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The protective mechanism of complete Freund’s adjuvant in type 1 diabetes Lee, I-Fang 2003

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THE  PROTECTIVE  MECHANISM OF  ADJUVANT  IN  TYPE  COMPLETE  1  FREUND'S  DIABETES  by I-Fang B . S c . ,  F u - J e n  Lee  C a t h o l i c  U n i v e r s i t y ,  R . O . C . , A  THESIS THE  SUBMITTED  IN  REQUIREMENTS MASTER  Taiwan,  1995 PARTIAL  FULFILMENT  FOR THE DEGREE OF  OF  OF  SCIENCE  i n THE  (Department  FACULTY OF  of  F a c u l t y We  P a t h o l o g y o f  accept to  THE  and  M e d i c i n e ; t h i s  /the  L a b o r a t o r y  M . S c .  t h e s i s  STUDIES  as  M e d i c i n e  Programme) conforming  required—&fe«iideird  UNIVER^IT>Y  OF^BRITISH  June ©  GRADUATE  I-Fang  2 0 03 Lee,  2 0 03  COLUMBIA  UBC  Rare Books and Special Collections - Thesis Authorisation Form  9/30/03 9:31 A M  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the head o f my d e p a r t m e n t o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department o f The U n i v e r s i t y o f B r i t i s h V a n c o u v e r , Canada  Columbia  Date  Page 1 of 1  ABSTRACT T y p e 1 d i a b e t e s is a multi-factorial d i s e a s e resulting from the destruction of (3c e l l s in the islets of L a n g e r h a n s of the p a n c r e a s , c a u s i n g a s e r i o u s d e r a n g e m e n t of g l u c o s e m e t a b o l i s m that c a n be fatal in patients d e p r i v e d of insulin treatment. It h a s b e e n reported that P-cell destruction in this d i s e a s e is mainly m e d i a t e d by s e l f - r e a c t i v e cytotoxic T l y m p h o c y t e s ( C T L ) . P r e v i o u s s t u d i e s h a v e s h o w n that a s i n g l e injection of c o m p l e t e F r e u n d ' s adjuvant ( C F A ) p r e v e n t s d i a b e t e s in n o n - o b e s e diabetic ( N O D ) m i c e , but the m e c h a n i s m ( s ) of protection r e m a i n unclear. In this t h e s i s I h a v e s h o w n that C F A i m m u n i z a t i o n of both N O D m i c e a s well a s c y c l o p h o s p h a m i d e a c c e l e r a t e d N O D m i c e m a r k e d l y r e d u c e d the i n c i d e n c e of d i a b e t e s a n d that this r e d u c e d i n c i d e n c e w a s a s s o c i a t e d with a d e c r e a s e in the n u m b e r of B-cell s p e c i f i c , a u t o r e a c t i v e C T L . In addition, the a d o p t i v e transfer of d i a b e t e s into s y n g e n e i c N O D / S C I D recipients w a s p r e v e n t e d by C F A i m m u n i z a t i o n a n d the protective effects of C F A w e r e lost w h e n cells e x p r e s s i n g the natural killer ( N K ) cell m a r k e r , a s i a l o G M 1 w e r e r e m o v e d from both d o n o r cells a n d recipient m i c e . R e t u r n i n g a population of C D 3 - D X 5 + c e l l s to the a d o p t i v e transfer r e s t o r e d the protective effects of C F A . T h e r e f o r e , N K cells m e d i a t e the protective effects of C F A p o s s i b l y through the d o w n r e g u l a t i o n of a u t o r e a c t i v e C T L a n d stimulation of N K cells r e p r e s e n t s a n o v e l a p p r o a c h to the prevention of a u t o i m m u n e d i a b e t e s .  ii  TABLE OF CONTENTS  ABSTRACT  II  TABLE OF CONTENTS  Ill  LIST OF FIGURES  VI  LIST OF TABLES  VII  ABBREVIATIONS  VIII  1 1.1  CHAPTER ONE: INTRODUCTION  1  Insulin-Dependent Diabetes Mellitus 1.1.1 The clinical presentation of IDDM 1.1.2 The etiology and cause of IDDM 1.1.3 The islets of Langerhans and f3-cells 1.1.4 IDDM is an autoimmune disease 1.1.5 Animal models of IDDM  1.2 1.2.1 1.2.2 1.2.3 1.2.4  Animal model: Non-Obese Diabetic (NOD) Mouse Characteristics and pathogenesis of NOD mouse Cell-Mediated Autoimmunity in the NOD mouse The role of CD4 vs. CD8 T cells in IDDM Preventive studies in the NOD mouse  1 1 2 2 3 4 6 6 8 9 10  1.3 Complete Freund's Adjuvant (CFA) 1.3.1 C F A effect in the NOD mouse 1.3.2 The Mechanism of C F A Protection  12 12 14  1.4  Autoreactive CD8+ Cytotoxic T Lymphocytes  16  1.5  Natural Killer (NK) Cells NK cells in Autoimmune Diabetes  18 19  2  CHAPTER TWO: OBJECTIVES  21  3  CHAPTER THREE: MATERIALS  22  3.1  Complete Freund's adjuvant  22  3.2  Cell Lines  22  3.3  Cell Culture Reagents  22  1.5.1  iii  3.3.1  R 1 0 medium  22  3.3.2  P h o s p h a t e buffered saline ( P B S )  23  3.4  Mice  23  3.5  Tissue Preparation Reagents  24  3.5.1  3.6  Erythrocyte lysis buffer (pH 7.4)  24  Immuno-stainging Reagents  24  3.6.1  Monoclonal antibodies  24  3.6.2  W a s h i n g buffer  24  3.6.3  Fixing buffer  25  3.7  Chromium Release assay (CRA)  25  3.8  ELISPOT assay  25  4  CHAPTER FOUR: METHODS  26  4.1  CFA immunizations and assessment of diabetes  26  4.2  Cell staining  26  4.3  NK cells enrichment  27  4.4  Cytotoxicity assay  27  4.5  ELISPOT assay  28  4.6  Immunostaining for NK cells in blood  29  4.7  Selective depletion of NK cells  29  4.8  Purification of NK cells  30  4.9  Adoptive transfer  30  4.10  Statistical analysis  31  5  CHAPTER FIVE: RESULTS AND DISCUSSION  32  5.1  CFA immunization prevents diabetes in NOD mice  32  5.2  CFA immunization prevents the accumulation of fJ-cell specific CTL in NOD mice  34  5.3  CFA immunization prevents cyclophosphamide-accelerated diabetes in NOD mice  36  5.4  CFA immunization decreased the proportion of NK cells in NOD mice  40  5.5  CFA induces rapid peripheral blood accumulation of NK cells in the early time point 42  iv  5.6  CFA activates cytotoxicity and cytokine secretion functions of NK in the early time point  46  5.7  CFA protection from diabetes is dependent on NK cells  49  6  CHAPTER SIX: SUMMARY  57  REFERENCES  58  v  LIST OF FIGURES  1. EFFECT OF CFA ON INCIDENCE OF DIABETES IN NOD MICE  33  2. EFFECT OF CFA ON |}-CELL SPECIFIC CTL  35  3. EFFECT OF CFA IN  CYCLOPHOSPHAMIDE-ACCELERATED  DIABETES  38  4. EFFECT OF CFA ON fi-CELL SPECIFIC CTL IN  CYCLOPHOSPHAMIDE-  ACCELERATED DIABETES  39  5. EFFECT OF CFA ON NATURAL KILLERCELLS  41  6. EFFECT OF CFA ON PERIPHERAL BLOOD NK CELLS  44  7. EFFECT OF CFA ON SPLENIC NK CELLS  45  8. EFFECT OF CFA ON NK IFN-y SECRETION  48  9. CHARACTERIZATION OF ASIALO GM1 POSITIVE SPLEEN CELLS  51  10. PREVENTION OF DIABETES BY CFA IS DEPENDENT ON A POPULATION OF ASIALO GM1 POSITIVE CELLS  vi  55  LIST OF TABLES  1. FUNCTIONAL T CELL DEFECTS IN NOD MICE  vii  ABBREVIATIONS AP  Alkaline phosphatase  APC  Antigen presenting cell  BB rat  Bio-breeding rat  BCG  Bacille Calmette-Guerin  BSA  Bovine serum albumin  CFA  Complete Freund's adjuvant  CTL  Cytotoxic T lymphocyte  CYP  Cyclophosphamide  DC  Dendritic cell  DM  Diabetes mellitus  FBS  Fetal bovine serum  HLA  Human leukocyte antigen  IDDM  Insulin-dependent diabetes mellitus  IFN-y  Interferon-gamma  IL-1, 6, 12  Interleukin -1, 6, 12  MHC  Major histocompatibility complex  MST  Median survival time  NK cell  Natural killer cell  NKT cell  Natural killer T cell  N O D mouse  Non-obese diabetic mouse  N S cell  Natural suppressor cell  PBS  Phosphate buffered saline  TCR  T cell receptor  TNF  Tumor necrosis factor  SFU  Spot forming unit  ix  1  1.1  CHAPTER ONE: INTRODUCTION  Insulin-Dependent Diabetes Mellitus  Diabetes Mellitus (DM) is a syndrome characterized by chronic hyperglycemia and disturbances of carbohydrate, fat, and protein metabolism. These disturbances are usually the result of absolute or relative deficiencies in insulin secretion and/or action. DM is divided into four subgroups: insulindependent (type 1), non-insulin-dependent (type 2), malnutrition-related, and other types of diabetes mellitus associated with certain conditions and syndromes (World Health Organization, 1999). This thesis is only concerned with the first subgroup: insulin-dependent diabetes mellitus (IDDM), which accounts for about 10% of the cases. In general, IDDM is known to affect 0.5% of the population of Western nations (1).  