T cells are crucial for the control of cytomegalovirus (CMV) in infected individuals. Although CMV-specific T cells can be quantified by various methods, clear correlates of protection from CMV disease have not been defined. However, responses to the pp65 protein are believed to play an important role. Here, the proportions of interferon γ–producing T cells following ex vivo activation with pools of overlapping peptides representing the pp65 and immediate early (IE)-1 proteins were determined at multiple time points and related to the development of CMV disease in 27 heart and lung transplant recipients. Frequencies of IE-1–specific CD8 T cells above 0.2 and 0.4% at day 0 and 2 wk, respectively, or 0.4% at any time during the first months discriminated patients who did not develop CMV disease from patients at risk, 50–60% of whom developed CMV disease. No similar distinction between risk groups was possible based on pp65-specific CD8 or CD4 T cell responses. Remarkably, CMV disease developed exclusively in patients with a dominant pp65-specific CD8 T cell response. In conclusion, high frequencies of IE-1 but not pp65-specific CD8 T cells correlate with protection from CMV disease. These results have important implications for monitoring T cell responses, adoptive cell therapy, and vaccine design.

No abstract

Antigen-specific, cytokine flow cytometry was used to analyze the prevalence and frequency of CD4 and CD8 memory T cells specific for the abundantly expressed cytomegalovirus (CMV) phosphoprotein 65 (pp65) in healthy CMV IgG-seropositive individuals. Stimulation of peripheral blood mononuclear cells with peptide pools and individual peptides derived from the pp65 amino acid sequence in 40 donors revealed that 63% of donors had a detectable CD4 T cell response and that 83% of donors had a detectable CD8 T cell response against this protein. The overall frequencies of T cells directed against pp65 were analyzed for 20 donors by stimulation with peptide pools covering the complete pp65 protein and were as high as 2 in 1000 and 9 in 1000 (median) peripheral blood CD4 and CD8 T cells, respectively. In addition, a comparison between CD4 responses to a CMV lysate containing various CMV proteins and pp65-specific responses in 9 donors indicated that pp65 was a dominant target of the CMV-specific CD4 T cell response in some, but not all, donors. Several new T cell epitopes were identified.

Antigen-specific T cells may be detected and enumerated by short-term ex vivo antigenspecific stimulation followed by cytokine flow cytometry. Most frequently, intracellular IFN-+ is used to identify T cells specific for cytomegalovirus (CMV), Epstein-Barr virus or HIV. Some researchers use whole blood, others peripheral blood mononuclear cells (PBMC) in this assay; however, the performance of the two systems has never been directly compared. Blood was drawn from previously characterized healthy CMV-positive donors, and CMVderived peptides or CMV lysate were used as stimulants. In an initial series of experiments, lithium-heparin was identified as the best coagulant to be used. Dose-response curves were established using concentrations between 0.1 and 40 ? g/ml of peptides and between 0.1 and 20 ? g/ml of virus lysate, respectively. IFN-+ -positive T cells were expressed as percent of the reference population, and frequencies measured in whole blood and PBMC were compared. Maximum responses were consistently higher in PBMC than in whole blood and were reached at lower concentrations of stimulant. In several instances, responses identified with PBMC were not at all detected with whole blood. In summary, studies using whole blood in this type of assay are likely to underestimate the frequencies of antigen-specific T cells.

Despite the success of antivirals in preventing clinically overt CMV disease in cardiac allograft recipients, sub-clinical active CMV infection remains a major concern because of its association with allograft rejection and vasculopathy. The measurement of CMV specific T-cell responses is a promising approach to assessing this situation. For simplicity, class-I MHC/peptide-multimers staining CD8 T-cells directly are often used but this ignores a much wider range of responses including the whole CD4 T-cell compartment. CD4 T-cells, however, were recently shown to be critical to reducing CMV load early after transplantation. To determine how extensive T-cell responses to CMV are, the responses to two dominant CMV proteins, IE-1 and pp65, were dissected in detail accounting for T-cell lineage, frequencies, epitope recognition and changes over time in more than 25 heart transplant recipients. Cross-sectional results from over 30 healthy CMV-carriers were analyzed for comparison. Responses were unexpectedly complex, with considerable inter-individual variation in terms of dominance, breadth, and recognized epitopes. Whereas the use of MHC/peptide-multimers for clinical CD8 T-cell response monitoring alone can be justified in some situations, short term T-cell activation combined with intracellular cytokine staining was clearly found to be of more general usefulness. The performance of IFN-gamma, TNF-alpha, or IL-2 as single read-outs in identifying activated T-cells was examined and confirmed that the frequently used IFN-gamma was best suited. These results should be used to inform the design of clinically applicable and diagnostically useful approaches to monitoring CMV specific responses in heart transplant recipients.

No abstract