,	 Bedford,	 MA).	 Production	 of	 the	 cancer	 cell-stimulated	 secretome	 biologic	 IA2	(IA2-HeLa)	was	done	using	a	modified	MLR	in	which	one	PBMC	donor	was	replaced	with	cultured	(allogenic)	HeLa	cells	(PBMC:HeLa	reaction;	Figure	1A).	In	brief,	freshly	isolated	human	PBMC	were	 co-cultured	with	HeLa	 cells	 at	 a	 ratio	 of	 PBMC:HeLa=50:1	 in	 24-well	tissue	culture	plates.	The	final	total	PBMC	number	was	1´106.	The	negative	control	Syngenic	(SYN)	secretome	was	prepared	from	untreated	single	donor	PBMC	seeded	at	2´106	cells	per	24-well	plate	well.	For	some	studies,	the	relative	effects	of	the	pro-inflammatory	IA1	and	IA2	were	compared	to	our	previous	described	Tolerogenic	Agent	1	(TA1)	preparation.[37]	TA1	manufacturing	 was	 accomplished	 using	 an	 in	 vitro	 human	 mPEG-MLR	 as	 previously	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 9  described.[35–42]	In	brief,	PBMC	from	one	of	the	human	donors	were	derivatized	using	20	kDa	succinimidyl	valerate	activated	methoxypoly	(ethylene	glycol)	(SVA-mPEG;	Laysan	Bio	Inc.	 Arab,	 AL)	 at	 a	 grafting	 concentration	 of	 2.0	mM	per	 4´106	 cells/ml.	 Post-production	processing	and	utilization	of	TA1	was	otherwise	identical	to	that	of	IA1.	In	all	experiments,	the	indicated	secretome(s),	or	secretome	derived	products,	were	compared	to	control	cells	treated	with	fresh	media	(denoted	as	‘Fresh’).	 		 Following	extensive	optimization	studies,	all	conditioned	media	were	collected	at	5	days	post	plating.	Post	collection,	the	media	was	processed	via	centrifugation	(400´g;	10	minutes)	to	remove	cells	and	cellular	debris	followed	by	ultrafiltration	using	a	0.2	µm	syringe	filter	(Pall	Corporation,	Port	Washington,	NY).	In	some	studies,	the	media	was	further	processed	as	described	below.	The	processed	media	were	aliquoted	and	stored	in	the	-80°	C	freezer.	Studies	(not	shown)	demonstrated	that	freezing	and	thawing	had	no	significant	impact	on	the	 immunomodulatory	 activity	 of	 the	 processed	 media.	 For	 tissue	 culture	 studies,	 the	processed	media	were	mixed	1:1	with	fresh	media	and	then	seeded	with	the	indicated	cells.	 		 	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 10  	Figure	 1.	 Allogeneic	 MLR-based	 secretome	 biomanufacturing	 process	 and	immunomodulatory	effects.	Panel	A:	Manufacturing	scheme	of	secretome	biotherapeutics.	SYN	was	derived	from	resting	PBMC	while	IA1	was	produced	using	an	allogenic	MLR.	 	 IA2	was	derived	from	a	PBMC-HeLa	cell	coculture.	 	 TA1	was	manufactured	form	a	mPEG-MLR	in	which	one	donor	population	was	modified	with	methoxy(polyethylene	glycol).	Secretomes	were	collected	at	day	5	and	further	processed	via	centrifugation	and	0.2	µm	ultrafiltration.	For	 some	 experiments,	 additional	 processing	 steps	 included	 size	 fractionation,	 exosome	purification	and	miRNA	isolation.	Panel	B:	The	T	lymphocyte	(CD3+)	proliferative	potential	of	IA1	was	assessed	for	both	complete	IA1	and	M.W.	restricted	subfractions	(<30,	30-100	and	³100	 kDa).	 Shown	 is	 the	 CFSE	proliferation	 of	 CD3+	 T	 cells	measured	 at	 day	 10	 via	 flow	cytometry.	 The	 proliferative	 potential	 of	 IA1	 was	 not	 associated	 with	 the	 cytokine-rich	fraction,	but	was	solely	observed	in	the	high	molecular	weight,	miRNA-containing,	fractions.	The	table	insert	indicates	the	M.W.	of	representative	cytokines	and	chemokines.	Results	of	independent	 experiments	 are	 shown	 using	 white	 circles.	 Panel	 C:	 Only	 IA1	 exosomes	induced	CD3+	T	 cell	proliferation.	Exosomes	 from	 fresh	PBMC	were	normalized	 to	100%.	Panel	 D:	 Analysis	 of	 subset	 differentiation	 of	 resting	 PBMC	 demonstrated	 that	 only	 IA1	induced	 a	 pro-inflammatory	 state	 as	 defined	 by	 the	 Th17:Treg	 ratio	 at	 10	 days	 post	treatment.	Panel	E:	Supporting	the	potential	 in	vivo	use	of	 IA1,	mice	treated	with	miRNA	prepared	from	IA1,	but	not	SYN	or	TA1,	had	significantly	elevated	Th17	levels	at	30	days	post	i.v.	 administration.	 However,	 RNase	 treatment	 of	 the	 IA1-miRNA	 abolished	 all	immunomodulatory	activity.	 	 Panels	B-E	show	the	mean	±	SEM.	Significance	was	calculated	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 11  in	comparison	to	fresh	unless	otherwise	specified.	For	all	studies	significance	was	denoted	as:	*	p<0.05;	**	p<0.01;	***	p<0.001;	and	****	p<0.0001.	N	³	4	for	all	samples.	 		Molecular	Weight	 Fractionation:	To	 characterize	 the	 active	 component(s)	 of	 the	 acellular	media,	 size	 fractionation	 studies	were	 done	 using	 the	 Amiconâ	 Ultra-0.5ml	 30/100	 kDa	fractionation	 tubes	 (EMD	 Millipore,	 Billerica,	 MA).	 Previous	 studies	 have	 similarly	 used	molecular	weight	 fractionation	to	generate	miRNA-enriched	fractions	(>90	kDa;	based	on	molecular	weight	of	the	miRNA	containing	Ago2	and	exosomes).	[43,	44]	 	 In	contrast,	the	majority	of	cytokines	and	chemokines	resides	in	the	<30	kDa	fraction	(Figure	1B)	allowing	for	 the	 delineation	 of	 their	 role	 in	 the	 IA1	 secretome.[45]	 Briefly,	 complete	 media	 was	centrifuged	at	14,000´g	for	15	minutes	to	collect	the	filtrate.	Another	2	minutes	of	1,000´g	spin	was	conducted	to	recover	the	concentrated	solute.	The	immunomodulatory	activity	of	both	 the	 filtrate	 and	 the	 concentrate	were	 assessed	 on	 resting	 human	PBMC	using	 a	 1:1	dilution	with	fresh	media	as	described	above.	To	assess	the	role	of	cytokines	and	chemokines	in	 the	 pro-inflammatory	 and	 anti-cancer	 effects	 of	 IA1,	 the	 proliferative	 effects	 of	 the	cytokine	rich	fraction	(<30	kDa)	was	compared	to	complete	media	and	≥30	kDa	fractions.	 	Exosome	 Preparation	 and	 Analysis:	 While	 significant	 amounts	 of	 stable,	 non-complex/encapsulated	miRNA	are	found	within	the	media,	additional	miRNA	are	bound	to	higher	molecular	weight	Ago2	(~97	kDa)	and	in	very	large	exosomes.	To	assess	the	role	of	exosome	encapsulated	miRNA,	exosomes	were	purified	from	the	processed	media	using	the	Total	Exosome	Isolation	Kit	(Cat.	No.	4478359;	Invitrogen	by	Life	Technologies,	Carlsbad,	CA).	Briefly,	 the	 indicated	acellular	preparations	were	mixed	with	 the	Total	Exosome	 Isolation	reagent	and	incubated	at	4°	C	overnight	followed	by	centrifugation	(10,000´g;	1	hour	at	4°	C).	The	pelleted	exosomes	were	 resuspended	 in	a	 sufficient	volume	of	 fresh,	 cell	 specific,	tissue	culture	media	to	the	volume	of	initial	acellular	product.	To	control	for	the	potential	xeno-stimulation	 of	 the	 IA1-human	 exosomes,	 exosomes	 from	 the	 human	 SYN	secretome	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 12  were	used	as	controls	in	the	cross-species	stimulation	studies	as	they	expressed	the	same	xenoantigen	disparity	as	the	IA1-derived	exosomes.	 	Functionality	of	Isolated	Secretome	miRNA:	 	 To	further	assess	the	potential	role	of	miRNA,	miRNA	 from	 the	 SYN,	 IA1	 and	 TA1	 secretome	media	were	 isolated	 using	 the	mirVanaTM	PARISTM	 kit	 (Cat.	No.	 AM1556,	 Ambion,	 Life	 Technologies;	 Grand	 Island,	NY).	 	 Following	processing,	 the	 highly	 enriched	 small	 RNA	 fraction	 containing	miRNA	was	 prepared.[37]	 	 	To	confirm	that	miRNA	was	the	active	component,	RNase	A	was	used	to	degrade	the	nucleic	acid.	 	 To	 assess	 the	 immunomodulatory	 activity	 of	 the	 miRNA	 preparations	 (±	 RNase	treatment),	 BALB/c	 mice	 were	 treated	 (i.v.	 200	 µl/mouse;	 N=5/group)	 with	 either	 the	control	or	RNase-treated	(50	ng	RNase	A;	10	minutes	at	37°C;	Life	Technologies)	miRNA	and	Th17	 levels	were	assessed	at	Day	30	post	 treatment.	All	murine	 studies	were	performed	following	protocol	approval	by	the	University	of	British	Columbia	Animal	Care	Committee	and	were	done	in	accordance	with	the	Canadian	Council	of	Animal	Care	guidelines.	 	 	Effect of IA1/IA2 Activation on Lymphocyte miRNA Expression: Total RNA was extracted from resting PBMC	±	treatment (SYN, IA1, IA2, TA1, anti-CD3/anti-CD28 and PHA) following 72 hours incubation using the mirVanaTM PARISTM kit (Ambion, Life Technologies, Grand Island, NY). Following processing, the highly enriched small RNA fraction containing miRNA was prepared using RNase/DNase free water.  