Autoreactive T cell response in CD25-negative fraction of peripheral blood mononuclear cells in established type 1 diabetes.

CD4(+)CD25(+) T cells (Tregs) play a critical role in maintaining dominant peripheral tolerance, and pathogenic autoreactive T cells may be frequent in the CD25-negative fraction of peripheral blood mononuclear cells (PBMCs) from patients with autoimmune disease. We therefore investigated whether T cell autoimmune responses to recombinant GAD65 can be detected by the use of ELISPOT assay in the CD25-negative fraction of PMBCs from Japanese type 1 diabetes (T1D) patients. The frequency of CD4(+)CD25(+) T cells was not different among patients with newly developed T1D, established T1D, and healthy controls. The CD25 positive cell-depleted fraction was obtained by negative selection with antihuman CD25 magnetic beads, reducing the number of CD4(+)CD25(+) T cells from 4-5% to less than 1%. In whole PBMC fraction, there was a significant elevation of IFN-gamma spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN-gamma spots from patients with established T1D was not significant. In the CD25-negative fraction, unlike whole PBMCs, we observed the significant IFN-gamma spots to GAD65 in the fraction from patients with established T1D (P < 0.05), but not in those with recently diagnosed disease. The phenomena were not observed for IL-4 spots. Our data suggest a possible role of Tregs maintaining dominant peripheral tolerance in T1D and application of further improved T cell assay detecting autoimmunity even in established T1D.