Comparative MHC tetramer staining, intracellular IFN-γ, and cytotoxicity analyses of CMV-specific T cells after sensitization with pentadecapeptides or peptide pools of pentadecapeptides. T lymphocytes of HLA-A*0201⁺ individuals were sensitized with pentadecapeptide 123 or peptide-pools 23 or 1. (A) T cells were analyzed for their capacity to bind MHC tetramer complexed with NLVPMVATV peptide. (B) Intracellular IFN-γ production by T lymphocytes was assessed in response to peptide-pulsed K562/A*0201 cells. 50% of the CD8⁺ cells of T lymphocytes sensitized with pentadecapeptide 123 bound MHC tetramer and 36% produced intracellular IFN-γ. 80% of the CD8⁺ cells of the T-cell line sensitized with the peptide pool 23 stained with the tetramer and 53% produced IFN-γ. T cells sensitized with pool 1 failed to stain with the MHC tetramer or to produce intracellular IFN-γ. Irrelevant MHC tetramers and target cells pulsed with irrelevant peptides did not produce results above background (data not shown). (C) T lymphocytes sensitized with either pentadecapeptide 123 (CD3 + penta 123), peptide pool 23 (CD3 + pool 23), or pool 1 (CD3 + pool 1) were analyzed for their capacity to induce lysis of peptide-pulsed K561/A*0201 cells or CMV-infected MRC-5 fibroblasts. Target cells were plated in triplicates. Results represent percentage of lysis ± standard deviation.