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The role of natural killer cells in autoimmune diabetes Lee, I-Fang 2010

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 THE ROLE OF NATURAL KILLER CELLS IN AUTOIMMUNE DIABETES  by  I-FANG LEE M.Sc., The University of British Columbia, 2003   A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES  (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  October 2010   © I-Fang Lee, 2010 
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 ABSTRACT  
 Type 1 diabetes (T1D) is caused by the autoimmune destruction of insulin-producing β- cells with consequent hyperglycemia and serious chronic complications. Both innate and adaptive immune responses are involved in the pathogenesis of this disease. Studies in animal models and in patients with T1D have shown that natural killer (NK) cells are involved both in disease progression and in disease protection, thus suggesting that NK cells can represent a potential therapeutic target in this disease, once the contribution of NK cells to islet immunity has been fully elucidated. Using the model of complete Freund’s adjuvant (CFA) injection, which has been reported to efficiently prevent diabetes in non-obese diabetic (NOD) mice, the role of NK cells in diabetes was investigated. Results showed that CFA immunization of NOD mice markedly increased frequency and function of NK cells. Notably, the adoptive transfer of diabetes into NOD/SCID recipients clearly implied that IFNγ secreted from NK cells mediated the protection effect of CFA, suggesting that the NK cell is a critical mediator for diabetes protection. Furthermore, investigation of the mechanism by which CFA activates NK cells found that this activation was through a CD1d-dependent but MyD88-independent pathway. These experiments also showed an up-regulation of NKG2D expression of NK cells and this might possibly be through decreased surface expression of NKG2D ligand.           Similar to the NOD mouse, in which NK cells exhibit numeric and functional abnormalities, experiments in peripheral blood mononuclear cells (PBMCs) from patients with T1D also demonstrated that NK cells from T1D patients are present at reduced frequencies and show diminished responsiveness to the cytokines IL-2 and IL-15. Interestingly, cell surface analysis reveals that NKG2D ligands are also expressed on 
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 human NK cells and that unlike in non-diabetic controls, T1D NK cells fail to down- regulate NKG2D ligands upon activation by cytokines. Furthermore, NK cells from patients exhibit decreased NKG2D-dependent cytotoxicity and cytokine secretion, as well as reduced NKG2D-mediated signaling pathways. Collectively, these results suggest that NK cell dysfunction and aberrant NKG2D signaling may contribute to the pathogenesis of T1D. 
 
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 TABLE OF CONTENTS ABSTRACT....................................................................................................................... ii TABLE OF CONTENTS ................................................................................................ iv LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES ......................................................................................................... ix LIST OF ABBREVIATIONS AND ACRONYMS ....................................................... xi ACKNOWLEDGEMENTS .......................................................................................... xiv 
  CHAPTER 1 INTRODUCTION......................................................................................1 1.1 THE IMMUNE SYSTEM .......................................................................................2   1.1.1 Cells comprising the immune system ................................................................3   1.1.2 Innate immunity ................................................................................................4       1.1.2.1 Receptors for innate immune response ..................................................5       1.1.2.2 Cells of the innate immune system.........................................................6  1.1.3 Link between innate and adaptive immunity......................................................8  1.1.4 Adaptive immunity .............................................................................................8       1.1.4.1 Cells of the adaptive immune system .....................................................8       1.1.4.2 Receptors for adaptive immune system..................................................9 1.2 AUTOIMMUNITY................................................................................................10 1.3 TYPE 1 DIABETES...............................................................................................12   1.3.1 The clinical presentation of T1D ....................................................................12   1.3.2 The etiology and cause of T1D .......................................................................12   1.3.3 T1D is an autoimmune disease .......................................................................12   1.3.4 The role of T and B cells in T1D.....................................................................13   1.3.5 Animal models of T1D ....................................................................................15 1.4 ANIMAL MODEL: THE NON-OBESE DIABETIC (NOD) MOUSE ............16   1.4.1 Characteristics and pathogenesis of NOD mouse ..........................................16 
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   1.4.2 Cell-mediated autoimmunity in the NOD mouse ............................................17   1.4.3 Preventive studies in the NOD mouse.............................................................18 1.5 COMPLETE FREUND’S ADJUVANT ..............................................................19   1.5.1 Mechanisms of CFA protection against autoimmune diabetes.......................19 1.6 NATURAL KILLER (NK) CELLS .....................................................................21   1.6.1 NK receptors ...................................................................................................22   1.6.2 NK cells in animal model of autoimmune diabetes.........................................24   1.6.3 NK cells in human T1D...................................................................................25 1.7 NATURAL KILLER T (NKT) CELLS ...............................................................26   1.7.1 NKT cells in autoimmune diabetes .................................................................27   1.7.2 NKT cells regulate immune responses............................................................28 1.8 THESIS OBJECTIVES.........................................................................................29   1.8.1 NK cells in the mouse model of autoimmune diabetes....................................29   1.8.2 NK cells in human patients with T1D .............................................................30  CHAPTER 2 MATERIALS AND METHODS.............................................................32 2.1 MATERIALS .........................................................................................................33   2.1.1 Cell lines .........................................................................................................33   2.1.2 Mice.................................................................................................................33   2.1.3 Antibodies .......................................................................................................33 2.2 METHODS .............................................................................................................34   2.2.1 CFA immunizations and assessment of diabetes ............................................34   2.2.2 Flow Cytometry...............................................................................................34   2.2.3 Purification and in vitro cultures of human NK cells .....................................35   2.2.4 Cytotoxicity .....................................................................................................35   2.2.5 ELISpot assay..................................................................................................36   2.2.6 BrdU treatment and immunostaining with anti-BrdU antibody .....................36   2.2.7 Selective depletion of mouse NK cells ............................................................37   2.2.8 Purification of mouse NK cells .......................................................................38   2.2.9 Adoptive transfer of mouse model...................................................................38   2.2.10 Human patient recruitment and sample collection.......................................38 
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   2.2.11 CFSE labeling...............................................................................................39   2.2.12 Cell signaling studies....................................................................................39   2.2.13 Lysate preparation, microarray production, and data acquisition ..............40   2.2.14 Array analysis ...............................................................................................41   2.2.15 Statistical analyses........................................................................................42  CHAPTER 3 REGULATION OF AUTOIMMUNE DIABETES BY COMPLETE FREUND’S ADJUVANT (CFA) IS MEDIATED BY NK CELLS.............................43 3.1 INTRODUCTION AND RATIONALE...............................................................44 3.2 RESULTS ...............................................................................................................44   3.2.1 CFA immunization prevents diabetes in NOD mice .......................................44   3.2.2 CFA immunization prevents the accumulation of β-cell-specific CTL in NOD            mice .................................................................................................................46   3.2.3 CFA induces NK cells to accumulate in blood ...............................................46   3.2.4 CFA increases cytotoxicity and IFNγ secretion by NK cells ..........................50   3.2.5 CFA increases NKG2D-mediated function of NK cells through down-            regulation of NKG2D ligand ..........................................................................50   3.2.6 CFA protection from diabetes is mediated by IFNγ secreted by NK cells......52   3.2.7 NK cytotoxicity is not required for the protective effects of CFA...................57  CHAPTER 4 CFA-MEDIATED STIMULATION OF NKT CELLS PROTECTS AGAINST AUTOIMMUNE DIABETES THROUGH THE SEQUENTIAL ACTIVATION OF NK CELLS......................................................................................61 4.1 INTRODUCTION AND RATIONALE...............................................................62 4.2 RESULTS ...............................................................................................................63   4.2.1 NKT cells are required for NK cell activation and mobilization by CFA ......63   4.2.2. NKT cells are activated by mycobacterial components of CFA ....................66   4.2.3 NKT cell activation by CFA is independent of MyD88...................................70   4.2.4 Activation of NKT cells by CFA is dependent on CD1d expression ...............72   4.2.5 NKT cells are required for CFA-mediated protection from autoimmune 
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            diabetes ...........................................................................................................74  CHAPTER 5 NATURAL KILLER CELLS FROM SUBJECTS WITH T1D HAVE DEFECTS IN NKG2D-DEPENDENT FUNCTION AND SIGNALING...................78 5.1 INTRODUCTION AND RATIONALE...............................................................79 5.2 RESULTS ...............................................................................................................80   5.2.1 NK cells from T1D patients are present at reduced frequencies ...................80   5.2.2. NK cells from T1D patients respond poorly to IL-2 and IL-15 .....................81   5.2.3 Activated T1D NK cells fail to down-regulate the NKG2D ligands MICA/B.....   ..................................................................................................................................85   5.2.4 T1D LAK cells exhibit reduced cytotoxicity, IFNγ secretion, and NKG2D           function ............................................................................................................87   5.2.5 T1D LAK cells exhibit defective NKG2D signaling........................................90 
 CHAPTER 6 DISCUSSIONS AND CONCLUSIONS .................................................94 6.1 DISCUSSIONS.......................................................................................................95   6.1.1 NK cells in autoimmune diabetes....................................................................95   6.1.2. NKG2D and NKG2D ligands in NK cell activation ......................................96   6.1.3 The role of innate receptors in the protective effect of CFA...........................98   6.1.4 The mechanism of CFA in autoimmune diabetes..........................................100   6.1.5 Mycobacteria in autoimmune diabetes .........................................................103 6.2 FUTURE DIRECTION.......................................................................................104 
 REFERENCES...............................................................................................................106 APPENDIX.....................................................................................................................121 ANIMAL CARE, BIOSAFETY AND ETHICS CERTIFICATES.......................121  

 
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 LIST OF TABLES Table 5.1   Characteristics of the T1D subjects and healthy control groups ....................80 
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 LIST OF FIGURES
 Figure 3.1   Effect of CFA on incidence of diabetes in NOD mice……………………...45 Figure 3.2   Effect of CFA on β-cell-specific CTL ...……………………………………47 Figure 3.3   Effect of CFA on NK cell frequency ……………………....……………….49 Figure 3.4   Effect of CFA on NK cell function ………………………………………...51 Figure 3.5   Effect of CFA on NKG2D ligand expression in NK cells …………………53 Figure 3.6   Characterization of asialo GM1-positive spleen cells ...……………………55 Figure 3.7   Prevention of diabetes by NK cells is dependent on IFNγ ....………………58 Figure 3.8   NK cytotoxicity is not involved in the protective effect of CFA in diabetes                    ……………………………………………………………………………….60 Figure 4.1   NKT cells are required for NK cell mobilization by CFA………………….65 Figure 4.2   NKT cells are required for NK cell activation by CFA …………………….67 Figure 4.3   NKT cells are activated by CFA ...…………………………………………69 Figure 4.4   NKT cell activation by CFA is independent of MyD88 ……………………71 Figure 4.5   NKT cell activation by CFA is independent of MyD88 ……………………73 Figure 4.6   The activation of NKT cells by CFA is dependent on CD1d expression …..75 Figure 4.7   NKT cells are required for CFA-mediated protection from autoimmune                     diabetes …………………………………………………………………….77 Figure 5.1   NK cells from PBMC of T1D subjects are present at reduced frequencies                    ……………………………………………………………………………….82 Figure 5.2   T1D NK cells are poorly responsive to IL-2/IL-15 stimulation ……………84 Figure 5.3   T1D NK cells fail to down-regulate the NKG2D ligands MICA/B upon                     activation ……………………………….…………………………………..86 
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 Figure 5.4   T1D LAK cells exhibit reduced cytotoxicity, IFNγ secretion, and NKG2D                     function ………………………………………….…………………………89 Figure 5.5   DAP10 phosphorylation at its YINM motif results in the activation of the                     PI3K pathway ………………………………………………………………90 Figure 5.6   T1D LAK cells exhibit defective NKG2D signaling ………………………92 Figure 5.7   T1D LAK cells exhibit defective NKG2D signaling ……………………….93 
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 LIST OF ABBREVIATIONS AND ACRONYMS 7AAD 7-amino-actinomycin αGC alpha-galactosylceramide Ab antibody APC antigen presenting cells ATCC American Type Culture collection BB rat Bio-breeding rat BCG Bacille Calmette-Guerin BM bone marrow BrdU 5-bromo-2-deoxyuridine BSA bovine serum albumin CD clusters of differentiation CFA Completer Freund’s adjuvant CTL cytotoxic T lymphocytes DC dendritic cells DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin DM diabetes mellitus E/T ratio effector/target ratio ELISpot Enzyme-linked immunosorbent spot ERK extracelluar signal-regulated kinase FBS fetal bovine serum GM-CSF granulocyte macrophage colony-stimulating factor HLA human leukocyte antigen HRP horseradish peroxidase HSP heat shock protein IDO indoleamine2,3-dioxygenase IFA incomplete Freund’s adjuvant IFN interferon Ig immunoglobulin IL interleukin 
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 ITAM immunoreceptor tyrosine-based activation motif ITIM immuno tyrosine-based inhibitory motif KIR killer cell immunoglobulin-like receptor LAK lymphokine-activated killers LPS lipopolysaccharide MAPK mitogen-activated protein kinase MEK mitogen-activated protein kinase kinase MFI mean fluorescent intensity MICA, B MHC class I-chain related protein A and B Mtb Mycobacteria tuberculosis MHC major histocompatibility complex MULT1 murine ULBP like transcript 1 MyD88 myeloid differentiation primary response gene 88 NCR natural cytotoxicity receptor NK natural killer NKG2D NK cell group 2D NKT Natural killer T NOD non-obsese diabetic NS natural suppressor PBMC peripheral blood mononuclear cells PBS phosphate-buffered saline PCR polymerase chain reaction PE phycoerythrin PI3K phophoinositide 3-kinase PLC phospholipase C PMA phorbol myristate acetate PAMP pathogen-associated molecular pattern PRR pathogen recognition receptor RAE-1 retinoic acid early inducible-1 gene RAG recombinantion-activating gene RBC red blood cells 
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 RNA ribonucleic acid RT room temperature SDS rodium dodecyl sulfate SOCS-1 suppressor of cytokine signaling-1 Syk spleen tyrosine kinase T1D type 1 diabetes T2D type 2 diabetes TCR T cell receptor Th T helper TLR toll-like receptor TNF tumor necrosis factor ULBP UL16 binding protein Y tyrosine 

 
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 ACKNOWLEDGEMENTS  I would like to sincerely thank my supervisor, Dr. Rusung Tan, for the opportunity to join his laboratory, to explore scientific research as well as for his guidance, insight and the constant support during my studies. I would also like to thank Dr. John P. Priatel and Dr. Peter van den Elzen for their intellectual contribution to this work. I wish to acknowledge the past and present members of Tan lab and van den Elzen lab for valuable discussions and technical support. There are many other people that I would like to thank for contributing in many different ways: my parents, Chin-Li Lee and Mei-Hsing Lee-Lin; my aunt, Cindy Hsing-Chi Lee; my sister, Yi-Jen Lee; Derek A. Birch, and also Claire Harrison and the animal unit staff, Dr. Jan P. Dutz, Dr. Dina Panagiotopoulos and Pamela Lutley. Each of you has supported and inspired. 
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        CHAPTER  1 INTRODUCTION              
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 1.1 THE IMMUNE SYSTEM The environment contains a wide variety of pathogenic microbes including viruses, bacteria, fungi, protozoa and multi-cellular parasites. These microbes can cause diseases and even kill their hosts. The immune system has developed to combat infectious agents, to protect the host from pathogens and to avoid permanent damage. It also monitors the cells of the host to recognize mutant cells and to avoid tumour development. Microorganisms come in many different forms, and different immune responses are required to deal with each type. The immune system developed multiple protective mechanisms consisting of soluble factors and cells with distinct roles in defense against infections. The site of the infection and the type of pathogen largely determine which immune response will be active.           Any immune response involves first recognizing the pathogen or other foreign, hazardous material, and second neutralizing or eliminating it. The coordinated cooperation of soluble mediators and cells leads to an adequate immune response by initiating inflammation and elimination of the pathogens or mutant cells.           The immune responses can be classified into two categories: innate immune responses and adaptive immune responses. The important difference between these two mechanisms is that an adaptive immune response is highly specific for a particular pathogen, while innate immunity recognizes a broad, conserved structure of pathogens. Innate responses are not altered on repeated exposure to one infectious agent, while the adaptive response improves with each successive encounter with the same pathogen leading to immunological memory. A key component in establishing and coordinating immune response is cytokines. Cytokines are soluble factors released by cells endowed 
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 with the property to initiate, modulate, and regulate immune responses.  1.1.1 Cells comprising the immune system Immune responses are mediated by a variety of specialized cells with diverse functions and by soluble molecules that immune cells secrete. These cells arise from a single progenitor, the pluripotent hematopoietic stem cell in the bone marrow. These pluripotent cells divide into two types of progenitor stem cells—the common lymphoid progenitor and the myeloid progenitor cell. The lymphoid progenitor cell develops into natural killer (NK) cells, T lymphocytes (T cells), and B lymphocytes (B cells). Unlike T and B cells, NK cells lack antigen specificity. The common myeloid progenitor cell gives rise to macrophages, neutrophils, dendritic cells (DCs), basophils / mast cells, eosinophils, and red blood cells. The number and localization of myeloid cells is dynamic and largely depends on the occurrence of an immune response and the cytokines and chemokines produced thereby. Myeloid immune cells circulate in the blood and enter tissues only at sites of infection or inflammation. Neutrophil numbers are massively increased during infection and are recruited to sites of infection and inflammation to phagocytose bacteria. Immature monocytes also enter tissues, where they differentiate into macrophages. Macrophages are found in all organs and tissues. Immature DCs travel through the blood to enter peripheral tissues. Upon encountering a potential antigen, they mature and migrate to lymphoid tissues, where they activate antigen-specific T cells. NK cells are important for eradication of pathogen-infected cells, especially during the early phase of infection (1). NK cells recognize cell surface changes that occur on a variety of virally infected cells and tumour cells and are believed to be involved in tumour surveillance (2). 
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           B cells combat extracellular pathogens in an antigen-specific way. Having recognized their specific antigen, B cells multiply and differentiate into plasma cells which produce large amounts of antibodies. T cells can be characterized by the cell surface receptors they express. The majority of T cells express a T cell receptor (TCR) composed of α and β chain (αβ T cells). A minor group of T cells displays a TCR composed of γ and δ chain (γδ T cells). γδ T cells seem to be important in immune responses to bacteria, but their role in adaptive immunity remains unclear (3, 4). T cells can be further characterized by the specific expression of the co-receptors CD4 and/or CD8. CD4+ and CD8+ T cells differ fundamentally from each other and perform distinct regulatory and effector functions. T and B cells are located in the central lymphoid organs, the thymus (T cells), and the bone marrow (B cells). It is in these compartments where they develop and differentiate. Mature T and B cells can be detected in the peripheral lymphoid organs (for example, spleen, lymph nodes, Peyer’s patches). Here, they develop further into effector cells. During infection, effector T cells are found notably at the site of inflammation. Cells of the lympoid and myeloid lineage interact and function in an orchestrated fashion to establish a successful immune response. Immune responses can be divided into innate and adaptive immune responses.  1.1.2 Innate immunity Innate immune responses are initiated instantaneously after recognition of foreign antigens. It is the first and rapid, although relatively unspecific, response of the immune system to an invading pathogen. Innate immune responses are essential for mounting an effective immune response and for establishing an adaptive immune response. 
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 1.1.2.1 Receptors for innate immune response Microorganisms display repeating patterns of molecular structure on their surface as well in their nucleic acids. Bacterial DNA, for example, contains unmethylated repeats of CpG. The innate immune system recognizes such pathogens by means of pattern recognition receptors (PRRs) that bind to features of these regular patterns. This strategy is based on the detection of a limited set of conserved molecular patterns (pathogen- associated molecular patterns (PAMPs) that are unique to the microbial world and invariant among entire classes of pathogens (5).           Innate recognition of PAMPs through toll-like receptors (TLRs) initiates an inflammatory response characterized by the recruitment of cells to sites of infection, which augments the killing of invading pathogens. Activation of TLRs induces the expression of genes encoding chemokines, chemokine receptors, and integrins that regulate cell migration to the sites of inflammation and initiate responses to inflammation (6, 7).           Stimulation of TLRs through pathogen products also induces the activation of macrophages and DCs and leads to the induction of several important mediators of innate immunity such as cytokines (8, 9). Additionally, surface expression of co-stimulatory molecules such as B7.1 (CD80) and B7.2 (CD86) on macrophages and DCs is evoked by the TLR pathway. Along with antigen presentation by MHC class II proteins on DCs and macrophages, these molecules activate CD4+ T cells and are needed to establish an adaptive immune response.           Most mammalian species have 10 to 15 TLRs that detect multiple PAMPs (10), including bacterial lipoproteins and lipoteichoic acids (detected by TLR2), double- 
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 stranded RNA (TLR3), lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), single- stranded viral RNA (TLR7), and the unmethylated cytosineguanine dinucleotide (CpG) DNA of bacteria and viruses (TLR9). TLRs 1, 2, 4, 5, and 6 seem to specialize in the recognition of mainly bacterial products that are not made by the host. Especially, TLR2 detects bacterial cell wall components like peptidoglycan, lipoteichoic acid, and lipoarabinomannan of gram-positive bacteria and mycobacteria (11). TLRs 3, 7, 8, and 9, in contrast, specialize in viral detection, are localized to intracellular compartments (12), and detect viral nucleic acids in late endosome-lysosome. Pattern recognition receptors activate conserved host defense signaling pathways that control the immune responses in a wide variety of cells. 

