Journal of Gastroenterology and Hepatology (1991) 6 , 574-579
SECOND INTERNATIONAL SYMPOSIUM ON PRIMARY BILIARY CIRRHOSIS Antigen presentation and effector mechanisms in 'auto-immune' diabetes YI WANG,* MARCIA M c D U F F I E ~A N D KEVIN J. LAFFERTY*+ Departments of *MicrobiologylImmunology and tPediatrics, Barbara Davis Center for Childhood Diabetes, Univtrsity of Colorado Health Sciences Center, Denver, Colorado, USA
INTRODUCTION Type I or insulin-dependent diabetes results from immunological destruction of insulin-producing islets of the pancreas. A number of auto-antibodies specific for pancreatic islets are found in the blood of the pre-diabetic patient and in newly diagnosed diabetic individual^.^,^ In addition, therapy that suppresses T lymphocyte function, such as cyclosporin, can prolong the function of residual insulinproducing cells in newly-diagnosed patient^.^" For these reasons the disease is considered to have an auto-immune aetiology. Animal models for this disease - the biobreedingmorcester (BB) rat and the non-obese diabetic (NOD) mouse - provide models that have allowed the analysis of this spontaneous, organ-specific, auto-immune disease.8 In this paper we review recent developments relating to the pathogenesis of type I diabetes both in humans and in animal models. Diabetes provides a model for tissue-specific auto-immunity that may be informative in the study of primary biliary cirrhosis (PBC).
Relevance of the model to primary biliary cirrhosis The first convincing link between Type I diabetes and the immune system came from the finding that the disease was associated with organ-specific 'auto-antibodies'. Its dependence on T lymphocytes has only been recognized since animal models for the disease process have become available. A similar evolution of concepts on the pathogenesis of PBC appears to be occurring, although the roles of immunoglobulins and T lymphocytes in the initiation of this disease process are still unclear.' Linkage to MHC haplotypes or alleles, which has been demonstrated in at least one population studied," strengthens the analogy of PBC to Type I diabetes. The most definitive studies await the development of a successful animal model of the disease process. In this regard, the recently reported transfer of peripheral blood
cells from patients with PBC into immunodeficient mice may provide such a model."
Genetics of diabetes Diabetes or, more correctly, the likelihood of developing this disease, is under genetic control; siblings of diabetic individuals have a 20-fold higher risk of developing this disease than individuals drawn from the general population." Although one recent study reported only a 36% concordance for identical twins of diabetic probands, demonstrating that in the case of diabetes we are not dealing with a strictly genetically defined disease,13 genetic factors clearly control the susceptibility of a particular individual to the development of this disease. Genes of the MHC play a major role in the control of diabetes susceptibility. At a population level, multiple studies since 1974 have documented both positive and negative associations between Type I diabetes and MHC.14 Further evidence has been provided by two recent family ~ t u d i e s . ' ~These , ' ~ studies show that one of six siblings who share both MHC alleles with a diabetic proband will develop Type I diabetes, while fewer than 1 in 100 siblings who share neither haplotype will develop overt disease. Siblings with a single shared MHC haplotype have an intermediate risk of roughly 1 in 20. If it is assumed that only one-third of those with genetic susceptibility will develop complete destruction of pancreatic beta cells and require insulin, as is indicated by the data on identical twins, then only about one in two individuals with the same HLA types as their diabetic siblings is genetically 'at risk' of the disease process. The simplest hypothesis developed to explain this finding is that a second gene in addition to MHC is required for disease susceptibility in most families. Actual evidence that genes other than MHC play a role in susceptibility to Type I diabetes has come from breeding studies using NOD mice. By mating NOD mice with mice from non-diabetes-prone inbred strains, several investiga-
Correspondence: K. J. Lafferty, Department of Microbiology, University of Colorado Health Sciences Center, 4200 E 9rh Avenue, Box B-140, Denver, Colorado 80262, USA. Accepted for publication 7 May 1991.
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tors have shown that the Fl generation is protected from development of diabetes. When more NOD genes are bred into these hybrid mice by mating FI with NOD animals, the frequency of diabetes in the offspring of this ‘backcross’ gives an indication of the number of these protective alleles. For example, Leiter et al. used the non-obese normal (NON) strain, a close relative of the NOD, as a non-diabetes-prone gene donor in such a breeding scheme.” In the first backcross generation, only 17% of the mice that were homozygous for the MHC haplotype of the NOD strain became diabetic during a 12 month observation period. (None of the MHC heterozygotes became diabetic.) During this period 75% of NOD mice from their colony would have become insulin-dependent. Using this percentage as the ‘penetrance’ of overt diabetes in genetically susceptible animals, the calculated fraction of backcross mice that were genetically susceptible is about one-quarter (0.17 x 0.75 = 0.23). If diabetes susceptibility could be prevented by a protective allele at a single gene locus that was inherited from the nondiabetes-prone parent, half the mice in the backcross should have been susceptible. If two unlinked alleles were present in the F I ,the probability of inheriting neither of the permissive alleles would be one-quarter (0.5 X 0.5). The actual rate of diabetes susceptibility, therefore, indicates that two protective alleles were independently transmitted in this breeding scheme. Wicker et al. confirmed these findings in breeding studies using C57BL/10 mice, a strain unrelated to the NOD.” Despite the complexity of genetic control of disease susceptibility, the major histocompatibility complex antigens, in particular class I1 MHC antigens, have been shown to play a critical role in the control of disease susceptibility. The Stanford group provided convincing evidence that resistance to diabetes is associated with the presence of asparagine at position 57 of the DQ /? chain of the class I1 MHC antigen in Caucasians.” This situation also appears to be the case in the NOD mouse, where diabetes prone animals are non-Asp at position 57 of the class I1 /3 chain.” This involvement of class I1 MHC antigen in the development of diabetes is likely to operate at an immunological level. We know that the major histocompatibility complex is a system of cell surface molecules that regulate immune function, both at the level of antigen presentation and in terms of shaping the reactive potential of each individual’s immune system.21>22 This contribution of the MHC to the development of auto-immunity (Type I diabetes) could be expressed at either of these levels.