Total cellular RNA of the samples was prepared using the Agilent RNA 6000 Nano Kit (Cat. No. 5067-1511; ). Sample RNA concentration and quality (e.g., integrity) was assessed using the Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA).  Samples were stored at -80° C until further use. To partially characterize and quantify the relative abundance the miRNA species present in the resting and differentially activated PBMC, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was done using the miScript miRNA PCR Array system (Qiagen, Frederick, MD) for the human immunopathology pathway.  These miRNA microarrays plates were run using an Applied Biosystems StepOnePlusTM Real Time PCR System (ThermoFisher Scientific, Grand Island, NY).  This human immunopathology array plate is pre-configured with the appropriate RNA and quality Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 13  controls and has been validated by Qiagen. This array profiles the expression of 84 miRNA differentially expressed during normal and pathological immune responses. It is worth noting that the 84 miRNA examined are not all inclusive and that other miRNA are likely to be present and could be of immunoregulatory importance. Threshold and baseline were defined and the resultant Ct (threshold cycle) values were calculated using the StepOnePlus software (v.2.1).  Ct values were exported and analyzed using the Qiagen GeneGlobe Online Analysis Center using the Relative Quantification qRT-PCR method for analysis (DDCt).  The data shown represent three biological replicates analyzed independently by qRT-PCR. Cross-Species	 Efficacy:	 Because	 miRNA	 are	 evolutionarily	 conserved,	 the	 cross-species	efficacy	of	human-	and	murine-sourced	IA1	on	murine	splenocytes	and	human	PBMC	was	examined.	All	murine	studies	were	done	in	accordance	with	the	Canadian	Council	of	Animal	Care	and	 the	University	of	British	Columbia	Animal	Care	Committee	guidelines	and	were	conducted	within	the	Centre	for	Disease	Modeling	at	the	University	of	British	Columbia.	For	production	of	murine	 IA1,	 two	MHC	 (H-2)	disparate	allogenic	 strains	of	mice	were	used:	BALB/c,	 H-2d;	 and	 C57BL/6,	 H-2b.	 Murine	 splenocytes	 were	 prepared	 from	 freshly	harvested	spleen	via	homogenization	into	a	cell	suspension	in	PBS	with	0.2%	bovine	serum	albumin	(BSA;	Sigma-Aldrich,	St.	Louis,	MO)	using	the	frosted	end	of	two	microscope	slides.	Red	 blood	 cells	 were	 removed	 by	 treating	 splenocytes	 using	 BD	 Pharm	 Lyse	 buffer	 (BD	Pharmingen,	San	Diego,	CA).	In	vitro	production	of	murine	IA1	was	done	as	described	above	for	the	human	IA1	except	for	resuspending	cells	in	RPMI	1640	media	supplemented	with	10%	heat-inactivated	Fetal	Bovine	Serum	(FBS;	Gemini	Bio-Products,	West	Sacramento,	CA),	1%	L-glutamine,	1%	penicillin-streptomycin	and	ß-mercaptoethaniol	(50	µM).	Resting	human	PBMC	 and	 murine	 splenocytes	 were	 treated	 with	 human-	 and	 murine-sourced	 IA1	respectively.	Cell	proliferation	and	the	Th17:Treg	ratio	were	determined	as	described	below.	In	addition,	the	effects	of	exosomes	isolated	from	human	IA1	on	murine	splenocytes	were	similarly	examined.	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 14  Comparative Effects of IA1 and IA2 to Existing T Cell Activation Approaches Effect	of	 IA1	on	Lymphocyte	Viability:	To	determine	whether	 IA1	exerted	direct	 toxicity	 to	treated	cells,	7-amino-actinomycin	D	(7AAD;	BD	Biosciences,	San	Jose,	CA)	viability	studies	were	done.	7AAD	is	a	fluorescent	nucleic	dye	that	is	excluded	from	viable	cells	but	can	enter	non-viable	cells	due	to	increased	membrane	permeability.	Cells	were	stained	with	7AAD	at	a	final	concentration	of	0.05	mg/ml	and	incubated	at	room	temperature	for	15	minutes	prior	to	flow	cytometric	analysis	as	previously	described.[35]	Effect	of	IA1	On	T	Cell	Proliferation	and	Phenotyping:	 	In	vitro	cell	proliferation	was	assessed	via	flow	cytometry	using	the	CellTrace	CFSE	(carboxyfluorescein	diacetate	succinimidyl	ester)	Cell	Proliferation	Kit	(Cat.	No.	C34554i;	CellTrace,	Molecular	probes,	Invitrogen,	Eugene,	OR).	Human	PBMC	labeling	was	done	according	to	the	product	insert	at	a	final	concentration	of	2.5	µM	CFSE	per	2´106	cells	total.	In	some	studies,	the	effects	of	IA1	induced	proliferation	relative	to	control	MLR,	mitogen	stimulation,	and	anti-CD3/anti-CD28	induced	proliferation	were	compared.	 	The	T	 cell	 lymphocyte	 subpopulations	 (CD3+CD4+	 and	CD3+CD8+)	were	measured	 by	flow	cytometry	using	fluorescently	labeled	anti-	CD3,	CD4	and	CD8	monoclonal	antibodies	(mAb;	BD	Pharmingen,	San	Jose,	CA).	Human	Th17	and	Treg	subsets	were	measured	using	the	BD	Th17/Treg	Phenotyping	Kit	(Cat.	No.	560762;	BD	Biosciences,	San	Jose,	CA).	 	 The	inflammatory	 Th17	 lymphocytes	 were	 CD3+CD4+IL-17+	 while	 T	 regulatory	 lymphocytes	(Treg)	were	CD3+CD4+Foxp3+.	After	 the	staining,	 the	cells	(1´106	total)	were	washed	and	resuspended	 in	phosphate	buffered	saline	(PBS	with	0.5%	BSA)	prior	to	 flow	acquisition.	Unstained	controls	were	used	to	determine	background	fluorescence.	The	role	of	Th17	cells	in	disease	(both	as	causative	Teff	and	in	the	anti-cancer	response)	has	evolved	significantly	over	the	last	several	years	and	may	be	underestimated	due	to	Treg-Th17	plasticity.[37,	46–49]	 	 	 Because	of	 interrelationship	between	Th17	and	Treg	cells,	 the	ratio	(Th17:Treg)	of	the	two	populations	serves	as	an	excellent	surrogate	for	assessing	the	inflammatory	state.	 	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 15  Indeed,	as	demonstrated	in	the	NOD	mouse,	Th17	cells	were	more	closely	aligned	with	both	the	 onset	 and	 prevention	 of	 Type	 1	 diabetes.[37]	 All	 samples	 were	 acquired	 using	 the	FACSCalibur	flow	cytometer	and	CellQuest	Pro	software	(BD	Biosciences,	San	Jose,	CA)	for	both	acquisition	and	analysis.	 	Comparison	of	IA1	to	Allorecognition	and	Pan	T	cell	Activation	Agents:	As	previously	noted,	past	clinical	studies	have	examined	the	anti-cancer	utility	of	allo-based	and	direct	T	cell	(anti-CD3/anti-CD28	 and	mitogen)	 activation	 therapies.	Hence,	 the	 resting	 PBMC	proliferation	potential	 of	 IA1	 was	 compared	 to	 MLR-based	 alloproliferation,	 anti-CD3/anti-CD28	 and	phytohemagglutinin	(PHA;	mitogen)	stimulation.	Allorecognition-based	proliferation	assays	(MLR)	were	conducted	as	described	above.	For	anti-CD3/anti-CD28	activation	assays,	freshly	isolated	 human	PBMC	were	 stimulated	with	plate-bound	 anti-human	CD3e	 (5	µg/ml;	BD	Pharmingen,	 San	 Diego,	 CA),	 in	 the	 presence	 of	 soluble	 anti-human	 CD28	 (1	 µg/ml;	 BD	Pharmingen,	San	Jose,	CA).	After	3	days	incubation,	the	T	cell	proliferation	and	differentiation	were	assessed	via	flow	cytometry.	For	mitogen	stimulation	studies,	PBMC	were	treated	with	phytohemagglutinin	(PHA,	Sigma-Aldrich,	St.	Louis,	MO)	at	an	amount	of	2	µg	per	1´106	total	cells;	following	4	days	incubation,	T	cell	proliferation	and	differentiation	were	measured	by	flow	cytometry.	Additionally,	the	effect	of	the	tolerogenic	miRNA-based	TA1	preparation	was	done	for	comparative	purposes.	 		Anti-Cancer	Efficacy	of	IA1-	and	IA2-	Activated	PBMC	on	Cancer	Cell	Proliferation	Cancer	Cell	 Lines:	To	 investigate	 the	anti-cancer	effects	of	 IA1	and	 IA2,	human	cancer	 cell	proliferation	assays	were	conducted	using	HeLa	and	SH-4	melanoma	cell	lines.	HeLa	cell	line	(CCL-2)	was	purchased	from	ATCC	and	cultured	under	5%	CO2	in	Dulbecco’s	modified	eagle’s	medium	(DMEM;	high-glucose	contains	4.5	g/L	D-glucose,	without	L-glutamine	or	sodium	pyruvate;	 Invitrogen,	Carlsbad,	CA)	supplemented	with	10%	heat-inactivated	FBS,	10	mM	HEPES,	 100U	 penicillin	 and	 100	 µg	 streptomycin	 (Invitrogen,	 Carlsbad,	 CA).	 Human	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 16  melanoma	SH-4	cell	line	(CRL-7724)	was	purchased	from	ATCC	and	cultured	under	5%	CO2	in	 DMEM	 supplemented	 with	 10%	 heat-inactivated	 FBS,	 4	 mM	 L-glutamine,	 1	 mM	 Na	pyruvate,	100U	penicillin	and	100	µg	streptomycin	(all	from	Invitrogen,	Carlsbad,	CA).	Both	cell	lines	were	used	at	~80%	confluence.	 	