{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T09:18:40Z","timestamp":1775639920951,"version":"3.50.1"},"reference-count":54,"publisher":"MDPI AG","issue":"22","license":[{"start":{"date-parts":[[2023,11,8]],"date-time":"2023-11-08T00:00:00Z","timestamp":1699401600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Stefan Morsch Stiftung","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"Clinician Scientist Program","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"F\u00f6rderverein f\u00fcr krebskranke Kinder T\u00fcbingen e.V., MaxCyte Inc.","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"University Children\u2019s Hospital of T\u00fcbingen","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]},{"name":"Open Access Publishing Fund of the University of T\u00fcbingen","award":["440-0-0"],"award-info":[{"award-number":["440-0-0"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.<\/jats:p>","DOI":"10.3390\/ijms242216065","type":"journal-article","created":{"date-parts":[[2023,11,8]],"date-time":"2023-11-08T01:54:30Z","timestamp":1699408470000},"page":"16065","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells"],"prefix":"10.3390","volume":"24","author":[{"given":"Tahereh","family":"Mohammadian Gol","sequence":"first","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miso","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ralph","family":"Sinn","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guillermo","family":"Ure\u00f1a-Bail\u00e9n","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah","family":"Stegmeyer","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paul Gerhard","family":"Gratz","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fatemeh","family":"Zahedipour","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0775-5820","authenticated-orcid":false,"given":"Alicia","family":"Roig-Merino","sequence":"additional","affiliation":[{"name":"MaxCyte Inc., Rockville, MD 20850, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Justin S.","family":"Antony","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0211-2343","authenticated-orcid":false,"given":"Markus","family":"Mezger","sequence":"additional","affiliation":[{"name":"Department of General Pediatrics, Hematology and Oncology, University Children\u2019s Hospital T\u00fcbingen, 72076 T\u00fcbingen, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,11,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Du, N., Guo, F., Wang, Y., and Cui, J. 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