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cos Figure 1 T-cell activation occurs when two signals are provided for the responsive T-cell: (1) antigen binding by the T-cell and (2) a co-stimulator (CoS) provided by the stimulator cell (S+). A control structure (c) on the surface of the S+ cell regulates the release of the co-stimulator. Co-stimulator is released when the responsive T cell interacts with the control structure in conjunction with antigen on the S + cell.
directly to an immunizing antigen ‘X’. This antigen is first processed by the active antigen presenting cells (APC) and presented on the cell surface in the form of peptide(s) designated as ‘x’ - in association with MHC molecules. On theoretical grounds we have proposed that this presentation as ‘c.x’, where c represents MHC antigen, is an absolute requirement for T cell activation because the complex operates as a control molecule regulating delivery of the CoS (Fig. l).23,24 That is, the only T cells that can be activated must be of specificity ‘c.x’, where c represents MHC antigen (class I or class 11) on the APC and x is the peptide product of the immunizing antigen, X . It is this requirement for ‘two signals’ for T cell activation by the APC that ensures T cell responses are restricted by MHC antigens of the One proposal of how APC function may regulate autoimmunity is that the presence of the amino acid asparagine at position 57 of the class I1 /? chain causes a change in the conformation of this antigen so that it is no longer capable of presenting the auto-antigen in question - in this case, peptide p the proposed initiating antigen in Type I diabetes (Fig. 2). Such a model of antigen presentation does not provide a complete solution to the auto-immunity problem. While T cell immunity of any kind requires appropriate antigen presentation, many self-antigens are presented on the surface of active APC without leading to auto-responses. Presentation of self-peptides is probably the normal rather than the abnormal situation.
Let us first consider the MHC contribution to antigen presentation. MHC antigens are of major importance in the Alteration of the reactive potential regulation of the T cell response, and diabetes in both MHC antigens also interact with T cells during the developanimal models is known to be a T cell dependent disease. T cell activation (Fig. 1) is a two-signal process requiring ment of the T cell repertoire in the thymus. Thymic maturaboth engagement of the T cell receptor by antigen and the tion of T cells matches the MHC of the individual with an provision of a second signal, the co-stimulator ( C O S ) . ~ ~ * * appropriate ~ range of T cell receptor specificities. In the first step of repertoire development, low levels of T cell receptor CoS activity is provided by metabolically active antigenmolecules are expressed on the surface of each thymocyte presenting cells (Fig. l).23124 Thus, T cells do not respond
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Effector mechanisms in auto-immune diabetes in the non-obese diabetic (NOD) mouse
non-Asp
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57
Asp
-
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Figure 2 The amino acid asparagine (Asp) at position 57 of the class I1 /j chain causes a change in the conformation of class I1 p chain so that the antigen presenting cell is no longer capable of presenting the islet-associated auto-antigen @).
that has completed effective rearrangement of both the a and /j genes. As each cell expresses a unique pair of receptor chains, the initial range of specificities expressed on these immature cells may be as large as 106.’5~z5 Unless the receptor molecules of a given cell are capable of binding to the MHC molecules expressed on thymic epithelial cells, the nascent cell appears to die very quickly. The survival of the immature T cells that are capable of interacting with selfMHC is known as ‘positive selection’. Selection of cells capable of interacting with self-MHC is important because T cells are constrained to recognizing and binding to foreign antigens only when these molecules bind to MHC molecules, as already discussed. This population of positively selected thymocytes has within it a subpopulation of cells capable of interacting very strongly with self-MHC antigens. Binding of the T cell receptors of these cells with MHC antigens, perhaps those expressed on cells other than thymic epithelial cells, results in the elimination of ‘self-reactive’ thymocytes before they are fully mature and functional. Known as ‘clonal deletion’, this process appears to be the major step in developing tolerance to self-antigens among the T cell pool of an individual positive and negative selection. Although there is no direct evidence for a failure of clonal deletion in Type I diabetes, the presence of ‘self-reactive’ T cells causing pancreatic infiltration and islet damage suggests that this process may be defective in these situations. A specific mechanism by which the unique MHC antigens associated with Type I diabetes could cause such a failure is unknown. It has been speculated, however, that alterations in MHC molecule structure, such as that induced by substitution of serine for asparagine at position 57, could prevent normal interactions between self-peptides, MHC and the receptors of developing T cells that result in deletion of selfreactive T cell clones.
The NOD mouse is an inbred mouse strain derived from an outbred Japanese mouse line.27 A number of colonies are now maintained in different parts of the world. Differences in the incidence of diabetes have been noted in different colonies. In our colony at Denver, diabetes occurs in female mice with an incidence rate of 75% by the age of 250 days. Males have 28% disease incidence by the age of 250 days. Disease starts out as an inflammatory response seen adjacent to the pancreatic islet; the inflammation is focused primarily in the periductular/perivascular region adjacent to the islet (Fig. 3a). With the passage of time, this inflammation increases and spreads to invade the islet tissue (Fig. 3b). The inflammatory lesion is made up of T cells - both CD4 and CD8 T cells -B cells, and macro phage^.^^'^^ The disease process is very specific. Initially, p cells, which make up the majority of cells in the islet, are intact and CL cells (glucagon producers) can be seen scattered neatly around the periphery of the islet. As the disease proceeds, p cells are destroyed while the a cells (and other minority cells of the islet) remain intact. Evidently the islet collapses into what appears to be an ‘a cell islet’. That is, the disease process is the result of very specific damage, leading to cell destruction.
