HIV Envelope Suppresses CD4⁺ T Cell Activation Independent of T Regulatory Cells

Cells and HIV-1

Human embryonic kidney 293T cells (American Type Culture Collection) were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS (HyClone) and glutamine (Invitrogen Life Technologies). PBMC from HIV-negative donors were obtained through an IRB-approved protocol and maintained in complete RPMI 1640 (Invitrogen Life Technologies) containing 10% FCS and glutamine. Aldrothiol-2 (AT-2) inactivated (18) HIV-1 NL4–3 virions were obtained from University of Pennsylvania Center for AIDS Research (CFAR) virology core.

Plasmid constructs and RNA synthesis

HIV Envs encoding IIIB, YU2, and 89.6 were cloned into the RNA expression plasmid pT7TS that contains a T7 promoter, 5′ and 3′ UTR sequences of Xenopus β-globin, and a 30-nuc long A tail (19). Plasmids were linearized with restriction enzymes and in vitro transcribed with T7 polymerase (mMessage T7 Ultra Kits (Ambion)), extended poly-A tail was added. RNA was maintained in nuclease-free water at a concentration of 1 μg/μl and stored at −20°C.

CD25⁺ cell depletion and T cell proliferation assay

CD25⁺ cells were depleted from PBMC using magnetic beads (Dynabeads CD25; Invitrogen Life Technologies). Briefly, PBMC and magnetic beads were washed twice with PBS containing 1% FBS. Then 1 × 10⁷ cells/ml were mixed with a 40:1 ratio of beads to CD25⁺ cells assuming 5% of cells expressed CD25, and rotated at 4°C for 45 min. A second round of depletion was performed with a decreased volume of beads (half of that used in the first round) to achieve near complete depletion of CD25⁺ cells. Whole and CD25-depleted PBMC were CFSE labeled, as described previously (5), and cultured with Env-expressing and control 293T cells in presence of TCR stimulation.

CD4⁺ T cell proliferation

Transcribed mRNA encoding HIV Env was complexed to lipofectin for delivery to cells as described previously (19). 293T cells seeded in 12-well plates with an approximate 90% confluence (5 × 10⁵ cells) were transfected with Env (IIIB, YU2, and 89.6) encoding mRNA (5 μg/ml) or control poly(AC) RNA (Sigma-Aldrich), incubated for 4 h to allow protein expression, and cocultured with 2 × 10⁶ CFSE loaded cells (PBMC or CD25-depleted PBMC) in the presence of stimulation with soluble anti-CD3 mAb (1 μg/ml; Ortho Biotech) and IL-2 (20 U/ml; AIDS Reference and Reagent Program) (20) or TSST-1 superantigen with or without soluble CD4 (sCD4) (10 μg/ml; AIDS Reference and Reagent Program) (21). Increasing or decreasing the ratio of Env-expressing 293T cells to PBMC did not affect suppression of CD4⁺ T cell proliferation. For exposure to free virions, 2 × 10⁶ CFSE⁺ PBMC were incubated with AT-2-inactivated HIV-1 NL4–3 virions at concentrations of 1, 0.3, or 0.1 μg HIV p24 Gag equivalents/ml, which corresponds to a gp120 concentration of ∼1, 0.3, and 0.1 nM, respectively (16). Four days after Env exposure, PBMC were collected, washed with PBS containing 1% FCS, and stained with anti-CD4-allophycocyanin (RPA-T4; BD Pharmingen). Cells were analyzed with a FACS-Calibur flow cytometer, and proliferation of CD4⁺ T cells was analyzed by CFSE dilution using Cellquest software (BD Pharmingen). A minimum of 10,000 events were analyzed. The percent of stimulated CD4⁺ T cells that proliferated was calculated as described (22).

