PD-1 blockade restored the GVHD-inducing capacity of Id3-cKO T cells. (A-E) Balb/b mice were subjected to total body irradiation (5 Gy on day −1 and 5 Gy on day 0) followed by infusion of 5 × 10⁶ B6 × B6/SJL F1(CD45.1⁺CD45.2⁺) TCD-BM alone or together with 1 × 10⁶ B6 (CD45.1⁻CD45.2⁺) naïve CD4⁺ and 5 × 10⁵ B6 (CD45.1⁻CD45.2⁺) naïve CD8⁺ WT or Id3-cKO T cells. Anti–PD-1 antibody (200 μg per mouse per injection) was administered intraperitoneally on days 0, 3, 5, and 7 after HSCT. In some experiment, isotype immunoglobulin G (IgG) was injected as control. (A) Survival rates of Balb/b mice. (B) Seventeen days after HSCT, cells were isolated from the spleen, liver, and intestine of recipients, enumerated and stained for flow cytometry analysis. (C) Graphs show the percentage of PD-1⁺ donor CD4⁺ T cells in the spleen and liver. (D) PD-1 protein levels on the surface of donor CD4⁺ T cells isolated from the spleen and liver of Balb/b recipients. (E) The number of IFN-γ⁺GM-CSF⁺ population in the spleen and liver. (F) Balb/c transplantation was performed as described in Figure 1. Anti-PD1 antibody was administered with the same regimen as in Balb/b recipients. Graph shows the survival rate. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, Student t test.