Michele P. Marron

HLA-A*0201-Restricted T Cells from Humanized NOD Mice Recognize Autoantigens of Potential Clinical Relevance to Type 1 Diabetes

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.

Functional evidence for the mediation of diabetogenic T cell responses by HLA-A2.1 MHC class I molecules through transgenic expression in NOD mice.

Particular major histocompatibility complex (MHC) class II alleles clearly contribute to T cell-mediated autoimmune type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice. However, studies in NOD mice indicate MHC class I-restricted T cell responses are also essential to T1D development. In humans, epidemiological studies have suggested that some common class I alleles, including HLA-A2.1 (A*02011), may confer increased susceptibility to T1D when expressed in conjunction with certain class II alleles. We show here that when HLA-A2.1 molecules are transgenically expressed in NOD mice, A2-restricted T cell responses arise against pancreatic β cells, leading to an earlier onset of T1D. The accelerated onset of T1D in the NOD.HLA-A2.1 transgenic mice is not due to nonspecific effects of expressing a third class I molecule, because a stock of NOD mice transgenically expressing HLA-B27 class I molecules showed no such acceleration of T1D, but rather were significantly protected from disease. These findings provide the first functional evidence that certain human MHC class I molecules can contribute to the development of T1D.

Genetic Disassociation of Autoimmunity and Resistance to Costimulation Blockade-Induced Transplantation Tolerance in Nonobese Diabetic Mice

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.

In vivo cytotoxicity of insulin-specific CD8+ T-cells in HLA-A*0201 transgenic NOD mice.

OBJECTIVE—CD8+ T-cells specific for islet antigens are essential for the development of type 1 diabetes in the NOD mouse model of the disease. Such T-cells can also be detected in the blood of type 1 diabetic patients, suggesting their importance in the pathogenesis of the human disease as well. The development of peptide-based therapeutic reagents that target islet-reactive CD8+ T-cells will require the identification of disease-relevant epitopes. RESEARCH DESIGN AND METHODS—We used islet-infiltrating CD8+ T-cells from HLA-A*0201 transgenic NOD mice in an interferon-γ enzyme-linked immunospot assay to identify autoantigenic peptides targeted during the spontaneous development of disease. We concentrated on insulin (Ins), which is a key target of the autoimmune response in NOD mice and patients alike. RESULTS—We found that HLA-A*0201-restricted T-cells isolated from the islets of the transgenic mice were specific for Ins1 L3–11, Ins1 B5–14, and Ins1/2 A2–10. Insulin-reactive T-cells were present in the islets of mice as young as 5 weeks of age, suggesting an important function for these specificities early in the pathogenic process. Although there was individual variation in peptide reactivity, Ins1 B5–14 and Ins1/2 A2–10 were the immunodominant epitopes. Notably, in vivo cytotoxicity to cells bearing these peptides was observed, further confirming them as important targets of the pathogenic process. CONCLUSIONS—The human versions of B5–14 and A2–10, differing from the murine peptides by only a single residue, represent excellent candidates to explore as CD8+ T-cell targets in HLA-A*0201–positive type 1 diabetic patients.

MHC Class II Molecules Play a Role in the Selection of Autoreactive Class I-Restricted CD8 T Cells That Are Essential Contributors to Type 1 Diabetes Development in Nonobese Diabetic Mice

Development of autoreactive CD4 T cells contributing to type 1 diabetes (T1D) in both humans and nonobese diabetic (NOD) mice is either promoted or dominantly inhibited by particular MHC class II variants. In addition, it is now clear that when co-expressed with other susceptibility genes, some common MHC class I variants aberrantly mediate autoreactive CD8 T cell responses also essential to T1D development. However, it was unknown whether the development of diabetogenic CD8 T cells could also be dominantly inhibited by particular MHC variants. We addressed this issue by crossing NOD mice transgenically expressing the TCR from the diabetogenic CD8 T cell clone AI4 with NOD stocks congenic for MHC haplotypes that dominantly inhibit T1D. High numbers of functional AI4 T cells only developed in controls homozygously expressing NOD-derived H2g7 molecules. In contrast, heterozygous expression of some MHC haplotypes conferring T1D resistance anergized AI4 T cells through decreased TCR (H2b) or CD8 expression (H2q). Most interestingly, while AI4 T cells exert a class I-restricted effector function, H2nb1 MHC class II molecules can contribute to their negative selection. These findings provide insights to how particular MHC class I and class II variants interactively regulate the development of diabetogenic T cells and the TCR promiscuity of such autoreactive effectors.

Prevention of “Humanized” Diabetogenic CD8 T-cell Responses in HLA-Transgenic NOD Mice by a Multipeptide Coupled-Cell Approach

OBJECTIVE Type 1 diabetes can be inhibited in standard NOD mice by autoantigen-specific immunotherapy targeting pathogenic CD8+ T-cells. NOD.β2mnull.HHD mice expressing human HLA-A2.1 but lacking murine major histocompatibility complex class I molecules develop diabetes characterized by CD8 T-cells recognizing certain autoantigenic peptides also targeted in human patients. These include peptides derived from the pancreatic β-cell proteins insulin (INS1/2 A2–10 and INS1 B5–14) and islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP265–273 and IGRP228–236). Hence, NOD.β2mnull.HHD mice represent a model system for developing potentially clinically translatable interventions for suppressing diabetogenic HLA-A2.1–restricted T-cell responses. RESEARCH DESIGN AND METHODS Starting at 4–6 weeks of age, NOD.β2mnull.HHD female mice were injected intravenously with syngeneic splenocytes to which various admixtures of the four above-mentioned peptides were bound by the cross-linking agent ethylene carbodiimide (ECDI). RESULTS Treatment with such cells bearing the complete cocktail of INS and IGRP epitopes (designated INS/IGRP-SPs) significantly inhibited diabetes development in NOD.β2mnull.HHD recipients compared with controls receiving splenocytes coupled with an irrelevant HLA-A2.1–restricted Flu16 peptide. Subsequent analyses found syngeneic splenocytes bearing the combination of the two ECDI-coupled IGRPs but not INS peptides (IGRP-SPs or INS-SPs) effectively inhibited diabetes development in NOD.β2mnull.HHD mice. This result was supported by enzyme-linked immunospot (ELISPOT) analyses indicating combined INS/IGRP-SPs diminished HLA-A2.1–restricted IGRP but not INS autoreactive CD8+ T-cell responses in NOD.β2mnull.HHD mice. CONCLUSIONS These data support the potential of a cell therapy approach targeting HLA-A2.1–restricted IGRP autoreactive CD8 T-cells as a diabetes intervention approach in appropriate human patients.