Inhibition	 of	 Cancer	 Cell	 Proliferation:	 The	 direct	 toxicity	 and	 anti-proliferative	 effects	 of	control	and	secretome-treated	(SYN,	IA1,	IA2	and	TA1)	PBMC	against	the	HeLa	(epithelial)	and	SH-4	(melanoma)	human	cancer	cell	 lines	were	assessed	using	an	ACEA	iCELLigence	instrument	(ACEA	Biosciences,	Inc.,	San	Diego,	CA).	The	iCELLigence	provides	a	continuous,	real-time,	measurement	of	cell	proliferation	using	changes	in	the	electrical	impedance	within	tissue	culture	wells.	 	 The	change	in	impedance	is	induced	by	the	increase	in	adherent	cells	and	is	unaffected	by	cells	(e.g.,	PBMC)	that	remain	non-adherent.	 	 	 All	studies	were	done	with	an	initial	seeding	density	of	5,000	HeLa,	or	20,000	SH-4,	cells	per	well	of	the	ACEA	E-8	electronic	microtiter	plate	in	DMEM	medium	with	an	acclimation	time	for	initial	adherence	for	60	minutes.	Cells	were	incubated	in	the	iCELLigence	instrument	maintained	at	37°	C	in	a	humidified	5%	CO2	incubator	for	7	days.	Direct	toxicity	of	the	secretome	products	(added	1:1	into	 DMEM)	was	 assessed	 by	measuring	 HeLa/SH-4	 cells	 proliferation	 in	 the	 absence	 of	PBMC.	The	effect	of	resting	and	secretome-activated	PBMC	on	cancer	cell	proliferation	was	assessed	by	overlaying	 the	PBMC	onto	 the	 seeded	cancer	 cells	 at	60	minutes.	 	 Sufficient	PBMC	were	added	to	the	wells	to	achieve	Effector:Target	ratios	of	0:1,	10:1,	25:1	and	50:1.	 	The	relatative	efficacy	of	the	SYN,	IA1,	IA2	and	TA1	activated	PBMC	were	then	compared.	In	some	HeLa	cell	experiments,	the	inhibition	of	cancer	cell	proliferation	by	purified	(resting	and	activated)	CD4+	and	CD8+	T	cells	was	assessed.	Purified	populations	of	CD4+	and	CD8+	T	cells	were	obtained	from	resting	PBMC	using	a	DynabeadsÒ	UntouchedTM	Human	CD4	or	CD8	 T	 Cell	 Isolation	 Kit	 (Cat.	 No.	 11352D	 and	 11348D,	 respectively;	 Invitrogen	 by	 Life	Technologies,	Carlsbad,	CA)	according	to	the	manufacturers	instructions.	The	purified	(³90%)	T	cells	were	pretreated	with	the	acellular	secretome	products	(SYN,	IA1	and	IA2)	for	24	hours	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 17  and	then	overlaid	on	cancer	cells	with	cell	proliferation	being	measured	continuously	for	7	days	(168	hours).	Lymphocyte:Tumor	 Conjugation:	 An	 important	 mechanism	 in	 the	 lymphocyte-mediated	killing	of	cancer	cells	is	direct	cell:cell	interaction.	To	quantitatively	assess	this	interaction,	flow	cytometric	cell	conjugation	assays	were	done.	Briefly,	PBMC	or	HeLa	cells	were	stained	with	amine	reactive	fluorescent	probes	at	a	final	concentration	of	0.5µM	CFSE	(PBMC)	or	0.2	µM	Far	Red-DDAO	(HeLa;	CellTrace,	Molecular	Probes,	 Invitrogen,	Eugene,	OR)	per	2´106	cells,	 respectively.	 Cells	were	washed	 3	 times	 in	 excess	RPMI	 1640	media	 to	 remove	 any	unincorporated	stain.	Stained	PBMC	and	HeLa	cells	were	co-cultured	in	RPMI	1640	media	at	a	 ratio	 of	 PBMC:HeLa=50:1	 to	 a	 final	 concentration	 of	 1´106	 cells/ml.	 Co-cultures	 were	centrifuged	briefly	at	100´g,	4°	C	for	1	minute	and	incubated	at	37°	C	for	20	minutes	to	allow	conjugation	of	 cells.	Cells	were	 fixed	by	addition	of	2%	methanol-free	 formaldehyde.	The	double-stained	 cell	 population	 (CFSE+Far	 Red-DDAO+)	 was	 examined	 to	 determine	 the	percentage	of	cell	conjugation	using	flow	cytometry	as	previously	described.	 		 In	addition	to	the	conjugation	assay,	time-lapse	photographies	of	the	PBMC:HeLa	cell	co-cultures	were	captured.	PBMC	and	HeLa	cells	(seeded	at	5,000	total)	were	co-cultured	 in	RPMI	1640	media	supplemented	with	25	mM	HEPES	and	0.01%	human	albumin,	at	a	ratio	of	 PBMC:HeLa=50:1,	 in	 a	 heated	 (37°	 C)	 humidity	 chamber	 (Becton	 Dickinson,	 Franklin	Lakes,	 NJ).	 Photomicrographs	 were	 taken	 at	 20X	magnification	 every	 10	 seconds	 for	 90	minutes	using	a	Nikon	Eclipse	Ti	microscope	mounted	with	a	camera	(Digital	sight	DS-U3)	and	 analyzed	 using	NIS-elements	 software.	 Representative	 photos	 at	 specific	 time	 points	were	then	extracted	for	presentation.	 		Results	 	Proliferation	and	Differentiation	Effects	of	IA1	and	IA2	on	Resting	CD3+	T	Cells		 Previous	studies	have	demonstrated	that	an	allogenic	cell-mediated	immune	response	can	exert	a	significant	anti-cancer	effect;	albeit	with	a	risk	of	GvHD	to	the	recipient.[16]	To	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 18  circumvent	the	GvHD	risk,	our	laboratory	has	utilized	an	acellular	cocktail	derived	from	the	secretome	 (i.e.,	 conditioned	 media)	 of	 an	 allogenic	 MLR.	 As	 diagrammatically	 shown	 in	Figure	1A,	acellular	cocktails	were	prepared	from	syngenic	(i.e.,	resting	cells;	SYN),	allogenic	(IA1),	allogenic-HeLa	(IA2)	and	tolerogenic	(TA1)	cell	culture	systems	after	a	5	days’	reaction.	The	TA1	preparation,	previously	shown	to	inhibit	alloproliferation	and	to	increase	Tregs	and	reduce	Teffs,	was	derived	from	an	allogenic	MLR	in	which	one	MHC-disparate	population	was	modified	by	grafting	mPEG	to	the	cells.[36,	37]	To	determine	if	a	pro-inflammatory	response	could	be	induced	by	these	acellular	cocktails,	resting	PBMC	were	treated	with	the	various	indicated	 preparations.	 As	 demonstrated	 in	 Figure	 1B,	 the	 IA1,	 but	 not	 SYN,	 induced	 a	significant	(p<0.0001)	increase	in	resting	CD3+	T	cell	proliferation	at	day	10	of	treatment.	Previously	we	hypothesized	that	soluble	cytokines	contained	in	the	IA1	or	TA1	conditioned	media	 mediated	 their	 respective	 immunomodulatory	 effects.[36,	 37]	 However,	 size	fractionation	 studies	 of	 the	 complete	 secretomes	 (Figure	 1B)	 found	 that	 the	cytokine/chemokine	rich	fraction	(<30	kDa)	exhibited	no	proliferative	effect	on	CD3+	T	cells.	In	contrast,	higher	molecular	weight	 fractions	containing	the	Ago2/miRNA	complex	(~97	kDa)	and	miRNA-rich	exosomes	(³100	kDa)	retained	significant	proliferative	activity	relative	to	the	complete	IA1	supporting	earlier	findings	of	biologic	activity	for	exosomes.[43,	50]	Of	note,	 if	 miRNA	 are	 purified	 (using	 the	 mirVanaTM	 PARISTM	 kit)	 from	 the	 secretome,	 the	immunomodulatory	 activity	 resides	 within	 the	 <10	 kDa	 fraction	 which	 contains	 the	miRNA	.[37]	In	contrast,	the	similarly	sized	fractions	from	the	Fresh	and	SYN	demonstrated	no	proliferative	activity.	Moreover,	exosome	isolation	studies	demonstrated	that	IA1,	but	not	the	SYN,	exosomes	induced	resting	PBMC	proliferation	(Figure	1C).	Importantly,	IA1	not	only	increased	 CD3+	 T	 cell	 proliferation	 (Figure	 1B)	 but	 also	 increased	 the	 Teff:Treg	 ratio	(Th17:Treg)	 resulting	 in	 a	 pro-inflammatory	 environment;	 crucial	 for	 cancer	 cell	 killing	(Figure	1D).	 	 Also	of	note,	IA1	exerted	an	immunomodulatory	effect	in	vivo.	Mice	treated	with	miRNA	extracted	 from	 IA1	exhibited	significantly	elevated	Th17	 levels	 even	30	days	post	i.v.	administration	(Figure	1E).	 	 Further	suggesting	a	central	role	for	secretome	miRNA,	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 19  RNase-treatment	of	the	extracted	IA1-miRNA	prior	to	its	administration	obviated	its	in	vivo	immunomodulatory	effect.	In	contrast,	SYN	had	no	effect	on	proliferation,	Teff:Treg	ratio,	or	in	vivo	murine	Th17	cells	levels	relative	to	fresh	media	or	saline	treatment.	 	 The	tolerogenic	TA1	was	characterized	by	decreased	Teff	and	increased	Treg	cells	(data	not	shown).	[36,	37]	 	 In	contrast	to	protein-based	signals	(e.g.	cytokines	and	chemokines),	miRNA	are	highly	conserved	 evolutionarily	 with	 well-established	 cross-species,	 and	 even	 cross-kingdom,	efficacy.[51]	To	 further	confirm	the	role	of	miRNA	in	the	pro-inflammatory	effects	of	 IA1,	cross-species	(Human	⬌	Mouse)	studies	were	conducted	using	resting	human	PBMC	and	murine	spleoncytes.	As	expected,	human-sourced	IA1	induced	a	significant	proliferation	of	resting	 human	 CD3+	 T	 cells	 (Figure	 2A).	 Importantly,	 murine-sourced	 IA1	 also	 induced	proliferation	(p<0.05)	of	human	CD3+	T	cells.	More	dramatically,	human	IA1	stimulated	a	significant	proliferation	(p<0.0001)	of	murine	splenocytes	at	a	level	comparable	to	murine-sourced	 IA1	 (Figure	2B).	 