Additional file 3 of CIS deletion by CRISPR/Cas9 enhances human primary natural killer cell functions against allogeneic glioblastoma

Additional file 3: Supplementary Fig. 1. Effect of CIS deletion on NKC receptor expression on the expanded NKCs. a Representative histogram of NK mock and NK dCIS analyzed by flow cytometry for NK activating and inhibitory receptors. Seven representative activating and inhibitory receptors are shown. The histograms were gated by the CD56-positive fraction. Blue, red, and gray histograms represent NK dCIS, NK mock, and negative background (NB) cells, respectively. b Graph depicts normalized MFI. Blue and red bars indicate NK mock and NK dCIS, respectively. Data are the mean ± SD, n = 4. The significance of differences was determined by the t-test or Mann-Whitney U test. n.s.: not significant, *P&lt; 0.05. Data are from at least two independent experiments. Supplementary Fig. 2. Induction of NK dCIS from two independent volunteers. (Left and right) NK dCIS data from Volunteer 1 and 2, respectively. (Top) Graph shows the NKC expansion ratio 3, 5, and 7 days after electroporation. Data are the mean ± SD (n = 5). The significance of differences was determined by one-way ANOVA followed by Tukey’s test. n.s.: not significant, **P &lt;0.01, *P &lt; 0.05. (Middle and bottom) Growth inhibition assays of NK mock and NK dCIS on T98G (middle) and U251MG cells (bottom). E:T ratios were 0.5:1 and 1:1 (0.5 × 106:1 × 106 and 1 × 106:1 × 106), respectively. Red, pink, green, light blue, and dark blue lines indicate GBM cells only (E:T = 0:1), NK mock (E:T = 0.5:1), NK dCIS (E:T = 0.5:1) NK mock (E:T = 1:1), and NK dCIS (E:T = 1:1), respectively. Data are the mean ± SD (n = 3–4). Supplementary Fig. 3. CIS mRNA expression in the CIS protein-deleted NKCs. Graph shows mRNA expression in the CIS protein-deleted NKCs extracted from microarray data. Blue and red graphs denote NK mock and NK dCIS, respectively. Data are the mean ± SD (n = 3). The significance of differences was determined by the t-test. *P&lt; 0.05. Supplementary Fig. 4. The influence of CIS expression and OT/OF effects in NK dCIS 14 days after electroporation of CIS exon 4 targeting-RNP. a Western blot analysis of CIS protein expression (top) and GAPDH expression (bottom) in NK mock and NK dCIS 14 days after RNP electroporation. CIS protein position in electrophoresis is indicated at 32 and 37 kDa. b OT (top) and OF (bottom) effects in in NK mock and NK dCIS 14 days after RNA electroporation. Right lanes depict 100-bp marker DNA. Supplementary Fig. 5. Representative