 1.1.2.2 Cells of the innate immune system Activation of innate immunity is initiated by recognition of antigenic, non-self structures by receptors expressed on immune cells. Macrophages located in the submucosal tissue are usually the first cells to encounter pathogens. They are supported by massive infiltration of neutrophils. Macrophages and neutrophils are specialized in phagocytosis and subsequent killing of the invading pathogens. Macrophages and neutrophils recognize pathogens by means of their cell surface receptors, such as mannose receptors or scavenger receptors, which enable them to discriminate between self and non-self structures. Further, they express receptors for antibody and complement, which increases phagocytosis of antibody- or complement-coated microbes.           DCs are, together with B cells and macrophages, professional antigen-presenting cells (APCs) and are thought to be key players in initiating an adaptive immune response 
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 (13). Immature DCs are specialized in antigen capture and processing. Upon antigen update, DCs mature, display large amounts of major histocompatibility complex (MHC)- peptide complexes at their surface, up-regulate their co-stimulatory molecules, and migrate to lymphoid organs, where they liaise with and activate antigen-specific T cells. DCs are the most potent antigen-presenting cells, and their activities can be induced by infectious agents and inflammatory products, so that DCs are mobile sentinels that bring antigens to T cells and express co-stimulators for the induction of immunity (14).           NK cells are innate immune cells that mediate cellular cytotoxicity and produce chemokines and inflammatory cytokines such as interferon-gamma (IFNγ) and tumor necrosis factor-alpha (TNFα) (15, 16). NK cells are usually constitutively active and, shortly after the initiation of infection, they undergo clonal expansion by proliferation. The resulting activated NK cells persist over a period of a few days (17). Although the expanded NK cell populations may help clear the virus, they do not, unlike T cells, provide long-lasting NK cell-mediated memory. NK cells also participate directly in adaptive immune responses, by interacting with DCs and by triggering T cell responses. For instance, induction of DC maturation to produce TNFα and IL-12 and up-regulation of co-stimulatory ligands are triggered by NK cells (18). Moreover, NK cells proliferate and acquire cytotoxic activity and the capacity to produce IFNγ through the interaction with DCs (19).           Apart from activation of other cells of innate immunity, NK cells also enhance induction of CD8+ T cell responses (20). T cell responses are influenced by IFNγ derived from NK cells, which promotes antigen processing and presentation to T cells and T helper type 1 (Th1) cell polarization. 
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 1.1.3 Link between innate and adaptive immunity Adaptive immunity commences with DCs capturing microbial antigens in the peripheral tissues. Subsequently, DCs migrate to the draining lymph nodes to present the processed antigens to naive T lymphocytes in the context of antigen-presenting molecules such as MHC molecules and CD1 molecules. During transit, DCs undergo a maturation program that provides the cells with the ability to stimulate naive T lymphocytes. Inside the lymph nodes, DCs contact antigen-specific T cells and induce their activation and differentiation into effector cells. T cell activation is achieved by co-presentation of MHC class II molecules with co-stimulatory receptors and the recognition of this complex by the appropriate TCR. The main pathway by which DCs become activated and mature to provide the second signal to naive T cells occurs via the TLR recognition of PAMPs (21). Induction of the co-stimulatory molecules CD80 and CD86 on the DC surface couples microbial recognition with the induction of co-stimulators that allows activation of pathogen-specific T cells. Since T cells receive the antigen-specific activation signal only in the context of a co-stimulatory signal, TLR-induced expression of co-stimulators transforms the pattern recognition signal into antigen-specific immune responses.  1.1.4 Adaptive immunity In contrast to innate immune responses, adaptive immunity is characterized by two hallmarks: specificity and memory.  1.1.4.1 Cells of the adaptive immune system The adaptive immune response is induced by activation of B and T cells, which leads to 
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 clonal expansion of B and T cells following recognition of their cognate antigen. B cells recognize soluble antigens via the B-cell receptor (BCR), a membrane-bound form of antibody. Upon antigen recognition, B cells start to proliferate and differentiate into IgM secreting effector cells, the plasma cells. In a T cell-dependent process, a subpopulation of activated B cells undergoes differentiation to produce other antibody isotypes, a process called isotype switch (22).           T cell activation is achieved by co-presentation of the MHC/antigen complex with co-stimulatory receptors and the recognition of this complex by the appropriate TCR/CD3 complex. Co-stimulatory receptors expressed on DCs trigger CD28 expressed on naive T cells, and activation leads to clonal expansion and the generation of effector T cells. A fraction of the activated B and T cells mature to long-living memory B and T cells and provide the basis for a fast and highly specific response to further infection with antigen.  1.1.4.2 Receptors for adaptive immune response The TCR and BCR are, unlike the receptors of innate immunity, highly specific for their cognate antigen. They are generated in immature B and T cells by recombination of different germline gene segments. This process provides the means for the generation of a highly diverse pool of T and B cells, each expression a unique TCR or BCR. To avoid the generation of TCRs and BCRs that recognize structures of the host, developing T and B cells are eliminated by negative selection in case their TCR or BCR recognizes host- derived structures. CD8 and CD4 molecules are co-receptors expressed on the surface of T cells and bind to MHC class I or MHC class II molecules, respectively. They support 
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 MHC molecule recognition by the TCR/CD3 complex and increase the sensitivity of antigen recognition by the T cell (23).   1.2 AUTOIMMUNITY The autoimmune diseases are a diverse group of conditions characterized by abnormal immune reactivity associated with autoreactive B and T cells responses. These autoimmune diseases affect hundreds of millions of people around the world. These systemic or organ-specific conditions are the third leading cause of morbidity and mortality in the industrial world after cancer and heart disease (24). Multiple factors are thought to contribute to the development of immune response to the self, including differences in genotypes, hormonal milieu, and environmental factors (25-28).           Non-genetic, probably environmental, factors are important in predisposing people to autoimmune diseases (29, 30). Clustering of autoimmune diseases in families suggests that shared genetic or environmental factors may be important in the pathogenesis of autoimmune diseases. The most powerful evidence in humans that immune-mediated diseases are due to environmental factors comes from the study of identical twins. Less than 50% of identical twins with an immune-mediated disease have an affected twin— that is, they are discordant for the disease (31). Nevertheless, even identical twins can differ genetically—for example, X-chromosome inactivation in females can lead to different patterns of mosaicism, methylation of CpG islands is associated with repression of transcription, and somatic rearrangements are involved in the development of T cell receptors and antibodies. Thus, discordance between identical twins may be determined 
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 by non-genetic factors operating on genetic expression. On the other hand, a common environmental effect operating in a genetically susceptible subject could lead to familial aggregation for a disease, so that non-identical twins have a similar concordance rate to their non-twin siblings, as with multiple sclerosis (32). Thus, there is an important distinction between environmental factors that determine the familial risk of a disease (most probably with limited influence compared with genetic factors) and environmental factors that act at the population level (and are strong determinants of disease risk).           To state that non-genetic or environmental factors are important in causing autoimmune disease does not preclude a role for genetic factors. Twin studies can be used to estimate the impact of genetic factors in leading to a disease. A genetic effect is suggested when the concordance rate in identical twins exceeds that in non-identical twins. In all twin studies of autoimmune diseases, identical twins are more often concordant for the disease than non-identical twins, indicating that genetic factors are important (31). Since the concordance rates in non-identical twins are also below 50%, it suggests that the genetic effect is not due to a single dominant gene. As increasing numbers of genes influence the disease susceptibility, the difference in concordance rates between identical and non-identical twins should increase above 2 : 1. Twin studies support such a polygenic model for the major immune-mediated diseases (31). However, even infectious diseases such as polio and tuberculosis show differences in identical and non-identical twin pair concordance rates (36 and 6%, respectively, for polio and 51 and 26%, respectively, for tuberculosis) (33). These studies taken together are consistent with genetic susceptibility influencing the appearance of clinical autoimmune disease even when environmental factors play a major role in causing it. 
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 1.3 TYPE 1 DIABETES 1.3.1 The clinical presentation of T1D T1D is defined by the presence of the classical clinical symptoms of thirst, polyuria, wasting, and in some cases ketoacidosis. Insulin injection is necessary to control hyperglycemia and to prevent the occurrence of ketoacidosis. If ketoacidosis is not treated, it can lead to diabetic coma and eventually death. The mean age for the onset of T1D is about 11 years (World Health Organization, 1999). The disease is most common in children and young adults of northern European origin, while it has a much lower incidence in their Asian and aboriginal counterparts. 
 1.3.2 The etiology and cause of T1D Although the exact etiology and cause of T1D are not known, it is generally accepted that the disease results from a complex interaction between host genetics and the environment, eventually leading to the destruction of the pancreatic islet insulin- producing cells, the β-cells. The destruction is selective for the β-cells. There are several lines of evidence suggesting that the destruction is the result of autoimmune processing. The near complete loss of β-cells and the concomitant insulin deficiency lead to the observed severe hyperglycemia and ketoacidosis and their related symptoms. 
 1.3.3 T1D is an autoimmune disease There is abundant evidence that T1D is a disease in which β-cells are destroyed by an autoimmune response directed against particular β-cell components. This autoimmune response occurs in the appropriate genetic background—that is, the predisposed 
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 individuals possess certain susceptibility alleles and lack other protective alleles that regulate the immune response. An important susceptibility locus that has been reported in T1D is linked to the human leukocyte antigen (HLA) class II genes lying within the major histocompatibility complex (MHC) region on the short arm of chromosome 6 (6p). It is now well known that both HLA DRB1*04-DQA1*0301-DQB1*0302 (DR4-DQ8) and DRB1*03-DQA1*0501-DQB1*0201 (DR3-DQ2) are positively associated with T1D development (34-38). However, genetic factors alone are not sufficient for the development of T1D, as the low concordance rate between monozygotic twins suggests that a large part of the susceptibility to T1D is non-genetic (39, 40). It has been suggested that non-genetic factors include antigenic bovine serum albumin (41, 42) and viral infections such as Coxsackie B4 virus and Cytomegalovirus (43-46). Antibodies to viral antigens have been reported to be increased in T1D patients (44). These viral antigens may mimic self antigens such that, following infection in genetically susceptible individuals, an immune response mounted against the foreign antigens by T cells results in damage to islet β-cells that express cross-reactive antigens (47). It has been suggested that the immune system participates in β-cell destruction through several of its components including B lymphocytes, NK cells, macrophages, DC, and antigen- presenting cells (APCs), but T1D is considered to be primarily a T-cell-mediated disease requiring both CD4+ and CD8+ cells.  1.3.4 The role of T and B cells in T1D Despite experimental factors, the possibility exists that there is a real difference in the frequency of immune cells infiltrating the islets of human T1D patients and non-obese 
 14
 diabetic (NOD) mice, an animal model of T1D. However, as mentioned above, the predominant cell types found infiltrating the islets of T1D patients, namely CD8+ T cells, CD4+ T cells, B cells, and macrophages, have been studied in NOD mice and found to play a crucial role in the development of autoimmune diabetes in this model as well (48). This is, of course, not evidence that the relative contribution of these cell types to the autoimmune process is the same, but it does provide support for the NOD mouse model in the study of the autoimmune process.           Although in NOD mice B cells are required for the development of diabetes (49, 50), a report identified a case of T1D development in an adult patient who lacked B cells due to a defect in the btk gene (51). This discrepancy does not entirely exclude the possibility that B cells play a critical role in progression to T1D in humans, nor that there exists diversity in disease pathogenesis in different patient populations (for example, adult versus pediatric patients).           Studies of the role of B cells in NOD mouse diabetes suggest that they play a critical role as antigen-presenting cells. NOD mice with B cells that have only a membrane-bound form of immunoglobulin still contribute to diabetes development (52). The ability of B cells to contribute to the autoimmune process in this system seems to be dependent on the expression of surface immunoglobulin, which allows these cells to bind antigen specifically and present it efficiently (53).          Even though many cell types have been implicated as critical for the development of autoimmune diabetes, the disease is considered to be a T cell-mediated disease in both humans and NOD mice. This is mainly due to mounting evidence suggesting that CD4+ and CD8+ T cells are the effectors leading to the demise of beta cells. In T1D patients, 
 15
 the study of the role of T cells has been limited by several factors including availability of samples and the stage of disease at which these can be obtained. In this regard, investigations of T cells in the NOD mouse model have allowed for a better understanding of their role in the development of this autoimmune disease.           Both CD4 and CD8 T cells are required for transferring disease to immunodeficient NOD mice (54). Treatment of NOD mice with anti-CD4 antibodies prevents disease (55) and, similarly, elimination of CD8+ T cells—by targeting expression of beta-2- microglobulin (required for the expression of class I MHC molecules), the CD8 co- receptor, or antibody treatment—prevents disease (56-58). The isolation of T cell clones that target islet antigens has been a very powerful tool for the study of pathogenic and regulatory T cell populations as well as therapeutic approaches.  
 1.3.5 Animal models of T1D Despite many years of research, T1D remains refractory to cure or prevention. This is probably due to the complex, multi-factorial nature of the disease, which depends on the intricate interaction between the genetic make-up of the individual and its environment. Therefore, there is enormous challenge in studying the disease and devising therapies for it.           There are several factors that contribute to the difficulty inherent in studying T1D. First, susceptibility to T1D is polygenetic, and as a result, studying the inheritance is more feasible when using animal models that exhibit less genetic diversity. Second, the sudden onset of disease in humans makes it difficult to identify causes prior to diagnosis and therefore does not allow investigation of the pathological events leading up to the 
 16
 clinical presentation of the disease. Third, the diseased organ (the pancreatic islets of Langerhans) is inaccessible by conventional methods for obtaining samples for investigation. Finally, new therapies cannot be readily tested in human patients unless their risks are minimal or negligible. This is because if T1D occurs, it is not lethal and can be treated with insulin. These factors have necessitated the development of animal models to study T1D.           Several animal models have been developed to study human T1D. The most widely studied animal models for the study of the pathogenesis of β-cell destruction are the NOD mouse and the Bio-Breeding (BB) rat, in which the disease occurs spontaneously, similarly to in humans (59-62). The literature is rich with data collected on the NOD mouse, more than for the BB rat, largely due to the wide availability of information and immunological reagents for mouse studies. In this thesis, the NOD mouse was used as the animal model for studying this disease.   1.4 ANIMAL MODEL: THE NON-OBESE DIABETIC (NOD) MOUSE 1.4.1 Characteristics and pathogenesis of the NOD mouse The NOD mouse, which was first observed and reported in Japan (59), has been widely used as a spontaneous model of autoimmune diabetes (63). This mouse model shares many features in common with human T1D (64), including the polygenic control reflected by the inheritance of particular MHC class II alleles and multiple non-MHC loci as genetic risk factors, the early appearance of an intra-islet inflammatory infiltrate (insulitis) and anti-islet cell anti-bodies, and the autoreactive T cell dependence of T1D 
 17
 pathogenesis and the ability to intervene in disease progression by modulation of T cell function (65-67). The appearance of insulitis and islet cell cytoplasmic and surface antibodies as well as many other cellular and humoral abnormalities have been observed before the onset of diabetes occurs between 13 to 30 weeks of age, with hyperglycemia, ketonuria, and a requirement for insulin treatment.           Pathogenesis of autoimmune diabetes in NOD mice is heralded by the infiltration—first by DCs and macrophages and then by T cells (both CD4+ and CD8+) and B cells—of the perivascular duct and peri-islet regions of the pancreatic islets of Langerhans (peri-insulitis) beginning at 3 to 4 weeks of age. This stage is following by the slow, progressive, and selective T cell-mediated destruction of insulin-producing islet β-cells by 4 to 6 months of age. While a nondestructive insulitis is observed in all female and male NOD mice, NOD females develop a more invasive and destructive insulitis and incur a higher incidence (70-80% by 30 weeks) of diabetes than males (10-40%). This pronounced gender difference is not observed in humans.  
 1.4.2 Cell-mediated autoimmunity in the NOD mouse Following Bottazzo’s demonstration in the early 1970s that T1D was associated with the development of islet-cell-reactive antibodies in the serum of diabetic individuals, much interest was generated in the potential immunological nature of this disease (68). Working with NOD mice, investigators subsequently were able to utilize the better characterized T-cell subsets of the mouse to demonstrate that the transfer of disease with spleen cells from diabetic NOD mice to disease-prone NOD mice was a T-cell- dependent phenomenon and that both CD4+ and CD8+ T cells were required for the 
 18
 acceleration of the disease process in the recipient animals (69). This finding was independently substantiated in a number of laboratories, and the conservative interpretation was that the destruction of islet β-cells in the disease process was similar to the destruction of tissue allograft. That is, the CD8+ T cell was the proximate effector of islet damage and the CD4+ T cell was functioning as a helper cell in the process. It is thought that CD4+ T cells first infiltrate the islets and then secrete a signal that allows the CD8+ T cells to enter the pancreas, eventually resulting in cellular damage.  
 1.4.3 Preventive studies in the NOD mouse Many interventions, such as neonatal thymectomy, anti-CD4 and anti-CD8 monoclonal antibodies, and immunosuppressive agents that target T cells, have been shown to avert diabetes in the NOD model (70). In particular, a number of immune manipulations of the NOD mouse are able to prevent development of diabetes. For instance, the incidence of hyperglycemia is highest in colonies housed in pathogen-free facilities (71), suggesting that infectious agents and immune dysregulation underly protection from disease. As well, many investigators have reported that modifying the immune response of the NOD mouse, from Th1 to Th2 bent, effectively prevents diabetes (72-75). Likewise, the administration of a single dose of CFA as well as Bacille Calmette-Guerin (BCG) has been shown to prevent diabetes in young NOD animals (76-78), and a similar protective effect has been noted in the BB rat (79). 
 
