HyTK-specific CD8⁺CTL recognize epitopes derived from both the Hy and TK domain of theHyTKfusion gene and are restricted by multiple class I MHC alleles. Samples of PBMCs were obtained from UPN 1574, UPN 4103, and UPN 7826 after the HyTK-positive T-cell infusion and stimulated twice 1 week apart with γ-irradiated HyTK-expressing donor T cells (A-C) or with HSV-TK–expressing donor T cells (UPN 7826 [D]). To map the HLA-restricting alleles, EBV-transformed LCL lines that matched at all 4 class I MHC alleles (Auto), allogeneic LCL lines that matched only at a single class I MHC allele, and mismatched LCLs (MM) were modified to express either the Hy (▦) or the HSV-TK (▪) domain of the HyTK fusion protein. Aliquots of the CD8⁺ CTL lines were then examined for recognition of nontransduced LCL- (□), Hy- (▦), or HSV-TK– (▪) modified target cells. Data are shown at an effector-target ratio of 5:1.