1.1.1 The clinical presentation of IDDM IDDM, which is also called type 1 diabetes, is defined by the presence of the classical clinical symptoms which include thirst, polyuria, wasting, and/or ketoacidosis and the necessity of insulin injection to control the 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 IDDM is about 11 years (World Health Organization, 1999). The disease is most common in children or young adults of northern European origin, while it has a much lower incidence in their Oriental or native American counterparts. l  1.1.2 The etiology and cause of IDDM Although the exact etiology and cause of IDDM 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 (3-cells. The destruction is selective for the (3-cells. There are several lines of evidence suggesting that the destruction is the result of an autoimmune process that is discussed below. The near complete loss of (3-cells and the concomitant insulin deficiency lead to the observed severe hyperglycemia and ketoacidosis and its related symptoms.  1.1.3  The islets of Langerhans and (3-cells  The islets of Langerhans are clusters of endocrine tissue scattered throughout the exocrine pancreas of vertebrates higher in evolution than the bony fish. In the adult human, the islets comprise 1-2% of the pancreatic mass (about 1 gm). However, the islet mass is dynamic, adjusting and adapting to meet the metabolic demands of the individual which vary depending on the size, age, and level of activity of the individual. When the islets, or more specifically the insulin-producing (3-cells, fail to meet those demands, diabetes occurs. The pancreatic islet is made up of four types of cells: insulin-producing (3-cells (60-80%), glucagon-producing cc-cells (15-20%) or pancreatic polypeptide producing PP-cells (about 5%), and somatostatin-producing 8-cells (3%). The 2  P-cells are polyhedral and heavily-granulated with 300nm-wide insulin secretory granules, where insulin maturation occurs. The islets are organized in a nonrandom fashion where the core of the islet is p-cells surrounded by a continuous mantle of non-P-cells, 1-3 cells thick. The main function of the islets of Langerhans, and their cells, is the control of metabolic homeostasis. For example, the P-cells function as "fuel-sensors", adapting the rate of insulin secretion in response to the variation in plasma levels of glucose and other substances such as amino acids, fatty acids, and ketone bodies. Insulin secretion is also stimulated by vagus nerve fibres and inhibited by sympathetic nerve fibres. In addition to nutrients and neural factors, p-cell function is influenced by other factors such as hormones.  1.1.4 IDDM is an autoimmune disease There is abundant evidence that IDDM is a disease in which P-cells are destroyed by an autoimmune response directed against particular P-cell components. This autoimmune response occurs in the appropriate genetic background, i.e., the predisposed individuals possess certain susceptibility alleles and lack other protective alleles which regulate the immune response. An important susceptibility locus that has been reported in IDDM 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  3  (6p). Almost all IDDM patients have HLA-DR3 and/or 4, or HLA-DQ3 and/or 4 genes (2). However, genetic factors alone are not sufficient for the development of IDDM as the low concordance rate between monozygotic twins suggests that a large part of the susceptibility to IDDM is non-genetic (3, 4). These non-genetic factors have been suggested to include antigenic bovine serum albumin (5, 6), as well as viral infections from Coxsackie B, mumps, rubella and cytomegalovirus (7-9). Antibodies to viral antigens have been reported to be increased in IDDM patients (7). One possibility is that 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 (primarily CD8+ that are involved in viral immunity) results in damage to islet p-cells that express cross reactive antigens (10). This immune reaction may then lead to B lymphocyte involvement resulting in the production of self-reactive antibodies leading to mass destruction of the islet p-cells. Alternatively, it has been suggested that some viruses can directly infect and kill the pancreatic P-cells (11).  1.1.5 Animal models of IDDM Despite many years of research, IDDM 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. 4  There are several factors that contribute to the difficulty inherent in studying IDDM. First, susceptibility to IDDM is polygenetic, and as a result, studying the inheritance is more feasible when using animal models which 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 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 IDDM occurs, it is not lethal and can be treated with insulin. These factors have necessitated the development of animal models to study IDDM. Several animal models have been developed to study IDDM. The most widely studied animal models for the study of the pathogenesis of |3-cell destruction are the non-obese diabetic (NOD) mouse and the Bio-Breeding (BB) rat in which the disease occurs spontaneously, similar to humans (12-15). 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 IDDM.  5  1.2  1.2.1  Animal model: Non-Obese Diabetic (NOD) Mouse  Characteristics and pathogenesis of NOD mouse  The NOD mouse was first observed and reported in Japan (12). It has been widely used as spontaneous model of IDDM (16) since this mouse model shares many features in common with human IDDM (17), 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 antibodies; and the autoreactive T cell dependence of IDDM pathogenesis and the ability to intervene with disease progression by modulation of T cell function (Table 1) (18 -20). 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 week of age, with hyperglycemia, ketonuria, and a requirement for insulin treatment. IDDM pathogenesis in NOD mice is heralded by the infiltration- first by dendritic cells 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 3-4 weeks of age. This stage is following by the slow, progressive, and selective T cell-mediated destruction of insulin-producing islet (3-cells by 4-6 months of age. Whereas a nondestructive insulitis is observed in all female and male NOD mice, NOD  6  females develop a more invasive and destructive insulitis and incur a higher incidence (70-85% by 30 weeks) of IDDM than males (10-40%). This pronounced gender bias is not observed in humans.  Table 1. Functional T Cell Defects in NOD Mice T CELL POPULATION Splenic T cells  DEFECT  ASSOCIATED WITH  RESTORED BY  SMLR response reduced IL-2 production  CD4+ T cell defect  IL-2  Thymocytes and Peripheral T cells  Response to TCR stimulation  Deficient PKC/Ras/MAPK signaling pathway Reduced IL-2/TL-4 production  IL-2 (partially) IL-4 (completely) Anti-CD28 monoclonal antibody  Intrathymic and peripheral NKlike T cells  Maturation  Reduced IL-4 production  IL-7  7  1.2.2 Cell-Mediated Autoimmunity in the NOD mouse Following Bottazzo's demonstration in the early 1970s that IDDM 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 (21). Working with NOD mice, investigators subsequently were able to utilize the more well characterized Tcell subsets of the mouse to demonstrate that the transfer of disease with spleen cells from diabetic NOD mice to disease-prone NOD animals was a Tcell- dependent phenomenon and that both CD4+ and CD8+ T cells were required for the acceleration of the disease process in the recipient animals (22). This finding was independently substantiated in a number of laboratories, and the conservative interpretation was that the destruction of islet P-cells in the disease process was similar to the destruction of tissue allograft. That is, the CD8+ T cell was the proximal 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, secrete a signal that allows the CD8+ T cells to enter the pancreas, eventually resulting in cellular damage.  