Figure 3 Pancreas from non-diabetic NOD mice was stained with haematoxylin and eosine. (a) The inflammation is focused primarily in the periductular/perivascular region adjacent to the islet. Note the presence of a lymphotic vessel. (b) Inflammation spreads to invade and damage the islet tissue. D: duct, I: islet, MNC: mononuclear cell infiltration ( x 160).
PBC and 'auto-immune' diabetes
T cell involvement in the disease process Both thymectomy at an early age, and T cell depletion can prevent the development of diabetes in the NOD mouse and in the BB rat.30-32The development of disease in both these models can also be arrested by treatment of animals with cyclosporine in the pre-diabetic p e r i ~ d . More ~ ~ , detailed ~~ investigations of the T cell involvement in the disease process have shown cells of both the CD4 and CD8 subset to be required for the transfer of disease from spontaneously diabetic animals to diabetes-prone NOD These findings have been reported independently by a number of groups. Such data have been interpreted in a classical immunological context as indicating that CD4 helper T cells are required for the activation of CD8 cytotoxic cells, specific for islet beta cells, and that it is these p cell specific CD8 cells that are the final effectors of immunological d a m a g ~ ~ ' - This ~ ' model (Fig. 4) would account for the specific destruction of islet p cells seen in this disease process. However, we tested some of the implications of this model (Fig. 4a) and found it to be in need of revision.
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Implications of the cytotoxic damage model The model that proposes that diabetes results from a direct attack of CD8 cytotoxic T cells on islet cells (Fig. 4a) has two testable implications: (1) The disease process, which involves a direct interaction of autoreactive T cells with target /3 cells, will be restricted by MHC antigens expressed on the islet tissue (Fig. 4a) - that is, allogeneic islet tissue will be resistant to auto-immune damage following transplantation to spontaneously diabetic animals (Fig 4a). (2) Although both CD4 and CD8 T cells would be required for the initiation of the disease process, the immunological effector cell will be the CD8 T cell (Fig. 4a). Both these implications of the direct cytotoxic model have now been tested.38
p
cell
Figure 4 (a) p-cell damage results from a direct cytotoxic attack by CD8 T cells. Primed T cells (T') are specific for the c.P complex and can recognize and kill self islet b-cells. T cells of c./3 specificity cannot interact with allogeneic p-cells presenting the complex. (b) p cell damage results from an indirect mechanism mediated by a CD4 T cell dependent inflammatory process. Primed CD4 T cells of specificity (c$) recognize the islet associated antigen p presented on host antigen presenting cells. Specific p-cell destruction results from the high sensitivity of these cells to inflammatory mediators (e.g. cytokines andor oxygen free radicals) released in the inflammatory focus.4z343 According to this model, syngeneic and allogeneic islets are equally sensitive to the immunological process. c: MHC antigen, p : p cell associated antigen.
Disease is not restricted by the MHC of grafted islet tissue We initially set out to test the first implication of the cytotoxic damage model: destruction of islet /3 cells is restricted by MHC antigens present on the islet tissue (Fig. 4). The model system used for these studies involves preculture of allogeneic (SJL) islet tissue in 95%O2to eliminate tissue immunogenicity .39 This allogeneic tissue was then transplanted to spontaneously diabetic NOD mice. The interpretation of such an experiment depends on the ability to differentiate between allograft immunity and disease recurrence as the cause of islet destruction. Unlike allograft immunity, the recurrence of spontaneous disease in an islet graft will have the following characteristics: (i) tissue specificity: islet tissue but not thyroid tissue will be destroyed; and (ii) disease association: islet grafts will be destroyed in diabetic-NOD mice but will survive in non-diabetic NOD mice. Therefore, we established a model system to determine whether the destruction of allogeneic islet grafts was
tissue-specific (islet versus thyroid destruction) and diseaseassociated (islet grafting in diabetic-NOD and non-diabetic NOD mice). To test tissue specificity of the destruction, cultured allogeneic islet tissue and thyroid tissue were transplanted to spontaneously diabetic animals. In this case the thyroid graft acted as a sentinel to indicate any activation of an allograft response during the study. Graft survival was determined by glycaemic control for islet graft or iodine uptake for thyroid graft and histological e~amination.~*-~' The results of this study were clear. All the cultured islet allografts were heavily infiltrated and damaged in diabetic NOD mice, whereas the thyroid grafts remained intact in all recipients at 3-4 weeks after grafting. Function analysis showed that the allogeneic thyroid grafts incorporated ''1 at levels similar to the control isografts, indicating thyroid graft function. Most of the cultured islet allografts failed to normalize blood
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glucose in diabetic NOD mice, whereas this cultured islet tissue can reverse chemical (streptozotocin)-induced Type I diabetes. The destruction of allogeneic thyroid tissue following the grafting of cultured thyroid and uncultured (immunogenic) islet tissue from the same donor showed the capacity of the thyroid graft to act as an immunological sentinel for the detection of an allograft response. In conclusion, allogeneic islet tissue was destroyed in a tissue-specific manner. Further confirmation that the damage seen following the transplantation of allogeneic cultured (non-immunogenic) islets to spontaneously diabetic animals was the result of disease comes from the demonstration that the destructive process is disease-associated. When cultured allogeneic islet tissue was grafted to non-diabetic NOD mice, the tissue remained intact, with little or no inflammatory response to the grafted tissue, which could be stained for intact insulin granules.38 But when the same tissue was transplanted to spontaneously diabetic animals; all grafts showed intense inflammation and partial or complete destruction of islet tissue. These studies show quite clearly that disease recurrence in grafted islet tissue is not restricted by MHC antigens expressed on the grafted tissue. Similar results were reported following grafting of allogeneic islet tissue to spontaneously diabetic BB rats.4’ This lack of MHC restriction of the disease process by islet MHC antigens is not consistent with the direct cytotoxic hypothesis (Fig. 4a).
