CMV-specific CD4⁺CD161⁺T cells are resistant to daunorubicin-induced lymphotoxicity in vitro, have drug-effluxing capability, and reside within the Th1 rather than Th17 subset. PBMCs were exposed to daunorubicin in vitro and apoptosis was assessed by annexin V staining. (A) Histograms show the percentage of annexin V+ cells in CD4⁺CD161⁻ and CD4⁺CD161⁺ subsets. (B) CD4⁺CD161⁺ T cells were significantly more resistant to apoptosis after 2 days of in vitro treatment with daunorubicin (P = .02), as assessed by annexin V staining compared with their CD161⁻ counterpart (n = 6). (C) CD161⁺Rh^loCD4⁺ T cells are resistant to daunorubicin-induced cytotoxicity in vitro. Sort-purified CD161⁺Rho^lo, CD161⁺Rho^hi, and CD161⁻ CD4⁺ T cell subsets were cultured for 48 hours in the presence and absence of daunorubicin. Bar plots show cumulative data comparing annexin V⁺ cell frequency within the CD4⁺CD161⁺Rho^lo, CD4⁺CD161⁺Rho^hi, and CD4⁺CD161⁻ populations. Data represent the mean ± SE from 3 separate experiments. (D) PBMCs from 7 CMV-seropositive healthy donors were incubated in vitro with daunorubicin for 48 hours and then stimulated with CMVpp65 peptide at a concentration of 1 µg/mL for 4 to 6 hours; cumulative data depict a relative increase in the frequencies of IFNγ-producing CD4⁺CD161⁺ CMV-specific T cells after in vitro treatment with daunorubicin; data from 7 different experiments are shown. *P = .0136. The absolute number of CMV-specific CD4⁺ T cells as measured by IFN-γ response in patients with AML recovering from daunorubicin-based induction chemotherapy compared with samples collected before chemotherapy. (E) PBMCs from CMV-seropositive healthy donors were stimulated with CMVpp65 peptide at a concentration of 1µg/mL for 4 to 6 hours, followed by incubation with Rho. IFN-γ secreting cells were enriched using the IFN-γ cell secretion and enrichment kit (Miltenyi). Rho pumping was assessed in CD4⁺CD161⁺IFN-γ secreting T cells compared with CD4⁺CD161⁻IFN-γ⁻ cells. Bar graph shows proportion of CD4⁺CD161⁺ IFN-γ⁺ T cells vs proportion of CD4⁺CD161⁻IFN-γ⁻ T cells capable of rapidly effluxing Rho (n = 5). (F) CMV-specific CD4⁺ T cells were enriched after in vitro stimulation with CMVpp65 pepmix, using an IFN-γ cell secretion and enrichment kit. IFN-γ-expressing cells were then sort-purified based on CD161 expression into CD4⁺CD161⁺IFN-γ⁺ and CD4⁺CD161⁻IFN-γ⁺T cells, and T-bet and RORC2 mRNA expression was analyzed by quantitative reverse transcription polymerase chain reaction. Bar graphs show cumulative results from 3 healthy individuals. *P < .05. (G) Sort-purified CD161⁺Rho^lo, CD161⁺Rho^hi, and CD161⁻ T-cell subsets from CMV-seropositive healthy donors (n = 3) were stimulated with CMVpp65 peptide-loaded monocytes at a 2:1 ratio for 6 hours. Cytokine production was analyzed by intracellular staining, assessing production of IFN-γ, IL-2, and IL-17A. (H) PBMCs were collected from healthy donors approximately 4 weeks and 2 years after influenza (Flu) vaccination, and Flu-specific CD4⁺ T cells were enumerated after in vitro stimulation with influenza antigen by intracellular IFN-γ assay. A representative experiment shows the frequencies of IFN-γ-producing Flu-specific CD4⁺ T cells before vaccination (left), 4 weeks (middle), and 2 years (right) after vaccination. Bar graph compares CD161 expression between Flu-specific-IFN-γ producing CD4⁺ T cells at 4 weeks and 2 years. **P < .01; ***P < .001).