It	must	be	noted	 that	 the	murine	 fresh	and	SYN	did	somewhat	elevate	human	and	murine	CD3+	proliferation	over	the	expected	baseline	levels;	perhaps	due	to	the	ongoing	immune	events	within	the	donor	mice.	Importantly,	in	the	context	of	cancer	cell	killing,	the	proliferation	induced	by	the	IA1	preparations	(human	and	murine)	resulted	in	a	pro-inflammatory	shift	in	the	Th17:Treg	ratio	both	intra-	and	inter-species	(Figure	2C-D).	Of	interest,	the	murine	IA1	induced	a	larger	increase	(p<0.05)	in	the	human	Teff:Treg	ratio	than	did	the	human	IA1.	In	mouse	splenocytes,	both	the	murine	and	human	IA1s	were	comparably	effective	at	increasing	the	Teff:Treg	ratio	relative	to	the	resting	and	SYN-treated	samples.	Moreover,	human	IA1-exosomes	similarly	showed	cross-species	efficacy	on	murine	CD3+	T	cell	proliferation.	As	shown	in	Figure	2E,	as	expected,	murine	IA1-exosomes	induced	murine	CD3+	T	cells	proliferation	and,	 in	accordance	to	what	was	observed	 in	Figure	2B,	human	 IA1-exosomes	 induced	 an	 even	 more	 potent	 increase	 in	 murine	 CD3+	 T	 cell	proliferation	 than	 did	 the	 murine	 IA1-exosomes.	 Human	 SYN-exosomes	 exerted	 no	proliferative	effect	while	murine	SYN-exosomes	showed	a	modest	increase	in	proliferation	similar	to	that	observed	with	murine	SYN-media	(Figure	2B).	Hence,	acellular	miRNA-based	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 20  preparations	derived	from	an	MLR	can	be	used	to	induce	a	pro-inflammatory	T	cell	response	while	obviating	the	risk	of	GvHD.	 		Figure	2.	IA1	and	IA1-derived	exosomes	demonstrate	cross-species	efficacy	on	resting	human	 and	 murine	 CD3+	 T	 lymphocyte	 proliferation	 and	 subset	 differentiation.	Panels	A-B:	Proliferation	(CFSE)	of	resting	human	and	murine	CD3+	T	lymphocytes	(10	and	7	days,	respectively)	treated	with	human-	or	murine-sourced	Fresh,	SYN	and	IA1.	Panels	C-D:	Effects	of	IA1	on	the	Th17:Treg	ratio	of	human	and	murine	CD4+	T	lymphocytes	at	day	10	or	7;	respectively.	Panels	E-F:	Murine	resting	splenocytes	were	treated	with	either	murine-	(E)	 and	 human-sourced	 (F)	 exosomes	 for	7	 days.	 	 IA1	 ,	 but	 not	 fresh	 or	 SYN,	 exosomes	demonstrated	 significant	 proliferative	 effects	 and	 cross-species	 efficacy.	 Shown	 are	 the	mean	±	SEM.	Significance	was	calculated	in	comparison	to	fresh	unless	otherwise	specified	and	denoted	as:	*	p<0.05;	**	p<0.01;	***	p<0.001;	and	****	p<0.0001.	N	³	3	for	all	samples.		Comparative Effects of IA1 and IA2 to Existing T Cell Activation Approaches 	 A	previous	problem	with	T	cell	activation	(e.g.,	mitogens	and	mAb)	strategies	has	been	an	overly	robust	response.	[11]	Hence,	we	examined	the	comparative	proliferative	efficacy	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 21  and	differentiation	profiles	of	 IA1	 relative	 to	 that	of	potent	T	 cell	 activators	such	as	anti-CD3/anti-CD28	and	PHA.	Moreover,	we	also	tested	a	PBMC-cancer	cell	 (HeLa;	Figure	1A)	generated	preparation	denoted	as	IA2	to	determine	if	a	cancer	cell	specific	agent	would	prove	more	efficacious	than	IA1.	As	shown	in	Figure	3A,	the	differential	effects	of	IA1	and	IA2	on	CD3+,	CD4+	and	CD8+	T	lymphocytes	were	compared	to	that	observed	in	a	control	MLR	as	well	as	direct	mAb	(anti-CD3/anti-CD28)	or	mitogen	(PHA)	stimulation.	Both	IA1	and	IA2	significantly	increased	the	CD3+	T	cell	proliferation	in	resting	PBMC	(12.2	±	1.21%	and	10.7	±	 0.47%,	 respectively),	 and	 this	 increase	 encompassed	 both	 CD4+	 and	 CD8+	 T	 cells.	Interestingly,	IA1	predominantly	increased	CD4+	while	IA2	predominantly	increased	CD8+	T	cell	 proliferation	 suggesting	 compositional	 and	mechanistic	 differences	 between	 the	 two	preparations.	As	anticipated,	the	acellular	IA1	and	IA2	induced	proliferation	of	resting	PBMC	was	slightly	less	than	50%	of	that	observed	in	a	control	two-way	MLR	(30.9	±	3.41%)	where	two	MHC-disparate	populations	are	both	proliferating.	In	contrast,	anti-CD3/anti-CD28	and	PHA	 resulted	 in	 very	 high	 levels	 of	 CD3+	 proliferation	 (78.1	 ±	 1.78%	 and	 94.4	 ±	 0.27%,	respectively).	 Indeed,	 these	 overly	 robust	 responses	 are	 indicative	 of	 the	 adverse	 clinical	events	 associated	 with	 mitogen/mitogen-like	 therapeutics.[11]	 Therefore,	 IA1	 and	 IA2 increased	resting	CD3+	T	cell	proliferation	in	a	more	restrained	manner	than	allo-	(i.e.,	MLR),	anti-CD3/anti-CD28,	 or	 PHA	 stimulation.	 In	 contrast	 to	 IA1,	 the	 TA1	 treatment	 had	 no	proliferative	effects	on	CD3+,	CD4+	and	CD8+	T	cell	proliferation	relative	to	the	resting	and	SYN-treated	cells.	 Indeed	as	 shown	previously,	TA1	shrank	pro-inflammatory	 subsets	and	dramatically	 decreased	 the	Th17:Treg	 ratio	 rescuing	NOD	mice	 from	 the	 development	of	Type	1	diabetes.[37]	Not	only	did	IA1	and	IA2	induce	differential	proliferation	of	CD4+	and	CD8+	 T	 cells,	 they	 also	 altered	 the	 CD4+	 subset	 (Th17	 and	 Treg)	 differentiation	 pattern	(Figure	3B).	IA1	significantly	increased	both	Th17	and	Treg,	but	the	increase	in	Th17	was	predominant	resulting	in	an	increased	Th17:Treg	ratio	(2.21-fold,	p<0.05).	In	contrast,	IA2	expanded	Th17	moderately	but	significantly	shrank	the	Treg	population;	the	loss	of	Tregs	further	elevated	the	Th17:Treg	ratio	(4.43-fold,	p<0.0001).	Therefore,	while	IA1	exhibited	a	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 22  pro-inflammatory	Teff:Treg	 ratio	 similar	 to	an	MLR,	 IA2	demonstrated	a	more	 significant	increase	(p<0.01)	in	the	Th17:Treg	ratio.	 			Figure	3.	IA1	and	IA2	promoted	differential	subset	proliferation	of	resting	PBMC	and	in	a	significantly	more	restrained	manner	than	pan	T	cell	activators.	Panel	A:	Shown	are	the	proliferation	of	resting	CD3+,	CD4+	and	CD8+	(top	to	bottom,	respectively)	human	T	lymphocytes.	Resting	CFSE	labeled	PBMC	were	treated	with	Fresh,	SYN,	IA1,	IA2	or	TA1	for	10	days.	The	acellular	secretome	preparations	were	added	1:1	 into	AIM	V	growth	media.	Shown	for	comparison	are	the	proliferative	response	of	a	control	allogeneic	MLR	(black	bar)	and	treatment	of	the	same	PBMC	(as	the	secretome	samples)	with	the	pan-T	cell	activators	anti-CD3/anti-CD28	 and	 PHA.	 	 Data	 for	 the	 MLR,	 anti-CD3/anti-CD28	 and	 PHA	 were	collected	 at	 days	 10,	 3	 and	 4	 respectively.	 CD3+,	 CD4+	 and	 CD8+	 T	 lymphocyte	 subset	proliferation	was	determined	via	flow	cytometry.	Panel	B:	The	pro-inflammatory	state	(i.e.,	ratio	of	Th17:Treg)was	determined	for	the	Fresh,	SYN,	IA1,	IA2	and	MLR	(black	bar)	at	day	10.	 	 The	Th17:Treg	(i.e.,	Teff:Treg)	ratio	of	PBMC	incubated	 in	 fresh	media	(grey	shaded	areas)	is	indicated	as	is	the	mean	±	SEM.	 	 Significance	was	calculated	in	comparison	to	fresh	unless	 otherwise	 specified	 and	 denoted	 as:	 *	 p<0.05;	 **	 p<0.01;	 ***	 p<0.001;	 and	 ****	p<0.0001.	N	³	4	for	all	samples.	 			 However,	in	cancers,	the	immune	system	is	not	in	a	“resting”	state	(i.e.,	as	modeled	by	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 23  resting	PBMC	used	above)	and,	while	ineffective	at	the	gross	level,	will	exhibit	some	degree	of	immune	activation.	To	determine	if	IA1	could	further	enhance,	or	jumpstart,	an	existing	immune	response,	MLR	studies	were	conducted	in	the	absence	and	presence	of	the	SYN	and	IA1	preparations.	As	shown	in	Figure	4A-B,	IA1	greatly	enhanced	the	alloresponse	within	an	MLR	 as	 demonstrated	 by	 human	 CD3+,	 (Panels	 A-B)	 CD4+	 and	 CD8+	 (Panel	 B)	 T	 cell	proliferation.	In	contrast,	SYN-treated	MLR	exhibited	no	significant	changes	in	CD3+	or	CD4+	T	cell	proliferation	relative	to	the	control	MLR.	However,	a	slight,	but	statistically	(p<0.05)	significant,	increase	in	CD8+	T	cell	proliferation	relative	to	the	control	MLR	was	observed.	Interestingly,	in	contrast	to	a	CD4+-centric	T	cell	proliferative	response	in	resting	PBMC,	IA1	had	a	greater	proliferative	effect	on	CD8+	T	cells	(p<0.0001	relative	to	CD4+	T	cells)	in	the	MLR	allorecognition	model.	Within	the	CD4+	T	cells,	IA1	significantly	increased	Th17	cells	relative	to	the	control	MLR	while	Treg	cells	remained	statistically	unchanged	(Figure	4C).	Interestingly,	the	SYN	actually	decreased	Th17	cells	(p<0.05)	but	had	no	effect	on	Treg	cells.	