Fig. 2.

Acute regulation of IFN-γ by Tregs but no inhibition of lineage commitment. Naïve DO11.10⁺CD25⁻Thy1.2⁺CD4⁺ T cells (targets) and DO11.10⁺Thy1.1⁺CD25⁺CD4⁺ cells (Tregs) were cultured with APC/OVAp under Th1-priming conditions (primary culture). On day 6, target T cells were sorted and restimulated, without Tregs, with APC/OVAp and rhIL-2 (A and B) or with rhIL-2 and rIL-4 (C) (secondary culture) for 6 d. Then cells were washed and restimulated for 6 h with plate-bound anti-TCRβ mAb (tertiary culture). Frequency of cytokine producers was assessed by intracellular cytokine staining. (A) Representative dot plot of cytokine production from target T cells in tertiary culture. (B) Percentage and fold expansion of IFN-γ producers in tertiary culture. (C) Percentage of IL-4 producers in tertiary culture after primary culture in Th1-priming conditions with or without Tregs and in secondary culture in Th2-priming conditions without Tregs. Labels in A to C refer to conditions in primary culture. (D) Thy1.1⁺CD4⁺ Th1 effector cells were stimulated with APC and anti-CD3 mAb with or without Tregs for 18–24 h and were assessed for IFN-γ using the cytokine secretion assay. Data are from at least three individual experiments. **P < 0.005, ***P < 0.0005, ns, not significant.