Characterization of CMV-specific CD4⁺T cells in healthy donors vs AML patients. PBMCs were stimulated with an overlapping pool of MHC class II CMVpp65 peptides. Cells were gated on IFN-γ-producing CD4⁺ T cells and examined for the coexpression of a panel of surface markers as outlined in “Materials and methods” (n = 11). (A) A representative FACS plot depicting IFN-γ-producing CMV-specific CD4⁺ T cells in a patient with AML before (baseline) and after recovery from chemotherapy (remission). (B) Significant fold increase in CMV-specific CD4⁺ T cells in patients with AML after daunorubicin treatment (*P = .036). (C) A representative FACS plot showing the phenotype of IFN-γ-producing CMV-specific CD4⁺ T cells (black) overlaid on total CD4⁺ T cells (gray). CMV-specific T cells are predominantly CD95⁺, CD45RA⁻, CD62L⁻, and CD127⁺. (D) A representative FACS plot showing that the majority of IFN-γ⁺ and TNF-α⁺ CMV-specific CD4⁺ T cells also express CD161. (E) CD161 expression on CMV-specific CD4⁺ and total CD4⁺ T cells. The majority of CMV specific CD4⁺ T cells coexpress CD161. (F) Representative FACS plots show the frequency of CMV-specific CD4⁺ T cells derived from CD161⁺ (top) and CD161⁻ (bottom) T cells after stimulation with CMVpp65-loaded autologous B cells in the presence of IL-4 and IL-7 (left) or IL-2 (right) for 10 days. Bar graphs depict cumulative results for absolute frequency of CMV-specific T cells from 4 independent experiments. Data are shown as mean ± SE.