ELISPOT assay for the determination of HLA restriction of the CTL lines generated from CMV seropositive donors. CTL lines generated after the third stimulation with pp65-transduced CD40-B cells were tested for HLA restriction. ELISPOT assays were performed by incubating the CTL line with one of the following: autologous LCL, pp65-transduced autologous LCL (□), 293T cells transfected with both the pp65 gene and the individual HLA cDNA (▪), or the pp65 gene alone (▧). In the case of P06 and P07, CTL lines were found to recognize 293T cells transfected with the pp65 gene alone, due to their endogenous expression of HLA-A*0201. Thus, 293T cells transfected with both mutant pp65 gene (pcDNA3-pp65^ΔNLV), lacking a dominant A*0201-restricted epitope (NLVPMVATV), and individual HLA cDNA were used in these cases (▤) (see “Materials and methods”). Each bar represents the number of spots per 10³ cells.