Cytotoxicity of IL-12 and IL-15–cultured ILC3s toward the classical NK-cell target K562 is perforin-mediated. ILC3s were expanded for 3 weeks with IL-2 and IL-7 or IL-12 and IL-15 and the cytolytic potential of bulk cultures against K562 targets was assessed after 18-hour stimulation at a 10:1 E:T ratio using a lactate dehydrogenase (LDH) cytotoxicity assay. (A) Cytotoxicity of expanded tonsillar and splenic NKp44⁺ and NCR⁻ ILC3s. For tonsils, N = 6 and N = 3 for NKp44⁺ and NCR⁻ ILC3s, respectively. For spleen, N = 3 and N = 6 for NKp44⁺ and NCR⁻ ILC3s, respectively; each sample is an individual HFL donor with 2 to 8 mice pooled. Expanded NK cells from the same donors were used as positive controls. (B) Cytolytic activity of IL-12 plus IL-15–expanded tonsillar NKp44⁺ ILC3s and NK cells in the presence of medium alone, mouse IgG1 isotype, anti-TRAIL antibody, CMA (effectors were preincubated for 2 hours) or a combination of the latter, N = 5 except for the last condition in which data were obtained from 3 donors. (C) Correlation between frequency of the above-stated populations and cytotoxicity for tonsillar NKp44⁺ ILC3 cultures. ns, P > .05; *P < .05; ***P < .001 using paired Student t test in panels A and B or Spearman correlation in panel C. r, Spearman rank correlation coefficient.