Consequently,	 as	 shown	 in	 Figure	 4D,	 the	 Th17:Treg	 ratio	 of	 the	 control	 MLR	 was	significantly	increased	(1.55-fold;	p<0.01)	by	IA1	further	supporting	its	role	as	a	potential	pro-inflammatory	agent.	In	contrast,	the	SYN	preparation	significantly	decreased	(0.59-fold;	p<0.05)	the	Teff:Treg	ratio.	Hence,	these	findings	suggested	that	IA1	could	enhance	the	anti-cancer	T	cell	mediated	immune	response.	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 24  	Figure	4.	IA1	accelerated	the	allorecognition	pathway	and	significantly	enhanced	the	alloproliferative	response	of	 the	MLR.	 	 Panel	A:	Representative	histograms	of	MLR	T	lymphocyte	proliferation.	M1	demonstrated	the	CFSE	dilution	upon	cell	proliferation	while	M2	 indicated	 the	 non-proliferative	 population.	 	 Panel	 B:	 IA1,	 but	 not	 SYN,	 significantly	enhanced	 the	 proliferation	of	 CD3+,	 CD4+	 and	 CD8+	 human	T	 lymphocytes	within	 a	MLR	alloproliferation	 model.	 Panel	 C:	 Flow	 cytometric	 studies	 demonstrated	 that	 IA1	significantly	upregulated	Th17	CD4+	T	cells	while	having	minimal	effects	on	Treg	cells.	Panel	D:	 Consequent	 to	 the	 IA1-mediated	 increase	 in	 Th17	 cells,	 the	 pro-inflammatory	 state	(Th17:Treg)	was	significantly	enhanced	relative	to	the	control	(Fresh)	MLR.	Each	acellular	preparation	was	added	1:1	into	AIM	V	growth	media.	Grey	shaded	horizontal	bar	(Panels	B-D)	 areas	 represent	 the	 control	 (i.e.,	 Fresh;	 mean	 ±	 SEM)	 MLR	 values	 for	 comparative	purposes.	Values	shown	are	the	mean	±	SEM	for	Day	7.	Significance	is	denoted	as:	*	p<0.05;	**	p<0.01;	***	p<0.001;	and	****	p<0.0001.	N	³	4	for	all	samples.	 		Anti-Cancer	Efficacy	of	IA1-	and	IA2-	Activated	PBMC	on	Cancer	Cell	Proliferation	The	anti-cancer	efficacy	of	IA1	and	IA2	activated	T	lymphocytes	was	assessed	using	two	in	vitro	cancer	models	(HeLa	and	SH-4	cell	lines).	Of	biologic	significance,	neither	the	IA1	or	IA2	 preparations	 exerted	 direct	 toxicity	 to	 resting	 PBMC	 following	 24	 hours	 of	 exposure	(Figure	 5A).	 Indeed,	 the	 viability	 of	 the	 resting	 PBMC	 was	 slightly,	 but	 statistically	significantly	(p<0.0001)	increased,	with	both	therapeutics.	Moreover,	IA1	and	IA1-activated	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 25  lymphocytes	demonstrated	minimal	bystander	toxicity	 to	primary,	non-cancerous,	cells	 in	vitro	(e.g.,	murine	myoblasts;	data	not	shown).	To	assess	the	anti-cancer	efficacy	of	IA1	and	IA2,	cancer	cell	growth	over	7	days	was	followed	using	a	real-time	proliferation	assay.	Similar	to	its	effect	on	PBMC,	IA1	(as	well	as	the	SYN	and	TAI)	had	no	direct	toxicity	to	HeLa	cells	(Figure	5B).	Surprisingly	however,	direct	treatment	of	HeLa	cells	with	IA2	demonstrated	an	almost	 total	 inhibition	 of	 growth.	 Indeed,	 by	 approximately	 30	 hours,	 no	 Hela	 cell	proliferation	was	noted	in	HeLa	cell	cultures	treated	directly	with	IA2.	The	direct	toxicity	of	IA2	to	HeLa	cells	(Figure	5B),	but	not	PBMC,	suggested	a	pro-apoptotic	or	necrotic	effect	on	the	HeLa	cells	that	was	not	induced	by	IA1.		Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 26  Figure	5.	HeLa	cell	cancer	model.	Panel	A:	Neither	IA1	nor	IA2	exerted	any	direct	PBMC	toxicity	over	the	24	hours	used	for	PBMC	pretreatment	as	assessed	at	24	hours	using	7AAD.	Shown	are	 the	mean	±	 SEM	 for	a	minimum	of	3	 independent	experiments.	Panel	B:	The	direct	HeLa	cell	toxicity	of	the	SYN,	IA1,	IA2	and	TA1	secretome	products	(ratio	of	1:1	with	HeLa	growth	media)	was	assessed.	As	shown,	SYN,	IA1	and	TA1	had	no	effect	on	HeLa	cell	proliferation	 relative	 to	 the	 control	 sample.	 In	 contrast,	 IA2	 exerted	 a	 potent	 and	 direct	toxicity	to	HeLa	cells.	Panels	C-D:	Shown	are	HeLa	cell	growth	curves	when	overlaid	at	a	25:1	or	50:1	ratio	(Panels	C	and	D,	respectively)	with	resting	PBMC,	or	the	same	donor	PBMC	pretreated	(24	hours)	with	SYN,	IA1,	IA2	or	TA1	prior	to	overlay.	 	 The	dotted	line	(indicated	by	*)	represents	to	direct	IA2	HeLa	cell	toxicity	shown	in	Panel	B.	Panel	E:	Supporting	the	findings	observed	in	Figure	1B,	PBMC	pretreatment	with	the	cytokine	poor,	miRNA-enriched,	≥30kDa	fraction	of	IA1	resulted	in	a	potent	anti-HeLa	effect.	In	contrast,	pretreatment	with	the	cytokine	rich	<30	kDa	fraction	of	IA1	was	indistinguishable	from	the	SYN	preparation.	Panel	 F-H:	 To	 assess	 the	 relative	 roles	 of	 CD4+	 and	 CD8+	 T	 cell	 subsets	 on	 HeLa	 cell	proliferation,	purified	CD4+	and	CD8+	T	cells	were	pretreated	(24	hours)	with	Fresh,	SYN,	IA1	or	IA2.	 	 As	shown	in	Panel	F,	CD8+	T	cells	solely	conferred	the	anti-Hela	effect	observed	with	 resting	 PBMC.	 	 In	 stark	 contrast,	 CD4+	 resting	 cells	 showed	 no	 anti-HeLa	 effects.	 	However,	pretreatment	of	purified	CD4+	T	cells	with	IA1,	and	to	a	slightly	lesser	extent	IA2,	resulted	in	a	potent	anti-HeLa	effect	relative	to	the	resting	CD4+	T	cells	or	pretreatment	with	the	SYN	agent	(Panel	G).	 	 Additionally,	 IA1	(in	particular)	and	IA2	greatly	enhanced	the	anti-HeLa	efficacy	of	CD8+	T	cells	as	well	(Panel	H).	 	 In	Panels	B-H	cell	proliferation	was	continuously	monitored	in	real-time	over	7	days	as	a	function	of	the	cell	impedance	index	using	an	ACEA	iCELLigence.	 	 The	proliferation	curves	shown	present	the	mean	±	SEM	of	a	minimum	of	4	independent	samples.	 	 HeLa	cells	were	seeded	at	an	initial	density	of	5,000	cells	per	well.	 	 PBMC	were	pretreated	with	the	indicated	agent	and	then	extensively	washed	prior	to	being	overlaid	onto	the	HeLa	cells.	 		 To	examine	the	effect	of	IA1	and	IA2	(as	well	as	the	SYN	and	TA1)	on	the	ability	of	resting	PBMC	to	recognize	and	attenuate	cancer	cell	growth,	resting	human	PBMC	were	treated	with	the	therapeutic	cocktails	for	24	hours,	washed,	and	overlaid	on	the	HeLa	cells	at	ratios	of	0:1,	25:1	 and	 50:1	 (PBMC:HeLa;	Figures	 5C-D).	 In	 the	 absence	 of	 PBMC,	 HeLa	 cells	 showed	continuous	growth	over	5-6	days	(Figure	5B-H).	Addition	of	untreated,	but	allogenic,	resting	PBMC	resulted	in	some	growth	retardation	by	day	4	and	the	eventual	killing	of	the	HeLa	cells	beginning	 at	 approximately	 day	 4	 or	 5	 consequent	 to	 allorecognition	 and	 an	 anti-HeLa	response.	 The	 SYN	 treated	 PBMC	 behaved	 similarly.	 In	 contrast,	 IA1	 pretreated	 PBMC	demonstrated	a	significantly	enhanced	anti-HeLa	effect	at	both	the	25:1	(Panel	C)	and,	most	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 27  significantly,	50:1	(Panel	D)	ratios.	Moreover,	as	also	shown	in	Figure	5C-D,	IA2	pretreated	PBMC	exerted	an	even	more	potent	(relative	to	IA1)	anti-HeLa	effect	at	both	ratios;	though	it	was	 actually	 less	 than	 the	 direct	 toxicity	 (*	 dotted	 line)	 of	 IA2	 to	HeLa	 cells.	 Of	 interest,	treatment	of	allogenic	PBMC	with	the	tolerogenic	TA1	demonstrated	no	anti-HeLa	effect;	in	fact,	TA1	reduced	the	inherent	alloresponse	and	actually	enhanced	HeLa	cell	proliferation.	This	was	particularly	apparent	at	the	25:1	ratio.	This	finding	agrees	with	previous	studies	which	have	demonstrated	that	TA1	increases	Treg	cells	and	reduces	Teff	cells	resulting	in	a	decreased	 Th17:Treg	 ratio	 and	 a	 tolerogenic	 microenvironment.[36,	 37,	 42]	 To	 further	characterize	 the	 active	 fraction	 of	 IA1,	 the	 anti-proliferative	 effects	 of	 resting	 PBMC	pretreated	with	either	the	<30	or	≥30	kDa	fractions	of	IA1	were	examined	(Figure	5E).	As	anticipated	by	Figure	1,	the	cytokine-rich	fraction	(<30	kDa)	pretreated	PBMC	showed	no	significant	variation	from	resting	PBMC	(dotted	blue	line).	In	contrast,	the	miRNA-containing	³30	kDa	 fraction	of	 IA1	mediated	 the	anti-HeLa	effects	and	was	almost	 indistinguishable	from	the	complete	IA1	preparation	(dotted	red	line)	at	the	50:1	ratio.	 	Purified	PBMC	consist	of	 lymphocytes	 (T	 cells,	B	 cells,	NK	cells)	 and	monocytes.	 It	 is	possible	that	IA1	could	directly	interact	with	each	of	these	subsets	and	enhance	their	anti-cancer	efficacy.	To	better	elucidate	the	effect	of	IA1	on	T	cells	within	the	PBMC,	CD4+	and	CD8+	subset	purification	studies	were	done	(Figure	5F-H).	