An Insulin-Chromogranin A Hybrid Peptide Activates DR11-Restricted T Cells in Human Type 1 Diabetes.

Hybrid insulin peptides (HIPs) formed through covalent cross-linking of proinsulin fragments to secretory granule peptides are detectable within murine and human islets. The 2.5HIP (C-peptide-chromogranin A [CgA] HIP), recognized by the diabetogenic BDC-2.5 clone, is a major autoantigen in the nonobese diabetic mouse. However, the relevance of this epitope in human disease is currently unclear. A recent study probed T-cell reactivity toward HIPs in patients with type 1 diabetes, documenting responses in one-third of the patients and isolating several HIP-reactive T-cell clones. In this study, we isolated a novel T-cell clone and showed that it responds vigorously to the human equivalent of the 2.5HIP (designated HIP9). Although the responding patient carried the risk-associated DRB1*04:01/DQ8 haplotype, the response was restricted by DRB1*11:03 (DR11). HLA class II tetramer staining revealed higher frequencies of HIP9-reactive T cells in individuals with diabetes than in control participants. Furthermore, in DR11+ participants carrying the DRB4 allele, HIP9-reactive T-cell frequencies were higher than observed frequencies for the immunodominant proinsulin 9-28 epitope. Finally, there was a negative correlation between HIP9-reactive T-cell frequency and age at diagnosis. These results provide direct evidence that this C-peptide-CgA HIP is relevant in human type 1 diabetes and suggest a mechanism by which nonrisk HLA haplotypes may contribute to the development of ß-cell autoimmunity.

Hybrid insulin peptide isomers spontaneously form in pancreatic beta-cells from an aspartic anhydride intermediate.

Hybrid insulin peptides (HIPs) form in beta-cells when insulin fragments link to other peptides through a peptide bond. HIPs contain nongenomic amino acid sequences and have been identified as targets for autoreactive T cells in type 1 diabetes. A subgroup of HIPs, in which N-terminal amine groups of various peptides are linked to aspartic acid residues of insulin C-peptide, was detected through mass spectrometry in pancreatic islets. Here, we investigate a novel mechanism that leads to the formation of these HIPs in human and murine islets. Our research herein shows that these HIPs form spontaneously in beta-cells through a mechanism involving an aspartic anhydride intermediate. This mechanism leads to the formation of a regular HIP containing a standard peptide bond as well as a HIP-isomer containing an isopeptide bond by linkage to the carboxylic acid side chain of the aspartic acid residue. We used mass spectrometric analyses to confirm the presence of both HIP isomers in islets, thereby validating the occurrence of this novel reaction mechanism in beta-cells. The spontaneous formation of new peptide bonds within cells may lead to the development of neoepitopes that contribute to the pathogenesis of type 1 diabetes as well as other autoimmune diseases.

Insulin B-chain hybrid peptides are agonists for T cells reactive to insulin B:9-23 in autoimmune diabetes.

Insulin is considered to be a key antigenic target of T cells in Type 1 Diabetes (T1D) and autoimmune diabetes in the NOD mouse with particular focus on the B-chain amino acid sequence B:9-23 as the primary epitope. Our lab previously discovered that hybrid insulin peptides (HIPs), comprised of insulin C-peptide fragments fused to other ß-cell granule peptides, are ligands for several pathogenic CD4 T cell clones derived from NOD mice and for autoreactive CD4 T cells from T1D patients. A subset of CD4 T cell clones from our panel react to insulin and B:9-23 but only at high concentrations of antigen. We hypothesized that HIPs might also be formed from insulin B-chain sequences covalently bound to other endogenously cleaved ß-cell proteins. We report here on the identification of a B-chain HIP, termed the 6.3HIP, containing a fragment of B:9-23 joined to an endogenously processed peptide of ProSAAS, as a strong neo-epitope for the insulin-reactive CD4 T cell clone BDC-6.3. Using an I-Ag7 tetramer loaded with the 6.3HIP, we demonstrate that T cells reactive to this B-chain HIP can be readily detected in NOD mouse islet infiltrates. This work suggests that some portion of autoreactive T cells stimulated by insulin B:9-23 may be responding to B-chain HIPs as peptide ligands.

Cathepsin D Drives the Formation of Hybrid Insulin Peptides Relevant to the Pathogenesis of Type 1 Diabetes.

Hybrid insulin peptides (HIPs) form in pancreatic ß-cells through the formation of peptide bonds between proinsulin fragments and other peptides. HIPs have been identified in pancreatic islets by mass spectrometry and are targeted by CD4 T cells in patients with type 1 diabetes (T1D) as well as by pathogenic CD4 T-cell clones in nonobese diabetic (NOD) mice. The mechanism of HIP formation is currently poorly understood; however, it is well established that proteases can drive the formation of new peptide bonds in a side reaction during peptide bond hydrolysis. Here, we used a proteomic strategy on enriched insulin granules and identified cathepsin D (CatD) as the primary protease driving the specific formation of HIPs targeted by disease-relevant CD4 T cells in T1D. We also established that NOD islets deficient in cathepsin L (CatL), another protease implicated in the formation of disease-relevant HIPs, contain elevated levels of HIPs, indicating a role for CatL in the proteolytic degradation of HIPs. In summary, our data suggest that CatD may be a therapeutic target in efforts to prevent or slow the autoimmune destruction of ß-cells mediated by HIP-reactive CD4 T cells in T1D.

