924 Disruption of m6A reader protein YTHDF2 enhances Th9 cell differentiation and potentiates the anti-tumor efficacy of CAR Th9 cells

Background YTHDF2, a well-known m6A reader protein, binds to and destabilizes m6A-modified mRNA. Previous research has demonstrated that YTHDF2 regulates NK cell anti-tumor immunity1, and tumor-associated macrophage reprogramming2. Recently, we uncovered a tumor-intrinsic role of YTHDF2 in immune evasion3. However, its role in T-cell immunity remains unclear Methods Naïve CD4+ T cells were isolated from Ythdf2f/f and Ythdf2f/fCd4Cre (Ythdf2 KO) mice and subjected to T helper (Th) cell differentiation assay. Bulk RNA-seq, m6A-seq, YTHDF2 RIP-seq, and RNA stability assay were used to elucidate the mechanism by which YTHDF2 regulates the mRNA stability of Gata3. For in vivo studies, we established a syngeneic model by implanting B16-OVA or MC38-OVA cells into C57BL/6 or Rag1–/– mice, followed by intravenous injection of WT or Ythdf2 KO OT2 Th9 cells. Tumors were analyzed by flow cytometry. To assess the antitumor efficacy of human CAR Th9 cells, we established a xenograft tumor model by subcutaneously implanting human A549 lung or Capan-1 pancreatic tumor cells in NSG mice and treating them with WT or YTHDF2-disrupted human CAR Th9 cells targeting EGFR or PSCA. Results YTHDF2 deficiency in T cells does not affect Th1, Th2, Th17, or Treg cell differentiation, but significantly enhances Th9 cell differentiation in both mice and humans. Mechanistically, YTHDF2 ablation in CD4+ T cells promote Th9 cell differentiation by stabilizing Gata3 mRNA in the presence of IL-4 signaling. Th9 cells possess unique properties that stimulate robust anti-tumor responses by engaging both adaptive and innate immunity4. Our findings reveal that YTHDF2-deficient antigen-specific Th9 cells produce higher levels of IL-9 and IL-21, enhancing the infiltration and cytotoxicity of NK cells and CD8+ T cells, thereby inhibiting tumor growth in mice. Leveraging these insights, we engineered YTHDF2-disrupted human CAR Th9 cells targeting EGFR or PSCA. These modified CAR Th9 cells produce more IL-9 and IL-21 compared to their wild-type counterparts. In vivo studies demonstrate that YTHDF2-disrupted CAR Th9 cells exhibit superior anti-tumor efficacy in lung and pancreatic cancer xenograft models compared to wild-type CAR Th9 cells. Conclusions Our findings highlight YTHDF2 as a critical regulator of Th9 cell differentiation and effector function, suggesting that targeting YTHDF2 could enhance the efficacy of CAR T cell immunotherapies. References Ma S, et al. The RNA m6A reader YTHDF2 controls NK cell antitumor and antiviral immunity. J Exp Med 2012;218. doi:10.1084/jem.20210279. Ma S, et al. YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8(+) T cells. Nat Immunol, 2023. doi:10.1038/s41590-022-01398-6. Xiao S, et al. The tumor-intrinsic role of the m(6)A reader YTHDF2 in regulating immune evasion. Sci Immunol 2024;9:eadl2171. doi:10.1126/sciimmunol.adl2171. Chen T, et al. Th9 cell differentiation and its dual effects in tumor development. Front Immunol 2020;11:1026. doi:10.3389/fimmu.2020.01026.