This	was	of	interest	as	studies	of	IA1	 on	 resting	 PBMC	 (Figure	 3)	 suggested	 a	 critical	 role	 for	 CD4+	 T	 cells	 while	 in	 an	allorecognition	(MLR;	Figure	4)	model	CD8+	T	cells	were	implicated.	As	shown	in	Figure	5F,	alloresponsive	resting	CD8+	T	lymphocytes	accounted	for	the	loss	of	HeLa	cell	proliferation;	indeed,	 resting	 CD4+	 T	 cells	 demonstrated	 no	 alloresponsive,	 nor	 anti-cancer,	 effect	 and	completely	 failed	 to	 inhibit	 HeLa	 cell	 proliferation.	 In	 contrast,	 when	 CD4+	 T	 cells	 were	pretreated	(24	hours)	with	IA1	or	IA2,	these	cells	demonstrated	significant	anti-HeLa	effects	(Figure	5G).	The	anti-HeLa	effect	was	most	pronounced	with	 IA1	 (as	was	anticipated	by	Figure	3).	Similarly,	IA1	greatly	enhanced	the	anti-HeLa	efficacy	of	CD8+	T	cells	(Figure	5H).	In	 contrast,	 IA2-activated	CD8+	T	 cells	had	minimal	effect	on	HeLa	cell	proliferation.	This	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 28  finding	was	somewhat	surprising	as	IA2	treatment	of	resting	PBMC	(in	the	absence	of	HeLa	cells)	increased	CD8+,	relative	to	CD4+,	T	cell	proliferation	(Figure	3).	Contrary	to	IA1	and	IA2,	SYN	treated	CD4+	or	CD8+	subsets	(Panels	G-H)	were	no	more	effective	than	resting	cell	subsets	(Panel	F)	at	inhibiting	HeLa	growth.	Of	note,	neither	IA1	or	IA2	pretreated	purified	CD4+	or	CD8+	T	cells	inhibited	HeLa	cell	proliferation	as	effectively	as	unfractionated	PBMC	(Figure	5C-D);	likely	consequent	to	the	well	described	synergistic	interaction	of	CD4+	and	CD8+	T	cells	and,	potentially,	a	small	additive	effect	of	monocytic	cells	found	within	the	PBMC	in	cancer	cell	killing.[52,	53]	 A	key	component	of	T	cell	mediated	inhibition	of	cancer	cell	proliferation	is	the	direct	interaction	of	the	immune	cell	with	the	target	cell.	To	investigate	the	interactions	between	control	and	IA1/IA2-activated	PBMC	with	HeLa	cells,	time-lapse	video	microscopy	and	cell	conjugation	studies	were	conducted.	As	illustrated	in	Figure	6A,	IA1	and	IA2	treated	PBMC	exhibited	 significantly	 greater	 interaction	 with	 HeLa	 cells	 than	 did	 resting	 PBMC.	Microscopically,	 this	was	most	pronounced	with	 IA1,	 versus	 IA2,	 as	noted	by	 the	greater	degree	 of	 clustering	 of	 PBMC	 (white	 arrows)	 and	 individual	 HeLa	 cells	 (asterisk).	Interestingly,	the	IA2-PBMC	treated	HeLa	cells	(black	open	arrows)	demonstrated	significant	cellular	vesiculating/blebbing	and	morphology	alterations	not	observed	 in	 the	 IA1-PBMC	sample.	 These	 morphological	 changes	 may	 be	 characteristic	 of	 IA2	 associated	apoptosis/necrosis.	These	microscopic	cell:cell	observations	were	further	confirmed	using	a	cell:cell	conjugation	assay	(Figure	6B).	As	noted,	both	IA1	and	IA2	significantly	increased	PBMC:HeLa	conjugation	while	 the	SYN	media	had	no	effect.	Consequent	 to	 the	enhanced	cell:cell	(PBMC:HeLa)	interactions,	HeLa	cell	proliferation	was	significantly	decreased	in	the	IA1	and	IA2,	but	not	SYN,	treatment	groups	(Figure	6C).	Hence,	both	IA1	and	IA2	activated	resting	PBMC	exerted	a	potent	anti-HeLa	effect.	 	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 29  	Figure	6.	IA1	and	IA2	differentially	affected	PBMC-HeLa	cell	interactions	as	shown	by	photomicroscopy	 and	 cell	 conjugation	 assays.	Findings	 shown	were	 obtained	 using	 a	PBMC:HeLa	ratio	of	50:1.	Panel	A:	Representative	images	of	PBMC-HeLa	conjugation	shot	at	0,	 30,	 60	 and	 90	 minutes	 during	 the	 time-lapse	 video.	 The	 black	 asterisks	 indicated	representative	 HeLa	 cells,	 the	 white	 arrows	 pointed	 at	 PBMC.	 Black	 open	 arrows	demonstrated	blebbing	of	HeLa	cells.	Time-lapse	video	was	acquired	for	90	minutes	after	72	hours	 of	 PBMC	 and	HeLa	 cell	 co-culture.	 Images	 shown	 are	 representative	 frames	 at	 the	indicated	times	from	one	of	three	independent	experiments.	Size	bar=10	µm.	Panel	B:	IA1	and	IA2	significantly	enhanced	conjugation	between	CFSE	labeled	PBMC	and	Far-Red	labeled	HeLa	cells	after	20	minutes’	co-culture	as	measured	by	flow	cytometry.	Panel	C:	HeLa	cell	proliferation	 index	 at	 72	 hours	 (derived	 from	 Figure	 5C)	 post	 overlay	 with	 IA1	 or	 IA2	pretreated	PBMC	correlated	with	the	microscopic	and	flow	cytometric	findings.	Panels	B-C:	Shown	are	the	mean	±	SEM.	The	individual	experiments	are	shown	with	the	white	circles	with	 a	 minimum	 of	 5	 independent	 experiments	 for	 each	 condition.	 Significance	 was	calculated	 in	comparison	to	 fresh	unless	otherwise	specified	and	denoted	as:	*	p<0.05;	**	p<0.01;	***	p<0.001;	and	****	p<0.0001.	For	all	samples	N	³	4.	 	 			Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 30  	 To	 further	 assess	 the	 anti-cancer	 utility	 of	 IA1	 and	 IA2,	 the	 highly	 metastatic	 SH-4	melanoma	cell	line	was	examined.	Interestingly,	as	shown	in	Figure	7A,	 in	the	absence	of	any	 PBMC,	 both	 IA1	 and	 IA2	 directly	 inhibited	 SH-4	 proliferation.	 The	 direct	 anti-proliferative	 effect	was	most	 apparent	 for	 IA2,	 and	 similar	 to	 that	 seen	with	 HeLa	 cells,	suggesting	that	IA2’s	direct	mechanism	of	action	could	be	broad	spectrum.	However,	despite	IA1’s	 lack	 of	 direct	 toxicity	 on	 HeLa	 cells,	 IA1	 significantly	 inhibited	 SH-4	 proliferation;	though	the	SH-4	cells	did	demonstrate	a	slow	but	consistent	proliferation	over	time	(Figure	7A).	As	shown	in	Figure	7B-C,	resting	PBMC	at	a	50:1	ratio	exhibited	an	allo-/anti-cancer	response	 resulting	 in	 the	 loss	 of	 SH-4	 proliferation	 and	 cell	 death	 starting	 after	approximately	72	hours.	Importantly,	both	IA1	and	IA2	pretreatment	of	same	donor	resting	PBMC	(Figures	7B	and	7C,	respectively)	enhanced	their	anti-proliferative	effects	on	SH-4	cells;	though	IA1	was	significantly	superior	to	IA2.	The	potency	of	IA1-activated	PBMC	was	readily	apparent	even	at	a	very	 low	10:1	ratio	and	was	even	more	dramatic	at	 increased	PBMC	 numbers	 (Panel	 B).	 Indeed,	 with	 IA1	 activation	 at	 both	 the	 25:1	 and	 50:1	 ratio,	minimal	SH-4	proliferation	was	noted	over	the	first	48	hours	and	the	SH-4	cell	death	(i.e.,	decreased	impedance)	became	readily	apparent	by	48	hours.	IA2-activation	also	enhanced	PBMC-mediated	SH-4	inhibition	but	to	a	much	lesser	degree	and	required	a	longer	time	(≥	72	hours)	until	cell	killing	(decrease	in	cell	impedance)	was	obvious.	The	results	of	IA1	and	IA2	 (both	 direct	 toxicity	 and	 PBMC	 activation)	 suggested	 that	 IA1	 and	 IA2	 were	 not	equivalent	and	functioned,	at	least	partially,	via	different	mechanisms.	 		Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 31  	Figure	7.	IA1	and	IA2	attenuated	SH-4	cell	proliferation	via	both	direct	SH-4	toxicity	as	well	as	enhancing	PBMC-mediated	growth	inhibition.	Panel	A:	In	contrast	to	HeLa	cells,	both	IA1	and	IA2	demonstrated	direct	toxicity/growth	arrest	to	SH-4	cells.	The	SYN	product	had	minimal	 inhibitory	effect	on	SH-4	proliferation.	Panel	B:	 IA1	pretreatment	of	resting	PBMC	significantly	increased	their	inhibition	of	SH-4	proliferation.	Shown	are	the	effects	of	IA1-activated	PBMC	(red	lines)	at	ratios	to	SH-4	cells	of	10:1,	25:1	and	50:1	relative	to	SYN-treated	PBMC	at	a	ratio	of	50:1	(shaded	area).	Indeed,	SH-4	proliferation	was	significantly	inhibited	by	IA1-activated	PBMC	within	a	few	hours	of	overlay.	Panel	C:	IA2-activated	PBMC	(10:1,	25:1	and	50:1	ratios)	also	inhibited	SH-4	cell	proliferation	though	to	a	much	lesser	extent	than	IA1.	Proliferation	curves	shown	represent	mean	±	SEM	of	a	minimum	of	three	independent	experiments.			 Immune	 cell	 activation	 occurs	 consequent	 to	 a	 number	 of	 factors	 including	 altered	intracellular	miRNA	expression	in	response	to	immunomodulatory	agents.	[54–56]	Indeed,	miRNA	are	key	regulators	of	cellular	differentiation	and	proliferation	and	can	serve	as	highly	sensitive	biomarkers	of	immune	activation,	tolerance,	or	quiescence.	[57–59]	To	examine	the	effect	 of	 the	 IA1	 and	 IA2	 secretome	 preparations	 on	 subsequent	 intracellular	 miRNA	expression,	resting	PBMC	were	examined.	As	shown	in	Figure	8A,	clustergram	expression	profiles	of	resting	PBMC	incubated	for	72	hours	in	fresh	and	SYN	media	were	similar	across	the	majority	of	the	84	miRNA	examined.	