IFNγR is required for Tregs to function appropriately in vivo. (A) Tregs also up-regulate both CXCR3 and IFNγR when activated (right panels). Mean and standard deviation of activated Tregs are as follows (n = 3). IFNγR⁺CXCR3⁻: 16.8% ± 7.0%, IFNγR-CXCR3⁻: 1.39% ± 1.2%, IFNγR⁺CXCR3⁺: 79.1% ± 6.1%, and IFNγR-CXCR3⁺: 2.7% ± 1.8%. (B) IFNγR Tregs were equally suppressive as WT Tregs in in vitro MLR assays ([³H]-thymidine incorporation was measured). Tregs were serial-diluted. The experiment was performed in triplicate or quadruplicate. Shown is 1 representative of 3 independent experiments with similar results. (C) IFNγR^−/− Tregs do not suppress GVHD (B6 → Balb/c). TCD BM (B6, 5 × 10⁶; CD45.1⁺) were injected into lethally irradiated Balb/c mice, followed by DLI of 2 × 10⁶ pan T (B6, CD45.2⁺) and 1.5 × 10⁶ Tregs (B6, CD45.2⁺). Data represent the pool of 2 independent experiments. (D) WT Tregs suppress GVHD induced by WT Tconvs but not by IFNγ^−/− Tconv. Both Tregs and Tconvs (5 × 10⁵ cells each) were injected along with TCD BM (5 × 10⁶ cells) at day 0. Data represent the pool of 2 independent experiments.