HSV-TK–specific CD8⁺and CD4⁺T cells can be directly visualized by CFC in samples of peripheral blood after HyTK-positive DLI. Samples of PBMCs obtained from the 3 patients after the first T-cell infusion were incubated for 6 hours with medium alone, the candidate 15-mer peptides, or with peptide pools that elicited responses from the HSV-TK–reactive T-cell lines. Cells were then stained with FITC-coupled anti–IFN-γ, PE-coupled anti-CD8β, and peridinin chlorophyll protein (PerCP)-Cy5.5–coupled anti-CD4 mAbs, respectively, and examined by flow cytometry. (A) Gated on CD8⁺ T cells. (B) Gated on CD4⁺ T cells. Values indicate the percentage of cells producing IFN-γ. Data are shown for representative HSV-TK peptides.