8  1.2.3 The role of CD4 vs. CD8 T cells in IDDM T cells are generally divided into two major groups: CD4+ and CD8+. CD denotes a cluster designation molecule that is a cell-surface antigen that allows phenotypic identification of the cells and in the case of CD4 and CD8, as co-receptor for the T-receptor (TCR). CD4+ T cells, sometimes referred to as "helper/inducer T cells", proliferate in response to antigen and stimulate other cells to become cytotoxic, and induce B cells to secrete antibodies. CD8+ T cells, on the other hand, are capable of both direct cellular cytotoxicity and immunosuppressive activities. In NOD mice, it is now clear that both CD4 and CD8 subsets of cells play a role in the development of disease. The fact that diabetes does not occur in the absence of CD4+ T cells, as shown by both studies using anti-CD4 antibodies (23), as well as in mice that lack CD4+ T cells (24), or in mice that are deficient in CD8+ T cells, either by anti-CD8 antibody injection into young mice (25) or mice in which few CD8+ T cells develop due to a genetic lack of [3-2 microglobulin (26-29) informs us that disease is a function of the action of both these types of cells. CD4+ T cell clones that cause diabetes have been isolated and found to recognize a number of different autoantigens including a |3-cell granule antigen (30), glutamic acid decarboxylase (31), insulin (32) and the more ubiquitous heat shock protein (33). But what of the CD8+ T cells? Although diabetogenic clones have been isolated from diabetic mice (34, 35), the peptides these cells recognize have not been fully identified. Susan Wong, working with Janeway and Flavel, isolated a CD8+ T cell clone by activating 9  NOD splenic T cells with B7-1 transgenic islet P-cells in vitro (36). This clone was diabetogenic when transferred to NOD/SCID recipient mice, and it was suggested that the clone destroyed islet cells through a Fas/Fas Ligand interaction (37). Santamaria's laboratory also isolated an autoreactive CD8+ T cell clone that accelerated the development of diabetes when transferred to disease-prone recipient NOD mice (34). In this latter case, active disease was only precipitated when the clone was transferred along with NOD CD4+ T cellrich splenocytes depleted of CD8+ T cells. The same laboratory has also demonstrated that transgenic animals (Tg 8.3) carrying the TCR derived from this autoreactive CD8+ T cell clone developed accelerated disease as well as P-cell damage.  1.2.4 Preventive studies in the NOD mouse Many interventions have been shown to avert diabetes in the NOD model such as neonatal thymectomy, by anti-CD4 and anti-CD8 monoclonal antibodies or by immunosuppressive agents that target T cells (38). In particular, a number of immune manipulations of the NOD mouse are able to prevent development of diabetes. For instance, the incidence hyperglycemia is highest in colonies housed in pathogen-free facilities (39) suggesting that infectious agents and immune dysregulation underlie protection from disease. As well, many investigators have reported that modifying the immune response of NOD mouse, from Th1 to Th2 bent, effectively prevents  10  diabetes (40-44). Likewise, the administration of a single dose of complete Freund's adjuvant (CFA) as well as Bacille Calmette-Guerin  (BCG) has been  shown to prevent diabetes in young NOD animals (45-47) and a similar protective effect has been noted in the BB rat (48).  n  1.3  Complete Freund's Adjuvant (CFA)  Complete Freund's adjuvant (CFA) is a preparation of heat-killed Mycobacterium  tuberculosis  in mineral oil emulsion. CFA is used as an  adjuvant to potentiate immune response to antigens, e.g. in vaccination protocols. CFA acts as an immunostimulant because it induces inflammatory cytokines such as IL-1, IL-6, and TNF-a.  1.3.1  CFA effect in the NOD mouse  Singh and colleagues (46) first discovered that a single injection of young (5 weeks) NOD mice with CFA led to protection from the development of diabetes. Histological examination revealed that this protection was associated with a substantial reduction of pancreatic insulitis. The levels of natural suppressor (NS) cells were discovered to be elevated in the spleens of CFA-treated mice, together with decreased splenic lymphocyte proliferative responses. These splenic NS cells were found to be Thy-1 negative nonadherent cells that appeared to have originated from the bone marrow. At the same time, another group (45) attempted the use of CFA as an adjuvant containing self-antigens (homogenates of pancreatic islets isolated from pre-diabetic NOD mice) with the intention of inducing the onset of diabetes prematurely in 8-9 weeks of age male and female NOD mice. To the investigators surprise, the results from those, and follow-up studies, showed  12  CFA conferred a state of protection against the development of diabetes in the CFA-treated animals. In addition, the incidence of histologically identifiable insulitis was also reduced in the CFA-treated mice by approximately 50%, compared to control animals that received no or saline injection. Upon further investigation the investigators reported failure of the splenic lymphocytes to respond to in vitro stimulation by the mitogen. Con-A, or by anti-CD3. They also reported the increase of splenic Thy-1 negative nonadherent cell populations (CD3-, CD4-, CD8-, nonphagocytic, esterase negative, and Mac-1+) in the CFA-treated animals. These cells suppressed Con-A- or anti-CD3-induced proliferation of T-lymphocytes derived from either the spleen or thymus of untreated NOD mice. Interestingly, in the CFA-treated animals, the thymus- and lymph node-derived cells did not have the same diminished response to stimuli as did the spleen cells, suggesting that the suppressive effect of CFA was restricted to the spleen. The role of CFA in the protection from diabetes was investigated further in syngeneic islet transplantation in diabetic NOD mice (49). When CFA was administrated at the time of islet transplantation, the median survival time (MST) of CFA-treated syngeneic islet recipients was extended beyond 107 days compared to 11 days in control islet recipient mice. On the other hand, CFA treatment had no effect on extending the MST in NOD mice transplanted with allogeneic islets, suggesting a different pathway for autoimmune destruction of islets from that of allograft immunity.  13  Another study (50) confirmed the effect of CFA treatment in the NOD mouse and indicated the importance of the timing of the CFA treatment in syngeneic islet transplantation into diabetic NOD mice. The study indicated that CFA should be administered after transplantation to confer protection against recurrent disease and not before. This is somewhat contradictory to findings of other investigators that, at least in primary autoimmune diabetes, CFA has to be administered early (i.e. at the time insulitis begins) to prevent the development of diabetes.  