Treg sorting and suppression culture

Whole PBMC or CD25-depleted PBMC were cultured with Env-expressing or control 293T cells. During Env exposure, whole PBMC were unstimulated, whereas CD25-depleted PBMC were stimulated with anti-CD3 and IL-2. A total of 48 h after Env exposure, PBMC were removed from 293T monolayer and stained with anti-CD4-FITC, anti-CD25-PE-Cy5, and anti-CD127-PE (BD PharMingen). CD4⁺, CD127⁻, CD25⁺ cell sorting was performed on a FACS/Aria (BDbioscience) to obtain the suppressors. A total of 2 × 10⁵ CFSE labeled CD25-depleted autologous PBMC responders were mixed with increasing amounts of FACS sorted CD4⁺, CD127⁻, CD25⁺ suppressors (Suppressor to Responder ratios of 1:3 and 1:6) in the presence of stimulation with anti-CD3 and IL-2. Four days after coculture, the proliferation of responder cells was determined by flow cytometry.

Treg analyses

PBMC were exposed to Env (either on 293T- or on AT-2-treated virions) as described above. For exposure to 293T cells, PBMC were either resting or stimulated by anti-CD3 and IL-2. For incubation with AT-2 inactivated virions, resting PBMCs were used. At various time points after Env exposure (days 1, 3, and 5), PBMC were collected, washed with PBS containing 1% FCS, and stained with anti-human CD4-FITC (SK3; BD Pharmingen) and anti-CD25-PE-Cy5 (M-A251; BD Pharmingen). Cells were then washed, fixed with 2% paraformaldehyde (eBiosicence), permeabilized with PBS 1% FCS containing 0.1% saponin (eBioscience), incubated with normal rat serum at 4°C for 15 min, and stained with anti-human FoxP3-allophycocyanin (PCH101; eBioscience) on ice for 30 min according to the manufacturer’s instructions. The appropriate isotype-matched Abs were used to define background staining. Expression of CD4, CD25, and FoxP3 in cells were determined by flow cytometry. A minimum of 10,000 events were analyzed.

IL-10 and TGF-β ELISA

IL-10 and TGF-β concentrations in culture supernatants were determined by commercially available ELISA kits (BD OptEIA) according to the manufacturer’s instructions.

Statistics

Mean, SEM, and student’s t test were performed using Microsoft Excel software.

Env expressed on free virions suppresses CD4⁺ T cell proliferation

In HIV infection, Env present on infected cells and free virions can contact CD4⁺ T cells and induce dysfunction (23). We previously reported that Env expression on cells, as occurs during vaccination with encoding vectors, such as DNA or viruses, induces an inhibition to Ag-specific CD4⁺ T cell proliferation in a CD4-dependent manner (5). This system used human PBMC as a source of CD4⁺ T cells that were cultured with Env-expressing mRNA transfected 293T cells or vaccinia virus infected DC in the presence of either anti-CD3 or superantigen (TSST-1) stimulation. To test whether Env on virions is also able to induce suppression of anti-CD3 stimulated CD4⁺ T cell proliferation, we incubated PBMC with increasing amounts of AT-2-inactivated HIV-1 (NL4–3), which is infection deficient but retains a functional (able to bind CD4) Env, modeling the noninfectious interactions between circulating free virions and CD4 in HIV infection (16). We found that inactivated virus could inhibit anti-CD3 stimulated CD4⁺ T cell proliferation in a dose dependent manner, which was overcome by sCD4 (Fig. 1⇓, A and B), similar to that observation for Env expressed on cells (Fig. 1⇓, C and D). Similar results were observed when TSST-1 superantigen was used as a stimulus. Variability in the amount of suppression was observed for different preparations of AT-2 virus ranging from 29% (Fig. 1⇓, A and B) to greater than 60% (data not shown). The data in Fig. 1⇓B shows the percentage of CD4⁺ T cells that were stimulated to proliferate in the culture at the initiation of stimulation (22). Deceased fluorescence intensity of anti-CD4 SK3 mAb staining for virion-incubated PBMC (24) verified a functional interaction between CD4 and Env (data not shown). Interestingly, we found that proliferation of CD4-negative cells was also abrogated after incubation with 1 μg/ml virions (Fig. 1⇓A), which could be due to impairment of CD4⁺ T cell helper functions by Env, leading to decreased growth cytokine production (IL-2 and others) and less activated accessory cells that, in turn, cause a decrease in Ag-stimulated CD8⁺ T cell proliferation.

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FIGURE 1.

