ELISPOT analysis of IL-4/IFN-γ-producing T cells and cytokine assay.

<p>Splenocytes from the same 10-week-old NOD mice used to examine the development of insulitis were pooled into a given treatment group. In the ELISPOT assay, splenocytes (10⁶/well) from CTB-Ins-GAD-, CTB-GAD-Ins-, CTB-GAD-, or WT-immunized mice were stimulated with insulin (A and B) or GAD65 peptide (C and D). T cells producing IL-4 (B and D) or IFN-γ (A and C) were detected. The number of spots was quantified in triplicate wells for each group. IFN-γ or IL-4 concentrations in serum (E and F) and pancreas (G and H) was measured via ELISA. Six mice per group were individually tested in two separate experiments. All data are expressed as the means ± SD. * p<0.05; ** P<0.01; *** P<0.001.