CMV-specific CD8⁺T cells from CLL patients are equally effective in cytokine production as those from HCs. (A) Enriched CD8⁺ T cells from CLL patients and HCs were stimulated with PMA/ionomycin, and cytokine production was measured within total CD3⁺CD8⁺ and CD3⁺CD8⁺CMV-tetramer⁺ cells after 6 hours by flow cytometry. Bars represent mean ± standard error of the mean (SEM) of n = 9 CLL and n = 5 HCs. Significant differences are presented as *.01 ≤ P < .05; **.001 ≤ P < .01; ***P < .001 (Mann-Whitney U test). (B-E) Enriched CD8⁺ T cells from CLL patients or HCs were incubated with HLA-I–matched EBV-LCLs, CLL cells, or B cells derived from HCs and loaded with increasing concentrations of the corresponding CMV pp65 peptide (HLA-A2 or HLA-B7) in the presence of αCD28, αCD29, brefeldin A, and GolgiStop. After 6 hours, cytokine production was analyzed by flow cytometry in CD3⁺CD8⁺tetramer⁺ cells. (B) Representative dot plots from 1 CLL patient and 1 HC. (C) Percentage of IFN-γ⁺, TNF-α⁺, and IL-2⁺ cells within CD3⁺CD8⁺tetramer⁺ cells and polyfunctional cells producing IFN-γ⁺, TNF-α⁺, and IL-2⁺ within the CD3⁺CD8⁺tetramer⁺ cells are presented. Results are shown as mean ± SEM of n = 9 CLL and n = 5 HCs. (D) Percentage of TNF-α⁺, TNF-α⁺IFN-γ⁺, TNF-α⁺IL-2⁺, and TNF-α⁺IFN-γ⁺IL-2⁺ (polyfunctional) cells within the CD3⁺CD8⁺tetramer⁺TNFα⁺ cells stimulated with CMV-peptide–loaded (10 ng/mL) EBV-LCLs. Results are shown as mean percentages of n = 9 CLL and n = 5 HCs. (E) Percentage of IFN-γ⁺, TNF-α⁺, and IL-2⁺ cells within the CD3⁺CD8⁺CMV-tetramer⁺ cells stimulated with 1.0 ng/mL CMV-peptide–loaded EBV-LCLs, stratified according to CMV-tetramer⁺CD8⁺ low and high cells defined as < or ≥ 5% CMV-tetramer⁺CD8⁺ cells within CD8⁺ cells, ALC, Rai stage, or IGHV mutation status. Bars represent mean ± SEM. conc, concentration; pos, positive.