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 1.5 COMPLETE FREUND’S ADJUVANT Complete Freund’s adjuvant (CFA) is a preparation of heat-killed Mycobacterium tuberculosis in a mineral oil emulsion. CFA is used as an adjuvant to potentiate immune response to antigens—for example, in vaccination protocols. CFA acts as an immunostimulant because it induces inflammatory cytokines such as IL-1, IL-6, and TNFα. 
 1.5.1 Mechanisms of CFA protection against autoimmune diabetes Evidence has been presented for CFA-induced protection against diabetes development in NOD mice in association with increases in or induction of antigen-nonspecific or natural suppressor (NS) cells (76, 77, 80). NS cells are generally considered to be large granular lymphocytes belonging to the T cell lineage but lacking mature T cell markers—that is, NS cells are CD4-CD8- (81). How NS cells exert their suppression of T and B cell functions is not known. However, the suppression may be effected by one or more factors and cytokines produced by NS cells (82, 83).           Most studies, however, have identified T regulatory cells and cytokines as mediators of the diabetes-protective effects of immune adjuvants (78, 84). T-cells induced after CFA treatment of NOD mice can prevent both the inductive and the effector phases of the autoimmune response that causes β-cell destruction and diabetes (78, 84). Thus, lymph node or splenic cells from CFA-treated NOD mice transferred protection from diabetes in young NOD mice; also, adoptive transfer of spleen cells from CFA-treated NOD mice, together with spleen cells from acutely diabetic NOD mice, delayed disease induction in irradiated recipient NOD mice (84). Depletion of the 
 20
 Thy1.2+ (total T) cells or the CD4+ (Th) cells from the CFA-treated NOD donor splenic cells abrogated the protective effects of these cells, indicating that the CFA-induced protective cells were CD4+ Th cells (84). In addition, CFA-treated old NOD mice were resistant to passive transfer of disease by spleen cells from acutely diabetic NOD mice; however, diabetes could be induced in the CFA-protected mice by cyclophosphamide treatment, which suggests that T regulatory cells (presumably deleted by cyclophosphamide) accounted for the protective effects of CFA against the autoimmune response.           The ability of CFA to inhibit the effector phase of diabetes in the studies above (84) confirmed an earlier report that treatment of already diabetic NOD mice with CFA at the time of syngeneic islet transplantation prevented β-cell destruction and disease recurrence (85). In these experiments, monocytic and lymphocytic cells still accumulated around the transplanted islets in the CFA-treated NOD mice, but these cells did not invade the islet, and insulin-containing β-cells remained intact even 200 days after islet transplantation (85). These findings suggest that autoreactive T-cells exist in the CFA- treated NOD mice but cannot function as effectors of β-cell destruction. Similar results were reported in another study, where autoreactive T-cells were considered to be dormant in the CFA-treated NOD mice (78).           Taken together, these studies suggest that the mycobacterial immune adjuvants CFA (and possibly other microbial agents and T-cell mitogens) may deliver tolerogenic signals—that is, activate regulatory (suppressor) T-cells that would render islet β-cell autoreactive T-cells nonresponsive. Later studies suggest that the regulatory T cells are of the CD4+ Th type and may belong to the Th2 subset that produces IL-4 and IL-10. 
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           Although many papers have discussed the possible mechanisms of CFA protection in autoimmune diabetes, none has mentioned the relation between CFA and NK cells. Therefore, in this thesis, one of our main goals was to investigate whether CFA prevented diabetes in NOD mice by regulating NK cells. 

 1.6 NATURAL KILLER (NK) CELLS NK cells are large granular lymphocytes that do not express B or T cell receptors and participate in the innate immune response. Under normal circumstances, they are primarily located in the peripheral blood, bone marrow, spleen, and liver (86-88). In humans, NK cells are identified by the lack of the surface marker CD3 and the presence of CD56, with or without CD16.           NK cells are innate immune cells that mediate cellular cytotoxicity and produce chemokines and pro-inflammatory cytokines such as IFNγ, TNFα, granulocyte macrophage colony-stimulating factor (GM-CSF), and macrophage inflammatory protein 1α and 1β (89). NK cells also participate directly in adaptive immune responses, by interacting with DCs and by triggering T cell responses. For instance, NK cells trigger the induction of DC maturation to produce TNFα and IL-12 and up-regulation of co- stimulatory ligands (18). Moreover, NK cells proliferate and acquire cytotoxic activity and the capacity to produce IFNγ through interaction with DCs (19). Apart from activation of other cells of innate immunity, NK cells also enhance induction of CD8+ T cell responses (20). T cell responses are influenced by IFNγ derived from NK cells, which promotes antigen processing and presentation to T cells and Th1 cell polarization. 
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 It has been shown that NK cells are activated by pro-inflammatory cytokines such as type I IFNs and IL-12 (90) or by bacterial unmethylated CpG-rich oligodeoxynucleotides (CpG-ODN) (91), Bacille Calmette-Guerin (BCG) (92), and CFA (93). Activation enhances both the cytotoxicity and cytokine production of NK cells.

 1.6.1 NK receptors Receptors on the surface of NK cells can trigger cell stimulation or inhibition. These receptors are coupled with intracellular signaling adaptors that contain activation or inhibition sites based on their tyrosine residues. These are called ITAM (immunoreceptor tyrosine-based activation motifs) and ITIM (immune tyrosine based inhibitory motifs) (94). Two main categories of NK inhibitory receptors have been identified: the heterodimer CD94 : NKG2A, specific for the HLA-E molecule, and the killer cell immunoglobulin-like receptors (KIR), which recognize HLA, B, and C molecules. On the other hand, NKG2D and natural cytotoxicity receptors (NCRs) are the main NK activating receptors. NCRs (NKp30, NKp44, NKp46, and NKp80) are expressed exclusively in NK cells and belong to the superfamily of immunoglobulin (95-97). NKG2D belongs to the type II C-type lectin-like family of transmembrane proteins and functions both as an activating and co-stimulatory receptor. It is encoded by a gene on human chromosome 12 mapping within the NK complex and chromosome 6 in mice. NKG2D is expressed as a homodimer on the majority of NK cells, but also in other lymphocytes such as γδ T cells as well as on subsets of CD8+ T cells (98). It is known that NKG2D recognizes a variety of ligands such as non-classic HLA class I molecule MICA/MICB (MHC class I-chain related protein A and B) and ULBP (UL16 binding 
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 protein), in humans, and RAE-1 (retinoic acid early inducible-1 gene), minor histocompatibility antigen H60, and MULT1 (murine ULBP like transcript 1), in mice (98).           T1D is an autoimmune disease, and NKG2D is implicated in its pathogenesis. While multifactorial, T1D is characterized by the destruction of insulin-producing β-cells by autoreactive T lymphocytes in the pancreas. Insulin-dependent, NOD mice, which have a defect in NK cell-mediated function, are widely used for the study of T1D (99). Genomic linkage analysis in NOD mice has revealed several insulin-dependent diabetes loci (100). The gene or genes conferring susceptibility to diabetes in NOD mice have been mapped to the NK complex region (101), where a cluster of genes preferentially expressed by NK cells are contained. The expression of RAE-1 by the pancreas in NOD mice and NKG2D by the autoreactive CD8+ cells infiltrating the pancreas led to the focus on NKG2D and its ligands in diabetes studies. One study showed the involvement of NKG2D in the disease progression, in that NKG2D blockade efficiently prevented the onset of autoimmune diabetes (99). In this study, treatment with a non-NK cell depleting anti-NKG2D mAb during the pre-diabetic stage completely prevented disease by impairing the expansion and function of autoreactive CD8+ T cells. This finding highlights a therapeutic potential of NKG2D blockade for autoimmune diseases where aberrant NKG2D recognition is known to participate.           Lanier et al. demonstrated that activated NK cells in NOD mice displayed a low level of NKG2D. In NOD mice, activated NK cells, but not from C57BL/6 mice, expressed NKG2D ligands, which resulted in down-regulation of the receptor NKG2D. Similar to the observation in the animal model of the disease (99), lower expression of 
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 NKG2D has also been observed in both recent-onset and long-standing diabetic patients, compared with healthy control subjects (102), thus triggering the hypothesis that perturbations in NKG2D ligands might be involved in the modulation of NK activity in both humans and mice. 










 1.6.2 NK cells in animal model of autoimmune diabetes The contribution of NK cells to the pathogenesis of autoimmune diabetes has been studied in well-established animal models of autoimmunity such as the BB rat and the NOD mouse; however, the results obtained are inconclusive and inconsistent. In the diabetes-prone BB rat, it has been shown that (i) deficiencies in gut NK cell number and function precede diabetes onset and (ii) splenic NK cells can induce β-cell destruction although NK cell depletion did not lead to prevention of spontaneous diabetes onset (103). In the NOD mouse, a study by Flodstrom et al. showed the involvement of NK cells in Coxsackie B4-induced diabetes (104). In this study of SOCS-1 transgenic NOD mice that hyperexpress SOCS-1 in pancreatic β-cells under the influence of the insulin promoter, it was clearly demonstrated that depletion of NK cells by treatment with anti- asialo GM1 prevented diabetes onset and reduced islet inflammation. Experiments performed on BDC2.5 transgenic mice on an NOD versus a C57BL/6.H-2g7 genetic background showed that NK-cell infiltration was increased in islets in the aggressive BDC2.5/B6.H-2g7 lesions and that depletion of NK cells was associated with diabetes protection, suggesting a role for NK cells in regulating the aggressiveness of β-cell destruction, at least in this experimental model of islet autoimmunity (105). 
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           On the other hand, some reports have indicated that NK cells are involved in protection from diabetes development. Beilke et al. demonstrated the crucial role of host MHC class I-dependent NK-cell reactivity in islet allograft tolerance, induced by either co-stimulation blockade using CD154 antibody therapy or by targeting CD11a, and also showed that this NK-mediated effect is perforin-dependent (106). In addition, Poly(I:C) treatment represents another approach that can induce diabetes protection in NOD mice. A recent study by Zhou et al. has shown that Poly(I:C) activates NK cells to exhibit an NK3-like phenotype and that these cells are involved in the induction of a Th2 bias of immune response to islet autoantigens (107).           In the animal models of autoimmune diabetes, NK cells have been reported to have both numerical and functional deficiencies (108, 109). However, it still remains unknown whether NK deficiencies affect immunoregulatory functions in NOD mice. As mentioned before, NK cell cytotoxicity and cytokine secretion can be increased by treatment with CFA. Moreover, it has been shown that a single injection of CFA into young NOD mice blocks the onset of diabetes. Therefore, in this thesis, the role of NK cells is studied using the model of CFA-mediated prevention of diabetes.

 1.6.3 NK cells in human T1D A few small studies in the 1980s and 90s have measured the frequency of NK cells in the peripheral blood in patients with T1D, especially in patients with recent onset (110-115). A few authors have found a numeric deficiency of NK cells independent of the disease duration and have suggested that this abnormality could be persistent and possibly genetically determined (115). On the other hand, other studies performed at that time did 
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 not find any abnormality in the frequency of NK cells in the peripheral blood (111, 116). Two important concerns with these early studies are the small cohort of patients and the use of non-specific markers such as H25, Leu7 (CD57), and Leu11a (CD16) to identify NK cells (116, 117). H25 has also been found bound to T cells. While Leu7 is not only expressed in T cells but also absent in some NK cells, Leu11a is presented on monocytes, macrophages, and some granulocytes in addition to NK cells.           Functional abnormalities of NK cells have also been reported in patients with T1D. A reduced lytic capacity, as determined by cytotoxicity assays, was demonstrated (118, 119). As for the numeric abnormalities, these results were not universally confirmed (115, 120).           Although NK defects have been suggested in early studies, few studies have addressed the impact of these defective NK cells in patients with T1D. In addition, not many studies have examined the effect of activating/inhibitory receptors on NK cells from patients with T1D. Therefore, one of the goals of this thesis is to investigate abnormalities in the frequency and function of NK cells in the peripheral blood of patients with T1D and, further, to investigate the possible role of NK receptors in the function of NK cells.   1.7 NATURAL KILLER T (NKT) CELLS NKT cell populations were originally described in humans as mycobacteria-reactive cells with specificity for cell wall glycolipid structures, in particular mycolic acids (121). Such cells are restricted by CD1 molecules that are non-classical MHC class I and non- 
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 polymorphic (122). Human CD1d-restricted NKT cells are characterized by the presence of an invariant Vα24JαQ TCR, which preferentially pairs with TCR Vβ11. They also express the C-type lectin NK receptors CD161 (NKRP1) and CD94. The equivalent population in mice also expresses CD161 and an invariant Vα14Jα28 TCR. CD1d- restricted NKT cells are thought to be regulatory T cells, principally in their ability to produce IL-4. Like innate effector cells, NKT cells show a lack of flexibility in their ability to recognize antigens.  1.7.1 NKT cells in autoimmune diabetes NKT cells have been implicated in modifying a broad range of immune responses including allergy, inflammation, infection, anti-tumor immunity, and autoimmunity (123). Moreover, a number of investigations in the NOD mouse model of T1D have suggested that NKT cells may play a pivotal role in the development of autoimmune diabetes. The relative number of invariant NKT cells from the pancreatic islet of NOD mice decreases around the time of conversion from peri-insulitis to invasive insulitis (124). Direct mechanistic evidence of a role for these cells in protection from diabetes is demonstrated in adoptive transfer experiments, in which the transfer of enriched fractions of CD1-restricted NKT cells in NOD mice led to a partial reduction in diabetes risk. The generation of NOD mice transgenic for Vα14Jα28 also resulted in a partial reduction in disease in some animals (125). However, generation of NOD mice with a germline deletion at the CD1 locus showed a further increase of disease risk over wild type or heterozygous controls indicating that endogenous NKT cells play a role in controlling disease even in susceptible animals (126, 127). This observation was supported by the 
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 reduced incidence of diabetes in wild type and in heterozygous, but not in homozygous, CD1-deficient NOD mice after treatment with α-galactosylceramide (αGC), which stimulates NKT cells in a CD1d-restricted manner (126).  1.7.2 NKT cells regulates immune responses Recently, it has become clear that NKT cells play an important role in bridging innate and adaptive immune responses and that activation of NKT cells triggers a chain of events leading to the activation of many cell types of the innate and adaptive immune systems. It has been shown that αGalCer activates NKT cells to express CD40 ligand (CD40L) and that through CD40-CD40L interaction, DC subsequently upregulate CD40 as well as
secrete a large number of cytokines such as IL-12 (128). In addition, NKT cells have been found to control the effector activity of autoreactive T cells by rendering them anergic, as demonstrated by lack of proliferation and cytokine production (129). This NKT-mediated control of effector cells was subsequently found to be dependent on cell- cell contact rather than cytokine (130). Further, studies in autoimmune myasthenia gravis showed that activated NKT cells modulate recruitment and/or expansion of T regulatory (Treg) cells through an IL-2-dependent mechanism (131). Several studies have shown that NKT activation also leads to the activation of NK cells. A study done by Hazlett et al. demonstrates the role of NKT and NK cells in the model of microbial infection. In their model, the immune responses of RAG-1-/- mice (NK cells present, but no NKT or T cells) and TCR Jα281-/- mice (NKT cell-deficient) were compared with those of wild type mice. They found that after bacterial infection, NK cells from both types of knockout mice produced significantly less IFNγ compared with those of wild type 
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 controls (132). Additionally, Carnaud et al. also found that NK cell activation and IFNγ production were not observed in RAG-deficient mice and, notably, that this NK activation by NKT cells is dependent on IFNγ produced from NKT cells (133). In this thesis, the role of NKT cells is studied using the model of CFA-mediated prevention of diabetes. 