Genetic homogeneity, high-resolution mapping, and mutation analysis of the urofacial (Ochoa) syndrome and exclusion of the glutamate oxaloacetate transaminase gene (GOT1) in the critical region as the disease gene

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disorder characterized by abnormal facial expression and urinary abnormalities. Previously, we mapped the gene to a genomic interval of approximately 1 cM on chromosome region 10q23-24, using families from Columbia. Here we demonstrate genetic homogeneity of the syndrome through homozygosity mapping in American patients with Irish heritage. We established a physical map and identified novel polymorphic markers in the UFS critical region. Haplotype analysis using the new markers mapped the UFS gene within one YAC clone of 1,410 kb. We also determined the precise location of the gene encoding for glutamate oxaloacetate transaminase (GOT1) within the new UFS critical region and determined its genomic structure. However, mutation analysis excluded GOT1 as a candidate for the UFS gene.

Through Regulation of TCR Expression Levels, an Idd7 Region Gene(s) Interactively Contributes to the Impaired Thymic Deletion of Autoreactive Diabetogenic CD8+ T Cells in Nonobese Diabetic Mice

When expressed in NOD, but not C57BL/6 (B6) genetic background mice, the common class I variants encoded by the H2g7 MHC haplotype aberrantly lose the ability to mediate the thymic deletion of autoreactive CD8+ T cells contributing to type 1 diabetes (T1D). This indicated some subset of the T1D susceptibility (Idd) genes located outside the MHC of NOD mice interactively impair the negative selection of diabetogenic CD8+ T cells. In this study, using both linkage and congenic strain analyses, we demonstrate contributions from a polymorphic gene(s) in the previously described Idd7 locus on the proximal portion of Chromosome 7 predominantly, but not exclusively, determines the extent to which H2g7 class I molecules can mediate the thymic deletion of diabetogenic CD8+ T cells as illustrated using the AI4 TCR transgenic system. The polymorphic Idd7 region gene(s) appears to control events that respectively result in high vs low expression of the AI4 clonotypic TCR α-chain on developing thymocytes in B6.H2g7 and NOD background mice. This expression difference likely lowers levels of the clonotypic AI4 TCR in NOD, but not B6.H2g7 thymocytes, below the threshold presumably necessary to induce a signaling response sufficient to trigger negative selection upon Ag engagement. These findings provide further insight to how susceptibility genes, both within and outside the MHC, may interact to elicit autoreactive T cell responses mediating T1D development in both NOD mice and human patients.

“Humanized” HLA Transgenic NOD Mice to Identify Pancreatic β Cell Autoantigens of Potential Clinical Relevance to Type 1 Diabetes

Abstract:  The mechanistic basis by which the H2g7 major histocompatibility complex (MHC) provides the primary risk factor for the development of T cell–mediated autoimmune type 1 diabetes (T1D) in non‐obese diabetic (NOD) mice involves contributions not only from the unusual Ag7 class II molecule, but also from the more common Kd and/or Db class I variants it encodes. Similarly, transgenic studies in NOD mice have confirmed the possibility first suggested in association studies that in the proper genetic context the common human HLA‐A2.1 class I variant can mediate diabetogenic CD8 T cell responses. T1D continues to develop in a further refined NOD stock that expresses human HLA‐A2.1, but no murine class I molecules (designated NOD.β2m‐.HHD). Islet‐specific glucose‐6‐phosphatase catalytic subunit–related protein (IGRP) is an important antigenic target of diabetogenic CD8 cells in standard NOD mice. Three IGRP‐derived peptides have also been identified that are presented by human HLA‐A2.1 molecules to diabetogenic CD8 T cells in NOD.β2m‐.HHD mice. At least one of these IGRP peptides (265‐273) can also be the target of autoreactive CD8 T cells in HLA‐A2.1‐expressing human T1D patients. Studies are currently under way to determine whether HLA‐A2.1‐restricted IGRP peptides can be used in a tolerance‐inducing protocol that inhibits T1D development in NOD. β2m‐.HHD mice. If so, this knowledge could ultimately lead to the development of a similar T1D prevention protocol in humans.

IL4 and IL4Rα genes are not linked or associated with type 1 diabetes

Previous studies have shown the immunoregulatory functions IL-4 in type 1 diabetes mellitus. Therefore, the genes involved in the IL-4 regulatory pathway are candidates for diabetes susceptibility genes. Here we have evaluated IL4 and the alpha subunit of the IL-4 receptor (IL4R α) genes using the affected sibpair (ASP) and transmission/disequilibrium test (TDT). We analyzed 309 diabetic families from the United States and 87 families from various European countries. There was no evidence that either of these two genes are linked or associated with type 1 diabetes. Means by which IL-4 directed signals could indirectly alter diabetes susceptibility are proposed.