Prevention of aGVHD by tolerogenic anti–IL-2 mAb (JES6-1A12) but not non-tolerogenic anti–IL-2 mAb (S4B6) requires PD-L1–dependent expansion of Tr1 cells. (A-E) Lethally irradiated WT or PD-L1−/− BALB/c recipients were given splenocytes (2.5 × 106) and TCD-BM cells (2.5 × 106) from C57BL/6 donors. Recipients were given a total of 3 i.p. injections of rat-IgG or anti–IL-2 mAb (JES6-1A12 or S4B6) (500 µg/mouse) at days 0, 2, and 4 after HCT. Day 6 after HCT, donor-type T cells from liver and colon were analyzed for Tr1 cells. (A-B) Representative staining pattern with percentage and yield of donor IL-10+ Foxp3– CD4+ T cells in liver and colon of WT recipients treated with anti–IL-2 mAb (JES6-1A12 or S4B6) or control IgG; n = 5 per group. (C) Representative staining pattern with percentage and yield of donor IL-10+ Foxp3– CD4+ T cells in liver and colon of PD-L1−/− recipients treated with anti–IL-2 mAb (JES6-1A12) or control IgG; n = 4 per group. (D-E) Blimp-1 and Eomes expression of donor CD4+ T cells in liver and colon of WT recipients treated with anti–IL-2 mAb (JES6-1A12 or S4B6) or control IgG; n = 4 per group. (F) Blimp-1 and Eomes expression of donor CD4+ T cells in liver and colon of PD-L1−/− recipients treated with anti–IL-2 mAb (JES6-1A12) or control IgG; n = 4 per group. (G) Lethally irradiated WT BALB/c recipients were given T cells (1.0 × 106) from WT or IL-10−/− C57BL/6 donors and TCD-BM cells (2.5 × 106) from WT C57BL/6 donors. Recipients were given a total of 4 i.p. injections of anti–IL-2 mAb (JES6-1A12) (500 µg/mouse) at days 0, 2, 4, and 6 after HCT. Plots of percent original body weight, diarrhea, and survival are shown. n = 8 per group. Data represent mean ± standard error combined from 2 replicate experiments. P values were calculated by using two-way analysis of variance (A-F) or log-rank test (G). *P < .05, **P < .01, ***P < .001, ****P < .0001. MFI, mean fluorescence intensity.