To determine the functional characteristics of the increased CD45RA− CD27− and CD45RA+ CD27− CD4+ T-cell populations in CMV-seropositive subjects we first examined their surface expression of markers

that were previously shown to be associated with migration (CCR7), co-stimulation (CD28), responsiveness to cytokines (IL7-Rα) and end-stage differentiation (CD57). We found that CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells both showed low CCR7, CD28 and Selleckchem MG-132 IL-7Rα but higher CD57 expression compared with naive CD45RA+ CD27+ and CD45RA− CD27+ populations indicating that they were more differentiated (Fig. 3a). In addition, on the basis of CD28, IL-7Rα and CD57 expression, the CD45RA+ CD27− subset was significantly more differentiated than the CD45RA− CD27− population (Fig. 3a). We next investigated Epigenetics inhibitor the functional properties of the CD45RA− CD27− and CD45RA+ CD27− subsets of CD4+ T cells. We showed that the expression of molecules associated with cytolytic potential such as granzyme B and perforin were not detectable in naïve CD45RA+ CD27+ and CD45RA− CD27+ CD4+ T cells (Fig. 3b). In contrast, both CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells expressed granzyme B and perforin, the levels of which were significantly higher in CD45RA+ CD27− cells when these populations were compared (Fig. 3b). Other

indicators of CD4+ T-cell functionality include production of cytokines such as IFN-γ, IL-2 and TNF-α, and the expression of the CD40 ligand. The co-expression of more than one function in individual Y-27632 2HCl cells may be associated with enhanced viral control.29

We therefore performed multiparameter flow cytometric analysis to identify simultaneously the relative expression of IFN-γ, IL-2, TNF-α and CD40 ligand in individual CD4+ T cells at different stages of differentiation defined by relative expression of CD45RA and CD27 (Fig. 3c; see Supplementary Information, Fig. S2 and Table S2). The CD45RA− CD27+, CD45RA− CD27− and CD45RA+ CD27− subsets contained more cells with three and four functions compared with the CD45RA+ CD27+ CD4+ naive T-cell population (functions expressed are detailed in Supplementary Information, Table S2). These differences were highly significant (Wilcoxon matched pairs test; for all comparisons naive versus other subsets P < 0·0001; Fig. 3c). Both CD45RA− CD27− and CD45RA+ CD27− CD4+ T cells showed equivalent multifunctionality (P = ns), which was higher than in the CD45RA− CD27+ and naive CD45RA+ CD27+ CD4+ T-cell populations (P < 0·01). This indicates that although CD45RA+ CD27− CD4+ T cells bear phenotypic characteristics of highly differentiated T cells, they are not exhausted functionally but instead are capable of potent effector function.