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. Author manuscript; available in PMC: 2017 Nov 15.
Published in final edited form as: Nat Biotechnol. 2017 May 15;35(8):765–772. doi: 10.1038/nbt.3860

Figure 3.

Figure 3

Protection of HLA-E-expressing cells from NK cell-mediated lysis. (a) Schematic representation of the hematopoietic differentiation protocol used for Elf-1 ESCs. Hematopoietic cells were harvested from day 24 to day 58. (b) Representative CD45 expression on day 38 suspension cells produced by B2M-/Etrimer ESC clone c5 as measured by flow cytometry (y axis, 7AAD; x axis, isotype control or CD45). Results for other B2M-edited lines are shown in Supplementary Figure 4A. (c) Flow cytometry of HLA-E and HLA-BC expression in ESC-derived CD45+ cells with the indicated B2M genotypes. (d) Expression of inhibiting and activating receptors NKG2A and NKG2C on NK cells derived from a healthy donor (donor 1). Isotype and specific antibody tracings shown in red and blue respectively. Percents shown are calculated by subtracting corresponding isotype control frequencies. (e) Chromium release assay results obtained with ESC-derived CD45+ cells of the indicated B2M genotype and normal NK cells (donor 1) at the indicated ratios (mean + SD, n=3). The p values (one-way ANOVA test) were <0.002 at all cell ratios. Asterisks indicate pair-wise comparisons with B2M+/+ cells that had p<0.05(*) or <0.01(**) after applying the post hoc Tukey HSD test. (f) Chromium release assays performed as in (e) in the presence of neutralizing antibodies against either HLA-E or NKG2A at a NK/CD45+ cell ratio of 10:1 (mean + SD, n=3). P values were calculated using the one-way ANOVA followed by Tukey HSD test; asterisks indicate statistical significant differences in regard to no Ab controls. (g) Luciferase imaging of 5 representative mice containing ESC-derived CD45+ cells of B2M-/Edimer(pre- Cre) (serves as class I-negative control) or B2M-/Etrimer genotypes, some of which also received NK-92 cells (− or + labels in the right panel). Results are shown for the same mice before NK-92 injection (Day 1) and one day afterwards (Day 2). (h) Quantitation of luciferase-expressing CD45+ cell levels in mice treated as in (g). The change in luminescence between Day 1 (pre NK-92 cell injection) and Day 2 or Day 4 (1 and 3 days after NK-92 cell injection) was measured for individual mice and divided by the average of control mice that did not receive NK-92 cells (n = 3 for both B2M-/Etrimer and B2M-/Edimer (pre-Cre) control groups). Horizontal black bars indicate the mean for each group. P values were calculated using the unpaired Student’s t test. For all panels, ** = p<0.01, and * = p<0.05.