优化 CAR-NK 细胞的转导和扩增:利用细胞因子调节提高性能

Tiziano Ingegnere, Benjamin Segain, Adeline Cozzani, Mattias Carlsten, Suman Mitra, Silvia Gaggero
{"title":"优化 CAR-NK 细胞的转导和扩增:利用细胞因子调节提高性能","authors":"Tiziano Ingegnere,&nbsp;Benjamin Segain,&nbsp;Adeline Cozzani,&nbsp;Mattias Carlsten,&nbsp;Suman Mitra,&nbsp;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. 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.</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":null,"pages":null},"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,&nbsp;Benjamin Segain,&nbsp;Adeline Cozzani,&nbsp;Mattias Carlsten,&nbsp;Suman Mitra,&nbsp;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. 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.</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\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-10-30\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Current protocols\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.70040\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.70040","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

摘要

细胞免疫疗法已成为治疗癌症患者最有效的方法之一。嵌合抗原受体(CAR)T 细胞的适应性转移以及单倍体自然杀伤(NK)细胞的使用可诱导淋巴瘤和白血病患者病情缓解。虽然 CAR T 细胞的应用已经确立,但由于存在严重不良事件的风险,包括细胞因子释放综合征和免疫效应细胞相关神经毒性综合征,目前这种方法的广泛应用受到限制。此外,如果不采用先进的 CRISPR 工程,异体 T 细胞输注引发移植物与宿主反应的风险也限制了自体 CAR T 细胞的使用。相比之下,基于 NK 细胞的癌症免疫疗法已成为一种安全的方法,即使在异体治疗中也是如此。然而,用T细胞修饰常用的水泡性口炎病毒G糖蛋白(VSV-G)伪型慢病毒高效转导原代血液NK细胞仍具有挑战性。本文介绍了一种详细的方法,通过在细胞因子补充培养基中进行短期培养,大大提高了 NK 细胞的转导效率。它还包括制备高滴度和高质量的慢病毒颗粒,以实现最佳的 NK 细胞转导。总之,本方案详细介绍了在细胞因子补充培养基中逐步培养 NK 细胞、用 VSV-G 慢病毒载体转导 NK 细胞以及随后扩增 NK 细胞进行功能测试的过程。© 2024 Wiley Periodicals LLC.基本方案 1:从人外周血单核细胞(PBMCs)中分离 NK 细胞基本方案 2:扩增 NK 细胞并用慢病毒转导生成 CAR-NK 细胞辅助方案 1:质粒扩增辅助方案 2:慢病毒制备辅助方案 3:慢病毒滴定
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
4.00
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信