Decreased cytotoxic functions in NK cell from patients with MM. (A) Heatmap of leading-edge genes standardized expression from “natural_killer_cell_mediated_cytotoxicity” KEGG pathway across NK clusters or disease state (see GSEA analysis in Figure 2G-H). (B-F) A spectral flow cytometry analysis of NK cells from 49 NDMM and 21 HD thawed BM samples was performed. (B) UMAP showing the identification of 5 main NK subsets by unsupervised clustering. (C-D) UMAP (C) and graphs (D) showing differences in cluster distributions among HD and NDMM. (E) Heat map showing the expression of the indicated markers by the different NK cell clusters. (F) FACS plots and graphs showing the mean fluorescence intensity (MFI) of the indicated markers by the different NK cell clusters. (G) Thawed PB HD or NDMM NK cells were incubated with P815 coated with anti-CD16, anti-NCR (NKp30, NKp44, NKp46), or control immunoglobulin G (IgG) for 6 hours. Representative FACS plots (G) and graphs (H) showing the expression of CD107a degranulation marker and the intracellular production of IFN-γ. (I-J) Thawed PB HD or NDMM NK cells were incubated with the indicated MM cell lines coated or not with isatuximab for 6 hours. Representative FACS plots and graphs showing the expression of CD107a degranulation marker by NK cells (I) and tumor cell apoptosis (J) Each dot represents an independent donor. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ANOVA (analysis of variance) with Tukey post hoc test analysis. ISA, isatuximab; NS, nonstimulated.