Differential expression of CXCL8, IL-22, and IFN-γ in human “stage III” NK cells both in vitro and in vivo. (A) Cytokine expression in in vitro derived NK cells. UCB CD34⁺ cells were purified and cultured in the presence of cytokine-mix. At day 20 of culture cells were harvested, washed and stimulated 4 hours in the presence of PMA 25ng/mL plus Ionomycin 1μg/mL and IL-23 50ng/mL in the presence of monensin. We assessed CXCL8, IL-22 and IFN-γ expression by flow cytometric gating on CD56⁺ cells. Representative experiment of 4 performed. (B-C): Cytokine expression in tonsil-derived stage III and stage IV NK cells. CD56⁺ cell were enriched by positive selection from fresh tonsil-derived cell suspensions and were stimulated 4 hours as described above. CXCL8, IL-22 and IFN-γ expression was analyzed by flow cytometry gating on Lin⁻(CD3⁻CD19⁻CD14⁻CD34⁻)CD56⁺ cells. Representative experiments of 6 performed. (D) Real-time RT-PCR analysis of CXCL8 expression in fresh tonsils-derived Lin⁻CD56⁺CD117⁻CD94⁺ and Lin⁻CD56⁺CD117⁺CD94⁻ cell subsets, purified by cell sorting. We calculated CXCL8 relative expression in Lin⁻CD56⁺CD117⁺CD94⁻ on the basis of CXCL8 expression level detected in Lin⁻CD56⁺CD117⁻CD94⁺, arbitrarily normalized to one. Data are expressed as mean ± SEM of relative expression obtained in 3 independent experiments. Samples were run in triplicate, we normalized gene expression levels to GAPDH mRNA, and performed relative quantification using the ΔΔC_T method.