HSV-TK–specific CD8⁺CTLs can be visualized by CFC and recognize multiple epitopes within the HSV-TK protein. (A) Design of peptide pools. The numbers of the pools (left column and top row) are shown in bold. Individual 15-mer peptides overlapping by 11 aa (n = 95) in these 20 pools correspond to the numbers in the respective columns and rows. Peptides 1 to 3 comprise the C-terminal aa sequence of the Hy protein (Hy_300-324), peptides 4 to 7 encompass the HyTK fusion site, and peptides 8 to 95 span the aa sequence of the HSV-TK protein (HSV-TK_10-376). The gray shading illustrates how a candidate peptide was chosen. (B) Identification of an immunogenic peptide from the peptide pools that induced IFN-γ production by CD8⁺ T cells. Samples of postinfusion PBMCs were obtained from UPN 4103 stimulated in vitro with γ-irradiated HyTK-positive donor T cells twice 1 week apart. Aliquots of the cultures were incubated for 6 hours with medium alone or with each 1 of the 20 peptide pools or the individual 15-mer peptides. Cells were then stained with FITC-coupled anti–IFN-γ and PE-coupled anti-CD8β mAbs and examined by flow cytometry. Cells were gated to identify CD8⁺ T cells and stained for intracellular IFN-γ. Values indicate the percentage of cells in the culture that produced IFN-γ in response to peptide stimulation. (C) Multiple 15-mer peptides throughout the sequence of the HSV-TK protein (boldface) are immunogenic. The underlined sequences indicate the 15-mer candidate peptides within the HSV-TK protein that were identified to induce IFN-γ production by CD8⁺ T cells in aliquots of the T-cell lines or postinfusion PBMCs from the 3 patients. Immunogenic peptides within the adjacent Hy protein (italics) or fusion site were not detected in these patients.