1.3.2 The Mechanism of CFA Protection The studies above discussed the effect of CFA administration on development of IDDM and on cellular subsets in the spleen of the treated animals. However, the mechanism by which CFA prevents IDDM is not fully understood. In NOD mice, a study has shown no protection in CFAimmunized NOD mice after treating with anti-mouse CD4 antibody (51). Another study reported that CFA immunization increased a population of Mac1+ (CD 11b) cells and this population might be responsible for mediating CFA protection in IDDM (45). Moreover, one study postulated that since CFA was known to induce the secretion of a variety of cytokines, it was possible that CFA could exert its protective effects through the induction of cytokines over a critical period in diabetes development, and it might lead to immune selftolerance and circumvention of autoimmunity. Finally, one study  14  indicated that CFA may prevent the development of diabetes by restoring TNF production of macrophages from the diabetes-prone BB rats (52). Subsequently, it has been shown that administration of CFA to NOD recipients of syngeneic islets prevented destruction of P-cells in the islet grafts; however, insulitis was not prevented. Importantly, a relative increase in IFN-y producing cells was observed in the untreated (control) mice, whereas the insulitis in the CFA-treated recipients was IL-4 rich (i.e. relative increase in IL-4 producing cells) and nondestructive, and P-cells were not destroyed. As a result, diabetes did not develop in these mice. Although many papers have discussed the possible mechanisms of CFA protection in IDDM, none has mentioned the relationship between CFA and cytotoxic lymphocytes (CD8+ cytotoxic T lymphocytes and natural killer cells). Therefore in this thesis, our main goal was to investigate whether CFA prevented diabetes in NOD mice by regulating cytotoxic lymphocytes.  15  1.4  Autoreactive CD8+ Cytotoxic T Lymphocytes  Although several facets of the immune system, notably defects of antigen presentation, inappropriate activation of CD4+ T cells and loss of suppressor cell activity, have been shown to play vital parts in the pathogenesis of IDDM, CD8+ cytotoxic T lymphocyte (CTL), by virtue of their cytotoxic effector function, have a decisive role in P-cell destruction (53-55). CD8+ CTL are activated to kill P-cells when their T cell receptors (TCR) recognize and form complexes with MHC class l-peptide molecules expressed on the surface of P-cells, thus bringing together CTL and target. Upon binding target cells with sufficient avidity, CTL secrete pre-formed granules containing perforin and granzyme B and upregulate expression of Fas ligand (FasL). Both mechanisms have been shown to induce apoptosis, although their relative importance in P-cell death is still unclear (56-59). Interestingly, most CD8+ CTL derived from islets of pre-diabetic and acutely diabetic NOD mice are restricted by the MHC class I molecule, H-2Kd and more importantly, most express highly homologous T cell receptor (TCR) a chains (60, 61). The high number of homologous TCR's observed in isletderived T cells suggests both that T cells are recruited to the islets by single antigenic structure and that expansion of T cells is occurring following antigen recognition. Indeed, approximately half of islet-derived CTL recognize a single peptide mimotope, designated NRP (KYNKANWFL) or analogs of  16  NRP, designed by mutational amino acid substitutions (e.g., NRP-A7 and NRP-V7 refer to analogs of NRP with an alanine (A) or a valine (V) substitution at the position 7 of the peptide, respectively) (62). In addition, TCR transgenic NOD mice that over-express one such islet-derived T cell receptor (Tg8.3 NOD mice) develop diabetes at an accelerated rate, providing further evidence that these particular CTL mediate P-cell damage in NOD mice (61).  17  1.5  Natural Killer (NK) Cells  NK cells are bone marrow-derived lymphocytes, many of which exhibit a granular morphology (63). They are found in the peripheral blood (comprising up to 15% of peripheral blood lymphocytes) and in tissues such as the spleen, lymph nodes, liver, placenta and peritoneal cavity. Human and rodent NK cells share many functions and features but differ in their expression of some surface molecules (64, 65). The typical human NK cell phenotype is CD3CD56+ CD16+ C D 2  dim  while in the mouse, mature NK cells express NK1.1,  CD2, CD16, DX5, 2B4 and members of the Ly-49 family of class I binding receptors. NK cells mediate a variety of functions that are important in human health and disease. In addition to their role in control of tumor metastasis and virus infections, NK cells participate in immuno-regulation, haematopoiesis, reproduction and neuroendocrine interactions. NK cells were initially characterized by their ability to mediate spontaneous cytotoxicity against tumor cell lines, in particular tumors of haematopoietic origin. They are capable of mediating the killing of target cells by several distinct mechanisms, most particularly via the granule exocytosis/perforin pathway (66, 67). Recent studies propose important functions for NK cells in directing and driving adaptive immune responses (64, 68-70). NK cells play a significant role in the host defense against certain microorganisms, in part through their ability to secrete cytokines such as IFN-y, TNF, and GM-CSF (71). NK cells also take  18  part in the development and regulation of adaptive immune responses. The mechanisms underlying these latter processes are less well understood; they most likely involve cytokines but may also involve direct interactions with APC such as dendritic cells (DCs) (72, 73). NK cells are activated by pro-inflammatory cytokines such as type I interferons (IFNs) and interleukin-12 (IL-12) (74) or by bacterial unmethylated cytosineguanine dinucleotides (CpG)-rich oligodeoxynucleotides (CpG-ODN) (75), eac/7/e Calmette-Guerin  (BCG) (76) and CFA (77). Activation enhances  both the cytotoxicity and cytokine production of NK cells.  1.5.1  NK cells in Autoimmune Diabetes  In the 1980s, several groups made a concerted effort to examine the number and function of NK cells in patients with type 1 diabetes mellitus. Many groups used different markers such as Leu7 (CD57) or Leu11a (CD16) to identify NK cells, but we now know that these markers are also present on other subsets of cells. One highly suggestive study, however, indicated that the use of both Leu7 and Leu11a was specific for NK cells since NK cells are the only population of leukocytes known to express both markers. Significantly, the authors reported "a clear tendency for striking low values (of Leu7+ and Leu11a+ cells) in patients with diabetes of short duration". Furthermore, these values were significantly correlated, suggesting that a population which expressed both markers (i.e. NK cells) was responsible for these deficiencies (78). 19  A defect in NK cell lytic activity in patients with type 1 diabetes was first reported in 1986 (79). Using Cr-labelled K562 targets, the author found that 51  the non-adherent leukocytes (principally T and NK cells) from diabetic patients killed fewer target cells than those of the controls. Two other groups subsequently confirmed this observation (80). In animal models of type 1 diabetes, NK cells have also been reported to have both numerical and functional deficiencies (81, 82). However, it still remains unknown if NK deficiencies affect immunoregulatory functions in NOD mice. As mentioned before, NK cell cytotoxicity and cytokine secretion can be increased by treating with CFA. Therefore, in this thesis, we hypothesized that CFA activates NK cells that then act to regulate diabetes.  20  2  CHAPTER TWO: OBJECTIVES  Complete Freund's adjuvant (CFA) treatment of NOD mice prevents autoimmune diabetes although the mechanism(s) of protection that is not fully understood. Based on observations described in chapter 1, we proposed the following specific objectives: (1) To determine the correlation of NRP-V7 reactive cytotoxic T lymphocytes and CFA by measuring the frequency of NRP-V7 reactive CTL in CFA- or PBS-injected NOD mice. (2) To determine the correlation of natural killer (NK) cells and CFA by measuring the population of NK cells in CFA- or PBS-injected NOD mice. (3) To determine the correlation of NK cell function and CFA administration by measuring cytotoxicity assay and cytokine secretion function in CFA- or PBS-injected NOD mice. (4) To determine the role of NK cells in CFA prevention mechanism by adoptive transfer experiment.  