Abstract

An important obstacle to a functional cure for HIV/AIDS is the persistence of viral reservoirs found throughout the body in various cells and tissues. Reservoirs can be latently infected cells, or in the case of follicular dendritic cells (FDC), non-infected cells that trap infectious virus on their surface through immune complexes (HIV-IC). Although several strategies have been employed to target and eliminate viral reservoirs, they are short-lived and ineffective. In an attempt to provide a long-term approach, chimeric antigen receptor T (CAR-T) cells were designed to eliminate native HIV on FDCs. Although effective at eliminating HIV-infected cells, and halting spreading infection, their ability to eliminate the viral reservoir found on (FDCs) remains unclear. We used a novel second-generation CAR-T cell expressing domains 1 and 2 of CD4 followed by the mannose binding lectin (MBL) to allow recognition of native HIV envelope (Env) to determine the capacity to respond to the viral reservoir found on FDCs. We employed a novel fluorescent lysis assay, the Carboxyfluorescein succinimidyl ester (CFSE) release assay, as well as flow cytometric based assays to detect functional CAR-T activation through IFN-γ production and CD107a (i.e., LAMP1) membrane accumulation to test cytolytic capacity and functional activation of CD4-MBL CAR-T cells, respectively. We demonstrated their efficacy at eliminating HIV-infected cells or cells expressing gp160. However, these CAR-T cells were unable to lyse cells bearing surface bound HIV-IC. We found that failed lysis was not a unique feature of a resistant target, but a limitation in the CAR-T recognition elements. CAR-T cells were inactive in the presence of free HIV or in the presence of concentrated, immobilized virus. Further experiments determined that in addition to gp120 recognition by the CAR-T, the adhesion molecule ICAM-1 was necessary for efficient CAR-T cell killing of HIV-infected cells. CAR-T cell activity and killing were inhibited in the presence of ICAM-1 blocking antibody. These results suggest that other factors, such as adhesion molecules, play a vital role in CAR-T responses to HIV-infected cells. In addition, our findings highlighted the necessity to consider all models of HIV reservoirs, including FDCs, when evaluating therapeutic efficacy.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

http://lib.byu.edu/about/copyright/

Date Submitted

2017-12-01

Document Type

Dissertation

Handle

http://hdl.lib.byu.edu/1877/etd9695

Keywords

HIV, chimeric antigen receptor, follicular dendritic cells

Language

english

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