HIV Env induces suppression of anti-CD3 stimulated CD4⁺ T cell proliferation through CD4 binding. Freshly isolated human PBMC were CFSE labeled and exposed to AT-2-treated HIV-1 virions (A and B) or Env (IIIB, 89.6, and YU2)-expressing 293T cells (C and D) in the presence or absence of sCD4 (10 μg/ml). The cultures were stimulated with anti-CD3 mAb and IL-2. Four days after Env exposure and stimulation, PBMC were stained for CD4 and the proliferation of CD4⁺ T cells was monitored by flow cytometry (A and C). The percent of input CD4⁺ T cells that proliferated (B and D) was calculated as described (22 ). Data are representative of three experiments. B, ¹p = 0.05. p values calculated using 2-tailed homoscedastic student’s t test comparing AT-2 virus-treated CD4⁺ T cell proliferation to no virus treated. D, ¹p < 0.01. p values calculated using 2-tailed homoscedastic student’s t test comparing 293T-expressed Env-treated CD4⁺ T cell proliferation to no Env-treated.

CD25⁺ Treg cells are not required for Env-induced suppression

We then investigated whether CD25⁺ Treg cells are involved in Env-induced suppression in our system. CFSE stained total or CD25-depleted PBMC were cultured with Env-expressing or control 293T cells in the presence of anti-CD3 and IL-2 stimulation. CD25 staining verified that with two rounds of magnetic bead depletion >95% of CD25⁺ cells were removed from PBMC (Fig. 2⇓A). As expected, CD25-depletion significantly enhanced the proportion of proliferating CD4⁺ T cells in both Env-exposed (from 2.8 to 5.2%) and control cultures (from 4.6 to 10.4%) (Fig. 2⇓, B and C). However, exposure to Env caused almost a 50% decrease in the proportion of proliferating CD4⁺ T cells in CD25-depleted PBMC (from 10.4 to 5.2%) (Fig. 2⇓, B and C), suggesting that pre-existing CD25⁺ Treg cells were not required for Env-induced suppression of CD4⁺ T cell proliferation. Similar results were observed when TSST-1 superantigen was used as a stimulus.

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FIGURE 2.

CD25⁺ Treg cells are not required for Env-induced suppression of proliferation. CD25⁺ cells were depleted from human PBMC using two rounds of CD25 magnetic beads. Flow cytometric analysis demonstrated depletion of greater than 95% of CD25⁺ cells (A). CFSE labeled whole (top, B) or CD25-depleted (bottom, B) PBMC were exposed or not to IIIB Env-expressing 293T cells in the presence of anti-CD3 and IL-2 stimulation. CD4⁺ T cell proliferation 4 days after Env exposure is shown. C, The percent of input CD4⁺ T cells that proliferated is shown for each condition. ¹p < 0.01. p values were calculated using a 2-tailed homoscedastic student’s t test comparing Env-treated CD4⁺ T cell proliferation to no virus-treated. Data are representative of at least four repetitions.

Effect of Env on frequency of CD4⁺, CD25⁺, FoxP3⁺ cells in PBMC

We show that CD25⁺ Treg cells are not required for Env to induce suppression of CD4⁺ T cell proliferation. One important question that remains highly controversial is whether and how HIV, or its associated gene products, regulate Treg cells. Both increased and reduced frequency of Treg cells have been reported in HIV⁺ patients compared with uninfected people (8, 10, 12). To test whether Env, as the major surface Ag of HIV, directly induces or activates Treg cells, we first determined and compared the percentage of CD4⁺, CD25⁺, FoxP3⁺ cells in both stimulated and resting human PBMC with or without exposure to Env expressed on 293T cells. Compared with resting PBMC, stimulation caused approximately a two-fold increase in the amount of CD4⁺, CD25⁺, FoxP3⁺ T cells in PBMC at all time points studied (Fig. 3⇓A), which is consistent with a study demonstrating activation induced FoxP3 expression in human T cells (25). Of importance, for both resting and stimulated PBMC, Env exposure did not induce significant changes in the percentage of CD4⁺, CD25⁺, FoxP3⁺ T cells (Fig. 3⇓A). Analyses of T cell proliferation in the activated cells demonstrated a significant reduction of CD4⁺ T cell divisions when cultured with Env mRNA transfected 293T cells, verifying Env expression on 293T cells and its inhibitory effect.

