Autoreactive T cells specific for insulin B:11-23 recognize a low-affinity peptide register in human subjects with autoimmune diabetes

Abstract

Previous studies in type 1 diabetes (T1D) in the nonobese diabetic mouse demonstrated that a crucial insulin epitope (B:9-23) is presented to diabetogenic CD4 T cells by IA g7 in a weakly bound register. The importance of antigenic peptides with low-affinity HLA binding in human autoimmune disease remains less clear. The objective of this study was to investigate T-cell responses to a lowaffinity self-epitope in subjects with T1D. HLA-DQ8 tetramers loaded with a modified insulin peptide designed to improve binding the lowaffinity register were used to visualize T-cell responses following in vitro stimulation. Positive responses were only detectable in T1D patients. Because the immunogenic register of B:9-23 presented by DQ8 has not been conclusively demonstrated, T-cell assays using substituted peptides and DQ8 constructs engineered to express and present B:9-23 in fixed binding registers were used to determine the immunogenic register of this peptide. Tetramer-positive T-cell clones isolated from T1D subjects that responded to stimulation by B:11-23 peptide and denatured insulin protein were conclusively shown to recognize B:11-23 bound to HLA-DQ8 in the low-affinity register 3. These T cells also responded to homologous peptides derived from microbial antigens, suggesting that their initial priming could occur via molecular mimicry. These results are in accord with prior observations from the nonobese diabetic mouse model, suggesting a mechanism shared by mouse and man through which T cells that recognize a weakly bound peptide can circumvent tolerance mechanisms and play a role in the initiation of autoimmune diseases, such as T1D.antigen presentation | self-antigen | MHCII tetramers T ype 1 diabetes (T1D) is a polygenic T-cell-mediated autoimmune disease with strong genetic linkages to the MHC class II and insulin promoter regions (1). Class II molecules are fundamental for CD4 + T-cell activation, whereas the insulin promoter polymorphism can modulate insulin levels in the thymus thereby influencing the threshold of selection for insulinspecific autoreactive T cells (2, 3). In the nonobese diabetic (NOD) mouse model of autoimmune mediated diabetes, insulinspecific IA g7 -restricted CD4 + T cells have been strongly implicated in β-cell destruction. In prediabetic NOD mice, 50% of the T-cell clones established from islet-infiltrating lymphocytes were insulin-specific and the majority of these clones recognized the insulin B:9-23 (B:9-23 SHLVEALYLVCGERG) epitope (4, 5). Moreover, substitution of a single residue in the B:9-23 region abrogated development of diabetes in a transgenic mouse model (6, 7). Thus, in the murine NOD model, the IA g7 -restricted B:9-23 epitope is considered to be pivotal in the development of diabetes.The position or "register" that B:9-23 occupies within the IA g7 binding groove has been a controversial but important question. At least three binding registers (R) have been considered, defined here by which B:9-23 amino acid occupies the first binding pocket (p1) position in the IA g7 ...

Regulatory vs. inflammatory cytokine T-cell responses to mutated insulin peptides in healthy and type 1 diabetic subjects

“…Therefore, we conclude that, taken together, these two studies point out the presence of a previously underappreciated response to insulin in T1D patients and provide useful tools to pursue the study of this response in the future. They also further reinforce the idea that the strong association of T1D in mice, rats, and humans with a similar MHCII polymorphism may be because of a similar usual mechanism of MHCII allele-specific autoantigen presentation (23).…”

“…However, in the course of these studies, several of our group collaborated with Kwok and coworkers (23) at the Benaroya Institute in Seattle. Kwok and coworkers used a different approach, in which T cells from established T1D patients with one or two DQ8 alleles were stimulated with WT or B22E mutant insulin peptides for a short time in vitro and then, single cell-sorted with DQ8 tetramers loaded with either peptide to establish a set of CD4 T-cell clones.…”

Peptide immunotherapy in experimental autoimmune encephalomyelitis

“…Can we identify these cells? Flow cytometric approaches using peptide-MHC tetramers, [86,87] or cell surface changes in response to brief in vitro stimulation with antigen [88] suggest that this will become increasingly possible. Can we purify sufficient numbers of autoantigen-responsive cells to allow transcriptional/epigenetic analyses?…”

Discovery of a Selective Islet Peptidome Presented by the Highest-Risk HLA-DQ8trans Molecule

“…The islet epitopes presented by HLA-DQ8trans are largely unknown. Importantly, HLA-DQ8cis/trans-restricted CD4 T-cell clones have been isolated from human insulitis lesions, and T-cell autoreactivity was confirmed for several proinsulin peptides, underscoring the potential relevance of preproinsulin (PPI) peptides presented by HLA-DQ in diabetogenesis (10)(11)(12)(13). We contend that knowledge of the HLA-DQ8trans islet peptidome provides insight in the mechanism by which HLA-DQ2/8 heterozygosity imposes excessive risk for T1D.…”