CD4 T cells are immunological effector cells in the diabetes of the NOD mouse Next, we set out to test the second implication of the cytoxic damage model - that the immunological effector cell of p cell destruction will be the CD8 T cell (Fig. 4a). Using the same antibody depletion protocol described for the disease transfer studies, we examined the T cell requirement for disease recurrence in syngeneic (NOD) islet tissue grafted to spontaneously diabetic NOD mice. In this study, disease was shown to be a CD4 T cell and not a CD8 T cell dependent process. Depletion of the CD4 subject facilitated a graft acceptance and function for as long as the CD4 T cells remained absent from the periphery. Disease recurrence was observed when the level of CD4 T cells rose above approximately 10% of peripheral lymphocytes. Depletion of CD8 T cells, on the other hand, while shown to be quite effective in blocking the transfer of disease from spontaneously diabetic to diabetes-prone animals, had no effect on disease recurrence following the grafting of islet tissue to spontaneously diabetic animals. In this latter situation the grafted tissue was heavily infiltrated by inflammatory cells and in most cases failed to function.38 On the basis of the above observations we find it necessary to abandon the direct cytotoxic hypothesis (Fig. 4a). These data show that both CD4 and CD8 T cells are required for the initiation of the disease process in diabetes prone animals. However, the immunological effector cell does not appear to be the CD8 T cell. The dependence of the disease process on the CD4 T cell led us to propose that autoimmune diabetes is the result of CD4 T cell dependent inflammatory tissue damage (the indirect process; Fig. 4b). One problem with this notion is the clear tissue specificity of
the disease process. If damage is simply the result of inflammatory mediators, how do we account for specific /j cell destruction in this process? Pancreatic islet tissue /3 cells are particularly sensitive to free radical damage,42 and one possibility is that in this disease we may be dealing with nonspecific inflammatory tissue damage that expresses itself as specific /?cell damage because of the target tissue’s sensitivity to oxygen radical^.^^'^^
Protection by superoxide dismutase Evidence for the involvement of the superoxide O2radical in the auto-immune diabetogenic process was obtained by examining the capacity of polyethelene glycol modified superoxide dismutase (PEG-SOD) to protect grafted islet In these tissue from damage in the diabetic studies, grafting of spontaneously diabetic NOD animals was effective in reversing the diabetes only when animals were treated with PEG-SOD plus PEG catalase, or PEGSOD alone for 10 days from the time of islet grafting. The use of PEG-modified enzymes in these studies is required to reduce the immunogenicity of the bovine enzymes and to provide enzymic activity with a prolonged half-life (approximately 36 h) in the recipient mouse.
Implications for therapy Although ‘auto-immune’ diabetes is dependent on at least a subset of circulating T cells in the NOD mouse and in humans, non-specific suppression of T cell function carries too many demonstrable and theoretical side effects to be useful as life-time therapy. The effectiveness of superoxide dismutase in preventing disease recurrence in transplanted pancreatic tissue suggests that methods of inhibiting inflammatory mediators, such as interleukins and oxygen radicals, may be effective in preventing damage to the pancreatic /i’ cells. Likewise, identification of specific subsets of CD4 T lymphocytes that are predisposed to react with pancreatic tissue may provide alternative ‘immunosuppressive’ therapy with significantly less life-time risk.
ACKNOWLEDGEMENT This work was supported by a NIH Program Project Grant no. P040144.
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Islet cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancer 1974; 2 : 1279-82. 2. BOTTAZZO G. F. cell damage in diabetic insulitis: are we approaching a solution? Diabetologia 1984; 26: 24 1-9. 3 . EISENBARTH G. S. Type I diabetes mellitus. A chronic autoimmune disease. N. Engl. 3. Med. 1986; 314: 1360-8. 4. NAYAK R. C., OMARM.A. K., RABIZADEH A., SRIKANTA S. & EISENBARTH G. S. ‘Cytoplasmic’ islet cell antibodies; Evidence that the target antigen is a sialoglycoconjugate. Diabetes 1985; 34: 617-9.
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6. STILLERC., DUPREJ., GENI M. et al. Effects of cyclosporine immunosuppression in insulin-dependent diabetes mellitus of recent onset. Science 1984; 223: 1362-7. 7. FEIJTREN G., ASSAN R., KAKSENTY G . el 01. Cyclosporin increases the rate and length of remissions in insulin-dependent diabetes of recent onset: Results of a multicentre double-blind trial. Lancet 1986; 19: 119-23. 8. ROSSINI A. A,, MORDES J. P. & LIKEA. A. Immunology of insulin-dependent diabetes mellitus. Ann. Rev. Immunol. 1985; 3: 289-320. I. R. & GERSHWIN M. E. Primary biliary cirrhosis: 9. MACKAY current knowledge, perspectives, and future directions. Semin. Liver Dis. 1989; 9: 149-57. L. D., POWELL F. C. & 10. GORESG . J., MOORES. B., FISHER DICKSON E. R. Primary biliary cirrhosis: associations with class I1 major histocompatibility complex antigens. Hepatology 1987; 7: 889-92. 11. KRAMS S. M., DORSHKIND K. & GERSHWIN M. E. Generation of biliary lesions after transfer of human lymphocytes into severe combined immunodeficient (SCID) mice. J. Exp. Med. 1989; 170: 1919-30. 12. GAMBLE D. R. An epidemiological study of childhood diabetes affecting two or more siblings. Diabetologia 1980; 19: 341-4. 13. THOMSON G., ROBINSON W. P., KUHNER M. K. etal. Genetic heterogeneity, modes of inheritance, and risk estimates for a joint study of Caucasians with insulin-dependent diabetes mellitus. Am. J . Hum. Genet. 1988; 43: 799-816. T. & MOLVIGJ. The HLA14. NERUPJ., MANDRUP-POULSEN IDDM association: implications for etiology and pathogenesis of IDDM. Diabetes Metab. Rev. 1986; 3: 779-802. A. N., SPENCERK. M., LISTER J., WOLF E., 15. GOKSUCH BoTTAzzO G. F. & CUDWORTH A. G. Can future type I diabetes be predicted? A study in families of affected children. Diabetes 1982; 31: 862-6. A. C., DEANB. M., SCHWARZ G., THOMAS J. M., 16. TARN INGRAM D., BOTTAZZO G. F. & GALEE. A. M. Predicting insulin-dependent diabetes. Lancet 1988; i: 845-50. D. V. & COLEMAN 17. PROCHAZKA M., LEITERE. H., SERREZE D. L. Three recessive loci required for insulin-dependent diabetes in nonobese diabetic mice. Science 1987; 237: 286-9. 