Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures

In an experimental study using the CRISPR/Cas9 system, “enhanced” NK cell lines with knockout of CISH , the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out β2-microglobulin gene, which provides membrane exposure of MHC class I, were obtaine...

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Bibliographic Details
Published in:	Molecular biology (New York) Vol. 56; no. 5; pp. 770 - 779
Main Authors:	Yusubalieva, G. M., Dashinimaev, E. B., Gorchakov, A. A., Kulemzin, S. V., Brovkina, O. A., Kalinkin, A. A., Vinokurov, A. G., Shirmanova, M. V., Taranin, A. V., Baklaushev, V. P.
Format:	Journal Article
Language:	English
Published:	Moscow Pleiades Publishing 01-10-2022 Springer Nature B.V
Subjects:	Biochemistry Biomedical and Life Sciences Brain cancer Cell lines CIS protein CRISPR Cytotoxicity Flow cytometry Glioblastoma Glioblastoma cells Human Genetics Immunotherapy Life Sciences Major histocompatibility complex Molecular Cell Biology Natural killer cells β2 Microglobulin glioblastoma multiforme NK cells YT CISH β2-microglobulin adoptive immunotherapy
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Summary:	In an experimental study using the CRISPR/Cas9 system, “enhanced” NK cell lines with knockout of CISH , the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out β2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1 + overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH –/– or B2M –/– knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH –/– has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1 + -CISH –/– and YT-VAV1 + B2M –/– lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1 + line. The lines of NK-like cells obtained may serve as a prototype for the creation of “enhanced” allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.
ISSN:	0026-8933 1608-3245
DOI:	10.1134/S0026893322050156