{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T09:02:02Z","timestamp":1781082122023,"version":"3.54.1"},"reference-count":8,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2024,10,30]],"date-time":"2024-10-30T00:00:00Z","timestamp":1730246400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,11]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell\u2010associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell\u2010based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV\u2010G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short\u2010term culture in cytokine\u2010supplemented medium. It also encompasses the preparation of high\u2010titer and high\u2010quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step\u2010by\u2010step culture of NK cells in cytokine\u2010supplemented medium, their transduction with VSV\u2010G lentiviral vectors, and subsequent expansion for functional assays. \u00a9 2024 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs)<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: NK cell expansion and transduction with lentivirus for generating CAR\u2010NK cells<\/jats:p><jats:p><jats:bold>Support Protocol 1<\/jats:bold>: Plasmid amplification<\/jats:p><jats:p><jats:bold>Support Protocol 2<\/jats:bold>: Lentivirus preparation<\/jats:p><jats:p><jats:bold>Support Protocol 3<\/jats:bold>: Lentivirus titration<\/jats:p>","DOI":"10.1002\/cpz1.70040","type":"journal-article","created":{"date-parts":[[2024,10,30]],"date-time":"2024-10-30T13:44:55Z","timestamp":1730295895000},"source":"Crossref","is-referenced-by-count":5,"title":["Optimizing CAR\u2010NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance"],"prefix":"10.1002","volume":"4","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3426-371X","authenticated-orcid":false,"given":"Tiziano","family":"Ingegnere","sequence":"first","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, UMR9020\u2010U1277 CANTHER  Lille France"},{"name":"These authors share first authorship"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin","family":"Segain","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, UMR9020\u2010U1277 CANTHER  Lille France"},{"name":"These authors share first authorship"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Adeline","family":"Cozzani","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, UMR9020\u2010U1277 CANTHER  Lille France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mattias","family":"Carlsten","sequence":"additional","affiliation":[{"name":"Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine Karolinska Institutet  Stockholm Sweden"},{"name":"Department of Cell Therapy and Allogenic Stem Cell Transplantation, Karolinska Comprehensive Cancer Center Karolinska University Hospital  Stockholm Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Suman","family":"Mitra","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, UMR9020\u2010U1277 CANTHER  Lille France"},{"name":"These authors share final authorship"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Silvia","family":"Gaggero","sequence":"additional","affiliation":[{"name":"Universit\u00e9 de Lille, CNRS, Inserm, CHU Lille, UMR9020\u2010U1277 CANTHER  Lille France"},{"name":"These authors share final authorship"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,10,30]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2015.00266"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.02873"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41573\u2010019\u20100051\u20102"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1080\/2162402X.2018.1470730"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1525\u20100016(16)33339\u20101"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.3389\/fimmu.2019.00957"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/0022\u20101759(86)90258\u20109"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2023.114689"}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70040","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,12,2]],"date-time":"2024-12-02T04:58:08Z","timestamp":1733115488000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70040"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,10,30]]},"references-count":8,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2024,11]]}},"alternative-id":["10.1002\/cpz1.70040"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70040","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,10,30]]},"article-number":"e70040"}}