2, E) and D. transcription; in addition, the unique Ag7 chain differs by 17 amino acids from the protective Ad allele (4). T1D development is reduced or abrogated in NOD mice transgenic for E or other H2-A alleles (3). MHCII glycoproteins normally present peptides to CD4+ T lymphocytes, and NOD mice harbor pathogenic CD4+ effector T cells that identify islet autoantigens offered by Ag7 (examined in (5)). Ag7 shares key polymorphisms, in particular a non-Asp residue at position 57, and peptide binding preferences with human HLA-DQB1 4-Aminophenol alleles that confer risk of T1D (6-8). Nonetheless, no unifying explanation for the link between these alleles and islet autoimmunity has yet emerged from considerations of peptide binding specificity (5). Moreover, the potential for Ag7-restricted T-cell autoreactivity extends to self antigens that are systemically expressed, both in the autoproliferation phenotype of NOD mice (9,10) and in the K/BN model of autoimmune arthritis (11,12). These considerations have prompted suggestions that Ag7 confers a broader tendency towards autoreactivity, perhaps mediated by unique biochemical properties other than their peptide binding specificity. Indeed, Ag7 Mouse Monoclonal to V5 tag exhibits several unusual biochemical properties, compared to other MHCII alleles (5). Many MHCII alleles remain stable as heterodimers in SDS at room temperature when loaded stably with peptides, but Ag7 dimers readily dissociate under these conditions (13). Genetic studies indicate that this relative instability represents an intrinsic structural house of the Ag7 heterodimer. SDS instability also correlates with the T1D risk or protection associated with human DQ alleles (14). In 35S-pulse/chase experiments performed (18), correlating with large differences in MHCII protein life span (19). Genetic deficiency in the peptide exchange cofactor, DM, reduces the surface expression of Ag7 and other MHCII alleles with low affinity for invariant chain peptides, and increases their turnover (20,21). Comparable effects on MHCII life span could result from unfavorable regulation of DM activity by HLA-DO/H2-DO (22), which is usually 4-Aminophenol differentially expressed during APC development (23-25). In NOD mice, MHCII associations with invariant chain may be abnormal, and the development and frequencies of B cells and DCs have been reported to be altered, with unknown effects for the regulation of MHCII protein life span (5). Moreover, T1D development is subject to environmental control. In NOD colonies worldwide, T1D incidence varies between from 20% to 80% in females at 30 weeks of age (26), suggesting that diabetes development is usually influenced by environmental and epigenetic factors. Environmental factors that contribute to this variance include multiple viral, bacterial, and eukaryotic pathogens, the resident intestinal microflora, as well as experimentally administered proinflammatory stimuli, all of which change host immune responsiveness (27). In addition, hormonal regulation is usually suggested by the much lower disease incidence in males; this, too, is usually influenced by the microbiome (28). Whether disease-relevant environmental factors affect MHCII protein life span remains unclear. A definitive assessment of the determinants of MHCII protein instability and its role in autoimmune pathogenesis requires quantification of turnover rates in APCs of living animals. Methodologies for addressing this problem have been lacking until recently. Radiolabeling, a standard approach for measuring protein life span (29), is hard to use for the first time. Surprisingly, our findings indicate that environmental and developmental variables, rather than structural polymorphism, are key determinants of accelerated turnover of Ag7 in NOD APCs, and that this phenotype is usually dispensable for development of T1D. Materials and Methods Mice and cell lines Animal studies were performed according to institutional and national guidelines under UK Home Office Project Licenses 80/2156 and 80/2440. For most studies, NOD mice (named NODlow where necessary to avoid ambiguity) were bred under specific pathogen-free conditions at the Centre for Biomedical Services, University or college of Cambridge, from founder stock kindly provided by Prof. Linda Wicker. NOD genotype (100%) was authenticated in a breeding trio using an array of 384 SNPs (Charles River Labs), 4-Aminophenol spaced 7 MB apart across the entire genome, which distinguish common inbred mouse strains. BALB/c.
