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From bench to patient and back - an interdisciplinary mixed-method patient-integrated approach to developing… Vent-Schmidt, Jens 2020

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FROM BENCH TO PATIENT AND BACK – AN INTERDISCIPLINARY MIXED-METHOD PATIENT-INTEGRATED APPROACH TO DEVELOPING A NEW POTENTIAL THERAPY FOR INFLAMMATORY BOWEL DISEASE  by Jens Vent-Schmidt  Diploma in Molecular Medicine, The University of Freiburg, Germany, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2020  © Jens Vent-Schmidt, 2020 ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: From Bench to Patient and Back – An Interdisciplinary, Patient-Integrated Approach to Developing a New Potential Therapy for Inflammatory Bowel Disease  submitted by Jens Vent-Schmidt in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Experimental Medicine  Examining Committee: Dr. Theodore S Steiner, Professor, Department of Medicine, UBC Supervisor  Dr. Laurie J Goldsmith, Adjunct Professor, Faculty of Health Sciences, SFU Supervisory Committee Member Dr. Deanna Gibsons, Associate Professor, Department of Biology, UBC-O University Examiner Dr. Paul A Keown, Professor, Department of Medicine, UBC University Examiner  Additional Supervisory Committee Members: Dr. Laura M Sly,  Associate Professor, Department of Pediatrics, UBC Supervisory Committee Member  iii  Abstract Inflammatory Bowel Disease (IBD) is life-changing because of recurrent intestinal inflammation. Current therapies are associated with mild to severe side effects, and none provide a cure. Recent research has provided pre-clinical and clinical data on cell-based therapy using the two best-characterized types of T regulatory cells, Foxp3+ Tregs and Foxp3- Type 1 regulatory (Tr1) cells. However, major hurdles exist. The ability of Tregs to regulate innate immunity is not well understood, and while high numbers of antigen-specific Tregs are needed, these cells are scarce. Studies showed that engineered chimeric antigen receptor-expressing Tregs (CAR-Treg) could curb intestinal inflammation, but these CARs were not relevant to human disease. CAR-Treg therapy is logistically and conceptually complex and patients might perceive an unacceptable risk to be associated with this therapy. Finally, people living with IBD have an increased risk of Clostridioides difficile infection (CDI), the most prevalent cause of nosocomial infectious diarrhea in Canada, and patient experiences with CDI have not been researched.   The purpose of this thesis was to elucidate how Tr1 cells and/or Tregs regulate innate immunity, which I addressed by investigating the suppressive effect of Tr1 and Tregs on the inflammasome. I then developed and tested a new CAR-Treg relevant to human IBD and conducted a survey to investigate patients’ willingness to try CAR-Treg. Finally, I analyzed a survey to describe the impact of CDI on patients in Canada.   My results demonstrate that Tr1 cells may have unique therapeutic effects in reducing inflammasome activation via Interleukin 10. I provide evidence that CAR-Tregs suppress proinflammatory T cells.  People with IBD indicated high willingness to try CAR-Treg therapy iv  in both a clinical trial and as a new treatment. Willingness to try was not correlated with disease state or medication history. Finally, CDI patients highlighted the symptom-related impact and long-lasting effect on quality of life. Patient priorities to attenuate impact include reducing time to diagnosis and improving patient education.   My research has implications on future development of Treg-based therapy for IBD and other immune-mediated diseases and demonstrates the promise of moving this therapy into clinical practice, as most patients indicated willingness to try.  v  Lay Summary Inflammatory Bowel Disease (IBD) is caused by flares of inflammation in the gut driven by overactive immune cells. Immune cells that usually stop inflammation do not work properly. Today, IBD medications stop this gut inflammation. However, as an unwanted side effect, they also stop inflammation throughout the body. This means people with IBD have a higher risk of infections including hospital-acquired infections such as Clostridioides difficile infection (CDI).  After discovering a new mechanism of how inflammation-stopping cells work, I genetically engineered these cells to stop the inflammation in the gut without affecting other sites of inflammation. To include IBD patients’ voices in this research, I surveyed IBD patients who showed willingness to try this therapy. Finally, I analyzed a CDI patients survey about their experience with the health care system, quality of life, and priorities. This work highlights the need to include patients early and throughout the research process. vi  Preface The work presented in this thesis stems from four main research projects that have been published or are currently being peer reviewed. Below, I highlight the details and a breakdown of my contributions to these projects:  Chapter 3: Tr1 Cells, but Not Foxp3 + Regulatory T Cells, Suppress NLRP3 Inflammasome Activation via an IL-10−Dependent Mechanism The entirety of this work is published, and I present only aspects that I have contributed to: Yao Y, Vent-Schmidt J, McGeough MD, Wong MQ, Hoffman HM, Steiner TS, Levings MK. Tr1 Cells, but Not Foxp3 + Regulatory T Cells, Suppress NLRP3 Inflammasome Activation via an IL-10–Dependent Mechanism. J Immunol. 2015;195(2):488-497. doi:10.4049/jimmunol.1403225  I devised the hypothesis that CD44hiFoxp3-CD4+ T cells are/give rise to Tr1 cells, modified the in vitro stimulation protocol and together with YY designed, conducted, and analyzed flow cytometry experiments to contribute data to the figures describing the phenotype of these cells. Together with YY, I conducted and analyzed inflammasome assay experiments and contributed data to the final figures. I designed, conducted, and analyzed all western blotting experiments. I did not contribute to the in vivo experiments described in the paper (conducted by YY, MMG, MQW) and did not include these in my thesis. HMM provided critical feedback, TSS and MKL conceived the study, supervised the work, provided feedback on the manuscript and secured funding. Data from this paper are included in Maggie (Yu) Yao’s and my own thesis and she is the first author on the paper. vii   Chapter 4: Developing Flagellin-Specific Chimeric Antigen Receptor Regulatory T Cell Therapy I conceived, designed, conducted, and analyzed all CAR-Treg experiments and wrote the entirety of the text presented in my thesis under supervision of TSS and MKL. These data are not yet submitted for publication.  The revised Treg expansion and retroviral transduction protocol has been accepted for publication:  Wu D, Wong MQ, Vent-Schmidt J, Boardman DA, Steiner TS, Levings MK. An optimized method for expansion and retroviral transduction of mouse regulatory T cells. J Immunol Methods. 2020. doi:10.1016/j.jim.2020.112931  I conceived the study, trained MW and DW and revised the manuscript. DW performed experiments, analysed data, interpreted results, and wrote the manuscript. MQW performed experiments, analysed data, wrote the manuscript. DAB performed experiments and revised the manuscript. TSS and MKL conceived and supervised the study, revised the manuscript, and secured funding.   viii  Chapter 5: Patients’ Willingness and Perspectives Towards Chimeric Antigen Receptor T-Regulatory Cell Therapy for Inflammatory Bowel Diseases The entirety of this work is published: Vent-Schmidt J, Goldsmith LJ, Steiner TS. Patients’ willingness and perspectives towards chimeric antigen receptor T- regulatory cell therapy for inflammatory bowel diseases. Crohn’s and Colitis 360. 2020. doi:010.1093/crocol/otaa085  I conceived and designed the study, analyzed data, interpreted results, wrote the manuscript, and secured funding. LJG and TSS assisted in survey design, interpretation of the analysis and provided feedback and revisions on the draft manuscript. TSS secured funding.  Chapter 6: Patient Experiences with Clostridioides Difficile Infection The entirety of this work is published: Vent-Schmidt J, Attara GP, Lisko D, Steiner TS. Patient experiences with Clostridioides difficile infection: Results of a Canada-wide survey. Patient Prefer Adherence. 2020;14:33-43. doi:10.2147/PPA.S229539  I developed methodology for quantitative and qualitative analysis, analyzed the survey, curated the data, wrote and revised the manuscript, GPA designed and implemented the survey and revised the manuscript, DL contributed to qualitative analysis and revised the manuscript, TSS helped design the survey, supervised the data analysis, and revised the manuscript.    ix  I also published these reviews and used them in the introduction section of this thesis:  • Vent-Schmidt J, Han JM, Macdonald KG, Levings MK. The role of FOXP3 in regulating immune responses. Int Rev Immunol. 2014;33(2):110-128. doi:10.3109/08830185.2013.811657 • Dawson NAJ, Vent-Schmidt J, Levings MK. Engineered tolerance: Tailoring development, function, and antigen-specificity of regulatory T cells. Front Immunol. 2017;8(NOV):1-8. doi:10.3389/fimmu.2017.01460  Ethics approval was obtained through the UBC Clinical Research Ethics Board (UBC CREB Number H17-01720 and H17-00747). Animal research protocols were approved by the UBC Animal Care Committee (A13-0194, A11-0272, A15-0051, A17-0177). x  Table of Contents  Abstract ................................................................................................................................... iii Lay Summary ............................................................................................................................ v Preface ...................................................................................................................................... vi Table of Contents ...................................................................................................................... x List of Tables ......................................................................................................................... xvii List of Figures.......................................................................................................................xviii List of Abbreviations ............................................................................................................... xx Acknowledgements ...............................................................................................................xxiii Dedication .............................................................................................................................. xxv Chapter 1: Introduction ............................................................................................................ 1 1.1 Inflammatory bowel disease.........................................................................................1 1.2 Treatment ....................................................................................................................2 1.2.1 Conventional therapy ...............................................................................................2 1.2.1.1 Corticosteroids and 5-aminosalicylic acid.........................................................3 1.2.1.2 Immunomodulators ..........................................................................................3 1.2.1.3 Surgery ............................................................................................................4 1.2.2 Biologics .................................................................................................................4 1.2.2.1 Tumor necrosis factor-blocking antibodies .......................................................5 1.2.2.2 Integrin-blocking antibodies .............................................................................6 1.2.2.3 IL-12/IL-23-blocking antibodies ......................................................................6 1.2.2.4 Janus kinase inhibitor .......................................................................................6 xi  1.3 IBD and the immune system ........................................................................................7 1.3.1 CD4+ T helper cells ..................................................................................................7 1.3.1.1 Type 1 immunity ..............................................................................................8 1.3.1.2 Type 2 immunity ..............................................................................................9 1.3.1.3 Type 3 immunity ..............................................................................................9 1.3.2 Regulatory T Cells ................................................................................................. 10 1.3.2.1 FOXP3+ regulatory T cells ............................................................................. 11 1.3.2.1.1 The role of FOXP3 ................................................................................... 11 1.3.2.1.2 Mechanism of Treg suppression ................................................................ 11 1.3.2.1.3 Treg plasticity ........................................................................................... 13 1.3.2.2 Type 1 regulatory cells ................................................................................... 13 1.3.3 Immunopathogenesis of IBD .................................................................................. 14 1.3.3.1 CD4+ T helper cells in IBD ............................................................................ 14 1.3.3.2 Mononuclear cells in IBD .............................................................................. 15 1.3.3.3 Inflammasomes in IBD .................................................................................. 16 1.3.4 Flagellin in IBD ..................................................................................................... 19 1.4 Regulatory T cells as treatment for IBD ..................................................................... 20 1.4.1 Chimeric antigen receptors to create antigen-specific regulatory T cells ................. 22 1.5 Patient’s perceived risk associated with chimeric antigen receptor regulatory T cell therapy........................................................................................................................................23 1.6 IBD increases risk for Clostridioides difficile infection .............................................. 25 1.7 Summary and synopsis of research questions ............................................................. 26 1.8 A reading guide for this interdisciplinary thesis ......................................................... 28 xii  Chapter 2: Methods and Materials ........................................................................................ 30 2.1 Basic science chapters ............................................................................................... 30 2.1.1 Animals ................................................................................................................. 30 2.1.2 Flow cytometry ...................................................................................................... 30 2.1.3 Enzyme-linked immunosorbent assay (ELISA) ...................................................... 31 2.1.4 Western blotting .................................................................................................... 31 2.1.5 RNA isolation and quantitative real-time polymerase chain reaction (RT-PCR) ..... 32 2.1.6 Immunohistochemistry........................................................................................... 33 2.1.7 CAR development ................................................................................................. 33 2.1.8 Bead binding assay ................................................................................................ 34 2.1.9 FITC labeling of recombinant proteins ................................................................... 35 2.1.10 Transient transfection ......................................................................................... 35 2.1.11 HEK 293T surface expression ............................................................................ 35 2.1.12 HEK 293T binding assay ................................................................................... 36 2.1.13 Virus production ................................................................................................ 36 2.1.14 Isolation of CD4+ T cells .................................................................................... 37 2.1.15 Isolation of bone marrow-derived antigen presenting cells ................................. 38 2.1.16 T cell stimulation ............................................................................................... 39 2.1.17 Coculture of T cells and bone marrow-derived antigen presenting cells and inflammasome assay .......................................................................................................... 39 2.1.18 T cell bead expansion, CAR transfection and phenotyping ................................. 40 2.1.19 Proliferation assay .............................................................................................. 41 2.1.20 Suppression assay .............................................................................................. 41 xiii  2.1.21 DNBS colitis ...................................................................................................... 42 2.1.22 T cell transfer colitis .......................................................................................... 43 2.1.23 Isolation of lamina propria lymphocytes (LPL) .................................................. 44 2.1.24 Statistical analysis .............................................................................................. 45 2.2 Survey research chapters ............................................................................................ 45 2.2.1 Inflammatory bowel disease survey ....................................................................... 45 2.2.1.1 Survey design ................................................................................................. 45 2.2.1.2 Primary outcome measures ............................................................................. 47 2.2.1.3 Secondary outcome measure .......................................................................... 47 2.2.1.4 Participant recruitment ................................................................................... 48 2.2.1.5 Statistical analysis .......................................................................................... 49 2.2.1.6 Thematic analysis ........................................................................................... 50 2.2.1.7 Ethical considerations .................................................................................... 50 2.2.2 Clostridioides difficile survey................................................................................. 50 2.2.2.1 Study design and recruitment ......................................................................... 50 2.2.2.2 Questionnaire and data analysis ...................................................................... 52 2.2.2.2.1 Statistical analysis..................................................................................... 52 2.2.2.2.2 Thematic analysis ..................................................................................... 53 2.2.2.3 Ethics approval............................................................................................... 53 Chapter 3: Tr1 Cells, but Not Foxp3 + Regulatory T Cells, Suppress NLRP3 Inflammasome Activation via an IL-10−Dependent Mechanism .......................................... 54 3.1 Introduction ............................................................................................................... 54 3.2 Results ....................................................................................................................... 54 xiv  3.2.1 Functional Tr1 cells can be derived from CD44hiFoxp3- T cells ............................. 54 3.2.2 CD44hiFoxp3- T cell-derived Tr1 cells express characteristic Tr1 cell surface markers and transcription factors ....................................................................................... 58 3.2.3 Tr1 cells but not Tregs suppress caspase-1-dependent IL-1 secretion in macrophages ..................................................................................................................... 60 3.2.4 Tr1 cell-mediated suppression of inflammasome activation is dependent on binding of secreted IL-10 to macrophage IL-10 receptor ................................................................ 62 3.2.5 Tr1 cells suppress Il1b transcription and caspase-1 activation via an IL-10R-dependent mechanism ....................................................................................................... 66 3.3 Discussion ................................................................................................................. 69 Chapter 4: Developing Flagellin-Specific Chimeric Antigen Receptor Regulatory T Cell Therapy ................................................................................................................................... 73 4.1 Introduction ............................................................................................................... 73 4.2 Results ....................................................................................................................... 74 4.2.1 Development of a FliC-specific CAR and detection of flagellin in mouse colons ... 74 4.2.2 FliC-CAR is expressed on the surface of HEK 293T cells and selectively binds to recombinant FliC ............................................................................................................... 77 4.2.3 Primary murine Tregs can be retrovirally transduced and express FliC-CAR.......... 79 4.2.4 Recombinant FliC activates FliC-CAR-Tregs to proliferate.................................... 82 4.2.5 FliC-activated FliC-CAR-Tregs suppress conventional Tcells ................................ 83 4.2.6 FliC-CAR-Tregs survive in immunocompetent host ............................................... 87 4.2.7 FliC-CAR-Tregs demonstrate potential to ameliorate DNBS colitis ....................... 89 4.2.8 Ex vivo but not expanded or FliC-CAR-Tregs protect from T cell transfer colitis ... 91 xv  4.2.9 Expanded Tregs started secreting pro-inflammatory cytokines and lost their Treg profile...................................................................................................................... ...............94 4.3 Discussion ................................................................................................................. 97 Chapter 5: Patients’ Willingness and Perspectives Towards Chimeric Antigen Receptor T-Regulatory Cell Therapy for Inflammatory Bowel Diseases ........................................... 100 5.1 Introduction ............................................................................................................. 100 5.2 Results ..................................................................................................................... 100 5.2.1 Response rate ....................................................................................................... 100 5.2.2 Demographics and disease history ........................................................................ 101 5.2.3 Perspectives on CAR-Treg therapy ...................................................................... 104 5.2.4 Preference on CAR-Treg fate ............................................................................... 106 5.2.5 Willingness to try CAR-Treg therapy ................................................................... 108 5.2.6 Influence of demographic factors on willingness to try CAR-Treg therapy ........... 110 5.3 Discussion ............................................................................................................... 114 Chapter 6: Patient Experiences with Clostridioides Difficile Infection ............................... 118 6.1 Introduction ............................................................................................................. 118 6.2 Results and discussion ............................................................................................. 120 6.2.1 Demographics ...................................................................................................... 120 6.2.2 Health care system exposure preceding diagnosis................................................. 121 6.2.3 Symptoms, diagnosis, and treatment .................................................................... 124 6.2.4 CDI impact on life ............................................................................................... 128 6.2.5 Most important priority to improve patient experience ......................................... 131 6.3 Conclusion............................................................................................................... 135 xvi  Chapter 7: Conclusion .......................................................................................................... 137 References.............................................................................................................................. 143 Appendices ............................................................................................................................ 167 Appendix A Focus group interview guide: patient’s perspectives on genetically Engineered cell therapy for IBD............................................................................................................. 167 Appendix B Questionnaire: patient’s perspectives to new IBD therapy development ........... 168 Appendix C Questionnaire: Clostridium difficile survey ...................................................... 179 Appendix D Codebook: thematic analysis for Clostridium difficile survey ........................... 185  xvii  List of Tables Table 5-1 Demographics and disease history of patient participants ........................................ 102 Table 5-2 Disease history of patient participants...................................................................... 102 Table 5-3 Participants’ first thoughts and concerns after reading the 1-page Firefighter Analogy ............................................................................................................................................... 104 Table 5-4 Participants’ most important consideration for choosing their preference of cells staying in their body in a dormant state vs cells to be eliminated from their body .................... 107 Table 5-5 Participants’ reasons for choosing their level of willingness to try CAR-Treg therapy as new treatment ..................................................................................................................... 109 Table 5-6 Participants’ reasons for choosing their level of willingness to try CAR-Treg therapy in a clinical trial ...................................................................................................................... 110 Table 5-7 Bivariate analysis of influence of demographic predictor variables on willingness to try CAR-Treg therapy either as new therapy or in a clinical trial to test efficacy ...................... 112 Table 5-8 Multivariable linear regression of demographic predictor variables on willingness to try CAR-Treg therapy as new therapy or in a clinical trial to test efficacy ............................... 113 Table 6-1 Gender and age distribution ..................................................................................... 121 Table 6-2 Survey qualifiers to query quality of life scores ....................................................... 131 Table 6-3 Emerging themes and distribution of responses ....................................................... 134  xviii  List of Figures Figure 1-1 Schematic of proposed CAR-Treg therapy ............................................................... 24 Figure 2-1 Sorting strategy and post-sorting purity .................................................................... 38 Figure 2-2 Schematic of survey design ...................................................................................... 46 Figure 3-1 Activated CD44hiFoxp3- T cells develop into Tr1 cells ............................................. 57 Figure 3-2 CD44hiFoxp3- T cells derived Tr1 cells suppress proliferation of T effector cells ..... 58 Figure 3-3 CD44hiFoxp3- T cells derived Tr1 cells express characteristic transcription factors and surface markers ......................................................................................................................... 60 Figure 3-4 Tr1 cells efficiently suppress inflammasome activation ............................................ 62 Figure 3-5 Inflammasome suppression by Tr1 cells depends on soluble factors ......................... 63 Figure 3-6 IL-10 secreted by Tr1 cells mediates inflammasome suppression ............................. 66 Figure 3-7 Tr1 cells inhibit il1b transcription and caspase-1 activation in an IL-10 receptor dependent mechanism ............................................................................................................... 68 Figure 4-1 Generation of a FliC-specific CAR........................................................................... 75 Figure 4-2 Availability and location of flagellin in mouse colons .............................................. 77 Figure 4-3 Expression of FliC-CAR on cell lines and specific binding to cognate antigen ......... 78 Figure 4-4 Treg and Tconv Expansion over 7 Days ................................................................... 80 Figure 4-5 Expression of FliC and control-CAR on primary murine Tregs ................................ 81 Figure 4-6 FliC-CAR expressing Tregs are specifically activated by FliC to proliferate............. 83 Figure 4-7 Schematic of antigen-specific suppression assay ...................................................... 84 Figure 4-8 FliC-CAR expressing Tregs suppress conventional Tcells upon stimulation though the CAR.......................................................................................................................................... 87 Figure 4-9 Adoptively Transferred FliC-CAR-Tregs Survive in an Immunocompetent Host ..... 89 xix  Figure 4-10 FliC-CAR-Tregs demonstrate potential to ameliorate DNBS colitis........................ 90 Figure 4-11 Ex vivo but not expanded Tregs Prevent T cell transfer colitis ................................ 94 Figure 4-12 Expanded Tregs secrete pro-inflammatory cytokines and enhance inflammasome activation .................................................................................................................................. 96 Figure 4-13 Addition of rapamycin to Treg expansion media preserves Treg phenotype. ........... 96 Figure 5-1 Willingness to try CAR-Treg therapy ..................................................................... 108 Figure 6-1 Medications and conditions prior to first CDI ......................................................... 122 Figure 6-2 Situations surrounding first C. difficile infection .................................................... 124 Figure 6-3 Symptoms and recurrence ...................................................................................... 125 Figure 6-4 Diagnosis and treatment ......................................................................................... 127 Figure 6-5 Impact of C. difficile infection on quality of life ..................................................... 