GVHD acceleration induced by PD-1/PD-1 ligand blockade resulted in increased activation and effector function of donor T-cells. (A-C) Lethally irradiated B10.BR recipients were given 10⁷ B6 BM cells with 5 × 10⁶ B6 splenocytes and treated with isotype-matched control antibody or anti-PD-L1 mAb. (A) Mice were killed on day 7 after BMT, and splenocytes (n = 5 mice/group) were analyzed by flow cytometry for LPAM-1 (α₄β₇) expression on donor CD4 and CD8 T-cells. (B) Common activation markers (CD25 and CD62L) on donor CD4 and CD8 T-cells were analyzed on day 7 (n = 4–5 mice/group) by flow cytometry. (C) Intracellular cytokine staining was performed on day 7 (n = 5 mice/group) and analyzed by flow cytometry to detect the percentage of donor CD4 and CD8 T-cells producing IFN-γ, IL-17, TNF-α, IL-2, IL-4, and IL-10 in spleen. (D) Lethally irradiated wt B6 recipients or PD-L1^−/− recipients were given 10⁷ BALB/c BM cells with 5 × 10⁶ BALB/c splenocytes. Mice were killed on day 7 after BMT (n = 5 mice/group), and splenocytes were analyzed by flow cytometry for intracellular expression of CD107a or granzyme B on donor CD4 and CD8 T-cells. Data are presented as mean fluorescence intensity (MFI). (A−D) Data are representative of 2 independent experiments. *P < .05; **P < .01; ***P < .001.