 1.8 THESIS OBJECTIVES T1D is characterized by an immuno-mediated progressive destruction of the pancreatic β cells. Although the precise mechanisms involved in its pathogenesis are still unclear, it is known that autoreactive T cells have a central role in the process. Due to the ability of NK cells to release cytokines, directly kill target cells as well as to interact with antigen- presenting and T cells, it has been suggested that they could be involved in the immune- mediated attack that leads to T1D or conversely that they inhibit the development of T1D. Therefore, the elucidation of the exact role of NK cells in the pathogenesis of T1D is important in order to explore whether possible related immune interventions may affect the risk of T1D or delay/block its age of presentation.  1.8.1 NK cells in the mouse model of autoimmune diabetes In the animal models of autoimmune diabetes, NK cells have been reported to have both numerical and functional deficiencies. However, it remains unclear whether NK deficiencies affect immunoregulatory functions in NOD mice. Several studies have shown that NK cells can be involved not only in disease progression but also in disease 
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 protection, suggesting that NK cells can represent a potential therapeutic target in T1D, once the contribution of these cells to islet autoimmunity has been fully elucidated. The function of NK cells can be increased after treatment with mycobacterial components such as CFA, and it has been shown that a single injection of CFA into young NOD mice blocks the onset of diabetes. Using the CFA model in the NOD mouse, we proposed the following specific objectives: (1) To determine the correlation of NK cell function and CFA by cytotoxicity assay and cytokine secretion function in CFA-injected or control NOD mice. (2) To determine the role of NK cells in the CFA prevention mechanism by adoptive transfer experiment. (3) To determine the mechanism(s) by which CFA activates NK cells.  1.8.2 NK cells in human patients with T1D Evidence from animal models suggests that natural killer (NK) cells can be important players in the development of T1D, although data on NK cells in human T1D remains scarce. In the 1980s, a few groups investigated the number and activity of NK cells in patients with T1D, often with conflicting results (111, 114-116, 118, 120). These conflicting results may be because the groups studied were small and the antibodies used to identify NK cells were limited and lacked specificity and hence could not represent the entire NK pool. Some reports have suggested a numeric deficiency of NK cells in the peripheral blood of patients with T1D (111, 118) or a functional abnormality (118, 119). In some of these cases there were permanent numeric and/or functional deficits (115, 119), while in others, transitory abnormalities were associated with the stage of disease 
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 (118, 119). Based on these observations, the aim of this study was to investigate possible abnormalities in the frequency or activation state of NK cells in the peripheral blood of patients with T1D. 
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  CHAPTER  2 MATERIALS AND METHODS 
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 2.1 MATERIALS 2.1.1 Cell lines           Target cell lines YAC-1 (for mouse experiments) and K562, Raji, and Daudi (for human experiments) were obtained from American Type Culture Collection (ATCC) for cytotoxicity assay.  2.1.2 Mice           Female NOD, NOD/SCID, NOD.IFNγ-/-, CD1d-/- NOD, and C57BL/6 (B6) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). CD1d-/- B6 and MyD88-/- NOD mice were kindly provided by Dr. Mark A. Exley (Harvard University) and Dr. Marc S. Horwitz (University of British Columbia), respectively. Mice were maintained in the specific pathogen–free facility at the Child and Family Research Institute. The Animal Care Committee, Faculty of Medicine, University of British Columbia, approved the care and use of all animals.  2.1.3 Antibodies           For mouse experiments, antibodies recognizing CD3ε (145-2C11), CD8 (53-6.7), TCRβ (H57-597), CD69 (H1.2F3), DX5 (HMα2), B220 (RA3-6B2), IFNγ (XMG1.2), and BrdU (3D4) were purchased from BD Biosciences. Anti-NKG2D (CX5) and anti- RAE-1 (CX1) antibodies were purchased from eBiosciences. Anti-NKp46 (259018) antibody was acquired from R&D Systems. CD1d tetramers, conjugated to either PE or allophycocyanin (APC), were provided by the NIH Tetramer Core Facility (Emory University). NRP-V7 tetramer, an H2-Kd tetramer bearing the superior agonist mimotope 
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 peptide NRP-V7 (KYNKANVFL) and conjugated to PE (134), was produced by Xiaoxia Wang in the Tan Laboratory.           For human experiments, antibodies against CD3ε (HIT3a), CD4 (RPA-T4), CD8 (HIT8a), CD19 (HIB19), CD25 (2A3), 2B4 (2-69), LAIR-1 (DX26), NKB1 (DX9), CD94 (HP3D9), CD56 (B159), CD122 (Mik-b2), and CD132 (AG184) were purchased from BD Biosciences. Antibodies recognizing IL-15α (eBioJM7A4; eBioscience), NKG2D (FAB139P; R & D Systems), MICA/B (H-300, Santa Cruz Biotech or clone 159207, R & D Systems), MICA (159227, R & D Systems), and MICB (236511, R & D Systems) were acquired from the indicated sources. Anti-rabbit IgG antibody conjugated to FITC was acquired from Cedarlane Laboratories.  2.2 METHODS 2.2.1 CFA immunizations and assessment of diabetes           Unless otherwise indicated, 5-week-old female NOD mice were given a single 100 µl injection of an emulsion of CFA in the base of their tails. Control mice were injected with either PBS (100 µl) or IFA (100 µl). The development of diabetes was monitored by testing blood glucose twice weekly with test strips. Mice with greater than 33 mM blood glucose measurement were considered diabetic and sacrificed.  2.2.2 Flow Cytometry           For mouse experiments, single cell suspensions were generated from spleens and treated with RBC lysis buffer containing ammonium chloride. For isolation of hepatic lymphocytes, livers were perfused with PBS via the portal vein, mashed through a metal 
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 mesh and enriched through ficoll-paque gradient centrifugation. Cells were fluorescently labeled by incubation with the indicated antibodies in FACS buffer (PBS with 2% FBS) for 30 minutes on ice. Subsequently, samples were washed and suspended in PBS containing 1% FCS and 2.5% paraformaldehyde.           For immunostaining in human experiments, cells were incubated with the indicated antibody for 30 minutes at 4° C and washed with FACS buffer three times. All data from mouse and human immunostaining were acquired on a FACSCalibur using CellQuest software (BD Biosciences) and analyzed with Flowjo (Tree Star, Inc).  2.2.3 Purification and in vitro culture of human NK cells           NK cells were isolated from peripheral blood using a human NK cell enrichment kit (StemCell Technologies) and typical isolation resulted in 95% or greater purity as determined by staining with anti-CD3 and anti-CD56 antibodies. Purified NK cells were expanded in RPMI 1640 medium supplemented with 10% AB human serum, 100 units/mL penicillin, 100 µg/ml streptomycin, 1 mM L-glutamine, 100 µM non-essential amino acids, 5.5 X 10-5 M β-mercaptoethanol, 1000 U/mL human rIL-2 (BD Biosciences), and 50 U/mL of rIL-15 (eBioscience). NK cells were expanded in vitro for 5-7 days prior to their use in cytotoxicity and IFNγ ELISpot assays.  2.2.4 Cytotoxicity           Target cell lines YAC-1 (for mouse experiments) and K562, Raji, and Daudi (for human experiments) were obtained from ATCC. One million target cells were labeled by incubating cells with 100 µCi of 51Cr for 90 minutes at 37° C, washed three times with 
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 PBS and seeded at 104 cells/well in round-bottom 96-well plates. Various numbers of effectors were added to each well, and plates were centrifuged at 500 rpm for 2 minutes and incubated at 37° C. After 4 hours incubation, 100 µl volumes of supernatant were collected and the amount of 51Cr-released measured using a gamma counter. For NKG2D stimulation in human experiments, NK cells were treated with 10 µg/mL of anti-NKG2D Ab (MAB139, R & D Systems) for 20 minutes at 37° C. After stimulation, cells were washed twice with complete RPMI medium before their use as effectors in CTL assays.  2.2.5 ELISpot assay           NK cell effectors were prepared using the same process as for CTL assays. ELISpots were performed in 96-well MAIP S4510 plates (Millipore) using a human IFNγ ELISpot kit from Mabtech, Inc. Immunospot plates were coated with 15 µg/ml of a capture anti-IFNγ mAb overnight at 4° C. Various numbers of NK cell effectors were setup with five thousand target cells (YAC-1, K562, Raji, or Daudi cells) and cultured for 24 hours at 37° C. Plate-bound cytokine was detected with biotinylated anti-IFNγ mAb, and spots were developed using streptavidin-ALP. A Bioreader-4000 (BIO-SYS GmbH) was used to enumerate ELISpots.  2.2.6 BrdU treatment and immunostaining with anti-BrdU antibody           To label proliferating NK cells, NOD mice were injected i.p. with 0.8 mg BrdU (5- bromo-2-deoxyuridine, Sigma Chemical Co.) 1 hour before being sacrificed. Peripheral blood and spleens were harvested for BrdU staining. BrdU-positive cells were stained 
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 using an already established technique (135). Cells were stained with surface markers on ice for 30 minutes. After surface staining, cells were fixed with fixing buffer for 10 minutes and permeabilized at 37°C for 1 hour using permeabilization buffer containing 0.5% BSA, 2 mM EDTA, and 0.1% Triton X-100. After being washed twice, cells were incubated in DNase solution at 37°C for 10 minutes and were subsequently stained with FITC-conjugated anti-BrdU antibody at room temperature in the dark. After staining, cells were washed twice and transferred into PBS containing 0.5% BSA and 2 mM EDTA. Immunostained cells were analyzed on a FACSCalibur flow cytometer.  2.2.7 Selective depletion of mouse NK cells           Depletion of NK cells was performed both in vitro and in vivo using anti-asialo GM1 (Wako Bioproducts). In vitro depletion of NK cells was performed by complement- mediated cytotoxicity. 2 x 107 spleen cells from diabetic NOD mice were incubated with a 1:200 dilution of rabbit anti-asialo-GM1 antibody for 1 hour at 4°C under constant agitation. The treated spleen cells were washed and then incubated with an appropriate dilution of rabbit complement (Sigma) for 1 hour at 37°C. After the cells were thoroughly washed, the resulting cell suspension was used as an NK-cell depleted suspension for adoptive transfer experiments. In vivo, NOD/SCID mice were injected intravenously with 50 µl of anti-asialo-GM1 or an equivalent amount of a rabbit serum (Sigma) a day before adoptive transfer experiments. The efficacy of such treatment was evaluated using standard YAC-1 cellular cytotoxicity assays and by flow cytometryfor NK cell markers. 
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 2.2.8 Purification of mouse NK cells           Spleen cells were pooled from NOD mice and stained with anti-CD3, -CD8 and -DX5 monoclonal antibodies on ice for 30 minutes. Cells were washed three times with PBS, and CD3-CD8-DX5+ cells were sorted on a FACSVantage SE Turbo cell sorter (Becton Dickinson) at the University of British Columbia Multi-User Flow Cytometry Facility. Purified cells were incubated in RPMI 1640 medium (Gibco) plus 10% of fetal bovine serum at 37°C overnight to detach antibodies. Cells were then washed twice with PBS before adoptive transfer.  2.2.9 Adoptive transfer of mouse model           Adoptive transfers were performed using an established technique (136). Recipient female NOD/SCID mice, 4 to 8 weeks of age, were injected intravenously with donor splenocytes (2 x 107 viable cells) suspended in 200 µl of PBS. Diabetic spleen cell donors were female NOD mice that had exhibited blood glucose levels greater than 33 mM for at least 2 weeks. Multiple diabetic donor spleens were pooled to produce sufficient cells for all of the hosts in a given experiment.  2.2.10 Human patient recruitment and sample collection           Human peripheral blood samples were collected from T1D patients with disease duration of more than 6 months and less than 2 years. Age-matched subjects with no autoimmune or metabolic diseases were used as controls. The University of British Columbia’s Clinical Research Ethics Board (Certificate # H07-01707) approved the collection of blood, and informed consent was obtained from all subjects. 
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  2.2.11 CFSE labeling           Human NK cells were purified from peripheral blood, washed with PBS to remove residual medium, suspended at a concentration of 1 x 107 cells/ml in PBS containing 1 µM of CFSE, and incubated for 10 minutes at room temperature. CFSE-labeling reactions were stopped by adding an equivalent volume of FCS and subsequently washing cells extensively with PBS.  2.2.12 Cell signaling studies           IL-2/IL-15 cultured human NK cells (10 x 106 cells) were stimulated with 10 µg/ml of anti-NKG2D Ab (MAB139, R & D Systems) for 15 minutes at 37° C. Cells were lysed in ice-cold lysis buffer (20 mM Tris-HCl pH 8.2, 100 mM NaCl, 10 mM EDTA) containing a protease inhibitor cocktail (Sigma). NKG2D was immunoprecipitated using anti-NKG2D Ab (1D11, eBioscience) and a combination of Protein G-Sepharose and anti-mouse IgG-agarose (Santa Cruz Biotec). Cell lysates and immunoprecipitates were analyzed by blotting with either anti-PI3-K (06-496, Upstate Biotec) or anti-NKG2D (1D11, eBioscience) Abs. Anti-mouse IgG antibody coupled with HRP (BioRad) and ECL (Pierce Biotec, Inc) was used to detect membrane-bound anti-PI3K and anti- NKG2D Abs. 
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 2.2.13 Lysate preparation, microarray production, and data acquisition           Purified human NK cells, expanded in a complete medium containing 1000 U/mL IL-2 and IL-15 (BD Biosciences) for 10 days, were serum-starved for 4 hours. For NKG2D signaling studies, NK cells (107 cells/mL) were incubated with 10 µg/ml of anti- NKG2D mAb (R & D Systems) for 10 minutes on ice, washed twice with PBS, and incubated for either 1 minute or 5 minutes in 37° C warmed PBS containing affinity- purified rabbit anti-mouse IgG F(ab')2 (Jackson Immunoresearch Labs). At indicated time points, cells were lysed in an equal volume of 2X lysis buffer containing 100 mM Tris pH 6.8, 4% SDS, 10% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol, 1X complete protease inhibitor cocktail (Roche), and 6X Halt phosphatase inhibitor cocktail (Pierce Biotechnology), and lysates were snap-frozen in dry ice to prevent any further changes in phosphorylation.  Upon collection of all of the samples, lysates were heated at 100° C for 15 minutes and centrifuged for 10 minutes.  The protein concentration of each lysate sample was determined using a Quant-It Protein Assay kit (Invitrogen).  Protein concentrations were then normalized to the sample with the lowest concentration within each timepoint using 1X lysis buffer.  The fabrication and processing of lysate arrays has previously been described in detail (137).  Using a robotic microarrayer (Bio-Rad) with solid spotting pins, lysates were contact-printed in triplicate features onto nitrocellulose- coated FAST slides (Whatman).  Slides were then blocked for 4 hours with 3% casein solution (Bio-Rad) and probed overnight at 4° C with anti-P(Y)-p85 PI3K, P(T308)- AKT, P(S473)-AKT, P(S473-193H12)-AKT, P(S71)-Rac1, P(S217/221)-MEK1/2, P(T202/Y204)-ERK, P(T180/Y182)-p38 MAPK, P(Y783)-PLCgamma1, P(Y1217)- PLCgamma2, P(S276)-p65-NFkB, P(S536)-p65-NFkB, P(S176/177)-IKK, P(S180/181)- 
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 IKK, P(S927)-p105-NFkB, and P(Y525/526)-Syk primary antibodies (all from Cell Signaling Technology) diluted 1:1000 in PBS supplemented with 10% fetal bovine serum and 0.1% Tween-20 (PBST-FBS).  Arrays were then thoroughly washed with PBST and proved with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Cell Signaling Technology) for 45 minutes.  To amplify the signal, slides were incubated in 1X Western Blot Amplification Module (Bio-Rad) for 10 minutes at room temperature.  The slides were then washed with PBST supplemented with 20% dimethylsulfoxide followed by PBST alone.  Slides were then probed with Alexa Fluor 647-conjugated streptavidin diluted 1:4000 in PBST-FBS.  The processed slides were scanned using a GenePix 4000A microarray scanner (Molecular Devices) at 10-micron resolution.  Photomultiplier tube (PMT) gain intensities were determined for each slide by optimizing the pixel intensity of the features to a range of between 3,000 and 60,000 units.  2.2.14 Array analysis           GenePix Pro 6.0 software (Molecular Devices) was used to determine the median pixel intensities for individual features and background acquired by the 635 nm laser and 670DF40 emission filter.  The background-subtracted median fluorescence intensities (MFI-B) were averaged for each sample printed in triplicate, and the standard deviation was calculated.  The intensity-fold change compared to the unstimulated sample was calculated as a ratio of MFI-B for each timepoint versus the MFI-B of the unstimulated sample, defined as the MFI ratio.  The log base 2 values of the MFI ratios were depicted in heatmap format using TMEV software (138). 
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 2.2.15 Statistical analyses      A Student’s t test was used to calculate statistical significance where indicated and a single factor ANOVA was used for multi-group comparison. Prism software (GraphPad Software) was used to create graphs and provided assistance with statistical tests. 
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        CHAPTER  3 REGULATION OF AUTOIMMUNE DIABETES BY COMPLETE FREUND’S ADJUVANT (CFA) IS MEDIATED BY NK CELLS 
 
 
 
 
 
 
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 3.1 INTRODUCTION AND RATIONALE In NOD mice, an animal model for T1D, a single injection of mycobacterial preparations such as BCG (M. bovis) or CFA between the ages of 4 and 10 weeks prevents the development of hyperglycemia (76, 77, 84, 139), but IFA, which does not contain mycobacterial products, is less protective (84). Thus, mycobacterial components appear to be an important part of the protective effect. However, despite the obvious preventive benefit of mycobacterial components in NOD mice, the mechanism(s) by which they protect β cells from autoimmune destruction are not fully understood.           Previously, abnormalities in activity of NK cells have been associated with autoimmune diabetes, and reports have shown that injection of a mycobacterial product such as BCG or CFA is able to activate NK cells. However, it remains unclear how NK cells exposed to CFA or the effects of CFA serve to regulate autoreactive CTL. Thus, in this thesis, the role of NK cells in the protective mechanism of CFA is studied.  3.2 RESULTS 3.2.1 CFA immunization prevents diabetes in NOD mice To confirm the protective effect of CFA in NOD mice, female NOD mice aged 5 weeks were immunized with CFA or PBS (n = 10 per group), and blood glucose was monitored twice weekly between 12 and 32 weeks of age. In agreement with previous studies (76, 77, 79, 84), a single injection of CFA either delayed or prevented diabetes in NOD mice (Fig. 3.1). By 32 weeks of age, nine PBS-injected mice were diabetic compared with three CFA-injected mice (p < 0.001).  
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 Figure 3.1 Effect of CFA on incidence of diabetes in NOD mice. Female NOD mice (n = 10) received a single injection of CFA (100 µl) to the tail base at 5 weeks of age. Control mice (n = 10) received a single injection of PBS. Blood glucose was monitored weekly, and any animal with a reading of ≥ 33mM was considered diabetic. 
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 3.2.2 CFA immunization prevents the accumulation of β-cell-specific CTL in NOD mice To determine the effect of CFA on a population of autoreactive β-cell-specific CTLs, the NRP-V7-reactive CTLs in NOD mice were quantified following immunization. A previous study has shown that NRP-V7-reactive CTLs are readily detected in the spleens of prediabetic NOD mice, and this population of CTLs participates in β-cell destruction (134). In the present study, female NOD mice (n = 5 per group) were immunized either with CFA or PBS at 5 weeks of age and were sacrificed between 14 and 18 weeks of age. Spleen cells were then analyzed for the presence of NRP-V7-reactive CTL (Fig. 3.2A). Mice that had been immunized with CFA had a significantly lower proportion of NRP- V7-reactive CTL than PBS-injected mice (p < 0.05), suggesting that CFA may prevent diabetes by regulating the number of diabetogenic NRP-V7 CTLs. This effect was specific to the NRP-V7 CTLs, as there was no change in the proportion of total CD8+ cells present in either CFA or PBS injected mice (Fig. 3.2B). 
 3.2.3 CFA induces NK cells to accumulate in blood Numerical and functional abnormalities in NK cells have been reported in NOD mice and been implicated in the etiology of T1D (140-142). Further, it has been shown that injection of CFA limits the development of diabetes in NOD mice (76, 77, 84, 139) by preventing the accumulation of β-cell-specific CTL in an NK cell-dependent manner. In this study, to determine whether CFA prevents the onset of diabetes through the activation of NK cells, the mechanism(s) by which NK cells mediate the effects of CFA was investigated. First, NK cell numbers were compared in female NOD mice treated with either CFA or PBS. NOD mice received CFA or PBS for 16 hours, and spleen cells 
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 Figure 3.2 Effect of CFA on β-cell-specific CTL. Female NOD mice were given a single injection of CFA (n = 20) or PBS (n = 20) at 5 wk of age. The mice were sacrificed at 14-18 wk of age, and the NRP-V7-reactive/CD8+ spleen cell population was determined (A). The total number of CD8+ cells in spleen was measured concurrently (B). The results are the mean value of five independent experiments, and the error bars refer to the SD generated from the repeated assays. The asterisk * indicates p values of less than 0.05. 
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 were harvested for staining. Immunostaining revealed an increased fraction of splenic NK cells among total splenocytes shortly after CFA injection (Fig. 3.3A and 3.3B). This rise in NK cell proportion is attributable to increases in their absolute numbers in the spleen after CFA injection (mean ± S.D., 0.65 ± 0.014 × 106 in PBS-injected group versus 1.94 ± 0.06 × 106 in the CFA-injection group). Temporal measurement of NK cell frequencies in peripheral blood showed a similar increase in the CD3-DX5+ NK population, peaking at 1 hour post-injection, and returning to pretreatment levels after 24 hours (Fig. 3.3C); the kinetics were more prolonged in the spleen (Fig. 3.3D). These data indicate that NK cell expansion, accumulation, or mobilization occurs rapidly following CFA treatment.           To determine whether NK cell proliferation is responsible for elevations in splenic cell numbers, NOD mice were injected with bromo-2-deoxyuridine (BrdU) intraperitoneally to measure NK proliferation following treatment with PBS or CFA. Peripheral blood and splenocytes were harvested to analyze BrdU incorporation using anti-BrdU antibody. The results showed that beginning 30 minutes after CFA treatment, BrdU incorporation in NK cells is detectable in blood (Fig. 3.3E), and a significant increase in BrdU population in the spleen at 16 hours after CFA injection is due to extra- splenic proliferation and mobilization to the spleen (Fig. 3.3F). Together, these data indicated that both recruitment and proliferation play a role in observed NK cell number increases following CFA injection.  
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 Figure 3.3 Effect of CFA on NK cell frequency. Female NOD mice received a single injection of CFA (n = 5) into the tail base at 6 weeks of age. Control mice received a single injection of PBS (n = 5). The mice were sacrificed 16 h after injection, and spleen cells were used to determine the frequency of NK cells. (A) Representative FACS dot plots of the CD3-/DX5+ cell population in the spleen. (B) Bar chart depicting the proportion of NK cells in PBS- or CFA- injected mice. Results are the mean value of three sets of independent experiments, and the error bars refer to the SD generated from the repeated assays. (C, D) The proportion of NK cells in NOD peripheral blood and spleen following a single injection of PBS (n = 6) or CFA (n = 6). (E, F) Female NOD mice received a single injection of CFA (n = 3) or PBS (n = 3), and 1 h prior to being sacrificed they received an intraperitoneal injection of BrdU. Peripheral blood and spleen cells were immunostained with anti-BrdU, anti-DX5, 7AAD, and anti-CD3 to determine the frequency of BrdU+ NK cells. Representative histograms of BrdU+ NK cells in peripheral blood and spleen from PBS- and CFA-injected NOD mice are shown, and bar charts depicting the proportion of BrdU+ NK cells in NOD peripheral blood and spleen following a single injection of PBS or CFA are presented to the right of the histograms. Error bars represent the SD. The asterisks *, **, and ***, indicate p values of less than 0.05, 0.01, and 0.0005, respectively. 
 
