Target structures of the CD8(+)-T-cell response to human cytomegalovirus: the 72-kilodalton major immediate-early protein revisited

Abstract

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.

FIG. 1

Design of peptide pools. The numbers of the pools (left column and top row) are shown in bold. Individual peptides (n = 81) in these 18 pools correspond to the numbers in the respective columns and rows, with peptide 1 = IE-1_1–15, peptide 2 = IE-1_7–21, etc., according to the complete sequence of the 72-kDa major IE protein.

FIG. 2

Identification of a CD8⁺-T-cell inducing peptide from peptide pools. Stimulation of PBMC from an HCMV IgG-seropositive donor with overlapping 15-amino-acid peptides originating from the IE-1 protein. The figure shows results obtained with six different peptide pools on day 1, two of which (pool 7 and pool 15) gave positive results, and stimulation with the candidate peptide IE-1_307–321 (EFCRVLCCYVLEETS), the only peptide contained in both positive pools, on day 2. IFN-γ-positive events are highlighted. Results for 50,000 CD3 T cells are displayed in each diagram. PBMC were stained with anti-IFN-γ–FITC, anti-CD69–PE, anti-CD8–PerCP, and anti-CD3–APC. Axes show log fluorescence intensity.

FIG. 3

Fine mapping of a 15-amino-acid peptide. According to the amino acid sequence of the HLA-B7-presented peptide IE-1_307–321 (EFCRVLCCYVLEETS), seven additional nonamer peptides were synthesized and tested on PBMC of an HLA-B7-positive donor whose CD8⁺ T cells were reactive to the original peptide (top). Several of these nonamer peptides led to IFN-γ induction in CD8⁺ T cells, with IE-1_309–317 giving the strongest response (middle and bottom). IFN-γ-positive events are highlighted. The activation marker CD69 is used to increase the specificity of the analysis. Results for 50,000 CD8⁺ T cells are displayed. PBMC were stained with anti-IFN-γ–FITC, anti-CD69–PE, anti-CD8–PerCP, and anti-CD3–APC. Axes show log fluorescence intensity.