Id3 deficiency leads to PD-1 upregulation on polyfunctional Th1 cells. (A-C) Approximately 6 to 8 days after allo-HSCT, cells were isolated from the spleen and liver of Balb/c recipients of donor B6 naïve T cells as described in Figure 1, and donor T cells were analyzed for effector function. Plots and graphs show the percentage of IFN-γ⁺ GM-CSF⁺ polyfunctional cells (A-B) and the number of Id3-cKO IFN-γ⁺GM-CSF⁺ T cells in the spleen and liver (C). (D-E) Plots and graphs show the frequency of lamina-propria lymphocyte (LPL) IFN-γ⁺GM-CSF⁺ cells in the intestine of Balb/c recipients 17 days after HSCT. (F-H) The IFN-γ⁺GM-CSF⁺ polyfunctional population was gated for the analysis of inhibitory molecule expression (F). The percentage of PD-1⁺ cells (G) and PD-1 level (mean fluorescence intensity [MFI]) on the surface of individual CD4⁺ polyfunctional T cells (H) isolated from the spleen and the liver of Balb/c recipients 6 and 8 days after HSCT. (I-J) Plots and graphs show the percentage of PD-L1–expressing donor CD4⁺ T cells isolated from the spleen and liver of Balb/c recipients. ∗P < .05; ∗∗∗P < .001, Student t test. At least 3 mice per group were analyzed.