Maki Nakayama

Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.

Insulin as an autoantigen in NOD/human diabetes

Although multiple islet autoantigens are recognized by T lymphocytes and autoantibodies before the development of type 1A (immune-mediated diabetes), there is increasing evidence that autoimmunity to insulin may be central to disease pathogenesis. Evidence is strongest for the NOD mouse model where blocking immune responses to insulin prevents diabetes, and insulin peptides can be utilized to induce diabetes. In man insulin gene polymorphisms are associated with disease risk, and autoantibodies and T cells reacting with multiple insulin/proinsulin epitopes are present. It is not currently clear why insulin autoimmunity is so prominent and frequent, and though insulin can be used to immunologically prevent diabetes of NOD mice, insulin-based preventive immunoregulation of diabetes in man is not yet possible.

The Stages of Type 1A Diabetes

Abstract: We can now predict the development of type 1 diabetes in man because it is a chronic autoimmune disorder with defined stages of disease. We can also readily prevent the disorder in animal models. A major goal is safe prevention in man, and for this we will almost certainly need a better understanding of pathogenesis, coupled with rigorous clinical trials.

Specificity and detection of insulin-reactive CD4+ T cells in type 1 diabetes in the nonobese diabetic (NOD) mouse

In the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D), an insulin peptide (B:9–23) is a major target for pathogenic CD4+ T cells. However, there is no consensus on the relative importance of the various positions or “registers” this peptide can take when bound in the groove of the NOD MHCII molecule, IAg7. This has hindered structural studies and the tracking of the relevant T cells in vivo with fluorescent peptide-MHCII tetramers. Using mutated B:9–23 peptides and methods for trapping the peptide in particular registers, we show that most, if not all, NOD CD4+ T cells react to B:9–23 bound in low-affinity register 3. However, these T cells can be divided into two types depending on whether their response is improved or inhibited by substituting a glycine for the B:21 glutamic acid at the p8 position of the peptide. On the basis of these findings, we constructed a set of fluorescent insulin-IAg7 tetramers that bind to most insulin-specific T-cell clones tested. A mixture of these tetramers detected a high frequency of B:9–23-reactive CD4+ T cells in the pancreases of prediabetic NOD mice. Our data are consistent with the idea that, within the pancreas, unique processing of insulin generates truncated peptides that lack or contain the B:21 glutamic acid. In the thymus, the absence of this type of processing combined with the low affinity of B:9–23 binding to IAg7 in register 3 may explain the escape of insulin-specific CD4+ T cells from the mechanisms that usually eliminate self-reactive T cells.

Transgenic Insulin (B:9-23) T-Cell Receptor Mice Develop Autoimmune Diabetes Dependent Upon RAG Genotype, H-2g7 Homozygosity, and Insulin 2 Gene Knockout

A series of recent studies in humans and the NOD mouse model have highlighted the central role that autoimmunity directed against insulin, in particular the insulin B chain 9-23 peptide, may play in the pathogenesis of type 1 diabetes. Both pathogenic and protective T-cell clones recognizing the B:9-23 peptide have been produced. This report describes the successful creation of BDC12-4.1 T-cell receptor (TCR) transgenic mice with spontaneous insulitis in F1 mice (FVB × NOD) and spontaneous diabetes in NOD.RAG−/− (backcross 1 generation). Disease progression is heterogeneous and is modified by a series of genetic factors including heterozygosity (H-2g7/H-2q) versus homozygosity for H-2g7, the presence of additional T-/B-cell receptor–rearranged genes (RAG+ versus RAG−/−), and the insulin 2 gene knockout (the insulin gene expressed in the NOD thymus). Despite lymphopenia, 40% of H-2g7/g7 BDC12-4.1 TCR+ RAG−/− Ins2−/− mice are diabetic by 10 weeks of age. As few as 13,500 transgenic T-cells from a diabetic TCR+ RAG−/− mouse can transfer diabetes to an NOD.scid mouse. The current study demonstrates that the BDC12-4.1 TCR is sufficient to cause diabetes at NOD backcross 1, bypassing polygenic inhibition of insulitis and diabetogenesis.

Structure-Based Selection of Small Molecules to Alter Allele-Specific MHC Class II Antigen Presentation

Class II major histocompatibility molecules are the primary susceptibility locus for many autoimmune disorders, including type 1 diabetes. Human DQ8 and I-Ag7, in the NOD mouse model of spontaneous autoimmune diabetes, confers diabetes risk by modulating presentation of specific islet peptides in the thymus and periphery. We used an in silico molecular docking program to screen a large “druglike” chemical library to define small molecules capable of occupying specific structural pockets along the I-Ag7 binding groove, with the objective of influencing presentation to T cells of the autoantigen insulin B chain peptide consisting of amino acids 9–23. In this study we show, using both murine and human cells, that small molecules can enhance or inhibit specific TCR signaling in the presence of cognate target peptides, based upon the structural pocket targeted. The influence of compounds on the TCR response was pocket dependent, with pocket 1 and 6 compounds inhibiting responses and molecules directed at pocket 9 enhancing responses to peptide. At nanomolar concentrations, the inhibitory molecules block the insulin B chain peptide consisting of amino acids 9–23, endogenous insulin, and islet-stimulated T cell responses. Glyphosine, a pocket 9 compound, enhances insulin peptide presentation to T cells at concentrations as low as 10 nM, upregulates IL-10 secretion, and prevents diabetes in NOD mice. These studies present a novel method for identifying small molecules capable of both stimulating and inhibiting T cell responses, with potentially therapeutic applications.

