Anti-IL-7Rα mAbs promote cytokine production but not antigen-specific Tregs after islet Ag-KLH immunization.

11–13 week old female prediabetic NOD mice were immunized with islet Ag-KLH or KLH in combination with anti-IL-7Rα antibodies or rat IgG (0.5 mg) administered on days 0 and 4. PLN and spleens were harvested on day 7 or 8. BDC2.5-specific T cells were enriched and identified with tetramers as in Fig 1. (A) 350 μg of islet Ag-KLH and 175 μg of KLH were used for the immunization. Contour plots are gated on CD4+BDC2.5+ T cells and show Foxp3 and CD25 expression within this population. The percentages of Foxp3+ Tregs within the BDC2.5+ T cells on day 8 are indicated. (B) Graph shows a summary of the percentages of Foxp3+ Tregs within the BDC2.5+ T cell population, as gated in A (n = 3). (C) Mice were immunized with 300, 30 or 3 μg of islet Ag-KLH and bar graph shows absolute numbers of BDC2.5+ T cells present in individual mice on day 7 ± SD (n = 3). (D) Bar graph shows the percentages of Foxp3+ Tregs within the BDC2.5+ T cells, as in B. (E, F) Mice were immunized with 350 μg of islet Ag-KLH or 175 μg of KLH and, on day 7, 1 x 106 splenocytes were used to determine cytokine-producing cells. Graphs show number of spots, representing cells producing IL-2 (E) and IFN-γ (F) as determined by ELISPOT assays with (closed symbols) or without (open symbols) adding a mixture of the islet Ags used for immunization to the in vitro cultures (n = 3). ANOVA with Bonferroni’s post test were used for statistical analysis, ** = p≤0.005, *** = p≤0.0005.