FOXP1 Knockdown Reprograms Th9 CAR‐T Cells to Overcome Antigen Escape

ABSTRACT Antigen‐loss variants (ALVs) are a major cause of relapse following chimeric antigen receptor (CAR) T cell therapy, particularly in solid tumors where antigen heterogeneity and immune suppression prevail. By integrating public single‐cell RNA sequencing analysis with experimental validation, we identify the transcription factor FOXP1 as a critical brake limiting Th9 CAR‐T cell differentiation and effector programming. FOXP1 knockdown reprograms Th9 CAR‐T but not Tc9 cells toward a metabolically active, cytotoxic, and exhaustion‐resistant phenotype, thereby enhancing their persistence and antitumor activity. CUT&Tag and transcriptomic profiling reveal that FOXP1 binds regulatory regions of Il9 , Spi1 , and Runx1 , as well as effector loci such as Tnf and Gzmb , repressing both Th9‐lineage and TCR‐downstream transcriptional programs. Its depletion releases this repression, broadly activating MAPK, PI3K‐Akt/mTOR, and NF‐κB pathways that sustain cytokine production and memory formation. Functionally, FOXP1‐deficient Th9 CAR‐T cells eradicate both antigen‐positive and antigen‐loss tumor populations by recruiting dendritic cells and promoting endogenous CD8 + T cell clonal expansion via the CD6‐Flt3L axis. Our findings establish FOXP1 as a transcriptional checkpoint integrating cytokine and signaling networks to control Th9 CAR‐T cell function and provide a mechanistic rationale for engineering CAR‐T therapies capable of overcoming antigen escape.