Production and Characterization of SIV-Specific CAR/CXCR5 T Cells

Abstract

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.