Prevention of aGVHD by tolerogenic anti–IL-2 mAb (JES6-1A12) requires PD-L1 expression by GVHD target tissues. 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 (Rec) were given a total of 4 i.p. injections of rat-IgG, anti–IL-2 mAb (JES6-1A12), or anti–IL-2 mAb (S4B6) (500 µg/mouse) at days 0, 2, 4, and 6 after HCT. Recipients given TCD-BM cells (2.5 × 10⁶) alone were used as controls. (A) Plots of percent original body weight, diarrhea, and percent survival are shown. n = 8 per group. Combined from 2 replicated experiments. (B) Mean ± standard error of the mean of histopathology scores of liver, small intestine, and colon are shown; n = 4 per group. Combined from 2 replicated experiments. (C-F) At day 6 after HCT, spleen, liver, and colon of recipients were harvested for analysis of donor CD4⁺ and CD8⁺ T-cell percentage and yield. Mean ± standard error of the mean of the percentage and yield of H-2K^b+TCRβ⁺ CD4⁺ or CD8⁺ T cells are shown; n = 4 to 11 per group. Combined from 2 to 3 replicated experiments. (G-I) Day 6 post-HCT, percentage of Eomes⁺PD1⁺ cells among donor CD4⁺ and CD8⁺ T cells in liver and colon of WT or PD-L1^−/− recipients treated with anti–IL-2 mAb (JES6-1A12), anti–IL-2 mAb (S4B6), or control IgG; n = 4 to 5 per group. (J) Day 6 post-HCT, percentage of Eomes⁺PD1⁺ cells among donor CD4⁺ and CD8⁺ T cells in liver and colon of WT and PD-L1^−/− recipients treated with anti–IL-2 mAb (JES6-1A12); n = 4 per group. Data represent mean ± standard error combined from 2 replicated experiments. P values were calculated by using the log-rank test (A), one-way analysis of variance (B), or two-way analysis of variance (C-J). *P < .05, **P < .01, ***P < .001, ****P < .0001.