NR2F6 functions as a key transcriptional checkpoint enforcing dysfunction of human CD3+ T cells and limiting CAR-T cell potency

Although nuclear receptor subfamily 2 group F member 6 (NR2F6) regulates lymphocyte activation in murine models, its function in human T cells remains incompletely defined. Here, we identify NR2F6 as a T cell receptor (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. Collectively, these findings position NR2F6 as a signaling-integrated transcriptional checkpoint that contributes to dysfunctional T cell states and may promote tumor immune evasion in humans.