21  3  3.1  CHAPTER THREE: MATERIALS  Complete Freund's adjuvant  CFA was purchased from Sigma, St. Louis, Missouri for the following experiments.  3.2  Cell Lines  YAC-1 was obtained from American Type Culture Collection (ATCC) as target cell line for cytotoxicity assay.  3.3  Cell Culture Reagents  3.3.1 R10 medium RPMI Medium 1640 (Gibco/BRL) Fetal bovine serum (FBS, Gibco/BRL) Heat-inactived FBS: Heat FBS at 60 °C for 30 minutes. Penicillin/ Streptomycin/ Neomycin (PSN, 1 mg/ml, Gibco/BRL): Use at 1% (v/v).  22  3.3.2 Phosphate buffered saline (PBS) 34.0g NaCl 4.28g N a H P 0 2  4  1.38g NaH P0 »H 0 2  4  2  Dissloved in 4L of H 0 and autoclave to sterilize. 2  3.4 Mice Female NOD mice were purchased from Taconic Farms (Germantown, NY) and female NOD/SCID were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in the specific pathogen-free facility at the animal care unit of the British Columbia Research Institute for Children's and Women's Health. The Animal Care Committee, Faculty of Medicine, University of British Columbia approved the care and use of all animals. The NOD mice were used as an established model of spontaneous type 1 diabetes and NOD/SCID mice have been reported to be resistant, and were used to examine host factors involved in the presentation of disease onset.  23  3.5  Tissue Preparation Reagents  3.5.1  Erythrocyte lysis buffer (pH 7.4)  4.145g NH CI 4  0.5g  KHCO3  18.6mg Na EDTA 2  Dissolved in 500ml H 0 2  3.6  Immuno-stainging Reagents  3.6.1  Monoclonal antibodies  FITC-conjugated anti-CD3; PE-conjugated DX5; PE-conjugated CD11c; PE-conjugated anti-CD4; PE-conjugated anti-CD8; Cy-Chrome-conjugated anti-CD8 and PerCP-conjugated B220 were purchased from Pharmingen. FITC-conjugated anti-CD8 and PE-conjugated CD11b were purchased from Cedarlane. PE-conjugated NRP-V7 tetramer was synthesized as previously described (83).  3.6.2 Washing buffer 100 ml Phosphate bovine serum (PBS) Bovine serum albumin (BSA): Use at 1% (v/v)  24  3.6.3 Fixing buffer 100ml Phosphate bovine serum (PBS) Fatal calf serum (FBS): Use at 1% (v/v) 2.5ml paraformaldehye  3.7  Chromium Release assay (CRA)  R10 medium Chromium-51  3.8  ELISPOT assay  Purified IFN-y antibody (final concentration: 15ug/ml) Biotin IFN-y antibody (final concentration: 1u.g/ml) Streptavidin-alkaline phosphatase (1:1000 dilution) Color-developing buffer: 25X AP color development buffer: 240uJ AP color reagent A: 60ul AP color reagent B: 60uJ H 0 : 5.76 ml 2  25  4  4.1  CHAPTER FOUR: METHODS  CFA immunizations and assessment of diabetes  Unless otherwise indicated, 5-week-old female NOD mice were given a single 100 uJ injection of an emulsion of CFA in the base of their tails. The development of diabetes was monitored by testing blood glucose twice weekly with test strips. Mice with greater than 23mM blood glucose measurement were considered diabetic and sacrificed.  4.2  Cell staining  Cells taken from experimental mice were removed erythrocytes and were washed in PBS and incubated with the indicated monoclonal antibodies conjugates with flurochrome for 30 minutes in a total volume of 25 uJ of washing buffer (PBS containing 3% bovine serum albumin). After incubation, cells were washed with washing buffer for three times and resuspended in PBS containing 1% fetal calf serum and paraformaldehyde. Immunostained cells were analyzed on a FACScalibur flow cytometer using Cellquest (Becton Dickinson, San Jose, CA).  26  4.3  NK cell enrichment  Anti-mouse Thy-1.2 monoclonal antibody (Phamingen) was used for depleting splenic T cells from mice. Briefly, 2 x 10 spleen cells from mice were 7  incubated with 5ug of anti-mouse Thy-1.2 antibody for 45 minutes at 4°C under constant agitation. The treated spleen cells were washed and incubated with a 1:6 dilution of rabbit complement (Sigma) for 45 minutes at 37°C. After the cells were thoroughly washed, the resulting cell suspension was used as a T-cell depleted suspension for following cytotoxicity assay and ELISPOT assay. The percentage of NK cells after T-cell depletion was determined by staining with CD3, DX5 and CD8 and was analyzed by flow cytometry.  4.4  Cytotoxicity assay  Target YAC-1 cells were labeled with C r for 90 minutes at 37°C and washed 51  twice. Labeled target cells (1 x 10 cells in 100 ul) were incubated with 4  different ratio of effector cells (Prepared from 4.3, 100 pi). Chromium release was measured in the supernatant (100 pi) harvested after 4-hour incubation at 37°C. The percent specific lysis was calculated as: 100 x [(experimental spontaneous release)/(total - spontaneous release)].  27  4.5  ELISPOT assay  The ELISpot assays were performed using 96-well MAIP S4510 0.45-u.m plates coated with a hydrophobic high protein binding Immobilon P membrane (Millipore, Watford, U.K.). ELISpot kits were supplied by Pharmingen (Pharmingen, San Diego, CA). The plate was coated with 15 u;g/ml antimouse IFN-y mAb (1-D1K, Pharmingen) for overnight at 4°C, washed six times with sterile PBS (Gibco), and blocked with 100 ul R10 (RPMI with 10% FCS) for 1 hour at room temperature. T cell-depleted spleen cells were resuspended in R10 and were applied to the plate with YAC-1 cells. The plate was incubated for 48 hours at 37°C, then washed six times using PBS. A second biotinylated mAb to IFN-y (7-B6-1-biotin, Pharmingen) was applied at 1 u.g/ml and incubated for 2 hours at room temperature. The plate was then washed with PBS for six times and incubated for an additional 1 hour at room temperature with 1:1000 dilution of streptavidin-ALP (Bio-Rad, Hercules, CA). The plate was then washed again as described above and developed with ALP conjugate substrate kit (Bio-Rad). The developing reaction was stopped after 10-20 minutes (according to the color intensity of spots) by flicking off the supernatant and running the wells under tap water. Spots were counted under the microscope.  28  4.6  Immunostaining for NK cells in blood  Peripheral blood was collected from tails of NOD mice before and 2, 6 and 24 hours after CFA or PBS immunization. Following lysis of erythrocytes, cells were incubated with antibodies to DX5 and CD3 on ice for 30 minutes and washed with PBS containing 3% bovine serum albumin twice. Immunostained cells were resuspended in PBS containing 1% fetal calf serum and paraformaldehyde and analyzed by flow cytometry as above.  4.7  Selective depletion of NK cells  Depletion of NK cells was performed both in vitro and in vivo using anti-asialo GM1 (Wako Bioproducts, Richmond, VA). In vitro depletion was performed by complement-mediated cytotoxicity to deplete NK cells. Briefly, 2 x 10 spleen 7  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, 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 ul of anti-asialo-GM1 or an equivalent amount of a rabbit serum (Sigma) one day before adoptive transfer experiments. The  29  efficacy of such treatment was evaluated using standard YAC-1 cellular cytotoxicity assays and by flow cytometry for NK cell markers.  4.8  Purification of 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 for three times with PBS and CD3-CD8-DX5+ cells were sorted on a FACSVantage S E Turbo cell sorter (Becton Dickinson, San Jose, CA) at the UBC Multi-User Flow Cytometry Facility. Purified cells were incubated in RPMI 1640 medium (Gibco, Grand Island, NY) plus 10% of fetal calf serum at 37°C overnight to detach antibodies. Cells were then washed twice with PBS before adoptive transfer.  