CD4+CD25+Foxp3+ regulatory T cells (Treg) are critical for controlling autoimmunity. Evidence suggests that Treg development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the Treg responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific Treg are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific Treg in NOD mice. Ag-specific Treg from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific Treg expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded Treg were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific Treg were more efficient than polyclonal Treg in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific Treg in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.

Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing β cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-Ag7 dimers for use in the nonobese diabetic mouse model of diabetes. I-Ag7 dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4+ T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-Ag7 dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4+ T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4+ T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-γ compared with cells from control I-Ag7 dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-Ag7 dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.