Deep Cellular and Molecular Characterization Reveal a Distinct Phenotype and Metabolic Signature of Human Senescent CD8+ T Cells: Implication for Cancer Cellular Therapy

Introduction: T cell-based cancer immunotherapy has demonstrated unprecedented efficacy in hematologic malignancies. However, only a minority of patients experienced long-term remissions with the existing treatment modalities. Dysfunction of infused T cells is regarded as one of the main reasons for failure of T cell therapies. Identification and insight into the mechanisms driving T cell dysfunction will offer additional tools to improve cellular and metabolic fitness of genetically engineered T cells for cancer therapy.

Aim: In contrast to exhaustion, aging and senescence-driven T cell dysfunction is less well understood and can impede cellular functions critical to efficient anti-tumor responses in elderly or pre-treated patients. Identification of senescent T cells has mainly relied on cell surface markers that have been used inconsistently in the literature. Here, we aim to provide a detailed molecular and functional signature of senescent T cells exhibiting all hallmarks of senescence including proliferation arrest, cellular phenotype, senescence-associated secretory phenotype (SASP) and β-galactosidase activity to abrogate and reprogram senescence-induced T cell dysfunction.

Methods: T cells were isolated from young (< 30 years old) and older (45-90 years old) healthy donors. CD8⁺ T cells were extensively analyzed for cellular phenotype (flow cytometry), mitochondrial function (metabolic flux assays, electron microscopy, flow cytometry), β-galactosidase activity and cellular functions including proliferation and apoptosis. Anti-tumor cytotoxic responses of different T cell subsets were tested by introduction of a tumor-antigen specific T cell receptor (TCR) via electroporation. Senescence was further characterized at the molecular level by analyzing the TCR repertoire and transcriptome by single-cell RNA sequencing.

Results: The detailed examination of different senescence hallmarks revealed a subset of T cells within a population of late-differentiated cells. Compared to other subsets, this T cell sub-population showed a complete lack of proliferation under different stimulation conditions and exhibited known surface markers of senescence such as CD57, KLRG1 and re-expression of CD45RA. Failure of proliferation was accompanied by signs of mitochondrial dysfunction such as low mitochondrial mass and membrane potential, altered shape in electron microscopy and reduced mitochondrial respiration. In turn, emergence of these features could be observed in non-senescent T cells upon senescence-inducing treatments. Importantly, senescent T cells exhibited superior cytotoxic potential as determined by degranulation assay. A more direct proof of tumor cell lysis by TCR gene transfer approach confirmed a potent cytolytic activity of these cells, despite their inability to proliferate. Finally, RNA sequencing on a single-cell level was performed and confirmed perturbations in mitochondrial gene expression and showed upregulation of signaling pathways linked to cytotoxicity and activation. Validation of these new findings are currently underway across different experimental approaches.

Conclusion: The deep characterization of senescence in T cells at the cellular and molecular level allows a more precise identification of these cells in patients undergoing adoptive T cell therapies. Furthermore, the results of this study suggest that these T cells could elicit strong anti-tumor cytotoxicity despite defective proliferation and might be useful for cancer treatment. The ongoing analysis of mechanisms involved in senescence induction can be used to restore proliferative potential in this T cell subset and hence improve T cell-based cancer immunotherapy.