NR2F6 Functions as a Key Transcriptional Checkpoint Enforcing Dysfunction of Human CD3+ T cells and Limiting CAR-T Cell Potency

Here, we identify NR2F6 as a TCR inducible transcriptional checkpoint that limits effector potency in human CD3⁺ T cells, including chimeric antigen receptor (CAR) T cells. In an anti EGFR CAR T cell-A549 co culture model, enforced NR2F6 expression, mimicking its upregulation in tumor infiltrating lymphocytes, markedly impairs killing under repeated tumor challenge.

In polyclonal T cells, NR2F6 overexpression and CRISPR/Cas9-mediated ablation produced directionally opposing effects, supporting its role as a negative regulator of activation, differentiation-associated programs, and cytokine production. Mechanistically, transcriptomic, epigenomic, and nuclear protein analyses are consistent with suppression of NFAT , AP 1 , and NF κB-associated pathways and reveal context dependent changes in TCF 1-associated readouts.