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 3.2.4 CFA increases cytotoxicity and IFNγ secretion by NK cells To determine whether NK cell function is enhanced by CFA, NK cytotoxicity and IFNγ production following CFA treatment were evaluated. Splenic NK cells were isolated from NOD mice 16 hours after injection of PBS or CFA and cultured in IL-2. The NK cells were subsequently used to determine cytotoxicity and IFNγ secretion upon recognition and stimulation by YAC-1 target cells. Consistent with earlier studies, only low-level cytotoxicity was observed against YAC-1 cells using PBS-treated NK effectors derived from NOD mice (Fig. 3.4A). However, NK cells from CFA-treated NOD mice had significantly increased lytic activity against target cells (Fig. 3.4A). In addition to cytotoxicity, the ability to secrete IFNγ was assessed by culturing IL-2-expanded NK cells from CFA- or PBS-treated mice for 4 hours at 37°C in the presence of the Golgi transport inhibitor GolgiStop. Intracellular staining of cells from CFA-treated mice demonstrated an augmented capacity to produce IFNγ (Fig. 3.4B). Together, these data indicate that the increased NK functions may play a role for the protective function of CFA in diabetes.  3.2.5 CFA increases NKG2D-mediated function of NK cells through down-regulation of NKG2D ligand It has been reported that the NK abnormality in NOD mice is a deficient activity of the receptor NKG2D (143). In these animals, upon IL-2 activation, NK cells from NOD mice but not from B6 mice expressed NKG2D ligands, which resulted in down-regulation of the receptor NKG2D and reduced NK function. To examine whether CFA-increased  
 
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 Figure 3.4 Effect of CFA on NK cell function. Female NOD mice received a single injection of CFA (n = 5) into the tail base at 6 weeks of age and control mice received a single injection of PBS (n = 5). Splenic NK cells were purified from PBS- or CFA-injected mice 16 h after injection and were subsequently cultured with recombinant mouse IL-2 for 5 days. The IL-2-activated NK cells were used as effector cells to determine cytotoxicity against YAC-1 target cells (A) and to assess IFN-γ secretion (B). Error bars represent the SD. The asterisks ** indicate p values of less than 0.005 in (A) and 0.01 in (B). 
 
 
 
 
 
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 IFNγ secretion in NK cells is through the restoration of NKG2D expression, IFNγ secretion was measured after treatment. Following CFA treatment (but not PBS), staining for intracellular IFNγ NK cells demonstrated a substantial increase in the frequency of IFNγ-producing cells (Fig. 3.5A). Moreover, a modest increase in NKG2D expression by NK cells from CFA-treated mice was also observed (mean fluorescent intensity (MFI) of NKG2D expression is 136.0 ± 4.435 (mean ± SEM) in the PBS-injected group and 193.8 ± 3.326 in the CFA-injected group) (Fig. 3.5B). To investigate whether CFA treatment also affected the expression of NKG2D ligands on NK cells, the surface levels of RAE-1 by flow cytometry and RNA expression of RAE-1α, RAE-1β and RAE- 1γ were analyzed. Immunostaining showed that the percentage of NK cells expressing RAE-1γ was significantly decreased in CFA-treated mice (Fig. 3.5C). Quantitative real- time PCR results revealed reduced levels of RAE-1α, RAE-1β and RAE-1γ messages in NK cells from CFA-treated mice (Fig. 3.5D). These results suggest that CFA may restore NKG2D-mediated function of NK cells through down-regulation of NKG2D ligand.  3.2.6 CFA protection from diabetes is mediated by IFNγ secreted by NK cells The mechanism by which CFA prevents diabetes in NOD mice is unclear, although the above data suggest that down-regulation of a population of autoreactive CTL or stimulation of NK cells may play a role. A previous study has suggested that the protective effect of CFA is dependent on a population of CD11b+ (Mac-1+) spleen cells (77), but the identity of the CD11b+ cells was unknown. Here it was hypothesized that CFA immunization may stimulate NK cells to mediate the protective effect. First, data in previous experiments indicated a rapid effect of CFA on a CD3-DX5+ subset of spleen 
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  Figure 3.5 Effect of CFA on NKG2D ligand expression in NK cells. (A) Spleen cells from PBS- or CFA-injected NOD mice were harvested and further cultured with GolgiStop at 37°C for 4 h before immunophenotyping for CD3, DX5, NKG2D, and intracellular IFNγ. The number shown in each quadrant of the density plots represents the proportion of cells in the gated CD3- DX5+ population. The bar chart depicts the proportion of IFNγ-secreting cells in gated NKG2D+ NK cell population from PBS- or CFA-injected mice. (B) Representative histograms of the expression level of NKG2D in NK cells from PBS- and CFA-injected NOD mice. (C) Spleen cells from PBS- or PBS-injected NOD mice were used to determine the frequency of NKG2D ligand-expressing cells. The RAE-1+ NK cell population in the spleen is shown as representative FACS dot plots and bar chart. The number shown in the quadrant of the plots represents the percentage of cells in gated CD3-DX5+ cells. (D) Expression of NKG2D ligand mRNA transcripts in CD3-DX5+ NK cells. Quantitative real-time PCR was performed and data were normalized to HPRT mRNA expression. Error bars represent the SD. The asterisks * and ** indicate p values of less than 0.05 and 0.005, respectively. 
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 cells (Fig. 3.3). Second, investigators have reported that CFA activates NK cells by increasing IFNγ secretion (93). Last, NOD mice have been shown to carry defects in NK cell activity (108, 109). To ascertain the role of NK cells in the protective effect of CFA, an adoptive transfer model of diabetes was used. Spleen cells from diabetic NOD mice have previously been shown to passively transfer diabetes to irradiated NOD mice (136), and CFA has been shown to inhibit the transfer of diabetes (84). The experiments to transfer diabetes to NOD/SCID mice were designed to prevent the transfer of disease by CFA immunization and to determine the effect of NK cell depletion on diabetes outcome. To separate the effects of NK cells thoroughly from the experiment, both NOD donor cells and NOD/SCID recipient mice were treated with anti-asialo GM1. It was necessary to first determine the phenotypic specificity of this Ab by costaining spleen cells from NOD mice and NOD/SCID mice with anti-asialo GM1 Ab and with markers for T cells (CD4 or CD8), macrophages, DC (CD11b or CD11c), and NK cells (DX5). Fig. 3.6A indicates that asialo GM1 is predominantly expressed by a population of CD11b+DX5+ spleen cells. Thus, as has been previously described (144), asialo GM1-positive cells are NK cells that co-express DX5 and CD11b. In addition, because asialo GM1 is also expressed on a subset of activated CD8 T cells, we costained the (activated) b-cell- specific CTL present in diabetic spleen cells with both NRP-V7 tetramer and CD8. Following anti-asialo GM1 depletion of spleen cells, there was no difference in the proportion of NRP-V7 cells present (Fig. 3.6B).           Next, whether NK cell depletion by anti-asialo GM1 would alter the outcome of adoptively transferred disease in mice immunized with CFA was investigated. Recipient NOD/SCID mice were pretreated with either anti-asialo GM1 Ab (to deplete NK cells) or 
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 Figure 3.6 Characterization of asialo GM1-positive spleen cells. (A) Spleen cells were harvested from 5- to 6-wk-old NOD/SCID or NOD mice and co-immunostained with rabbit anti- asialo GM1 and the indicated PE-conjugated mAbs. Histogram plots of asialo GM1–positive spleen cells are shown (PE, PE fluorescence). (B) The same spleen cell subset was costained with anti-CD8 and NRP-V7 tetramer before and after depletion with anti-asialo GM1. 
 

 
 
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 rabbit serum (control) and were then immunized with CFA after 24 hours. Donor spleen cells pooled from diabetic NOD mice were also depleted of NK cells using asialo GM1 Ab. After NK cell depletion, spleen cells were intravenously injected to the NOD/SCID recipients, and the blood glucose of recipients was measured weekly, beginning on the day of adoptive transfer.           All PBS-immunized NOD/SCID recipients receiving spleen cells developed diabetes within 5 weeks of adoptive transfer (Fig. 3.7A). In contrast, the group of adoptively transferred mice immunized with CFA did not develop disease until between 6 and 10 weeks following adoptive transfer, with two (33%) mice remaining diabetes-free beyond 10 weeks (Fig. 3.7A and 3.7B; p < 0.0001, log-rank test). However, CFA- immunized mice that were pretreated with anti-asialo GM1 and received anti-asialo GM1-depleted spleen cells developed hyperglycemia between 3 and 4 weeks post transfer (Fig. 3.7B). To confirm the specificity of the anti-asialo GM1 effect, a group of NOD/SCID recipients was pretreated with rabbit IgG and subsequently injected with CFA before the adoptive transfer of diabetic spleen cells. These mice developed diabetes at the same rate as mice treated with CFA alone (data not shown).           To verify that NK cells and not NKT cells expressing asialo GM1 were mediating the effects of CFA, a population of CD3-DX5+ cells (5 x 105 cells) obtained by sorting NOD spleen cells was returned to the anti-asialo GM1-treated donor spleen cells before adoptive transfer. These cells were greater than 99% CD3-DX5+ and thus did not include any NKT cells (data not shown). The protective effect of CFA was restored in mice that received a put-back of CD3-DX5+ cells, with the onset of hyperglycemia delayed beyond 8 weeks. This result was highly significant when compared with mice that did not receive 
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 a put-back of CD3-DX5+ cells (Fig. 3.7B; p < 0.005, log-rank test). Altogether, these data suggest that NK cells are important for the protective effect of CFA in diabetes.           Other investigators have shown that IFNγ has a protective role in diabetes and is also necessary for the protective effects of CFA (145, 146). To determine the contribution of IFNγ to NK-mediated protection from disease, NK cells obtained from NOD.IFNγ KO mice were co-transferred with diabetogenic spleen cells to NOD/SCID recipients. As described in the previous paragraph, NK-depleted recipients injected with CFA and adoptively transferred with diabetogenic cells all developed diabetes within 5 weeks. Restoration of NK cells from NOD mice delayed onset of disease in CFA-treated animals, confirming the previous finding that the protective effect of CFA can be mediated by NK cells. However, when NK cells derived from NOD.IFNγ KO mice were co-adoptively transferred, a rapid onset of disease occurred, similar to that in NK- depleted recipients (Fig. 3.7B). Together, these data indicate that NK cells require IFNγ to mediate the protective effects of CFA.  3.2.7 NK cytotoxicity is not required for the protective effects of CFA The experiments in Figure 3.7 do not exclude a protective role for NK cytotoxicity in addition to IFNγ secretion, as the possibility remains that NK cytotoxicity is impaired in NOD.IFNγ KO mice. To assess a possible role for NK cytotoxicity, the lytic capacity of purified NK cells derived from CFA-injected NOD or NOD.IFNγ KO mice was assayed by chromium release assay. The results indicated that, while NK cells from IFNγ KO mice are unable to secrete IFNγ with or without CFA treatment, their cytotoxic capacity  
 
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  Figure 3.7 Prevention of diabetes by NK cells is dependent on IFN-γ . (A, B) Pooled spleen cells from diabetic NOD mice (2 × 107 cells) were adoptively transferred to NOD/SCID recipient mice that were injected with PBS (filled triangles, n = 6), CFA (filled square, n = 6) or were injected with CFA 24 h after depletion of NK cells from both donor cells and recipient mice with anti-asialo GM1 (empty circles, n = 6). Two groups of NOD/SCID recipient mice that were also pre-treated of 50 µL asialo GM1 antibody and injected with 100 µL CFA received adoptive transfer of asialo GM1-depleted diabetogenic spleen cells combined with 5 × 105 CD3-/DX5+ spleen cells from either NOD mice (empty triangles, n = 7) or NOD.IFNγ KO mice (filled circles, n = 7). Blood glucose was monitored weekly for all experiments and diabetes was defined as a single reading of 33 mM. 
 
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 was unimpaired and remained similar to that of wild-type NOD NK cells (Fig. 3.8). Because adoptive transfer IFNγ KO NK cells did not restore protection, these data suggest that CFA-induced NK cytotoxicity does not participate in the protective mechanism. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 Figure 3.8 NK cytotoxicity is not involved in the protective effect of CFA in diabetes. Wild type NOD mice and IFNγ-/- (IFNγ KO) NOD mice received a single injection of CFA (n = 5) into the tail base at 6 weeks of age. The mice were sacrificed, and NK cells were purified from spleen. Purified NK cells were incubated with 2000 U/mL of mouse IL-2 for 5 days, and IL-2 activated cells were subsequently used for cytotoxicity against YAC-1 cells. N.s. indicates not significant.    
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       CHAPTER  4 CFA-MEDIATED STIMULATION OF NKT CELLS PROTECTS AGAINST AUTOIMMUNE DIABETES THROUGH THE SEQUENTIAL ACTIVATION OF NK CELLS 
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 4.1 INTRODUCTION AND RATIONALE Invariant NKT (iNKT) cells comprise a unique subset of T cells that expresses an invariant TCR along with surface markers characteristic of NK cells. NKT cells recognize hydrophobic lipid and glycolipid antigens presented by the non-polymorphic MHC class I-like molecule CD1d (122) and rapidly secrete large amounts of Th1 and Th2 cytokines upon TCR stimulation.           NKT cells have been implicated in modifying a broad range of immune responses varying from allergy, inflammation, infection, anti-tumor immunity and autoimmunity (123). Moreover, a number of investigations in the NOD mouse model of T1D have suggested that NKT cells play a critical role in the regulation of autoimmune diabetes (72, 126, 127, 147-150). Comparison of NKT cell clones derived from T1D subjects versus at-risk non-diabetic siblings has demonstrated a Th1 bias correlated with diabetes in human twin studies (75). By contrast however, an independent study failed to discern NKT cell differences between T1D subjects and healthy controls when their cell frequencies and capacity to secrete IL-4 were assessed directly ex vivo (151). Collectively, these findings indicate that NKT cells regulate the development of autoimmune diabetes in mice and that their involvment in the diabetogenesis of humans remains controversial.  The injection of a mycobacterial preparation such as BCG or CFA prevents the onset of autoimmune diabetes in NOD mice. Findings in Chapter 3 demonstrated that CFA rapidly activates NK cells and that NK cells are required for protection from disease in this mouse model. However, the mechanism by which CFA activates NK cells and prevents diabetes onset remains unknown. Recently, it has become clear that NKT cells 
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 have an important role in bridging innate and adaptive immune responses and that activation of NKT cells triggers a chain of events leading to the activation of many cell types of the innate and adaptive immune systems such as DCs, B cells, and NK cells (133). In addition, several studies have shown that NKT cells are activated after treated with mycobacterial products (152-154). Based on these observations, it is postulated that NKT cells may participate in CFA-mediated NK activation and further that NKT cells may also be required for the protective effect of CFA in diabetes. In this chapter, the role of NKT cells in both CFA-mediated NK cell activation and protection from diabetes is investigated.  4.2 RESULTS 4.2.1 NKT cells are required for NK cell activation and mobilization by CFA Observation of rapid NK cell activation, including CD69 expression and IFNγ secretion, following the treatment of alpha-galactosylceramide (αGC), a superagonist for NKT cells, suggests the possibility that CFA-induced NK cell activation may be mediated through the action of NKT cells (133, 155). To test the hypothesis that CFA-mediated activation of NK cells is dependent on CD1d-restricted NKT cells, wild type and CD1d- deficient B6 mice were treated with a single injection of IFA or CFA and the frequency and activation status of their resident splenic and hepatic NK cells assessed 16 hours later (Fig. 4.1). Consistent with our previous findings on NOD mice, electronic gating on TCRβ-DX5+NKG2D+ cells of wild type B6 mice revealed that CFA-treatment results in a sharp increase in the proportion of NK cells among hepatic lymphocytes (2-fold; CFA: 20.9 ± 2.9 % versus IFA: 10.4 ± 1.5 %; p < 0.01; Fig. 4.1A), while a less dramatic rise is 
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 observed in the spleen of IFA-treated or untreated mice (1.2-fold change; CFA: 2.5 ± 0.2 % versus IFA: 2.0 ± 0.1 %; p < 0.05; Fig. 4B). Similar findings are also seen when NK cell discrimination is based on reactivity to anti-pNK46 (a specific antibody only expressed on NK cells), anti-NKG2D, and anti-TCRβ antibodies (Fig. 4.1C). In contrast to wild type mice, the frequency of NK cells in CD1d-/- mice was not found to change upon CFA injection (Fig. 4.1A and 4.1B). Collectively, these observations suggest that inactivated mycobacterial components, the only component difference between CFA and IFA, are responsible for the altered NK cell frequencies observed after CFA administration and that these changes require CD1d expression.           To evaluate the capacity of mycobacterial components to trigger NK cell activation, CD69 expression and IFNγ production in wild type and CD1d-/- B6 mice were monitored 16 hours post-treatment with CFA or IFA (Fig. 4.2). These experiments found that hepatic and splenic NK cells in wild type mice induced CD69 expression (Fig. 4.2A) and produced IFNγ (Fig. 4.2B) after CFA treatment, while those in CD1d-/- mice did not. By contrast, IFA treatment did not affect NK cell expression of CD69 or IFNγ regardless of mouse genotype (data not shown). The inability of CFA to activate NK cells in CD1d-/- mice is not likely due to an intrinsic NK cell defect, since NK cell number and development appear to be normal in these mice (156). Further, a previous study has shown that NK cells from CD1d-/- mice can respond normally to poly (I:C), initiating proliferation and acquisition of effector function at a level comparable to that of NK cells from wild type mice (155). Together, these findings suggest that CFA may activate NK cells through an NK cell-extrinsic mechanism possibly involving CD1d-restricted NKT cells. 
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 Figure 4.1 NKT cells are required for NK cell mobilization by CFA. Wild type (+/+) and CD1d-/- C57BL/6 mice received a single injection of CFA or IFA into the tail base at 6-8 wks of age. Sixteen hours post-injection, mice were sacrificed and lymphocytes harvested from livers and spleens. NK cell frequencies in the liver (A) and spleen (B) of wild type and CD1d-/- mice were determined by assessing the frequency of TCRβ- cells that are positive for both DX5 and NKG2D expression. Representative dot plots and cumulative data are shown. (C) Hepatic NK cells were detected with anti-DX5 or anti-NKp46 and anti-NKG2D antibodies. The frequency of NK cells is shown as representative (dot plots) and cumulative (bar graphs) data. The asterisks *, **, and *** indicate p values of less than 0.05, 0.01, and 0.001, respectively. The error bars represent the SD.  
 
