Extended Data Fig. 7: IL-15 and CD16, NKp30, and NKp46 stimulations of NK cells lead to CREB phosphorylation; and CREM regulation by STAT3 and STAT5.
From: CREM is a regulatory checkpoint of CAR and IL-15 signalling in NK cells
(a) Representative FACS plots of NK cells showing pCREB levels under the denoted conditions; (b) pCREB expression by phospho-flow cytometry in NK cells that were either untreated (−) or treated (+) with plate-bound anti-CD16, anti-NKp30, or anti-NKp46 for stimulation for 30 min, the PKA inhibitor H89, or the calcium chelator EGTA as indicated prior to profiling (n = 3 donors); (c) PKA activity assessed in whole cell lysates from NT NK cells that were either unstimulated or stimulated for 30 min with increasing concentrations of IL-15 (n = 3 donors); (d,e) pCREB expression by phospho-flow cytometry in NT NK cells that were either unstimulated or stimulated for 30 min with increasing concentrations of IL-15 (50, 500, and 5000 pg/ml) in the presence or absence of the PKA inhibitor H89 for up to 2 h prior to stimulation (n = 5 donors); FSK: forskolin; gMFI FC: geometric mean fluorescence intensity fold change; control: unstained; (f) pCREB expression by phospho-flow cytometry in NT NK cells that were either unstimulated or stimulated for 30 min with increasing concentrations of IL-15 in the presence or absence of the calcium chelator EGTA for up to 2 h prior to stimulation (n = 3 donors); (g) CREM expression as assessed by qPCR in NT NK cells that were either unstimulated or stimulated for 24 h with IL-15 (500 pg/ml) in the presence or absence of EGTA during the stimulation time (n = 3 donors); (h,i) Whole cell lysates from NT NK cells following 30 min stimulation with increasing doses of IL-15 as analyzed by western blot for phospho- and total STAT3 (h) and STAT5 (i) with β-actin serving as a loading control (n = 3 donors); (j,k) Whole cell lysates from NT, CAR70/IL-15, and CAR70 NK cells following stimulation of the CAR for 30 min as analyzed by western blot for phospho- and total STAT3 (j) and STAT5 (k) with β-actin serving as a loading control (n = 2 donors); (l,m) Whole cell lysates from NK cells that were either wild-type (WT) or knockout (KO) for STAT3 (l) or STAT5a/STAT5b (m) as analyzed by western blot for total STAT3 (l) and STAT5 (m) with β-actin serving as a loading control for STAT3 (l) and STAT5a (m) gels (n = 3 donors); (n) CREM expression in NK cells that were either WT, STAT3 KO, STAT5a/b KO, or CREM KO that were either unstimulated or stimulated with IL-15 (5000 pg/ml), assessed by qPCR (n = 5 donors); (o,p) ChIP-qPCR for the enrichment for pSTAT3 (o) and STAT5b (p) in the promoter region of CREM as well as a positive control promoter (STAT3 for both) in NT NK cells incubated for 1 h in the absence or presence of IL-15 (n = 4 donors). ns: non-significant; unstim: unstimulated. Statistical comparisons were performed using two-way ANOVA with Tukey correction (b,n), one-way ANOVA (Fisher’s LSD test, c), multiple t-tests with Holm-Šídák correction (e,f), one-way ANOVA with Tukey correction (g), and t-tests (o,p). Data are represented as mean ± SEM.
