Loss of YTHDF2 enhances Th9 programming and CAR-Th9 cell antitumor efficacy

CD4+ T cells differentiate into various subsets, including T helper 1 (Th1), Th2, Th9, Th17, and regulatory T cells, which are essential in immune responses and cancer immunotherapy. However, the role of RNA N6-methyladenosine (m6A) modification in this differentiation remains unclear. Here, we demonstrate that YTHDF2, a key m6A reader protein known to destabilize m6A-modified mRNA, negatively regulates Th9 cell differentiation. Ablation of Ythdf2 in both mouse and human naïve CD4+ T cells significantly promotes Th9 differentiation by stabilizing Gata3 and Smad3 mRNA under IL-4 and TGF-ß signaling. Ythdf2-deficient Th9 cells produce higher levels of IL-9 and IL-21, leading to increased tumor infiltration and cytotoxicity by CD8+ T and NK cells, thereby improving antitumor activity versus wild-type Th9 cells. Moreover, YTHDF2 depletion in CAR-Th9 cells enhances immune activation, reduces terminal differentiation, and augments antitumor efficacy. Targeting YTHDF2 thus represents a promising strategy for enhancing Th9 and CAR-Th9 cell-based cancer immunotherapies. Overall design: Total RNA was isolated by TRIzol reagent from fifty million WT and Ythdf2-KO Th9 cells. m6A MeRIP sequencing was performed to detect the m6A modification alterations in two groups. YTHDF2 RNA immunoprecipitation sequencing was performed to map the target transcripts bound by YTHDF2 in Th9 cells.