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 4.2.2 NKT cells are activated by mycobacterial components of CFA Since it was found that CD1d expression is critical for NK cell activation by mycobacterial components of CFA (Fig. 4.1 and 4.2), and previous studies have shown that NKT cells are activated and proliferate in response to M. bovis infection in mice (154), whether NKT cell frequencies and NKT cell activation are affected by exposure to CFA was next investigated. Wild type B6 mice were left untreated (0 h) or subcutaneously injected with CFA for the indicated periods of time, and hepatic and splenic NKT cells were analyzed by gating on αGC/CD1d tetramer+ CD3+ cells (Fig. 4.3). Although FACS analysis detected a similar proportion of NKT cells in the spleens of CFA-treated and control mice (data not shown), the frequency of NKT cells in liver was three-fold reduced in CFA-treated mice as early as 6 hours post-injection (2.9-fold change, from 31.9 ± 3.9 % before injection to 11.1 ± 1.6 % after; Fig. 4.3A). However, the NKT cell proportion among hepatic lymphocytes had almost returned to normal, resembling frequencies observed in naïve mice, by 24 hours post-CFA treatment (26.5 ± 2.1 % versus 31.9 ± 3.9 %; Fig. 4.3A). Collectively, these kinetic studies with CFA describe a preferential and transient modulation of hepatic NKT cell abundance. Although the mechanism responsible for the diminished hepatic NKT cell proportions has not been determined, this phenomenon induced by CFA could be a consequence of the NKT cells trafficking to extrahepatic sites besides the spleen, activation-induced cell death, as has been reported after αGC administration (157), or the failure to detect them due to TCR/NK1.1 down-regulation (158). 

 
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 Figure 4.2 NKT cells are required for NK cell activation by CFA. Wild type (+/+) and CD1d-/- C57BL/6 mice received a single injection of CFA or IFA into the tail base at 6-8 wks of age. Sixteen hours post-injection the mice were sacrificed, and lymphocytes were harvested from livers and spleens. The monitoring of NK cell activation was based on measurements of CD69 levels (A) and IFNγ secretion (B). The asterisks * indicate p values of less than 0.05. The error bars represent the SD. 
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  Next, to determine whether CFA influences NKT cell immune activation, the surface expression levels of the early activation marker CD69 were measured (Fig. 4.3B). The kinetic analyses revealed that hepatic NKT cells elevated CD69 expression as early as 6 hours post CFA-treatment (MFI = 160.3 ± 17.5 versus 65.3 ± 2.8 in naïve mice) and that their levels peaked by 16 hours post-injection (MFI = 213 ± 54). By contrast, CFA was found to have a very minor effect on surface levels of CD69 expressed by splenic NKT cells (data not shown). Because activated NKT cells have been shown to produce both Th1 and Th2 cytokines including IFNγ and IL-4 (158), whether CFA provoked NKT cells to secrete either of these two cytokines was next explored using intracellular FACS (Fig. 4.3C). CFA injection resulted in a marked increase in the percentage of NKT cells synthesizing IFNγ (7.4-fold change; 6.7 ± 1.0% versus 0.9 ± 0.1% in naïve mice). However, IL-4 production by NKT cells following CFA injection could not be detected (data not shown). As a positive control, NKT cells from mice treated with the powerful NKT cell agonist αGC were found to produce high levels of IFNγ. Together, these experiments demonstrate that CFA injection quickly induces NKT cells to express CD69 and produce IFNγ.  
 
 
 
 
 
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  Figure 4.3 NKT cells are activated by CFA. Six- to eight-week old female C57BL/6 mice received a single injection of CFA into the base of the tail. At indicated time points, mice were sacrificed and lymphocytes harvested from the liver. (A) NKT cell frequencies (αGC/CD1d tetramer+ CD3+) among hepatic lymphocytes are indicated within dot plots. Cumulative data is shown as a bar graph. (B) Expression of CD69 by hepatic NKT cells is shown in representative histograms and cumulative data. (C) C57BL/6 mice were treated with IFA, CFA, or alpha- galactosylceramide (αGC), and the fraction of hepatic NKT cells secreting IFNγ 16 h post- treatment was determined by intracellular flow cytometry. Representative dot plots and cumulative results are shown. Error bars represent the SD. The asterisks ** and *** indicate p values of less than 0.01 and 0.001, respectively. 
 
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 4.2.3 NKT cell activation by CFA is independent of MyD88  CFA's mycobacterial antigens may activate NKT cells by a number of potential mechanisms. For instance, NKT cells can be activated directly, through their TCR recognition of self− or foreign−lipid antigens presented by CD1d molecules on the surface of an APC, or indirectly, via cytokines such as IL-12 produced by APCs upon toll-like receptor (TLR)-induced activation (159). Additionally, it is also possible that NKT cells are directly activated through cell-intrinsic recognition of TLR agonists, a phenomenon recently suggested for conventional CD8 T cells (160). The fact that heat- killed Mycobacterium tuberculosis (Mtb), the mycobacterial products present in CFA, contains a number TLR2-agonists (161, 162) suggests that it may activate NKT cells through a TCR-independent pathway.           To investigate whether the activation of NKT cells by the mycobacterial products of CFA is mediated through TLR signaling, mice deficient with MyD88 gene were utilized. MyD88 is a signaling adaptor molecule required for signal transduction and inflammatory cytokine production induced by TLR2 as well as most other TLRs (10). First, wild type and MyD88-/- NOD mice were injected with IFA or CFA and the frequency and activation status of hepatic NKT cells was assessed 16 hours later (Fig. 4.4). In contrast to the hepatic NKT cell frequencies of untreated and IFA-treated mice, those of wild type and MyD88-/- mice plummeted following administration of CFA (Fig. 4.4A and data not shown). In addition, CFA-treatment was found to induce similar levels of CD69 and IFNγ expression by hepatic NKT cells in MyD88-/- mice relative to wild type (Fig. 4.4B and 4.4C). Together, these findings demonstrate that Mtb products of CFA activate NKT cells through a MyD88-independent mechanism. 
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 Figure 4.4 NKT cell activation by CFA is independent of MyD88. Female NOD and MyD88-/- NOD mice received a single injection of CFA or IFA into the base of the tail, and hepatic lymphocytes were harvested 16 h later. (A) Percentage of NKT cells (CD3+ and αGC/CD1d tetramer+) in the livers of treated mice. (B) CD69 expression on hepatic NKT cells following the indicated treatment. (C) Percentage of liver NKT cells secreting IFNγ in treated mice. The error bars represent the SD. The asterisks * and ** indicate p values of less than 0.05 and 0.01, respectively.   









 
 
 
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To determine whether NKT cells can be directly activated by Mtb-stimulated DC and whether MyD88 can modulate this interaction, an in vitro assay was designed using the NKT cell line DN32, a murine CD1d-restricted, Vα14Jα18 invariant TCR-expressing T cell hybridoma (163). DN32 cells were stimulated with BM-derived DCs (BM-DCs) from wild type and MyD88-/- mice treated with medium alone, heat-killed Mtb (strain H37Ra from CFA), or αGC (positive control), and NKT cell activation was assessed by measuring CD69 expression and IL-2 secretion (Fig. 4.5A and 4.5B). Notably, wild type and MyD88-/- DCs were found to be equally capable of inducing DN32 cells to elevate CD69 and IL-2 expression. Together, these findings suggest that Mtb-treated DCs can directly activate NKT cells through a MyD88-independent pathway.  4.2.4 Activation of NKT cells by CFA is dependent on CD1d expression NKT cells can become activated during various microbial infections through the recognition of exogenous- or endogenous-lipid antigens in a CD1d-dependent manner (164). To determine if Mtb-induced NKT cell activation requires TCR recognition of CD1d molecules, the NKT cell line DN32 was stimulated with BM-DCs as treated above (Fig. 4.5), except this time BM-DCs were generated from wild type or CD1d-/- B6 mice and stimulations were performed in the presence of control rat or blocking anti-CD1d antibody. Similar to the findings with NOD DCs (Fig. 4.5), DN32 cells stimulated with Mtb-treated DCs from B6 mice up-regulated surface CD69 expression and produced more IL-2 compared to NKT cells incubated with untreated-DCs (Fig. 4.6). Regarding to 
 
 
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  Figure 4.5 NKT cell activation by CFA is independent of MyD88. (A, B) The mouse NKT cell line DN32 was stimulated with DCs from either NOD wild type or MyD88-deficient mice, in medium alone or in the presence of heat-killed Mtb (H37RA) or αGC. (A) NKT cell activation was measured via CD69 expression after DC stimulation. Representative histograms and cumulative data are shown. (B) NKT cell activation was monitored through IL-2 secretion upon DC stimulation. Twenty-four h post-stimulation, culture supernatents were harvested and IL-2 concentrations measured by ELISA. The error bars represent the SD. The asterisks *, **, and *** indicate p values of less than 0.05, 0.01, and 0.001, respectively.  
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 the mechanism of NKT cell activation, Mtb-induced elevations in CD69 and IL-2 levels by DN32 cells were found to be sensitive to the addition of blocking anti-CD1d antibodies. Further, Mtb-treated CD1d-/- DCs failed to induce CD69 expression or IL-2 secretion by DN32 cells. As expected, treatment with αGC resulted in the strong induction of CD69 expression and IL-2 secretion by DN32 cells, and this was dependent on CD1d expression and blocked by anti-CD1d antibodies (Fig. 4.6). Together, these results suggest that heat-killed Mtb cannot directly activate NKT cells and that it is dependent on TCR recognition of CD1d molecules.  4.2.5 NKT cells are required for CFA-mediated protection from autoimmune diabetes Findings from my previous experiment, demonstrating that NK cells and their capacity to secrete IFNγ are critical in CFA-mediated protection of NOD mice from diabetes (Fig. 3.4), along with those in Figures 4.1 and 4.2, indicating that NK cell activation by CFA is dependent on CD1d, led me to speculate that CFA-mediated protection of NOD mice from diabetes requires CD1d expression. To address this hypothesis, wild type and CD1d-/- female NOD mice were injected with a single dose of CFA, IFA, or PBS, and blood glucose levels were monitored weekly for the onset of diabetes (Fig. 4.7A, 4.7B). As expected, treatment of wild type NOD mice with CFA at 5 to 6 weeks of age protected from the development of disease, while treatment with IFA or PBS showed no demonstrable effect on the occurrence of disease (Fig. 4.7A and data not shown). In accordance with two groups (126, 150) and in contradiction to another group’s finding that CD1d-deficiency exacerbates disease (127), analyses of CD1d-/- female NOD mice  

 
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  Figure 4.6 The activation of NKT cells by CFA is dependent on CD1d expression. The mouse CD1d-restricted NKT cell line DN32 was cultured with DCs derived from either wild type or CD1d-/- C57BL/6 mice. NKT cell stimulations were performed in medium alone or in the presence of heat-killed Mtb (H37Ra) or αGC. For blocking of TCR-CD1d interactions, cultures were treated with anti-CD1d mAb or isotype control rat IgG. CD69 expression on DC-stimulated NKT cells is shown as representative histogram plots (A) and cumulative data (B). (C) DC- stimulated NKT cells were cultured under the indicated conditions and their IL-2 secretions measured by ELISA. Error bars represent the SD. The asterisks * and ** indicate p values of less than 0.05 and 0.01, respectively. 
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 revealed no significant differences in disease incidence and age of diabetes onset as compared to wild type NOD females.  Remarkably, the incidence of diabetes in the CD1d-/- cohort was largely unaffected by administration of CFA (Fig. 4.7B). Together, these experiments demonstrate that CD1d is essential for CFA-mediated protection of NOD mice from diabetes. 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
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 Figure 4.7 NKT cells are required for CFA-mediated protection from autoimmune diabetes. Female NOD mice (A) and CD1d-/- NOD mice (B) received a single injection of CFA, IFA, or PBS into the base of the tail at 5 to 6 weeks of age. Blood glucose was monitored weekly, and mice exhibiting a blood glucose measurement of greater than 33 mM were considered diabetic. 







 
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       CHAPTER  5 NATURAL KILLER CELLS FROM SUBJECTS WITH T1D HAVE DEFECTS IN NKG2D-DEPENDENT FUNCTION AND SIGNALING 
 
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 5.1 INTRODUCTION AND RATIONALE Recent studies suggest that NK cells may be both important regulators and inducers of autoimmune diseases (104, 105, 117, 165, 166), and several reports have documented that NK cell functions in NOD mice are impaired compared to those of healthy mice (108, 109, 140, 141). Recent research has demonstrated that NOD NK cells exhibit decreased NKG2D-dependent functioning and that this deficit may contribute to T1D in NOD mice (143). Activated NOD NK cells, but not C57BL/6 NK cells, were found to maintain NKG2D ligand expression, resulting in the down-modulation of the NKG2D receptor through a mechanism dependent on the YxxM motif of DAP10 (143). As a consequence, reduced NKG2D levels on NOD NK cells led to decreased NKG2D- dependent cytotoxicity and cytokine production (143). My research has shown that administration of CFA to NOD mice causes NK cells to down-regulate NKG2D ligand expression and is correlated with improved NKG2D receptor levels and heightened NK cell function. Moreover, NK cells rejuvenated by CFA-treatment were found to protect NOD/SCID mice from the development of T1D following the adoptive transfer of these hosts with diabetogenic splenocytes. Collectively, these findings suggest that chronic exposure of NOD NK cells to NKG2D ligands results in their desensitization and that augmentation of NK cell function protects NOD mice from disease.  Although investigations of humans with T1D have also described NK cell alterations (111, 115, 116, 118, 120), these studies have been limited in size, the findings controversial, and the molecular mechanisms underlying the phenotype not identified (1, 111, 115, 116, 118, 120). Given the important roles that NK cells play in diabetes of the NOD mouse, it was important to determine whether numeric and/or functional deficits 
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 are also present among NK cells from T1D patients and whether NK cells participate in the regulation of T1D.  5.2 RESULTS 5.2.1 NK cells from T1D patients are present at reduced frequencies To address whether numerical and/or functional NK cell deficiencies are present in human T1D patients, peripheral blood mononuclear cells (PBMCs) from patients with longstanding T1D (> 0.5 years and < 2 years; mean age: 9.3 ± 4.5 years; mean T1D duration: 1.4 ± 0.5 years) and age-matched non-diabetic controls (mean age: 10.7 ± 4.0 years) were analyzed using standardized processing of blood samples under exacting experimental conditions (Table 5.1).   Table 5.1  T1D Subjects Healthy Controls Numbers 103 145 Females/males               42/61 90/55 Mean age (y)             9.3± 4.5           10.7± 4.0 Mean age of onset (y)            8.9 ± 4.2                N/A Mean duration of T1D (y)            1.4 ± 0.5                N/A Mean A1C            7.8 ± 1.2                N/A 
 Characteristics of the T1D subject and healthy control groups from British Columbia's Children's Hospital are presented. Data are shown as the means ± standard deviation. All T1D subjects were being treated with insulin and did not show evidence of other autoimmune diseases.   
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           We rationalized that if NK cell dysfunction were an intrinsic property of the T1D immune system, longstanding measurable defects would still be present in subjects following presentation of that disease. We also limited our subjects to those whose onset of diabetes was no greater than two years in order to minimize the potential effects of chronic hyperglycemia on lymphocyte function. Frequencies of NK (CD3-CD56+), NKT (CD3+CD56+), CD4 T (CD3+CD56-CD4+), CD8 T (CD3+CD56-CD8+), and B cell (CD3-CD19+) subsets among PBMCs were assessed using standard flow cytometric techniques (Fig. 5.1A and 5.1B). In contrast to the modestly elevated proportions of CD4 T, CD8 T, and B cells in the peripheral blood of T1D patients, the NK cell fraction in T1D subjects was markedly reduced, about 37%, relative to normal age-and sex-matched controls (control: 6.58 ± 2.93 % versus T1D: 4.18 ± 1.66 %, P < 0.0005). To ascertain whether T1D subjects exhibit decreased NK cell numbers, complete blood cell counts were performed on fresh blood samples from T1D and non-diabetic subjects to calculate their abundance (Fig. 5.1C). As lymphocyte numbers were modestly reduced in T1D subjects relative to controls (T1D: 2.21 ± 0.74 x 109/L, n=11 versus control: 2.96 ± 0.74 x 109/L, n=10; P < 0.02), NK cell numbers per blood volume were calculated to be decreased about 2-fold in T1D subjects relative to controls (T1D: 0.92 ± 0.37 x 109/L versus control: 1.94 ± 0.86 x 109/L, P < 0.0001).  5.2.2 NK cells from T1D patients respond poorly to IL-2 and IL-15 The critical roles of the cytokines IL-2 and IL-15 in NK cell homeostasis (167, 168) led us to hypothesize that a lack of responsiveness by T1D NK cells to IL-2 and IL-15 could underlie their decreased representation and cell numbers. To address this question, 
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 Figure 5.1 NK cells from PBMC of T1D subjects are present at reduced cell frequencies and numbers. (A) Flow cytometric analyses of non-diabetic control and T1D PBMC using antibodies against CD3 and CD56 markers. (B) PBMC obtained from T1D subjects (black bars, n = 11) and age-matched, non-diabetic controls (white bars, n = 14) were analyzed by flow cytometry to identify the proportions of NK cells (CD3-CD56+), B cells (CD3-CD19+), T cells (CD3+CD19-), CD4 T cells (CD4+CD19-), CD8 T cells (CD8+CD19-), and NKT cells (CD3+CD56+). The asterisk *** indicates a p value of less than 0.0005. Frequencies of other lymphocyte subpopulations when compared between T1D subjects and age-matched controls were not found to be significantly different. (C) T1D subjects possess fewer NK cells than normal controls. Complete blood cell counts were used to determine NK cell numbers present per L of blood. 
 
