(A) PBMCs from a seropositive donor are inducible to become HCMV-specific cytotoxic effectors using peptide-loaded fibroblasts. Autologous fibroblasts were incubated with 10 μmol/L pp65_495-503 , and fresh PBMCs were added to the well for a 7-day stimulation repeated three times as described in Materials and Methods. Two days before harvest of the PBMCs, autologous fibroblasts were treated with IFN-γ, and infected with HCMV strain AD169. Additional IFN-γ–treated fibroblasts were incubated with peptide or medium, and a CRA was conducted at the indicated time points using 2,000 targets in each case. To verify that the target fibroblasts could function as APC, at each time point they were evaluated against the T-cell clone 3-3F4 (data not shown). The CRAs were conducted at each time point using PBMCs that had been depleted of CD4⁺ T cells. (), Day 0; (▪), second stimulation; (), third stimulation. (B) PBMCs from a seropositive donor are inducible to become HCMV-specific cytotoxic effectors using peptide-loaded PHA-blasts. Fresh PBMCs were incubated with peptide-loaded PHA-blasts for two rounds of stimulation. The effectors were incubated with CMV-infected or peptide-sensitized fibroblasts as described in (A). Both autologous and completely HLA mismatched fibroblast targets were used in CRAs with the peptide-stimulated PBMCs. (), Matched; (▪), mismatched.