A Natural Cyclic Peptide Enhances Cytotoxic Th9 Differentiation and Antitumor Immunity

Adoptive cell therapy (ACT) relies on durable and functional T cells to mediate tumor clearance. Th9 cells are a metabolically fit CD4? T cell subset with strong persistence but limited cytotoxicity. Here, we identify Endomelipaneptide A (EpA), a cyclic peptide from Ganoderma lucidum–associated endophytic fungi, as a potent enhancer of Th9 differentiation. EpA promotes a cytotoxic Th9 phenotype with enhanced mitochondrial function and metabolic fitness. Overall design: Naïve CD4+ T cells were isolated from splenocytes of C57BL/6J, and were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin-streptomycin and 0.1% ß-Mercaptoethanol. Naïve CD4+ T cells were differentiated under plate-bound anti-CD3 (Bioxcell, 17A2, 5 µg/mL) and soluble anti-CD28 (Bioxcell, 37.51, 1 µg/mL) conditions with specific cytokines for Th9 differentiation: IL-4 (peprotech 400-04,10 ng/mL), TGF-ß (EPOTO Biotech, 1 ng/mL), anti-IL-12 (Bioxcell, C17.8, 5 µg/mL), and anti-IFN-? mAbs (Bioxcell, XMG1.2, 10 µg/mL). After the initial 3-day culture, cells were continue culture: IL-4 (10 ng/mL), TGF-ß (1ng/mL). RNAex Pro RNA Reagent was used to obtain total RNA of Th9 cells on day 4 for RNAseq.