130  xx  List of Abbreviations  5-ASA 5-aminosailcylic acid APC antigen presenting cell APC-dye allophycocyanin ATP adenosine triphosphate B6 C57BL/6 (mouse strain) BD becton dickinson BMDAPC bone marrow-derived antigen presenting cells BSA bovine serum albumin CAR chimeric antigen receptor CARD caspase recruitment domain family member CAPS cryopyrin-associated periodic syndrome CD Crohn’s disease CBir C3H/HeJBir (mouse strain) C.diff Clostridioides difficile CD cluster of differentiation CDI Clostridioides difficile infection CPD cell proliferation dye CTLA-4 cytotoxic T-lymphocyte-associated protein 4 DAMP damage-associated molecular pattern DMEM Dulbecco’s modified eagle medium DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DC dendritic cell DSS dextran sodium sulfate eGFP enhanced green fluorescent protein EDTA Ethylenediaminetetraacetic acid ELISA enzyme linked immune-absorbance assay FACS fluorescence activated cell sorting FCS fetal calf serum FITC fluorescein isothiocyanate FOPX3 forkhead box P3 FVD fixable viability dye eFluor-780 HRQoL health-related quality of life GFP green fluorescent protein Glu glutaMAX GM-CSF Granulocyte monocyte colony stimulating factor HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid xxi  HEK human embryonic kidney Her2 human epidermal growth factor receptor 2 IBD inflammatory bowel disease IBDU IBD unclassified IC indeterminate colitis IFN interferon Ig immunoglobulin IL interleukin i.p. intra peritoneal IPEX immunodysregulation polyendocrinopathy enteropathy X-linked syndrome IRES internal ribosomal entry site JAK janus kinase LPL lamina propria lymphocytes LPS lipopolysaccharide MHC major histocompatibility complex mRNA messenger RNA MFI mean fluorescence intensity MLN  mesenteric lymph nodes NGFR delta low-affinity nerve growth factor receptor p75mut NLR NOD-like receptor NOD mice non-obese diabetic mice NOD nucleotide-binding oligomerization domain OVA chicken egg albumin peptide 257-264 PAMP pathogen-associated molecular patterns PBS phosphate-buffered saline PCR polymerase chain reaction PD-1 programmed cell death protein 1 PE phycoerythrin Pen/Strep penicillin/ streptomycin Plat-E Platinum E PMA 12-O-tetradecanoylphorbol-13-acetate RCF relative centrifugal force RNA ribonucleic acid RORγt retinoic acid-related orphan receptor γt RPMI Roswell park memorial institute medium RT room temperature RT-PCR quantitative real-time polymerase chain reaction scFv single-chain antibody comprised of fused variable regions of immunoglobulin heavy and light chain xxii  SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis T1D type 1 diabetes TCR T cell receptor Teff T effector cell Th T helper cell TLR toll-like receptor Tn naïve T helper cell TNF tumor necrosis factor TNBS 2,4,5-trinitrobenzene sulfonic acid TNP 2,4,6-trinitrophenol Tr type 1 regulatory cell Treg regulatory T cell Tconv conventional T cell TGFβ transforming growth factor β TSDR Treg-specific demethylated region UC ulcerative colitis WT wild type xxiii  Acknowledgements I conducted this research and wrote this dissertation on the unceded traditional ancestral territories of the Coast Salish peoples including the Qayqayt (the only First Nation without a land base), the xʷməθkʷəy̓əm (Musqueam), the skwxwú7mesh (Squamish) and the səl̓ilwətaɁɬ (Tsleil-Waututh). As a settler and new Canadian, I am grateful to live, learn and play on this land. I have started my lifelong learning about the history of First Nations and my own practice in decolonization.  I offer my sincere gratitude to my family for your ongoing support throughout the years. It was not easy to finish this PhD as a father of two and a husband. I thank my wife Ru for taking care of our kids and for providing me with the much-needed time and space to write my thesis. I thank my kids for distracting me and helping me to appreciate what is truly important in life! I thank my parents for your emotional and financial support throughout my education. I know that it was difficult for you to accept my move to Canada and I thank you for accepting and supporting me in my own path. It is this support that I will pay forward to my own children.  I thank my supervisor Dr. Ted Steiner for your continuous trust, guidance, and ongoing brainstorming sessions. Your open-mindedness and support allowed me to learn a new discipline outside both of our expertise’s and without your support I would not have been able to pursue my research interest and passion. I thank my committee member Dr. Laurie Goldsmith, for providing mentorship and training above and beyond what I expect from a supervisory committee member. It was through your insights and questions that I learned to conduct survey xxiv  research. I thank my committee member Dr. Laura Sly for your attention to detail and critical questions and especially for your ongoing support during the rough patches of my PhD.  I thank the UBC Public Scholarship Initiative for the grant support to leave the lab and conduct patient-oriented research. The institutional support through the PSI, enabled me to take on the challenge of learning a new discipline and to conduct this non-traditional PhD. I thank the Vanier Canada Graduate Scholarship, the UBC Four Year Fellowship and the CIHR Transplantation Training Program for providing me with salary support throughout my PhD research.   I engaged in many conversations across campus and across disciplines and found many open doors and open minds, brainstormed research ideas and received feedback. Engaging in these conversations across disciplines shaped my reflexivity and sharpened my research focus. Thank you to all those members of the UBC community for supporting me. A special thanks to all members of the CTLT Grad Team for the great and fun work in providing Teaching Development for graduate students and postdocs. Our interdisciplinary and insightful conversations were transformative in my thinking. I learned a lot and my learning continues.  I thank all members of Dr. Megan Levings’ lab who helped me during my time in her lab. I especially thank Jon, Jana, and Adele for providing a warm welcome and fun atmosphere in the lab and May for her great work as a technician.   Finally, I thank all my friends and especially Pete and Nicole for supporting me throughout my PhD, for listening to me when things where difficult and for pushing me over the finish line. xxv  Dedication I dedicate this thesis to my children Denali and Linden who are the most important people in my life and who I love unconditionally. Thank you for bringing sunshine to me every day of my life  and for making me a better person.  1  Chapter 1: Introduction 1.1 Inflammatory bowel disease Inflammatory bowel disease is the umbrella term for Crohn’s disease (CD), ulcerative colitis (UC), and indeterminate colitis (IC), also known as IBD unclassified (IBDU). Canada has the highest prevalence and incidence of inflammatory bowel disease (IBD) globally, with around one in 140 people affected, and an annual economic cost of ~$2.6 billion1. Despite this significant burden of disease, the public lacks awareness of the impact of IBD2. While disease presentation differs between the conditions and etiology is unclear3,4, disease symptoms are ultimately caused by recurrent flares of intestinal inflammation. IBD symptoms can include chronic diarrhea, bleeding, weight loss, and abdominal pain, and patients with UC are at increased risk for colorectal cancer3,4. Current therapies control symptoms rather than providing a cure. Furthermore, most are not gut-specific and have many side effects (see Chapter 1.2)2.  As a result of the devastating symptoms, people living with IBD, especially those suffering from active disease or chronic CD, report significantly reduced health-related quality of life (HRQoL) compared to people not affected by this disease5,6. In a recent systematic review studying how UC affects patient well-being, the authors report a clinically relevant reduction of HRQoL in patients with active disease. In contrast, HRQoL scores of those in remission where comparable to the background population7,8. A similar systematic review on CD highlighted the decrease of HRQoL scores of patients with active disease9. The impact of IBD on HRQoL and the lack of safe and effective gut-specific therapy underscore the need for development of new treatments.   2   1.2 Treatment 1.2.1 Conventional therapy Patients are typically diagnosed when exhibiting signs of active disease and treatment with “induction therapy” focuses on inducing remission which includes normal stool frequency, complete mucosal healing (defined as complete absence of ulcers and inflammatory lesions) in CD and endoscopic healing in UC, no rectal bleeding in UC, and the lack of other symptoms3,4. Once in remission, patients with CD are placed on “maintenance therapy” to prolong remission3. Patients with UC receive maintenance therapy if they experience more than one annual relapse4. The overall goal of maintenance therapy for IBD is to prolong steroid-free remission3,4. Conventional therapy induces and maintains remission through anti-inflammatory or immune suppressive mechanisms. Treatment targets are nonspecific and therapies are associated with mild to severe side effects. High-quality evidence on the effectiveness of conventional therapy is rare10.   Induction therapy for CD follows one of two therapeutic strategies, either “step-up” or the more recently developed “step-down”, while UC is typically treated in a “step-up” approach3,4 and the treatment regimen depends on the site and severity of IBD. The “step-up” starts with milder drugs and escalates to more aggressive therapy as needed. The “step-down”, which has been shown to be more effective, starts with aggressive treatment and a reduction of doses or alteration of medication once the patient is in remission3.  3  1.2.1.1 Corticosteroids and 5-aminosalicylic acid Corticosteroids are recommended as first-line treatment to induce remission in active CD and in moderate to severe UC3,4. They act by inhibiting the immune system through several pathways including inhibiting inflammatory cytokine expression and adhesion molecules and inducing apoptosis pathways in activated lymphocytes. However, corticosteroids often lead to systemic side effects and are not recommended as maintenance therapy.   Mild to moderate UC is treated with 5-aminosalicylic acid (5-ASA) to induce as well as maintain remission4. Patients with mild to moderate UC who don’t respond to 5-ASA may receive topical steroids to induce remission, however these are inferior compared to 5-ASA in UC4. While many clinicians prescribe 5-ASA for CD, its use is not recommended since clinical trials failed to demonstrate effectiveness compared to placebo in CD10–12. Furthermore about 25% of patients that receive 5-ASA stop the use due to side effects which for some patients include worsening of their CD symptoms. Antibiotics are often prescribed for the treatment of CD, however evidence of effectiveness is lacking and antibiotics lead to side effects3.   1.2.1.2 Immunomodulators Immunomodulators such as thiopurines and methotrexate are effective in inducing remission of moderate UC and maintaining steroid-free remission of both CD and UC3,11. Thiopurines are metabolized into purine antagonists and inhibit proliferation of lymphocytes by directly interfering with DNA and RNA synthesis. Development of severe side effects including acute pancreatitis and hepatotoxicity, and increased risk of lymphoma and skin cancer lead to around 10% of patients stopping treatment3. Methotrexate, a folate antagonist, is a better tolerated 4  alternative to thiopurines and unlike thiopurines is also effective in inducing remission. However, methotrexate is a teratogen and abortifacient and increased dosage could lead to hepatic fibrosis.  1.2.1.3 Surgery Patients with severe IBD and bowel obstruction are hospitalized and fed through parenteral nutritional support to allow their bowels to rest and recover. Up to 60% of patients with CD require one type of surgery during their life time and a significant number of patients require multiple surgeries3. Common procedures for CD include drainage of abscess and stricturoplasty, a less invasive method to treating bowel-obstructing strictures compared to resection. These procedures are nowadays conducted via laparoscopy which has reduced post-operative complications and hospital length of stay compared to open surgery3. Resections or partial resection of the affected portion of the bowel might be necessary depending on the severity of disease. While bowel resection typically does not cure a patient, UC and CD colitis can be technically cured by complete resection of the colon and creation of a j-pouch during which the terminal end of the ileum is connected to the anus while a j-shaped pouch is created which acts as a reservoir3,4. A less commonly used alternative to j-pouch is the introduction of a permanent ileostomy in which the terminal end of the ileum is connected to an external ostomy bag4.   1.2.2 Biologics Moderate to severe cases of both CD and UC are treated with biologics as both induction and maintenance therapy3,4,11. Biologics are antibodies or kinase inhibitors directed against pro-inflammatory targets such as tumor necrosis factor (TNF) and provide clinicians with therapies 5  against very specific targets. These agents are used for both, induction and maintenance therapy, are effective at stopping active disease and result in mucosal healing in around 30% of CD and 45% of UC patients13. After induction of remission, patients either continue to receive regular doses of the biologic used to induce therapy, or switch to conventional maintenance therapy such as immunomodulators. While very effective, many commonly used biologics are not gut-specific and side effects include risk of serious infections and blood cancer. Furthermore, all are significantly more expensive than conventional therapy14 which underscores the need for development of new therapies.  1.2.2.1 Tumor necrosis factor-blocking antibodies TNF inhibitors were the first class of biologics to be approved for IBD therapy. Most TNF inhibitors (adalimumab (Humira), golimumab (Simponi) and certolizumab pegol (Cimzia)) are injected subcutaneously with the notable exception of infliximab (sold as Remicade, Inflectra) which is injected intravenously. Anti-TNF antibodies are administered systemically and are not gut specific; patients who receive these drugs are prone to developing adverse effects including an elevated risk of severe infections. Furthermore, some patients develop antibodies against the anti-TNF antibodies which renders the therapy ineffective3. The recently developed and currently tested anti-TNF antibody AVX-470 is administered orally and might thus act more gut specific with less systemic adverse effects, however larger clinical trials are needed to demonstrate efficacy15.  6  1.2.2.2 Integrin-blocking antibodies The newer integrin blocking antibody vedolizumab (Entyvio) prevents immune cell adhesion and migration into the inflamed area of the bowel. Integrins are cell adhesion molecules that facilitate binding of cells to other cells or the extracellular matrix. By preventing recruitment of more leukocytes, these molecules are effective in inducing and maintaining remission. Vedolizumab targets integrin-47, which is expressed specifically in activated leukocytes in the gut which renders this molecule gut-specific with lower risk of systemic adverse reactions3,15. Comparative studies suggest that vedolizumab is as least as effective as adalimumab in inducing and maintaining remission of IBD15.  1.2.2.3 IL-12/IL-23-blocking antibodies A more recent addition to approved biologic therapy for UC and CD is ustekinumab, a monoclonal interleukin (IL)-12 and IL-23 blocking antibody15. Ustekinumab targets p40, a common subunit of IL-12 and IL-233. Both interleukins have pro-inflammatory effects; IL-12 promotes T helper cell 1 (Th1) differentiation which ultimately results in TNF production  (see 1.3.1.1, Type 1 immunity) while IL-23 promotes Th17 differentiation which ultimately results in chemokine production and recruitment of innate immune cells (see 1.3.1.3, Type 3 immunity). Clinical studies with ustekinumab indicate that it is a very safe drug that induces and maintains long-term remission of CD and UC15.  1.2.2.4 Janus kinase inhibitor The first small-molecule Janus kinase (JAK) inhibitor Tofacitinib was approved for use in UC, but not CD in 2018 and more JAK inhibitors are being tested for both UC and CD. Tofacitinib is 7  administered as an oral immediate-release tablet and provides a new treatment option for patients with UC that do not respond to other biologics4. JAKs are intracellular signal transduction molecules that phosphorylate and activate receptors for a range of over 50 pro- and anti-inflammatory cytokines15. Tofacitinib therefore inhibits cytokine-induced signal transduction and results in an inhibition of cytokine expression and lymphocyte function4,15. Side-effects include elevated risk of infections such as herpes zoster and reactivation of cytomegalovirus15. To prevent herpes zoster, patients typically receive vaccination shortly before treatment with Tofacitinib15.  1.3 IBD and the immune system 1.3.1 CD4+ T helper cells CD4+ T helper cells are a major component of the cell-mediated adaptive immunity and can be subdivided into four distinct lineages associated with different types of immunity. T helper type 1 cells (Th1) mediate type 1 immunity, T helper type 2 cells (Th2) mediate type 2 immunity and T helper type 17 (Th17) cells mediate type 3 immunity16,17. Regulatory T cells (Treg) represent a distinct lineage that promote tolerance and modulate immune responses (discussed below)17.   Each lineage is characterized by a specific set of cytokines required for their differentiation, expression of distinct master transcription factors and secretion of a defined set of key cytokines. These cytokines in turn recruit other immune cells and each type of immunity is specifically activated to fight different types of infections and conditions. While Th cells differentiate into distinct subsets and their associated cytokines and transcription factors inhibit other lineage 8  differentiation, there is some level of plasticity depending on the surrounding cytokine milieu, and partially differentiated cells can acquire the ability to express cytokines associated with another lineage17,18.  For the purpose of the thesis, I will refer to CD4+ T helper cells which are not Treg as conventional T cells (Tconv), as none of my experimental protocol included use of differentiated Th lineages.  1.3.1.1 Type 1 immunity Type 1 immunity is associated with fighting intracellular pathogens such as Leishmania major and Mycbacterium tuberculosis, viruses such as Herpes simplex virus, and cancerous cells and is carried out by Th1 cells and CD8+ cytotoxic T cells among others16,19. Cells involved in type 1 immunity are characterized by expression of the master transcription factor T-bet and secretion of interferon gamma (IFNγ) which plays an essential role in activation of mononuclear phagocytes16. Type 1 immunity tends to cause tissue damage due to involvement of cytolytic cells which are recruited to lyse infected or cancerous cells. This cytolysis in turn further perpetuates the immune response16. A misguided type 1 response can result in autoimmune diseases including rheumatoid arthritis and multiple sclerosis and is associated with Crohn’s disease16,20.   9  1.3.1.2 Type 2 immunity Type 2 immunity is activated in the presence of extracellular parasites such as intestinal nematodes, insect venoms and other xenobiotics and is associated with allergy, asthma and ulcerative colitis16,20. Th2 cells and other activated cells express their master transcription factor GATA-3 and secrete their signature cytokines IL-4, IL-5 and IL-13, which activate B-cells to produce IgE and recruit mast cells, basophils and eosinophils16,19. The cytokine IL-4 is critical to the activation of type 2 immunity16.  1.3.1.3 Type 3 immunity Type 3 immunity is directed to fight extracellular bacteria such as Staphyloccus aureus and fungi such as Candida albicans and, similar to type 1 immunity, is associated with some autoimmune disease19. Many autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and type 1 diabetes mellitus (T1D) as well as Crohn’s disease were originally thought to be caused exclusively by type 1 immunity and more recently attributed to type 3 immunity16. More recent evidence demonstrated a complex interplay between cells associated with type 1 and type 3 immunity and plasticity of Th17 cells to produce type 1 IFN which led to speculation about the clear distinction between these cell types. It is unclear whether the type 1 immune response in these autoimmune diseases could play a protective role16. Cells associated with type 3 immunity express the master transcription factor retinoic acid-related orphan receptor γt (RORγt) and secrete the key cytokines IL-17 and/or IL-22 which recruit macrophages as well as neutrophils and stimulate epithelial cells to mount antimicrobial responses16.  10  1.3.2 Regulatory T Cells Tregs are key regulators of the immune system and reduce harm to self by dampening innate and adaptive immune responses during infections and preventing autoimmunity through maintaining self-tolerance21,22. The best-known subsets of Tregs are forkhead box P3 (FOXP3)+cluster of differentiation (CD)25hi cells, herein referred to as Treg, and FOXP3- IL-10 producing type 1 regulatory (Tr1) cells21,23. Tregs stem from both thymic and peripheral origin and employ various suppressive mechanisms (see below) whereas Tr1 are exclusively peripheral derived and primarily act via IL-1023,24. Similar to Tconv, Treg home to the inflamed tissue where they suppress the local immune responses25.   In IBD, peripheral Tregs and Tr1 cells are most relevant in maintaining gut homeostasis, and patients with active IBD were found to have reduced numbers of Tregs and elevated numbers of Th17 cells in their blood26,27. Conversely, levels of Foxp3, IL-17 and IL-1 mRNA are heightened in the mucosa of patients with IBD28. This indicates possibly dysfunctional Tregs with a more Th17-like phenotype. Both Treg and Tr1 can secrete IL-10, which was shown to prevent colitis in mouse models of IBD22,29,30. Consequently, cells derived from IL-10-/- mice were unable to inhibit colitis30. Recent research found that children carrying mutations in either IL-10 or IL-10 receptor (IL-10R) subunits IL-10RA and IL-10RB develop severe, early-onset IBD, which can be treated by hematopoietic stem cell transplantation31,32.    11  1.3.2.1 FOXP3+ regulatory T cells 1.3.2.1.1 The role of FOXP3 Tregs require high and stable levels of FOXP3, their master-regulator, to keep their suppressive phenotype33. Constitutive FOXP3 expression is facilitated by de-methylation of the FOXP3 gene and the so called Treg-specific demethylated region (TSDR) is currently the only unique marker of Treg33. The critical impact of FOXP3 on Treg development and suppressive function, is highlighted in the phenotype of patients with immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome33,34. IPEX patients either lack circulating FOXP3+ Tregs, or their Tregs are unable to suppress due to a dysfunctional FOXP3 transcriptional program35–37. As a result of this loss of Treg functioning, patients suffer from severe autoimmunity presenting as several diseases including IBD-like disease, T1D, and psoriasis caused by unregulated and overactive T cells32,38. Regardless of its importance to Treg functioning, expression of FOXP3 is not sufficient to confer a Treg phenotype in human T cells and activated Th cells express low amounts of FOXP325. In contrast, murine Tregs can be induced by expression of Foxp3 and Foxp3 acts as defining marker for murine Tregs25. In this thesis, I used a strain of C57BL/6J (B6)xFoxp3-eGFP mice in which green fluorescent protein (GFP) is expressed when Foxp3 is expressed and used to identify Tregs.   1.3.2.1.2 Mechanism of Treg suppression Tregs suppress proliferation, activation and production of pro-inflammatory cytokines such as IL-2, IL-4 and IFNγ by different types of innate and adaptive immune cells including CD4+ and CD8+ T cells, B cells, macrophages and mature dendritic cells25,39,40. This suppression is mediated through several mechanisms including contact-mediated suppression through  12  co-inhibitory molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1(PD-1), surface-bound transforming growth factor β (TGFβ) local depletion of IL-2, release of cytolytic molecules such as granzyme A, granzyme B and perforin, and expression of inhibitory cytokines including IL-10, IL-35 and secretory TGF25. Foxp3 expression directly suppresses expression of proinflammatory cytokines by Tregs33.  Release of inhibitory cytokines such as IL-10, IL-35 and secretory TGFβ allows Tregs to directly inhibit target cells through a contact-independent mechanism25. TGF plays a role in Treg maintenance and a microenvironment rich in TGFβ directs naïve T cells to become Tregs after activation which further aids in the regulation of the immune response28. All three cytokines are implicated in IBD. Suppression by IL-10 plays a critical role in the intestinal mucosa, where it regulates the immune response to microbial antigens and ultimately maintains a healthy mucosal immune homeostasis41. As alluded to above, IL-10 released by Tregs is necessary to prevent colitis in a mouse model30. TGF was implicated in Treg mediated suppression and prevention of colitis in an IBD model as Tregs were unable to regulate Th cells resistant to TGF25. Finally, IL-35 plays a key role in Treg functioning and expression of this heterodimeric cytokine by naïve T cells was sufficient to result in a suppressive phenotype. Deletion of either component of IL-35 resulted in Tregs failing to cure IBD in a mouse model of colitis25.   13  1.3.2.1.3 Treg plasticity Recent findings show that, depending on the surrounding environment, Treg can upregulate transcription factors characteristic for the Tconv type that is being suppressed42. While this phenotypic adaptation may play a role in immune homeostasis, this modification of the Treg expression profile can lead to a downregulation of FOXP3 expression and acquisition of a proinflammatory phenotype, a process known as Treg plasticity42. For example, it was found that more Tregs convert to an IFN-γ producing effector phenotype in non-obese diabetic (NOD) mice compared to wild type (WT) mice43. Furthermore, IFN-γ producing Tregs with reduced suppressive capacity were discovered in the blood of patients with multiple sclerosis and autoimmune diabetes33.   1.3.2.2 Type 1 regulatory cells Tr1 cells arise in the periphery from either naïve T cells or memory T cells44 and express high levels of IL-10 and TGF Similar to Tregs, secretion of these molecules regulate Th cells and suppress colitis in a mouse model. Furthermore, a recent study demonstrated the ability of human Tr1 to suppress Th cells, to inhibit myeloid cells from releasing proinflammatory cytokines IL-1 and TNF and to induce goblet-cell differentiation. Goblet-cell function then improves barrier function through mucus secretion in intestinal organoid cultures45. While Tr1 and Treg secrete the same inhibitory cytokines, their function is spatially distinct. Tr1 are the major producer of IL-10 in the small intestine and Treg produce most IL-10 in the colon44. The IL-10 released by CD4+ T cells is critical to maintain intestinal homeostasis and mice that carry a CD4+ specific deletion of IL-10 develop spontaneous colitis similar to IL-10 knockout mice. This phenotype is less severe when IL-10 is deleted only in Tregs41.  14  1.3.3 Immunopathogenesis of IBD 1.3.3.1 CD4+ T helper cells in IBD In healthy individuals, intestinal immune homeostasis depends on a proper balance between Tconvs, which drive immune responses that clear pathogens, and Tregs which suppress inflammation and prevent inappropriate immune responses40,46. IBD disrupts the delicate balance between Tregs and Tconvs, allowing Tconvs to be activated by self-, food- and commensal antigens and thus cause intestinal inflammation47–49. This disturbed balance of Tregs and Tconvs is recapitulated in experimental models of IBD, providing a tractable model for mechanistic and therapeutic studies of Tregs50.  CD and UC have some degree of overlapping disease presentation, immune perturbation, and treatment strategies. Thus, IBD research often does not discriminate between CD and UC. However, the underlying immune response is quite different and these differences might mean that different treatment strategies are warranted. Traditionally, CD was defined as a Th1-driven immune condition associated with high levels of IFN-secreting Th1 cells. On the other hand, UC was defined as a non-typical Th2 response with high levels of IL-5- and IL-13-secreting Th2 cells20,44. Furthermore, CD is associated with higher levels of IL-2 compared to UC, which indicates a more activated T cell response20.  While this dichotomy might be clinically useful for discerning CD from UC51, the actual immune response is more complex with a key role played by Th17 cells. IL-17 is elevated in patients with both CD and UC but levels are higher in CD. Furthermore, mixed Th1/Th17 that secrete both IL-17 and IFN were discovered in CD44. Both, CD and UC are associated with elevated levels 15  of the proinflammatory cytokine IL-1. While peripheral Treg frequencies in IBD are reduced, they are more abundant in the lamina propria and function when isolated and tested in vitro. Furthermore, levels of IL-10 are elevated in the intestine of patients with IBD compared to individuals without IBD20.  This observation could indicate that Th cells are less responsive to Treg suppression or that Treg function is reduced in the lamina propria20. Similarly, frequencies of peripheral Tr1 in IBD are comparable to healthy individuals and, while isolated Tr1 show the same phenotype in healthy individuals and people with IBD, a subset of  IFN producing Tr1 cells was described to lower IL-10 levels in IBD44.  1.3.3.2 Mononuclear cells in IBD Dendritic cells (DC) include various types and function to control local microenvironments by sensing pathogens and sampling for antigens20. In response, DCs either initiate appropriate proinflammatory immune responses and promote differentiation of lymphocytes or promote tolerance against non-harmful antigens. DCs thus orchestrate the interplay between innate and adaptive immunity and intestinal DCs contribute to maintaining mucosal homeostasis. Mucosal DCs in patients with CD and UC display an activated phenotype associated with inflammation20.   Intestinal macrophages are responsible for recognition and clearance of pathogenic bacteria as well as orchestrating the immune response. Based on their cytokine profile and activation state, macrophages have been distinguished as classically activated M1 macrophages, associated with proinflammatory activity including development of IBD, and as alternatively activated M2 macrophages, associated with immunoregulatory activity20,52. This dichotomy is no longer considered standard nomenclature as these states are functional in nature, depend on the local 16  microenvironment, and macrophages can convert between different states on a spectrum between M1 and M253. Intestinal macrophages in healthy people do not get activated by commensal bacteria, do not express the innate-immune receptor CD14 and are able to promote Treg differentiation through IL-10 expression while inhibiting dendritic cell-driven Th1 and Th17 differentiation54. In contrast, intestinal macrophages in mouse models of IBD and patients with CD, but not UC, express CD14, secrete proinflammatory cytokines and promote differentiation of Th1 and Th17 cells54.   Both DCs and macrophages sense microbial pathogen-associated molecular patterns (PAMP) using toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLR), which are types of pattern-recognition receptors39. Expression of these receptors as well as sensing are tightly regulated which avoids unwarranted immune responses against commensal bacteria and thus promotes tolerance. Mucosal macrophages and DCs isolated from patients with IBD display elevated expression of TLRs and genome-wide association studies identified polymorphisms and mutations in genes encoding for NLRs and TLRs to be associated with elevated risk for IBD39.  1.3.3.3 Inflammasomes in IBD Inflammasomes are cytosolic multiprotein complexes expressed in immune cells including macrophages, monocytes and DCs and are activated in response to the integration of danger signals that include PAMPs and damage-associated molecular patterns (DAMPs)55–57. For this Thesis, I will focus on the NOD-like receptor protein (NLRP)3 inflammasome. To activate the inflammasome, two danger signals are needed. Signal 1, for example LPS binding to TLR4, 17  primes the inflammasome by stimulating expression of inflammasome-related components such as inactive NLRP3, pro-IL1 and pro-IL1858. Signal 2 activates the inflammasome and is mediated via different pathways including adenosine triphosphate (ATP) binding to its receptor P2X7 or lipid raft formation triggered by interaction of charged particles, such as monosodium urate (MSU) crystals or alum, with the membrane lipids. Activation starts with inflammasome assembly by forming a complex of NLRP3, the adaptor protein ASC and procaspase-1. Once assembled, pro-caspase-1 is cleaved and active caspase-1 in turn cleaves pro-IL-1 and pro-IL-18 to form the active pro-inflammatory cytokines IL-1 and IL-18. IL-1 alongside IL-6 then induces naïve T cells to differentiate into Th17 cells. Inflammasome activation is tightly regulated and inappropriate activation, for example through gain-of-function mutations, result in inappropriate production of IL-1 Hyperactive NLRP3 inflammasomes are associated with immune pathologies such as the autoinflammatory cryopyrin-associated periodic syndrome (CAPS), type 2 diabetes, neurodegenerative disease and IBD55,59–64.   