Id3 represses genes associated with T-cell effector differentiation and inhibitory signaling. WT and Id3-cKO naïve CD4⁺ T cells were activated and cultured under Th1 polarization condition. Cells were harvested 24 hours (TCR primed) and 96 hours after activation. 96 hour-cultured cells were sorted into CD62L^hi and CD62L^lo (Th1 cells) populations before library preparation. (A) Gene set enrichment analysis using Hallmark gene set showed significant enrichment of tumor necrosis factor α signaling via NF-κb, IFN-γ response, IL-2–STAT5 signaling, IL-6–STAT3 signaling, allograft rejection, IFN-α response, and apoptosis comparing transcriptome from 4-day cultured Id3-cKO Th1-like cells with WT. (B) Id3-regulated gene signature in TCR-primed CD4⁺ T cells and effector Th1 CD4⁺ T cells. (C-G) Heat maps demonstrate 5 categories of differential expressed genes: TFs critical for effector proliferation and differentiation (C); cytokines that distinguish Th1 from Th2 and Th17 cells (D); cell survival and death molecules (E); costimulatory molecules important for GVHD T-cell proliferation and survival (F); and inhibitory receptors (G). Data were collected from 3 independent experiments. (H) Flow cytometry analysis shows that loss of Id3 leads to increased expression of PD-1 (H, I) and PD-L1 (J) in Th1 cells. ∗∗∗P < .001. Experiments were performed >5 times.