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 purified NK cells from T1D patients and age-matched controls were labeled with the mitotic tracker CFSE and cultured in vitro either in media alone or with addition of IL-2 and IL-15 (Fig. 5.2A). After one week, measurements of cellular proliferation indicated that very few T1D NK cells had proliferated. By contrast, significant numbers of control NK cells had undergone one or more cell divisions. The vast majority of NK cells, whether patient- or control-derived, failed to proliferate in the absence of exogenous cytokines, demonstrating that cell division was dependent upon cytokine stimulation (data not shown). To determine whether the lack of proliferation by T1D NK cells was associated with a decreased cellular recovery, equivalent numbers of control and T1D NK cells were placed into culture with IL-2 and IL-15 (Fig. 5.2B). One week later, cell counts of cultures revealed that the yield from wells containing T1D NK cells was decreased two-fold relative to control group. These findings indicate that reduced frequencies of NK cells in T1D subjects are correlated with poor responsiveness to IL-2 and IL-15.           To address whether poor IL-2/IL-15 responsiveness by T1D NK cells is a result of insufficient cytokine receptor expression, expression levels of IL-2 and IL-15 receptor subunits were compared (Fig. 5.2C). Flow cytometric analyses revealed that T1D NK cells expressed modestly reduced levels, as determined by comparison of mean fluorescence intensity values (MFI), of IL-2Rβ/IL-15Rβ (CD122), and of IL-2Rγ/IL- 15Rγ (CD132 or common-γ chain) relative to control (CD122: T1D = 65.3 ± 5.8 versus control = 75.2 ± 12.5; CD132: T1D = 26.1 ± 5.9 versus control = 29.8 ± 2.7). CD122 and CD132 interact with CD25 to form the high affinity IL-2 receptor, while these two subunits are thought to bind IL-15 through trans-presentation by IL-15Rα chain on an 
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 Figure 5.2 T1D NK cells are poorly responsive to IL-2/IL-15 stimulation. (A) Purified NK cells from peripheral blood of T1D patients (n = 8) and age-matched controls (n = 4) were labeled with CFSE and cultured for one week with rIL-2 and rIL-15. Representative (histograms) and cumultative data (bar graphs) are shown for the cell division history of cultured NK cell populations. (B) One million purified NK cells from T1D patients (n = 8) and age-matched controls (n = 4) were cultured for one week with rIL-2 and rIL-15 and then counted. (C) Expression of IL-2Rβ/IL-15Rβ (CD122) and common-g chain receptor (CD132) on NK cells was determined directly ex vivo by flow cytometry. Cumulative data was plotted out as MFI values. Error bars represent the SD. 
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 accessory cell (168). Regardless of the NK cell origin, we were unable to detect significant expression of either of the unique subunits of these two cytokine receptors, IL- 2Ra (CD25) and IL-15Ra (data not shown). These results indicate that the hypo- responsiveness of T1D NK cells to IL-2/IL-15 stimulation is not due to a lack of cytokine receptor expression.  5.2.3 Activated T1D NK cells fail to down-regulate the NKG2D ligands MICA/B To investigate the surface phenotype of T1D NK cells and potential causes of their dysfunction, we analyzed the expression of different NK markers on cells directly ex vivo and after in vitro activation with IL-2/IL-15 (Fig. 5.3A). T1D NK cells were found to express normal levels of MICA/B, 2B4, CD94, LAIR, and NKB-1 directly ex vivo as judged by percent positive and MFI values (Fig. 5.3A and data not shown, respectively). In addition, T1D and control NK cells induced the expression of the C-type lectin CD94 and extinguished the SLAM family receptor 2B4. However, the levels of NKG2D ligands MICA/B on control NK cells were almost completely lost upon activation in vitro, while T1D-derived NK cells maintained higher MICA/B expression (Control = 4.8 ± 0.6 MFI; T1D = 24.1 ± 5.0 MFI; 5-fold change in MFI; Fig. 5.3B, 5.3C). Despite retaining high MICA/B levels, activated T1D NK cells expressed NKG2D levels that were comparable to control NK cells (Fig. 5.3B, 5.3C). Together, these experiments reveal that T1D NK cells exhibit dysregulated MICA/B but normal CD94 and 2B4 expression upon stimulation with IL-2/IL-15.  
 
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  Figure 5.3 T1D NK cells fail to down-regulate the NKG2D ligands MICA/B upon activation. (A) Surface marker analyses were performed on T1D (n = 10) and age-matched control (n = 10) NK cells either directly ex vivo (NK) or after one-week of in vitro activation with IL-2/IL-15 (LAK). Cells were stained with antibodies specific for MICA/B, NKG2D, 2B4, CD94, LAIR and NKB-1 antibodies, electronically gated on CD56+CD3- cells and the percent positive for the indicated marker determined. The triple asterisk *** represents p < 0.001 using a one-tailed, Student’s t test. (B) Representative histograms illustrating MICA/B and NKG2D expression on freshly isolated (NK) and one-week activated NK cells (LAK) from the peripheral blood of T1D and age-matched control subjects. Shaded histogram represents isotype-control antibody staining. (C) Cumulative data comparing MICA/B and NKG2D expression, as mean fluorescence intensity values, on T1D (n = 10) and age-matched control (n = 10) NK cells directly ex vivo and one-week after activation with IL-2/IL-15. Error bars represent the SD. 
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 5.2.4 T1D LAK cells exhibit reduced cytotoxicity, IFNγ secretion, and NKG2D function To evaluate their effector function, purified T1D and control NK cells were expanded with IL-2 to generate lymphokine-activated killer (LAK) cells and assessed for their ability to kill either HLA-negative, NK cell-sensitive K562 or NK cell-resistant, LAK- sensitive Raji targets using standard 51Cr-release assays (Fig. 5.4A). T1D LAK cells were found to be less efficient killers of K562 cells on a per cell basis than control LAKs (T1D LAK = 70.4 ± 3.0 % kill at 10:1 E/T ratio kill versus control LAK = 80.4 ± 2.6 kill at 5:1 E/T ratio). In addition, a similar deficit in T1D LAK cytotoxicity was also observed against Raji targets (T1D LAK = 78.6 ± 1.8 % kill at 10:1 E/T ratio kill versus control LAK = 80.5 ± 5.7 kill at 5:1 E/T ratio). Next, the ability of control and T1D LAK cells to produce IFNγ upon exposure to target cells were assessed (Fig. 5.4B). Control or T1D LAK cells were incubated with either K562 or Raji cells for 24 hours and IFNγ secretion enumerated by ELISpot assays. In findings similar to those of the cytotoxicity experiments, T1D LAK cells demonstrated a two-fold decreased capacity to produce IFNγ when stimulated with K562 targets (T1D LAK = 220 ± 13 spots at 10:1 E/T ratio versus control LAK = 210 ± 29 spots at 5:1 E/T ratio). Similarly, T1D LAK cells also displayed marked reductions in IFNγ secretion relative to controls when treated with Raji stimulators in either a ten (220 ± 12 versus 320 ± 18) or a five (140 ± 10 versus 190 ± 4) to one ratios of Raji stimulators. Together, these findings demonstrate that LAK cells derived from T1D subjects display reduced effector function compared those derived from non-diabetic controls.  Previous work in NOD mice has suggested that the expression of NKG2D ligands on activated NK cells affects NKG2D signaling and results in decreased NKG2D- 
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 dependent cytotoxicity and cytokine production (143). Because activated T1D NK cells possess unusually high levels of NKG2D ligands, whether these cells also exhibited defects in NKG2D function needs to be examined (Fig. 5.4C). To address this question, T1D and control LAK cells were treated with either anti-NKG2D Ab or control mouse Ab for 20 minutes, washed, and then incubated with 51Cr-labeled Daudi targets, a cell line known to express NKG2D ligands and to be sensitive to NKG2D-mediated killing (169, 170). Stimulation of control LAK cells with anti-NKG2D Ab resulted in markedly improved killing of targets compared to that of the control mouse Ab (70.1 ± 2.8 % versus 88.4 ± 1.7 %; 26.1 % increase, p < 0.0001) whereas T1D LAK cells were unaffected by treatment (54.5 ± 5.1 % versus 59.0 ± 7.4 %; 8.2 % increase, p < 0.39). Using ELISpot assays, the effect of anti-NKG2D Ab treatment on the ability of non- diabetic control and T1D LAK cells to secrete IFNγ following incubation with Daudi stimulators was assessed (Fig. 5.4D). As with the cytotoxicity results, anti-NKG2D Ab stimulation had a more profound and significant effect on IFNγ production by control LAK cells (183 ± 19 versus 241 ± 19 spots; 31.7 % increase, p < 0.031). In comparison, T1D LAK cells treated with anti-NKG2D Ab displayed an insignificant rise (96 ± 8 versus 116 ± 12; 20.8 % increase, p > 0.096). These results suggest that a defect in the NKG2D-dependent activation pathway of T1D NK cells may be responsible for their diminished effector functions.  
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  Figure 5.4 T1D LAK cells exhibit reduced cytotoxicity, IFNγ  secretion, and NKG2D function. LAK cells were generated by treating purified NK cells with rIL-2/rIL-15 and were tested for effector function. (A) The cytotoxicity of LAK cells from T1D subjects (n = 8) and age-matched controls (n = 14) was assessed using standard chromium-release assays with either K562 or Raji cell lines as targets and the indicated numbers of effector: target (E:T) ratios (**, p < 0.01; ***, p < 0.001). (B) The capacity of LAK cells of T1D (n = 8) and age-matched controls (n = 14) to produce IFNγ following stimulation with either K562 or Raji cell lines was measured with ELISpot assays using the indicated effector: target (E:T) ratios (**, p < 0.01; ***, p < 0.001). Error bars represent the standard deviation. (C) T1D (n = 6) and control LAK cells (n=6) were cultured with 51Cr-labeled Daudi target cells at a 5:1 effector:target (E:T) ratio and cytolytic activity was assessed at the end of a 4 h incubation. Data are presented in both dot-graph (control Ig, filled square; NKG2D, filled triangle) and bar-graph (control Ig, open bars; NKG2D, filled bars) formats. The triple asterisk *** indicates a p value of less than < 0.001. (D) T1D (n = 6) and control LAK cells (n = 6) were treated with Daudi stimulators and IFNγ production measured by ELISpot assay. Data are presented in the same way as in C, in dot- and bar-graph formats. The asterisk * indicates a p value of less than 0.05. 
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 5.2.5 T1D LAK cells exhibit defective NKG2D signaling NKG2D-mediated effector functions are triggered through NKG2D’s association with the transmembrane-adaptor molecule DAP10 (171, 172). Coupling of NKG2D with DAP10 leads to formation of a multi-molecular signaling complex and the activation of multiple downstream signaling cascades including the PI3K-AKT pathway (summarized in Fig. 5.5), which is critically involved in effector function, cell growth, and cell survival (171, 173).  
  Figure 5.5 DAP10 phosphorylation at its YINM motif (blue box) results in the activation of the PI3K pathway.    
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         To investigate whether NKG2D signaling is altered in T1D subjects, PI3K in association with NKG2D-DAP10 complexes in control and T1D LAK cells was first measured following treatment with either anti-NKG2D Ab or control mouse Ab (Fig. 5.6A, 5.6B). After Ab stimulation, NKG2D-DAP10 complexes were pulled down by immunoprecipitation and probed with either anti-NKG2D or anti-p85 subunit of PI3K antibodies. Strikingly, NKG2D stimulation resulted in the efficient association of PI3K with NKG2D-DAP10 complexes in LAK cells from three non-diabetic controls but not T1D subjects.           To determine the activation status of downstream signaling molecules of NKG2D, reverse phase protein lysate microarrays were used to measure their phosphorylation with phospho-(P)-specific antibodies as in a previous study (137). NK cells expanded from six T1D subjects and six non-diabetic controls were serum-starved for 4 hours and then stimulated with anti-NKG2D antibodies for 5 minutes, and their lysates were probed with specific antibodies that recognize phosphorylated signaling proteins (Fig. 5.7). Robust phosphorylation of signaling proteins was detected in all six control samples over the sampled times. By contrast, 5/6 T1D samples showed no evidence of stimulation-induced phosphorylation and 3/6 in this group exhibited stimulation-induced dephosphorylation. Together with the findings shown in Figure 5.6, these findings suggest that impaired effector functions by T1D LAK cells may be a consequence of aberrant signaling through the NKG2D receptor. 



 
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  Figure 5.6 T1D LAK cells exhibit defective NKG2D signaling. (A) LAK cells generated from T1D and age-matched control NK cells were pre-activated with either a stimulating mouse anti- NKG2D Ab or control mouse Ig for 20 min and tested for killer activity and cytokine secretion. (B) T1D (n = 3; C052, C053, and C068) and control LAK cells (n = 3; D068, D069, and D088) were stimulated with either anti-NKG2D Ab or mouse IgG for 15 min. NKG2D was pulled down with anti-NKG2D Abs and blotted with either anti-NKG2D or anti-PI3K Abs. Densitometric measurements are presented on NKG2D and PI3K band intensities and ratios of cumulative means ± SD. The asterisk * indicates a p value of less than 0.05. 



   
 
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 Figure 5.7 T1D LAK cells exhibit defective NKG2D signaling. (A) Purified NK cells from TID subjects (n = 6, labeled “D”) and non-diabetic controls (n = 6, labeled “C”) were stimulated with anti-NKG2D Abs for a time course. Reverse phase protein lysate microarrays were used to detect phosphorylation of downstream signaling molecules of NKG2D. Results are expressed as log base 2 MFI ratio values and are presented as heatmaps of phosphorylation changes over time, with yellow reflecting an increase, blue reflecting a decrease, and black representing no change, compared to baseline time zero.      
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        CHAPTER  6 DISCUSSION AND CONCLUSIONS 
 
 
 
 
 
 
 