Excessive Th1 responses due to the absence of TGF-β signaling cause autoimmune diabetes and dysregulated Treg cell homeostasis

TGF-β signaling in T cells is critical for peripheral T-cell tolerance by regulating effector CD4+ T helper (Th) cell differentiation. However, it is still controversial to what extent TGF-β signaling in Foxp3+ regulatory T (Treg) cells contributes to immune homeostasis. Here we showed that abrogation of TGF-β signaling in thymic T cells led to rapid type 1 diabetes (T1D) development in NOD mice transgenic for the BDC2.5 T-cell receptor. Disease development in these mice was associated with increased peripheral Th1 cells, whereas Th17 cells and Foxp3+ Treg cells were reduced. Blocking of IFN-γ signaling alone completely suppressed diabetes development in these mice, indicating a critical role of Th1 cells in this model. Furthermore, deletion of TGF-β signaling in peripheral effector CD4+ T cells, but not Treg cells, also resulted in rapid T1D development, suggesting that conventional CD4+ T cells are the main targets of TGF-β to suppress T1D. TGF-β signaling was dispensable for Treg cell function, development, and maintenance, but excessive IFN-γ production due to the absence of TGF-β signaling in naive CD4+ T cells indirectly caused dysregulated Treg cell homeostasis. We further showed that T cell–derived TGF-β1 was critical for suppression of Th1 cell differentiation and T1D development. These results indicate that autocrine/paracrine TGF-β signaling in diabetogenic CD4+ T cells, but not Treg cells, is essential for controlling T1D development.

Islet-derived CD4 T-cells targeting proinsulin in human autoimmune diabetes

Type 1 diabetes results from chronic autoimmune destruction of insulin-producing β-cells within pancreatic islets. Although insulin is a critical self-antigen in animal models of autoimmune diabetes, due to extremely limited access to pancreas samples, little is known about human antigenic targets for islet-infiltrating T cells. Here we show that proinsulin peptides are targeted by islet-infiltrating T cells from patients with type 1 diabetes. We identified hundreds of T cells from inflamed pancreatic islets of three young organ donors with type 1 diabetes with a short disease duration with high-risk HLA genes using a direct T-cell receptor (TCR) sequencing approach without long-term cell culture. Among 85 selected CD4 TCRs tested for reactivity to preproinsulin peptides presented by diabetes-susceptible HLA-DQ and HLA-DR molecules, one T cell recognized C-peptide amino acids 19–35, and two clones from separate donors responded to insulin B-chain amino acids 9–23 (B:9–23), which are known to be a critical self-antigen–driving disease progress in animal models of autoimmune diabetes. These B:9–23–specific T cells from islets responded to whole proinsulin and islets, whereas previously identified B:9–23 responsive clones from peripheral blood did not, highlighting the importance of proinsulin-specific T cells in the islet microenvironment.

Gamma Delta T Cell Receptors Confer Autonomous Responsiveness To The Insulin Peptide B:9–23

The range and physical qualities of molecules that act as ligands for the gammadelta T cell receptors (TCRs) remain uncertain. Processed insulin is recognized by alphabeta T cells, which mediate diabetes in non-obese diabetic (NOD) mice. Here, we present evidence that gammadelta T cells in these mice recognize processed insulin as well. Hybridomas generated from NOD spleen and pancreatic lymph nodes included clones expressing gammadelta TCRs that responded specifically to purified islets of Langerhans and to an insulin peptide, but not to intact insulin. The gammadelta TCRs associated with this type of response are diverse, but a cloned gammadelta TCR was sufficient to transfer the response. The response to the insulin peptide was autonomous as demonstrated by stimulating single responder cells in isolation. This study reveals a novel specificity for gammadelta TCRs, and raises the possibility that gammadelta T cells become involved in islet-specific autoimmunity.

Conserved T cell receptor α-chain induces insulin autoantibodies

A fundamental question is what are the molecular determinants that lead to spontaneous preferential targeting of specific autoantigens in autoimmune diseases, such as the insulin B:9–23 peptide sequence in type 1 diabetes. Anti-insulin B:9–23 T cell clones isolated from prediabetic NOD islets have a conserved Vα-segment/Jα-segment, but no conservation of the α-chain N region and no conservation of the Vβ-chain. Here, we show that the conserved T cell receptor α-chain generates insulin autoantibodies when transgenically or retrogenically introduced into mice without its corresponding Vβ. We suggest that a major part of the mystery as to why islet autoimmunity develops relates to recognition of a primary insulin peptide by a conserved α chain T cell receptor.