Analysis of CMV-specific CD8⁺ T cells using peptide stimulation and tetramer binding. (A) Patients who were positive for HLA-A2, -B7, or -B8 were analyzed for the frequency with which they bound to appropriate CMV-specific tetramers. Representative flow cytometry analysis is shown for −CMV (left) and +CMV (right) patients. Cells were gated as follows: lymphocytes were identified in a FSC/SSC plot, out of these, singlets were isolated. From these, CD14⁻/CD20⁻/CD3⁺ T cells were identified and further gated on CD8⁺ T cells, which were analyzed for their binding to matching tetramers. (B) Left, bar graph illustrating the frequency of tetramer-binding cells from −CMV (blue) and +CMV (red) patients. Right graph, Tetramer⁺ cells from +CMV patients were further separated into their respective memory subsets. (C) Representative flow cytometry analysis from −CMV and +CMV patients of intracellular cytokine expression after stimulation with CMV-specific peptides. CD8⁺ T cells were gated as described in Figure 2A and then analyzed for the production of IFNγ and TNF. (D) Frequency of IFNγ⁺/TNF⁺ CD4⁺ and CD8⁺ T cells of +CMV (n = 6) and −CMV (n = 4) patients (left). Also the frequency of IFNγ⁺/TNF⁺ CD8 T cells in each of the 4 memory subsets is shown for +CMV and −CMV patients (right). *P ≤ .05; **P ≤ .01 Wilcoxon rank-sum test.