Extended Data Fig. 2: CREM is induced by CAR-CD3ζ and interleukin 15 (IL-15) stimulation.
From: CREM is a regulatory checkpoint of CAR and IL-15 signalling in NK cells
(a) Transduction efficiency of CAR70, CAR70.3ζ.Y6F and CD27 ECD NK cells; ECD: extracellular domain; (b) Whole cell lysates from NT, CAR70, CAR70.3ζ.Y6F and CD27 ECD NK cell groups were analyzed by western blot for phospho-CD3ζ (Y142; pCD3ζ). NK cells were unstimulated (−) or stimulated (+) with CD70 antigen (Ag) for 30 min. β-actin was used as loading control. Representative blot is shown; (c) Densitometry analysis quantifying the relative band intensity of CAR-specific pCD3ζ normalized to loading control (n = 3 donors); (d) CREM expression in NK cells that were either unstimulated or stimulated with IL-2 or IL-15 at increasing concentrations (50, 500, and 5000 pg/ml) for 24 h as assessed by qPCR (n = 3 donors); (e) CREM expression in NK cells stimulated with IL-15 (500 pg/ml) for 24 h in the presence or absence of an IL-15 antagonist (IL-15 Ab) as assessed by qPCR (n = 4 donors); (f) CREM expression in NT and CAR70 NK cells that were either unstimulated or stimulated with either IL-15 (500 pg/ml) or CD70 antigen (Ag) or both for 24 h as assessed by qPCR (n = 3 donors); (g) Representative FACS plot of CREM and IL-15R expression in NK cells stimulated with IL-15 (5000 pg/ml) for 24 h compared to unstimulated cells; (h) Percentage CREM expression in IL-15R+ vs. IL-15R- NK cells stimulated with increasing concentrations of IL-15 (n = 3 donors); (i,j) Longitudinal analysis of CREM (i) and Ki67 (j) expression in NK cells following stimulation with IL-15 (5000 pg/ml), assessed by flow cytometry (n = 3 donors); gMFI FC: geometric mean fluorescence intensity fold change; (k) Longitudinal analysis of CREM expression in NK cells following stimulation with IL-15 (5000 pg/ml), as assessed by qPCR (n = 6 donors); (l) CREM expression in NT and CAR70/IL-15 NK cells both 48 h post-transduction (Before) as well as one week later following expansion with universal antigen presenting cells (uAPCs) and IL-2 (After) as assessed by qPCR (n = 2 donors). ns: non-significant. Statistical comparisons were performed using one-way ANOVA with Tukey correction (a,c,e), two-way ANOVA with Tukey correction (d), and two-way ANOVA (Fisher’s LSD test, f,h,i,j,k,l). Data are represented as mean ± SEM.