As inflammasomes are involved in the interaction between microbiota and the mucosal immune system including the crosstalk between components of the innate and adaptive immune system, they are of particular interest to IBD immunopathogenesis. While IL-1 is a critical pro-inflammatory cytokine in driving IBD-related inflammation and increased levels are associated with increased severity of IBD, evidence suggests that IL-1 does not act by itself but in concert with other cytokines including TNF and IL-665. Despite the clear role of IL-1 in IBD pathogenesis, the role of inflammasomes in IBD pathogenesis is controversial. Some studies suggest that inflammasomes perpetuate experimental colitis while others indicate a protective 18  effect20,65. Most of these studies were performed in either the dextran sodium sulfate (DSS) or the trinitrobenzene sulfonic acid (TNBS) colitis models and evidence suggests that the contradicting results depended on factors such as the gut microbiome composition as well as effects of the inflammasome other than activation of IL-1. For example, there is evidence that IL-18 as well as IL-1 can mediate regeneration of the epithelial barrier. Furthermore, some evidence suggests that inflammasome activity regulates gut microbiota which in turn regulate Tregs. A deletion of NLRP3 could thus correspond to lower Treg activity and IL-10 and in turn result in increased disease while increased NLRP3 inflammasome activity would ultimately increase numbers of Tregs which provide protection from disease65.  To date, no studies have directly investigated the role of inflammasomes in IBD pathobiology. Circumstantial evidence demonstrates their importance though as levels of IL-1, IL-18 and caspase-1 activity were increased in immune cells isolated from patients with CD20. Furthermore, patients with IL-10 receptor-deficiency suffer from IL-1 mediated early onset IBD and IL-10 uniquely downregulates inflammasome activity55,65. As IL-1 affects release of a range of pro-inflammatory cytokines, it could be an interesting treatment target for some patients with IBD. Indeed, reduced levels of pro-inflammatory cytokines were found in colonic sections from patients with CD cultured with an inhibitor of signal 265. Patients with IBD-causing mutations in inflammasome inhibitors such as IL-10 receptor- or caspase recruitment domain family member 8 (CARD8)-deficiency (a direct inflammasome inhibitor) were successfully treated with the IL-1 receptor-antagonist anakinra while unresponsive to standard treatments. Patients with other forms of IBD however did not respond to anakinra65. 19  1.3.4 Flagellin in IBD The uncontrolled inflammatory response in IBD ultimately leads to a breakdown of the intestinal wall and commensal bacteria further activate pro-inflammatory responses resulting in an inflammatory positive-feedback loop66. A common feature of commensal and pathogenic bacteria is the expression of flagella, which are motility organelles67. Flagellin is the main structural component of flagella and is comprised of highly conserved N- and C-terminal hairpin forming domains which are separated by a hypervariable domain.  The conserved domains of flagellin are molecular patterns recognized by TLR 5 and elicit a potent inflammatory response of the innate immune system68. In addition, antibodies against flagellin were found in patients suffering from Crohn’s disease69. The same study has identified flagellin to elicit antibody and Th1 cell responses in the colitic mouse strain C3H/HeJBir (CBir), and flagellin specific Tconv cells were sufficient to induce colitis upon adoptive transfer into immunocompromised mice69. The flagellin responsible in this study was subsequently named CBir1 and antibodies directed against CBir1 are important diagnostic tools for CD70. Subsequent studies have described Fla2, another antigenic flagellin in murine models of colitis and in  CD71–73.   In contrast to the pro-inflammatory response described above, a recent study published by Steimle et al74 found that symbiotic Escherichia coli strain Nissle 1917-derived flagellin elicits an anti-inflammatory response via TLR5 signalling74. This strain is used to extend clinical remission in UC as active ingredient of the probiotic drug Mutaflor®75 and was previously thought to elicit its anti-inflammatory effect in live-form only74. While different types of 20  flagellin, derived from symbiotic and pathobiontic bacteria activate TLR5 with opposing outcomes, it is evident that flagellin is implicated in IBD and could be used as therapeutic target74.  1.4 Regulatory T cells as treatment for IBD The Treg function to suppress active immune responses has raised interest in their use as cellular therapy to treat autoimmune diseases and to prevent transplant rejection26,76. Previous studies have demonstrated that Tregs are able to prevent IBD in animal models and can even cure established colitis77. The overall response rate in a clinical trial of antigen-specific Tr1 therapy in human CD was 40% and treatment was well tolerated. However, with only 20 patients, this was a small scale study, treatment benefit did not last beyond 12 weeks and the suppressive capacity of these Tr1 cells might be unstable78,79. Later studies identified a more stable Treg subpopulation in blood isolated from patients with CD which can also be expanded in vitro. These CD45RA+ cells express the gut specific 47 integrin (see also 1.2.2.2), reduce inflammation in the lamina propria of active CD mucosa and home to gut tissue in a mouse model80. The TRIBUTE trial aims to test these in vitro expanded Tregs in CD patients81.  Furthermore, studies in murine models of T1D have shown that Treg therapy with either isolated, polyclonal or antigen-specific Treg derived from transgenic animals can prevent T1D82. A recent study successfully showed prolonged remission of recently diagnosed T1D in children after infusion of autologous Tregs and demonstrated safety of this therapy83. Pre-clinical transplant studies with solid organs or pancreatic islet suggest that induction and maintenance of in vivo tolerance depends on the presence of Tregs at the time of transplantation and that adoptive 21  transfer of Treg was sufficient to prevent allograft rejection82. These studies in other disease settings are relevant to development of Treg-based therapy as they demonstrate feasibility of using Treg as a means to specifically dampen the immune response.  Despite promising pre-clinical data, some major hurdles have to be overcome to translate these results into clinical settings. The lack of a unique surface marker of Treg combined with the fact that activated Tconv upregulate CD25, a marker associated with Treg, makes it challenging to acquire a pure population of human Treg82. In addition, pre-clinical data from animal models show that high numbers of Treg cells are needed but only about 2% of the CD4+ T cells in the blood are Tregs and an even smaller number of those Tregs are antigen specific and would be activated upon encounter of their cognate antigens in vivo at the site of inflammation82. The remaining Tregs would remain unstimulated, thus significantly reducing their suppressive potency. Ex vivo expansion protocols are being developed but contaminating Tconv cells expand more effectively than Tregs, increasing the number of contaminating cells82. Another potential drawback of Treg therapy is the possibility that a small subpopulation of transferred Treg loses their FOXP3 expression and “Treg-ness” and converts to a Tconv phenotype84. This phenomenon would be even more dangerous, when transferring antigen-specific Tregs. A feasible approach to retain stable FOXP3 expression is transduction of CD4+ T cells with a lentiviral vector85.   22  1.4.1 Chimeric antigen receptors to create antigen-specific regulatory T cells Naturally, the antigen-specificity of every T cell receptor (TCR) is the product of gene rearrangement, manipulation of this process would be difficult86. Chimeric antigen receptors (CAR) are a way to circumvent this gene rearrangement process and to engineer T cells specific for any antigen of interest87. CARs are comprised of an extracellular domain which originates from a single-chain antibody comprised of the fused variable regions of immunoglobulin heavy and light chain (scFv) specific for the antigen of interest. This domain is linked by a transmembrane “spacer” sequence to the intracellular signalling domains from the TCRζ-chain and from the co-stimulatory receptor CD2887. The CAR is then expressed in the T cell via a lentiviral or retroviral vector system.   Activation of the CAR by cross-linking via binding to its antigen therefore provides both the TCR stimulatory and the necessary co-stimulatory signal to trigger TCR-like signalling cascades that ultimately activate the T cell activation. This activation is independent from the intrinsic TCR of the T cell and from antigen presentation by antigen presenting cells (APCs). CARs have been used successfully in clinical trials in cancer patients, with the objective to direct cytotoxic T cells to tumor-specific antigens in a major histocompatibility complex (MHC)-independent mechanism88. In 2017, the FDA approved tisagenlecleucel or Kymirah® for B-cell acute lymphoblastic leukemia and in 2018 for refractory diffuse large B-cell lymphoma71. Kymirah was subsequently approved in Canada, Australia, Japan, the European Union and Switzerland90. In addition, several studies have investigated the use of CAR in various disease models in mice.   23  Importantly, Tregs engineered to express a CAR were found to be protective in the mouse models of experimental autoimmune encephalitis, as well as in T-cell-transfer-, and TNBS-induced colitis85,91–93. In addition, Treg with a CAR directed against 2,4,6-trinitrophenol (TNP) were able to ameliorate oxazolone induced colitis upon addition of low doses of TNBS via the so called “bystander effect”92. While the latter study provides an important proof-of-principle that antigen specific Treg are able to cure experimental colitis, the CAR was directed against a synthetic antigen with no relevance to human disease.  1.5 Patient’s perceived risk associated with chimeric antigen receptor regulatory T cell therapy CAR-Treg therapy is seen as promising approach to bring Treg therapy into clinical practice to treat autoimmune disease and prevent transplant rejection. It would involve purifying Tregs from the patient’s blood, genetic modification with CAR to make them specific for targeting intestinal inflammation, and injection back into the patient’s blood (Figure 1-1). The specificity for intestinal inflammation would lower risks associated with CAR-Treg therapy compared to untargeted Tregs which have shown their therapeutic potential in animal models but have high risk for side effects94. The risk associated with CAR-Treg therapy is anticipated to be similar to that of biologic therapy with global immunosuppression driven by the CAR-Tregs native TCR being the most severe anticipated possible adverse effect76,91–93,95.  24    Figure 1-1 Schematic of proposed CAR-Treg therapy CAR-Treg therapy involves drawing a unit of blood from the patient, isolating, and activating Treg cells, virally transfecting Tregs with the CAR and expanding CAR-Tregs to generate sufficient numbers for re-infusion into the patient’s blood. CAR-Tregs then home to the site of inflammation, get activated by flagellin and suppress the local immune response.  Since the treatment is conceptually and logistically complex, and includes terms such as genetic engineering and virus, patients might perceive an unacceptable risk to be associated with this therapy96. It has been shown that people with IBD accept medication risk in exchange for treatment benefit, and past clinical trials using autologous stem cell transplantation to treat refractory CD suggests patients’ willingness to try a toxic and risky therapy97. Nevertheless, some risks, particularly risks associated with new or unusual therapy, may not be acceptable to patients98,99. Understanding of patient’s willingness to accept and perspectives on CAR-Treg therapy ultimately helps patient-oriented therapy development and reduces acceptance-related barriers to moving a new therapy into clinical practice.  25   1.6 IBD increases risk for Clostridioides difficile infection Clostridioides difficile infection (CDI) is the leading cause of nosocomial infectious diarrhea in adults in Canada. While Canadian infection rates for hospital-acquired CDI have been increasing between 1999 to 2013/2014 and trending downward since, community-acquired CDI rates are still trending upwards100–102. Symptoms range from mild diarrhea to potentially lethal pseudomembranous colitis100. Typically, colonic microbiota act as barrier in the gut, providing resistance to CDI. However, disruption of host microbiota, most commonly through antibiotic treatment, results in increased susceptibility to infection103,104. Furthermore, up to 20% of adults over the age of 65 may be asymptomatically colonized by C. difficile, which increases their chance of CDI and may act as potential vehicles of transmission in a healthcare setting105,106. The rate of asymptomatic carriers is increased among people living with IBD107.  People living with IBD are have an almost 5-fold increased risk of contracting CDI which in turn complicates the progression of their IBD107. Risk of contracting CDI is especially elevated in younger IBD patients, within two years post-diagnosis, which may be related to increased antibiotic usage, in individuals seeking ambulatory care or hospitalization and in individuals receiving corticosteroids or TNF inhibitors to manage their IBD. When acquired during an IBD-related hospitalization, CDI increases the duration of that hospitalization, risk of requiring colectomy and mortality107.   26  1.7 Summary and synopsis of research questions IBD is a life-changing disease resulting from recurrent intestinal inflammation. In IBD, the balance between proinflammatory and tolerogenic components of both the adaptive and innate mucosal immune system is disrupted. This loss of balance results in an activation of proinflammatory and a concomitant reduction of tolerogenic responses to components of commensal bacteria which contribute to the intestinal inflammation28,108. Current treatments for IBD aim to induce and maintain remission through anti-inflammatory or immune suppressive mechanisms. Treatment targets are nonspecific and therapies are associated with mild to severe side effects, and high-quality evidence on the effectiveness of conventional therapy is rare10. As a result, IBD leads to a significant reduction in health-related quality of life (HRQoL). Furthermore, immunosuppressive medication and regular ambulatory care and hospitalization increase the risk for nosocomial infections such as CDI. The impact of IBD on HRQoL and the lack of safe and effective gut-specific therapy underscore the need for development of new treatments.  In Chapter 3: Tr1 Cells, but Not Foxp3 + Regulatory T Cells, Suppress NLRP3 Inflammasome Activation via an IL-10−Dependent Mechanism, I describe a collaborative study into the ability of Tregs or Tr1 cells to regulate inflammasome activity. Inflammasomes play a key role in the interaction between microbiota and components of the innate and adaptive immune system and there is evidence that inflammasome activity influences regulatory T cells. As chronic inflammatory diseases caused by NLRP3 deficiency are associated with changes in immunoregulatory cells including Tregs and Tr1 cells, I was interested in understanding a possible role of these cells in modulating inflammasome activity. These insights furthered our 27  understanding of mechanisms of Treg suppression and have implications on the development of Treg-based therapy for IBD. I wrote Chapter 3 in first person plural as the research was conducted in collaboration with Yu Yao, published as Yao et al. (2015)55 and included in both her and this PhD thesis. In this thesis, I reproduce only the aspects of the research that we conducted collaboratively.  In Chapter 4: Developing Flagellin-Specific Chimeric Antigen Receptor Regulatory T Cell Therapy, I describe the development and testing of flagellin-specific CAR-Tregs. Recent studies investigated the use of subsets of Tregs as therapeutic means to stop intestinal inflammation in IBD and animal models and clinical trials have demonstrated the potential and safety of Treg therapy in IBD94. Proof-of-concept studies indicated feasibility of creating CAR-Tregs to ensure site-specific suppression of conventional T cells but those model CARs were not relevant to IBD88–9078,94,109. I designed an optimized method for expansion and retroviral transduction of mouse Tregs which was published as Wu et al. (2020)110 and developed and tested the first CAR-Treg directed against an antigen relevant to human IBD (unpublished). I transferred this research project to Dan Wu and Dominic Boardman to concentrate on my survey research.  In Chapter 5: Patients’ Willingness and Perspectives Towards Chimeric Antigen Receptor T-Regulatory Cell Therapy for Inflammatory Bowel Diseases, I describe the results from surveying people living with IBD to gauge their willingness to try CAR-Treg therapy. My overall hypothesis was that people living with advanced stages of IBD or running out of treatment options (i.e. receiving biologics) would indicate a higher willingness to try CAR-Treg 28  therapy than would other people living with IBD. The manuscript related to this chapter was recently published as Vent-Schmidt et al. (2020)111.  In Chapter 6: Patient Experiences with Clostridioides Difficile Infection, I describe the results of an exploratory Canada-wide survey to understand the impact of CDI on patients in Canada. Previous research on the impact of CDI centered around the health care system, clinician perspectives and economic burden, but not on patient experiences and their priorities in the health care system. The results of this study have implications on improving patient experience with the health care system and developing a patient-centered health care system. The results of this chapter are published in Vent-Schmidt et al. (2020)112.   1.8 A reading guide for this interdisciplinary thesis I present this thesis as an interdisciplinary mixed-method thesis, drawing from basic biomedical methodologies in Chapters 3 and 4 and from survey research methodologies in Chapters 5 and 6 which I integrate in Chapter 7. The purpose of sharing this reflection on my process from a positivist basic scientist to a pragmatist mixed-method researcher is to provide readers with a guide for how to approach my thesis. The primary target audience for my thesis are health scientists and clinicians.   I started my PhD with M.Sc. level training as a basic researcher and a worldview that acknowledges the existence of one objective, observable and quantifiable external reality, independent of the individual. With this worldview, I created knowledge that added to the collectivity of facts describing that one reality. To me, quantitative research meant working with 29  objectively gathered data and qualitative research meant working with data gathered through subjective inputs which included quantitative instruments such as Likert-scales.    Throughout my PhD, I immersed myself in experiences that honed my reflexivity, shaped my worldview, and altered my understanding of knowledge and truth. Working in pedagogy, I experienced constructivism and interpretivism and learned to see truth and reality as something subjectively experienced. Individuals impacted by and involved in my research live in their own subjective realities shaped by their life experiences. These subjective truths are not in competition and therefore untrue claims, but different aspects and descriptions of the phenomenon I research; my data does not contain outliers. As a researcher with a pragmatist worldview, it is my responsibility to interpret different perspectives shared by my research participants rather than seeking one average perspective. My research goal is to create a complex and multifaceted description, rather than one description reduced to common denominators.  The reader of this thesis will follow my path as a researcher and will leave with my perspective of how integrating research methodologies from different disciplines could work.  30  Chapter 2: Methods and Materials 2.1 Basic science chapters 2.1.1 Animals C57Bl/6 mice (Jackson Laboratory, 000664), C57Bl/6 Thy1.1 mice (Jackson Laboratory, 000406), B6.129S2-Il10rb tm1Agt/J (referred to as il10rb-/-) mice (Jackson Laboratory, 005027), IL-10GFP mice (Jackson Laboratory, 008379), Foxp3-mRFP mice (Jackson Laboratory, 008374), Thy 1.2 Foxp3EGFP mice (Jackson Laboratory, 006772), B6.Cg-Tg(TcraTcrb)425Cbn/J (OT-II) and Tcrb-/- mice (Jackson Laboratory, 002118), were purchased from Jackson Laboratory and bred at the BC Children’s Hospital Research Institute animal facility. Thy1.1 Foxp3EGFP mice were generated by crossing C57Bl/6 Thy1.1 with Thy 1.2 Foxp3EGFP mice, and Foxp3RFPIL-10GFP mice were generated by crossing Foxp3RFP with IL-10GFP mice in house. Caecal sections of Muc2-/- mice where kindly donated by Dr. Bruce Vallance, UBC who maintains and studies these mice as published113. All animal experiments described in this thesis were approved by the University of British Columbia Animal Care Committee.  2.1.2 Flow cytometry To detect extracellular cytokines, cells were stained in PBS for 25 min at RT with antibodies labeled with fluorescent dyes in the presence of fixable viability dye (FVD) to distinguish dead from live cells. Cells were washed once and resuspended in FACS buffer for detection on BD FACSCanto I or BD LSRFortessa (both: BD Biosciences). To detect intracellular cytokines, cells were restimulated for 1h in 100l RPMI/HEPES containing 100ng/ml PMA and 1g/ml Ionomycin at 37ᵒC. Subsequently, 2l brefeldin A was added and cells were incubated for 5h at 37ᵒC. Extracellular proteins were stained as described above. Prior to staining for intracellular 31  markers, cells were fixed by applying eBioscience GFP-retaining Fix/Perm 20 min at RT. Cells were washed and centrifuged for 3 min at 2300 rpm and stained with antibodies against intracellular proteins for 25 min at RT in eBioscience perm buffer. Flow cytometry data were analyzed using FlowJo 10 software.  2.1.3 Enzyme-linked immunosorbent assay (ELISA) To determine cytokine levels in culture supernatant, sandwich ELISA was employed. Primary antibody was coupled to Nunc Maxisorb plates followed by incubation with supernatant, either diluted for detection of IL-2 and IFN, or undiluted for IL-10. Subsequently, biotinylated secondary antibody was added coupled with streptavidin-bound horseradish peroxidase. Cytokine concentration was detected through color-change in TMB substrate, read at 650nm. Analysis was performed by 5 parameter linear regression through Microsoft Excel or GraphPad Prism based on a standard curve prepared in parallel to samples. To determine FliC concentration in stool samples, one fecal pellet was mashed in 500l ELISA coating buffer followed by centrifugation at 13000 rpm for 5 min. Supernatant was sterile filtered and diluted 1:100 followed by a 1:2 dilution series. This dilution series was added to Nunc Maxisorb plate and incubated over-night. FliC was detected by monoclonal 5D4 antibody, followed by goat anti-mouse HRP coupled secondary antibody and detection as described above.  2.1.4 Western blotting Macrophages were scraped off the plates and lysed in Laemmli buffer. Proteins were then denatured and separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For analysis of IL-1 or caspase-1, a 12% or 15% SDS-PAGE was used, respectively. 32  Following SDS-PAGE, separated proteins were transferred to a membrane which was probed with antibodies directed against IL-1 (R&D Systems), caspase-1 (Santa Cruz Biotechnology), and -actin (Cell Signaling Technology). Primary antibodies bound to target proteins were then marked using horseradish peroxidase (HRP)-conjugated donkey anti-goat (Santa Cruz Biotechnology) or goat anti-rabbit (Dako) secondary antibody and visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) on CL-X Posurer Films (Thermo Scientific). Manufacturers’ instructions were followed.  2.1.5 RNA isolation and quantitative real-time polymerase chain reaction (RT-PCR) RNA from BMDAPC was isolated using E.N.Z.A Total RNA isolation kit following the manufacturer’s instructions (OMEGA bio-tek, Norcross, GA, USA). Following RNA isolation, reverse transcription was performed using qscript cDNA synthesis kit (Quanta Biosciences, Gaithersburg, MD, USA) and RT-PCR was conducted using Applied Biosystems 7500 Fast Real-Time PCR System. The primer sequences were as follows (5’ to 3’): il1b, 5’-TGTAATGAAAGACGGCACACC-3’ and 5’-TCTTCTTTGGG- TATTGCTTGG-3’; maf, 5’-AGGATGGCTTCAGAACTGGC-3’ and 5’- GGTCTCCACCGGTTCCTTTT-3’; ahr, 5’-AGCCGGTGCAGAAAACA- GTA-3’ and 5’-CCAGGCGGTCTAACTCTGTG-3’; prdm1, 5’-CTGTA- CAAGCTGCCCCCAAG-3’ and 5’-TAAGGATGCCTCGGCTTGAA-3’; tgfb1, 5’-GGTGGACCGCAACAACGCCAT-3’ and 5’-GGGGTTCGGGC- ACTGCTTCC-3’; and 18s, 5’-CAAGACGGACCAGAGCGAAA-3’ and 5’- GGCGGGTCATGGGAATAAC-3’. Data were normalized to 18s rRNA using the 22-ddCt method  33  2.1.6 Immunohistochemistry Paraffin-embedded caecal sections were deparaffinized through heating to 80ᵒC for 10 min, followed by four subsequent 2 min incubation in xylene solution, two 3 min incubation in 100% ethanol, one 3 min incubation in 95% ethanol, 1 3 min incubation in 75% ethanol and one 5 min incubation in water. Antigen retrieval was performed by incubating slides submerged in preheated buffer for 30 min in the steamer, followed by PBS wash. Endogenous biotin was blocked with biotin-blocking kit (Life technologies #E21390) according to the manufacturer’s recommendation. Tissue sample was incubated in blocking buffer (dilution buffer plus 2% serum) for 1h at RT or overnight at 4ᵒC. Following a wash, slides were incubated in primary biotin-coupled antibody diluted in dilution buffer (0.1% Triton X-100, 0.1% BSA, 0.01% Na-azide, 0.04% EDTA) overnight at 4ᵒC or for 2h at RT. Streptavidin-coupled secondary antibody was applied for 1h at RT and slides were subjected to final washing steps in PBS and water. Slides were mounted in mounting media containing DAPI and fluorescence was detected by microscopy.  2.1.7 CAR development FliC-specific hybridoma strains were produced by the University of Virginia Lymphocyte Culture Center (Charlottesville, VA). Hybridoma were grown in RPMI complete (10% FCS, 1% Glu, 1% Pen/Strep) and antibody specificity was confirmed by ELISA and bead binding assay (see below). FliC antibody from hybridoma “5D4” was selected and the isotype was confirmed as IgG2b. RNA was isolated from 5 million cells with EZNA total RNA animal cell protocol. cDNA was synthesized from 4µg RNA using IgG2b specific primers.   34  Degenerate PCR was performed as described (Babcook 1996). PCR product, light and heavy chains were separated on a 1% agarose gel @95V for 40 min and purified with QIAquick Gel Extraction Kit. Correct size of PCR product was verified by GeneRuler 1kb Plus DNA ladder. Both light and heavy chains were ligated into Kanamycin resistant vector (pBKCMV) and transfected into NEB 5-alpha Competent E. coli (New England Biolabs, ref C2987H) employing high efficiency transformation protocol. Colonies were picked and plasmids were purified with EZNA Plasmid Mini Kit followed by test digestion with HincII. Positive clones were sent to sequencing. Heavy and light chains, separated by a glycine linker, were then cloned into a human epidermal growth factor 2 (Her2)-CAR containing murine stem cell virus retroviral expression vector kindly gifted by Dr. Jonathan Brahmson. The original selectable marker GPF was replaced with a truncated nerve growth factor receptor (NGFR) sequence114 to distinguish CAR expression in Tregs isolated from Foxp3-eGFP reporter mice. The CAR contains an internal myc-tag as expression control.  2.1.8 Bead binding assay Beads (ThermoFisher FluoSphere Sulfate Microspheres 4.0µm, ref F8859),  were washed in PBS and incubated at 1M/ml in 1µg/ml cognate antigen, FliC, Fla2 or PBS control, on a V-bottom plate for 1h at RT vigorously shaking. Following a wash, beads were blocked in 5% BSA for 1h at RT. Supernatants from FliC-specific hybridoma was added in 1:100 dilution in PBS and incubated for 1h at RT vigorously shaking. After washing, beads were incubated with secondary goat anti-mouse IgG (H+L) APC 1:2000 (Invitrogen, clone 83B1-1) and incubated for 30 min at RT in the dark. Binding of antibody to bead-coupled antigen was determined by readout on BD FACScanto I. 35  2.1.9 FITC labeling of recombinant proteins FITC-labeled FliC and BSA were produced via a modification of the method described by Riggs JL et al. 1958 and Hidaka and Fidge et al. 1999. FITC powder (kindly gifted by Dr. Zakaria Hmama) was dissolved at 1mg/ml in DMSO, mixed 1:10 ratio with either recombinant FliC or BSA and incubated for 2h in the dark at RT while mixing followed by removal of excess FITC by buffer exchange via centrifugal filtration (Millipore Amicon Ultra-4 Centricugal Filter Unit with Ultracel-10 membrane, ref  UFC801024) at 3000rpm. FITC-labelled FliC and BSA were coupled to beads as described above and fluorescent labeling was verified by flow cytometry.   2.1.10 Transient transfection For surface expression and binding experiments, HEK 293T cells were grown to confluence in DMEM complete (10% FCS, 1% Glu, 1% Pen/Strep) and plated at 70K per well in 1ml DMEM complete without antibiotics overnight. Transient transfection was performed either via Lipofectamine 2000 Transfection Reagent (Thermofisher, ref  12566014) in Opti-MEM (Thermofisher, ref 31985070) or via jetPRIME (Polypus-transfection, ref 114-07), according to manufacturers’ recommendation.   2.1.11 HEK 293T surface expression  HEK 293T cells transfected as described above were incubated for 48h at 37°C in 5% CO2. Cells were detached by pipetting up and down and surface expression of CAR was tested with antibodies directed against NGFR coupled to PE (BD Pharmingen, clone C40-1457) and myc-tag coupled to APC (Cell Signalling, ref 9402) and read on BD FACScanto I. 36  2.1.12 HEK 293T binding assay HEK 293T cells were transfected and incubated as described above. After 48h, cells were detached and incubated with FITC-labelled FliC or BSA in the dark on ice. After 1h, anti-NGFR-PE was added and incubation was continued for another 30 min. Cells were analyzed by flow cytometry and binding was assessed by analyzing FITC positive cells within the NGFR positive (CAR+) and NGFR negative (CAR-) fraction. Mean fluorescence intensity (MFI) of CAR+FITC+ was divided by MFI of CAR-FITC+ and analyzed with Graphpad Prism 5.01.  2.1.13 Virus production For virus production, 1.15M Platinum-E (Plat-E) cells (Morita 2000) were plated in 10ml Plat-E maintenance media (DMEM, 10% FBS, 1% L-glutamine, 10mM HEPES, 10ug/ml blasticidin, 1ug/ml puromycin) in a T75 flask three days prior to transfection, to achieve 70% confluence while transfection. Three hours prior to transfection, media was replaced with 10ml Plat-E transfection media (DMEM, 10% FBS, 1% L-glutamine, 1% HEPES). Plat-E cells were transfected with CAR-encoding plasmid alongside viral envelope-protein encoding plasmid (kind gift Dr. Jonathan Bramson) via jetPRIME following the manufacturer’s protocol. At 24h after transfection, media was replaced with 10ml fresh transfection media. Virus was harvested at 48h and again at 72h by filtering all media through a 45µm syringe filter unit (Pall, ref 4614) and concentrating it 10x via Amicon Ultra 100K centrifugal filter (Millipore, ref UFC910024) at 3000rpm. At 48h, 10ml fresh transfection media was added to the Plat-E cells for 72h virus production.  37  2.1.14 Isolation of CD4+ T cells Male and female six to eight-week-old C57/Bl6JxFoxp3-eGFPxThy1.1 or –Thy1.2 mice were euthanized according to animal ethics guidelines. Inguinal, axillary, brachial, superficial cervical and mesenteric lymph nodes and spleen were harvested and dispensed by straining through a 100µm (Falcon, ref 352360) and subsequently a 40µm cell strainer (Falcon, 352340) using the thumb pad of a 1ml syringe (BD, ref 309659).   CD4+ cells were then isolated by negative selection (EasySep™ Mouse CD4+ T Cell Isolation Kit, Stemcell Technologies, ref 19852) and either magnetically sorted as CD4+CD25- Th cells and CD4+CD25+ Treg by labeling with CD25-PE and anti-PE magnetic beads using a magnetic column according to the manufacturers instruction (Milteny Biotec), or stained with eF450-conjugated antibody directed against CD4 (eBio, clone RM4-5) for FACS sorting. Following straining through a 30µm cell strainer (Milteny pre-separation filter, ref 130-041-407), Tregs and Tconv were purified by FACS sorting as CD4+Foxp3+ and CD4+Foxp3-, respectively. To separate Tconv, Tregs, and Tr1, negatively isolated CD4+ cells (see above) were stained using CD4-efluor450, CD44-APC and purified by FACS sorting as CD4+Foxp3-CD44int/lo Tconv, CD4+Foxp3+CD44+ Treg, and CD4+Foxp3-CD44hi Tr1 to >95% purity (Figure 2-1).  38    Figure 2-1 Sorting strategy and post-sorting purity  2.1.15 Isolation of bone marrow-derived antigen presenting cells Bone marrow-derived antigen presenting cells (BMDAPC) were derived on D0 of the experiment from the same mice that were used as T cell donors. Femurs were cleared from surrounding tissue and flushed with MODC medium (RPMI, 10% FCS, 1% GlutaMax, 10mM HEPES, 50µM 2-ME 1% Pen/Strep). Cells were filtered through 100µm cell strainer, washed, and grown in 40ml MODC per bone in the presence of 50 ng/ml recombinant GM-CSF (eBioscience, ref 34-8311-82). Media was replaced after 2 and 5 days, pelleting and keeping the detached cells. GM-CSF was added back assuming that all had been spent. BMDAPC were 39  pulsed over night with 1µg/ml OVA 257-264 (Invivogen, Vac-Sin) in the presence of 100 ng/ml LPS. After incubation, BMDAPCs were washed with PBS.   2.1.16 T cell stimulation  FACS sorted CD4+Foxp3-CD44int/lo Tconv, CD4+Foxp3+CD44+ Treg, and CD4+Foxp3-CD44hi Tr1 were diluted to 1x106/ml and stimulated using immobilized anti-CD3 (10μg/ml), soluble anti-CD28 (2μg/ml), and rhIL-2 (200 unit/ml). After 3 days, cells were carefully resuspended and re-diluted to at 1x106/ml in new media with replenished cytokines. Supernatants used for experiments were harvested on either day 4 or 5.  