The non-obese diabetic (NOD) mouse is an excellent animal model of autoimmune diabetes associated with insulitis. The progression of insulitis causes the destruction of pancreatic beta cells, resulting in the development of hyperglycemia. Although it has been well documented that T cells are required for the development of insulitis and diabetes in NOD mice, the importance of B cells remains unclear. To clarify the role of B cells in the pathogenesis of NOD mice, we therefore generated B cell-deficient NOD (B-NOD) mice. Surprisingly, none (of 13) of the B-NOD mice developed diabetes by 40 weeks of age, while the control littermates with B cells (B+NOD) suffered from a high proportion (43 of 49) of diabetes. The insulin reactivity of B+NOD mice was significantly impaired, while the B-NOD mice showed a good insulin response, thus suggesting the pancreatic beta cell function to be well preserved in B-NOD mice. Although B-NOD mice did develop insulitis, the extent of insulitis was significantly suppressed. These data thus provide the direct evidence that B cells are essential for the progression of insulitis and the development of diabetes in NOD mice.

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Omenn syndrome is a severe primary immunodeficiency with putative autoimmune manifestations of the skin and gastrointestinal tract. The disease is caused by hypomorphic mutations in recombination-activating genes that impair but do not abolish the process of VDJ recombination, leading to the generation of autoreactive T cells with a highly restricted receptor repertoire. Loss of central tolerance in genetically determined autoimmune diseases, e.g., autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy, is associated with defective expression by medullary thymic epithelial cells of AIRE, the transcription activator that induces thymic expression of tissue-specific antigens. Analysis of AIRE expression in the thymi of 2 Omenn syndrome patients and 1 SCID patient, by real-time RT-PCR and immunohistochemistry, demonstrated a profound reduction in the levels of AIRE mRNA and protein in patients as compared with a normal control subject. Lack of AIRE was associated with normal or even increased levels of keratin and lymphotoxin-beta receptor mRNAs, while mRNAs of the self-antigens insulin, cytochrome P450 1a2, and fatty acid-binding protein were undetectable in thymi from immunodeficiency patients. These results demonstrate that deficiency of AIRE expression is observed in severe immunodeficiencies characterized by abnormal T cell development and suggest that in Omenn syndrome, the few residual T cell clones that develop may escape negative selection and thereafter expand in the periphery, causing massive autoimmune reactions.

BackgroundRecently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2).MethodsPolymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese.FindingsA TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody.InterpretationThe TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans.FundingMinistry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.

SUMMARYThe role of antibody and cell-mediated immunity in the resistance of adult mice to intracutaneous infection with herpes simplex virus type I (HSV-x) was studied in nu/nu and nu/+ mice. In nu/+ mice, local skin lesions began to appear at the site of inoculation on the 4th day after intracutaneous challenge with the virulent Hayashida strain of HSV-I. Zosteriform skin lesions were observed in some animals. Almost complete regression of the lesions had occurred by the I6th day p.i. In contrast, all of the nu/nu mice that developed local skin lesions died after development of severe zosteriform skin lesions. After repeated intraperitoneal inoculations with the avirulent SKa strain of HSV-I, nu/nu mice did not produce detectable amounts of neutralizing antibody and succumbed to infection, indicating no development of resistance.Passively transferred neutralizing antibody prevented nu/nu mice from developing zosteriform skin lesions by the Hayashida strain of HSV-I, as long as the minimum concentration of serum antibody was maintained and prolonged their survival time. Adoptive transfer of I-o × lO 7 immune nu/+ spleen cells to nu/nu mice provided almost complete recovery from infection with production of sporadic low levels of anti-HSV antibody. The protective action of the immune spleen cells was lost after pre-treatment with anti-0 serum and fresh guinea pig serum prior to transfer of the cells. These data indicate that T cell-mediated cellular immunity plays a major role in recovery from intracutaneous HSV infection in mice, while antibody-mediated protection due to passive administration of HSV antibody is effective only in limiting the spread of virus.

Co-stimulatory molecules of CD28, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and the newly identified inducible co-stimulator (ICOS) are expressed on cell surfaces and provide regulatory signals for T-cell activation. Their genes are candidate susceptibility genes for type 1 diabetes because they co-localize to Chromosome 2q33 with the IDDM12 locus. After determining the genomic structure and screening for polymorphisms of the ICOS gene, we performed association studies between newly identified polymorphisms of the ICOS gene, together with known polymorphisms of CD28 and CTLA-4 genes, and type 1 diabetes. The 49A/G dimorphism in exon 1 and the (AT)n in the 3' untranslated region of the CTLA-4 gene were significantly associated with type 1 diabetes. Evaluation of the CTLA-4 49A-3'(AT)n 86-bp haplotype frequency in patients and controls confirmed the results from the analysis of each polymorphic site. Dimorphism in intron 3 of the CD28 gene was associated with type 1 diabetes only in the early-onset group. In contrast, there was no association with the microsatellite polymorphisms in the ICOS gene or dimorphisms in the promotor region of CTLA-4. Of the three genes encoding co-stimulatory molecules, the CTLA-4 gene appears to confer risks for the development of type 1 diabetes.

IFN-α inhibits B lymphocyte development, and the nuclear protein Daxx has been reported to be essential for this biological activity. We show in this study that IFN-α inhibits the clonal proliferation of B lymphocyte progenitors in response to IL-7 in wild-type, but not in tyk2-deficient, mice. In addition, the IFN-α-induced up-regulation and nuclear translocation of Daxx are completely abrogated in the absence of tyk2. Therefore, tyk2 is directly involved in IFN-α signaling for the induction and translocation of Daxx, which may result in B lymphocyte growth arrest and/or apoptosis.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.