18. WICKERL. S., MILLERB. J., COKERL. Z. et al. Genetic control of diabetes and insulitis in the non-obese diabetic (NOD) mice. Pedigree analysis of a diabetic H-2""Vb heterozygote. J. Exp. Med. 1987; 165: 1639-54. J. A., BELLJ. I. & MCDEVITT H . 0. HLA-DQ/3 gene 19. TODD contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 1987; 329: 599-604. 20. ACHA-ORBEA H. & MCDEVITTH. 0. The first external domain of the non-obese diabetic mouse class I-A fi chain is unique. Proc. Natl Acad. Sci. U S A 1987; 84: 2435-9. 2 1. GERMAIN R. G. Analysis of the expression and function of class-I1 major histocompatibility complex-encoded molecules by DNAmediated gene transfer. Ann. Rev. Immunol. 1986; 4: 281-315. 22. BRACIALE T . J., MORRISON L . A , , SWEETSER M. T., SAMBROOK J., GETHINCM-J. & BRACIALE V. L . Antigen presentation pathways to class I and class I1 MHC-restricted T lymphocytes. Immunol. Rev. 1987; 98: 95-114. A. J. A new analysis of 23. LAFFERTY K. J. & CUNNINGHAM allogeneic interactions. Aust. J. Exp. Biol.Med. Sci. 1975; 53: 27-42. 24. LAFFERTY K. J., PROWSE S. J. & SIMEONOVIC C. J. Immunology of tissue transplantation: a return to the passenger leukocyte concept. Ann. Rev. Immunol. 1983; 1: 143-73. 25. DAVISM. M. & BJORKMAN P. J. T-cell antigen receptor genes and T-cell recognition. Nature 1988; 334: 395-402. 26. BLACKMAN M., KAPPLER J . & MARRACK P.The role of the T
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SECOND INTERNATIONAL SYMPOSIUM ON PRIMARY BILIARY CIRRHOSIS Antigen presentation and effector mechanisms in 'auto-immune' diabetes YI WANG,* MARCIA M c D U F F I E ~A N D KEVIN J. LAFFERTY*+ Departments of *MicrobiologylImmunology and tPediatrics, Barbara Davis Center for Childhood Diabetes, Univtrsity of Colorado Health Sciences Center, Denver, Colorado, USA
INTRODUCTION Type I or insulin-dependent diabetes results from immunological destruction of insulin-producing islets of the pancreas. A number of auto-antibodies specific for pancreatic islets are found in the blood of the pre-diabetic patient and in newly diagnosed diabetic individual^.^,^ In addition, therapy that suppresses T lymphocyte function, such as cyclosporin, can prolong the function of residual insulinproducing cells in newly-diagnosed patient^.^" For these reasons the disease is considered to have an auto-immune aetiology. Animal models for this disease - the biobreedingmorcester (BB) rat and the non-obese diabetic (NOD) mouse - provide models that have allowed the analysis of this spontaneous, organ-specific, auto-immune disease.8 In this paper we review recent developments relating to the pathogenesis of type I diabetes both in humans and in animal models. Diabetes provides a model for tissue-specific auto-immunity that may be informative in the study of primary biliary cirrhosis (PBC).
Relevance of the model to primary biliary cirrhosis The first convincing link between Type I diabetes and the immune system came from the finding that the disease was associated with organ-specific 'auto-antibodies'. Its dependence on T lymphocytes has only been recognized since animal models for the disease process have become available. A similar evolution of concepts on the pathogenesis of PBC appears to be occurring, although the roles of immunoglobulins and T lymphocytes in the initiation of this disease process are still unclear.' Linkage to MHC haplotypes or alleles, which has been demonstrated in at least one population studied," strengthens the analogy of PBC to Type I diabetes. The most definitive studies await the development of a successful animal model of the disease process. In this regard, the recently reported transfer of peripheral blood
cells from patients with PBC into immunodeficient mice may provide such a model."
Genetics of diabetes Diabetes or, more correctly, the likelihood of developing this disease, is under genetic control; siblings of diabetic individuals have a 20-fold higher risk of developing this disease than individuals drawn from the general population." Although one recent study reported only a 36% concordance for identical twins of diabetic probands, demonstrating that in the case of diabetes we are not dealing with a strictly genetically defined disease,13 genetic factors clearly control the susceptibility of a particular individual to the development of this disease. Genes of the MHC play a major role in the control of diabetes susceptibility. At a population level, multiple studies since 1974 have documented both positive and negative associations between Type I diabetes and MHC.14 Further evidence has been provided by two recent family ~ t u d i e s . ' ~These , ' ~ studies show that one of six siblings who share both MHC alleles with a diabetic proband will develop Type I diabetes, while fewer than 1 in 100 siblings who share neither haplotype will develop overt disease. Siblings with a single shared MHC haplotype have an intermediate risk of roughly 1 in 20. If it is assumed that only one-third of those with genetic susceptibility will develop complete destruction of pancreatic beta cells and require insulin, as is indicated by the data on identical twins, then only about one in two individuals with the same HLA types as their diabetic siblings is genetically 'at risk' of the disease process. The simplest hypothesis developed to explain this finding is that a second gene in addition to MHC is required for disease susceptibility in most families. Actual evidence that genes other than MHC play a role in susceptibility to Type I diabetes has come from breeding studies using NOD mice. By mating NOD mice with mice from non-diabetes-prone inbred strains, several investiga-
Correspondence: K. J. Lafferty, Department of Microbiology, University of Colorado Health Sciences Center, 4200 E 9rh Avenue, Box B-140, Denver, Colorado 80262, USA. Accepted for publication 7 May 1991.