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 6.1 DISCUSSIONS 6.1.1 NK cells in autoimmune diabetes It has been reported that in animal models of T1D, NK cells have numeric and functional defects (140, 141, 174), but few studies have addressed the impacts of this dysfunction. Data in this thesis indicate that NK cells present in NOD mice, while deficient in number and function, may be rescued by CFA injection and that this restoration of NK activity may be through up-regulation of NKG2D. Additionally, the adoptive transfer experiments clearly imply that IFNγ produced by NK cells is a critical mediator of CFA protection. The importance of IFNγ is also supported by a report from Serreze’s group showing that BCG or CFA treatment fails to inhibit diabetes development in IFNγnull mice (145). In that report, the subset of IFNγ secreting cells important for protection was not identified. This thesis clearly shows that NK-secreted IFNγ plays a key role for the protective effect of CFA, because NK cells from the IFNγ KO mouse transferred back to CFA-treated recipients were unable to rescue mice from disease. Although the lytic capacity of NK cells is also increased after CFA injection, results in this study show this CFA-induced NK cytotoxicity does not participate in the protective mechanism.           Previous work done by Tian and colleagues has also demonstrated a protective role for NK cells (107). In their report, NOD mice received a series of poly (I:C) injections, a well-known NK activator, and the onset of diabetes was delayed or blocked. The adaptive transfer model indicated that without NK cells, the protective effect of poly (I:C) was abrogated, which is similar to the findings in this thesis. Nevertheless, Tian’s group suggested that unlike NK1- or NK2-like phenotype (175-177), long-term poly (I:C)- treated NK cells regulate disease by exhibiting an NK3-like phenotype that is able to 
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 produce increased levels of TGF-β and IL-10. In addition, Tian’s group further hypothesized that these regulatory cytokines act to inhibit the Th1 response to islet autoantigens. Experiments in this thesis do not rule out this hypothesis. Altogether, these findings suggest that NK cells may be a potential therapeutic target for diabetes prevention.  6.1.2 NKG2D and NKG2D ligands in NK cell activation This thesis investigated the involvement of NKG2D and NKG2D ligands in T1D. Mouse experiments showed that the enhancement of NK cell function through CFA treatment results in improved NKG2D receptor levels and decreased NKG2D ligand expression, reduces autoreactive CTL numbers, and protects NOD mice from disease.           Lanier and colleagues have suggested that defective NKG2D expression underlies NK defects in NOD mice (143). They found that NKG2D expression was dramatically decreased in activated NK cells from diabetes-prone NOD but not diabetes-resistant B6 mice and that reduced expression of NKG2D was likely due to co-expression of an NKG2D ligand, RAE-1, at the cell surface, resulting in self-modulation of NKG2D expression and functional impairment of NK cells. Similarly, Courdert et al. (178) have reported a decreased function of NKG2D in NK cells after a long period of exposure to NKG2D ligand, and Reyburn and his colleagues have also shown that when human NK cells are recipients of NKG2D-ligand membrane transfer following synaptic encounter with NKG2D ligand-bearing cells, NKG2D ligand transfer and co-localization with NKG2D on NK cell membrane resulted in abrogated NKG2D function and reduced cytotoxic function (179). These findings suggest that co-expression of NKG2D and its 
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 ligands in the NK cell surface decrease NK activity. Data in this thesis show that CFA increases the frequency of NKG2D-positive NK cells and, notably, that this cell population is able to up-regulate IFNγ production, suggesting that promotion of NKG2D function is one mechanism of CFA protection. Moreover, the expression of RAE-1 on NK cells is decreased following CFA treatment, further strengthening the argument that the effect of CFA on NKG2D ligand may mediate NK function.           Lanier et al. have shown a decreased expression level of NKG2D on NK cells. A later study done by the same group suggested that NKG2D is involved in the progression of diabetes, since NKG2D blockage prevents autoimmune diabetes in NOD mice. However, their report focused mainly on the correlation of NKG2D on autoreactive CD8+ T cells and NKG2D ligands on pancreatic islets. They showed that there was an increased expression of RAE-1 on prediabetic pancreatic islets and that infiltrating autoreactive CD8+ T cells in the pancreas from 16-week-old NOD mice expressed NKG2D. In addition, when NOD mice were treated with neutralizing anti-NKG2D antibody, diabetes onset was prevented. These results suggest that the interaction of NKG2D with its ligands may be one mechanism by which pancreatic islet cells were destroyed by autoreactive cells, possibly CD8+ T cells. Interestingly, data in this thesis find that CFA injection increases NKG2D expression on NK cells and that these cells are protective against diabetes in NOD mice. It is possible that the administration of CFA increases NKG2D-dependent function in NK cells to produce IFNγ and that the IFNγ produced down-regulates autoimmunity. Moreover, because NK cells from CFA-treated mice are protective, it is unlikely that NKG2D-expressed NK cells would cause the destruction of islet cells. 
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           In addition to mouse experiments, human experiments show that NKG2D ligands MICA/B can also be detected on human NK cells. In addition, T1D NK cells retain a high level of NKG2D ligands after IL-2 stimulation. Moreover, down-regulation of MICA/B expression is linked with proliferation of human NK cells induced by cytokine stimulation. These experiments suggest that the expression of NKG2D ligands on activated T1D NK cells could result in their chronic stimulation through the NKG2D receptor, inducing NK cell hyporesponsiveness.           Despite aberrant maintenance of NKG2D ligand expression on activated T1D NK cells, the NKG2D receptor down-modulation is not observed. The finding that surface levels of NKG2D on T1D NK cells continued to match closely those of non-diabetic control NK cells suggested that impaired NKG2D function by T1D NK cells was a consequence of downstream (intracellular) signaling rather insufficient receptor expression. Consistent with this interpretation and with the diminished NKG2D-mediated effector function observed (Fig. 5.5), T1D NK cells were found to possess an intracellular signaling transduction defect proximal to the NKG2D receptor affecting the PI3K-AKT pathway (Fig. 5.7 and 5.8)  6.1.3 The role of innate immune receptors in the protective effect of CFA The action of Mtb to induce inflammatory immune responses has long been hypothesized to be a consequence of its ability to stimulate the innate immune system and then the antigen-specific adaptive immune system. Several studies have reported that components in Mtb interact with different members of the TLR family. For example, mycobacterial heat shock protein (HSP) 65 signals exclusively through TLR4, while HSP70 is known to 
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 signal through TLR2 and TLR4 (180). In addition, TLR9 has also been shown to play a role in host defense against infection with Mtb (181). These data implicated a role of TLR for the initiation and coordination of host responses to Mtb. Nevertheless, through the study of mice lacking the two adaptor proteins MyD88 and Trif that are essential for TLR signal transduction, it has been found that a TLR-independent pathway may also be used to induce the immune response by mycobacterial products. Myd88-/-; Trif Lps2/Lps2 mice, which cannot transmit signals through TLRs, and control C57BL/6 mice were immunized with the T cell dependent antigen given in CFA, and the role of TLR signaling in induced antibody responses was evaluated. Antibody responses of Myd88-/-; Trif Lps2/Lps2 mice were comparable to those of control mice, indicating that signals transmitted by MyD88 and Trif made no appreciable contribution to the antibody response (182). Therefore, CFA's adjuvant activity must include at least one mechanism that is independent of the actions of TLR agonists. Consistent with this conclusion, in this thesis both in vitro and in vivo experiments with CFA in MyD88-/- mice suggest a model in which CFA activates NKT cells via a TLR-independent route. At this time, it is not known whether Mtb activates NKT cells through provision of microbial lipids acting as cognate antigens, stimulation of APCs resulting in cytokine production, or modulation of self-lipid antigen presentation by CD1d molecules.           Although the TLR family has been shown to be important for the recognition of Mtb, other membrane-bound pattern-recognition receptors, including the mannose receptor, DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin), and Dectin-1, also contribute to the propagation of Mtb inflammatory signals (183). Phagocytosis of Mtb by human macrophages is primarily via the mannose 
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 receptor and is associated with an anti-inflammatory program (184). Recent reports showed that Mtb binds to and is internalized by human DCs through DC-SIGN, which recognizes ligands within the mycobacterial envelop (184). Moreover, in splenic DC, Dectin-1-dependent signaling was shown to be important for Mtb-induced IL-12 production (184). How the different receptors contribute to the proinflammatory response to administration of CFA needs to be further elucidated.  6.1.4 The mechanism of CFA in autoimmune diabetes A possible mechanism by which mycobacterial components mediate protection of autoimmune diabetes is proposed in this thesis. In CFA-treated NOD mice, NKT cells are rapidly activated in a TCR-CD1d-dependent manner. Subsequently, these NKT cells are able to trigger NK function, possibly through DC or through IFNγ. Some studies have shown that NKT cells induce DC maturation, as evidenced by increased expression of CD86, production of IL-12, and priming of T cell responses. These mature DCs produce various cytokines such as IL-12, IL-15, IL-2, and IFN-α/β, which contribute to the activation of different NK cell functions (18). Other studies have shown that activated NKT cells, by producing IFNγ, potently induce NK cytotoxicity as well as IFNγ secretion (133). Once NK cells are activated in this process, possibly through increasing their NKG2D-dependent function, they produce IFNγ, and the produced IFNγ may directly decrease the population of autoreactive T cells or indirectly regulate other immune cells.           How does IFNγ secreted by NK cells regulate autoimmune diabetes? Several studies provide supporting evidence for the role of IFNγ in down-regulation of immune cells. First, in vitro experiments have shown that IFNγ induces apoptosis of activated 
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 CD4 T cells in mycobacterial infection (185) and in vivo experiments by another group have shown that BCG immunization causes apoptosis of diabetogenic T cells in NOD mice by a mechanism involving IFNγ and TNFα (174). Second, recent studies have shown the association of CD4+ IL-17-producing cells (Th17 cells) in the pathogenesis of autoimmune diseases including diabetes. Harrington et al. and Park et al. have reported that the development of Th17 cells from naïve precursor cells was potently inhibited by IFNγ (186, 187). In addition, it has been found that IFNγ regulates the induction and expansion of IL-17-producing cells during BCG infection (188). Moreover, a paper showed that IFNγ restores normoglycemia in NOD mice, most likely by localized bystander suppression of pathogenic IL-17-producing cells (189). These findings suggest that suppression of development of Th17 cells may be one of the mechanisms by which IFNγ regulates diabetes. Finally, indoleamine 2,3-dioxygenase (153) may be involved in IFNγ-dependent protection from diabetes development. IDO is an IFNγ inducible enzyme for tryptophan metabolism, and the IDO pathway is a potential mechanism for suppression T cell response (190, 191). In NOD mice, several studies have reported that impaired tryptophan catabolism in DCs correlates with enhanced autoimmune responses and that expression of IDO in DCs down-regulates T1D (192-194). Therefore, it is possible that CFA-induced IFNγ secretion triggers the activation of the IDO pathway, which subsequently suppresses autoreactive T cell response.           Similar to our result, a recent paper published by Mori et al. also supports importance of IFNγ in CFA-mediated protection (195). In their study, CFA failed to induce long-term protection in IFNγ signaling deficient NOD (IFNγR-/-) mice. Using IFNγR-/- mice, they showed no difference of Th1/Th17 balance after CFA injection. In 
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 addition, the frequency or function of T regulatory cells was not changed compared to wild type NOD mice. However, Mori et al. demonstrated that autoreactive T cells lacking IFNγR are more resistant to the suppressive function of Treg cells. Although the mechanism by which Treg cells suppress autoreactive T cells were not elucidated in that study, their experiments suggested that IFNγ signaling in pathogenic T cells may account for their different susceptibility to Treg-mediated suppression. Our study does not rule out this possibility.           Other than cytokine production, direct lysis of target cells is also a key effector function of NK cells. Several possibilities arise in which NK cytotoxicity might play a suppressive role in the modulation of autoimmunity. It has been reported that NK cells are able to kill autoreactive lymphocytes or immature DCs. The immunoregulatory role of NK cells in suppressing colitis in a murine CD4+ T cell transfer model was found to be dependent on perforin, suggesting that NK cells were directly killing autoreactive T cells or some other intermediate effector cells such as DCs (196). In addition, several studies have shown that NK cells are potentially able to influence the subsequent adaptive immune response by lysing immature DCs (18, 197) or developing T cells (198). However, in our adoptive transfer model, which transferred diabetic splenocytes to induce rapid development of diabetes, we showed that NK cytotoxicity appears not to be important in disease protection. Nevertheless, in NOD mice or human T1D patients, it is still possible that NK cells down-regulate the autoimmune response to β-cell destruction through direct cytotoxicity of autoreactive T cells or of DCs. Therefore, it is necessary to further investigation the role of NK cytotoxicity in the progression of disease development. 
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 6.1.5 Mycobacteria in autoimmune diabetes In experimental immunostimulaton therapies, some agents such as macrophage activators (199), CFA (76-79), and BCG (200, 201) have prevented the onset of autoimmune diabetes in mice or rats. Despite successful use of mycobacterial adjuvants in prevention and reversal of diabetes in animal models, vaccination with BCG in young children with type 1 diabetes was not shown to reduce disease incidence or progression. In a study of patients with newly-diagnosed type 1 diabetes, clinical remission occurred in 65% of 17 patients who received a single intracutaneous administration of BCG vaccine compared with 7% of 29 patients who did not receive BCG vaccine (202). No adverse effects were reported. In a large multicentre trial involving 72 patients with newly-diagnosed type 1 diabetes the effect of BCG plus nicotinamide was compared to that of nicotinamide alone (203). No significant differences were found between the 2 groups; the rate of clinical remission was 41% vs 46% in the BCG plus nicotinamide and nicotinamide only group, respectively, and the length of remission was similar in these 2 groups. In Sweden, BCG vaccination of newborns had no significant effect on the incidence of childhood-onset type 1 diabetes (204). Additionally, in a Canadian study of 2 different case-control series of type 1 diabetes cases and their matched control participants (205), the overall vaccination rates among type 1 diabetes cases and control participants were quite similar (21.5% vs 22.3% and 17.7% vs 15.1%, respectively). Together, these results suggest that BCG vaccination does not prevent type 1 diabetes in human. These differences between human and animal model may reflect different pathogenic mechanisms in the animal model compared to type 1 diabetes in humans, or they may reflect the differences between BCG and CFA. In CFA preparations, the mycobacterial components are 
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 suspended in an oil emulsion, and upon injection into animals results in chronic TLR stimulation. On the other hand, BCG is an aqueous preparation with a half-life considerably less than CFA. Therefore, CFA may be able to provide a long-term and stronger suppression to autoimmunity, whereas BCG-induced protection is likely to only last for short-time period. In animal model of diabetes, the time frame to negatively regulate autoimmune responses to β-cell destruction is relatively shorter than that required in human T1D patients and this might be the reason that a single dose of BCG is adequate to provide the protection in animals but not in humans.           As a key regulator of inflammation, Th1 cells have a critical role in autoimmune diabetes. The mycobaterial products can influence the induction of these cells, alter the balance in the local cytokine microenvironment and may lead to modulation of inflammtion. Therefore, the stimulation of innate immune responses and regulation of inflammatory responses by these products could potentially act as new therapeutic agents in prevention and regulation of autoimmunity. It should be kept in mind that mycobacterial strains, time of treatment, dosage and administration route are factors that must be considered to optimize adjuvant therapy.   6.2 FUTURE DIRECTION In this study, abnormalities in the frequency and activity of NK cells have been described both in animal models and in patients with T1D, and it has been identified that the NK defect is linked to the alteration of NKG2D/NKG2D ligand expression level in NK cells. By treatment with CFA, this alteration can be compensated for and the onset of diabetes 
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 further prevented. These findings provide insights into possible treatment of autoimmune diseases. However, questions remain to be addressed from this study. First, the mechanism by which CFA down-regulates NKG2D ligands needs to be elucidated. Reports from studies of viral infection have shown that IFNγ was able to inhibit the expression of NKG2D ligands in cells (206) and that inhibition of NKG2D ligand expression is through the increased level of microRNA (miR-520b) (207). Based on these findings, it is possible that CFA-induced IFNγ may increase the level of microRNA, especially miR-520b, in NK cells and subsequently inhibit the expression of NKG2D ligands.           Many studies have suggested that NKT cells are important against bacteria, viruses, tumors, and autoimmune diseases (208). This study demonstrates that NKT cells can be activated by CFA; however, CFA contains a mixture of mycobacterial components and mineral oil and therefore it is important to explore the main mycobacterial component that enables CFA to enhance the function of NKT cells.           Although it has been shown in this study that IFNγ produced from NK cells is critical to the protection of diabetes, how this cytokine regulates the population of autoreactive lymphocytes is unclear. Several possible mechanisms have been described in 6.1.4, but whether one or more of these mechanisms participates in IFNγ-mediated regulation remains to be further elucidated.    
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   APPENDIX ANIMAL CARE, BIOSAFETY AND ETHICS CERTIFICATES 
 122
 12/01/10 11:49 AMhttps://rise.ubc.ca/rise/Doc/0/TNV5M0TFLMM4VCL7FJROFE2128/fromString.html Page 1 of 1   THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE BREEDING PROGRAMS Application Number:  A05-1111 Investigator or Course Director: Rusung Tan Department: Pathology & Laboratory Medicine Animals:  Mice NOD 200 Mice NODscid 150 Mice SAP knockout 150 Mice IFN-gamma knockout 30 Mice IDD4-NOD 100  Approval Date: January 21, 2009 Funding Sources:  Funding Agency:  Canadian Institutes of Health Research (CIHR) Funding Title: The function and manipulation of autoreactive cytotoxic T lymphocytes in Type 1 diabetes  Funding Agency:  National Cancer Institute of Canada Funding Title:  Natural killer T cell dysfunction in SAP knockout mice  Funding Agency:  Canadian Diabetes Association Funding Title:  Mechanism of autoimmune regulation by natural killer cells   Unfunded title:  N/A The Animal Care Committee has examined and approved the use of animals for the above breeding program. This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures.  Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 
 123
 12/01/10 11:50 AMhttps://rise.ubc.ca/rise/Doc/0/9VFL5LRA49EKHDE1IVOH8397A1/fromString.html Page 1 of 1   THE UNIVERSITY OF BRITISH COLUMBIA ANIMAL CARE CERTIFICATE Application Number: A05-1251 Investigator or Course Director: Rusung Tan Department: Pathology & Laboratory Medicine Animals:  Mice Non-obese diabetic (NOD) 164 Mice NODscid 32 Mice Balb/c 100 Mice IFN-gamma knock out 16  Start Date:  September 1, 2006 Approval Date:  January 5, 2009 Funding Sources: Funding Agency:  Canadian Institutes of Health Research (CIHR) Funding Title:  Cellular mechanisms of childhoold autoimmunity: from T1D to SLE and back  Funding Agency:  Juvenile Diabetes Research Foundation International Funding Title:  Mechanisms of autoimmune regulation by natural killer cells  Funding Agency:  Juvenile Diabetes Research Foundation International Funding Title:  Mechanisms of autoimmune regulation by natural killer cells  Funding Agency:  Canadian Institutes of Health Research (CIHR) Funding Title:  CIHR Team in Childhood Autoimmunity  Funding Agency:  Canadian Institutes of Health Research (CIHR) Funding Title:  Cellular mechanisms of childhoold autoimmunity: from T1D to SLE and back  Funding Agency:  Canadian Institutes of Health Research (CIHR) Funding Title:  CIHR Team in Childhood Autoimmunity    Unfunded title:  N/A The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures.  Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093 
 124
 12/01/10 11:09 AMhttps://rise.ubc.ca/rise/Doc/0/1F46PL4JR3K43C1N0I1FLLIGFC/fromString.html Page 1 of 1   The University of British Columbia     Biohazard Approval Certificate  PROTOCOL NUMBER: B07-0136   INVESTIGATOR OR COURSE DIRECTOR: Rusung Tan   DEPARTMENT: Medical Microbiology   PROJECT OR COURSE TITLE: Cellular mechanisms of childhood autoimmunity: from T1D to SLE and back   APPROVAL DATE: September 1, 2009 START DATE: September 13, 2007   APPROVED CONTAINMENT LEVEL: 2 with Containment Level 3 Operating Procedures    FUNDING TITLE: CIHR Team in Childhool Autoimmunity FUNDING AGENCY: Canadian Institutes of Health Research (CIHR)  FUNDING TITLE: Mechanisms of autoimmune regulation by natural killer cells FUNDING AGENCY: Juvenile Diabetes Research Foundation International  FUNDING TITLE: Cellular mechanisms of childhood autoimmunity: from T1D to SLE and back FUNDING AGENCY: Canadian Institutes of Health Research (CIHR)   UNFUNDED TITLE: Cellular mechanisms of childhood autoimmunity: from T1D to SLE and back  The Principal Investigator/Course Director is responsible for ensuring that all research or course work involving biological hazards is conducted in accordance with the University of British Columbia Policies and Procedures, Biosafety Practices and Public Health Agency of Canada guidelines. This certificate is valid for one year from the above start or approval date (whichever is later) provided there are no changes. Annual review is required. A copy of this certificate must be displayed in your facility. Office of Research Services 102, 6190 Agronomy Road, Vancouver, V6T 1Z3 Phone: 604-827-5111 FAX: 604-822-5093 
 125
 
 Room A2-136, 950 West 28 th Avenue Vanc ouver, BC V5Z 4H4 Phone: 604-875-3103 Fax: 604-875-2496 Research ReviewCommittee Feb 16 2009 Certificate of Approval -- RENEWAL -- PRINCIPAL INVESTIGATOR Tan, Rusung DEPARTMENT Pathology NUMBER CW03-0027 /H03-70046 CO-INVESTIGATORS: Dutz, Jan; Tucker, Lori; Van Den Elzen, Peter; Panagiotopoulos, Constadina; Turvey, Stuart; Cabral, David; Utz, Paul; C&W DEPARTMENTS, PATIENT BASED PROGRAMS AND ADMINISTRATIVE JURISDICTIONS IMPACTED BY THIS STUDY: Medical Specialties and General Pediatrics; Pathologyand Laboratory Medicine; SPONSORING AGENCIES: Canadian Institutesof Health Research; Juvenile Diabetes Foundation International; TITLE Mechanisms of Autoimmune Regulation by Natural KillerCells, CIHRTeam in Childhood Autoimmunity. TERMS OF RENEWAL Feb 16 2009 - Feb 08 2010 CERTIFICATION: The protocol for the above-named project has been reviewed by the Research Review Committee and has been found to be appropriate with respect to ethics, methodology, patient impact and availability of C&W resources _________________________________________________________________ Approval of the C&W Research Review Committee Dr. M. Levine, Chair Dr. M. Bond, Associate Chair This Certificate of Approval is valid for the above term provided there is no change in the research protocol

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