Ginsenoside F3 alleviates T cell exhaustion via RIPOR2-mediated immunometabolic reprogramming to potentiate anti-PD-1 therapy

Background: T cell exhaustion (Tex) induced by chronic antigen exposure critically limits Cancer Immunotherapy. Mechanistic understanding remains incomplete, largely due to inefficient, costly in vivo models and a lack of effective interventions. Improved in vitro models are therefore urgently needed for mechanism discovery and drug screening.

Methods: An optimized in vitro T cell exhaustion model was established via chronic anti-CD3 antibody stimulation, achieving robust reproducibility, high cell yields (∼10⁸), and suitability for high-throughput analyses. Exhaustion was validated functionally and by proteomic profiling across multiple timepoints. Rho family-interacting cell polarization regulator 2 (RIPOR2) was identified as a key regulator and functionally verified through siRNA knockdown assays. Molecular docking screened Ginsenoside F3 (GF3) targeting RIPOR2, and GF3 was tested both in vitro and in a mouse NSCLC model with anti-PD-1 therapy.

Results: Our model accurately recapitulated clinical exhaustion phenotypes, notably reproducing diminished cytotoxicity (∼60% reduction compared to controls). Proteomics dynamically revealed significant protein alterations during exhaustion, including a ∼14-fold downregulation of RIPOR2 mRNA in exhausted T cells. RIPOR2 depletion further exacerbated exhaustion (e.g.,Interferon-gamma (IFN-γ) secretion reduced ∼30%, Programmed cell death protein 1 (PD-1) increased ∼20%). GF3 robustly bound RIPOR2, significantly reversed exhaustion phenotypes (reducing PD-1⁺TIM-3⁺(Hepatitis A virus cellular receptor 2) cells by ∼40%), and restored cytokine production. In vivo, GF3 enhanced CD8⁺T cell infiltration, synergized with anti-PD-1 therapy, and significantly reduced tumor burden (∼40% decrease).

Conclusion: RIPOR2 is identified as a critical immunometabolic regulator of T cell exhaustion. GF3-mediated RIPOR2 restoration effectively reverses exhaustion, presenting a novel immunotherapeutic strategy. Our optimized in vitro model provides an efficient platform for future mechanistic and pharmacological research.