Validation of function of T_regs and T_effs isolated from donors. (A) Experimental outline. Donor mice received 10⁶ A20HA intravenously, followed 10 days later by 2.5 × 10⁶ CFSE-labeled, CD4⁺-enriched HA-specific T cells. At 14 days after T-cell transfer, mice were vaccinated with 10⁷ pfu VaccHA (intraperitoneally) and were killed 5 days later. (B) Spleens and LNs were harvested and analyzed by fluorescence-activated cell sorter (FACS). T_regs (CFSE^lowGITR⁺) or T_effs (CFSE^lowGITR⁻) were sorted. (C-E) A total of 10 000 sorted T_regs or T_effs were cultured in vitro either alone or with 10 000 naive CD4⁺ cells from a 6.5/Rag^−/− mouse (naive responder [N]), along with 200 000 splenocytes from a WT BALB/c mouse, and stimulated with 10 μg/mL HA peptide. Proliferation (C) and cytokine production (D,E) were measured as described in “Methods.” Data represent mean (± SE) of triplicate cultures.