Induction of HIV-1 Replication in Latently Infected CD4⁺ T Cells Using a Combination of Cytokines

Six HIV-1–seropositive patients who were naive to antiretroviral therapy, and seven patients who were receiving HAART (Table 1) were subjected to apheresis in order to obtain PBMCs according to protocols approved by the National Institute of Allergy and Infectious Diseases and the University of Washington Institutional Review Boards.

Resting CD4⁺ T cells were isolated from peripheral blood of HIV-1–infected individuals using a combination of magnetic bead depletion and fluorescent activated cell sorting techniques as described previously (27).

To determine the frequency of latently infected, resting CD4⁺ T cells from patients carrying replication competent HIV-1, micro coculture assays were carried out as described previously (27).

After isolation of resting CD4⁺ T cells from HIV-1–infected individuals, cells (2.0–10 × 10⁶) were incubated with complete medium consisting of RPMI supplemented with 10% FCS, penicillin–streptomycin, and l-glutamine in a tissue culture plate with the following cytokines: IL-2 (100 U/ml; Boehringer Mannheim, Indianapolis, IN), IL-1β (5 ng/ml; R&D Systems, Minneapolis, MN), IL-4 (3 ng/ml; R&D Systems), IL-6 (5 ng/ml; R&D Systems), TNF-α (2.5 ng/ml; R&D Systems), or with the combination of IL-2, IL-6, and TNF-α. Purified resting CD4⁺ T cells were also incubated with complete medium in the absence of any cytokines as a negative control. As a positive control, the same number of cells were incubated with anti-CD3 antibody (OKT3) in the presence of 2 × 10⁶ irradiated PBMCs from HIV-1–seronegative individuals as feeder cells. To examine the inhibitory role of glucocorticoids in the induction of HIV-1 replication in latently infected, resting CD4⁺ T cells by cytokines, dexamethasone (10⁻⁸ M; Calbiochem, La Jolla, CA) was added to cultures containing the combination of three cytokines or anti-CD3 antibody. Cultures were incubated in a 37°C CO₂ incubator for 9 d, and supernatants from each culture were removed on days 3, 6, and 9 for determination of HIV-1 p24 by ELISA (Coulter Corp., Miami, FL). To examine the effects of in vitro antiretroviral agents on the combination of cytokines or anti-CD3 induced viral replication, zidovudine (AZT; 4 μM; Sigma Chemical Co., St. Louis, MO), lamivudine (3TC; 5 μM; gift from Dr. Raymond Schnazi, Emory University, Atlanta, GA), and the protease inhibitor indinavir (10 μM; Merck, Rahway, NJ) were added to the media.

To measure cytokine-induced cellular activation in latently infected, resting CD4⁺ T cells, flow cytometric analysis of the expression of activation markers and thymidine incorporation assays were carried out. Resting CD4⁺ T cells that had been incubated with various cytokines were stained with PE-conjugated anti-CD25 (Becton Dickinson, San Jose, CA) and PE-conjugated anti–HLA-DR antibodies (Becton Dickinson) along with FITC-conjugated anti-CD4 antibody (Becton Dickinson) 6 d after incubation. On day 6, [³H]thymidine incorporation was measured in resting CD4⁺ T cells that had been incubated with the above cytokines or with anti-CD3 antibody.

