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. 2021 Jan 29;11:586168. doi: 10.3389/fimmu.2020.586168

Figure 1.

Figure 1

Knockout of B2M and simultaneous overexpression of sc-HLA-E in primary NK cells (A) Outline of the lentiviral expression cassettes used for CRISPR/Cas9 mediated B2M-knockout and expression of the sc-HLA-E molecule. Cas9 and gB2M are expressed from the same lentiviral vector, driven by an EFS and an U6 promoter, respectively. The HLA-E*03:01 heavy chain is linked to B2M and the HLA-G leader peptide by G4S linkers and expression is driven from an SFFV promoter. (B) Knockout of B2M in SKM1 cells abrogates surface expression of HLA class I detected via flow cytometry on day 10 after transduction. (C) Frequencies of HLA class I and sc-HLA-E expressing primary NK cells 4 days after transduction with lentiviral particles encoding gB2M and Cas9, sc-HLA-E or a combination of both compared to an untransduced control. (D) Frequencies of modified cells measured on day 4, 7 and 14 after transduction (mean ± SD; n = 6 for sc-HLA-E, n = 5 for sc-HLA-E/B2M-KO and B2M-KO). (E) Growth of NK cell cultures measured 4, 7 and 14 days after transduction. Shown is the cumulative cell count of all NK cells within the culture. The NK cells showed a fold-expansion from the day of transduction of 15.2 ± 5.72 for parental, 9.36 ± 1.29 for sc-HLA-E and 6.74 ± 0.31 for sc-HLA-E/B2M-KO NK cells (mean ± SD; n = 3).