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Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4? CAR from HIV-1 infection and alleviates impairment of cell proliferation.

ABSTRACT: Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4? CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4? on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4?-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4? as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4?-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4?. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4?. In addition, the number of CD4?-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4?-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.

SUBMITTER: Kamata M

PROVIDER: S-EPMC4686265 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation.

Kamata Masakazu M Kim Patrick Y PY Ng Hwee L HL Ringpis Gene-Errol E GE Kranz Emiko E Chan Joshua J O'Connor Sean S Yang Otto O OO Chen Irvin S Y IS

Biochemical and biophysical research communications 20150519 3

Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hy ...[more]

PMID: 25998390

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Project description:CD8+ and CD4+ T cells from HIV infected patients with HIV-RNA viremia of >50 copies/ml and CD8+ and CD4+ T cells from healthy controls were isolated by negative selection (Miltenyi Biotech, Auburn, CA). Cell sorting of the CD4 and CD8 T cell subsets were performed based on surface staining of CD3+CD8+ or CD3+CD4+ and na ve CD45RA+CD27+CD127highHLA-DRlow, and CD3+CD8+ or CD3+CD4+ and memory CD45RA-CD27+CD127highHLA-DRlow. Sorted cell populations were spun down and stored as dry pellets at -80M-BM-0C. Samples analyzed by transcript levels of genes related to cytokine signaling were determined by the JAK/STAT Signaling Pathway microarray. CD8+ and CD4+ T cells from HIV infected patients with HIV-RNA viremia of >50 copies/ml and CD8+ and CD4+ T cells from healthy controls were isolated by negative selection (Miltenyi Biotech, Auburn, CA). Cell sorting of the CD4 and CD8 T cell subsets were performed based on surface staining of CD3+CD8+ or CD3+CD4+ and naM-CM-/ve CD45RA+CD27+CD127highHLA-DRlow, and CD3+CD8+ or CD3+CD4+ and memory CD45RA-CD27+CD127highHLA-DRlow. Sorted cell populations were spun down and stored as dry pellets at -80M-BM-0C. Samples analyzed by transcript levels of genes related to cytokine signaling were determined by the JAK/STAT Signaling Pathway microarray (http://www.sabiosciences.com/rt_pcr_product/HTML/PAHS-039A.html, SABiosciences Frederick, MD). Briefly, total RNA was harvested from each individual T cell subset from each patient or healthy control and contaminating DNA was digested with DNase. Messenger RNA was converted to cDNA and loaded onto PCR array plates for quantitative real-time PCR. Quantification of transcript levels was determined by normalizing to 5 housekeeping genes from each individual sample. Relative gene expression levels for each subset were averaged and compared between cell populations from the patient group and healthy controls. Because of the multiple comparisons only p values M-bM-^IM-$ 0.01 were considered significant.

Dataset Information

Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4? CAR from HIV-1 infection and alleviates impairment of cell proliferation.

Publications

Ectopic expression of anti-HIV-1 shRNAs protects CD8(+) T cells modified with CD4ζ CAR from HIV-1 infection and alleviates impairment of cell proliferation.

Similar Datasets

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