The replication of HIV-1 is intimately associated with the state of cellular activation of susceptible cell populations (2, 28). In this regard, we first investigated the role of individual cytokines or combinations of cytokines on the activation of highly enriched, resting CD4⁺ T cells from HIV-1–seropositive patients by examining the expression of cell surface activation markers CD25 and HLA-DR. As shown in Table 2, in the absence of cytokines or any other activating stimuli, purified resting CD4⁺ T cells from infected individuals expressed no measurable CD25 or HLA-DR on their cell surfaces. After a 6-d incubation in vitro, only IL-2 and IL-4 induced slight increases in activation markers on resting CD4⁺ T cells. However, the combination of IL-2, IL-6, and TNF-α showed marked increases in CD25 and HLA-DR expression. The induction of cellular activation in purified resting CD4⁺ T cells by the combination of three cytokines was further confirmed by the cellular incorporation of DNA precursor thymidine (Fig. 1). With the exception of IL-2, which induced a slight degree of cellular proliferation, no individual cytokine resulted in active proliferation of resting CD4⁺ T cells. Cells stimulated with anti-CD3 antibody showed profound cellular activation as judged by both the expression of activation markers and [³H]thymidine incorporation, confirming that purified resting CD4⁺ T cells are fully capable of undergoing cellular proliferation.

Given the fact that the combination of IL-2, IL-6, and TNF-α potently activates resting CD4⁺ T cells as described above, we first examined the inducibility of replication-competent HIV-1 in latently infected, resting CD4⁺ T cells from antiretroviral therapy–naive, HIV-1–seropositive individuals using individual cytokines and combinations of cytokines in vitro. Despite the fact that highly purified, resting CD4⁺ T cells from all patients carried replication-competent HIV-1 with a frequency determined by micro coculture assay ranging from 8.1 to 205.8 infectious units per 10⁶ resting CD4⁺ T cells (Table 1), measurable p24 was not detected in cultures containing neither cytokines nor anti-CD3 antibody throughout the entire incubation period (Fig. 2). Among the individual cytokines, only IL-2 had a slight effect on the induction of HIV-1 expression in latently infected, resting CD4⁺ T cells (Fig. 2). However, cultures incubated with the combination of IL-2, IL-6, and TNF-α had a dramatic increase in HIV-1 replication. The level of viral production induced by this cytokine combination was equivalent to that of cells stimulated with anti-CD3 antibody. When a combination of three antiretroviral drugs was added to the cultures incubated with the three cytokine combination or with anti-CD3 antibody, viral replication was completely suppressed as shown in Fig. 2.

We and others have recently reported that HIV-1–infected individuals receiving HAART for prolonged periods of time with no detectable plasma viremia carry a small number of latently infected, resting CD4⁺ T cells that produce infectious HIV-1 upon mitogenic stimulation in vitro (24–26). In this regard, we sought to determine whether activation of HIV-1 replication can be achieved by cytokines alone in the absence of a potent mitogenic stimulus in latently infected, resting CD4⁺ T cells from patients who had been receiving HAART for considerable periods of time and in whom plasma viremia was markedly suppressed. The frequency of latently infected, resting CD4⁺ T cells carrying replication-competent HIV-1 from these patients ranged from 0.5 to 40.1 infectious units per 10⁶ resting CD4⁺ T cells (Table 1). When purified resting CD4⁺ T cells from these patients were incubated with individual cytokines, including IL-2, no significant HIV-1 replication was detected in the supernatants (Fig. 3). However, the combination of IL-2, IL-6, and TNF-α effectively induced HIV-1 replication in this population of cells from seven out of seven patients receiving HAART, three of whom (patient nos. 10, 12, and 13) had no detectable plasma viremia as determined by the ultrasensitive reverse transcriptase PCR assay (Roche Labs., Nutley, NJ) with a detection limit of 50 copies of HIV-1 RNA per milliliter of plasma. As in patients receiving no antiretroviral drugs, triple combination of antiretroviral drugs in vitro completely abolished HIV-1 replication induced by either the cytokine combination or anti-CD3 stimulation.

Glucocorticoids, a class of hormones with broad antiinflammatory and immunoregulatory properties (34–36), have been shown to block HIV-1 replication in vitro and in vivo (37, 38). One of the major effects of glucocorticoids is to inhibit the secretion and effects of cytokines (39–41). In addition, it has been shown that glucocorticoids can block nuclear factor κB– mediated cellular activation in vitro (42). To examine the inhibitory role of glucocorticoids in the induction of HIV-1 replication in latently infected, resting CD4⁺ T cells by cytokines, dexamethasone was added to cultures containing the combination of three cytokines or anti-CD3 antibody. As shown in Fig. 4, cultures containing dexamethasone markedly inhibited replication of HIV-1 in latently infected, resting CD4⁺ T cells from antiretroviral drug–naive patients when cultures were stimulated with either the combination of IL-2, IL-6, and TNF-α or with anti-CD3 antibody.

