Rohan Vinayak Sami

Background:  CAR-T cell immunotherapy serves as a major new treatment method in oncology, but T-cell exhaustion is the main barrier to usage in a clinical setting.¹² Tex occurs when T-cells undergo chronic antigen exposure within tumor microenvironments.³ This leads to a progressive loss of effector functions and self-replicative ability.¹² This state has upregulated inhibitory receptors such as PD-1 and CTLA-4.³ The progression to exhausted cells occurs slowly as the first step is the creation of TPEX (progenitor exhausted T-cells) which allow the T-cells to still have self-replicative properties but with reduced effectiveness.¹² Chronic activation of antigens eventually causes these cells to become terminally exhausted.¹² This state is characterized by an epigenetic scar that makes exhaustion nearly irreversible.¹² Understanding what causes this transition is essential to creating new advances in CAR-T therapy to counteract and prevent this same transition.¹²

Objectives: This literature review’s purpose was to evaluate molecular and metabolic mechanisms that lead the progression from progenitor to terminal T-cell exhaustion.

Search Methods: A literature search was conducted using the PubMed database to find peer-review studies within the last 7 years. Search strategies included using specific terms such as T-cell exhaustion and Car-T cell therapy.

Results:  Chronic stimulation of the T-cell receptor activates the calcineurin pathway that leads to persistent calcium signaling.³ This leads to an imbalance of transcription factors where there is more NFAT than AP-1 allowing NFAT to act independently leading to upregulation of the TOX and NR4A transcription factors.¹² TOX starts early epigenetic events that initial Tex specific changes in cells.¹¹ Also, chromatin remodeling complexes like cBAF members (Arid1a) modulate T-cell persistence.³ This transcriptional change in the T-cells is related to a severe metabolic shift.¹¹ Tex cells have a significantly higher rate of aerobic glycolysis compared to TPEX cells (2.5x). This shift is due to the leakage of ROS and mitochondrial deterioration.³

To overcome these obstacles, the use of CRISPR/Cas9 to perform a NR4A triple knockout leads to decreasing transcription constraints and enhances the production of TNF and IFN-gamma leading to increased effector function, which is a problem with exhausted T-cells.² Also, implementing logic-gated synthetic Notch (synNotch) significantly reduced tonic signaling which results in decreased chronic antigen stimulation of the T-cells.⁹ Finally, clinical data has shown that combining Car-t cells with immune checkpoint inhibitors decreases the rate of exhaustion and increases the production of effector molecules.³

Conclusion: T-cell exhaustion is a major road block in Car-T cell therapy; however interventions such as CRISPR NR4A knockouts protect these cells from turning into this state.² These findings suggest that future clinical treatment of tumors will include individual specific immunotherapy augmented with other therapies that prevent Car-T therapy from becoming ineffective due to cellular exhaustion.³

Works Cited

1. Zhu T, et al. T-cell exhaustion from a multiomics perspective. Clin Transl Med. 2026.
2. Chen J, et al. NR4A transcription factors limit CAR T cell function in solid tumours. Nature. 2019.
3. Chow A, et al. Clinical implications of T cell exhaustion for cancer immunotherapy. Nat Rev Clin Oncol. 2022.
4. Khan O, et al. TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion. Nature. 2019.
5. Belk JA, et al. Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors. Cancer Cell. 2022.
6. Wu H, et al. Mitochondrial dysfunction promotes the transition of precursor to terminally exhausted T cells. Nat Commun. 2023.
7. Richter FC, et al. Take my breath away-mitochondrial dysfunction drives CD8+ T cell exhaustion. Genes Immun. 2024.
8. Dong W, et al. Exhaustion-like dysfunction of T and NKT cells in SCID. Int J Mol Med. 2023.
9. Garcia-Robledo JE, et al. Engineering the next generation of CAR T-cells. Front Oncol. 2026.
10. Zhou Y, et al. Ponatinib alleviates CAR-T cell exhaustion. Blood. 2025.
11. Qin D, et al. Supercharging CAR-T cells through transcriptional and epigenetic armoring. Theranostics. 2025.
12. Ahn T, et al. Decoding and overcoming T cell exhaustion. Mol Ther. 2024.