4.9  Adoptive transfer  Adoptive transfers were performed as previously described (84). Recipient female NOD/SCID mice, 4-8 weeks of age, were injected intravenously with donor splenocytes (2 x 10 viable cells) suspended in 200 uJ of PBS. Diabetic 7  spleen cell donors were female NOD mice that typically had exhibited blood glucose level greater than 33mM for at least 2 weeks. Multiple diabetic donor spleens were pooled to produce sufficient cells for all of the hosts in a given experiment.  30  4.10 Statistical analysis Student's t test was used to calculate statistical significance in most experiments and ANOVA analysis was used for multi-group comparisons. A Log Rank test was applied to compare survival curves.  31  5  5.1  CHAPTER FIVE: RESULTS AND DISCUSSION  CFA immunization  prevents diabetes in NOD mice  To confirm the protective effect of CFA in NOD mice, five-week-old female NOD mice were injected with 100 u,l of 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 (45, 46, 50, 51), a single injection of CFA both delayed and prevented diabetes in NOD mice (Figure 1). By 32 weeks of age, 9 PBS-injected mice were diabetic compared to 3 CFA-injected mice. In addition, the level of insulitis in 14-18 week old NOD mice observed from the immunostaining slides for both CFA- and PBS-immunization showed that CFA-immunization group had mild level of insulitis compared to PBSimmunization group (data not shown).  32  "lOOffl-  .2  -*-CFA  -a-PBS  80-  4-*  o S n  c o z  60-  6-  6-  40-  6-  20-  6-12  —i— 16  —i— 20  —i— 24  —i— 28  --o ~32  A g e (weeks)  Figure 1. Effect of CFA on incidence of diabetes in NOD mice. Female NOD mice (n=10) received a single injection of CFA (100uJ) 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 > 23mM was considered diabetic.  33  5.2  CFA immunization prevents the accumulation  of (J-cell specific  CTL  in NOD mice  As mentioned in chapter 1, one population of CTL that appears to be important for P-cell destruction in NOD mice is characterized by its recognition of the NRP-V7 peptide. To determine the effect of CFA on a population of autoreactive P-cell specific CTL, the NRP-V7 reactive CTL in NOD mice were quantified following immunization. Our laboratory has reported previously that NRP-V7 reactive CTL are readily detected in the spleens of pre-diabetic NOD mice (83). In this study, female NOD mice (n = 5 per group) were immunized either with CFA or PBS at 5 weeks of age and sacrificed between 14 and 18 weeks of age. Spleen cells were then analyzed for the presence of NRP-V7 reactive CTL (Figure 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 CTL. This effect was specific to the autoreactive CTL, as there was no change in the proportion of total CD8+ cells present in either CFA or PBS injected mice (Fig. 2B). Since the NRP-V7 reactive CTL populations in pancreatic islets, lymph nodes and spleen have been shown to be significantly increased in pre-diabetic NOD mice between 11-14 weeks of age and CFA-immunization prevents diabetes, it was not unexpected that this P-cell specific CTL population decreased after CFA treatment. 34  A. J2 o> o  1  - ' 5 0  1.25-  + >  1-ooH  | 0.75H g  0.50-  •  o 0.00-  CFA  PBS  B.  30-j  "o5  25-  a. » 15+ 00 Q 10-  O  |  ~ T ~  PBS  CFA  I  5-  °  Figure 2. EfTecr of CFA on B-cell specific CTL. Female NOD mice were given a single injection of C F A (n=20, 100uJ) or P B S (n=20, 1 OOLII) at 5 weeks of age. The mice were sacrificed at 14-18 weeks of age and the NRP-V7 reactive / CD8+ spleen cell population was determined (A). (* P < 0.05, Student's t-Test). The total number of CD8+ cells in spleen was also measured concurrently (B). The results are the mean value of five independent experiments and the error bars refer to the standard deviation generated from the repeated assays.  35  5.3  CFA immunization prevents  cyclophosphamide-accelerated  diabetes in NOD mice  Previous studies have shown that upon cyclophosphamide (CYP) administration, NOD mice (about 70%) rapidly progress to overt diabetes with severe insulitis within two to three weeks (85, 86). Moreover, tuberculosis  or Bacille Calmette-Guerin  Mycobacterium  (BCG) administration has been shown  to prevent this cyclophosphamide-induced diabetes (87). Here we determined the effect of CFA immunization on the progression of cyclophosphamideaccelerated diabetes in NOD mice and monitored its effect on P-cell specific CTL. Eight-week-old female NOD mice were separated to three groups for different treatment: (A) Cyclophosphamide (250mg/kg mouse weight) injection three days before immunizing with 100uJ of PBS. (B) Cyclophosphamide (250mg/kg mouse weight) injection three days before immunizing with 1 OOULI of CFA. (C) 100uJ of PBS immunization only. Blood glucose was monitored twice weekly for two weeks.  As expected, 4 out of 5 NOD mice from group A became diabetic or hyperglycemia two weeks later, whereas none of NOD mice from group B or C developed diabetes (Figure 3, only show data from group A and B). To determine if there was a difference in the number of NRP-V7 reactive CTL 36  between three groups of mice, we sacrificed mice two weeks after treatment and analyzed this population by flow cytometry (Figure 4). NOD mice from group A had a significantly increased proportion of NRP-V7 reactive CTL compared to PBS-immunized group (group C). However, after CFA immunization, the proportion of NRP-V7 reactive population was significantly decreased. Cyclophosphamide is an alkylating agent used in cancer therapy because it blocks cell cycle progression. It is still unclear how cyclophosphamide accelerates diabetes; however, our data show that cyclophosphamide somehow results in the expansion of autoreactive CTL. In addition, consistent with our previous result that CFA decreased the population of NRP-V7 autoreactive CTL, CFA prevented the expansion of this autoreactive CTL population in cyclophosphamide model.  37  Figure 3. Effect of CFA in cyclophosphamide-accelerated diabetes. 2 groups of female NOD mice (n=5 per group) were given a single injection of C F A (100ul) or P B S (100ut) followed by a single dose of cyclophosphamide (250mg/kg mouse weight) at 8 weeks of age. Blood glucose was monitored twice weekly and any animal with a reading of > 23mM was considered diabetic.  38  1-5-  j  •  PBS  O +  r-.  >  I] C Y P ICYP+CFA  1.0-  I  tt  + to Q  I——I  0.5-  O 0.0PBS  F i g u r e 4.  CYP  Effect of CFA on p-cell specific  accelerated  CYP+CFA  CTL in  cyclophosphamide-  diabetes.  2 g r o u p s of f e m a l e N O D m i c e (n=5 p e r g r o u p ) w e r e g i v e n a s i n g l e injection of (100uJ) o r P B S (100ut) f o l l o w e d by a s i n g l e injection of c y c l o p h o s p h a m i d e  CFA  (250mg/kg  m o u s e w e i g h t ) at 8 w e e k s o f a g e . T h e t h i r d g r o u p o f m i c e w a s t r e a t e d w i t h P B S u,l) o n l y . T h e m i c e w e r e t h e n s a c r i f i c e d 2 w e e k s l a t e r a n d t h e N R P - V 7 r e a c t i v e / C D 8 + s p l e e n cell population w a s d e t e r m i n e d . (P = 0 . 0 6 7 2 for C Y P CYP+CFA,  Student's t-Test).  39  versus  (100  5.4  CFA immunization produces a late decrease in splenic NK cells of NOD mice  CFA administration has been shown to stimulate both the cytotoxicity and IFN-y secretion of NK cells in C57BI6 mice (77). In order to determine whether CFA also affects NK cells in NOD mice, spleen cells from CFA- or PBSimmunized NOD mice (as 5.3) were sacrificed 10-14 weeks after immunization and stained for CD3, a T cell marker, and DX5, a pan-NK cell marker (Figure 5), to determine the NK population. Surprisingly, mice injected with CFA had a significantly lower proportion of CD3-DX5+ (represented NK cell) spleen cells than PBS-injected mice (p < 0.001) suggesting that CFA has an effect on number or distribution of NK cells. NK cells have been shown to have both numerical and functional deficiencies in NOD mice and NOD/SCID mice, and our data showed that CFA-immunized mice had even lower NK events comparing to PBS-immunized mice. Shi etal have shown that NK cytotoxicity and IFN-y secretion are increased as early as one week after CFA immunization. Therefore, it is possible that NK cells in NOD mice were activated by CFA at an early time point but were subsequently eliminated by apoptosis following regulation of the immune response.  