Fig. 2: ML-NK differentiation and behavior.

A Protocol describing cytokine-induced memory-like NK cell generation. Primary NK cells were isolated form healthy donors and cultured for 30 hours in NK media supplemented with IL12, IL15, and IL-18. Next, primary NK cells were cultured for 7 days to allow ML-NK cell differentiation. B Gene expression analysis comparing primary NK cells with ML-NK cells. ML-NK cells showed upregulation of multiple NK cell activation markers. Data were analyzed using paired t test. C Gene-ontology analysis showing biological processes (green graph), molecular function (blue graph), and cellular compartment (purple graph) affected in ML-NK cells according to the gene expression results. Data were generated using GSEA (https://www.gsea-msigdb.org/). D Clustergram showing differentially expressed genes in ML-NK cells compared with naive NK cells. E Graphs showing the percentage of genes upregulated or downregulated in the cellular functions identified in the GSEA analysis. F, G Primary NK and ML-NK cells were co-cultured for 24 h with the HNSCC cell line SCC-6 to analyze their cytotoxic potential against established cell lines. ML-NK cells showed increased cytotoxic capacity compared with primary NK cells. Data were analyzed using Brown-Forsythe and Welch ANOVA tests. H, I Images show ML-NK cell cytotoxicity against patient-derived HNSC tumor cells after 3 days in co-culture (SCC-6 are included as comparison). Fluorescence images show a representative experiment. The experiment was repeated three independent times, acquiring five images per experiment. Images from the same experiment were averaged and used as an independent replicate. Graphs show average of these three independent replicates ± standard deviation. Data were analyzed using Brown–Forsythe and Welch ANOVA tests. P value was set to 0.05. Bar graphs show average ± standard deviation. *, **, *** denote P value < 0.05, <0.01, <0.005, respectively.