Pharmacologic HIV-1 Nef blockade promotes CD8 T cell–mediated elimination of latently HIV-1–infected cells in vitro
Shariq Mujib, … , Thomas E. Smithgall, Mario A. Ostrowski
Shariq Mujib, … , Thomas E. Smithgall, Mario A. Ostrowski
Published September 7, 2017
Citation Information: JCI Insight. 2017;2(17):e93684. https://doi.org/10.1172/jci.insight.93684.
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Research Article AIDS/HIV Immunology

Pharmacologic HIV-1 Nef blockade promotes CD8 T cell–mediated elimination of latently HIV-1–infected cells in vitro

Abstract

Eradication of the HIV-1 latent reservoir represents the current paradigm to developing a cure for AIDS. HIV-1 has evolved multiple mechanisms to evade CD8 T cell responses, including HIV-1 Nef–mediated downregulation of MHC-I from the surface of infected cells. Nef transcripts and protein are detectable in samples from aviremic donors, suggesting that Nef expression in latently HIV-1–infected CD4 T cells protects them from immune-mediated clearance. Here, we tested 4 small molecule inhibitors of HIV-1 Nef in an in vitro primary CD4 T cell latency model and measured the ability of autologous ex vivo or HIV-1 peptide–expanded CD8 T cells to recognize and kill latently infected cells as a function of inhibitor treatment. Nef inhibition enhanced cytokine secretion by autologous CD8 T cells against latently HIV-1–infected targets in an IFN-γ release assay. Additionally, CD8 T cell–mediated elimination of latently HIV-1–infected cells was significantly enhanced following Nef blockade, measured as a reduction in the frequency of infected cells and Gag protein in cultures following viral outgrowth assays. We demonstrate for the first time to our knowledge that Nef blockade, in combination with HIV-specific CD8 T cell expansion, might be a feasible strategy to target the HIV-1 latent reservoir that should be tested further in vivo.

Authors

Shariq Mujib, Aamir Saiyed, Saleh Fadel, Ardalan Bozorgzad, Nasra Aidarus, Feng Yun Yue, Erika Benko, Colin Kovacs, Lori A. Emert-Sedlak, Thomas E. Smithgall, Mario A. Ostrowski

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Figure 2

Validation of the primary CD4 T cells in vitro latency model.

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Validation of the primary CD4 T cells in vitro latency model. (A) CD4 T ...
(A) CD4 T cells from an HIV-1–uninfected donor were exogenously infected with X4 tropic HIV-1 NL4-3 (top panel) or an R5 tropic primary HIV-1 isolate J32228M4 (bottom panel) to generate latently infected cells as described in the Methods. Latently HIV-1–infected cells were cultured in medium alone or with IL-15 for 6 days to induce virus production in the presence of HAART. HIV-1 Gag p24–producing cells were enumerated by flow cytometry. Representative flow cytometry plots and summary data from 8 experiments are shown; Wilcoxon matched pairs tests were performed to determine statistical significance. Latently infected CD4 T cells generated via this method were utilized for all further experiments. (B) Primary CD4 T cells were isolated and stimulated with anti-CD3 and anti-CD28 antibodies (1 μg/ml each) and 100 U/ml IL-2 for 2 days and subsequently infected with HIV-1 NL4-3 in the presence or absence of the HAART cocktail used in the study. The frequency of HIV-1–infected cells was determined via flow cytometry 5 days later. Cells receiving antiretrovirals were not permissive to infection. (C) Resting HIV-1–infected (bottom panel) or mock-infected (top panel) CD4 T cells were treated with each of the Nef inhibitors for a period of 4 days in medium with HAART and 0.5 ng/ml IL-7. Surface MHC-I expression was then analyzed via flow cytometry. The mean fluorescence intensity (MFI) of MHC-I, indicative of the molecules of MHC-I on the surface of CD4 T cells, for total mock-infected and only the HIV-1 Gag–expressing latently HIV-1–infected cultures (bottom panel) were determined. The MHC-I MFI ratio between the DMSO control (red lines; NT DMSO) to those receiving Nef inhibitors (blue lines) is reported as the MFI fold (MFI Inhibitor/MFI DMSO). Nef blockade did not alter MHC-I expression on uninfected cells. (D) Histograms depicting the MHC-I expression of the Gag+ and Gag– cells within the same culture with each inhibitor and DMSO control are shown and reported as the MHC-I ratio (MFI Gag+/MFI Gag–). Also shown is the percentage increase in the MHC-I ratio following Nef blockade relative to the DMSO control. Inhibitor treatment restored the MHC-I MFI ratio to a greater degree relative to the DMSO control samples. Overall, these results indicate that all 4 compounds restored MHC-I expression on latently HIV-1–infected CD4 T cells.