Expansion of CMV-specific CD4⁺ and CD8⁺ T cells from UCBT recipients and characterization of CD8 T-cell specificity. (A) Frequency of CMV-specific CD8⁺ T cells by CFC for IFN-γ performed on 2 representative T-cell lines expanded after IFN-γ capture. (B) CMV-specific T cells derived from UCBT recipients recognize pp65 peptides. Polyclonal T-cell lines were incubated with a peptide pool of 15-mer peptides with 11 amino acid overlaps spanning the entire pp65 protein and evaluated by CFC to detect IFN-γ⁺ cells. Results of 5 patients and 1 ND are shown. (C) Viral genome scan to determine the antigen specificity of CD8⁺ T cells derived from UCBT recipients at different times after transplant. Polyclonal T-cell lines were incubated with COS cells that were transfected without or with a plasmid encoding a patient specific HLA DNA alone (gray bars), with a plasmid encoding a single CMV-ORF alone (white bars) or cotransfected with both plasmids encoding the CMV ORF and the HLA allele (black bars). Supernatants were collected at 24 hours and assayed for IFN-γ by enzyme-linked immunosorbent assay. Results for the genome scan for T-cell lines derived from blood obtained on day 80, 180, and 365 post-UCBT from 2 representative patients are shown. (D) Deep sequencing of TCR Vb receptor genes in CMV-specific polyclonal T-cell lines from UCBT recipients (patient no. 2 top graph and patient 4 bottom graph) was performed. Results show the percentage of total clones of the top 10 clones tracked over time in both patients.