Linkage disequilibrium (association) analysis was used to evaluate a candidate region near the CTLA4/CD28 genes using a multi-ethnic collection of families with one or more children affected by IDDM. In the data set unique to this study (Spanish, French, Mexican-American, Chinese and Korean), the transmission/disequilibrium test (TDT) revealed a highly significant deviation for transmission of alleles at the (AT)n microsatellite marker in the 3' untranslated region (P = 0.002) and the A/G polymorphism in the first exon (P = 0.00002) of the CTLA4 gene. The overall evidence for transmission deviation of the CTLA4 A/G alleles is also highly significant (P = 0.00005) in the combined data set (669 multiplex and 357 simplex families) from this study and a previous report on families from USA, Italy, UK, Spain and Sardinia. Significant heterogeneity was observed in these data sets. The British, Sardinian and Chinese data sets did not show any deviation for the A/G polymorphism, while the Caucasian-American data set showed a weak transmission deviation. Strong deviation for transmission was seen in the three Mediterranean-European populations (Italian, Spanish and French) (P = 10(-5)), the Mexican-American population (P = 0.002) and the Korean population (P = 0.03). These results suggest that a true IDDM susceptibility locus (designated IDDM12) is located near CTLA4.

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic β cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

Type 1 diabetes in both humans and nonobese diabetic (NOD) mice results from T-cell-mediated autoimmune destruction of insulin-producing pancreatic β cells. Linkage studies have shown that type 1 diabetes in NOD mice is a polygenic disease involving more than 15 chromosomal susceptibility regions. Despite extensive investigation, the identification of individual susceptibility genes either within or outside the major histocompatibility complex region has proven problematic because of the limitations of linkage analysis. In this paper, we provide evidence implicating a single diabetes susceptibility gene, which lies outside the major histocompatibility complex region. Using allelic reconstitution by transgenic rescue, we show that NOD mice expressing the β 2 microglobulin (β 2 M) a allele develop diabetes, whereas NOD mice expressing a murine β 2 M b or human allele are protected. The murine β 2 M a allele differs from the β 2 M b allele only at a single amino acid. Mechanistic studies indicate that the absence of the NOD β 2 M a isoform on nonhematopoietic cells inhibits the development or activation of diabetogenic T cells.

Curing type 1 diabetes by islet transplantation requires overcoming both allorejection and recurrent autoimmunity. This has been achieved with systemic immunosuppression, but tolerance induction would be preferable. Most islet allotransplant tolerance induction protocols have been tested in nonobese diabetic (NOD) mice, and most have failed. Failure has been attributed to the underlying autoimmunity, assuming that autoimmunity and resistance to transplantation tolerance have a common basis. Out of concern that NOD biology could be misleading in this regard, we tested the hypothesis that autoimmunity and resistance to transplantation tolerance in NOD mice are distinct phenotypes. Unexpectedly, we observed that (NOD × C57BL/6)F1 mice, which have no diabetes, nonetheless resist prolongation of skin allografts by costimulation blockade. Further analyses revealed that the F1 mice shared the dendritic cell maturation defects and abnormal CD4+ T cell responses of the NOD but had lost its defects in macrophage maturation and NK cell activity. We conclude that resistance to allograft tolerance induction in the NOD mouse is not a direct consequence of overt autoimmunity and that autoimmunity and resistance to costimulation blockade-induced transplantation tolerance phenotypes in NOD mice can be dissociated genetically. The outcomes of tolerance induction protocols tested in NOD mice may not accurately predict outcomes in human subjects.

T ype 1 diabetes is an autoimmune disease characterized by the T-cell-mediated destruction of the insulin-secreting -cells of the pancreatic islets (1,2). It is a complex disease involving numerous susceptibility genes and environmental factors, which may act as triggers of the autoimmune response. Much research has been focused on identifying type 1 diabetes susceptibility genes in an effort to enhance the prediction of which individuals are at high risk of developing the disease, as well as to understand the pathogenesis in order to prevent the disease. Thus far, only two susceptibility regions have been well characterized. I D D M 1 maps to the HLA region and actually contains multiple susceptibility genes (3-11). I D D M 2 h a s been identified as the variable number of tandem repeats immediately 5 to the insulin gene (12-19), which appears to play a role in the level of gene expression (13,14,(20)(21)(22). Many additional susceptibility intervals have been suggested by complete or partial genome scans conducted by multiple labs (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). Several of these intervals (I D D M 4, I D D M 5, I D D M 8, and I D D M 1 2) have been confirmed in additional data sets (33-35), but the exact genes responsible for increased susceptibility to type 1 diabetes have not been i d e n t i fied. Other proposed susceptibility intervals remain to be confir m e d .One of the confirmed type 1 diabetes susceptibility loci is I D D M 1 2 , located on chromosome 2q33. Initial studies provided evidence of linkage and association of a polymorphic marker in the C T L A 4 gene with type 1 diabetes in Italian and Spanish families (35). These results were confirmed in an additional data set that included French, Spanish, MexicanAmerican, and U.S. Caucasian families (34). Case-control studies of a Belgian and German data set provided additional c o n firmation of association between C T L A 4 p o l y m o r p h i s m s and type 1 diabetes (35,36). There is also association of C T L A 4 polymorphisms with Graves' disease and Hashimoto's thyroiditis, both autoimmune thyroid diseases (36)(37)(38)(39)(40).Although these studies of I D D M 1 2 provided convincing evidence for I D D M 1 2, the precise location of the gene responsible for the disease is not well defined. Although C T L A 4 is an ideal candidate gene for autoimmune diseases, the association between type 1 diabetes and CTLA4 p o l ymorphisms could be due to linkage disequilibrium of the RESEARCH DESIGN AND METHODSS u b j e c t s . Genomic DNA was obtained from members of 178 simplex and 350 multiplex (with two or more affected children) type 1 diabetes families (Ta b l e 1). The collection includes U.S. Caucasian, Mexican-American, French, Spanish, Italian, Korean, and Chinese families. All diabetic patients participating in this study were diagnosed using the criteria of the National Diabetes Data Group. The clinical information on the patients has been described in previous publications (34,44). G e n o t y p i n g . Microsatellite repeats were ge...