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< 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 <0.01, *P < 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< 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 < 0.01. All data were from at least two independent experiments.