Extended Data Fig. 8: CREM KO improves CAR-NK cell function in multiple tumor models.
From: CREM is a regulatory checkpoint of CAR and IL-15 signalling in NK cells
(a,b) Spheroid killing assay of GFP+ UMRC3 cells by CREM WT NT, CREM WT CAR70/IL-15, and CREM KO CAR70/IL-15 NK cells at an E:T ratio of (a) 1:1 and (b) 2:1 (n = 3 donors per group; the assays at the different E:T ratios were performed as part of the same experiment using the same donors as the ones used in Fig. 3a). The spheroid green image mean determines the spheroid growth over time. Data were normalized to the spheroid green image mean at T = 0 when the spheroid was formed and before adding the NK cells (black arrow); the bar graphs show the area under the curve (AUC) for the normalized spheroid green image mean (n = 3 donors); (c) Spheroid killing assay of GFP+ BCX.010 cells by CREM WT NT, CREM WT CAR70/IL-15, and CREM KO CAR70/IL-15 NK cells (n = 2 donors). Data were quantified and normalized as in (a,b); (d) Impedance killing assay of SKOV3 cells by CREM WT NT, CREM WT CAR.TROP2/IL-15, and CREM KO CAR.TROP2/IL-15 NK cells over time by xCELLigence device (n = 2 donors). The cell index which reflects tumor growth was normalized to the time of NK cell addition (black arrow); (e) KO efficiency of CREM in NT, IL-15, CAR70/IL-15 and CAR70 NK cells as assessed by PCR followed by gel electrophoresis; (f) Histograms of CD70 expression by flow cytometry in CD70 WT vs. CD70 KO UMRC3 cells; FMO: fluorescence minus one (negative control); (g) Impedance killing assay of CD70 WT or CD70 KO UMRC3 cells by NT, IL-15, CAR70/IL-15, or CAR70 NK cells that are either CREM WT or CREM KO (n = 2 donors). The cell index which reflects tumor growth was normalized to the time of NK cell addition (black arrow); (h) Impedance killing assay of UMRC3 cells by CREM WT or CREM KO NT NK cells that were either unstimulated or stimulated by incubation with increasing concentrations of IL-15 (50, 500, or 5000 pg/ml) for 24 h prior to adding them to UMRC3 cells plated the day before in xCELLigence device (n = 3 donors). The cell index which reflects tumor growth was normalized to the time of NK cell addition (black arrow). ns: non-significant. Statistical comparisons were performed using one-way (a-right,b-right) or two-way ANOVA with Tukey correction (a-left,b-left,c,d,g,h), Data are represented as mean ± SEM.