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tors have shown that the Fl generation is protected from development of diabetes. When more NOD genes are bred into these hybrid mice by mating FI with NOD animals, the frequency of diabetes in the offspring of this ‘backcross’ gives an indication of the number of these protective alleles. For example, Leiter et al. used the non-obese normal (NON) strain, a close relative of the NOD, as a non-diabetes-prone gene donor in such a breeding scheme.” In the first backcross generation, only 17% of the mice that were homozygous for the MHC haplotype of the NOD strain became diabetic during a 12 month observation period. (None of the MHC heterozygotes became diabetic.) During this period 75% of NOD mice from their colony would have become insulin-dependent. Using this percentage as the ‘penetrance’ of overt diabetes in genetically susceptible animals, the calculated fraction of backcross mice that were genetically susceptible is about one-quarter (0.17 x 0.75 = 0.23). If diabetes susceptibility could be prevented by a protective allele at a single gene locus that was inherited from the nondiabetes-prone parent, half the mice in the backcross should have been susceptible. If two unlinked alleles were present in the F I ,the probability of inheriting neither of the permissive alleles would be one-quarter (0.5 X 0.5). The actual rate of diabetes susceptibility, therefore, indicates that two protective alleles were independently transmitted in this breeding scheme. Wicker et al. confirmed these findings in breeding studies using C57BL/10 mice, a strain unrelated to the NOD.” Despite the complexity of genetic control of disease susceptibility, the major histocompatibility complex antigens, in particular class I1 MHC antigens, have been shown to play a critical role in the control of disease susceptibility. The Stanford group provided convincing evidence that resistance to diabetes is associated with the presence of asparagine at position 57 of the DQ /? chain of the class I1 MHC antigen in Caucasians.” This situation also appears to be the case in the NOD mouse, where diabetes prone animals are non-Asp at position 57 of the class I1 /3 chain.” This involvement of class I1 MHC antigen in the development of diabetes is likely to operate at an immunological level. We know that the major histocompatibility complex is a system of cell surface molecules that regulate immune function, both at the level of antigen presentation and in terms of shaping the reactive potential of each individual’s immune system.21>22 This contribution of the MHC to the development of auto-immunity (Type I diabetes) could be expressed at either of these levels.
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cos Figure 1 T-cell activation occurs when two signals are provided for the responsive T-cell: (1) antigen binding by the T-cell and (2) a co-stimulator (CoS) provided by the stimulator cell (S+). A control structure (c) on the surface of the S+ cell regulates the release of the co-stimulator. Co-stimulator is released when the responsive T cell interacts with the control structure in conjunction with antigen on the S + cell.
directly to an immunizing antigen ‘X’. This antigen is first processed by the active antigen presenting cells (APC) and presented on the cell surface in the form of peptide(s) designated as ‘x’ - in association with MHC molecules. On theoretical grounds we have proposed that this presentation as ‘c.x’, where c represents MHC antigen, is an absolute requirement for T cell activation because the complex operates as a control molecule regulating delivery of the CoS (Fig. l).23,24 That is, the only T cells that can be activated must be of specificity ‘c.x’, where c represents MHC antigen (class I or class 11) on the APC and x is the peptide product of the immunizing antigen, X . It is this requirement for ‘two signals’ for T cell activation by the APC that ensures T cell responses are restricted by MHC antigens of the One proposal of how APC function may regulate autoimmunity is that the presence of the amino acid asparagine at position 57 of the class I1 /? chain causes a change in the conformation of this antigen so that it is no longer capable of presenting the auto-antigen in question - in this case, peptide p the proposed initiating antigen in Type I diabetes (Fig. 2). Such a model of antigen presentation does not provide a complete solution to the auto-immunity problem. While T cell immunity of any kind requires appropriate antigen presentation, many self-antigens are presented on the surface of active APC without leading to auto-responses. Presentation of self-peptides is probably the normal rather than the abnormal situation.
Let us first consider the MHC contribution to antigen presentation. MHC antigens are of major importance in the Alteration of the reactive potential regulation of the T cell response, and diabetes in both MHC antigens also interact with T cells during the developanimal models is known to be a T cell dependent disease. T cell activation (Fig. 1) is a two-signal process requiring ment of the T cell repertoire in the thymus. Thymic maturaboth engagement of the T cell receptor by antigen and the tion of T cells matches the MHC of the individual with an provision of a second signal, the co-stimulator ( C O S ) . ~ ~ * * appropriate ~ range of T cell receptor specificities. In the first step of repertoire development, low levels of T cell receptor CoS activity is provided by metabolically active antigenmolecules are expressed on the surface of each thymocyte presenting cells (Fig. l).23124 Thus, T cells do not respond
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Effector mechanisms in auto-immune diabetes in the non-obese diabetic (NOD) mouse
non-Asp
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Figure 2 The amino acid asparagine (Asp) at position 57 of the class I1 /j chain causes a change in the conformation of class I1 p chain so that the antigen presenting cell is no longer capable of presenting the islet-associated auto-antigen @).
