Prevention of GVHD by tolerogenic anti–IL-2 mAb (JES6-1A12) but not non-tolerogenic anti–IL-2 mAb (S4B6) requires expression of PD-L1 by GVHD target tissues to inhibit activation of IL-2-Stat-5 and AKT-mTOR pathways in donor T cells. Lethally irradiated WT and 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.v. injections of rat-IgG or anti–IL-2 mAb (JES6-1A12 or S4B6) (500 µg/mouse) at days 0, 2, and 4 after HCT. At day 6 after HCT, spleen and colon were harvested for analysis. (A) Representative Gene Set Enrichment Analysis plots are shown of IL-2-STAT5 pathway-related gene set expression of CD4⁺ T and CD8⁺ T cells in the spleen or colon of the WT recipients treated with anti–IL-2 mAb (JES6-1A12) vs IgG cohorts. P values were calculated by using the bioconductor package “clusterProfiler” version 3.10.1. (B-C) p-AKT and phosphorylated mTOR (pmTOR) expression of donor CD4⁺ T cells in the spleen (SPL) and colon of WT recipients treated with anti–IL-2 mAb (JES6-1A12 or S4B6) or control IgG; n = 5 per group. (D) p-AKT and pmTOR expression of donor CD4⁺ T cells in the SPL and colon of PD-L1^−/− recipients treated with anti–IL-2 mAb (JES6-1A12) or control IgG; n = 4 per group. (E) p-AKT and pmTOR expression of donor CD4⁺ T cells in the SPL and colon of WT or PD-L1^−/− recipients treated with anti–IL-2 mAb (JES6-1A12); n = 4 to 5 per group. Data represent mean ± standard error combined from two replicate experiments. P values were calculated by unpaired two-tailed Student t tests. *P < .05, **P < .01, ****P < .0001. MFI, mean fluorescence intensity.