Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines

Abstract

Although it has been demonstrated that certain cytokines, particularly proinflammatory cytokines, can enhance ongoing viral replication in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected individuals, it is unclear what role these cytokines play in the induction of HIV-1 replication in latently infected, resting CD4(+) T cells. This study demonstrates that the in vitro combination of the proinflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha together with the immunoregulatory cytokine IL-2 are potent inducers of viral replication in highly purified, latently infected, resting CD4+ T cells derived from HIV-infected individuals who are antiretroviral therapy-naive as well as those who are receiving highly active antiretroviral therapy (HAART). Viral replication induced by this combination of cytokines was completely suppressed in the presence of HAART in vitro. Given that an array of cytokines, including IL-6, TNF-alpha, and IL-2, are copiously expressed in the microenvironment of the lymphoid tissues, which harbor the latent viral reservoirs, induction of HIV by this combination of cytokines may in part explain the commonly observed reappearance of detectable plasma viremia in HIV-infected individuals in whom HAART was discontinued. Moreover, since it is likely that these infected cells die upon activation of virus and that HAART prevents spread of virus to adjacent cells, the observation that this combination of cytokines can markedly induce viral replication in this reservoir may have important implications for the activation-mediated diminution of the latent reservoir of HIV in patients receiving HAART.

Figure 1

Cytokine-induced cellular proliferation in latently infected, resting CD4⁺ T cells. Highly enriched (>99%) resting CD4⁺ T cells from one HIV-1–seronegative and two HIV-1–infected individuals were incubated with the indicated individual cytokines, a combination of cytokines, or anti-CD3 antibody for 6 d, and [³H]thymidine incorporation by 10⁴ resting CD4⁺ T cells was measured. A representative set of results from patient no. 2 is shown.

Figure 2

Induction of HIV-1 replication by the combination of IL-2, IL-6, and TNF-α in latently infected, resting CD4⁺ T cells from antiretroviral drug–naive patients. Resting CD4⁺ T cells from infected patients were isolated and further incubated with no cytokine, individual cytokines as indicated, the combination of IL-2, IL-6, and TNF-α, or anti-CD3 antibody with irradiated PBMCs from HIV-1–seronegative individuals. In addition, cells were incubated with the combination of IL-2, IL-6, and TNF-α or anti-CD3 antibody in the presence of three drugs (AZT, 3TC, and Indinavir; see Materials and Methods). Supernatants from each culture were removed on days 3, 6, and 9, and HIV-1 p24 was measured by ELISA.

Figure 3

Induction of HIV-1 replication by the combination of IL-2, IL-6, and TNF- α in latently infected, resting CD4⁺ T cells from patients receiving HAART. Resting CD4⁺ T cells from infected patients were isolated and further incubated with no cytokine, individual cytokines as indicated, the combination of IL-2, IL-6, and TNF-α, or anti-CD3 antibody with irradiated PBMCs from HIV-1–seronegative individuals. In addition, cells were incubated with the combination of IL-2, IL-6, and TNF-α or anti-CD3 antibody in the presence of three drugs (AZT, 3TC, and Indinavir; see Materials and Methods). Supernatants from each culture were removed on days 3, 6, and 9, and HIV-1 p24 was measured by ELISA.

Figure 4

Suppression of induction of HIV-1 replication in latently infected, resting CD4⁺ T cells stimulated with the combination of cytokines or anti-CD3 antibody by in vitro glucocorticoids. Resting CD4⁺ T cells were incubated with IL-2, IL-6, and TNF-α, or with anti-CD3 antibody with irradiated feeders in the presence or absence of dexamethasone (10⁻⁸ M). Supernatants from each culture were removed on days 3, 6, and 9, and HIV-1 p24 was measured by ELISA.