Tiziano Ingegnere, Benjamin Segain, Adeline Cozzani, Mattias Carlsten, Suman Mitra, Silvia Gaggero
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In contrast, NK cell-based 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-G) 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-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs)</p><p><b>Basic Protocol 2</b>: NK cell expansion and transduction with lentivirus for generating CAR-NK cells</p><p><b>Support Protocol 1</b>: Plasmid amplification</p><p><b>Support Protocol 2</b>: Lentivirus preparation</p><p><b>Support Protocol 3</b>: Lentivirus titration</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 11","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Optimizing CAR-NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance\",\"authors\":\"Tiziano Ingegnere, Benjamin Segain, Adeline Cozzani, Mattias Carlsten, Suman Mitra, Silvia Gaggero\",\"doi\":\"10.1002/cpz1.70040\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. 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引用次数: 0
Optimizing CAR-NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance
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-associated 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-based 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-G) 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-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC.
Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs)
Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells
Support Protocol 1: Plasmid amplification
Support Protocol 2: Lentivirus preparation
Support Protocol 3: Lentivirus titration