...We have previously shown that the expression of CD57, CD300a, and CD161 was differentially affected by age and chronic CMV infection, indicating that these markers are a hallmark of CMV infection and T cell ageing. The aim of this present study was to clarify whether these three markers define distinct T cell subpopulations with a specific functional and molecular signature. Specifically, we analyzed the effect of age and chronic CMV infection on the functionality of T cells according to CD161, CD300a, and CD57 expression. We found that these markers defined different T cell subsets, both at the phenotypic and functional levels. CD57 was the best biomarker for CD4+ T cell cytotoxicity and was a hallmark of CMV infection. CD300a+ T cells were heterogeneous and included different cell subsets. The population of CD161+ T cells dramatically decreased with age, independently of CMV infection, and represented a sign of age-associated immune system alterations. The latter could contribute to an increased risk of autoimmune disease and infection in older adults. Our results underline the importance of better understanding the factors involved in the immunosenescence process to be able to uncover new biomarkers and open new avenues for the investigation and development of novel age-related disease therapies.