2.1.17 Coculture of T cells and bone marrow-derived antigen presenting cells and inflammasome assay Activated Tregs, Tr1 or Tconv cells were cocultured with BMDAPC for 30 minutes or supernatant isolated from these cultures (=conditioned media) (see 2.1.16) was added to BMDAPC for 16 hours. Coculture ratios and supernatant concentration is indicated in the figure legend. Where relevant, 10μg/ml of IL-10 blocking antibody (JES5-2A5, eBioscience) or isotype control (IgG1κ, eBRG1, eBioscience) was added 30 minutes prior to the coculture. To activate the NLRP3 inflammasome, BMDAPCs were stimulated with LPS (10ng/ml) for 5 hours, and ATP (5mM) for 1 hour after coculture. To analyze inflammasome activity, BMDAPCs were lysed for analysis of protein expression or mRNA and supernatants were collected for analysis by ELISA (see 2.1.3).   40  2.1.18 T cell bead expansion, CAR transfection and phenotyping Purified Tregs and Tconv were seeded at 1M/ml and expanded either in Tang expansion media (DMEM 4.5g/l glucose, 10% FCS, 10mM HEPES, 2mM Glutamax, 1% NEAA, 1% Na-Pyruvate, 10µg/ml Ciprofloxacin, 50µM 2-ME) or Vent-Schmidt expansion media (RPMI, 10% FCS, 10mM HEPES, 2mM Glutamax, 1% Na-Pyruvate, 1x Pen/Strep, 50µM 2-ME) in the presence of either 1000U, or 100U IL-2 for Treg or Tconv, respectively, using a modification of the Tang and Bluestone et al. method115. For the purpose of this study, IL-2 used in media refers to recombinant human IL-2.   After 48h and 72h activation, 600µl media-supernatant was taken off. To transfect the cells, 100µl concentrated viral supernatant, or 100µl Treg expansion media for untransfected condition was added alongside 2µg/ml Lipofectamine 2000 and 1.6µg/ml Polybrene and cells were subjected to spinoculation at 805 RCF and 32⁰C. The transfection method was refined later in the study. Cells were transduced only one time at 48h and supernatant of 5D4 transduced Plat-E cells was diluted 1/50 to adjust CAR expression between Her2-CAR and 5D4-CAR. After spinoculation, cells were incubated for 2-4h at 37⁰C in 5% CO2 followed by addition of 500µl media. Cells were counted every day starting on D4 (96h) and adjusted to 1M/ml or 0.5M/ml over the weekend.   Phenotype and CAR expression was verified by flow cytometry on D7. Cells were stained with fixable viability dye eFluor-780 (FVD) (eBioscience, ref. 65-0865-14) to differentiate live from dead cells. Treg phenotype was assessed via Foxp3-GFP and CAR expression was determined by co-expression of ∆NGFR and c-myc with the antibodies described above. 41  2.1.19 Proliferation assay Tregs were expanded for 4 to 7 days after transduction with CAR. Cells were washed twice in PBS to eliminate residual IL-2 and Treg cytokines in the media and labelled in eFluor 680 cell proliferation dye (CPD)(eBioscience 65-0840-85) according to the manufacturer’s protocol. Tregs were then subjected to restimulation by either plate-bound α-CD3 plus soluble α-CD28, by 10µg/ml recombinant FliC, or PBS control, all in the presence of 100U IL-2. After 3 to 4 days, Treg were washed in PBS and stained with FVD and antibodies directed against CD4 and ∆NGFR. Proliferation was assessed via flow cytometry by CPD dilution in the live cell fraction.  2.1.20 Suppression assay Tregs were expanded for 4 days after transduction with the CAR and washed twice with PBS prior to setting up the experiment. Tconv were isolated from OT-II mice by CD4 negative selection as described above, followed by MACS CD25+ cell depletion by positive isolation (CD25 MicroBead Kit mouse, Milteny Biotec ref. 130-091-072) according to the manufacturer’s protocol. The flow-through CD25- fraction was used as Tconv and labeled in CPD as described above.   For the suppression assay, 50k Tconv were co-cultured with Treg at a 1:1, 1:2, 1:4, 1:8 and 1:16 ratio and at 1:0 as control. To assess antigen-specific suppression, this co-culture was incubated in the presence of 10µg/ml plate-bound FliC and 200k OVA-pulsed BMDAPC. This would stimulate Tconv through their TCR and Treg only if they express the FliC-CAR. As control, the Tconv-Treg co-culture was incubated with plate-bound α-CD3 plus soluble α-CD28 which activates all T cells present in the culture. After incubating for 3 to 4 days, cells were stained 42  with FVD and -CD4 and proliferation of Tconv was determined via flow cytometry. Analysis was performed with the proliferation program of FlowJo7.  2.1.21 DNBS colitis Male C57Bl/6 mice were placed on a Styrofoam pad and lightly anesthetized by administering 1.5% isoflurane via nose cone such that animals retained blink and swallow reflexes and displayed regular respiration. Tear gel was applied to their eyes to avoid the eyes drying out. For T cell transferred animals: 1M expanded Tregs were washed in PBS and stained with eF450 Proliferation dye according to manufacturer’s protocol, resuspended in 100µl and administered i.p.  To initiate DNBS colitis, remaining stool was removed from distal colon by gently apply pressure with fingertips to the posterior end of the animal. DNBS was dissolved at 6mg/100µl in 50% ethanol and drawn into a 1ml syringe. Then, a 26 G needle with a PE-50 polyethylene catheter fastened to its end was attached to the syringe. To administer the catheter intrarectally, a small amount of ethanol solution was released while inserting the catheter to lubricate and allow for easier insertion. This was performed very carefully and stopped if resistance was felt to avoid perforation of the colon. The catheter was inserted until approximately 3-4 cm proximal to the anus and 100µl of either DNBS solution or EtOH for negative control was slowly injected. After injection, animals were positioned head-down while resting on Styrofoam for 90s to avoid loss of DNBS and placed under a heat lamp to wake up. Animals were carefully observed during and shortly after waking period and daily monitored for weight loss and signs of dehydration, or distress for the duration of the experiment. 43  Animals were euthanized 3-5 days after DNBS administration. For clinical scoring, weight and colon length was measured and colon was macroscopically scored as ulceration (0-10), diarrhea (0 or 1), adhesions (0,1,2), and colonic wall thickness in mm. For histology, ~1.5cm portion of distal colon was collected and cut longitudinally. Formalin fixed tissue was sliced longitudinally and stained with H&E. Slides were microscopically scored as cell infiltrate (0-3), loss of mucosal architecture (0-3), crypt abscess (0 or 1), and goblet cell depletion (0-3). Spleen, MLN, and LPL were collected separately from animals that received Treg adoptive transfer and lymphocytes were analyzed by flow cytometry.  2.1.22 T cell transfer colitis On day 0 of colitis, ex vivo Treg and Tconv were isolated as described above. Expanded and CAR-Tregs were created as described above. To induce colitis in C57/Bl6JxThy1.2xTCR-/- animals, Tconv were injected i.p. either alone or in the presence of equal amount of Treg. Control animals received equal volume of PBS i.p. Animals were fed non-irradiated chow and tap water and were monitored every other day until weight loss was observed. Following initial weight loss, animals were monitored more closely on a daily basis. Endpoint was determined by either 20% weight loss or 8 weeks, whichever was reached first. Clinical severity was determined by weight loss and colon length. Histological score was determined from H&E sections as goblet cell depletion (0-4), hyperplasia (0-3), crypt abscess (0-1), cell infiltration (0-3) and epithelial integrity (0-4) and was scored separately for mid and distal colon. Scoring was performed blinded by at least two independent assessors.  44  2.1.23 Isolation of lamina propria lymphocytes (LPL) Colon was removed from animals and faeces and mucus were cleaned off by scraping in cold PBS, followed by cutting into ~5mm sections. Sections were added to 10ml PBS/BSA, inverted several times and allowed to sediment. Supernatant was discarded and replaced with 10ml 37ᵒC RPMI/EDTA (RPMI, 10% FCS, 5mM EDTA) and incubated for 15 min at 37ᵒC while shaking. Solution was vortexed thoroughly for 10 sec; this step was critical for successful isolation. Supernatant was removed and the RPMI/EDTA procedure repeated once or until supernatant stayed clear. RPMI/HEPES (RPMI, 10% FCS, 15mM HEPES) was added to 15ml and solution was incubated for 10 minutes at RT. Supernatant was discarded and tissue was cut into ~1mm sections while submerged in residual medium.   To digest the tissue, 0.2mg/ml Sigma type VIII collagenase was added to 10 ml RPMI/HEPES and tissue was incubated for 20 min at 37ᵒC while shaking. Solution was vortexed thoroughly for 10 sec and supernatant was filtered through a 20m cell strainer and placed on ice. Digestion was repeated once or twice until supernatant was clear. All digestion-supernatant fractions were combined and centrifuged at 2000 rpm for 10 min. Supernatant was pipetted off and discarded. Cell pellet was resuspended in 3 ml Percoll 30, filtered through a 30m cell strainer and layered over Percoll 75/40 gradient. Percoll gradient was prepared by creating Percoll 100 with 9 volume Percoll plus 1 volume 10x BSA and diluting with sterile PBS/BSA (0.1%). Phenol red (100l) was added to Percoll 40 to improve interphase visibility. Gradient was centrifuged for 20 min at 1800 rpm, 10ᵒC without brakes.   45  To recover LPL at the interphase between Percoll 40 and 75, Percoll 30 and 40 layers were removed by pipetting and the desired interphase collected. Cells were washed twice in cold PBS/BSA (0.1%) at 2000 rpm. In preparation for FACS analysis and to reduce background, cells were incubated with Fc-block in parallel to FVD for 10 min at RT followed by washing for 5 min at 1500 rpm. Cells were subsequently prepared as described above under “flow cytometry”.  2.1.24 Statistical analysis Statistical analysis was performed in GraphPad Prism 5 and 6 using one-way ANOVA followed by Tukey’s multiple comparison tests unless otherwise as indicated. Significant differences were set at p>0.05.  2.2 Survey research chapters 2.2.1 Inflammatory bowel disease survey 2.2.1.1 Survey design The survey was designed to understand the perspectives of persons living with IBD on this hypothetical therapy. In the description of CAR-Treg therapy in the survey, I positioned CAR-Treg therapy as an alternative to injectable biologics. I also developed a “firefighter” analogy to explain the new therapy to survey participants. The remainder of the survey consisted of questions based on the research questions and hypotheses.  I refined the draft survey using patient input and conducted two iterative focus groups using a semi-structured interview approach (Appendix A) to ensure the firefighter analogy and survey questions relayed the intended meaning (Figure 2-2). 46    Figure 2-2 Schematic of survey design Arrows between survey design and focus groups indicate exchange of insights. Arrows from left to right indicate researchers’ voices, arrows from right to left indicate patients’ voices.  In both focus groups, participants were asked to read the firefighter analogy and complete key draft survey questions. Participants then discussed their thoughts on CAR-Treg therapy, the firefighter analogy, and the meaning of the draft survey questions. The draft survey was revised before the second focus group based on feedback from the first focus group. Revisions included rephrasing to eliminate confusion, reframing of jargon, eliminating irrelevant questions, and adding new questions, one of which became the secondary outcome (described below). The final survey was then completed by the focus group participants to identify any remaining concerns. The final survey design was published online using Qualtrics. The complete questionnaire can be found in Appendix B.  47  Both focus groups were facilitated by me, audio recorded and transcribed verbatim. I also collected participants’ written responses to the draft survey questions.  2.2.1.2 Primary outcome measures I informed participants that I see CAR-Treg therapy as an alternative to injectable biologics and employed two primary outcome measures: (1) patients’ willingness to try CAR-Treg as a new treatment after it has been established to work well, and (2) patients’ willingness to try CAR-Treg in a clinical trial testing how well it works.  Patients’ willingness to try CAR-Treg under the two hypothetical conditions was measured using a continuous rating scale anchored at three points using both numbers and phrases: (1) 0 “definitely not”, (2) 50 “somewhat likely”, and (3) 100 “I would have definitely tried it”. This scale was created for this study and pilot-tested by patients in the focus groups without concern. No additional reliability or validity testing was performed on the scale. Survey participants were also provided an open-ended text box to share why they rated their willingness the way they did for the two outcome measures.  2.2.1.3 Secondary outcome measure The secondary outcome measure was participants’ preferences over CAR-Treg fate after initial use. This secondary outcome measure reflects my interest in guidance for further CAR-Treg development—specifically, whether patients would want CAR-Tregs to remain dormant in their bodies, ready to be reactivated in a future flare of IBD, or prefer CAR-Tregs to be eliminated from their bodies after successful induction of remission. Participants were told that the former 48  option included a possible long-term risk for the cells to change. In a follow-up open-ended question, participants were asked to share their most important consideration in choosing a preference over CAR-Treg fate.    2.2.1.4 Participant recruitment I aimed to conduct two focus groups with 5 – 8 participants each to inform the survey design. To recruit participants, I collaborated with the Gastrointestinal Society (GI Society) of Canada and advertised the study at an educational event and via posting on their website. Due to illness and last-minute cancellations, I conducted the first group with 2 participants and the second group with 6.  Survey participants were recruited through two emails, one in English, one in French, to the proprietary mailing list created and owned by Discover Therapies, a Canada-based patient-driven group that aims to connect patients with ongoing clinical trials and focuses on IBD. Additionally, survey participants were recruited through two postings to the Facebook account of Discover Therapies. I expected that there would be some overlap between individuals targeted in the email list and the Facebook group, but had no way to assess overlap between the two recruitment methods or to prevent people from completing the survey more than once. We do not have a list of participants for these reasons. Survey recruitment occurred in October and November 2018 and the online survey was available between October 2018 and April 2019.  49  2.2.1.5 Statistical analysis I analyzed the survey data through descriptive and inferential statistics. The results for the primary outcome measures, willingness to try CAR-Treg therapy, were non-normally distributed. To identify any demographic and disease-related factors that might contribute to the level of willingness, I used the non-parametric Mann-Whitney and Kruskal-Wallis rank-based statistical analyses and performed Dunn’s multiple comparison post-hoc test to identify any difference in rank sums between groups. Due to the non-symmetrical nature of my results, I report these as testing for difference between medians of willingness between groups. I chose to compare medians over dichotomizing the outcome variable in order to represent the variability in responses. I contracted age groups to reduce the number of age categories as well as levels of education to reduce the number of education categories. I conducted Chi-square statistics to identify statistical significance of preference of cell fate compared to null hypothesis that there would not be a preference of cells to stay vs to be eliminated after reducing the flare. I used Prism 5, GraphPad Prism Software, Inc. for these analyses.  To analyze the influence of multiple independent variables on my primary outcome measures (willingness to try CAR-Treg therapy as new treatment or in a clinical trial), I conducted multiple linear regression analysis using SPSS Version 26, IBM. First, I conducted Shapiro-Wilk normality testing which confirmed that the dependent variables were not normally distributed with both outcome variables being negatively skewed with identified ceiling effect. As all independent variables were categorical, and most contained multiple categories, I created dummy variables for categorical independent variables. The regression analysis compared groups to a randomly assigned reference category (male, age 14-39, Crohn’s disease, never took 50  immunomodulators or biologics, no IBD complications, preference of cells to stay after treating the flare, living in a rural community in Canada, highest education of high school or less). As missing values appeared to be at random, I deleted missing values pairwise to avoid reducing overall sample size. I tested for multicollinearity by using a matrix of Pearson’s bivariate correlations. I interpreted the intercept as conditional mean for this group and report the slope of the predictor variables as units of increase or decrease of the conditional mean.   2.2.1.6 Thematic analysis  I analyzed open ended answers to survey questions by inductive thematic analysis. Codes were created as new concepts emerged and later summarized and assigned to two themes. Multiple codes were possible per answer and I selected exemplary quotes for inclusion in this manuscript.  2.2.1.7 Ethical considerations I pursued this study under ethics approval from the UBC Clinical Research Ethics Board (UBC CREB Number H17-01720).  2.2.2 Clostridioides difficile survey 2.2.2.1 Study design and recruitment The GI (Gastrointestinal) Society developed a mixed-method (quantitative and qualitative) survey with guidance from their medical advisory board as part of their ongoing quality control. Participants were recruited via the English (www.badgut.org) and French (www.mauxdeventre.org) websites as well as Facebook (www.facebook.com/GISociety/) and Twitter (@GISociety) accounts of the GI Society. Additionally, the GI Society collaborated with 51  five internal medicine physicians across Canada who shared the survey link with their patients. To qualify, participants had to self-identify as either an individual who had experienced CDI or a non-physician caregiver of someone with CDI.    52  2.2.2.2 Questionnaire and data analysis The questionnaire was divided into two portions, non-identifiable personal information (six questions total) and questions on C. difficile (18 questions total). Twenty-three questions were multiple-choice and the final two questions were open-ended and focused on the most important priority regarding improved patient experience, as well as an opportunity to share additional comments. The complete questionnaire can be found in Appendix C.  I included a total of 167 qualifying responses: 119 patient responses and 48 caregiver responses. Caregivers were given the instruction to “answer as the patient, or on behalf of the patient for whom you are responding”. Thus, caregivers might have responded in lieu of a patient who was too sick to respond themselves, or by imagining the patient perspective. I analyzed each question from the entire dataset, as well as stratified by patient and caregiver responder. Data are presented from the entire dataset unless otherwise specified.   2.2.2.2.1 Statistical analysis Quantitative parameters were analyzed through descriptive and comparative statistics using GraphPad Prism 5. Quality of life questions yielded non-normal results and were analyzed by Friedman test and Dunn’s multiple comparison post-hoc. These data are presented stratified by patient and caregiver.    53  2.2.2.2.2 Thematic analysis Open ended questions were analyzed through inductive thematic analysis. I created the initial codebook and my co-author Daniel Lisko and I applied the codebook to all responses. Following independent analysis, we collaboratively refined the codebook and added new codes where applicable. I then re-analyzed all data using the final codebook, which is provided in Appendix D. Percentages of responses are calculated by the total number (104) of respondents who answered at least one open-ended question.  2.2.2.3 Ethics approval The original survey was hosted through the GI Society as quality control for their ongoing programming. Survey respondents were provided with a cover letter to the survey that stated reasons for conducting the survey, eligibility criteria, and information on how the results will be used. Consent to participate was provided by selecting one of the two eligibility criteria. All participants were 19 years of age and older. The GI Society was not required to obtain ethics approval for this quality control study and did not explicitly ask participants to provide informed consent. Prior to sharing the anonymous data for scientific analysis, I obtained ethics approval for secondary use of data by the UBC Clinical Research Ethics Board (UBC CREB Number H17-00747) to ensure it was ethical to analyze and publish this data. 54  Chapter 3: Tr1 Cells, but Not Foxp3 + Regulatory T Cells, Suppress NLRP3 Inflammasome Activation via an IL-10−Dependent Mechanism 3.1 Introduction Tregs and Tr1 cells regulate adaptive and innate immune responses through various mechanisms and their role in controlling adaptive immune responses has been characterized comprehensively. In terms of controlling innate immune responses, it was known that both cell types down-regulate APC maturation and antigen presentation through CTLA-4 and/or LAG-3 dependent inhibition of CD80/86 expression23,25. Furthermore, Tr1 cells and Tregs kill myeloid APCs using CD226 and granzyme B, and granzyme A, B and perforin, respectively25,116–118. Given the role of both the inflammasome and Tr1 and Treg cells in chronic inflammatory diseases, we hypothesized that Foxp3+ Tregs and/or Tr1 cells have a role in modulating inflammasome activity.  3.2 Results 3.2.1 Functional Tr1 cells can be derived from CD44hiFoxp3- T cells Prior to our publication of this chapter, there had been no published efficient method to isolate and/or differentiate Tr1 cells, which represented a barrier to investigating their biology. Previously, Tr1 cells were differentiated from naïve Th cells in vitro through TCR stimulation, either in the presence of cytokines such as IL-10, IL-27, IL-6, or IL-12, by co-stimulation of CD46 or ICOS-L, or through co-culture with tolerogenic DCs23,119–122. A common limitation to these methods were the production of only small (i.e. <30%) proportions of IL-10-producing cells which were not pure Tr1 cells as they co-produced high levels of IFN-γ. A recent study 55  identified that exposure of effector memory T cells to the ligand for CD44 can also induce Tr1 cells identified by IL-10 production123. Based on that finding, we hypothesized that CD44 expression could identify cells as Tr1 precursor cells.   To investigate whether CD44hi cells could differentiate into Tr1 cells, we sorted CD44hiFoxp3- T cells (Tr1), CD44int/loFoxp3- naïve T cells (Tn), and Tregs from spleens and lymph nodes of foxp3RFPx il10GFP reporter mice. We then activated these cells using anti-CD3/28 mAbs in the presence of IL-2 and analyzed fold expansion and expression of IL-10 and Foxp3 after four days. The fold expansion of Tr1 cells was comparable to Tregs, and significantly less than Tn cells (Figure 3-1A). Over 80% of cells in the putative Tr1 cell population were IL-10+ and only cultures of Tregs expressed Foxp3. We did not detect a significant proportion of IL-10+ cells in the Treg and Tn cell populations (Figure 3-1B). IL-10+ cells emerged from the Tr1 population after one day of stimulation and their proportion increased over time (Figure 3-1C). We measured high levels of IL-10 in supernatants of Tr1 cultures after 4 days of stimulation and confirmed the fidelity of the il10GFP reporter (Figure 3-1D).  56     57  Figure 3-1 Activated CD44hiFoxp3- T cells develop into Tr1 cells (A) CD4+Foxp3+ Tregs (Tregs), CD4+CD44int/loFoxp3- naïve conventional T cells (Tn), or CD4+CD44hiFoxp3- Tr1 cells (Tr1) were FACS-sorted from Foxp3RFPx IL-10GFP reporter mice, representative FACS plot. (B,C) Sorted Tregs, Tn, or Tr1 were stimulated with anti-CD3/28 mAbs and IL-2 for 4 days. (B) Fold expansion of cells after 4 days of stimulation (n=6, *p<0.05, **p<0.01). (C) Representative histograms show Foxp3 and IL-10 expression in live CD4+ lymphocytes (n=3). (D) Representative histograms of daily expression of IL-10 in Tr1 cells; n=3. (E) IL-10 in T cell supernatants measured by ELISA (mean ± SEM, n=7, ** p<0.001). (F) IL-17 and IFN-γ expression in Tregs, Tn, Tr1, or Th1 cells (derived from Tn cells by culturing in the presence of IL-12). Representative plots of n=5. (G,H) CD44int/loFoxp3‾ naïve T conventional cells were FACS sorted from Foxp3rfpIL10gfp mice followed by stimulation with anti-CD3/28 ± 50ng/ml of IL-27 for 4 days. Representative FACS plots show expression of (G) IL-10 and (H) IFNγ and IL-17 in FVD‾CD4+ cells; n=3 (I) T cell subsets cultured for 4 days as described above, washed and cultured in media alone (no addition of anti-CD3/28 and IL-2) for 24 hours. Amounts of secreted cytokines were analyzed by cytokine bead array (mean ± SEM, n=5, * p<0.05, ** p<0.01, **** p<0.0001, two way ANOVA followed by Tukey’s Test comparing different T cell expression of each cytokine).   We characterized the overall cytokine profile to confirm that our method indeed generated Tr1 cells. Tr1 cells were reported to produce some IFN-, albeit less than Th1 cells23 and our activated Tr1 cells produced significantly less IFN- than in vitro polarized Th1 cells (Figure 3-1E) or Tr1 cells differentiated from Tn cells using IL-27 (Figure 3-1F&G). Levels of IL-2, IL-4, IL-17, TNF-α, and IL-6 were similar to those secreted by Tregs or Tn cells (Figure 3-1H). To confirm the suppressive capacity of CD44hiFoxp3- derived Tr1 cells, we conducted suppression assays with CD4+ T cells. Both, Tr1 cells and Tregs suppressed T effector (Teff) cells, however Tregs did so more potently (Figure 3-2).  58    Figure 3-2 CD44hiFoxp3- T cells derived Tr1 cells suppress proliferation of T effector cells Treg and Tr1 cell suppressive capacity was measured by analyzing proliferation of CD4+CD25- T cells cultured with or without Tregs or Tr1 cells at decreasing ratios. Depicted is a representative plot and the average percent suppression as calculated using division indices (mean ±SEM, n=3, *p<0.05).   3.2.2 CD44hiFoxp3- T cell-derived Tr1 cells express characteristic Tr1 cell surface markers and transcription factors To further confirm the Tr1 phenotype of Tr1 cells derived from CD44hiFoxp3- T cells, we tested for Tr1 cell-associated surface markers and transcription factors124. A significantly higher proportion of Tr1 cells compared to Tn cells or Tregs expressed LAG-3 and/or CD49b (Figure 3-3A). Furthermore, CD44hiFoxp3--derived Tr1 cells had higher expression of additional Tr1-associated markers including CD39, and TIM-3 (Figure 3-3B) compared to Tn cells23. Tregs expressed significantly higher levels of LAP, CD73, or Helios than either Tr1 or Tn cells (Figure 3-3B).  Finally, we analyzed expression of Tr1-associated transcription factors23,120,125,126 both ex vivo and after 4 days of activation. Compared to Treg and Tn cells, CD44hiFoxp3--derived Tr1 cells showed significantly higher expression of maf both ex vivo (Figure 3-3C) and after activation 59  (Figure 3-3D). Compared to Tn cells, the gene encoding for Blimp-1, prdm1, was significantly higher expressed after activation (Figure 3-3D) while expression of ahr was significantly higher ex vivo (Figure 3-3C). Collectively, our data demonstrate that a population of CD44hiFoxp3- cells is poised to up-regulate IL-10 and differentiate into Tr1 cells without the need to be exposed to additional polarizing cytokines. This finding translates into a new and efficient method to generate Tr1 cells for further investigation of their biology.     60  Figure 3-3 CD44hiFoxp3- T cells derived Tr1 cells express characteristic transcription factors and surface markers Expression of (A) CD49b, LAG-3, and (B) other Treg-associated molecules was determined in T cell subsets sorted from Foxp3GFP reporter mice after 4 days of activation; gated on FVD-CD4+ cells. (A) Left: representative contour plots and average percentage of CD49b+LAG-3+ T cells; right: average MFI from 3-4 independent experiments (mean ± SEM, *** p<0.001, **** p<0.0001). (B) Top: representative histogram; bottom: average MFI from 3-4 independent experiments (mean ± SEM, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001) (C) mRNA expression of the indicated genes, expressed as fold increase to Tn, was analyzed by qPCR immediately after sorting. (mean ± SEM, n=3, *p<0.05, ** p<0.01 (D) mRNA expression of the indicated genes, expressed as fold increase to Tn cells, was analyzed by qPCR after 4 days of activation. (mean ± SEM, n=4-6, * p<0.05, ** p<0.01, ***p<0.001)  3.2.3 Tr1 cells but not Tregs suppress caspase-1-dependent IL-1 secretion in macrophages Once we established the new method of isolating Tr1 cells, we were interested to answer our initial hypothesis that Tregs and/or Tr1 cells regulate the inflammasome. To address this, we co-cultured T cells and macrophages overnight and activated the inflammasome by stimulation with LPS for 4 hours followed by ATP for 1 hour. Co-culturing macrophages with T cells did not affect macrophage viability (Figure 3-4A). We found that Tr1 cells significantly suppressed the secretion of mature IL-1β (Figure 3-4B&C) in a dose dependent manner (Figure 3-4D). Treg did not affect IL-1β secretion, while co-culture with Tn cells increased IL-1β secretion. This increase was not observable after stimulation with either LPS or ATP alone, which indicates that Tn cells do not express factors that could directly stimulate the inflammasome (Figure 3-4B). The observed IL-1β secretion was caspase-1-dependent and completely abrogated by caspase-1-inhibitor YVAD-FMK (Figure 3-4B).  61      62  Figure 3-4 Tr1 cells efficiently suppress inflammasome activation Indicated cell types were stimulated with anti-CD3/28 for 5 days in the presence of YVAD where indicated, co-cultured with BMDM at a ratio of 5:1 (200,000 T cells : 40,000 macrophages) for 30 minutes followed by stimulation with LPS for 4 hours and ATP for 1 hour. (A) Adherent cells were collected and viability was determined by fixable viability dye (FVD) using flow cytometry. Percentage indicates viable cells, representative plot of n=2. (B, C) IL-1β in supernatants was measured by ELISA. (B) Representative data from one experiment (mean ± SD). (C) Average percent suppression of IL-1β secretion, normalized to media (mean ± SEM, n=6, * p<0.05 *** p<0.001). (D) Coculture of BMDM with decreasing ratios of Tr1 or Tn cells. IL-1β in supernatants was measured by ELISA (mean percent suppression ± SEM, n=3, multiple t-test with Sidak-Bonferroni multiple comparisons correction).  3.2.4 Tr1 cell-mediated suppression of inflammasome activation is dependent on binding of secreted IL-10 to macrophage IL-10 receptor As Tr1 cells suppress their target cells through secretion of inhibitory cytokines, we tested the effect of T cell-conditioned media on IL-1 secretion. Indeed, Tr1 cell-conditioned media potently suppressed IL-1β (Figure 3-5A&B) in a dose-dependent manner (Figure 3-5C), while Treg-conditioned media had little effect and Tn cell-conditioned media enhanced IL-1β (Figure 3-5A&B). Taken together, we observed that Tr1 but not Treg potently suppressed caspase-1-dependent secretion of IL-1β independently of cell contact.  63    Figure 3-5 Inflammasome suppression by Tr1 cells depends on soluble factors (A,B) BMDM were incubated with or rIL-10 (100ng/ml) or conditioned media from stimulated Treg, Tr1 or Tn cells at a 1:1 ratio to non-conditioned media and stimulated as indicated. (S=supernatant) (A) Representative data and (B) average percent suppression (mean ± SEM, n=8, * p<0.05, ****p<0.0001). (C) BMDM were cultured and stimulated as indicated above with decreasing ratios of conditioned media (mean percent suppression ± SEM n=4, *p<0.05, multiple t-test with Sidak-Bonferroni multiple comparisons correction) (D) BMDM were cultured with decreasing amounts of rIL-10 and stimulated as above (mean percent suppression ± SEM, n=3). (E) Indicated cell types were stimulated with anti-CD3/28 for 4 days in the presence of IL-2 and IL-10 in supernatants was measured by ELISA (n=3, **p<0.01). 64  Since Tr1 cells are known to release large amounts of the anti-inflammatory cytokine IL-10 (Figure 1-1H&Figure 3-5E), we tested the effect of recombinant IL-10 (rIL-10) on the inflammasome and observed a similar, dose-dependent suppressive effect (Figure 3-5B&D). This suggested that Tr1 cells might mediate their suppressive effect via release of IL-10. However, while Tregs released ~26 ng/ml IL-10 (Figure 3-5F), which should result in ~80% suppression of the inflammasome (Figure 3-5E), we did not observe Treg conditioned media-mediated IL-1β inhibition.   