Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN, USA.
Laboratory of Viral Diseases, NIAID, The National Institutes of Health, Bethesda, MD, USA.
Methods Mol Biol. 2022;2421:171-185. doi: 10.1007/978-1-0716-1944-5_12.
HIV-specific chimeric antigen receptor (CAR) T cells that target lymphoid follicles have the potential to functionally cure HIV infection. CD8 T cells, NK cells, or peripheral blood mononuclear cells (PBMC) may be modified to express HIV-specific CARs as well as follicular homing molecules such as CXCR5 to target the virally infected T follicular helper cells that concentrate within B cell follicles during HIV infection. This chapter outlines methods utilizing a simian immunodeficiency virus (SIV) rhesus macaque model of HIV to produce transduced T cells from primary PBMCs. Methods are presented for production of an SIV-specific CAR/CXCR5-encoding retrovirus used to transduce primary rhesus macaque PBMCs. Procedures to evaluate the functionality of the expanded CAR/CXCR5 T cells in vitro and ex vivo are also presented. An in vitro migration assay determines the ability of the T cells expressing CAR/CXCR5 to migrate to the CXCR5 ligand CXCL13, while an ex vivo migration assay allows measurement of the transduced T cell migration into the B cell follicle. Antiviral activity of the CAR/CXCR5 transduced T cells is determined using a viral suppression assay. These methods can be used to produce T cells for immunotherapy in SIV-infected rhesus macaques and to evaluate the functionality of the cells prior to infusion. Similar procedures can be used to produce HIV-specific CAR/CXCR5 T cells.
针对淋巴滤泡的 HIV 特异性嵌合抗原受体 (CAR) T 细胞有可能从功能上治愈 HIV 感染。CD8 T 细胞、NK 细胞或外周血单核细胞 (PBMC) 可被修饰以表达 HIV 特异性 CAR 以及滤泡归巢分子，如 CXCR5，以靶向 HIV 感染期间集中在 B 细胞滤泡内的病毒感染的滤泡辅助 T 细胞。本章概述了利用 HIV 恒河猴猴免疫缺陷病毒 (SIV) 模型从原代 PBMC 产生转导 T 细胞的方法。介绍了用于转导原代恒河猴 PBMC 的 SIV 特异性 CAR/CXCR5 编码逆转录病毒的生产方法。还介绍了评估体外和体外扩增的 CAR/CXCR5 T 细胞功能的程序。体外迁移测定确定表达 CAR/CXCR5 的 T 细胞向 CXCR5 配体 CXCL13 迁移的能力，而体外迁移测定允许测量转导的 T 细胞向 B 细胞滤泡中的迁移。通过病毒抑制测定来确定 CAR/CXCR5 转导的 T 细胞的抗病毒活性。这些方法可用于生产 SIV 感染恒河猴的免疫疗法 T 细胞，并在输注前评估细胞的功能。类似的程序可用于生产 HIV 特异性 CAR/CXCR5 T 细胞。