that has completed effective rearrangement of both the a and /j genes. As each cell expresses a unique pair of receptor chains, the initial range of specificities expressed on these immature cells may be as large as 106.’5~z5 Unless the receptor molecules of a given cell are capable of binding to the MHC molecules expressed on thymic epithelial cells, the nascent cell appears to die very quickly. The survival of the immature T cells that are capable of interacting with selfMHC is known as ‘positive selection’. Selection of cells capable of interacting with self-MHC is important because T cells are constrained to recognizing and binding to foreign antigens only when these molecules bind to MHC molecules, as already discussed. This population of positively selected thymocytes has within it a subpopulation of cells capable of interacting very strongly with self-MHC antigens. Binding of the T cell receptors of these cells with MHC antigens, perhaps those expressed on cells other than thymic epithelial cells, results in the elimination of ‘self-reactive’ thymocytes before they are fully mature and functional. Known as ‘clonal deletion’, this process appears to be the major step in developing tolerance to self-antigens among the T cell pool of an individual positive and negative selection. Although there is no direct evidence for a failure of clonal deletion in Type I diabetes, the presence of ‘self-reactive’ T cells causing pancreatic infiltration and islet damage suggests that this process may be defective in these situations. A specific mechanism by which the unique MHC antigens associated with Type I diabetes could cause such a failure is unknown. It has been speculated, however, that alterations in MHC molecule structure, such as that induced by substitution of serine for asparagine at position 57, could prevent normal interactions between self-peptides, MHC and the receptors of developing T cells that result in deletion of selfreactive T cell clones.
The NOD mouse is an inbred mouse strain derived from an outbred Japanese mouse line.27 A number of colonies are now maintained in different parts of the world. Differences in the incidence of diabetes have been noted in different colonies. In our colony at Denver, diabetes occurs in female mice with an incidence rate of 75% by the age of 250 days. Males have 28% disease incidence by the age of 250 days. Disease starts out as an inflammatory response seen adjacent to the pancreatic islet; the inflammation is focused primarily in the periductular/perivascular region adjacent to the islet (Fig. 3a). With the passage of time, this inflammation increases and spreads to invade the islet tissue (Fig. 3b). The inflammatory lesion is made up of T cells - both CD4 and CD8 T cells -B cells, and macro phage^.^^'^^ The disease process is very specific. Initially, p cells, which make up the majority of cells in the islet, are intact and CL cells (glucagon producers) can be seen scattered neatly around the periphery of the islet. As the disease proceeds, p cells are destroyed while the a cells (and other minority cells of the islet) remain intact. Evidently the islet collapses into what appears to be an ‘a cell islet’. That is, the disease process is the result of very specific damage, leading to cell destruction.
Figure 3 Pancreas from non-diabetic NOD mice was stained with haematoxylin and eosine. (a) The inflammation is focused primarily in the periductular/perivascular region adjacent to the islet. Note the presence of a lymphotic vessel. (b) Inflammation spreads to invade and damage the islet tissue. D: duct, I: islet, MNC: mononuclear cell infiltration ( x 160).
PBC and 'auto-immune' diabetes
T cell involvement in the disease process Both thymectomy at an early age, and T cell depletion can prevent the development of diabetes in the NOD mouse and in the BB rat.30-32The development of disease in both these models can also be arrested by treatment of animals with cyclosporine in the pre-diabetic p e r i ~ d . More ~ ~ , detailed ~~ investigations of the T cell involvement in the disease process have shown cells of both the CD4 and CD8 subset to be required for the transfer of disease from spontaneously diabetic animals to diabetes-prone NOD These findings have been reported independently by a number of groups. Such data have been interpreted in a classical immunological context as indicating that CD4 helper T cells are required for the activation of CD8 cytotoxic cells, specific for islet beta cells, and that it is these p cell specific CD8 cells that are the final effectors of immunological d a m a g ~ ~ ' - This ~ ' model (Fig. 4) would account for the specific destruction of islet p cells seen in this disease process. However, we tested some of the implications of this model (Fig. 4a) and found it to be in need of revision.
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Implications of the cytotoxic damage model The model that proposes that diabetes results from a direct attack of CD8 cytotoxic T cells on islet cells (Fig. 4a) has two testable implications: (1) The disease process, which involves a direct interaction of autoreactive T cells with target /3 cells, will be restricted by MHC antigens expressed on the islet tissue (Fig. 4a) - that is, allogeneic islet tissue will be resistant to auto-immune damage following transplantation to spontaneously diabetic animals (Fig 4a). (2) Although both CD4 and CD8 T cells would be required for the initiation of the disease process, the immunological effector cell will be the CD8 T cell (Fig. 4a). Both these implications of the direct cytotoxic model have now been tested.38
p
cell
Figure 4 (a) p-cell damage results from a direct cytotoxic attack by CD8 T cells. Primed T cells (T') are specific for the c.P complex and can recognize and kill self islet b-cells. T cells of c./3 specificity cannot interact with allogeneic p-cells presenting the complex. (b) p cell damage results from an indirect mechanism mediated by a CD4 T cell dependent inflammatory process. Primed CD4 T cells of specificity (c$) recognize the islet associated antigen p presented on host antigen presenting cells. Specific p-cell destruction results from the high sensitivity of these cells to inflammatory mediators (e.g. cytokines andor oxygen free radicals) released in the inflammatory focus.4z343 According to this model, syngeneic and allogeneic islets are equally sensitive to the immunological process. c: MHC antigen, p : p cell associated antigen.
