DYS-specific CD4⁺ T cells have an effector memory membrane phenotype. PBMCs from HLA-DRB*0701–positive, CMV-seropositive donors were stimulated with DYS peptide or CMV lysate, and antigen-specific CD4⁺ T cells were determined by intracellular IFN-γ expression. The expression of CD45RA, CD45RO, CD28, CD27, and CD57 then was determined on this population. (A) Representative flow cytometric profiles from 3 donors (07, 08, and 02) showing membrane phenotype of DYS-specific cells. The percentage of (B) DYS-specific or (C) CMV lysate–specific CD4⁺ T cells expressing each marker is shown from individual donors. The mean value is represented by a horizontal line. (D) DYS-specific CD4⁺ T cells demonstrate direct ex vivo cytotoxic activity. Freshly isolated PBMCs were CD8⁺ depleted by magnetic selection and incubated with autologous LCL targets at an effector-target ratio of 25:1 in chromium release assays. Target cells were CMV lysate pulsed, peptide pulsed, or unpulsed; MVA-gB infected or control MVA infected.