Pemetrexed potentiates γδ T cell-based immunotherapy in NSCLC through ATM-STING-NF-κB-mediated induction of NKG2D ligands.

Adoptive T-cell therapy is a promising strategy for cancer immunotherapy; however, its efficacy is often limited by the immunosuppressive tumor microenvironment. γδ T cells, particularly the Vγ9Vδ2 subset, exhibit innate-like cytotoxicity and are emerging candidates for adoptive immunotherapy. Pemetrexed, an antifolate chemotherapeutic agent, has documented immunomodulatory effects in αβ T-cell settings; however, its impact on γδ T-cell antitumor responses remains insufficiently defined. Here, Vγ9Vδ2 T cells were isolated and expanded from healthy-donor peripheral blood mononuclear cells (PMBCs) and co-cultured with non-small cell lung cancer (NSCLC) cells with or without pemetrexed pretreatment. Pemetrexed significantly enhanced γδ T cell-mediated cytotoxicity compared with either treatment alone. Mechanistically, pemetrexed increased the expression of NKG2D ligands, including MHC class I chain-related proteins A/B (MICA/B) and UL16-binding proteins (ULBPs), through the ataxia-telangiectasia mutated (ATM)-stimulator of interferon genes (STING)-nuclear factor-κB (NF-κB) signaling axis. ATM activation triggered cyclic GMP-AMP synthase-independent STING signaling and preferentially activated NF-κB rather than interferon regulatory factor 3 (IRF3), thereby promoting transcriptional upregulation of NKG2D ligands and improving tumor recognition by γδ T cells. Consistent with the cell-line findings, pemetrexed increased MICA/B and ULBP2/5/6 expression in NSCLC patient-derived organoids. Furthermore, in an in vivo NSCLC animal model, combined pemetrexed and adoptive γδ T-cell therapy suppressed tumor growth more effectively than either treatment alone and was accompanied by increased NKG2D ligand expression. Collectively, these findings reveal a tumor-sensitizing mechanism by which pemetrexed potentiates γδ T-cell antitumor function and support combining pemetrexed with γδ T cell-based immunotherapy for NSCLC.