Disease is not restricted by the MHC of grafted islet tissue We initially set out to test the first implication of the cytotoxic damage model: destruction of islet /3 cells is restricted by MHC antigens present on the islet tissue (Fig. 4). The model system used for these studies involves preculture of allogeneic (SJL) islet tissue in 95%O2to eliminate tissue immunogenicity .39 This allogeneic tissue was then transplanted to spontaneously diabetic NOD mice. The interpretation of such an experiment depends on the ability to differentiate between allograft immunity and disease recurrence as the cause of islet destruction. Unlike allograft immunity, the recurrence of spontaneous disease in an islet graft will have the following characteristics: (i) tissue specificity: islet tissue but not thyroid tissue will be destroyed; and (ii) disease association: islet grafts will be destroyed in diabetic-NOD mice but will survive in non-diabetic NOD mice. Therefore, we established a model system to determine whether the destruction of allogeneic islet grafts was
tissue-specific (islet versus thyroid destruction) and diseaseassociated (islet grafting in diabetic-NOD and non-diabetic NOD mice). To test tissue specificity of the destruction, cultured allogeneic islet tissue and thyroid tissue were transplanted to spontaneously diabetic animals. In this case the thyroid graft acted as a sentinel to indicate any activation of an allograft response during the study. Graft survival was determined by glycaemic control for islet graft or iodine uptake for thyroid graft and histological e~amination.~*-~' The results of this study were clear. All the cultured islet allografts were heavily infiltrated and damaged in diabetic NOD mice, whereas the thyroid grafts remained intact in all recipients at 3-4 weeks after grafting. Function analysis showed that the allogeneic thyroid grafts incorporated ''1 at levels similar to the control isografts, indicating thyroid graft function. Most of the cultured islet allografts failed to normalize blood
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glucose in diabetic NOD mice, whereas this cultured islet tissue can reverse chemical (streptozotocin)-induced Type I diabetes. The destruction of allogeneic thyroid tissue following the grafting of cultured thyroid and uncultured (immunogenic) islet tissue from the same donor showed the capacity of the thyroid graft to act as an immunological sentinel for the detection of an allograft response. In conclusion, allogeneic islet tissue was destroyed in a tissue-specific manner. Further confirmation that the damage seen following the transplantation of allogeneic cultured (non-immunogenic) islets to spontaneously diabetic animals was the result of disease comes from the demonstration that the destructive process is disease-associated. When cultured allogeneic islet tissue was grafted to non-diabetic NOD mice, the tissue remained intact, with little or no inflammatory response to the grafted tissue, which could be stained for intact insulin granules.38 But when the same tissue was transplanted to spontaneously diabetic animals; all grafts showed intense inflammation and partial or complete destruction of islet tissue. These studies show quite clearly that disease recurrence in grafted islet tissue is not restricted by MHC antigens expressed on the grafted tissue. Similar results were reported following grafting of allogeneic islet tissue to spontaneously diabetic BB rats.4’ This lack of MHC restriction of the disease process by islet MHC antigens is not consistent with the direct cytotoxic hypothesis (Fig. 4a).
CD4 T cells are immunological effector cells in the diabetes of the NOD mouse Next, we set out to test the second implication of the cytoxic damage model - that the immunological effector cell of p cell destruction will be the CD8 T cell (Fig. 4a). Using the same antibody depletion protocol described for the disease transfer studies, we examined the T cell requirement for disease recurrence in syngeneic (NOD) islet tissue grafted to spontaneously diabetic NOD mice. In this study, disease was shown to be a CD4 T cell and not a CD8 T cell dependent process. Depletion of the CD4 subject facilitated a graft acceptance and function for as long as the CD4 T cells remained absent from the periphery. Disease recurrence was observed when the level of CD4 T cells rose above approximately 10% of peripheral lymphocytes. Depletion of CD8 T cells, on the other hand, while shown to be quite effective in blocking the transfer of disease from spontaneously diabetic to diabetes-prone animals, had no effect on disease recurrence following the grafting of islet tissue to spontaneously diabetic animals. In this latter situation the grafted tissue was heavily infiltrated by inflammatory cells and in most cases failed to function.38 On the basis of the above observations we find it necessary to abandon the direct cytotoxic hypothesis (Fig. 4a). These data show that both CD4 and CD8 T cells are required for the initiation of the disease process in diabetes prone animals. However, the immunological effector cell does not appear to be the CD8 T cell. The dependence of the disease process on the CD4 T cell led us to propose that autoimmune diabetes is the result of CD4 T cell dependent inflammatory tissue damage (the indirect process; Fig. 4b). One problem with this notion is the clear tissue specificity of
the disease process. If damage is simply the result of inflammatory mediators, how do we account for specific /j cell destruction in this process? Pancreatic islet tissue /3 cells are particularly sensitive to free radical damage,42 and one possibility is that in this disease we may be dealing with nonspecific inflammatory tissue damage that expresses itself as specific /?cell damage because of the target tissue’s sensitivity to oxygen radical^.^^'^^
Protection by superoxide dismutase Evidence for the involvement of the superoxide O2radical in the auto-immune diabetogenic process was obtained by examining the capacity of polyethelene glycol modified superoxide dismutase (PEG-SOD) to protect grafted islet In these tissue from damage in the diabetic studies, grafting of spontaneously diabetic NOD animals was effective in reversing the diabetes only when animals were treated with PEG-SOD plus PEG catalase, or PEGSOD alone for 10 days from the time of islet grafting. The use of PEG-modified enzymes in these studies is required to reduce the immunogenicity of the bovine enzymes and to provide enzymic activity with a prolonged half-life (approximately 36 h) in the recipient mouse.
Implications for therapy Although ‘auto-immune’ diabetes is dependent on at least a subset of circulating T cells in the NOD mouse and in humans, non-specific suppression of T cell function carries too many demonstrable and theoretical side effects to be useful as life-time therapy. The effectiveness of superoxide dismutase in preventing disease recurrence in transplanted pancreatic tissue suggests that methods of inhibiting inflammatory mediators, such as interleukins and oxygen radicals, may be effective in preventing damage to the pancreatic /i’ cells. Likewise, identification of specific subsets of CD4 T lymphocytes that are predisposed to react with pancreatic tissue may provide alternative ‘immunosuppressive’ therapy with significantly less life-time risk.
ACKNOWLEDGEMENT This work was supported by a NIH Program Project Grant no. P040144.
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