CMV-specific CD8⁺ T cells have a cytotoxic phenotype and are elevated in CMV-seropositive B-CLL. (A) A representative dot plot of CMV-specific CD8⁺ T cells of a healthy control (upper panel) and a patient with Rai stage 1 B-CLL (lower panel) using CD8-PercP (y-axis) versus HLA-A2 or HLA-B7 CMV-tetramer-APC (x-axis) gated on lymphogate via FSC and SSC. (B) Composition of the different T-cell subsets of CD8⁺ CMV-specific T cells was determined based on 4-color immunostaining of CD27-FITC, CD45RA-PE, CD8-PercP, and CMV-tetramer-APC. Cells were gated on lymphocytes via FSC/SSC, and CMV-specific CD8⁺ T cells were additionally gated on both CD8- and CMV-tetramer positivity as represented by the square box in upper right quadrant of the dot plot in panel A. CMV-specific CD8⁺ T cells consist of the cytotoxic cell phenotype and show an increase in both the percentage (C) and absolute number (P = .0225; D) of CMV-specific CD8⁺ T cells in patients with B-CLL (squares, n = 10) compared with controls (circles, n = 6).