mRNA expression values in NK mock and NK dCIS obtained by Clariom™ S array microarray. The mRNA expression of the receptors and cytotoxic granules whose protein expression (in parentheses) was obtained by flow cytometric analysis are as follows: IL-2RA (CD25), IL2RB (CD122), IL2RG (CD123), GZMB (granzyme B), PFR1 (perforin), ITGAL (CD11a), CD226 (DNAM-1), KLRK1 (NKG2D), NCR1 (NKp46), NCR2 (NKp44), NCR3 (NKp30), FCGR3A (CD16), PDCD1 (PD1), LAG3 (LAG3), HAVCR2 (TIM3), TIGIT (TIGIT), CD96 (CD96), and KLRC1 (NKG2A). The mRNA signal intensity (log2) was corrected by calculating the characteristic control signal values. The significance of differences was determined by the t-test. n = 3, **P &lt; 0.01. All data were from at least two independent experiments.附加文件3：补充图1。CIS缺失对扩增NK细胞表面NK细胞受体表达的影响。a 流式细胞术（Flow Cytometry）检测NK mock与NK dCIS细胞的NK激活型与抑制型受体表达的代表性直方图。展示了7种典型的激活型与抑制型受体。所有直方图均以CD56阳性群设门。蓝色、红色与灰色直方图分别代表NK dCIS、NK mock与阴性背景（Negative Background, NB）细胞。b 归一化平均荧光强度（Mean Fluorescence Intensity, MFI）统计图。蓝色与红色柱形分别代表NK mock与NK dCIS组。数据以平均值±标准差表示（n=4）。差异显著性采用t检验（t-test）或曼-惠特尼U检验（Mann-Whitney U Test）进行分析。n.s.：无显著性差异，*P<0.05。所有实验至少独立重复2次。 补充图2：从2名独立健康志愿者中诱导获得NK dCIS细胞。（左、右）分别为志愿者1与志愿者2的NK dCIS相关实验数据。（上方）统计图展示了电穿孔（Electroporation）后第3、5、7天的NK细胞扩增倍数。数据以平均值±标准差表示（n=5）。差异显著性采用单因素方差分析（One-way Analysis of Variance, ANOVA）结合图基检验（Tukey's Test）进行分析。n.s.：无显著性差异，**P<0.01，*P<0.05。（中、下方）分别为NK mock与NK dCIS对T98G细胞（中）及U251MG细胞（下）的生长抑制实验。效靶比（Effector:Target, E:T）分别设置为0.5:1与1:1（对应细胞数为0.5×10^6:1×10^6与1×10^6:1×10^6）。红色、粉色、绿色、浅蓝与深蓝色曲线分别代表仅胶质母细胞瘤（Glioblastoma, GBM）细胞组（E:T=0:1）、NK mock组（E:T=0.5:1）、NK dCIS组（E:T=0.5:1）、NK mock组（E:T=1:1）与NK dCIS组（E:T=1:1）。数据以平均值±标准差表示（n=3~4）。 补充图3：CIS蛋白敲除NK细胞中的CIS mRNA表达水平。统计图展示了从基因芯片数据中提取的CIS蛋白敲除NK细胞的mRNA表达量。蓝色与红色曲线分别代表NK mock与NK dCIS组。数据以平均值±标准差表示（n=3）。差异显著性采用t检验（t-test）进行分析，*P<0.05。 补充图4：靶向CIS外显子4的核糖核蛋白（Ribonucleoprotein, RNP）经电穿孔转染NK细胞14天后，NK dCIS中CIS表达水平及OT/OF效应的影响。a 蛋白质印迹（Western Blot）分析RNP电穿孔转染14天后NK mock与NK dCIS细胞的CIS蛋白（上方）与GAPDH（甘油醛-3-磷酸脱氢酶，Glyceraldehyde-3-Phosphate Dehydrogenase, GAPDH，下方）表达水平。电泳中CIS蛋白的迁移位置对应32 kDa与37 kDa。b RNA电穿孔转染14天后NK mock与NK dCIS细胞的靶向（On-Target, OT）与脱靶（Off-Target, OF）效应。右侧泳道为100碱基对（bp）DNA分子量标记。 补充图5：通过Clariom™ S基因芯片微阵列检测获得的NK mock与NK dCIS细胞的代表性mRNA表达量。经流式细胞术分析获得蛋白表达的受体与细胞毒性颗粒对应的mRNA表达如下：IL-2RA（CD25）、IL2RB（CD122）、IL2RG（CD123）、GZMB（颗粒酶B，Granzyme B）、PFR1（穿孔素，Perforin）、ITGAL（CD11a）、CD226（DNAM-1）、KLRK1（NKG2D）、NCR1（NKp46）、NCR2（NKp44）、NCR3（NKp30）、FCGR3A（CD16）、PDCD1（PD1）、LAG3（LAG3）、HAVCR2（TIM3）、TIGIT（TIGIT）、CD96（CD96）及KLRC1（NKG2A）。通过计算特征性对照信号值对mRNA信号强度（log2转换后）进行校正。差异显著性采用t检验（t-test）进行分析，n=3，**P<0.01。所有实验至少独立重复2次。