Figure 1. A small molecule library screening approach identifies Cbl-b inhibitors enhancing the activity of primary human NK cells. (a) Experimental design of the small molecule library screening approach on primary human NK cells in co-culture with A549 β2m^−/− (created with BioRender.com). NK cells were enriched from healthy donors’ PBMCs and primed with a suboptimal concentration of IL-15 (0.2 ng/mL). Following compound treatment, NK cells were co-cultured with A549 β2m^−/− overnight. Screening hits were identified by measurement of IFN-γ secretion from supernatants. (b) Small molecule screening compounds target classes. (c) Volcano plot representing screening hits based on modified Z score value. Top four screening hits were identified based on a threshold of normalized modified Z score ≥3. (d) Heatmap of IFN-γ, granzyme B, and TNF-α concentrations for screening hits validation. Primary human NK cells from healthy donors PBMCs (n=3) were treated in dose response with the top four screening compounds and cytokines measured from the supernatants. (e) Representative flow cytometry plots for NK cell degranulation (CD107a) and NK cell cytotoxicity. NK cells from healthy donors PBMCs (n=9) were primed with a suboptimal concentration of IL-15 (0.2 ng/mL) and co-cultured with A549 β2m^−/− (CFSE+) at an E:T ratio of 2:1 for 6 hours (p values are from paired t-test). (f) CellTrace Violet dilution and statistics of primary human NK cells after treatment with Cbl-b inhibitor (p values are from paired t-test). (g) Volcano plot showing differentially expressed proteins on primary human NK cells after overnight treatment with Cbl-b inhibitor in the presence of a suboptimal concentration of 0.2 ng/mL IL-15 (n=6; gray=NS, green=log2 FC, blue=adjusted p value, red=log2 FC and adjusted p value. For statistical analyses see “Material and methods”). Cbl-b, Casitas B-lineage lymphoma; CFSE, carboxyfluorescein succinimidyl ester; DMSO, dimethylsulfoxide; E:T, effector to target; IFN, interferon; IL, interleukin; NK, natural killer; PBMCs, peripheral mononuclear cells; TLR, toll-like receptor; TNF, tumor necrosis factor.