Tissue-infiltrating alloreactive T cells require Id3 to deflect PD-1–mediated immune suppression during GVHD

• Id3 limits PD-1 expression and exuberant effector differentiation of Th1 cells, protecting them from PD-1–mediated suppression during GVHD.

• Id3 reduces chromatin accessibility of transcription factors that drive T-cell PD-1 transcription, differentiation, and dysfunction.

Persisting alloreactive donor T cells in target tissues are a determinant of graft-versus-host disease (GVHD), but the transcriptional regulators that control the persistence and function of tissue-infiltrating T cells remain elusive. We demonstrate here that Id3, a DNA-binding inhibitor, is critical for sustaining T-cell responses in GVHD target tissues in mice, including the liver and intestine. Id3 loss results in aberrantly expressed PD-1 in polyfunctional T helper 1 (Th1) cells, decreased tissue-infiltrating PD-1⁺ polyfunctional Th1 cell numbers, impaired maintenance of liver TCF-1⁺ progenitor-like T cells, and inhibition of GVHD. PD-1 blockade restores the capacity of Id3-ablated donor T cells to mediate GVHD. Single-cell RNA-sequencing analysis revealed that Id3 loss leads to significantly decreased CD28- and PI3K/AKT-signaling activity in tissue-infiltrating polyfunctional Th1 cells, an indicator of active PD-1/PD-L1 effects. Id3 is also required for protecting CD8⁺ T cells from the PD-1 pathway–mediated suppression during GVHD. Genome-wide RNA-sequencing analysis reveals that Id3 represses transcription factors (e.g., Nfatc2, Fos, Jun, Ets1, and Prdm1) that are critical for PD-1 transcription, exuberant effector differentiation, and interferon responses and dysfunction of activated T cells. Id3 achieves these effects by restraining the chromatin accessibility for these transcription factors. Id3 ablation in donor T cells preserved their graft vs tumor effects in mice undergoing allogeneic hematopoietic stem cell transplantation. Furthermore, CRISPR/Cas9 knockout of ID3 in human CD19–directed chimeric antigen receptor T cells retained their antitumor activity in NOD/SCID/IL2Rg^−/− mice early after administration. These findings identify that ID3 is an important target to reduce GVHD, and the gene-editing program of ID3 may have broad implications in T-cell–based immunotherapy.