40  ** w  aJ o  +  X Q •  3H  ro  Q O  CFA  PBS  Figure 5. Effect of CFA on natural killer cells. F e m a l e N O D mice w e r e given a single injection of C F A (n=20, 100uJ) or P B S (n=20, 100u.l) at 5 w e e k s of age. T h e mice w e r e sacrificed at 14-18 w e e k s of a g e and the C D 3 - / D X 5 + s p l e e n cell population w a s determined. (* P < 0.0001, Student's t-Test).  41  5.5  CFA induces rapid peripheral blood accumulation  of NK cells  From the observation in 5.4 that the NK cell population was decreased 14-18 weeks after CFA immunization, we therefore hypothesized that CFA may affect or activate NK cells at an earlier time point following immunization. To test our hypothesis, NOD female mice were injected with CFA or PBS and peripheral blood was assayed for CD3-DX5+ cells before (0) and at 2, 6 and 24 hours after immunization (Figure 6). In NOD mice injected with CFA, there was a significant accumulation of CD3-DX5+ cells, peaking at 6 hours postimmunization. By 24 hours, the proportion of CD3-DX5+ cells in the peripheral blood had returned to pre-immunization levels. In contrast, there were no significant differences in the population of CD3-DX5+ cells of PBS-immunized NOD mice. These data suggest that CFA acts to increase the proportion of NK cells in peripheral blood shortly after immunization. Immunization of NOD mice with CFA produced an expansion of CD3-DX5+ cells in peripheral blood as early as 6 hours after receiving a single administration of CFA. The same pattern was also seen in spleen cells (Figure 7). While other groups have reported that BCG stimulates NK cells to proliferate (88), it is more likely, given the rapid accumulation of NK cells, that the increased proportion of cells seen following CFA immunization represents a trafficking phenomenon, rather than NK cell proliferation. Since CFA was administered subcutaneously in the tail base, NK cells may have been responding and trafficking from the spleen to the tail in response to bacterial  42  stimulation.  43  40-  1  jf>  C F A (n=7)  ~o o 30-  P B S (n=3)  + uo X  9  JL  20-  I  to Q O  tOH 2  6  24  Hours after injection  Figure 6. Effect of CFA on peripheral  blood NK cells.  F e m a l e N O D mice w e r e given a single injection of C F A (n=7, 1 OOLII) or P B S (n=3, 1 OOLLI) at 8 w e e k s of age. Peripheral blood w a s collected before a n d at 2 , 6 and 2 4 hours after C F A or P B S injection. T h e N K cell population w a s determined by staining m o n o n u c l e a r cells with antibodies to C D 3 and D X 5 surface markers. (* P < 0.05, S i n g l e Factor A N O V A ) .  44  8n CO  0  6  12  24  Hours after CFA treatment  Figure 7. Effect  of CFA on splenic NK cells.  Female NOD mice were given a single injection of CFA (n=3, 100uJ) at 5 weeks of age. Spleen cells were harvested 0, 6, 12 and 24 hours after immunization and the NK cell population was determined by staining mononuclear cells with antibodies to CD3 and DX5 surface markers.  45  5.6  CFA activates the cytotoxicity and cytokine secretion functions of  NK cells  Previous result indicates an effect of CFA on NK cells. To determine whether CFA acts on the function of NK cells in NOD mice, female NOD mice were immunized withlOOuJ of CFA or PBS, spleen cells were harvested at 2, 4, 6 and 24 hours following immunization and assayed for cytotoxicity and IFN-y secretion. NOD mice have been shown to possess defects in NK cell number; therefore, to obtain enough NK cells for this experiment, we depleted T cells by using anti-mouse Thy-1.2 monoclonal antibody and pooled NK cells before functional assays. Flow cytometric analysis confirmed that after T cell depletion, 90% of T cells were depleted (data not shown). For cytotoxicity assay, T-cell depleted spleen cells were subsequently mixed with chromium51 labeled YAC-1 target cells with 100:1, 50:1 and 25:1 dilution and incubated at 37 °C for 4 hours. Released chromium-51 was detected by a y-counter. We used 50:1 and 25:1 dilution for T-cell depleted spleen cells and YAC-1 target cell in IFN-y ELISPOT assay and incubated at 37 °C for 48 hours. IFN-y secretion was detected by anti-IFN-y antibody and an alkaline phosphatase colour readout. The result of the NK cytotoxicity assays showed a slightly increased killing by CFA-immunized NK cells but the difference were not statistically significant between CFA and PBS groups (data not shown). This may have been because the time points that we choose (2, 6 and 24 hours) were not the optimal for the cytotoxicity assay, or perhaps the number of NK  46  cells was insufficient for the cytotoxicity. However, IFN-y secretion by CFAimmunized NK cells significantly increased compared to PBS-immunized mice (Figure 8A and B). In conjunction with the rapid cell mobilization shown previously, our results indicated that NK cells are stimulated by CFA. These findings also suggest that a defect of IFN-y secretion exists in NOD NK cells that may be overcome by administration of CFA.  47  PBS  CFA  YAC-1 CFA/SC  PMA  B. * 50  •  CFA(n=7)  •  P B S (n=4)  o CFA  PBS  Figure 8. Effect of CFA on NK IFN-y secretion. F e m a l e N O D m i c e w e r e given a single injection of C F A (n=7, 100u,l) or P B S (n=6, 1 OOJJ.1) a n d sacrificed 4 hours after injection. S p l e e n cells w e r e depleted of T cells before performing IFN-y E L I S p o t a s s a y s (500,000 effector cells per well a n d 10,000 Y A C - 1 target cells per well). (A) R e p r e s e n t a t i v e wells are s h o w n (from left to right): T cell-depleted s p l e e n cells from C F A or P B S i m m u n i z e d N O D m i c e incubated with Y A C - 1 cells, cells from C F A i m m u n i z e d mice incubated with P M A (5 ng/ml) a n d ionomycin (0.4 ug/ml), Y A C - 1 cells in m e d i u m alone, a n d T - d e p l e t e d s p l e e n cells from C F A i m m u n i z e d m i c e in m e d i u m a l o n e ( C F A / S C ) . (B) S u m m a r y of E L I S p o t data. T h e m e a n n u m b e r of s p o t s produced by T-depleted s p l e e n cells obtained from C F A or P B S i m m u n i z e d N O D m i c e is s h o w n a s spot forming units ( S F U ) per 5 x 1 0 cells (* P < 0.01, Student's t-Test).  48  5  5.7  CFA protection from diabetes is dependent on NK cells  The mechanism by which CFA prevents diabetes in NOD mice is unclear, although the above data suggest that downregulation of a population of autoreactive CTL or activation of NK cells may play a role. A previous study has suggested that the protective effect of CFA is dependent on a population of CD11 b+ (Mac-1 +) spleen cells (45) but the identity of the CD11 b+ cells was unclear. For the following reasons, we hypothesized that CFA immunization stimulated NK cells to mediate the protective effect. First, our data showed an early effect of CFA on a CD3-DX5+ subset of spleen cells (Figure 6 and 8). Second, investigators have reported that CFA activates NK cells both by increasing IFN-y secretion and by increasing cytolytic activity (77). Lastly, NOD mice have been shown to carry defects in NK cell activity (81). To ascertain a role for NK cells in the protective effect of CFA, we used an adoptive transfer model of diabetes. Spleen cells from diabetic NOD mice have previously been shown to passively transfer diabetes to irradiated NOD mice (89) and CFA has been shown to inhibit the transfer of diabetes (50). We designed experiments to adoptively transfer diabetes to NOD/SCID mice, to prevent the transfer of disease by CFA immunization and to determine the effect of NK cell depletion on diabetes outcome. In order to remove the effects of NK cells thoroughly from the experiment, we treated both NOD donor and NOD/SCID recipient mice with anti-asialo GM1. We first determined the phenotypic specificity of this antibody by co-staining spleen cells from NOD mice and NOD/SCID mice with anti-asialo GM1 and markers for T cells (CD4 49  or CD8), macrophages / dendritic cells (CD11b or CD11c) and NK cells (DX5). Figure 9 indicates that asialo GM1 is predominantly expressed by a population of CD11b+/DX5+ spleen cells. Thus, asialo GM1 positive cells are NK cells that co-express DX5 and CD11b as has been previously described (90).  