Quantitative and functional assessment of CMV-specific T-cell responses.

(A) Quantitative assessment of CMV-specific CD8⁺ T cells using HLA-peptide tetramers. HLA class I–peptide tetramers and monoclonal antibodies specific for CD8 were used to determine the frequency of CMV-specific CD8⁺ T cells specific for peptides derived from CMV pp65. Representative examples of staining using the A2-pp65 tetramer (10.5% of CD8⁺ T cells, left) and the B7-pp65 tetramer (1.86% of CD8⁺ T cells, right) are illustrated. (B) The function of CMV-specific CD8⁺ T cells may be assessed via a combination of tetramer staining and cytokine flow cytometry. Peripheral blood mononuclear cells were stained first with HLA-pp65 tetramers and then stimulated with the antigenic HLA-restricted peptide derived from pp65. The fraction of tetramer-staining cells producing intracellular cytokines was then determined by flow cytometry using antibodies specific for intracellular TNF-α. Representative examples of this analysis are shown for 2 subjects, who had relatively higher (70%, left) or lower (27%, right) functional fractions of tetramer-staining cells. (C) The proportion of CD4⁺ T cells responding to the CMV pp65 protein was assessed using cytokine flow cytometry following stimulation with a mixture of 138 overlapping peptides spanning the entire pp65 protein. CMV-specific CD4⁺ T cells were determined by the fraction of cells that were CD69⁺ that also produced intracellular TNF-α following stimulation. A representative example is shown for an individual with 6.8% of CD4⁺ T cells specific for CMV pp65.