To confirm whether Tr1 cell-mediated suppression of the inflammasome was dependent on the release of IL-10, we repeated the inflammasome assays in macrophages derived from il10rb-deficient mice. Compared to WT macrophages, IL-10R-/- macrophages were resistant to Tr1 cell- (Figure 3-6A&B) and Tr1 conditioned media-mediated inflammasome suppression (Figure 3-6C&D). Furthermore, addition of neutralizing IL-10 antibodies to Tr1-macrophage co-cultures reversed the suppressive effect of Tr1 cells (Figure 3-6E&F). Taken together, we observed that Tr1 cells, but not Tregs, potently suppressed inflammasome activation and IL-1 secretion in an IL-10R-dependent mechanism.    65     66  Figure 3-6 IL-10 secreted by Tr1 cells mediates inflammasome suppression BMDM from il10rb-/- or WT or mice were co-cultured with (A, B) T cells or (C, D) T cell conditioned media (S=supernatant) and stimulated as indicated, IL-1 was measured by ELISA. (A, C) Representative data (mean ± SD) and (B, D) averaged data normalized to BMDM alone (mean ± SEM, *p<0.05, multiple t-test with Sidak-Bonferroni multiple comparisons correction). (E,F) Indicated cell types were cocultured with BMDM in media alone, or in the presence of anti-IL-10 blocking antibody, or isotype control. (E) Representative experiment and (F) averaged IL-1β fold increase normalized to media control (mean ± SEM, n=3, *p<0.05).  3.2.5 Tr1 cells suppress Il1b transcription and caspase-1 activation via an IL-10R-dependent mechanism We then investigated the mechanism by which Tr1 cells inhibit secretion of IL-1β. Inflammasome activation and IL-1β secretion are tightly regulated and depend on two independent signals56,57. TLR (for example via LPS) or cytokine-receptor mediated activation of NF-B results in transcription and translation of pro-IL-1β. Signal 2 (for example ATP) then leads to inflammasome assembly and activation which in turn cleaves pro-caspase-1 resulting in active caspase-1. Active caspase-1 in turn cleaves pro-IL-1β to produce active IL-1β. To elucidate the mechanism of Tr1 cell-mediated inflammasome suppression, we tested for relevant mRNA and protein signals. For these tests, we used T cell conditioned media instead of co-cultures to ensure that all mRNA and protein were macrophage derived.  We confirmed that Tr1-conditioned media, as well as rIL-10, inhibited transcription of il1b mRNA in macrophages (Figure 3-7A). This translated into reduced pro-IL-1β protein expression as demonstrated by Western blotting (Figure 3-7B). It is worth noting that neither Tr1-conditioned media, nor rIL-10, completely inhibited the transcription and translation of 67  pro-IL-1β as would be expected based on the observed reduction of IL-1β secretion. To elucidate additional effects of Tr1 cells on the inflammasome, we tested for caspase-1 activation. Indeed, we found Tr1-conditioned media to inhibit the generation of mature caspase-1 (Figure 3-7C). Furthermore, we observed that the activation and cleavage of caspase-1, and thus the activation of the inflammasome, was inhibited, as we did not observe an effect of Tr1-conditioned media on the expression of pro-caspase 1 (Figure 3-7C). In line with our findings on the necessity of IL-10R on Tr1-cell mediated secretion of IL-1β, we observed that Tr1-conditioned media was unable to suppress the transcription of il1b mRNA (Figure 3-7D) and cleavage of caspase-1 (Figure 3-7E) in macrophages derived from il10rb-deficient mice. In summary, Tr1 cells suppress pro- IL-1β production and inflammasome activation in an IL-10 dependent manner.  68    Figure 3-7 Tr1 cells inhibit il1b transcription and caspase-1 activation in an IL-10 receptor dependent mechanism BMDM from WT or il10rb-/- mice cultured in the presence or absence of conditioned media (as indicated) or with rIL-10 (100ng/ml). (A) il1b mRNA in WT BMDM and (D) il10rb-/- BMDM, represented as fold increase normalized to stimulated BMDM alone (mean ± SEM, n=3, * p<0.05). WT or il10rb-/- BMDM were lysed protein levels were analyzed by Western blotting. Representative blots (n=3) for (B) pro-IL-1β (p31 as indicated by arrow), (C,E) pro-caspase 1 (short exposure), pro- and mature caspase 1 (long exposure), and β-actin (loading control).  69  3.3 Discussion Recent research as focused on developing and testing Treg- and Tr1 cell-based therapy for a variety of immune mediated disease including inflammatory bowel disease and transplant rejection and first clinical trials have been conducted78,127. The hope is that cell-based therapies will provide more specific and longer-lasting treatment or even a cure. Inflammasome activation contributes to the uncontrolled inflammation in these diseases and it was not known whether Treg or Tr1 cells can regulate the inflammasome. In this Chapter, I provided evidence that Tr1 cells but not Tregs can regulate inflammasome activity through inhibiting transcription of il1b mRNA and activation of caspase-1. The complete findings of this project are published in Yao et al. (2015)55. Our findings indicate that Tr1 cells might have unique therapeutic potential not shared by Tregs and development of cell-based therapies should take both cell types into consideration.  Furthermore, we developed a new method to efficiently generate large populations of Tr1 cells. The lack of such method has been a barrier to studying Tr1 biology. We discovered that isolated CD4+CD44hiFoxp3- T cells from unmanipulated mice contain a population of cells which is poised to differentiate into Tr1 cells. In vitro activated CD4+CD44hiFoxp3- T cells express specific characteristics of Tr1 cells including expression of a Tr1-associated cytokine profile (IL-10hiIFN-loIL-2-IL-4-) and cell surface markers including CD49b, LAG-323. While no Tr1-specific transcription factors have been described, CD4+CD44hiFoxp3- T cell-derived Tr1 cells expressed elevated levels of cMaf, Blimp1 and Ahr, transcription factors associated with IL-10 production23 and over 80% of these cells were positive for IL-10. Furthermore, expression of CD39, ICOS, CTLA-4, and PD-1 was significantly higher compared to naïve T cells and 70  remained elevated after 4 days of activation when activation-induced expression of some of these markers would be diminished in naïve T cells.   CD44 was described as a marker for T cell memory, however, CD44+ cells isolated from laboratory-raised mice show distinct characteristics including rapid antigen-independent proliferation compared to antigen-experienced CD44+ memory T cells. Their development is independent of commensal bacterial antigens as they can be isolated in similar frequencies from germ-free mice and their function remained unkown128. We suggest that these CD4+CD44hiFoxp3- cells are poised to mount an immunoregulatory response with a distinct cytokine expression pattern. In contrast, antigen-experienced memory T cells would mount immunostimulatory responses.   Tr1 cells are known to rely heavily on IL-10 to mediate their suppressive function and we found IL-10R signaling to be crucial for the Tr1 mediated inhibition of inflammasome activation. Tr1 cells were unable to inhibit the inflammasome in macrophages isolated from il10rb-deficient mice. Previous studies reported inconsistent results which may have resulted from different times of delivery and degradation of IL-10. While some groups reported IL-10 mediated inhibition of il1b in human monocytes, murine peritoneal derived macrophages and macrophage cell lines129–132, others were unable to repeat these findings133–135. While we found recombinant IL-10 to be just as potent as Tr1 cells in suppressing IL-1 in vitro, we suggest that Tr1 cells in vivo would be more effective than recombinant IL-10 as they could continuously deliver IL-10 in a site-directed manner.   71  Our findings expand on recent studies in IL-10 knockout mice that developed severe colitis or antigen-induced arthritis133,136. While the authors postulate these phenotypes to be caused by  increased inflammasome activation and IL-1 production, they could not rule out an effect of increased production of pro-inflammatory cytokines. Our findings add to these studies in that we demonstrate an IL-10 driven regulation of inflammasome priming and caspase-1 activation. Interestingly, an earlier study investigating memory T cells, isolated as CD4+CD44hiCD25-, found these cells to inhibit the inflammasome through a contact-dependent, but not soluble-mediator driven mechanism. Indirect evidence suggested a putative role for TNF family ligands137. While that group employed a similar cell sorting strategy as us, they employed a different cell stimulation method. Taken together, these findings suggest that different cells derived from a similar starting population employ different mechanisms that could be used in a cell-based therapeutic approach to reduce innate inflammation.   In our system, Tregs were unable to suppress the inflammasome even though they produced more IL-10 than the minimal dose of recombinant IL-10 needed. Tregs employ different methods to suppress their target cells, including cell-contact and cytokine mediated suppression, and while some studies found IL-10 to be critical, others found it to be not necessary for Treg mediated suppression in disease models and in vitro assays25. Studies in mice with Treg-specific il10 deletion highlighted the essential role of IL-10 in establishing tolerance at mucosal surfaces. On the other hand, these studies did not find an essential role of IL-10 in settings of autoimmunity138. One possible explanation for the failure of Tregs to inhibit the inflammasome in our system stems from findings that adenosine perpetuates inflammasome activation139. Murine Tregs express high levels of the surface-bound nucleotidases CD39 and CD73 which 72  hydrolyze ATP to adenosine. As we activated the inflammasome with LPS and ATP, it is conceivable that the adenosine produced by Treg CD39 and CD73 could counteract the IL-10 driven inflammasome inhibition. Further research is needed to explore whether Treg may inhibit the inflammasome in other experimental settings or not.  73  Chapter 4: Developing Flagellin-Specific Chimeric Antigen Receptor Regulatory T Cell Therapy 4.1 Introduction Tregs and Tr1 cells have long been considered candidates to develop a cell-therapy for IBD and other immune-driven diseases (see 1.4). Major hurdles to overcome include the need for high numbers of antigen specific Tregs combined with their scarcity82. While isolation of antigen-specific Tregs would be challenging due to their very small numbers and the need to know their TCR specificity, antigen-specific Tregs can be generated using CARs (see 1.4.1). Efficient protocols for ex vivo expansion of polyclonal human Tregs under good manufacturing practice conditions have recently been established140. However, while pre-clinical research tends to be conducted in mouse models, efficient protocol for expansion of polyclonal murine Tregs are lacking and repeated stimulation in vitro is associated with loss of Foxp3 expression and suppressive capacity141.   In this chapter, I describe the development of a murine CAR directed against the E. coli H18 FliC flagellin (see 1.3.4). I also describe a new method for effectively expanding and in vitro transducing ex vivo murine Tregs as outlined in paragraph 2.1.18. A further refinement of my method has recently been published as Wu et al. (2020)110.  74  4.2 Results 4.2.1 Development of a FliC-specific CAR and detection of flagellin in mouse colons To conduct preclinical testing and assess the therapeutic potential of FliC-specific CAR-Tregs, I created mouse FliC- and Her2-CARs starting from a human FliC-CAR that was developed from a FliC-specific hybridoma (5D4) available in our lab. The CARs I developed included two selectable markers, NGFR and myc (Figure 4-1A). For the experiments described in this chapter, I used Tregs transduced with three different retroviral constructs; FliC-CAR – the CAR under investigation; Her2-CAR – a control CAR directed against an irrelevant antigen (the human receptor tyrosine-protein kinase erbB-2); and NGFR – a no CAR control expressing only NGFR (Figure 4-1B). Prior to testing the FliC-CAR, I assessed the specificity of the FliC antibody to FliC flagellin by bead-binding assay. I incubated sulfate microspheres with either recombinant FliC flagellin, recombinant A4-Fla2 flagellin (from Lachnospiraceae A4) or BSA control, added supernatant from cultures of the 5D4 hybridoma and tested binding via secondary antibody and readout using flow cytometry. The FliC antibody used to create the FliC-CAR was highly specific for only FliC flagellin and did not detect control A4-Fla2 flagellin (Figure 4-1C).   75    Figure 4-1 Generation of a FliC-specific CAR (A) Schematic of the domains in the FliC-CAR (scFV = single chain variable fragment, heavy = heavy chain, light = light chain, gly = glycine-serine linker, myc = myc-tag, TM = CD-28 derived transmembrane domain, signal 2 = CD28 costimulatory domain, signal 1 = CD3 stimulatory domain). (B) Schematic of the viral constructs encoding for the FliC and Her2-CAR and the NGFR selectable marker, separated by an internal ribosomal entry site (IRES), and the vector alone control (NGFR). (C) Anti-FliC-antibody specificity was determined by binding to BSA, FliC or A4-Fla2 coated fluosphere sulphate beads and detection with goat anti-mouse-APC via flow cytometry. Data are representative of 4 independent experiments.  In order for FliC CAR-Tregs to be functional, they would need exposure to the FliC antigen in vivo. To determine feasibility of this, I assessed the location of flagellin in colons from mouse models for intestinal inflammation by immunostaining with the 5D4 antibody used to create the FliC-CAR (Figure 4-1D). Two models, chemically induced colitis with flagellin enemas and mice colonized with enteroaggregative E. coli served as positive control as flagellin was expected to be present in high amounts. Importantly, I also detected immunoreactive flagellin 76  near lamina propria cells in colons isolated from spontaneously colitic Muc2-/- mice. This demonstrates that flagellin is able to cross the disrupted intestinal barrier and enter the surrounding tissue – the location where CAR-Tregs are expected to get activated by flagellin. Furthermore, to ensure feasibility of testing FliC-CAR-Tregs in the TCRß-/- T cell transfer colitis model, I confirmed the availability of flagellin in stool pellets using ELISA (Figure 4-2).   77  Figure 4-2 Availability and location of flagellin in mouse colons (A) Colonic sections from indicated models of intestinal inflammation were incubated with biotinylated anti-FliC mAb, visualized with streptavidin fluorochrome (red), and counterstained with DAPI (blue). Adjacent sections were mounted side by side on the same slide, and one section was used as a secondary antibody control (no anti-FliC). Mice treated with oral DSS plus flagellin enemas (left panel), or colonized with E. coli (centre panel) show strong luminal staining for flagellin. Spontaneously colitic Muc2-/- mice (right panel) show staining in the lamina propria, indicative of flagellin translocation across the luminal barrier. (B) FliC concentration in stool samples. Fecal pellets from TCRß-/- mice were resuspended and clarified, supernatant was diluted as indicated (grey bars). Recombinant FliC was diluted as indicated (white bars). FliC presence in stool and standard curve was determined with anti-FliC antibody by ELISA. Results representative of n=3.  4.2.2 FliC-CAR is expressed on the surface of HEK 293T cells and selectively binds to recombinant FliC A major milestone in the development of CARs is the ability to get processed intracellularly and expressed on the cell surface. To determine this, I transiently transfected HEK 293T cells with FliC-CAR, Her2-CAR and NGFR and assessed surface expression by flow cytometry and staining for NGFR and myc tag. Positive staining for NGFR indicates successful transduction but not expression of the CAR, as this marker is separated by an internal ribosomal entry site (IRES) (see Figure 4-1A). Positive staining for myc tag confirms surface expression of the CAR as the myc tag is located on the extracellular side of the CAR adjacent to the transmembrane domain (see Figure 4-1A). All three constructs were successfully transfected and both NGFR and myc tag were expressed at high levels on the surface of HEK 293T cells (Figure 4-3A), indicating that HEK 293T cells are able to express both FliC-CAR and Her2-CAR on the cell surface. 78    Figure 4-3 Expression of FliC-CAR on cell lines and specific binding to cognate antigen (A) HEK 293T cells were transiently transfected with all indicated constructs and surface expression of the CAR was assayed by flow cytometry with antibodies directed against NGFR (CD271) and myc tag. (B) FliC and BSA were coupled to FITC and incubated with 293T cells transiently transfected with indicated constructs. Binding of fluorescent FliC and BSA was determined by flow cytometry after gating on NGFR expression. Representative histograms from 1 of 4 experiments (C) Mean fluorescent intensity pooled from N=4 repetitions (N=2 for NGFR) for the binding assay described in B; * p = 0.0133 derived from an unpaired, two-tailed t-test between BSA and FliC.  While surface expression of a CAR is a promising milestone in CAR development, it does not ensure proper folding of the functional domains, extracellular antigen-binding and intracellular signaling. Furthermore, even properly folded domains might not be functional due to steric 79  inhibition, for example the antigen-binding site might be physically blocked by the myc tag or components of the plasma membrane. To assess antigen binding, I expressed the FliC-CAR as well as control plasmids in HEK 293T cells, incubated these cells with FITC-coupled, bead-bound recombinant FliC and BSA control and tested NGFR positive cells for FITC signal by flow cytometry (Figure 4-3B). Importantly, only FliC-CAR expressing HEK 293T cells were positive for FITC, indicating that this CAR recognizes recombinant FliC which makes it a promising candidate for further development. As a side note, I developed two FliC-CAR from different anti-flagellin hybridomas in parallel and, while both CARs were expressed on the surface of HEK 293T cells, only the 5D4-CAR detected FliC (data not shown). I used this CAR for the remainder of this project.  4.2.3 Primary murine Tregs can be retrovirally transduced and express FliC-CAR After confirming surface expression and antigen binding on cell lines, I had to determine whether primary murine Tregs can express this FliC-CAR and the control Her2-CAR. To do this, I first modified a protocol for expansion of polyclonal Tregs originally developed by Tang et al (2004)115 as described in paragraph 2.1.18. In brief, I harvested inguinal, axillary, brachial, superficial cervical and mesenteric lymph nodes and spleens of either C57/Bl6JxFoxp3-eGFPxThy1.1 or –Thy1.2 mice, isolated CD4+ cells by negative selection and purified Foxp3+ Tregs and Foxp3- Tconv via FACS sorting. I then activated these cells in the presence of 1000U (Tregs) or 100U (Tconv) hIL-2 for 48 hours, followed by spinoculation (on D2 and D3) with or without virus (see paragraph 2.1.13) in the presence of Lipofectamine and Polybrene. I then continued expansion, until D7-10 post-sorting. During the expansion, I counted cells daily and adjusted dilution to 1M/ml. A typical round of expansion would start with 400K (Treg) or 200K 80  (Tconv) cells and result in 10-20M cells at the end of the 7-10 day expansion period (Figure 4-4). Following cell expansion, I collected the cells and set up pre-clinical assays to determine the therapeutic capacity of FliC-CAR-Tregs (Figure 4-5A).    Figure 4-4 Treg and Tconv Expansion over 7 Days Foxp3+ Tregs and Foxp3- Tconv were isolated as described and expanded using anti-CD3/28 dynabeads in the presence of 1000U (Tregs) or 100U (Tconv) hIL-2. After 48h, cells were infected with retrovirus as indicated (UT: untransduced; Her2: Her2-CAR; 5D4: FliC-CAR). Cell numbers were counted daily and cells were diluted to 1M/ml. Counts on D0, D4 and D7 were plotted to demonstrate a representative expansion pattern (n=15).  81    Figure 4-5 Expression of FliC and control-CAR on primary murine Tregs (A) Schematic of the experimental set up for production and testing of CAR-Tregs (B) FOXP3 expression was determined by flow cytometry for eGFP. Surface expression of ∆NGFR control, FliC- and Her2-CAR transduced Treg was visualized with antibodies directed against ∆NGFR and myc tag after gating on eGFP.  To assess whether expanded primary murine Tregs retain their Foxp3 expression and are able to express the FliC- and Her2 CAR on the surface, I stained expanded Tregs for NGFR and myc tag. Indeed, viable cells retained their Foxp3 expression (Figure 4-5B, left panel). Importantly, expanded Tregs expressed both NGFR and the CAR on their surface (Figure 4-5B). Expression levels of FliC-CAR were generally higher than Her2-CAR and typically between 80 and 95%. I 82  also tested expression of the respective CARs in Tconv and conducted some experiments with Tr1 cells, to build on my insights reported in Chapter 3. Both Tconv and Tr1 cells are able to express FliC- and Her2-CAR, however Tr1 cells do not survive the strong expansion protocol (data not shown). My initial Tr1 findings are now being followed-up on in a spin-off project by another PhD student.  4.2.4 Recombinant FliC activates FliC-CAR-Tregs to proliferate As mentioned above, surface expression of a CAR does not guarantee binding to its cognate antigen and antigen-binding does not guarantee function. To assess whether FliC-CAR-Tregs can be activated through their CAR, I used a proliferation assay. After 7 to 10 days of expansion, I labeled Tregs with CPD and re-stimulated the cells either via recombinant FliC, through their TCR using anti-CD3/28 (positive control) or in PBS (negative control), all in the presence of 100U IL-2 for 3 to 4 days, and assessed proliferation (CPD dilution) by flow cytometry. Indeed, only FliC-CAR-Tregs were stimulated by FliC while none of the control Tregs were stimulated via FliC (Figure 4-6). All cells were re-stimulated through their TCR and it is important to note that this re-stimulation led to extensive cell death resulting in low numbers of surviving proliferating cells (Figure 4-6A). While FliC-CAR-Tregs in two experiments retained residual activity in the PBS control, FliC nevertheless restimulated these cells (Figure 4-6A,C)  83    Figure 4-6 FliC-CAR expressing Tregs are specifically activated by FliC to proliferate (A) Tregs were transduced and cultured for 7 to 10 days as described above. Tregs were then labeled with proliferation dye and re-stimulated in the presence of 100U IL-2 using either PBS control, plate-bound -CD3 and soluble α-28 or FliC. Figure shows representative data from 7 independent experiments (B) Quantification of N=7 experiments; * p < 0.05  (one-way ANOVA followed by Tukey’s post-test). (C)  Paired comparison of FliC-CAR expressing Tregs cultured in the presence of FliC or PBS control * p < 0.05(paired, two tailed t-test between PBS and FliC),  = experiment depicted in Figure 4A.  4.2.5 FliC-activated FliC-CAR-Tregs suppress conventional Tcells The ultimate goal for CAR-Treg therapy is that activation through the CAR enables Treg to suppress the local immune response. In vitro suppression assays are well established and test the capacity of Tregs to suppress responder Tconvs142. However, as Tconv are typically activated 84  through polyclonal TCR stimulation using anti-CD3/28, this would also activate Treg via their TCR and it would be impossible to separate suppression mediated by CAR activation from suppression mediated by TCR activation.   To overcome this hurdle, I developed an antigen-specific suppression assay using Tconv isolated from OT-II mice, which have a transgenic TCR specific for OVA (see paragraph 2.1.20). In brief, BMDAPC were incubated with OVA in order to process this peptide and present it on their MHC II. OT-II Tconv were stained with CPD and then activated via their TCR through the MHC-OVA complex. Simultaneously, FliC-CAR-Treg were activated by recombinant FliC. As a control, I set up a positive control suppression assay in which I stimulated both OT-II Tconv and Treg using anti-CD3/28. A schematic of this experiment is depicted in Figure 4-7.    Figure 4-7 Schematic of antigen-specific suppression assay (A) Positive control suppression assay. Treg and responder Tconv are activated with plate bound anti-CD3 and soluble anti-CD28 via their native TCR. (B) Antigen-specific suppression assay. Treg are activated by soluble flagellin via their FliC-CAR, while responder OT-II Tconv are activated by OVA presented on BMDAPC-MHC II via their transgenic TCR.  85   All three types of Treg—FliC-CAR, Her2-CAR and untransduced control—retained their suppressive capacity following spinoculation with or without viral expression and 7 days of expansion (Figure 4-8A,B). In all cases, the suppression levels depended on the ratio of Treg/Tconv. Suppression levels by FliC-CAR Tregs were higher compared to control Tregs which may have been a result of their high baseline auto-activation. The two repetitions of the suppression assay were conducted with the Tregs derived from the same expansions that showed high auto-activation (Figure 4-6A,C). Importantly, only FliC-CAR-Treg were able to suppress responder OT-II cells in the antigen-specific suppression assay. As I only conducted two repetitions of the suppression assay, I report these data as preliminary.  86     87  Figure 4-8 FliC-CAR expressing Tregs suppress conventional Tcells upon stimulation though the CAR Tregs were transduced and cultured as described. Tregs and APC-proliferation-dye labelled OT-II Tconv were co-cultured in the indicated ratios on (A,B) plate-bound -CD3 in the presence of soluble -CD28, or (C,D) plate-bound FliC in the presence of bone marrow derived antigen presenting cells (BMDAPC), pulsed with OVA 323-339 overnight. Tconv proliferation was detected after 3 or 4 days via FACS and analysis was performed with FlowJo 7 by division index. (A) FACS plots of n=2 experiments (n=1 per row), (C) Representative FACS plot of n=2 experiments; (B,D) combined data from n=2 experiments.  4.2.6 FliC-CAR-Tregs survive in immunocompetent host A key requirement for Tregs to work as cell therapy is their ability to survive in an immunocompetent host. If the host’s immune system were to attack and eradicate transferred Tregs, the host would have to require an immunosuppressive regimen such as chemotherapy or irradiation. This requirement would defeat the purpose for cell therapy and the prospect of avoiding global immunosuppression. To test whether FliC-CAR-Tregs might survive in a host, I stained 2M expanded FliC-CAR or untransduced Tregs with a CPD and injected them i.p. into TLR5-/- mice. TLR5-/- mice are immunocompetent, but they lack TLR5 and host cells would thus not get activated by injected FliC, which could lead to systemic immune activation. After 8h, I challenged half of the FliC-CAR recipients with recombinant FliC i.p. I harvested spleen, mesenteric lymph nodes (MLN) and LPL on D4 days post-transfer and assayed the cells by flow cytometry. While the absolute cell numbers recovered were very small, I found transferred cells in the spleens harvested from all conditions (Figure 4-9A, top row). Importantly, I only found FliC-CAR-Tregs in MLN isolated from mice that received FliC (Figure 4-9A, centre row). As expected, I was unable to recover adoptively transferred Tregs from the LPL fraction (Figure 4-9A, bottom row). To test homing of FliC-CAR-Tregs to the lamina propria in future 88  experiments, I would disrupt the intestinal barrier and apply FliC via enema to ensure FliC availability within the lamina propria. Interestingly, I only found FliC-CAR-Tregs isolated from MLN to be actively proliferating. This might indicate that these cells came in contact with FliC. I conducted one round of transfer into two recipient mice per condition and report these data as preliminary.     89  Figure 4-9 Adoptively Transferred FliC-CAR-Tregs Survive in an Immunocompetent Host Indicated cell types were adoptively transferred i.p. into TLR5-/- mice. Where indicated, hosts received i.p. challenge with recombinant FliC 48h post transfer (+FliC). Indicated tissues were harvested on D4 post-transfer and isolated cells were assayed by flow cytometry. (A) Flow cytometry plots. Numbers indicate cell count of Foxp3+ cells. (B) Proliferation of transferred cells was assessed by CPD dilution.  4.2.7 FliC-CAR-Tregs demonstrate potential to ameliorate DNBS colitis I tested the therapeutic potential of FliC-CAR-Tregs in two different models of colitis, DNBS colitis and T cell transfer colitis. DNBS is a chemical that is dissolved in ethanol and administered via enema into immunocompetent mice, in my case C57Bl/6. While the exact mechanism of pathogenesis is not fully understood, it is thought that ethanol disrupts the intestinal barrier and DNBS haptenizes intestinal proteins to trigger an immune response143. DNBS colitis typically develops 2-3 days after application. To test FliC-CAR-Tregs, I adoptively transferred 1M expanded Tregs i.p. directly after applying DNBS. I report these data as preliminary findings as I only conducted one experiment with three animals per group. Over the course of 5 days, animals that received either no Tregs or UT Tregs lost weight and recovered after three days, while animals that received FliC-CAR-Tregs barely lost weight indicating possible prevention of disease (Figure 4-10A). This potential for FliC-CAR-Tregs to prevent or ameliorate disease is further highlighted by the lower clinical (Figure 4-10B) and histological scores (Figure 4-10C,D). It is important to notice that the animals were euthanized after the period of heaviest weight loss and might have recovered partially. Further experiments to elucidate the role of FliC-CAR-Tregs in DNBS colitis are warranted.  90    Figure 4-10 FliC-CAR-Tregs demonstrate potential to ameliorate DNBS colitis C57Bl/6 mice received DNBS and ethanol intrarectally via enema and indicated types of Treg i.p. where indicated at the same time point. (A) Weights were monitored daily and plotted as percent of initial body weight. (B) Clinical score was assessed by two independent researchers based on colonic gross anatomy. (C) H&E stained histological sections (D) Histological score was assessed by two independent researchers. 91   4.2.8 Ex vivo but not expanded or FliC-CAR-Tregs protect from T cell transfer colitis Studies in T cell transfer colitis demonstrated the crucial role for Tconv in promoting colitis as well as the capacity of Tregs to prevent and reverse established colitis, and this model is well suited to research Treg-based therapies for IBD77,143,144. In contrast to DNBS colitis, T cell transfer colitis is a labor- and time-intensive model. Colitis is established by adoptive transfer of Tconv into a host with compromised adaptive immune system. While many studies employ Rag-/- mice that lack both T and B cells, I used Tcrb-/- mice which only lack T cells. My initial experiments to establish this model in our animal facility indicated that the model required 6-8 weeks before colitis was established and that it was necessary to feed recipient mice non-irradiated chow and tap water. I tested transfer of 250K, 500K or 1M Tconv and found that 500K Tconv seemed to be most effective at establishing colitis (Figure 4-11A,G). Furthermore, I found that co-transfer of ex vivo Tregs prevented colitis at ratios of Tconv/Treg of 1:1 and 1:2 (Figure 4-11B-F,H). Strikingly, neither expanded and untransduced, nor FliC-CAR-Tregs prevented disease in this model (Figure 4-11D,F,I). The observed weight loss coincided with clinical scores (data not shown) and histology (Figure 4-11J).   92   93     94  Figure 4-11 Ex vivo but not expanded Tregs Prevent T cell transfer colitis Indicated numbers of indicated cell populations derived from Thy1.1 Foxp3EGFP mice were injected into Thy1.2 Tcrb-/- mice to establish colitis. After transfer, mice were fed non-irradiated chow and tap water and monitored daily for weight loss and symptoms of colitis. Mice were euthanized after reaching 20% weight loss or 8 weeks whichever came first. (A-F) Weight loss graphs from different experiments, total n=5 (graphs E and F: same experiment, different cages, cage E: weight increase after initial measurement) (G) Comparison of different numbers of Tconv (Th) required to establish disease, (H) co-transfer of indicated numbers of ex vivo Treg at different ratios, (I) co-transfer of indicated numbers of expanded untransduced (UT) and FliC-CAR-Tregs (FliC Treg). (G,H,I) Compiled data from n=5 experiments (excluding cage E), total 57 mice, each dot represents one mouse. (D) H&E stained histological section from one experiment.  