Expression of the CTLA-4 gene is absolutely required for immune homeostasis, but aspects of its molecular nature remain undefined. In particular, the characterization of the soluble CTLA-4 (sCTLA-4) protein isoform generated by an alternatively spliced mRNA of CTLA4 lacking transmembrane-encoding exon 3 has been hindered by the difficulty in distinguishing it from the transmembrane isoform of CTLA-4, Tm-CTLA-4. In the current study, sCTLA-4 has been analyzed using novel mAbs and polyclonal Abs specific for its unique C-terminal amino acid sequence. We demonstrate that the sCTLA-4 protein is secreted at low levels following the activation of primary human CD4(+) T cells and is increased only rarely in the serum of autoimmune patients. Unexpectedly, during our studies aimed to define the kinetics of sCTLA-4 produced by activated human CD4(+) T cells, we discovered that Tm-CTLA-4 is associated with microvesicles produced by the activated cells. The functional roles of sCTLA-4 and microvesicle-associated Tm-CTLA-4 warrant further investigation, especially as they relate to the multiple mechanisms of action described for the more commonly studied cell-associated Tm-CTLA-4.

By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.

Type 1 diabetes (T1D) is a polygenic disease with multiple insulin-dependent diabetes (Idd) loci predisposing humans and NOD mice to disease. NOD.B10 Idd9 congenic mice, in which the NOD Idd9 chromosomal region is replaced by the Idd9 from T1D-resistant C57BL/10 mice, are significantly protected from T1D development. However, the genes and pathways conferring T1D development or protection by Idd9 remain to be fully elucidated. We have developed novel NOD.B10-Idd9 (line 905) congenic mice that predominantly harbor islet-reactive CD4(+) T cells expressing the BDC2.5 TCR (BDC-Idd9.905 mice). To establish functional links between the Idd9 genotype and its phenotype, we used microarray analyses to investigate the gene expression profiles of ex vivo and Ag-activated CD4(+) T cells from these mice and BDC2.5 (BDC) NOD controls. Among the differentially expressed genes, those located within the Idd9 region were greatly enriched in islet-specific CD4(+) T cells. Bioinformatics analyses of differentially expressed genes between BDC-Idd9.905 and BDC CD4(+) T cells identified Eno1, Rbbp4, and Mtor, all of which are encoded by Idd9 and part of gene networks involved in cellular growth and development. As predicted, proliferation and Th1/Th17 responses of islet-specific CD4(+) T cells from BDC-Idd9.905 mice following Ag stimulation in vitro were reduced compared with BDC mice. Furthermore, proliferative responses to endogenous autoantigen and diabetogenic function were impaired in BDC-Idd9.905 CD4(+) T cells. These findings suggest that differential expression of the identified Idd9 genes contributed to Idd9-dependent T1D susceptibility by controlling the diabetogenic function of islet-specific CD4(+) T cells.