Tissue-infiltrating Alloreactive T Cells Require Id3 to Deflect PD-1-mediated Immune Suppression during GVHD

1.Id3 limits PD-1 expression and exuberant effector differentiation of Th1 cells, protecting them from PD-1-mediated suppression during GVHD.2.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 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, Prdm1) that are critical for PD-1 transcription, exuberant effector differentiation, and IFN 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-versus-tumor effects in mice undergoing allo-HSCT. Furthermore, CRIPSR/Cas9 knockout of ID3 in human CD19-directed CAR-T cells retained their anti-leukemia activity in NOD/SCID/IL2Rg−/− mice early after administration. These findings identify that ID3 is an important target to reduce GVHD, and gene-editing program of ID3 may have broad implications in T cell-based immunotherapy.