4.2.9 Expanded Tregs started secreting pro-inflammatory cytokines and lost their Treg profile The Tregs that showed residual stimulation in the PBS control of my proliferation assays (Figure 4-6) came from the same rounds of expansion as the expanded Tregs that failed to prevent T cell transfer colitis (Figure 4-11C). These expansions coincided with a batch change of anti-CD3/28 beads, media, as well as a breakdown of the CO2 sensor in the incubator. I hypothesized that something had changed that led Tregs to lose their phenotype and acquire pro-inflammatory profile. To investigate this, I analyzed cytokines in expansion media (data not shown) as well as in the supernatants from the proliferation assays (Figure 4-12). I also tested expansion media in inflammasome assays to investigate whether these expanded Tregs might enhance the inflammasome and thus contribute to intestinal inflammation (see Chapter 3). As hypothesized, expanded Tregs secreted IFN at comparable levels as Tconv (Figure 4-12A). Interestingly, both expanded Tregs and Tconv secreted high levels of IL-10 (Figure 4-12B). Strikingly, IL-10 95  secreted by expanded Treg or Tconv was unable to suppress the inflammasome and inflammasome IL-1 secretion was enhanced in macrophages incubated with expansion media (Figure 4-12C). Recombinant IL-10 suppressed the inflammasome as expected. Furthermore, expanded Tregs lost their TSDR (data not shown). Importantly, FliC stimulation clearly activated only FliC-CAR-Treg and -Tconv, further demonstrating that my FliC-CAR functions as anticipated. From these results, I conclude that the observed inability of expanded Treg to prevent T cell transfer colitis is linked to the acquisition of a pro-inflammatory phenotype.   96  Figure 4-12 Expanded Tregs secrete pro-inflammatory cytokines and enhance inflammasome activation (A,B) Supernatant from proliferation assays was tested for presence of indicated cytokines by ELISA. Representative of n=4 (D) BMDAPCs  were incubated with indicated expansion media from Treg expansion and inflammasome was activated as described above. IL-1 secretion was tested by ELISA. Representative of n=4.   Having discovered the loss of Treg phenotype after expansion, I further refined the Treg expansion method. Based on human Treg expansion protocols, I added rapamycin at different time points and at different concentrations to the expansion media. My preliminary results showed that addition of rapamycin indeed reduces production of IFN (Figure 4-13). However, rapamycin also slowed down the expansion and reduced the final numbers of Tregs available for further experimentation.   Figure 4-13 Addition of rapamycin to Treg expansion media preserves Treg phenotype. Tregs and Tconv were subjected to the expansion protocol with or without addition or rapamycin (rapa) at the indicated concentration and on indicated time points. Culture supernatants were analyzed by ELISA. Sample plot from n=6 troubleshooting experiments using different concentrations and timepoints. 97  4.3 Discussion Recent research has focused on the development of Treg-based cell-therapies for chronic immune-mediated diseases including inflammatory bowel disease. While pre-clinical studies produced promising results, translation into clinical practice has been impacted by the need to produce high numbers of antigen specific Tregs94. Furthermore, pre-clinical testing relies on animal models of colitis, and reliable protocols for expanding mouse Tregs are missing141. To overcome the challenge of isolating antigen specific Tregs, I developed and tested a CAR directed against the FliC flagellin. Flagellin is a bacterial protein readily present in the gut and able to cross the intestinal barrier at the site of inflammation in a mouse model of colitis. Furthermore, antibodies and Th1 cell responses against flagellin were found in people living with CD and in mouse models of colitis69–73. The availability and implication in IBD make flagellin an ideal target for Treg-therapy. Previous studies on CAR-Tregs for IBD demonstrated proof-of-principle but neither of these CARs were relevant to human IBD85,91–93. To test the FliC-CAR, I also developed a new method to expand and retrovirally transduce mouse Tregs.  A key requirement for a functional CAR is its ability to get processed intracellularly and trafficked to the cell membrane. Design considerations include the length it protrudes from the cell membrane which could affect binding to and synapse formation with its antigen145.  I confirmed that my FliC-CAR met the basic design requirements through experiments in cell lines and primary mouse T cells. In both systems, the FliC-CAR was expressed on the cell surface and able to bind to its cognate antigen. Furthermore, re-stimulation of expanded CAR-Tregs through the CAR activated Tregs to proliferate and to secrete an array of cytokines. I confirmed the suppressive capacity of CAR-Tregs through an antigen specific suppression assay 98  in which I individually activated transgenic Tconv responder cells through their cognate antigen and FliC-CAR-Treg through the CAR. Overall, these FliC-CAR-Tregs demonstrated their potential as therapeutic candidate in the in vitro assays.  Only four other studies testing CAR-Treg in IBD were published and the authors tested their CARs in two animal models of colitis, chemically induced colitis, and T cell transfer colitis. A CAR-Treg redirected against TNP, the agent used to induce TNBS colitis was able to prevent both TNBS colitis and oxazolone colitis when mice were given trace amounts of TNBS91,92. The same group demonstrated that CAR-Tregs redirected against carcinoembryonic antigen (CEA) prevented T cell transfer colitis in CEA-transgenic mice93. These three seminal studies laid the groundwork for my project and provided important proof-of-concept results that CAR-Treg therapy could be feasible in IBD. I tested FliC-CAR-Tregs in both the DNBS colitis model (which is similar to TNBS) and in the T cell transfer colitis model. In my preliminary data, I saw potential that FliC-CAR-Tregs could suppress DNBS colitis. I was also able to re-isolate FliC-CAR-Tregs from an immunocompetent host. My data from the T cell transfer colitis model were not as promising. In these experiments, neither FliC-CAR-Tregs, nor expanded UT Tregs were protective. In fact, mice that received FliC-CAR-Tregs lost more weight compared to those that received expanded UT Tregs. This might indicate that expanded Tregs not only lost the capacity to protect from disease but played an active role in promoting disease and FliC-CAR signaling perpetuated this role.  After finding expanded Tregs unable to protect from T cell transfer colitis as well as showing high levels of autoactivation, I tested whether they still maintained their Treg phenotype. This 99  concern appeared after I was forced to change batches of reagents including the anti-CD3/28 dynabeads which might have led to an increased activation. As described previously, repeated in vitro stimulation of mouse Tregs can lead to loss of Foxp3 expression and suppressive function141. Indeed, expanded Tregs from these experiments had lost their TSDR and acquired the capacity to secret IFN and IL-2. I did not see a reduction in GFP expression; however, it is conceivable that Foxp3 expression was reduced and I detected residual GFP. These Tregs also enhanced secretion of IL-1 in the inflammasome assay described in Chapter 3. Overall, these findings indicated that some changes in my expansion protocol led to a loss of Treg-specific properties. As human Treg expansion protocols rely on the addition of rapamycin146, I tested addition of various doses and at different timepoints. I passed on the confirmation of these preliminary findings to a junior PhD student and this refined Treg expansion protocol is now published as Wu et al. (2020)110.  Overall, my findings demonstrate the therapeutic potential of using flagellin as a target for CAR-Tregs. I confirmed the ability of the FliC-CAR to activate Tregs in vitro and I have preliminary data suggesting that these cells protect from DNBS colitis. I also developed a new model for expanding and retrovirally transducing Tregs which will enable further pre-clinical testing of my FliC-CAR-Tregs as well as allow other researchers to test CAR-Tregs either for IBD or in other autoimmune settings. 100  Chapter 5: Patients’ Willingness and Perspectives Towards Chimeric Antigen Receptor T-Regulatory Cell Therapy for Inflammatory Bowel Diseases 5.1 Introduction In Chapter 4: Developing Flagellin-Specific Chimeric Antigen Receptor Regulatory T Cell Therapy, I describe the development of a CAR-Treg therapy as a new potential therapy for IBD. Given the complex concept and logistics involved in CAR-Treg therapy, and the use of genetic tools such as a genetically engineered virus, I hypothesized that patients might perceive an unacceptable risk with this therapy which could limit future clinical application96. To answer this question, I surveyed people living with IBD to gauge their willingness to try CAR-Treg therapy given two hypothetical scenarios—in a clinical trial or as a new treatment. My overall hypothesis was that people living with advanced stages of IBD or running out of treatment options (i.e. receiving biologics) would indicate a higher willingness to try CAR-Treg therapy than would other people living with IBD.  5.2 Results 5.2.1 Response rate The advertisement email was sent twice (once in English, once in French) to an average of 4798 subscribers, and the Facebook posts reached 1922 people. The combination of the two recruitment methods were accessed by 1680 people (1603 from advertisement emails and 77 from Facebook posts; extent of overlap not known). Six hundred and ninety-seven (697) patients participated in the survey (41% of clicks on recruitment links), with 534 patients completing the survey (32% of clicks on recruitment links; 77% of patients who started the survey).  101  5.2.2 Demographics and disease history The majority of survey participants identified as female, had more than high school education, and resided in Canada (Table 5-1). Participants came from all age groupings between 14 and 79 years of age and resided in a variety of community sizes. The majority of participants reported having CD (58%) (Table 5-2). The mean age of symptom onset in this cohort was 24.8 years and mean age of diagnosis was 30.1 years (28 years for CD, 33 years for UC and 31 years for IC). Most participants experienced their last symptoms within three months before responding to my survey. Over half (57%) of the respondents reported at least one IBD complication, and 37% reported more than one IBD complication with the most frequent complications being stricture and surgery. Most participants who had surgery had part of their bowel removed. As expected, most participants had tried first line treatment while fewer tried second- and third-line treatments.   102  Table 5-1 Demographics and disease history of patient participants Data are presented as counts and as percent of total respondents (n=534) or as mean with minimum and maximum values.  n % Gender   Female 354 66% Male 176 33% Other 0 0% Missing data 4 1% Age   14-18 1 0% 19-29 95 18% 30-39 112 21% 40-49 127 24% 50-59 104 19% 60-69 69 13% 70-79 20 4% 80-89 2 0% 90+ 0 0% Missing data 4 1% Education   High school or less 74 14% Some college/university 197 37% Undergraduate degree 137 26% Graduate degree 119 22% Missing data 7 1% Community of Residence   Rural 132 25% Suburban 214 40% Urban 178 33% Missing data 10 2% Country of Residence   Canada 289 54% USA 183 34% Other 62 12%  Table 5-2 Disease history of patient participants Data are presented as counts and percent of total respondents (n=534) or as mean with minimum and maximum values. 103   n % Disease   Ulcerative colitis 203 38% Crohn's Disease 312 58% Indeterminate colitis 19 4% Age at onset   Years, mean (min, max)  24.8 (0, 75) n/a Age at diagnosis   Years, mean (min, max)  30.1 (8, 75) n/a Interval diagnosis-onset   Years, mean (min, max) 5.3 (0, 56)  Last symptoms   Within the last 3 months 402 75% 3 to 6 months ago 39 7% 6 months to 1 year ago 41 8% More than 1 year ago 52 10% Severity of last symptoms   Mean, scale 0-100 (min, max) 56.5 (0, 100) n/a All IBD medication a patient has tried (multiple allowed)  Corticosteroids 439 82% 5-ASA 426 80% Immunomodulators 294 55% Biologics 325 61% Antibiotics 294 55% Probiotics 322 60% Exclusive Enteral Nutrition  138 26% Other 70 13% IBD complications (multiple allowed)   Abscess 127 24% Fistula 150 28% Stricture 193 36% Surgery 179 34% Reported one complication 111 21% Reported two complications 89 17% Reported three complications 52 10% Reported all complications 51 10% Did not report any complications 231 43% Surgery because of IBD (multiple allowed)   Resection 147 84% J-Pouch 11 6% Temporary Ostomy 39 22% Permanent Ostomy 17 10% Other 36 20% 104  5.2.3 Perspectives on CAR-Treg therapy Responses from the 445 participants who completed the open-ended question about the firefighter analogy describing the proposed CAR-Treg therapy fell under three broad themes (Table 5-3). The first theme was focused on the promises of CAR-Treg therapy. This included explicitly stating their interest, hope, and willingness to try the therapy. Other participants highlighted that they appreciated the personalized aspect of this therapy and a belief that this therapy would be better than their current medication.   The second theme focused on concerns participants had with the CAR-Treg approach. These included participants who thought CAR-Treg therapy sounded scary or mentioned concerns about the genetic engineering aspect of therapy development. Participants were also concerned about side effects in general, side effects caused specifically by the virus used, and mistakes during lab handling. Additional concerns included worries about cost or whether their insurance would cover the treatment.  The third theme focused on participants’ questions about the CAR-Treg therapy. Participants wanted to learn more about the therapy—both in general and with respect to the specifics of the application, often related to time and amount. Other participants were interested in whether CAR-Treg therapy also treats systemic manifestations of their IBD, such as arthritis, and wanted to see more research done before making a decision regarding their willingness to try.  Table 5-3 Participants’ first thoughts and concerns after reading the 1-page Firefighter Analogy Data are presented as emerging themes (bold) and categories and representative quotes. 105  Themes Representative quotes Promises  Interesting   It is an interesting concept. I'm very interested, as this is not something, I am familiar with despite having done much research into Crohn's treatments, immunology, and inflammation. Hope  Great hope it will work. Sounds too good to be true, but hope it is. I want to try this No concerns. I’d try it today if were available. Personalized therapy   I would rather have my own cells rather than a synthetic drug or chemical fighting my disease. To be designed specifically for me, great! Better than current treatment    Everything would be better than Humira or Remicade. I’m eager to find any type of treatment that works better than what I have had. Sounds like less harmful side effects are possible than with current immunosuppressant therapies. Concerns  This sounds scary It sounds a little scary and futuristic. Genetic engineering and GMOs    Introducing modified cells back into my body sounds like a bad idea. These cells make me think of GMOs and the negative connotation surrounding them. My concern is that the genetically engineered cells may trigger unknown illnesses in the patient. Use of a virus  I was concerned that the virus would not be contained to the inflamed area. I would worry the virus would become active. Mistakes during lab handling  Concerned about getting blood injected back in me, what if it has not been stored correctly or properly labeled? Side effects That it will cause an inflammatory response that will end up making my Crohn’s worse. Cost and insurance coverage  Sounds expensive. Sounds great also sounds expensive, what are the other downsides besides cost? Open questions  More information on therapy    How long does it take to engineer and put the blood back into your body? How long does it take to start working? And how large of a dose of cells would it be? A vial? A pint? Do you need to redo this with every flare? More research needed   What are some of the potential side effects? Is it possible that these cells could mutate/become harmful? Sounds promising but would like to see more research done. Does this treat systemic symptoms  I have systemic inflammation specifically IBD related arthritis. Would this treatment be able to help that as well? 106  5.2.4 Preference on CAR-Treg fate When asked to choose between two options of what could happen to the cells after treating their flare, significantly more participants indicated a preference for cells to stay in their body in a dormant state, ready to fight a new flare when needed, over having the cells eliminated from their bodies after successful induction of remission (57% and 39%, respectively; p<0.0001). When prompted to share their most important consideration in choosing CAR-Treg fate, those that indicated preference of cells to stay considered how debilitating their flares were; their wish to live flare-free; their worries that repeated treatment might lose effectiveness; their dislike of needles; their residence in a remote area; their concern around cost of treatment; their wish for improved quality of life; and their old age (Table 5-4). In contrast, those participants that indicated a preference for the cells to stop existing in their bodies highlighted that their flares were manageable; that flares were something known; the lack of long-term data on side-effects; their worries about mutations; their young age; and that this was not yet a cure.   107  Table 5-4 Participants’ most important consideration for choosing their preference of cells staying in their body in a dormant state vs cells to be eliminated from their body Data are presented as categories (bold) and representative quotes. Themes Representative quotes Reasons for indicating preference for cells to stay in a dormant state  Flares are debilitating  Dealing with a flare is terrible and the thought of having a severe flare is scary. I'd rather risk possibly side effects to not experience UC symptoms. Wish to live flare-free  Of course, I would love to be "cured" of my CD. I want to try anything to help prevent the flare ups. Worry about repeated treatment I have stopped treatment and started it again and not effective. Dislike needles I don't like needles and want a normal life. Living in remote area  Because our remoteness having a flare up and waiting for cells would put a strain in my ability to work. Cost of treatment The considerable cost of having to go in for multiple treatments. Improved quality of live  I am done with the years of pain and missing out on life. So that I can live a normal life without worrying about my Crohn’s 24/7. Old age  Flares are hard at my age. My age, where "long-term" may be moot. Reasons for indicating preference for cells to be eliminated  Flares are manageable  I can usually control my flare from progressing to an unmanageable state. My flares are not usually so severe that I couldn’t handle a bit of a wait. Flares are something known   Waiting for a next flare is how I live currently so I am okay with staying that way. I'd prefer to stick with the known as opposed to the unknown. Lack of long-term data on side-effects  Unintended long-term consequences that cannot be tested in the short-term. Lack of information/longitudinal studies on the possible risks. Worry about mutations  Not knowing what the dormant cells might “change” into. The risk of the cells changing/mutating into harmful cells in my body.  Young age  I’m still relatively young, so the long-term risks concern me more than the inconvenience of repeated treatments. No cure yet I just wish for a cure.   108  5.2.5 Willingness to try CAR-Treg therapy When asked to rate willingness to try CAR-Treg therapy as a new treatment, participants indicated a median willingness of 93.5 [IQR 82-100] (Figure 5-1Figure 5-1). Given the prompt to elaborate on why a certain level of willingness was chosen, respondents that indicated willingness to try highlighted the need to trust their GI doctor; the personalized nature of this therapy; the wish to change their current treatment, and their desperate wish for a cure (Table 5-5). Those that did not indicate willingness to try highlighted their concern about this being a new therapy, and the success of their current therapy.    Figure 5-1 Willingness to try CAR-Treg therapy New Treatment indicates willingness to try CAR-Treg therapy as a new treatment that was shown to work well and is safe in people. Clinical Trial indicates willingness to participate in a clinical trial to test the efficacy of CAR-Treg therapy after it has been shown to be safe in people. Red bars indicate median and interquartile range. *** p<0.0001 109  Willingness to try CAR-Treg therapy in a clinical trial was significantly lower compared to willingness to try as new treatment (p < 0.0001) with a median of 77.5 [IQR 60-91] (Figure 5-1). Respondents who indicated willingness to participate in a clinical trial mentioned the necessity of contributing to research and past experience with clinical trials. Additionally, they mentioned their non-response to biologics and need for a new therapy; their worries about biologics; and the promise of site-directed personalized therapy (Table 5-6). Respondents who did not indicate willingness to participate in a clinical trial highlighted the success of their current therapy; the availability of other options for them, the wish for demonstrated long-term safety and efficacy and a general decision not to participate in research.  Table 5-5 Participants’ reasons for choosing their level of willingness to try CAR-Treg therapy as new treatment Data are presented as categories (bold) and representative quotes. Themes Representative quotes Reasons for indicating willingness to try as new treatment  Trust in their GI doctor  I trust my gastrointestinal doctor, he knows me. I trust his [my doctor’s] judgment; he hasn’t steered me wrong yet. Personalized nature of the therapy Specific to gut, made for me with my own cells. Wish to change current treatment  I am on a biologic and the side effects are scary. [I want] to stop with weekly Humira injections. Desperate wish for a cure I would try anything. Reasons for indicating non-willingness to try as new treatment  Concern that this is a new therapy   I’d be hesitant due to the newness of the therapy. I have to be out of options before blindly taking something with no data proving long-term use. Success of current therapy   My symptoms are being managed well enough. My present medication is working well and I experience little side effects. I’d only use it after I fail biologics.  110  Table 5-6 Participants’ reasons for choosing their level of willingness to try CAR-Treg therapy in a clinical trial Data are presented as categories and representative quotes. Themes Representative quotes Reasons for indicating willingness to try in a clinical trial  Calling to participate in research    I would risk a long-term flare up for the greater good. Innovation is not possible without testing hypotheses. I believe in supporting research, trying cutting edge approaches, and doing the best I can for my health. Positive experience with participation in clinical trials I have had experience and success with clinical trials in the past.  Need for other treatment options  I've already failed with all the biologics. Humira stopped working and I’m having nasty side effects from the Remicade. Worries about using biologics So many terrible known side effects of biologics. Promise of site-directed personalized therapy  More targeted than biologicals. It is using my own blood cells to improve my health rather than a biologic. Reasons for indicating non-willingness to try in a clinical trial  Success with current treatment  Biologics have been keeping my Crohn’s in check. Would not stop them unless necessary. Availability of other treatment options  Because I am not out of options. I can still use biologics which are proven safe to use. Wish for demonstrated long-term safety and efficacy  I would be a little hesitant not knowing if it works and taking a risk on the side effects. I prefer more established medications that were tested for long time. General unwillingness to participate in clinical trials I choose not to be a lab rat.   5.2.6 Influence of demographic factors on willingness to try CAR-Treg therapy Overall, bivariate analyses did not identify clear demographic predictor variables for willingness to try CAR-Treg therapy in either scenario. As shown in Table 5-7 (new treatment), I observed a significantly higher willingness (p=0.0001) to try CAR-Treg therapy in participants that indicated preference of the cells to stay (median 95, IQR 84-100) compared to those who 111  preferred the cells to die after treating a flare (median 90, IQR 77-100). Furthermore, participants residing in other countries (median 100, IQR 89-100) indicated a significantly higher level of willingness (H = 14.9, p=0.006) compared to Canada (median 91, IQR 77-100) and the USA (median 93, IQR 82-100). In line with the bivariate analysis, the multiple regression analysis identified preference of cells to stay and residence in another country as significant predictor variables and there was no significant effect of any other predictor variables on the willingness (Table 5-8, new treatment). There was no correlation between any of the predictor variables (data not shown).  In contrast to what I found for willingness to try CAR-Treg as new treatment, the bivariate analyses on willingness to try CAR-Treg therapy in a clinical trial revealed a significant influence of gender. Females indicated significantly lower willingness than men (median 73, IQR 58-90 and median 80, IQR 71-91, respectively, p=0.0041) (Table 5-7, clinical trial). In line with my findings on willingness to try CAR-Treg as new treatment, I observed a significantly higher willingness for participants who preferred cells to stay compared to those participants who preferred cells to die after treating the flare (median 80, IQR 68.25-93.5 and median 71, IQR 56.25-82, respectively, p<0.0001). Furthermore, I observed a trend (p=0.0531) that willingness to participate in a clinical trial for CAR-Treg therapy appears to decline with increased level of education. The multiple regression analysis corroborated these findings: female gender and preference of cells to stay were identified to have a significant impact on willingness to accept CAR-Treg therapy as a clinical trial while level of education was not significant (Table 5-8, clinical trial).  112  Table 5-7 Bivariate analysis of influence of demographic predictor variables on willingness to try CAR-Treg therapy either as new therapy or in a clinical trial to test efficacy Crosstab parameters include median, interquartile range (IQR), minimum and maximum values (min, max) and Mann-Whitney or Kruskal-Wallis p-values  New Treatment Clinical Trial   median IQR min, max p-value median IQR min, max p-value Gender       0.1102       0.0041 Female 92 80.75-100 0, 100  73 58-90 0, 100  Male 93 82.25-100 5, 100  80 71-91 8, 100  Age    0.9800     14-39 92 81-100 0, 100  73.5 59.75-97.25 0, 100  40-69 92 82-100 0, 100  79 62.5-92 0, 100  70-90+ 91.5 79.75-100 60, 100  73 69.25-81 7, 100  Education       0.2432       0.0531 High school or less 91.5 81.75-100 21, 100  81 61.75-92.75 9, 100  Some college 95 82.5-100 0, 100  78 64.50-92 0, 100  Undergraduate Degree 92 80.5-100 19, 100  73.5 60-100 0, 100  Graduate Degree 91 79-100 18, 100  70 54.5-86.5 0, 100  Community       0.4329       0.1721 Rural 94 82.25-100 20, 100  78.5 62-91 0, 100  Suburban 92 81-100 0, 100  76.5 62-90 7, 100  Urban 91 80-100 29, 100  72 57-90 7, 100  Country       0.0060       0.0817 Canada 91 77-100 0, 100  75 60-89.5 0, 100  USA 93 82-100 18, 100  76 60-91 0, 100  Other 100 89-100 70, 100  81 71-94 0, 100  Medications tried       0.2983       0.3057 Biologics 92 81.5-100 18, 100  78 61-90 0, 100  Immunomodulators 90.5 71-100 0, 100  73 52-82.5 0, 100  Other 94 82-100 0, 100  76 65-91 0, 100  Complications       0.8880       0.5675 Yes 92 81-100 5, 100  78 61-91 0, 100  No response 93 81-100 0, 100  76 60.25-89 0, 100  Fate of CAR-Treg       0.0001       <0.0001 Cells should stay 95.5 84-100 7, 100  80 68.25-93.5 0, 100  Cells should die 90 77-100 0, 100  71 56.25-82 0, 100   113  Table 5-8 Multivariable linear regression of demographic predictor variables on willingness to try CAR-Treg therapy as new therapy or in a clinical trial to test efficacy   New Treatment Clinical Trial  Beta p-value Beta p-value (Constant) 85.970  79.945  Gender     Female -3.116 0.069 -5.802 0.010 Age     40-69 0.500 0.786 3.149 0.194 70-90+ -0.763 0.746 -0.921 0.766 Education     Some college 4.408 0.076 0.607 0.853 Undergraduate degree 2.636 0.330 -1.998 0.575 Graduate degree 2.882 0.294 -3.876 0.284 Community     Urban -0.325 0.884 -1.925 0.511 Suburban -2.045 0.328 1.558 0.571 Country     USA 3.519 0.094 1.718 0.474 Other country 9.101 <0.0001 6.781 0.046 Disease     Ulcerative colitis 0.286 0.878 -0.406 0.869 Indeterminate colitis 4.915 0.275 -3.785 0.523 Medications tried     Immunomodulators -3.421 0.234 -6.205 0.101 Biologics 1.088 0.585 -2.069 0.430 Complications     Yes 1.162 0.508 0.972 0.674 Fate of CAR-Treg     Cells stay -6.350 <0.0001 -8.054 <0.0001    114  5.3 Discussion To the best of my knowledge, this survey represents the first study to research willingness of people living with IBD to try CAR-Treg therapy. Participants indicated high median willingness to try CAR-Treg as treatment and slightly lower median willingness to participate in a clinical trial to test efficacy. Significantly more participants preferred CAR-Tregs to stay dormant in their bodies to prevent future flares compared to being eliminated with a need for renewed future treatment. In contrast to my hypothesis that willingness would be correlated with a more advanced disease state, and/or biologic treatment, I did not find any demographic or clinical parameters to predict willingness to try CAR-Treg therapy as new treatment.   Participant willingness to participate in a clinical trial for CAR-Treg was lower compared to trying it as new treatment but higher than anticipated. Recent studies on motivations and barriers to participate in IBD clinical trials found similar and slightly lower levels of willingness to participate in hypothetical clinical trials147–149. These studies identified a clear correlation between advanced or active disease and willingness. I did not find similar correlations in my survey; however, participants that indicated willingness highlighted their need for other treatment options while those who did not indicate willingness highlighted the success they had with their current treatment and the availability of other treatment options. I found participants highlighting a calling to participate in research, a sentiment found throughout the IBD literature147–149. Furthermore, respondents that indicated willingness to participate highlighted past clinical trial experience as a reason. While Gehrmann et al (2016) and Larussa et al (2020) reported a correlation between past clinical trial experience in IBD and willingness to participate, Ravikoff et al (2012) found the opposite result. In line with Ravikoff’s et al (2012) findings, 115  female participants in my survey were significantly less likely to indicate willingness to participate in a clinical trial. Studies in other clinical areas have also found gender differences in participation in hypothetical clinical trials with females less likely when side effects are unknown and when the research is not well explained150,151. The scenario in my survey specified that the treatment was safe in people, however survey participants commented on lack of long-term safety data and I did not provide an explanation of the trial protocol.  Willingness to try a new treatment or participate in a clinical trial might be affected by thoughts, concerns, and understanding of that treatment. When I asked about participants’ first thoughts and concerns after reading the firefighter analogy, participants mentioned concerns around mistakes in lab handling and that it involves genetic engineering and a virus. This could indicate a general mistrust towards anything created in the lab based on news reports and highlights the importance of avoiding sample mix-ups in hospital settings. Additional comments indicated a need for more refined educational material for patients once this therapy is ready for clinical application. This is in line with Veilleux’ et al (2017) report that patients’ understanding of information provided by their physician resulted in reduced anxiety, and increased treatment satisfaction and adherence152.  