50  NOD/SCID DX5  _ o  CD11b  o  CD11c  o  CD8  o  CD4  Figure 9. Characterization ofasialo GM1 positive spleen cells. S p l e e n cells w e r e harvested from 5-6 w e e k old N O D / S C I D or N O D mice a n d c o immunostained with rabbit anti-asialo GM1 and the indicated phycoerythrinconjugated monoclonal antibodies. Histogram plots o f a s i a l o GM1 positive s p l e e n cells are s h o w n . P E , phycoerythrin fluorescence.  51  We next investigated whether NK cell depletion by anti-asialo GM1 would alter the outcome of adoptively-transferred disease in mice immunized with CFA. Recipient NOD/SCID mice were pre-treated with either anti-asialo GM1 antibody (to deplete NK cells) or rabbit serum (control), then immunized with CFA 24 hours later. Donor spleen cells pooled from diabetic NOD mice were also depleted of NK cells by asialo GM1 antibody. After NK cell depletion, spleen cells were intravenously injected to the NOD/SCID recipients and the blood glucose of recipients was measured once weekly beginning on the day of adoptive transfer.  Figure 10A shows that all PBS-immunized NOD/SCID recipients receiving spleen cells developed diabetes within 5 weeks of adoptive transfer. In contrast, the group of adoptively transferred mice that were immunized with CFA did not develop disease until between 6 and 10 weeks following adoptive transfer, with 2 (33%) mice remaining diabetes-free beyond 10 weeks. However, CFA-immunized mice pre-treated with anti-asialo GM1, that received anti-asialo GM1 depleted spleen cells, developed hyperglycemia between 3 and 4 weeks post-transfer. To confirm the specificity of the antiasialo GM1 effect, a group of NOD/SCID recipients were pre-treated 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 (Figure 10B).  52  To exclude the possibility that natural killer T (NKT) cells expressing asialo GM1 were mediating the effects of CFA, a population of CD3-DX5+ cells (5 x 10 cells) obtained by sorting NOD splenocytes was returned to the anti-asialo 5  GM1 treated donor spleen cells prior to adoptive transfer. 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 (Figure 10C). Previous results have shown that NK cells are activated and play a role in regulating the immune response after CFA immunization. How might NK cells be stimulated by CFA? We suggest that the effects of CFA immunization are likely mediated first by antigen presenting cells, in particular dendritic cells (DCs) and possibly by the intermediary actions of NKT cells. There are several lines of evidence to support this model. First, DCs by virtue of their anatomical locations (mucosal and epithelial antigen exposure sites) and expression of innate receptors are one of the first cells to recognize and respond to foreign stimuli including vaccines and mycobacterial antigens (91, 92). Second, DCs are known to express CD1 and to activate NKT cells in a CD1 dependent fashion (93). Finally, activated NKT cells rapidly "cross-talk" to stimulate NK cells, primarily through the secretion of IFN-y (94). Moreover, NK cells themselves have recently been shown to directly suppress the afferent limb of the primary immune response (95) providing a potential mechanism by which NK cells limit the expansion of P-cell specific CTL. Alternatively, DC may activate NK cells directly without the need for an NKT cell intermediary possibly through the secretion of IL-12 (96). Many recent  53  reports have highlighted the importance of the interaction between human dendritic cells and natural killers based on a pioneering study describing the existence of reciprocal control during NK-DC interactions in the mouse system. These studies have shown that DCs can prime resting NK cells, which in turn, after activation, might induce DC maturation. However, NK cells negatively regulate the function of DCs also by killing immature DCs in peripheral tissues (97-99). Gerosa  etal  have shown that in the presence of  inflammatory mediators such as lipopolysaccharide or mycobacteria, human NK cells (CD3-CD16+CD56+) were activated by dendritic cells. Because NK cells are known to be both functionally and numerically deficient in NOD mice (as well as NOD/SCID mice) (81, 82, 100), we hypothesize that autoimmunity in these mice may be partly a consequence of the inability of NK cells to regulate DC. Interestingly, defects in NK cells have previously been suggested in humans with type 1 diabetes (78, 80, 101). However, these observations have not recently been pursued in the context of current knowledge and it may be worthwhile to revisit these observations in light of our findings.  54  A. O-CFA  100 fr o  .a re  - A - C F A & asialo GM1  80  Oi  •  c o  z  -•-PBS  i  60  6-  40  6—o  I I I  20  •4-  0  —i  10  W e e k s after adoptive transfer  B. 100* fl>  C F A & asialo GM1 - C F A & rabbit s e r u m  80  J3  CO  '•5 • c o  z  60 H 40-  20 I  0-  4  6  8  W e e k s afetr adoptive transfer  55  10  c. --0--CFA M  o .o  — A - C F A & asialo GM1  80-1  - • - C F A & asialo GM1 & NK 6-  c  0 - - 0  204  02  4  6  8  10  Weeks after adoptive transfer  Figure 10. Prevention of diabetes by CFA is dependent on a population ofasialo GM1 positive cells.  (A) Pooled spleen cells from diabetic NOD mice (2 x 10 cells) were adoptively 7  transferred to NOD/SCID recipient mice that were immunized with PBS (•, n=8), CFA (O, n=8) or with CFA after depletion of NK cells from donor cells and recipient mice with anti-asialo GM1 (•, n=10). (B) Recipient mice immunize d with CFA were pre-treated with either 50 uJ of rabbit anti-asialo GM1 antibody or rabbit serum IgG ( • , n=3) before adoptive transfer. (C) One group of NOD/SCID recipient mice that was also pre-treated with 50 \i\ of asialo GM1 antibody and immunized with 100 uJ of CFA received adoptive transfer with asialo GM1-depleted diabetogenic spleen cells mixed with 5 X 10 CD3- / DX5+ spleen cells (•, n=4). Blood glucose was monitored 5  weekly for all experiments and diabetes was defined as a single reading of > 33mM.  56  6  CHAPTER SIX: SUMMARY  The overall objective of this thesis was to elucidate the protective mechanism of complete Freund's adjuvant in non-obese diabetic mice. Our first hypothesis is that, since the number of NRP-V7 reactive CTL in NOD mice correlates with progression to overt disease, prevention of diabetes by CFA immunization should prevent the accumulation of these autoreactive cells. Second, CFA has been reported to augment both NK cytolytic function and cytokine secretion in C57BL/6 mice and similar effects have been described in human patients using another mycobacterium-containing adjuvant, BCG. Therefore, we hypothesized that the function of NK cells in NOD mice may be augmented by immunization with CFA. As expected, the proportion of NRPV7 reactive CTL was significantly decreased in NOD mice by a single immunization of CFA compared to a PBS-immunized group. As well, we demonstrated that in cyclophosphamide-accelerated diabetes model, CFA prevention of diabetes was also associated with a decrease in NRP-V7 reactive CTL. 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