Ginsenoside Rg3 enhances antitumor effects of PD-1 mAb in lung adenocarcinoma by promoting T-cell mitophagy via TBC1D15

Abstract Objectives Although anti-PD-1 monoclonal antibodies represent a cornerstone in modern non-small cell lung cancer (NSCLC) management, sustained clinical efficacy remains restricted to a limited subset of patients. T-cell exhaustion and an immunosuppressive tumor microenvironment are major limiting factors. Some traditional Chinese medicine modalities have been developed to modulate the tumor immune microenvironment. Specifically, ginsenoside Rg3 serves as a potent dual-action agent, actively modulating the intratumoral niche while concurrently exerting strong inhibitory effects against tumor expansion. This study was designed to investigate whether Rg3 could augment the antitumor immunity elicited by anti-PD-1 blockade within non-small cell lung malignancies, while simultaneously deciphering the underlying molecular cascades. Methods We employed an in vitro tumor cell and tumor-specific cytotoxic T cell co-culture system, flow cytometry, quantitative PCR, Western blotting, whole-transcriptome sequencing (RNA-seq), and a mouse tumor xenograft model with adoptive CTL transfer to study the effects of Rg3 and PD-1 mAb combination therapy on cytotoxic T lymphocytes (CTLs) and the underlying molecular mechanism. The functions of key molecular mediators were validated by gene knockdown and overexpression approaches. Results Rg3 enhanced the cytotoxic activity of lung adenocarcinoma cell-specific CTLs, reducing A549 cell viability by 45.9 % at 10 μmol/L compared with the untreated control (p<0.05). Combination treatment with Rg3 and anti-PD-1 mAb showed significantly greater cytotoxic activity than either monotherapy, with CTL-mediated killing rates reaching 71.9 % compared with 50.7 % for anti-PD-1 mAb alone and 48.2 % for Rg3 alone (all p<0.05). Transcriptomic analysis and experimental validation revealed that the combination treatment induced upregulation of TBC1D15 in CTLs, which promoted mitophagy, improved mitochondrial integrity, and was associated with enhanced cytolytic activity of CTLs. Conclusions Co-administration of Rg3 with anti-PD-1 potentiates antitumor immunity by modulating CTL mitochondrial homeostasis via TBC1D15-mediated mitophagy, thereby enhancing CTL cytotoxic function. These findings support further investigation of Rg3 as an adjuvant to PD-1 blockade.