Detection of IFN-γ⁺ CMV-specific CD8⁺ T cells after UCBT by CFC of PBMCs and after in vitro expansion of T cells. (A) PBMCs from a normal donor (ND) and from 2 representative CMV-seropositive UCBT recipients were analyzed for CMV-specific CD8⁺ T cells by CFC. The number in the top right quadrant indicates the percentage of IFN-γ⁺ T cells in the CD8⁺ subset. (B) CD8⁺ T-cell lines were generated from PBMCs obtained at the indicated times by a single in vitro stimulation with RV798-infected fibroblasts and then assayed for IFN-γ by CFC. Data are shown for a normal donor and a representative UCBT patient at multiple time points. Mock-infected autologous fibroblasts served as a negative control in the assay. (C) IFN-γ CFC assay for CMV-specific T cells in T-cell lines from each of 19 UCBT recipients at intervals after transplant (15 CMV positive and 4 CMV negative) and from 2 normal donors. The data shows the percentage of IFN-γ⁺ T cells in the gated lymphocyte population after restimulation with RV798-infected fibroblasts. White bars, day 56; light gray bars, day 80; dark gray bars, day 180; black bars, day 365; asterisk (*) indicates not done. (D) Lysis of autologous fibroblasts infected with RV798 (light gray bars), mock-infected (white bars), or HLA-mismatched fibroblasts either RV798-infected (dark gray bars) or mock-infected (black bars) by CMV-specific T-cell lines from UCBT recipients. The graph shows the mean specific lysis of cell lines from different time points after UCBT in 5 patients. The effector-to-target (E:T) ratio was 10:1.