Similarly,	the	tolerogenic	TA1	therapeutic	was	also	very	closely	aligned	to	resting	cells	in	the	fresh	media	despite	being	generated	in	an	mPEG-MLR	system	biologically	capable	of	allorecognition	(blocked	only	by	the	membrane	grafted	polymer).	In	contrast,	both	IA1	and	IA2	demonstrated	significant	variances	from	the	profile	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 32  of	the	control	PBMC	(Figure	8B).	Moreover,	as	indicated	by	the	yellow	boxes	in	Figure	8B,	IA1	and	IA2	were	very	dissimilar	to	each	other	over	a	broad	range	of	miRNA	demonstrating	that	the	induced	T	cell	activation	pathways	were	not	equivalent	(IA1≠IA2)	as	anticipated	by	the	findings	shown	in	Figures	3	and	5-7.	As	can	be	observed,	the	expression	of	numerous	miRNA	was	vastly	altered	by	IA1	treatment	while	IA2	treated	PBMC	more	closely	resembled	the	 control	 sample.	 Interestingly,	 despite	 the	 disparities	 noted	 in	 miRNA	 expression	 of	treated	 resting	 PBMC,	 IA1	 and	 IA2	 increased	 CD3+	T	 cell	 proliferation	 to	 a	 similar	 level;	however,	a	significant	(p<0.05)	discrepancy	of	CD4+	and	CD8+	T	cell	proliferation	(Figures	3	and	 8C)	 was	 noticed	 as	 IA1	 was	 CD4+-centric	 while	 IA2	 was	 CD8+-centric.	 All	 these	observations	could	contribute	to	the	differential	functions	between	IA1	and	IA2.	In	contrast	to	the	more	restrained	proliferation	seen	with	IA1	and	IA2,	activation	of	resting	PBMC	using	anti-CD3/anti-C28	 or	 PHA	 resulted	 in	 distinctly	 divergent	miRNA	 expression	 patterns	 to	either	 IA1	 or	 IA2;	 likely	 consequent	 to	 these	 agents	 near	 universal	 activation	 and	proliferation	of	CD3+	T	cells	(Figure	3).	 		Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 33  	Figure	 8.	 IA1	 and	 IA2	 pretreatment	 induced	 differential	 intracellular	 miRNA	expression	 profiles	 in	 resting	 PBMC.	 To	 partially	 assess	 the	 effect	 of	 the	 secretome-derived	products	on	PBMC,	miRNA	arrays	were	conducted	on	resting,	SYN,	IA1,	IA2,	anti-CD3/anti-CD28	 and	PHA	 stimulated	 PBMC	 at	 72	 hours	 post-treatment.	 Total	 RNAs	were	extracted	 from	treated	cells	 for	 the	profiling	of	84	miRNA	differentially	expressed	during	normal	 and	 pathological	 immune	 responses.	 Panel	 A:	 miRNA	 clustergram	 expression	profiles	 of	 PBMC	 treated	with	 SYN	 and	 TA1	 in	 comparison	 to	 Control.	Panel	 B:	 miRNA	expression	 profiles	 of	 PBMC	 treated	 with	 IA1,	 IA2,	 anti-CD3/anti-CD28	 or	 PHA	 in	comparison	to	Control.	Yellow	boxed	indicated	differential	miRNA	patterns	between	IA1	and	IA2	treatment.	Yellow	(*)	represented	pro-apoptotic	miRNA	differentially	upregulated	in	IA2	relative	to	IA1.	Clustergram	data	in	A-B	represent	three	independent	experiments.	Panel	C:	Proliferation	rates	of	CD3+,	CD4+	and	CD8+	T	cells	in	the	indicated	conditions	(derived	from	Figure	3).	Shown	are	the	mean	±	SEM	of	a	minimum	of	4	independent	experiments.	¢	p<0.05.	 		 Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 34  Discussion	 The	theory	and	practice	of	 ‘modern’	pro-inflammatory	(i.e.,	 increased	Teff:Treg	ratio)	immunotherapy	 arguably	 originated	 in	 1891	 with	 William	 Coley’s	 treatment	 of	 cancer	patients	with	bacteria	(and	 later	other	toxins)	to	 induce	an	 immune	response	that	would	exert	a	 toxic	bystander	effect	on	a	 tumor	mass.[60–63]	Despite	some	clinical	success,	and	their	 availability	 until	 1962,	 Coley’s	 Toxins	 garnered	 criticisms	 from	within	 the	medical	community	 and	 were	 eventually	 supplanted	 by	 the	 newer,	 and	 ‘safer’,	 developments	 of	radiation	and	chemo-	therapy;	which	themselves	pose	significant	short-	and	long-term	risks	to	 the	 patient.[60–64]	 Today,	 almost	 130	 years	 later,	 immunotherapy	 has	 refocused	 on	Coley’s	core	principles	of	inducing	the	endogenous	immune	response.	Ironically,	similar	to	Coley’s	use	of	bacteria,	genetically	modified	strains	of	Salmonella	sp.,	as	well	as	recombinant	polioviruses,	have	 been	 used	 to	 induce	 an	 inflammatory	microenvironment	 at	 the	 tumor	site.[65–67]	 Additionally,	 tumor-specific	 immunotherapy	 has	 been	 explored	 in	 which	autologous	cancer	cells	are	isolated,	modified,	and	re-infused	into	the	patient	in	an	attempt	to	enhance	anti-cancer	immune	cell	activation.[68–72]	More	recently,	autologous	or	allogenic	adoptive	 cell	 transfer	 (ACT)	 immunotherapy,	 especially	CAR-T	cell	 therapy,	have	become	important	clinical	tools.[15, 73, 74]	However,	while	ACT,	and	CAR-T	cells	in	particular,	will	prove	to	be	a	crucial	tools	in	cancer	immunotherapy,	they	are	accompanied	by	significant	issues	including	cost,	manufacturing	time	(weeks-months)	and	safety	(e.g.,	cytokine	release	syndrome	 induction).[14, 15, 75, 76] But are there other, safer, faster, and lower-cost ACT-immunomodulatory approaches that could be used to stimulate a patient’s autologous immune response?	 As evidenced in this study, and previous publications, the secretome of immunological cells can be used to exert potent immunomodulatory effects both in vitro and in vivo.[35–37] Importantly, the biomanufacturing conditions dictate the secretome generated allowing for the reproducible production of either tolerogenic or pro-inflammatory agents. Indeed, a previous study from our laboratory demonstrated that a tolerogenic miRNA-based secretome (TA1; Figures 1 Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 35  and 3) could enhance the production of Treg cells (and simultaenosuly decrease Teff cells) and effectively inhibit the onset of Type 1 diabetes in NOD mice.[35–37] Moreover, as	demonstrated	in	the	current	report,	the	allorecognition-based	acellular	IA1	secretome	preparation	can	be	used	 as	 a	 pro-inflammatory	 adjuvant	 for	 autologous	 ACT	 therapy	 by	 enhancing	 the	proliferative	response	of	resting	lymphocytes	and	increasing	the	Teff:Treg	ratio	and	thereby	increasing	PBMC	anti-cancer	activity.	 		 Of	note,	despite	IA1	being	biomanufactured	from	the	secretome	of	the	allorecognition	response,	 IA1	 activated	 lymphocytes	 are	 not	 MHC-restricted	 and	 the	 acellular	 IA1	therapeutic	 poses	 no	 risk	 of	 GvHD.	 Efficacy	 of	 IA1	 is	mediated	 by	 a	 complex	mixture	 of	soluble	 and	 exosome-encapsulated	 miRNA	 arising	 from	 the	 MLR	 allorecognition	microenvironment;	not	residual	cytokines	within	the	IA1	preparation	(Figure	1).	The	use	of	a	bioreactor	system	to	produce	the	therapeutic	miRNA	was	critical	due	to	the	complexity	and	low	fidelity	of	miRNA	bioregulatory	pathways.	Based	on	the	complicated	regulatory	action	of	 miRNA,	 we	 consciously	 chose	 an	 anti-reductionist	 approach	 to	 produce	 a	 complex	pattern	 of	 miRNA	 expression	 that	 mimics	 normal	 biology	 in	 order	 to	 achieve	 maximal	biological	 functionality.	 In	 biology,	 it	 is	 important	 to	 note	 that	 activation	 of	 some,	 and	inhibition	of	other,	pathways	interplay	to	produce	a	biological	response	to	stimuli.	Thus,	a	pattern	 of	 expression,	 comprising	 both	 INCREASED	 and	 DECREASED	 miRNA	 species,	 is	essential	 for	 effective	 immunomodulation	 of	 the	 recipient	 –	 making	 the	 miRNA	 cocktail	similar	 to	 therapeutic	 intravenous	 immunoglobulin	 (IVIG)	which	 consists	 of	hundreds	of	thousands	of	unique	IgGs	from	thousands	of	donors.	Due	to	the	evolutionary	conservation	of	miRNA,	and	the	miRNA	bioregulatory	process,	significant	cross-species	(human	⬌	mouse)	efficacy	was	noted	with	the	IA1	biotherapeutic	(Figure	2).	 		 Importantly,	the	secretome	produced	IA1	exerted	no	ex	vivo	toxicity	to	PBMC	(Figure	5A),	 or	 primary	 cells,	making	 the	 product	 suitable	 for	 autologous	 ACT	 therapy.	 IA1	 did,	however,	 induce	 a	 significant	 pro-inflammatory	 response	 within	 resting	 T	 lymphocytes	(Figures	1-3),	increase	the	Teff:Treg	ratio	(Figures	1-3)	and	enhance	an	existing	immune	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 36  response	(MLR;	Figure	4);	effects	that	would	be	beneficial	in	individuals	with	absent	or	weak	endogenous	anti-cancer	responses.	The	enhancement	of	an	endogenous	response	can	also	be	 observed	 in	our	 cancer	 cell	 line	models	which	 employed	 allogenic	 PBMC.	