Production and Characterization of SIV-Specific CAR/CXCR5 T Cells.
Production and Characterization of SIV-Specific CAR/CXCR5 T Cells.
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SIV 特异性 CAR/CXCR5 T 细胞的生产和表征。
DOI：
10.1007/978-1-0716-1944-5_12
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Skinner,PamelaJ
中科院分区：
文献类型：
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作者：
Pampusch,MaryS;Hajduczki,Agnes;Mwakalundwa,Gwantwa;Connick,Elizabeth;Berger,EdwardA;Skinner,PamelaJ
靶向淋巴滤泡的HIV特异性嵌合抗原受体（CAR）T细胞具有功能性治愈HIV感染的潜力。可以修饰CD 8 +T细胞、NK细胞或外周血单核细胞（PBMC）以表达HIV特异性汽车以及滤泡归巢分子如CXCR 5，以靶向在HIV感染期间集中在B细胞滤泡内的病毒感染的T滤泡辅助细胞。本章概述了利用猴免疫缺陷病毒（SIV）恒河猴模型的HIV产生转导的T细胞从原代PBMC的方法。提供了用于产生SIV特异性CAR/CXCR 5编码逆转录病毒的方法，所述逆转录病毒用于扩增原代恒河猴PBMC。还提供了在体外和离体评估扩增的CAR/CXCR 5 T细胞的功能性的程序。体外迁移测定确定表达CAR/CXCR 5的T细胞迁移到CXCR 5配体CXCL 13的能力，而离体迁移测定允许测量转导的T细胞迁移到B细胞滤泡中。使用病毒抑制测定法测定CAR/CXCR 5转导的T细胞的抗病毒活性。这些方法可用于在SIV感染的恒河猴中产生用于免疫治疗的T细胞，并在输注前评估细胞的功能。类似的程序可用于产生HIV特异性CAR/CXCR 5 T细胞。
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.