Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers

Abstract

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.

FIG. 1.

MHC stabilization on T2 cells using potential HLA A2-binding peptides from HCMV antigens (pp65, pp71, pp150, IE-1, gB, pp50, pp28, gH, US3, US2, UL16, and UL18). T2 cells were initially incubated with 100 μg (final concentration) of each of the peptides/ml for 1 h at 37°C and then for 14 to 16 h at 26°C, followed by incubation at 37°C for 2 to 3 h. HLA A2 expression on these cells was analyzed by flow cytometry using the BB7.2 antibody. The dotted line indicates the mean + 3 SEM of the fluorescence intensity for HLA A2 on T2 cells incubated at 26°C without peptide, which was the cutoff for a positive result.

FIG. 2.

Recognition of HCMV peptide epitopes by T cells from healthy seropositive donors. PBMC were cocultivated with peptide-sensitized (20 μg/ml) autologous PBMC at a ratio of 2:1 for 7 days. On day 7 these cultures were restimulated with autologous γ-irradiated Epstein-Barr virus-transformed LCLs sensitized with peptide epitopes. On day 10 these T-cell lines were used as polyclonal effectors in a standard ⁵¹Cr release assay against peptide-sensitized autologous PHA blasts. An effector-to-target ratio of 10:1 was used in these assays. Panel A shows data for HLA A2-restricted epitopes, while panel B shows data for HLA A1-, A24-, B8-, B27-, B35-, and B44-restricted epitopes. T-cell lines were established from donors SB and SE (A) and CS, SC, TC, MB, PP, RE, RK, and MW (B). Results are expressed as percent specific lysis. Complete T-cell epitope sequence, source of the antigen, and the HLA restriction for each of these epitopes is shown on the y axis. Representative data from a minimum of three different sets of experiments are shown.

FIG. 3.

Specific lysis by HCMV-specific T cells of autologous LCLs infected with recombinant vaccinia virus encoding individual HCMV antigens (Vacc.pp65 and Vacc.IE-1) (A) or HCMV-infected HLA-matched fibroblasts (B). LCLs were infected for 12 to 14 h (MOI, 10:1) with vaccinia constructs and processed for standard ⁵¹Cr release assay. T-cell clones or polyclonal lines from donors SB (pp65 specific, SB 62.3 and IE-1 specific, SB 75.20) and SC (IE-1 specific, SC ELRR) were used as effectors in the assay (A). Fibroblasts were infected with AD-169 strain of HCMV for 14 to 16 h (MOI, 5:1) and used as targets in the CTL assay. CTL clones or polyclonal lines from donors SB (IE-1 specific, SB 75.20; pp65 specific, SB NLVP), PP (pp50 specific, PP VTEH), and TC (IE-1 specific TC, ELRR and IE-1 specific, TC-QIKV) were used as effectors in this assay (B). Results are expressed as percent specific lysis observed at an effector-to-target ratio of 5:1. Representative data from a minimum of three different sets of experiments are shown.