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Updated: Apr 29, 2026
Measuring Mitochondrial Function of Naïve and Effector CD8 T Cells
Published on: March 28, 2025
Ginsenoside F3 alleviates T cell exhaustion via RIPOR2-mediated immunometabolic reprogramming to potentiate anti-PD-1
Menglin Jiang1, Weiqian Bao1, Lewei He1
1Chinese Medicine Guangdong Laboratory /State Key Laboratory of Traditional Chinese Medicine Syndrome, The Second Clinical College of Guangzhou University of Chinese Medicine (Guangdong Provincial Hospital of Chinese Medicine), Guangzhou, Guangdong, China.
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.
Insights
An optimized in vitro T cell exhaustion model identifies Rho family-interacting cell polarization regulator 2 (RIPOR2) as a key target. Ginsenoside F3 (GF3) reverses exhaustion, enhancing cancer immunotherapy efficacy.
Area of Science:
- Immunology
- Cancer Biology
- Drug Discovery
Background:
- T cell exhaustion limits cancer immunotherapy effectiveness.
- Current in vivo models are inefficient and costly.
- Need for improved in vitro models for research and drug screening.
Purpose of the Study:
- Develop and validate an optimized in vitro T cell exhaustion model.
- Identify key regulators of T cell exhaustion.
- Screen and test potential therapeutic interventions.
Main Methods:
- Chronic anti-CD3 antibody stimulation for T cell exhaustion model.
- Proteomic profiling and siRNA knockdown for regulator identification.
- Molecular docking and in vitro/in vivo testing of Ginsenoside F3 (GF3).
Main Results:
- Model accurately recapitulated exhaustion phenotypes with reduced cytotoxicity.
- RIPOR2 identified as a critical regulator, downregulated in exhausted T cells.
- GF3 reversed exhaustion markers, restored cytokine production, and reduced tumor burden in vivo.
Conclusions:
- RIPOR2 is a critical immunometabolic regulator of T cell exhaustion.
- GF3 effectively reverses T cell exhaustion by targeting RIPOR2.
- Optimized in vitro model serves as an efficient platform for research and drug discovery.
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