Journal: PLoS Pathogens
Article Title: A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes
Figure Lengend Snippet: ALT-803 induces HIV transcription from both a primary cell model of latency and from ex vivo patient samples. Primary CD4 + T-cells from healthy donor PBMC were activated, infected with a luciferase reporter HIV virus, and allowed to return to a resting state. This is similar to a post-activation latency model that has been previously described [ 17 ] with additional minor modifications (see Methods ). After 1 week, infected cells were treated with the agents at concentrations indicated for 2 days. A. Shown are luminescence values from luciferase, indicating dose-dependent HIV reactivation by each of the compounds tested. B. Compound associated cytotoxicity was determined in latently-infected cells in parallel with the virus activation assay using Cell Titer Glo (CTG) Dose dependent cytotoxicity was observed with SAHA and romidepsin but not with IL-15 or ALT-803. C and D. PBMC were isolated from HIV-infected subjects who had been suppressed by ART for at least 2 years. Cultures were maintained in the presence of ARVs. Either 1 nM of ALT-803 or 5 ng/ml PMA + 500 ng/ml ionomycin were added for 7 days. HIV activation was measured by quantitating viral RNA in cell-free supernatant using COBAS qPCR. C. The geometric mean of viral copies/ml for each donor are indicated for control (DMSO) and ALT-803 treated wells, following 7 days of treatment. D . The fold HIV activations of all donors are plotted as the ratio of the viral copies/ml for ALT-803 treated or PMA/ionomycin treated to control wells. These results indicate that ALT-803 reactivates virus from the natural patient reservoir, though to a lesser degree than PMA/ionomycin. E-I. Cryopreserved CD4 + T-cells from ARV-treated HIV-infected subjects were thawed, CFSE-labeled, and treated with either 1 nM ALT-803 or 200 ng/ml of PHA for 7 days in the presence of ARVs. E . Activation of CD4 + T-cells within PBMCs was measured at day 7 by flow cytometry, gating on CD3 + CD4 + T-cells and assessing CD69 expression. Each line represents a different subject. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. F . Proliferation of CD4+ T-cells within PBCs was measured at day 7 by flow, gating on CD3 + CD4 + T-cells and assessing proliferation as %CFSE dim cells. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. G-I. Cell associated DNA was isolated from purified CD4 + T-cells following 6 days of stimulation. Absolute copies of HIV-Gag and RPP30 were determined by droplet digital PCR and used to calculate copies of HIV per 10 6 CD4 + T-cells. Shown are results from three different subjects. Quantifications were determined in triplicate ( G ) or quadruplicate ( H I ). Means and SEM are shown and the P value was calculated by one-way ANOVA with Holm-Sidak’s multiple comparison test.
Article Snippet: Productively-infected cell killing assays Primary CD4+ T-cells were enriched from PBMC by negative selection (Easysep, Stemcell Technologies) and then activated for 48 hours with 1 μg/ml each of anti-CD3 (OKT3, ebioscience) and anti-CD28 (CD28.2, ebioscience) in RPMI-10 supplemented with 50 U/ml IL-2.
Techniques: Ex Vivo, Infection, Luciferase, Activation Assay, CTG Assay, Isolation, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cytometry, Expressing, Purification, Digital PCR