Human Virtual Memory CD8+ T cells: the role of cytokines on cell development and functionality

Virtual memory T cells (TVM) are a subset of TCRαβ+CD8+ T cells recently identified in mice that, despite lacking prior antigen exposure, express typical markers of conventional memory T cells and play a protective role during the early phase of inflammatory/infectious processes. They are characterized by a high expression of the transcription factor Eomesodermin (Eomes). TVM cells have been recently described also in human peripheral blood. Similar to their murine counterparts, human TVM express high levels of Eomes. Unlinke murine TVM, they are divided into two subsets characterized by mutually exclusive expression of NKG2A and KIR. While murine TVM development has been extensively studied, little is known about the differentiation of these cells in humans. Evidence suggests that, in mice, IL-4, IL-15, and type I interferons (IFN-I) may play a role in this process. To investigate the impact of these cytokines on human thymic and peripheral TVM development, we cultured human pediatric thymocytes and adult peripheral blood mononuclear cells (PBMCs) in vitro with or without these cytokines and analyzed their phenotype using a 27-color spectral flow-cytometry panel. TCRαβ+CD8+Eomes+ cells developed among thymocytes only in the presence of IL-4 or IL-4+IL-15 after 9 days of in vitro stimulation. Interestingly, these cells were characterized by a naïve phenotype (CD45RA+CD27+CCR7+), lacking the expression of both NKG2A and KIRs. On the other hand, IL-4+IL-15 in vitro-stimulated PBMCs resulted in the expansion of two subsets of TCRαβ+CD8+Eomes+ cells characterized by the expression of either KIRs or NKG2A with a TEMRA (CD45RA+CCR7-) and TEM (CD45RA-CCR7-) phenotype respectively. Notably, we observed that the stimulation with IL-15+IFN-I is necessary by PBMCs to acquire a cytotoxic-potential, evidenced by a high expression of Granzyme B and Perforin. Overall, our findings indicate that human TVM differentiation is dependent on IL-4+IL-15, while cytotoxic-potential of these cells are sustained by IL-15+IFN-I stimulation.