My finding that more participants preferred prevention of future flares while accepting long-term risk of the CAR-Treg cells to change is in line with previous reports which found that people with IBD were willing to accept an elevated risk of potentially serious side effects in exchange for an improvement of their IBD99. Furthermore, other research has demonstrated that people with IBD would accept long-term risk of serious side effects in exchange for an extension of 116  remission98. My participants further qualified their most important consideration for choosing a preference for fate by stating that “flares can be debilitating” and that they would “rather have risks than flares” after selecting a preference for CAR-Treg cells to stay dormant in their bodies. Those participants who preferred CAR-Tregs to be eliminated while accepting the risk of a future flare tended to indicate their ability to “manage their flare” and that their “flares are not too severe” and would thus have less clinical benefit from accepting the risk. These findings are in line with Johnson et al’s report (2007)99 that risk-acceptance increases with clinical benefit.  I report my results as exploratory as I employed a convenience sampling approach and was unable to precisely identify my population of interest. While this limits the generalizability of my findings, the demographic, disease, and medication history of the participants matched clinical patterns across IBD for most characteristics. My respondents reported a mean age of diagnosis of 28 years for CD and 33 years for UC. Previous studies found the peak incidence for CD and UC to be 20-30 years and 30-40 years, respectively153,154. Thirty four percent of my respondents experienced surgery and 84% of these were colectomies. Clinical observations reported rates of colectomy range around 25-30% after 25 years of disease153. There is no sex-difference in prevalence and incidence in IBD155,156 overall, although UC is slightly more common in men while CD is more common in women153. Nonetheless, I received more responses from participants who identify as female. This skewing has been observed by others157,158 and could be due to an increased propensity for women to talk about their health and seek health-related information159,160.  117  With respect to study limitations, my estimated survey response rate was 32% which is similar to previous IBD online surveys157,161,162. Since I was unable to rule out multiple openings of the email and Facebook post by individuals, the real response rate might be slightly higher. I also found that 30% of those who provided consent dropped out. I anticipated a high dropout rate because my survey encompassed 29 questions, multiple open-ended questions, and the one-page written firefighter analogy. Furthermore, while I did not meet the assumption of normally distributed data, my survey results contained 34 records per independent variable, which is more than the various rules-of-thumb recommendations of 10-20 subjects per variable and far more than the recently identified 2 subjects per variable163. Given the exploratory nature of my survey, I was comfortable with conducting and interpreting the analysis. 118  Chapter 6: Patient Experiences with Clostridioides Difficile Infection 6.1 Introduction Several research groups and national programs have surveyed health care sites and treating physicians, to evaluate incidence, prevalence, infection control practices and economic burden of CDI in Canada. In 1997, two independent groups surveyed Canadian hospitals and showed a national average of 3.06 cases per 1,000 admissions in large hospitals with over 200 beds164 and 5.9 cases per 1,000 patient admissions across 19 Canadian Hospital Epidemiology Committee member sites165. A follow-up study in 2009 by the Canadian Nosocomial Infection Surveillance Program found similar numbers with 4.6 cases per 1,000 admissions166. While this latter study emphasized patient outcomes using a prospective surveillance method, they did not directly survey patients. In 2011, Wilkinson et al.167 surveyed 2,880 health care facilities across Canada, 943 (33%) of which responded to the survey, and identified a broad variation in infection prevention and control practices that were implemented to curb CDI. Overall, acute care sites were more likely to submit liquid stools for testing, compared to mixed care and long term care sites167. In 2015, Levy et al.168, analyzed direct and indirect medical cost due to CDI in Canada based on nation-wide rates of CDI associated with hospital visits. They estimated the total cost of CDI to the Canadian economy to be over CAD $280 million, 90% of which was directly related to in hospital cost168. A recent Ontario population-based matched cohort study based on personal health information identified increased risk for all-cause mortality and higher cost compared to uninfected control subjects169.   119  Despite a push toward improved patient experience and a patient-centered health care system170, no research group has surveyed Canadian patients living with CDI to understand their perspectives and experiences. Madeo at al.171 conducted a pilot study on patient-reported knowledge, awareness and beliefs on nosocomial infections. In their small mixed-method survey of 110 patients, they concluded that patients were aware of the risk of nosocomial infections. However, patients lacked knowledge on routes of infection and prevention. Patients’ main source of knowledge were television and newspaper, with MRSA being named most often as a source of nosocomial infection171. In an interview-based study with 15 patients, Madeo and Boyack172 researched the needs and lived experiences of elderly patients with CDI and identified four key themes of experiences — “physical suffering and impact on daily activities of living, lack of control over bowel function, lack of understanding of the illness, and issues around privacy and dignity”. They also found patients lacked an understanding as to how they became infected. As practice points, the group recommended increased patient education and improved staff-patient communication172. Overall, only a few studies investigated patient experiences with nosocomial infections, none focused on CDI and none have been conducted in the Canadian context.  To address this knowledge gap, the Gastrointestinal (GI) Society developed a web-based survey to collect lived experiences and perspectives from patients with CDI. Questions were focused on disease severity and management, quality of life. The survey also included an open-ended question on the most important priority to improve the patient’s experience with CDI. I then partnered with the GI Society to analyze the survey responses and write the manuscript.  120  6.2 Results and discussion 6.2.1 Demographics A total of 267 people responded to the survey. Of these, 85 did not complete all demographic and at least one survey question and 15 were non-Canadian residents. After removing these non-eligible participants from the sample set, I analyzed a total of 167 responses, 119 of which came from people infected with C. difficile and 48 from caregivers/loved ones of an infected person. There are no publicly available recent data on CDI incidence in Canada. I analyzed responses stratified by patients and caregivers and compiled the data. Most results found no statistical differences between groups and are presented compiled in this report, except when looking at questions based on quality of life (QoL). Of the total cohort, 77% of respondents were female and 22% were male. The majority (77%) were between 30-69 years old with a median age grouping of 50-59 years (Table 6-1)  Respondents included residents of all Canadian provinces, with the majority of respondents primarily from Ontario (32%), British Columbia (24%,) and Alberta (16%). While the survey was offered in French and English, uptake in Quebec (the second most populated province in the country) was low with only 8% of respondents. I did not receive responses from any of the territories. A total of 19% of surveyed respondents identified as health care professionals with the majority (49%) working in a hospital setting. Furthermore, I observed a bias toward female respondents, an observation made by other researchers when employing online surveys158.    121  Table 6-1 Gender and age distribution Data are presented as percent of total respondents  n % Gender   Male 36 22 Female 129 77 Prefer not to say 2 1 Age   0-18 4 2 19-29 13 8 30-39 22 13 40-49 31 19 50-59* 42 25 60-69 34 20 70-79 12 7 80-89 7 4 90-100 2 1 * median age group  6.2.2 Health care system exposure preceding diagnosis  CDI has been most commonly correlated with exposure to antibiotics and prior hospitalization for non-CDI related reasons173. In line with these correlations, 63% of surveyed respondents indicated taking antibiotics in the three months prior to being diagnosed (Figure 6-1A). Most antibiotics were prescribed by physicians, whereas 12% of respondents were prescribed antibiotics by their dentist (Figure 6-1B). A total of 73% of respondents indicated to have experienced at least one concurrent situation, for example a medical condition or childbirth. Of these, 29% were hospitalized for non-CDI-related reasons, 17% were suffering from an existing severe illness, 11% suffered from a flare of inflammatory bowel disease, and 10% had received abdominal surgery just prior to their first experience with CDI (Figure 6-1C). CDI is a GI disease 122  and 60% of survey respondents lived with at least one additional GI condition prior to contracting C. difficile. The majority had irritable bowel syndrome (34%), inflammatory bowel disease (18%), or gastroesophageal reflux disease (17%) (Figure 6-1D).     Figure 6-1 Medications and conditions prior to first CDI (A) Distribution of participants that took antibiotics prior to diagnosis, and (B) health professional who prescribed the antibiotics. (C) Accompanying situations at the time of C. difficile infection and (D) concurrent gastrointestinal conditions.   123  While exposure to health care settings has been identified as a risk factor for contracting CDI, 62% of surveyed respondents indicated their symptoms began while living at home (Figure 6-2A). Only 25% indicated that they were hospitalized or living in a long-term care home. Furthermore, 8% indicated “other” and further specified they had been in contact with a health facility prior to developing symptoms. Examples of health facility contact were “shortly after being discharged from the hospital”, “working as RN in ER” and “visiting a patient in hospital”. Of those hospitalized, 69% stated that their hospital stay was prolonged because of their CDI and 33% stated they required hospitalization for their first infection. When asked for the length of their first hospital stay, 30% indicated 1-6 days, 25% indicated 1-2 weeks, 26% indicated 3-4 weeks, and 19% reported “other” and further specified times between 6 weeks and 6 months (Figure 6-2B). Overall, these findings confirm previous observations that most respondents had been exposed to either antibiotics, health care settings, or both prior to their first experience of CDI.     124  Figure 6-2 Situations surrounding first C. difficile infection (A) Exposure to health care facilities and travel preceding development of first symptoms. (B) Length of hospital stay because of first C. difficile infection.  6.2.3 Symptoms, diagnosis, and treatment CDI can be a severe infection and the most commonly reported symptoms in the survey mirror those described in the clinical literature: watery, severe diarrhea (72%), diarrhea (65%), fatigue (63%), weight loss (54%) and loss of appetite (53%) (Figure 6-3A). Most respondents experienced two or more symptoms from a list of 12 with the majority reporting between four and seven (Figure 6-3B). Past clinical trials estimated the expected recurrence rate for CDI to be between 25% and 30%174,175 which is in line with my observation that 26% of respondents indicated CDI recurrence (Figure 6-3C). Importantly, 33% of respondents indicated having CDI more than once and 13% could not remember (Figure 6-3D). This might mean that survey participants used different definitions of recurrence than clinicians, or that not all repeated CDI experiences were diagnosed as recurrence. Of the respondents who had CDI more than once, 27% experienced CDI two to four times while 7% experienced CDI five or more times (Figure 6-3E). Respondents were given the option to specify the number of recurrences if they selected 5+ times and I found the maximum to be 12 times. These data demonstrate that this participant population is representative compared to the expected symptoms and recurrence rates.  125    Figure 6-3 Symptoms and recurrence Symptoms and complications experienced with CDI, (A) symptoms as percent of total responses and (B) number of symptoms experienced per individual as percent of total responses. (C) Recurrence of C. difficile infection compared to (D) reinfection with C. difficile and (E) number of C. difficile reinfections per respondent.  To provide prompt treatment for CDI, time to diagnosis is critical. Nevertheless, only 13% of respondents received their diagnosis within the first 48 hours after the onset of their symptoms (Figure 6-4A). In contrast, 29% had to wait between 2 to 5 days and 29% between 6 and 30 days. Surprisingly, 10% of respondents indicated they had to wait over 30 days between the onset of 126  symptoms and diagnosis. The mean time to resolution of diarrhea after initiation of antibiotics in CDI has been reported as 3-4 days176, but some patients take longer. Current guidelines recommend extending the standard 10d treatment course to 14d if diarrhea has not resolved by day 10177,178. Interestingly, only 32% of respondents indicated that their symptoms resolved within 7 days. While 26% experienced resolution between 8-14 days, 22% were treated for longer than 14 days and 14% did not experience resolution (Figure 6-4B). As expected, the majority (94%) of respondents were treated with antibiotics for their first CDI (Figure 6-4C), but 6% indicated they did not receive treatment for their CDI at all. Of these, 27% indicated they concurrently used probiotics while 8% of all respondents used only probiotics to treat their first CDI. In contrast, 96% of respondents treated their subsequent CDI with antibiotics and 47% of these used probiotics in addition to antibiotics (Figure 6-4E). I did not ask whether people received their probiotics after consultation with a health care professional.   127    Figure 6-4 Diagnosis and treatment (A) Time between onset of symptoms and diagnosis and (B) time between commencement of treatment and symptom resolution, or no resolution (NR). Antibiotic (abx), probiotic (pbx) or combination (abx+pbx) treatment used to overcome first (C) and any subsequent (D) C. difficile infections.  If antibiotic therapy fails to resolve recurrent CDI, fecal microbial transplantation (FMT) is reported to be a safe and effective alternative179. One respondent out of the 167 participants indicated receiving FMT for their first CDI episode. More people received FMT for subsequent CDI, albeit much less than expected. A total of five respondents out of 67 who had experienced subsequent CDI indicated treatment by FMT. The open-ended section of the survey revealed that 128  these participants experienced FMT to be the cure to their CDI with one participant commenting they would have liked to try FMT, but it was not available to them. All respondents who received FMTs also indicated they were treated with antibiotics. Overall, these responses demonstrate that most patients were treated with antibiotics as first line of therapy and more are turning to probiotics during recurrent infections.   6.2.4 CDI impact on life CDI can have a devastating impact on patients and I was interested whether there may be any lasting impact on self-assessed quality of live (QoL). To address this, three questions on the survey accessed participants’ QoL with scores ranging from the “6” (able to carry on normal activity including work; no special care needed, social activities not restricted) to “1” (unable to care for self and requiring institutional or hospital care). Detailed qualifiers for all QoL numbers are listed in Table 6-2. The majority (77%) of responding patients (patient group) who identified as “person who has been infected with the C. difficile bacterium” reported a QoL of 6 before their first CDI experience (Figure 6-5A). In contrast, respondents who identified as “caregiver/loved one of a person who has been infected with the C. difficile bacterium, but not in a health care provider capacity” (caregiver group) and responded in lieu of the patient, indicated a lower median QoL of 5 before the patient’s first CDI experience, with interquartile ranges of QoL of 3 and QoL of 6 (Figure 6-5B). The impact that CDI has on patients was evident from the reduced QoL participants indicated for their worst experience of CDI (Figure 6-5A). The patient group reported a median QoL of 4 with 37% reporting to be unable to care for self (QoL of 1 and 2), 35% unable to work and/or needing some assistance with normal activities (QoL of 3 and 4) and 28% with minor or no restrictions to their normal activities (QoL of 5 and 6). Again, the 129  caregiver group indicated a lower QoL compared to the patient group with a median QoL of 3 (Figure 6-5B). Interestingly, while 41 % of this group reported a QoL of 1, compared to only 23% in the patient group, 30% of caregivers indicated a QoL of 6 at the worst stage of CDI compared to only 10% of the patient group. Taken together, these observations suggest that someone who experienced a reduced quality of life prior to CDI also experiences on average a more severe impact on their quality of life during active disease. I do not know enough about the conditions under which caregivers responded to the survey. It is conceivable that they responded in lieu of a sicker patient (pre-CDI QoL was lower in this group), or that they responded from imagining the patient’s perspective. In the latter case, the difference between the patients and caregiver groups could indicate that caregiver’s imagination of the patient perspective differs from the actual patient perspective, underscoring the importance of interviewing patients themselves.     130  Figure 6-5 Impact of C. difficile infection on quality of life Data separated by (A) patient and (B) caregiver response. Data presented as violin plot of individual survey responses. Thickness of plot corresponds to number of responses whereas all responses are plotted.  Finally, I was interested in the long-term impact of CDI on people’s lives. To answer this, I asked participants for their current quality of life. I acknowledge that this survey question does not conclusively address long-term impact as participants might have answered the survey at different stages after recovering. I found a dramatically reduced median QoL of 1 in the caregiver group but cannot conclude that this QoL is related to CDI or another disease. I also observed a slightly reduced median QoL of 5.5 and broader interquartile ranges in the patient group. Those patients who received FMT to treat their CDI reported current QoL at the same level as before their first CDI experience and perceived FMT to have cured their CDI. Current clinical trials on FMT include assessing health related quality of life measures and will likely be able to provide further insights into this observation.  For the purpose of my analysis, I assumed that people would not have filled out this survey while experiencing CDI symptoms and therefore “currently” means post-CDI. This is a limitation to my analysis and follow-up research should address this point. While 77% indicated a QoL of 6 pre-CDI, 50% indicated this post-CDI. As analyzed by paired analyses, these respondents now indicate a QoL of 5 (24%), of 4 (18%), and of 3 (8%). From the open-ended question, I learned that many respondents suffer from long-term consequences after their CDI was cured. I did not have enough participants to further stratify my analysis based on time between symptom onset and diagnosis or treatment. Overall, the data demonstrate a significant impact of CDI on patient’s 131  self-assessed QoL and the lasting impact CDI has on a subgroup of patients, highlighting the importance of early diagnosis and a rapid cure.  Table 6-2 Survey qualifiers to query quality of life scores Score Qualifier 1 able to carry on normal activity including work; no special care needed, social activities not restricted 2 able to carry on with most normal activities and able to work, but social activities restricted 3 able to carry on with most activities but unable to work 4 some assistance needed with normal activities 5 unable to care for self but able to stay at home 6 unable to care for self and requiring institutional or hospital care  6.2.5 Most important priority to improve patient experience In the final two survey questions, participants were asked to highlight their most important priority to improving the experience with CDI and to share further comments. I found their answers to overlap sufficiently, compiled both questions and analyzed the answers using inductive thematic analysis. While reading the answers, four major themes emerged, each of which I sub-divided into two to six individual codes (Appendix D). The key emerging themes in my survey were concerns about the healthcare system (63%), concerns about antibiotics (17%), needs from healthcare providers (45%) and impact on life (49%) (Table 6-3). The denominator for percentages represents number of people who responded to the open-ended questions and not all survey respondents. Most of those who answered the question on important priorities provided several themes in their response. After finishing my analysis, I compared the insights from this survey with the key insights described by Madeo and Boyack172 in their 15-patient interview study and found each of their themes reflected across my themes and codes.  132  Of the comments about the healthcare system, I found the most frequent important priority to be time to diagnosis (26%) and speed and selection of treatment (10%), which relates to the importance of timely diagnosis and treatment discussed above. Importantly, 5% highlighted that they received a wrong initial diagnosis while 4% were not referred to a specialist fast enough. A total of 13% observed insufficient prevention protocols such as “I witnessed cleaning personnel not cleaning hospitals properly, while I have been in Emergency at all hours”. Furthermore, 6% experienced poor attitudes by doctors and hospital staff to patients, for example. “I had a nurse berate me in emerge because I "contaminated" the bathroom, screaming that in front of all the patients”. This insight is reflected in Madeo and Boyack’s172 theme as “issues around privacy and dignity”. Of the respondents indicating concerns about antibiotics, five mentioned their CDI was related to a prescription for clindamycin by a dentist.   133  Needs from health care providers were focused around patient education (12%) which aligns with Madeo and Boyack’s172 themes “lack of understanding of the illness” and “lack of knowledge on how the patients got infected”. Furthermore, respondents highlighted the importance of getting cured (9%) which was related to ongoing bowel concerns and recurrence (12%). Many respondents indicated their preference for using pro/prebiotics (9%) and some considered seeing alternative providers (2%) most important to improving their experience.  Half of the respondents commented on the impact of CDI on their lives. Specifically, 12% commented on the severity of symptoms which Madeo and Boyack further sub-divided into “physical suffering” and “lack of control over bowel function”172. Furthermore, 11% indicated changes in daily habits, such as “ensure you always wash hands and clean areas you touch” which is mirrored in “impact on daily activities of living” by Madeo and Boyack172. Finally, 22% of respondents highlighted the emotional impact of CDI on either patients or family and caregivers. One significant answer summarized this as “My mother was isolated in the hospital once she contracted the disease and became so depressed that she decided to stop all medications and dialysis and die”. Overall, the responses support and mirror my insights from the quantitative portion of the survey and highlight a need for further education of health care professionals to compassionately deal with CDI in a fast and effective manner.   134  Table 6-3 Emerging themes and distribution of responses Data presented as percentages of respondents who answered to open ended questions (104 total responses) and percentage of themes Themes n % Concerns about health care system 65 63% Time to diagnosis 27 26% Wrong initial diagnosis 5 5% Speed and selection of treatment 10 10% Faster referral to specialist 4 4% Poor attitudes towards patient 6 6% Insufficient prevention protocols 13 13% Concerns about antibiotics 18 17% Clindamycin 5 5% Antibiotics in general 13 13% Needs from health care providers 47 45% Patient education 12 12% Pro/Prebiotics 9 9% Fecal transplant 3 3% Alternative providers 2 2% Importance of cure 9 9% Ongoing bowel concern and recurrence 12 12% Impact on life 51 49% Severity of symptoms 12 12% Complaints about treatments 6 6% Change in daily habits 11 11% Emotional impact on patient 12 12% Emotional impact on Family/Caregiver 10 10%  Abbreviations: abx, antibiotic; C. difficile, Clostridioides difficile; CDI, Clostridioides difficile infection; NR, no resolution; pbx, probiotic  135  6.3 Conclusion This report is the first survey of Canadian residents with lived experience of CDI and among the first internationally. I recognize that a limitation of this study is potential selection bias, as survey recruitment targeted followers of the GI Society. Patients who experience recurrence or additional gastrointestinal conditions might be more likely to follow the GI Society which might have skewed my results toward those more severe cases. As CDI is a notifiable disease, follow-up studies to this exploratory survey could aim to collaborate with the Public Health Agency of Canada to get a random sample with a larger sample size drawn from all Canadians who experienced CDI to minimize selection bias and improve generalizability.  My results confirm expected clinical parameters and report important insights regarding the lasting impact of CDI on patients’ lives. As previously reported, contraction of CDI was correlated with prior exposure to antibiotics and/or health facilitates and two-thirds of this survey’s respondents experienced at least one other GI condition. This population might be more likely to visit health facilities than the general public, thereby increasing their risk for infection with C. difficile. Time to diagnosis is critical for fast and effective treatment; however, only a minority of respondents received their diagnosis within 7 days after symptom onset. A similar-sized group experienced complete resolution of symptoms within seven days after starting treatment. My sample size was too small to stratify treatment success based on time to diagnosis.     136  Clearly, the acute impact of CDI on patient’s lives is related to the severity of symptoms which bring upon physical suffering and precluded patients from participating in their daily activities. As CDI is a GI condition with symptoms such as severe diarrhea, patients are concerned with their privacy and dignity. Several patients experienced poor attitudes of hospital staff related to their bowel issues and highlighted a need for improved patient-staff communication.  Finally, patients identified a lack of education in multiple groups. They indicated the need for understanding the illness and its potential impact on their future lives. Greater awareness among hospital staff and adherence to cleaning protocols could potentially reduce future infections with C. difficile. Several patients observed poor hygiene and cleaning in their respective health care settings. Additionally, as many patients experienced slow diagnosis and referral to a specialist, which translated to a delay in treatment commencement, I see a need for education of physicians to correctly identify the symptoms. Several patients reported misdiagnosis of CDI for anxiety during ER visits which could be addressed through communication and education strategies within hospitals across Canada. 137  Chapter 7: Conclusion To the best of my knowledge, this thesis represents the first interdisciplinary mixed-method patient-integrated research on developing a new therapy for people living with IBD. I approached my research using methodologies from basic science and mixed-method health research and my findings inform each discipline. Through this work, I highlight the importance of integrating patient-oriented research with basic science.   With my published work55 presented in Chapter 3, I contributed key insights to our understanding of suppressive mechanisms used by two types of regulatory T cells, Tr1 cells and Tregs. This study was the first to demonstrate that Tr1 cells, via secretion of IL-10, directly inhibit inflammasome activation and IL-1 secretion. Surprisingly, Treg were unable to modulate inflammasome activation despite their secretion of IL-10. This might indicate non-redundant immunoregulatory functions of these cell types. Taken together, these data provide further mechanistic insight into Tr1-mediated cell suppression and their potential to uniquely modulate inflammation caused by tissue injury and inflammasome activation. These findings have implications in development of Tr1 cell-based therapy for chronic inflammatory diseases including IBD. I also describe a new protocol for effective isolation and activation of Tr1 cells which will facilitate future research into the biology of these cells. Future development of regulatory T cell-therapies should take this into account when choosing the cell type to be used as therapy.    138  In Chapter 4, I present the first CAR relevant to human IBD. My pre-clinical research findings highlight the potential for this CAR to be translated into clinical practice in the future. From previous studies, I knew that flagellin is readily present and immunogenic in IBD69–72. I confirmed the availability of flagellin in inflamed mouse lamina propria, a key requirement for testing CAR-Tregs in mouse models of IBD. In vitro, I demonstrated that binding of recombinant FliC to FliC-CAR activates CAR-Tregs to proliferate and to suppress Tconv cells. I also present preliminary data on the potential of these CAR-Tregs to survive in an immunocompetent mouse and to protect from DNBS colitis. Drawing from my insights presented in Chapter 3, I conducted preliminary testing of this FliC-CAR in Tr1 cells and found that Tr1 cells are able to express the FliC-CAR. This has opened the path for a new study currently conducted by another graduate student. I also presented a new effective protocol to expand and retrovirally transduce mouse Tregs, the refined version of which is published110. My findings have paved the way for continued development and testing of the FliC-CAR, currently conducted by a postdoctoral fellow. Should the mouse CAR be able to protect from colitis in mouse models, a final approach to testing the therapeutic potential could include humanizing the CAR and testing it in human Tregs expanded under a GMP compliant protocol in a humanized mouse model of colitis109.  While CAR-T cells have been clinically used and FDA-approved in several forms of cancer88–90, no one has conducted clinical studies with CAR-Treg in any disease setting. Given that this FliC-CAR-Treg showed therapeutic potential, I saw room to include patients’ voices to inform future research into CAR-Treg therapy. Engaging patients early in the research process ensures that therapeutic interventions address their needs. With my published work111 in In Chapter 5, I present my findings from a survey to gauge willingness of people living with IBD to try CAR-139  Tregs under two hypothetical settings, as new treatment and in a clinical trial. To the best of my knowledge, this is the first survey on patient willingness and perspectives on CAR therapy. My findings have implications on the development of CAR-Treg therapy for IBD, for autoimmune disease, and as therapy to prevent transplant rejection. In my exploratory survey, I found clear evidence for the acceptability of this treatment approach by people living with IBD, which indicates the feasibility of further therapy development. I suggest refining educational materials to address areas of concern around the process of creating CAR-Tregs and the application of this therapy, such as questions around dose and time of infusion and hospitalization. I further recommend continuous patient involvement throughout the stages of therapy development to ensure the final therapy would be accepted in clinical practice and to address patients’ needs and emerging concerns during clinical testing.   People living with IBD are in regular contact with the health care system and CAR-Treg is likely to be applied in ambulatory care. While rates are falling, people living with IBD experience higher risk of contracting CDI compared to the general population. When acquired during an IBD-related hospitalization, CDI increases the duration of that hospitalization, the risk of requiring colectomy, the likelihood and mortality107. Based on these data, I became interested in understanding people’s lived experience with CDI in the Canadian health care setting. From the survey I analyzed, it was clear that while many people who experienced CDI had to wait for their diagnosis, most perceived time to diagnosis as critical for fast and effective treatment112. During their hospital stay, several patients experienced poor attitudes of hospital staff related to their bowel issues which affected their dignity. It was suggested that this could be overcome by improved patient-staff communication and provider education. Finally, I identified a lasting 140  impact of CDI after resolution on people’s self-assessed QoL. My data also clearly indicate a need for further standardizing prevention and control practices across Canada. Reducing infection rates and the spread of CDI would benefit patient’s lives and reduce the cost of CDI to the Canadian economy as well as reduce the risk of contracting CDI for people living with IBD.  