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

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.