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Chronic antigenic stimulation has been associated with peripheral blood expansions of CD8pos. T cells characterized by CD57 expression, loss of CD27 expression, and reversal of the CD45RObright /RAdim phenotype usually associated with immunological memory towards a CD45ROdim /RAbright phenotype. However, the relationship and functional significance of these subset(s) has remained controversial. Here, this issue was addressed using a novel flow cytometric technique that allows simultaneous detection of human cytomegalovirus (HCMV)‐specific CD8pos. memory T cells by rapid (< 6 h) HCMV peptide‐specific induction of cytokine synthesis, and their phenotypic characterization, including CD57, CD27 and CD45RA/RO. The vast majority of resting CD8pos. T cells capable of rapid induction of IFN‐γ and TNF‐α synthesis in response to HCMV peptides were found in a subset characterized by intermediate to high expression of CD57, down‐regulation/loss of CD27, and varying degrees of reversal of the classical “memory” CD45RObright /RAdim phenotype. This subpopulation likely includes the fully differentiated memory cells responsible for the long‐term immune defense against HCMV reactivation.

Cell-mediated immunity plays an essential role in the control of infection with the human cytomegalovirus (HCMV). However, only a few CD8+-T-cell epitopes are known, with the majority being contained in the pp65 phosphoprotein, which is believed to dominate the CD8+-T-cell response to HCMV. Here, we have readdressed the issue of CD8+ T cells specific for the 72-kDa major immediate-early protein (IE-1), which is nonstructural but is found very early and throughout the replicative cycle. Using a novel flow-cytometric assay, we were able to identify CD8+-T-cell epitopes (by IE-1 peptide-specific induction of cytokine synthesis) and simultaneously measure the frequency of cells directed against them. For this purpose, 81 pentadecamer peptides covering the complete 491-amino-acid sequence of IE-1 were tested on peripheral blood mononuclear cells of anti-HCMV immunoglobulin G-seropositive donors. At least 10 new epitopes were identified, and the fine specificity and presenting HLA molecule of the first of them was determined. The frequencies of CD8+ T cells directed against IE-1 were similar to those directed against pp65 in donors tested with known pp65-derived peptides. Importantly, additional testing of a corresponding set of peptides covering the complete sequence of pp65 on 10 of these donors identified individuals whose CD8+ T cells recognized IE-1 but not pp65 and vice versa, clearly illustrating that either protein may be a major target. In summary, our results suggest that IE-1 is far more important as a CD8+-T-cell target than current opinion suggests.

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