Hybrid Insulin Peptides Are Autoantigens in Type 1 Diabetes.

We recently established that hybrid insulin peptides (HIPs) are present in human islets and that T cells reactive to HIPs are found in the residual islets of organ donors with type 1 diabetes (T1D). Here, we investigate whether HIP-reactive T cells are indicative of ongoing autoimmunity in patients with T1D. We used interferon-γ enzyme-linked immune absorbent spot analyses on peripheral blood mononuclear cells (PBMCs) to determine whether patients with new-onset T1D or control subjects displayed T-cell reactivity to a panel of 16 HIPs. We observed that nearly one-half of the patients responded to one or more HIPs. Responses to four HIPs were significantly elevated in patients with T1D but not in control subjects. To characterize the T cells reactive to HIPs, we used a carboxyfluorescein succinimidyl ester-based assay to clone T cells from PBMCs. We isolated six nonredundant, antigen-specific T-cell clones, most of which reacting to their target HIPs in the low nanomolar range. One T-cell clone was isolated from the same patient on two different blood draws, indicating persistence of this T-cell clone in the peripheral blood. This work suggests that HIPs are important target antigens in human subjects with T1D and may play a critical role in disease.

Mapping I-A(g7) restricted epitopes in murine G6PC2.

G6PC2, also known as islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP), is a major target of autoreactive CD8(+) T cells in both diabetic human subjects and the non-obese diabetic (NOD) mouse. However, in contrast to the abundant literature regarding the CD8(+) response to this antigen, much less is known about the potential involvement of IGRP-reactive CD4(+) T cells in diabetogenesis. The single previous study that examined this question in NOD mice was based upon a candidate epitope approach and identified three I-A(g7)-restricted epitopes that each elicited spontaneous responses in these animals. However, given the known inaccuracies of MHC class II epitope prediction algorithms, we hypothesized that additional specificities might also be targeted. To address this issue, we immunized NOD mice with membranes from insect cells overexpressing full-length recombinant mouse IGRP and measured recall responses of purified CD4(+) T cells using a library of overlapping peptides encompassing the entire 355-aa primary sequence. Nine peptides representing 8 epitopes gave recall responses, only 1 of which corresponded to any of the previously reported sequences. In each case proliferation was blocked by a monoclonal antibody to I-A(g7), but not the appropriate isotype control. Consistent with a role in diabetogenesis, proliferative responses to 4 of the 9 peptides (3 epitopes) were also detected in CD4(+) T cells purified from the pancreatic draining lymph nodes of pre-diabetic female animals, but not from peripheral lymph nodes or spleens of the same animals. Intriguingly, one of the newly identified spontaneously reactive epitopes (P8 [IGRP(55-72)]) is highly conserved between mice and man, suggesting that it might also be a target of HLA-DQ8-restricted T cells in diabetic human subjects, an hypothesis that we are currently testing.

Phosphorylation of SNAP-23 regulates exocytosis from mast cells.

Regulated exocytosis is a process in which a physiological trigger initiates the translocation, docking, and fusion of secretory granules with the plasma membrane. A class of proteins termed SNAREs (including SNAP-23, syntaxins, and VAMPs) are known regulators of secretory granule/plasma membrane fusion events. We have investigated the molecular mechanisms of regulated exocytosis in mast cells and find that SNAP-23 is phosphorylated when rat basophilic leukemia mast cells are triggered to degranulate. The kinetics of SNAP-23 phosphorylation mirror the kinetics of exocytosis. We have identified amino acid residues Ser(95) and Ser(120) as the major phosphorylation sites in SNAP-23 in rodent mast cells. Quantitative analysis revealed that approximately 10% of SNAP-23 was phosphorylated when mast cell degranulation was induced. These same residues were phosphorylated when mouse platelet degranulation was induced with thrombin, demonstrating that phosphorylation of SNAP-23 Ser(95) and Ser(120) is not restricted to mast cells. Although triggering exocytosis did not alter the absolute amount of SNAP-23 bound to SNAREs, after stimulation essentially all of the SNAP-23 bound to the plasma membrane SNARE syntaxin 4 and the vesicle SNARE VAMP-2 was phosphorylated. Regulated exocytosis studies revealed that overexpression of SNAP-23 phosphorylation mutants inhibited exocytosis from rat basophilic leukemia mast cells, demonstrating that phosphorylation of SNAP-23 on Ser(120) and Ser(95) modulates regulated exocytosis by mast cells.