A limitation to the analyses presented in this dissertation stems from the use of statistical hypothesis testing using p-values throughout my published research chapters. Given that my research has been peer-reviewed and published, I discuss these as limitation to my dissertation and recommend future research to adhere to the new recommendations as outlined by the American Statistical Association.   While still standard practice in my discipline, the American Statistical Association has recommended to abandon the practice of solely relying on p-values to determine signifiance180,181. Limitations of the dichotomized interpretation of p < 0.05 inferring statistical significance and thus scientific significance vs p > 0.05 inferring non-significance are plentiful. These include, but are not limited to, the arbitrary choice of the value 0.05, the risk of overstating effects that lead to selective publication and policy decisions, and the continuity of the p-value itself implying that p = 0.049 and p = 0.051 are not statistically different for all but the most extreme cases with very little uncertainty. Furthermore, focusing on the p-value obscures model effect sizes and associated uncertainties, which are both more relevant to interpretation of results.    141  Recommendations for addressing limitations to the p-value by the American Statistical Association focus on the idea of a thoughtful researcher. One that considers their results in light of what has already been known about the topic; one that anticipates practical importance of results and what would be considered a “meaningful effect size” prior to conducting experiments181. If p-values are to be reported, then it should be as numerical values rather than dichotomously and indicated by *. Proposed alternatives to p-values include the reporting of some quantitative measure of uncertainty, such as confidence or credibility intervals, in light of the practical meaningfulness in relation to the research question and hypothesis. In regression analyses, the recommendation is to report the standard error of coefficients (effects) to indicate precision of the estimate produced by the model rather than the p-value181. In summary, I have reflected on the statistical implications of my data in light of these new guidelines and will implement these in future research projects.  Overall, my thesis advances the field of cell-therapy for IBD and has implications for the development of cell-therapy for other immune-mediated disease. I contributed to a better understanding of the mechanism of action of regulatory T cells and developed a new Treg-based therapy with a clear mandate of people living with IBD to continue this development. I recommend continued patient-engagement in any therapy development to ensure early buy-in and the flexibility to be able to address patients’ concerns. I also highlighted shortcomings of how people with CDI, a nosocomial infection, experience the Canadian health care system related to this infection and provided clear recommendations for how to address their concerns.  142  Through conducting interdisciplinary work, I gained valuable insights into disciplinary language and the importance of finding common vocabulary and translating between experts in their respective disciplines. A key recommendation for any future PhD student who aims to embark on an interdisciplinary research journey is to communicate their goals early on, find supportive committee members specializing in different disciplines and to be open to change the composition of their committee throughout the course of their PhD to reflect the disciplinary needs at any given stage. Open-mindedness and the willingness to learn new approaches and appreciate different perspectives is key to interdisciplinary work. 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Remind what’s part of the focus group (incl. feedback on final survey) Remind about privacy 5’ Explain study goals & purpose for focus group Learn about their thoughts regarding the potential therapy we develop. Receive feedback on web survey questions and later improve our questions. Topic 1: IBD experience and treatment -15 min 13:15-13:30 10’ Round robin (try faster than 10’)        Quick introduction by name, type of IBD and how long you’ve had it. then hand out demographic survey for IBD details 5’   Demographic questions survey:  Reminder: answers will be kept confidential.  This will help us better understand your experience with IBD in relation to the later part of the focus group   Topic 2: CAR-Treg therapy -55 min 13:35-15:25 We are currently working on a new therapy and are interested in your thoughts. We also created a web survey and look forward to your feedback to make sure the questions are relevant to the community.  5’ Read over firefighter story (individual) 5’ Answer Q 1 - 4 10’ Based on what you just saw, what are your first thoughts? Brainstorm as group 5’ Answer Q 5 10’ Discuss answers, other emerging side effect concerns? 5’ Answer Q 6 – 9 10’ Discuss phrasing of the questions, any suggestions? What did they understand? 5’ Discuss Q 10 plus segue into final comments Conclusion -5 min 14:25-14:30 Do you have any final comments or suggestions you would like to share with us? Thank participants for their time, participation in discussion and insights. Remind everyone not to share the content of the FG for privacy reasons. Hand out cash envelopes; collect signatures   168  Appendix B  Questionnaire: patient’s perspectives to new IBD therapy development Survey Flow Block: Introduction (2 Questions) Branch: New Branch If If To complete this survey, you must reside in Canada and meet either of the following criteria. Ple... Person living with inflammatory bowel disease Is Selected Standard: Questions about impact of IBD (11 Questions) Standard: Firefighter Story (1 Question) Standard: Concerns/Insights (5 Questions) Standard: Willingness (6 Questions) Standard: Demographic Questions (6 Questions) Standard: Tablet Link (1 Question) Branch: New Branch If If To complete this survey, you must reside in Canada and meet either of the following criteria. Ple... Caregiver/loved one of a person living with inflammatory bowel disease Is Selected Standard: Caregiver Questions about impact of IBD (11 Questions) Standard: Firefighter Story (1 Question) Standard: Caregiver Concerns/Insights (5 Questions) Standard: Caregiver Willingness (6 Questions) Standard: Caregiver Demographic Questions (6 Questions) Standard: Tablet Link (1 Question)    Start of Block: Introduction  Letter  PATIENTS’ PERSPECTIVES TO NEW IBD THERAPY DEVELOPMENT  Thank you for your interest in participating in our survey study. We want to learn about your opinions and thoughts on a new inflammatory bowel disease (IBD) treatment that is being developed. Your insights will help us to design further IBD therapy research and guide development of educational materials for people living with IBD.  You can participate if you either have IBD (Crohn’s disease, ulcerative colitis, or indeterminate colitis), or are a caregiver/loved one of someone with IBD, live in Canada, and are over 14 years of age. Both, patients, and their caregiver/loved ones can participate separately.  You will spend about 7-10 minutes of your time to answer this survey. You may skip questions you are not comfortable answering or end the survey at any time with no negative consequences. At the end of the survey, you will have the opportunity to have your name entered into a draw for one of three tablet computers valued around $300 each.   169  Our survey will be collected anonymously and we will not collect any personal information. If you choose to enter the draw, your personal information will be entered without association to your survey responses. We will erase all personal information after notification of the winners.  If you have any concerns or complaints about your rights as a research participant and/or your experiences while participating in this study, contact the Research Participant Complaint Line in the UBC Office of Research Ethics at 604-822-8598 or if long distance e-mail RSIL@ors.ubc.ca or call toll free 1-877-822-8598. Please reference the study number [H17-01720] when calling so the Complaint Line staff can better assist you.  Thank you in advance for your time. By continuing to the survey, you are consenting to participate in our survey study.  If you have any questions about this survey, you may contact the research team:  Principal Investigator: Dr. Theodore Steiner Professor and Associate Head, Division of Infectious Diseases, University of British Columbia,  Vancouver Coastal Health Research Institute, and BC Children's Hospital Research Institute tsteiner@mail.ubc.ca   PhD candidate: Jens Vent-Schmidt, Dipl. Mol. Med University of British Columbia jensvs@mail.ubc.ca  By continuing to the survey, you are consenting to participate in this research.    Q1 To complete this survey, you must reside in Canada and meet either of the following criteria. Please indicate whether you are: o Person living with inflammatory bowel disease  (1)  o Caregiver/loved one of a person living with inflammatory bowel disease  (2)  o I don’t identify as either  (3)   Skip To: End of Survey If Q1 = 3 End of Block: Introduction  Start of Block: Questions about impact of IBD  Q8 The following questions will talk about your IBD and the impact it has on your life.    170  Q9 Which type of IBD have you been diagnosed with? o Crohn’s Disease  (1)  o Ulcerative Colitis  (2)  o Inflammatory Bowel Disease Unclassified (IBDU) or Indeterminate Colitis (IC)  (3)     Q10 How old (full years) were you when you first experienced symptoms related to IBD? ________________________________________________________________    Q11 How old (full years) were you when you were formally diagnosed with IBD? ________________________________________________________________    Q12 When did you last experience IBD symptoms? o Currently, or within the last 3 months  (1)  o 3-6 months ago  (2)  o 6 months to 1 year ago  (3)  o Greater than 1 year ago  (4)     Q13 How bad were your most recent IBD symptoms? Use the slider bar below to place the slider near the number that best fits your symptoms.  No symptoms Significant symptoms but able to manage Worst Symptoms I can Imagine   0 10 20 30 40 50 60 70 80 90 100  1 (1)      171  Q14 Have you ever had (or do you currently have) any of the following because of your IBD (select all that apply)? ▢ Abscess (pocket of pus in the intestine)  (1)  ▢ Fistula (abnormal connection between two parts of the body, for example bowel and skin)  (2)  ▢ Stricture (narrowing in the intestine)  (3)  ▢ Any type of intestinal surgery  (4)    Display This Question: If Q14 = 4  Q15 What types of surgery have you had performed because of your IBD (select all that apply)?  ▢ Removal of a part of my bowel  (1)  ▢ J-Pouch (after removal of colon and rectum in UC)  (2)  ▢ Temporary ostomy (pouch bag connected to bowels)  (3)  ▢ Permanent ostomy  (4)  ▢ I don’t remember/know  (5)  ▢ Other  (6)  ▢ I have not had intestinal surgery because of my IBD  (7)    Display This Question: If Q15 = 6  Q16 If "other", please specify ________________________________________________________________    172  Q17 Which of the following medications have you tried specifically to treat your IBD at any point since your diagnosis (select all that apply)? ▢ Antibiotics (metronidazole (Flagyl), ciprofloxacin (Ipro))  (1)  ▢ 5-ASA (mesalazine/mesalamine such as Asacol, Mezavant, Salofalk, Lialda, or Pentasa), sulphalazine (Salazopyrin), or olsalazine (Dipentum))  (2)  ▢ Injectable Biologics (infliximab (Remicade, Inflectra), adalimumab (Humira), vedolizumab (Entyvio), ustekinumab (Stelara), golimumab (Simponi))  (3)  ▢ Corticosteroids (prednisone (Deltasone), budesonide (Entocort or Cortiment), methylprednisolone (Solu-Medrol), betamethasone (Betnesol), hydrocortisone (Cortenema))  (4)  ▢ Immunomodulators (azathioprine (Imuran), 6-MP (Purinethol), methotrexate, mycophenolate mofetil (CellCept), cyclosporin(Neoral/Sandimmune), tacrolimus (Prograf))  (5)  ▢ Probiotics   (6)  ▢ Exclusive enteral nutrition (EEN, such as Boost or Ensure without any food)  (7)  ▢ Other  (8)  Display This Question: If Q17 = 8  Q18 If "other", please specify ________________________________________________________________ End of Block: Questions about impact of IBD  Start of Block: Firefighter Story  Story Inflammatory Bowel Disease occurs in flares of inflammation and, as analogy, you can imagine these flares to be like a forest fire. We will use this analogy to explain the concept of the therapy we are discussing.   We don’t know why these fires happen, but we do know that some cells, the pro-inflammatory immune cells, continue to feed the fires. We also know that other types of cells, the anti-inflammatory cells, which can be imagined as firefighter cells, are not able to put out the fires. This might be because there are not enough firefighter cells to stop the fires, the existing firefighter cells might be too exhausted, or they are lacking the special tools to put out these fires.   Most current medications stop not only the inflammation in your gut but will also stop other inflammation that 173  happens. This is why side effects include increased susceptibility to infection. In our fire analogy, this can be imagined as opening a dam and flooding the entire area. The fires are off, but there could be considerable damage to everything in the water’s way. In contrast, the therapy we are discussing takes advantage of very specific genetic approaches to give your anti-inflammatory firefighter cells the right tools to stop only the inflammation in your gut.    For this therapy, we will draw a unit of the patient's blood and isolate the anti-inflammatory firefighter cells. We will then activate them in the laboratory and let them grow so we have enough cells available. In the next step, we will mix the cells with an inactive virus that contains the genetic tools to make the cells specific, grow them in the lab and then re-inject them into the patient. The virus is necessary to deliver this tool to the cells but it is modified so it can never spread to other cells. The tool we bring into the firefighter cells is so specific, that these cells will find the inflammation in the patient's gut, get activated, and reduce all surrounding inflammation, specifically stopping only that forest fire. This genetic approach has been tested successfully in cancer patients and is called “chimeric antigen receptor”.  These specialized anti-inflammatory firefighter cells cannot get activated anywhere else in the patient's body and remain inactive if they don’t find the inflammation.   End of Block: Firefighter Story  Start of Block: Concerns/Insights  Q19 Having read the description and analogy, what are your first thoughts and concerns about our proposed therapy (genetically engineered anti-inflammatory firefighter cells) for IBD?  ________________________________________________________________    Q20 There are two possible options that could happen to these cells, after they successfully treated your flare. Which one would you prefer? o Cells stay in your body in a dormant state, ready to fight a new flare when needed. There might be long-term a risk for them to change.  (1)  o Cells stop existing in your body. You will need a new treatment when you enter the next flare. There may be a risk of developing a severe flare while waiting for the cells to be ready.  (2)     174  Q21 What was your most important consideration in choosing your answer to the last question? ________________________________________________________________   Q22 Many IBD therapies increase the chance of developing side effects of varying severity. We do not know what the side effects of our proposed therapy will be and we anticipate it to be offered as an alternative to injectable biologics (for example Humira, Remicade). Below are some of the known side-effects of injectable biologics. Please let us know how important each of these is to you when choosing a new therapy.  Not at all important Very important   0 10 20 30 40 50 60 70 80 90 100  Allergic reaction to therapy (1)  Anaemia (low red blood cell count) (22)  Arthritis (23)  Bacterial sepsis (infection of whole body) (24)  Blood cancer (leukaemia, lymphoma) (25)  Chronic heart failure/heart attack (26)  Fatigue (27)  Headaches (28)  Hepatitis (29)  Liver failure (30)  Low blood count (31)  Making IBD worse (32)  Nausea or vomiting (33)  Pneumonia (lung inflammation) (34)  Reduced immune system function (35)  Risk of loss of fertility (36)  Serious infections (37)  Skin cancer (38)  Skin rash (39)  Stroke (40)  Sudden blindness (41)    175  Q23 Please list any other side-effects you are concerned about. ________________________________________________________________  End of Block: Concerns/Insights  Start of Block: Willingness  Q24 Please answer the next two questions based on the information we provided, and assuming we addressed your safety concerns in our research.  If, during your last flare-up of IBD symptoms, your GI specialist had offered you a choice between biologics and our therapy with specialized anti-inflammatory firefighter cells.     Q25 Assume this is a clinical trial, our therapy was shown to be safe in people but we don't yet know how well it works. How likely would you have been willing to choose our therapy?     Definitely not Somewhat likely I would have definitely tried it   0 10 20 30 40 50 60 70 80 90 100  1 (1)      Q26 Please let us know why. ________________________________________________________________    Q27 Assume your GI specialist told you that this is a new treatment that was shown to work well and is safe in people.  How likely would you have been willing to choose our therapy?  Definitely not Somewhat likely I would have definitely tried it   0 10 20 30 40 50 60 70 80 90 100  1 (1)     176   Q28 Please let us know why? ________________________________________________________________    Q29 Do you have any additional comments? ________________________________________________________________  End of Block: Willingness  Start of Block: Demographic Questions  Q2 With the following questions, we aim to learn more about the people taking our survey and the population of people living with IBD. We won’t collect any information that will allow us to figure out who you are from your answers.    Q3 How old are you? o 14-18  (1)  o 19-29  (2)  o 30-39  (3)  o 40-49  (4)  o 50-59  (5)  o 60-59  (6)  o 70-79  (7)  o 80-89  (8)  o 90+  (9)     177  Q4 What is your gender? o Female  (1)  o Male  (2)  o Other  (3)     Q5 Which Canadian province of territory do you reside in? o British Columbia  (1)  o Alberta  (2)  o Saskatchewan  (3)  o Manitoba  (4)  o Ontario  (5)  o Quebec  (6)  o New Brunswick  (7)  o Nova Scotia  (8)  o Newfoundland and Labrador  (9)  o Yukon  (10)  o Northwest Territories  (11)  o Nunavut  (12)  o I don’t currently reside in Canada  (13)     Q6 How would you describe the community you live in? o Rural  (1)  o Suburban  (2)  o Urban  (4)    178   Q7 What is the highest level of education you have completed? o Did not complete high school  (1)  o High school diploma or equivalent  (2)  o Some college or university  (3)  o Undergraduate degree  (4)  o Graduate degree  (6)   End of Block: Demographic Questions   179  Appendix C  Questionnaire: Clostridium difficile survey Gastrointestinal Society www.badgut.org Clostridium difficile Survey  Preamble The Gastrointestinal Society represents patients with gastrointestinal diseases and disorders on a variety of health care fronts. We have designed this survey to help us understand your opinions and outlook regarding the effects that infection with the C. difficile bacterium has on the lives of patients. We will use this information anonymously and in aggregate to shape future programming and to inform community members, health care professionals, and health policy decision-makers.  Clostridium difficile (C. difficile) is a virulent, toxin- and spore-producing pathogen that causes inflammation of the inner lining of the colon, incapacitating diarrhea that frequently requires hospitalization and, in the most serious cases, death. The spore form of the pathogen is resistant to gastric acid, antibacterial soaps, and alcohol-based hand sanitizers, and can survive for months on surfaces. C. diff has been associated with numerous outbreaks across Canada.  1. To complete this survey, you must meet one of the following criteria: • person who has been infected with the C. difficile bacterium • caregiver/loved one of a person who has been infected with the C. difficile bacterium, but not in a health care provider capacity [Can’t continue if one of these categories is not selected]  The following questions are asking for the patient experience, so please answer as the patient, or on behalf of the patient for whom you are responding.  Personal Information Please note: we will keep all personal information strictly confidential and use it only for the purposes of facilitating this survey.  2. Age [CHECK ONE] • <19 • 19-29 • 30-39 • 40-49 • 50-59 • 60-69 • 70-79 • 80-89 • >89   180  3. Gender [CHECK ONE] • female • male • other • prefer not to say  4. In which Canadian province or territory do you live? [DROP-DOWN BOX] • Alberta • British Columbia • Manitoba • New Brunswick • Newfoundland and Labrador • Northwest Territories • Nova Scotia • Nunavut • Ontario • Prince Edward Island • Quebec • Saskatchewan • Yukon • I do not live in Canada (please specify country)  5. Are you a health care professional? [CHECK ONE] • yes • no  6. If yes, do/did you work in a: [MULTICHECK] • long-term care facility • hospital • clinic/physician’s office • dental office • other health care setting (please specify) • none of the above • not a health care professional    181  C. difficile Questions 7. What symptoms or complications have you experienced at any time while infected with C. difficile? [MULTICHECK] • diarrhea • watery, severe diarrhea • fecal incontinence • fever • loss of appetite • weight loss • nausea • abdominal pain/tenderness • fatigue • pseudomembranous colitis • bowel perforation • bowel surgery • sepsis • C. difficile infection recurrence • resulted in the death of the person you were caring for • other (please specify)  8. How many times have you had C. difficile infection? [CHECK ONE] • 1 • 2 • 3 • 4 • 5+ (please specify) • I don’t remember/know  For the remaining questions on this page, answer based on your experience with your FIRST C. difficile infection only.  9. How long was it from onset of diarrhea until you received a diagnosis? [CHECK ONE] • <48 hours • 2-5 days • 6-10 days • 11-30 days • >30 days • I don’t remember/know  10. Had you been taking antibiotics in the three months preceding diagnosis? [CHECK ONE] • yes • no • I don’t remember/know   182  11. Who prescribed you the antibiotics in the three months preceding diagnosis? [CHECK ONE] • physician in clinic or office • physician in hospital • naturopath • dentist • pharmacist • other (specify) • I wasn’t taking antibiotics in the three months preceding diagnosis  12. Did your first experience with C. difficile infection begin following any of these situations: [MULTICHECK] • an existing severe illness • an abdominal surgical procedure • having another gastrointestinal infection • childbirth • a flare of Crohn’s disease or ulcerative colitis • bone marrow/stem cell transplant • undergoing cancer chemotherapy • taking antibiotics for a chronic condition unrelated to C. difficile infection (especially over a prolonged period of time) • taking medication to reduce stomach acid • taking medication to suppress your immune system, e.g., for an autoimmune disease or due to an organ transplant • in hospital for some other reason • none of the above  13. Which treatment(s) did you use to combat your first C. difficile infection? [MULTICHECK] • antibiotics: (e.g., metronidazole, vancomycin, fidaxomicin) • fecal transplant from a relative • fecal transplant from a non-relative • probiotics • other (please specify) • I don’t remember/know  14. How many days after starting treatment did your symptoms completely resolve (even if you relapsed later, answer with how long it took to resolve initially)? • <4 • 4-7 • 8-14 • >14 • did not resolve • did not treat  15. Did your symptoms begin while: [MULTICHECK] • in a hospital • in a long-term care home • living at home • travelling • other (please specify)   183  16. If your symptoms began while in a hospital, did your first experience with C. difficile infection prolong your stay? [CHECK ONE] • yes • no • not applicable  17. Did your first experience with C. difficile infection require hospitalization? [CHECK ONE] • yes • no • I was already in the hospital  18. How long was your hospital stay? [CHECK ONE] • 1-3 days • 4-6 days • 1-2 weeks • 3-4 weeks • other (please specify) • I was never hospitalized  C. difficile Questions Continued 19. Do you have any of these gastrointestinal conditions? [MULTICHECK] • diverticular disease • irritable bowel syndrome • gastroesophageal reflux disease (GERD) • inflammatory bowel disease (e.g., Crohn’s disease, ulcerative colitis) • celiac disease • none of the above  20. How would you rate your quality of life before your first C. difficile infection? [CHECK ONE] • able to carry on normal activity including work; no special care needed, social activities not restricted • able to carry on with most normal activities and able to work, but social activities restricted • able to carry on with most activities but unable to work • some assistance needed with normal activities • unable to care for self but able to stay at home • unable to care for self and requiring institutional or hospital care  21. How would you rate your quality of life at your worst with C. difficile infection? [CHECK ONE] • able to carry on normal activity including work; no special care needed, social activities not restricted • able to carry on with most normal activities and able to work, but social activities restricted • able to carry on with most activities but unable to work • some assistance needed with normal activities • unable to care for self but able to stay at home • unable to care for self and requiring institutional or hospital care   184  22. How would you rate your quality of life currently? [CHECK ONE] • able to carry on normal activity including work; no special care needed, social activities not restricted • able to carry on with most normal activities and able to work, but social activities restricted • able to carry on with most activities but unable to work • some assistance needed with normal activities • unable to care for self but able to stay at home • unable to care for self and requiring institutional or hospital care  23. Thinking of any or all subsequent experience(s), which treatments have you used to combat C. difficile infection? [MULTICHECK] • antibiotics: (e.g., metronidazole, vancomycin, fidaxomicin) • fecal transplant from a relative • fecal transplant from a non-relative • probiotics • other (please specify) • I don’t remember/know • I only had C. difficile infection one time  24. In your opinion, what is the most important priority regarding improving your experience with C. difficile infection? [BOX]  Finish Thank you for taking part in this survey!  If you would like to be entered into a draw to win an iPad mini, please provide the information requested below. We will keep all of your information confidential and will never share it or sell it to any outside party. Your name and address will be detached from your answers before we conduct the survey analysis. To accept, please complete this section. If you are not interested, please leave this section blank. [TEXT BOXES] Salutation First Name Last Name Address Line 1 Address Line 2 City Province Postal Code Email Phone  25. If you have any additional comments, you can enter them here: [BOX]  Thank you for completing our survey!  185  Appendix D  Codebook: thematic analysis for Clostridium difficile survey Themes Codes   Inclusion Exclusion Examples Concerns about healthcare system Time to diagnosis T Comments regarding time to initial diagnosis Comments on time between first diagnosis and subsequent diagnosis of CDI or other illnesses Much quicker diagnosis    Wrong initial diagnosis W Comments regarding quality of diagnosis or specific statement of wrong diagnosis   Better faster diagnosis Cdiff was misdiagnosed for an anxiety attack    Speed and selection of treatment S Comments referring to correct treatment choice (or mentioning of wrong treatment). Comments on the importance of short time between diagnosis and treatment Comments on medication for pain or other conditions Having a Doctor with knowledge of the right antibiotic for this condition Identifying the possible infection and expediting lab work to confirm a diagnosis and move to treating sooner   Faster referral to specialist F Comments indicating experience with slow referral to a specialist. Comments indicating necessity for fast referral to specialist Comments on seeing a specialist without mentioning time of referral being referred to an internal disease specialist immediately   Poor attitudes towards patient P Comments on patients being mistreated or not cared for appropriately by a healthcare provider   Attitudes of nurses at hospital. They acted almost repulsed I was treated awfully because I had c-diff, even had a nurse berate me in emerge because I "contaminated" the bathroom 186  Themes Codes   Inclusion Exclusion Examples   Insufficient prevention protocols I Comments regarding the lack of proper prevention protocols in the Canadian healthcare setting Comments regarding the lack of prevention outside the healthcare setting or in healthcare settings outside of Canada Hospital staff have to be more proactive in making sure extra care is given to stop any out breaks Prevention is key Concerns about antibiotics Clindamycin C Comments linking clindamycin to perceived reason for CDI   Was prescribed clindamycin by a dentist.   Antibiotics in general A Comments linking antibiotics as reason for having contracted CDI or recurrent flare off CDI Comments on antibiotic use as therapy for CDI. Specific comments on clindamycin. c diff outbreak occurred after being prescribed a second-round antibiotics for a severe hand injury Needs from healthcare providers Patient education E Comments on patient or family education, for example on CDI, long term consequences, or suggested changes of life habits Comments on educating healthcare providers or the public Education of families and caregivers of risks to vulnerable people in hospital setting  Education about potential long-term effects after clearing the infection   Pro/Prebiotics PR Comments on probiotics either during CDI therapy or post CDI   Dad should have been given a prescription for Probiotics immediately    Fecal transplant FT Comments mentioning fecal transplants a cure   Identifying it and having the transplant. That stopped it   Alternative providers ALT Comments and recommendations on seeing alternative healthcare providers   See a naturopath immediately   Importance of cure CU Comments highlighting the importance of being cured or Comments coded as "severity of symptoms" such as diarrhea Getting rid of it   187  Themes Codes   Inclusion Exclusion Examples stopping recurrence Finding a permanent solution to be free of potential re-occurrence   Ongoing bowel concern and recurrence O Comments on long-term consequences either directly related to CDI or related symptoms such as fatigue Any comment on recurrence my grandma had it she died due to the long-term effects of c diff   However, not sure that my energy level has ever been the same since Impact on life Severity of symptoms SY Comments on CDI symptoms such as diarrhea Comments on the emotional impact of CDI The most important thing is to be able to control the Diarrhea and stomach pain   Complaints about treatments CT Comments on side effects or low efficiency of treatments   The medication made me really sick   The side effects of medication basically wiped out my white blood cell count   Change in daily habits D Comments on change, delay and return to daily activities due to CDI Comments referring to CDI symptoms such as fatigue, ongoing bowel issues Ensure you always wash hands and clean areas you touch   Emotional impact EI Comments interpreted as emotional impact on the patient Comments on emotional impact on anyone else than the patient once she contracted the disease and became so depressed    EF Comments interpreted as emotional impact on relatives of the patient Comments interpreted as emotional impact on the patient  It has affected our social life, ..., worry   c diff had a devastating effect on the family  

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