Tumour-associated ligands of the activating NKG2D (natural killer group 2, member D; also called KLRK1) receptor-which are induced by genotoxic or cellular stress-trigger activation of natural killer cells and co-stimulation of effector T cells, and may thus promote resistance to cancer. However, many progressing tumours in humans counter this anti-tumour activity by shedding the soluble major histocompatibility complex class-I-related ligand MICA, which induces internalization and degradation of NKG2D and stimulates population expansions of normally rare NKG2D+CD4+ T cells with negative regulatory functions. Here we show that on the surface of tumour cells, MICA associates with endoplasmic reticulum protein 5 (ERp5; also called PDIA6 or P5), which, similar to protein disulphide isomerase, usually assists in the folding of nascent proteins inside cells. Pharmacological inhibition of thioreductase activity and ERp5 gene silencing revealed that cell-surface ERp5 function is required for MICA shedding. ERp5 and membrane-anchored MICA form transitory mixed disulphide complexes from which soluble MICA is released after proteolytic cleavage near the cell membrane. Reduction of the seemingly inaccessible disulphide bond in the membrane-proximal alpha3 domain of MICA must involve a large conformational change that enables proteolytic cleavage. These results uncover a molecular mechanism whereby domain-specific deconstruction regulates MICA protein shedding, thereby promoting tumour immune evasion, and identify surface ERp5 as a strategic target for therapeutic intervention.

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 ...

Posttranslational modification (PTM) of self-proteins has been shown to elicit clinically relevant immune responses in rheumatoid arthritis and celiac disease. Accumulating evidence suggests that recognition of modified self-proteins may also be important in type 1 diabetes. Our objective was to identify posttranslationally modified GAD65 peptides, which are recognized by subjects with type 1 diabetes, and to assess their disease relevance. We show that citrullination and transglutamination of peptides can enhance their binding to DRB1*04:01, a diabetes-susceptible HLA allele. These and corresponding modifications to amino acids at T-cell contact positions modulated the recognition of multiple GAD65 peptides by self-reactive T cells. Using class II tetramers, we verified that memory T cells specific for these modified epitopes were detectable directly ex vivo in the peripheral blood of subjects with type 1 diabetes at significantly higher frequencies than healthy controls. Furthermore, T cells that recognize these modified epitopes were either less responsive or nonresponsive to their unmodified counterparts. Our findings suggest that PTM contributes to the progression of autoimmune diabetes by eliciting T-cell responses to new epitope specificities that are present primarily in the periphery, thereby circumventing tolerance mechanisms.

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