Methods for Characterization of Senescent Circulating and Tumor-Infiltrating T-Cells: An Overview from Multicolor Flow Cytometry to Single-Cell RNA Sequencing

Immunosenescence is the general term used to describe the aging-associated decline of immunological function that explains the higher susceptibility to infectious diseases and cancer, increased autoimmunity, or the reduced effectiveness of vaccinations. Senescence of CD8+ T-cells has been described in all these conditions. The most important classical markers of T senescent cells are the cell cycle inhibitors p16ink4a, p21, and p53, together with positivity for SA-βgal expression and the acquirement of a peculiar IFNγ -based secretory phenotype commonly defined SASP (Senescence Associated Secretory Phenotype). Other surface markers are the CD28 and CD27 loss together with gain of expression of CD45RA, CD57, TIGIT, and/or KLRG1. However, this characterization could not be sufficient to distinguish from truly senescent cells and exhausted T-cells. Furthermore, more complexity is added by the wide heterogeneity of T-cells subset in aged individuals or in the tumor microenvironment. A combined analysis by multicolor flow cytometry for surface and intracellular markers integrated with gene-expression arrays and single-cell RNA sequencing is required to develop effective interventions for therapeutic modulation of specific T-cell subsets. The RNASeq offers the great possibility to reveal at single-cell resolution the exact molecular hallmarks of senescent CD8+ T-cells without the limitations of bulk analysis. Furthermore, the comprehensive integration of multidimensional approaches (genomics, epigenomics, proteomics, metabolomics) will increase our global understanding of how immunosenescence of T-cells is interlinked to human aging.