In this study, we have examined the synergistic role of cytokines in the reactivation of HIV-1 replication in latently infected, resting CD4⁺ T cells from patients who were receiving no antiretroviral therapy as well as from patients who were receiving HAART and in whom plasma viremia was markedly suppressed, and in certain patients even undetectable by the ultrasensitive reverse transcriptase PCR. Despite the fact that certain proinflammatory and immunoregulatory cytokines have been shown to upregulate the expression of HIV-1 in chronically infected cell lines and in the PBMCs of infected individuals (3, 10–13), it has been unclear what effect they have on latently infected, resting CD4⁺ T cells. This study clearly demonstrates a synergistic role of the combination of IL-2, IL-6, and TNF-α in the induction of HIV-1 replication in latently infected, resting CD4⁺ T cells. The fact that individual cytokines did not induce significant replication of HIV in these cells, whereas combinations of cytokines were potent inducers, is consistent with previous observations of the synergistic effects of certain cytokines in the induction of expression of HIV from chronically infected cell lines (12). HIV-1 replication in latently infected, resting CD4⁺ T cells induced by the three cytokine combination and anti-CD3 stimulation was effectively suppressed by dexamethasone at modest concentrations (10⁻⁸ M), which agrees with previous findings that glucocorticoids can block HIV-1 replication by inhibiting the secretion of certain cytokines, particularly proinflammatory cytokines (39–41) or by inhibiting nuclear factor κB–mediated cellular activation (42).

The findings in our study suggest that combinations of cytokines secreted in response to nonspecific stimuli or as a result of a specific antigenic stimulus could explain at least in part the reappearance of plasma viremia in patients receiving HAART in whom HIV-1 replication was successfully contained initially but in whom therapy was subsequently interrupted (33). Given the fact that the latently infected pool of resting CD4⁺ T cells resides predominantly in lymphoid tissue (27), and since cells situated in the microenvironment of lymphoid tissue are continually exposed to the secretion of proinflammatory and immunoregulatory cytokines (43), it is highly likely that these cytokines readily induce HIV replication in latently infected, resting CD4⁺ T cells in the absence of HAART or when HAART is ineffective in completely suppressing virus replication (33). The fact that latently infected, resting CD4⁺ T cells from individuals receiving HAART and in whom plasma viremia was markedly suppressed, and in certain patients even undetectable, could be readily induced to replicate virus when cells were taken ex vivo and cultured in the presence of the combination of cytokines is highly consistent with their ability to become reactivated when in vivo HAART is discontinued. The ability of in vitro HAART to again suppress cytokine-induced virus replication underscores this observation.

Finally, given the relatively long half-life of these latently infected, resting CD4⁺ T cells (24–26) and the fact that they are constantly within an environment capable of providing the stimuli for reactivation, it is not unreasonable to explore strategies aimed at deliberately diminishing the size of this pool of cells. In this regard, since cytokine-mediated induction of HIV-1 replication in these cells with subsequent release of virus probably results in death of the cell (44–46), and since the presence of HAART in vitro prevents spread of released virus, it is conceivable that in vivo administration of cytokines together with HAART may have such an effect.

We thank Joseph Adelsberger, Randy Stevens, and Dr. Michael Baseler for their help with cell sorting experiments; and Shuying Liu and Julie Metcalf for providing patient laboratory data. We also thank Drs. Lawrence Corey, M. Michelle Berry, and Jo Ann M. Mican for providing PMBCs from patients.

HAART

highly active antiretroviral therapy