As	 noted	 in	Figures	5-7	resting	PBMC	would,	after	~4-5	days	of	co-culture,	inhibit	and	eventually	kill	the	HeLa	cells;	 likely	due	to	both	allogenic	and	anti-cancer	responses.	However,	 the	anti-proliferative	response	was	vastly	accelerated	(i.e.,	pre-primed)	and	enhanced	by	treating	the	resting	PBMC	for	24	hours	with	IA1	prior	to	overlaying	onto	the	cancer	cells.	Importantly,	IA1	 induced	 a	 significantly	more	 restrained	 proliferative	 response	 than	 either	mAb	 (e.g.,	anti-CD/anti-CD28)	or	mitogen	(e.g.,	PHA)	induced	activation	of	resting	PBMC.	Indeed,	the	clustergram	analysis	of	IA1	(or	IA2)	on	miRNA	expression	of	treated	cells	was	dramatically	different	than,	and	often	inverse	to,	that	of	the	anti-CD3/anti-CD28	or	PHA	stimulated	cells	(Figure	8).	 		 How	could	IA1	be	utilized	in	ACT	therapy?	As	diagrammatically	shown	in	Figure	9,	the	bioproduction	of	IA1	(and	IA2)	from	the	secretome	is	both	inexpensive	and	rapid	(5	days)	and	the	IA1	can	be	stored	for	long	periods	(several	months	frozen	in	the	laboratory;	data	not	shown).	Moreover,	neither	IA1	(nor	IA2)	production	actually	requires	tissues	(PBMC	or,	for	IA2,	cancer	cells)	derived	from	the	patient	making	it	an	‘off-the-shelf’	immune	adjuvant.	Most	importantly	for	patient	care,	ex	vivo	activation	of	lymphocytes	is	rapid	(24	hours);	in	stark	contrast	to	the	weeks	to	months	necessary	for	production	and	expansion	of	CAR-T	cells.	The	IA1-activated	cells	exhibited	dramatically	enhanced	immune	recognition	of	cancer	cells	over	resting	 PBMC	 as	 evidence	 in	 photomicrographs	 and	 proliferation	 assays	 (Figure	 9B-C).	Hence,	 IA1	 activation	 of	 autologous	 PBMC	 could	 employed	 as	 a	 first	 line	 therapy	 or,	potentially,	be	used	in	an	immunotherapeutic	bridge	while	CAR-T	cells	are	produced.	Due	to	the	simplicity	and	low	cost	of	the	approach,	multiple	rounds	could	be	used	as	necessary	with	large	 numbers	 of	 autologous	 PBMC	 employed.	 Indeed,	 due	 to	 the	 ability	 to	 infuse	 large	numbers	of	IA1	treated	autologous	cells,	enhanced	recognition	of	not	only	the	primary	tumor	but	metastatic	sites	as	well	could	be	achieved	thus	improving	long-term	survival.	Of	note,	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 37  similarly	to	our	previous	study	on	the	tolerogeneic	TA1	(Figure	1A	and	3A)	in	the	inhibition	of	 Type	 1	 diabetes	 in	NOD	mice,	 IA1	 or	 IA2	 could	 be	 directly	 injected	 into	 the	 recipient	yielding	a	systemic	reset	of	the	immune	system.[37] 		Figure	9.	Schematic	presentation	of	use	and	mechanism	of	action	for	IA1	secretome	therapeutic.	Panel	A:	Bioreactor	production	of	IA1	secretome	is	readily	accomplished	using	an	allogenic	MLR.	Source	materials	include	PBMC	donors	(A	and	B),	autologous	cells	(a;	as	one	 donor),	 lymphocytic	 cell	 lines,	 or	 leukoreduction	 filters	 from	 blood	 collection	 bags.	Acellular	supernatant	is	collected	at	day	5	for	processing	into	IA1	(Figure	1).	IA1	is	stable	for	months	when	aliquoted	and	frozen.	Panels	B-C:	Weak	to	absent	 immune	response	to	both	the	primary	tumor	and	metastatic	sites	allows	for	cancer	progression.	PBMC	(D)	from	the	patient	are	treated	ex	vivo	for	24	hours	with	IA1	and	then	reinfused	into	the	individual	where	they	show	enhanced	recognition	and	killing	of	the	primary	tumor	(b)	and,	potentially,	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 38  improved	 immune	 surveillance	 at	 metastatic	 sites	 (c).	 This	 is	 supported	 by	photomicrographs	of	 allogenic	PBMC	responding	 to	HeLa	cells.	As	 shown,	 after	72	hours	incubation,	resting	(weak	responders;	left)	PBMC	show	limited	interaction	when	overlaid	on	HeLa	cells.	In	contrast,	the	same	PBMC,	when	treated	for	24	hours	with	IA1,	show	a	robust	enhanced	interaction	(right)	with	the	HeLa	cell	monolayer.	 			 Finally,	in	an	attempt	to	determine	if	the	anti-cancer	efficacy	of	the	alloresponse-based	IA1	could	be	improved	upon,	the	anti-HeLa	acellular	secretome	product	IA2	was	similarly	studied.	Somewhat	surprisingly,	despite	the	broad	similarity	in	the	bioproduction	of	IA1	and	IA2	(Figure	1A),	the	resultant	secretome-based	agents	exhibited	significant	immunological	and	anti-cancer	differences.	As	noted	in	Figure	5,	IA2-treated	PBMC	were	more	effective	at	inhibiting	HeLa	cell	proliferation	than	were	the	same	PBMC	pretreated	with	IA1	–	suggesting	a	HeLa	cell	specificity.	Indeed,	this	observation	was	supported	by	the	finding	that	IA2-treated	PBMC	were	less	effective	than	IA1-treated	PBMC	in	inhibiting	SH-4	proliferation	(Figure	7).	At	 the	 T	 cell	 level,	 upon	 stimulation	 of	 resting	 CD3+	 T	 lymphocytes,	 IA1	 predominantly	increased	CD4+	while	IA2	predominantly	increased	CD8+	T	cell	proliferation.	Among	CD4+	subsets,	both	IA1	and	IA2	expanded	the	Th17	subset	but	IA2	simultaneously	shrank	the	Treg	resulting	in	a	larger	magnitude	of	increase	in	the	Th17:Treg	ratio	in	the	IA2	treated	PBMC.	Intriguingly,	despite	 the	enhanced	killing	of	HeLa	cells	by	the	IA2	treated	PBMC,	purified	CD4+	 or	 CD8+	 T	 cell	 subpopulations	 treated	with	 IA2	were	 not	 as	 effective	 as	 the	 same	subpopulations	pre-treated	with	IA1	(Figure	5).	Evidence	of	these	immunologic	disparities	may	be	 seen	 in	 the	 inverse	expression	patterns	of	several	miRNA	noted	 in	 resting	PBMC	treated	with	IA1	versus	IA2	suggesting	differential	activation	pathways	(Figure	8).	 		 More	interestingly,	the	IA2	biotherapeutic	demonstrated	significant	direct	toxicity	to	not	only	HeLa	cell	 (from	which	 it	was	derived)	but	also	SH-4	cells	 (Figures	5-7).	This	direct	toxicity	was	morphologically	suggestive	of	IA2	induced	apoptosis	with	significant	blebbing	observed	in	the	HeLa	cells	(Figure	6A).	This	observation	was	supported	by	the	observation	that	in	IA2,	but	not	IA1,	treated	PBMC,	upregulation	of	several	miRNA	(Figure	8;	e.g.,	miR-Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 39  29b-3p,	miR-186-5p,	and	miR-16-5p)	associated	with	apoptosis	was	apparent.[77–79]	Hence,	IA1≠IA2	 suggesting	 that	 the	 cell	 types	 [lymphocyte:lymphocyte	 versus	lymphocyte:epithelial	(i.e.,	HeLa)]	utilized	in	the	bioproduction	alters	the	composition	of	the	secretome	and	the	derived	miRNA-enriched	product.	This	would	not	be	surprising	since	cells	produce	and	export	free	and	exosome	encapsulated	miRNA,	the	composition	of	which	will	vary	based	on	cell	 type,	 activation	 state	and	 function.[27, 28, 33, 34]	Ongoing	 studies	are	investigating	 the	differential	 effects	of	 cell	 types	on	bioreactor	produced	secretomes	and	miRNA.			 In	 sum,	 bioreactor	 production	 of	 the	 lymphocyte	 allorecognition	 secretome	 yields	 a	miRNA-based,	 MHC-independent,	 biotherapeutic	 (IA1)	 that	 can	 be	 reproducibly	manufactured	 and	 exhibits	 potent	 immunomodulatory	 activity.	 The	 IA1	 biotherapeutic	 is	both	easy	and	inexpensive	to	produce	and	can	be	used	ex	vivo	to	induce	a	rapid	(24	hour	of	incubation)	pro-inflammatory	response	 in	resting,	patient	sourced,	autologous	PBMC	that	may	 dramatically	 enhance	 their	 anti-cancer	 efficacy	 upon	 reinfusion	 into	 the	 donor.	Furthermore,	by	altering	the	type	of	tissue	(e.g.,	cancer	cell	versus	lymphocyte)	to	which	the	lymphocyte	 population	 is	 responding	 to,	other	 secretome-biotherapeutics	 can	 be	 derived	that	may	be	capable	of	inducing	apoptosis	in	targeted	tissues.	Successful	development	of	this	secretome	biotherapeutic/manufacturing	approach	may	prove	useful	in	both	treating	cancer	and	in	preventing/reducing	the	metastatic	potential	of	existing	cancers.	 		 	Yang et al. Pro-Inflammatory Secretome Immunotherapy Page 40  Acknowledgements	The	authors	would	like	to	thank	Dr.	Duncheng	Wang	for	his	technical	contributions	to	this	 study.	 This	 work	 was	 supported	 by	 grants	 from	 the	 Canadian	 Institutes	 of	 Health	Research	 (Grant	 No.	 123317;	 MDS),	 Canadian	 Blood	 Services	 (MDS)	 and	 Health	 Canada	(MDS).	 The	 views	 expressed	 herein	 do	 not	 necessarily	 represent	 the	 view	of	 the	 federal	government	of	Canada.	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