Disulfiram reactivates latent HIV-1 in a Bcl-2-transduced primary CD4+ T cell model without inducing global T cell activation

Abstract

Highly active antiretroviral therapy (HAART) can reduce plasma HIV-1 levels to below the detection limit. However, due to the latent reservoir in resting CD4(+) cells, HAART is not curative. Elimination of this reservoir is critical to curing HIV-1 infection. Agents that reactivate latent HIV-1 through nonspecific T cell activation are toxic. Here we demonstrate in a primary CD4(+) T cell model that the FDA-approved drug disulfiram reactivates latent HIV-1 without global T cell activation. The extent to which disulfiram reactivates latent HIV-1 in patient cells is unclear, but the drug alone or in combination may be useful in future eradication strategies.

Figures

Fig. 1.

Screening of small-molecule libraries identifies disulfiram as an agent that reactivates latent HIV-1. (A) Summary of screening results from the MicroSource Spectrum library. The results were normalized to the response to 10 ng/ml phorbol myristate acetate. (B) Chemical structures of disulfiram and its metabolites, DDTC and DDTC-Me. (C) Representative flow cytometry data sets for untreated, disulfiram-treated, and anti-CD3 plus anti-CD28 antibody-treated cells. The dot plots in the first row are forward scatter-side scatter (FSC-SSC) plots. Histographs in the second row show cells gated in R1 in FSC-SSC plots. The GFP-FL2 dot plots in the third row show viable cells gated in R2 in propidium iodide histographs. Cells that appear in R3 (GFP+) are reactivated latently infected cells. The percentage of GFP+ cells was calculated based on the number of cells in R3 divided by the number of cells in R2.

Fig. 2.

(A) Effects of disulfiram on latently infected Bcl-2-transduced CD4+ T cells from different donors. (B) Kinetic profile for the reactivation of latent HIV-1 by different concentrations of disulfiram. (C) Effects of disulfiram and costimulation on levels of HIV-1 transcripts in latently infected Bcl-2-transduced cells after 16 h of treatment. The fold change is shown relative to levels in untreated cells. Data are means plus standard deviations of triplicate samples from 1 of 2 independent experiments, both of which produced similar results. (D) Kinetic profile of the effect of 0.5 μM disulfiram on levels of HIV-1 transcripts in latently infected Bcl-2-transduced cells. The fold change is shown relative to levels in untreated cells. Data are means plus standard deviations of triplicate samples from 1 of 2 independent experiments, both of which produced similar results. (E) Comparison of disulfiram, DDTC, and DDTC-Me for reactivating latent HIV-1. (F) Toxicity of disulfiram and DDTC treatment, measured in a propidium iodide exclusion assay.

Fig. 3.

Disulfiram does not cause CD4+ T cell activation. (A) Effects of disulfiram and anti-CD3 plus anti-CD28 treatment on the sizes of resting CD4+ T cells. Cell size was measured by flow cytometry based on forward scatter at 24 h and 48 h. (B) Effects of disulfiram and DDTC on the expression of activation markers in primary resting CD4+ T cells, compared to the effect of anti-CD3 plus anti-CD28 costimulation. Data are means plus standard deviations of triplicate samples from 1 of 2 independent experiments. (C) Effects of disulfiram and DDTC on global DNA and RNA levels in primary resting CD4+ T cells, compared to the effect of anti-CD3 plus anti-CD28 costimulation. Hoechst 33342 and pyronin Y were used to stain DNA and RNA, respectively, and results were read using a flow cytometer.