Effects of prostratin on T-cell activation and human immunodeficiency virus latency

Abstract

Human immunodeficiency virus (HIV) replication is linked to cellular gene transcription and requires target cell activation. The latent reservoir of HIV-1 in quiescent T cells is thought to be a major obstacle to clearance of infection by highly active antiretroviral therapy (HAART). Thus, identification of agents that can induce expression of latent virus may, in the presence of HAART, allow elimination of the infected cells by the immune response. We previously used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis. Latently infected mature thymocytes can be exported into the periphery, providing an efficient primary cell model to determine cellular activation signals that induce renewed expression of latent virus. Here we characterized the effects of prostratin, a non-tumor-promoting phorbol ester, on primary human peripheral blood lymphocytes (PBLs) and assessed its ability to reactivate latent HIV infection from thymocytes and PBLs in the SCID-hu (Thy/Liv) model. Prostratin stimulation alone did not induce proliferation of quiescent PBLs; however, it could provide a secondary signal in the context of T-cell receptor stimulation or a primary activation signal in the presence of CD28 stimulation to induce T-cell proliferation. While prostratin alone was not sufficient to allow de novo HIV infection, it efficiently reactivated HIV expression from latently infected cells generated in the SCID-hu mouse. Our data indicate that prostratin alone is able to specifically reactivate latent virus in the absence of cellular proliferation, making it an attractive candidate for further study as an adjunctive therapy for the elimination of the latent HIV reservoir.

FIG. 1.

Proliferative response of T-cells to prostratin. Highly purified quiescent T cells were cultured in the presence of the indicated concentrations of prostratin alone (⋄) or with the additional stimulation signals provided by anti-CD3 antibodies (□). At days 2, 3, and 4, cells were harvested in triplicate and assayed for thymidine incorporation as described in Materials and Methods. Data from day 3 poststimulation are presented. Results are the averages of triplicate wells. These results are representative of three experiments.

FIG. 2.

Expression levels of activation markers following stimulation with various concentrations of prostratin. Highly purified quiescent T cells were cultured in the presence of the indicated concentrations of prostratin alone (A), prostratin plus anti-CD3 (B), and prostratin plus costimulation with anti-CD3 and anti-CD28 (C). Following 2 days in culture, cells were stained and analyzed as described in Materials and Methods. The cells used for this experiment were the same as those used for the experiment illustrated in Fig. 1, and results are representative of three experiments.

FIG. 3.

(A) Induction of activation markers following stimulation with prostratin plus antibodies. Highly purified T cells were cultured under the indicated conditions in the absence (upper panels) or presence (lower panels) of 1 μM prostratin. Following 3 days in culture, cells were stained and analyzed as described in Materials and Methods. US, unstimulated; αCD3, stimulated with anti-CD3 antibodies; αCD28, stimulated with anti-CD28 antibodies; αCD3+αCD28, costimulated with anti-CD3 and anti-CD28. (B) Cell cycle progression following stimulation with prostratin. Highly purified T cells were cultured under the indicated conditions in the absence (upper panels) or presence (lower panels) of 1 μM prostratin. Following 3 days in culture, cells were stained and analyzed for DNA/RNA content as described in Materials and Methods. US, unstimulated; αCD3, stimulated with anti-CD3 antibodies; αCD28, stimulated with anti-CD28 antibodies; αCD3+αCD28, costimulated with anti-CD3 and anti-CD28. 7AAD, 7-amino-actinomycin D; PY, pyronin Y.

FIG. 4.

(A) Effect of prostratin on HIV latency. Upper panels, mock- and HIV_JR-CSF-infected thymocytes were harvested 5 weeks postinfection (day 0), stained with antibodies to CD4 and CD8, and analyzed by flow cytometry. Lower panels, thymocytes were then cultured without stimulation, costimulated with antibodies to CD3 and CD28, or cultured in the presence of 1 μM prostratin. p24 in the supernatant was assessed as described in Materials and Methods. The dotted line indicates the level of detection of the ELISA. (B) Effect of prostratin on HIV gene expression. NL-r-HSAs-infected CD4 SP thymocytes were isolated as described for Fig. 3 and cultured under the described conditions. Three days following stimulation, cells were stained with anti-mouse CD24 to detect HIV-encoded receptor gene expression. Gates were set according to mock-infected thymocytes cultured in parallel so that the background was <1%. Percentages indicate reporter expression and induction from latency.