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FIGURE 3.

Effect of Env delivered by cell surface expression or on virions on FoxP3⁺, CD25⁺ Treg cells in unstimulated and stimulated PBMC. Unstimulated or anti-CD3 stimulated PBMC were analyzed by flow cytometry for CD25 and FoxP3 expression on CD4⁺ T cells at days 1, 3, and 5 after exposure to HIV Env expressed by 293T cells (A) or AT-2-treated virions (B). Histograms shown are gated on live CD4⁺ cells. Data are representative of at least four repetitions.

We then investigated whether Env present on free virions affected the percentage of FoxP3⁺ Treg cells in PBMC, as we observed inhibition of CD4⁺ T cell proliferation by free virions (Fig. 1⇑, A and B). PBMC were incubated with AT-2-inactivated HIV-1 virions at 1 μg/ml p24 Gag protein equivalent. Surface and intracellular staining demonstrated no significant difference in the number of CD4⁺, CD25⁺, FoxP3⁺ cells between virion incubated and control PBMC at all studied time points (days 1, 3, and 5) (Fig. 3⇑B), which is in contrast with the observation that exposure of purified CD4⁺ T cells to AT-2-treated HIV resulted in significantly increased FoxP3 expression at both mRNA and protein levels, resulting from Env-induced enhancement in cell viability and consequent accumulation of FoxP3⁺ cells (16). Taken together, our data suggested that neither Env delivered on cells as would be found during infection or after encoding vector delivery as part of a vaccine nor Env present on free virions could directly induce or activate T regulatory cells in resting or stimulated PBMC.

IL-10 and TGF-β production in Env-exposed PBMC

Immune suppression mediated by Treg cells can occur through either cell contact or suppressive cytokines, particularly IL-10 and TGF-β (26, 27, 28). To test whether Env exposure, rather than inducing an increased number of FoxP3⁺ cells, stimulates an enhanced production of IL-10 and/or TGF-β by Tregs, supernatants of cultures with and without Env exposure were collected at various time points (days 1, 3, and 5) after initiation of coculture, followed by quantitation of IL-10 and TGF-β by ELISA. TGF-β was undetectable at any of the studied time points in both Env exposed and control supernatants (data not shown). Significant amounts of IL-10 were produced and accumulated in the supernatants starting at 12 h after stimulation. However, no difference was observed in the amount of IL-10 production between Env-exposed and control cultures (Fig. 4⇓). Considered together, these data suggest that Env does not induce a more activated phenotype of Treg cells leading to increased suppressive cytokine production.

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FIGURE 4.

Effect of Env exposure on IL-10 production. 293T cells expressing IIIB Env or control transfected were cultured with PBMC in the presence or absence of anti-CD3 and IL-2 stimulation. Supernatants were collected at the indicated time points, and supernatant associated IL-10 was determined by ELISA. Data are representative of at least five experiments. Error bars are the SEM for triplicate measurements.

Env does not enhance the suppressive activity of CD4⁺, CD25⁺, CD127⁻ T cells

We demonstrate that Env induces neither an increased number of CD4⁺, D25⁺, FoxP3⁺ cells nor enhanced IL-10 and TGF-β production in PBMC, and their depletion does not negate Envs ability to suppress CD4⁺ T cell proliferation. To test whether Env activates Treg cells, resulting in an enhanced suppressive activity independent of IL-10 or TGF-β, an in vitro suppression assay was developed in which Treg cells isolated from Env-exposed and control PBMC were cultured with autologous responder cells in the presence of anti-CD3 stimulation (29). The suppressive activity of Treg cells, on a per-cell basis, was evaluated by their capacity to inhibit anti-CD3 stimulated responder cell division. In addition to CD25, CD127, a subunit of the IL-7R, has also been shown to be a useful cell surface marker for defining Tregs (30, 31). We sorted Tregs from control RNA or Env RNA-transfected 293T cell exposed PBMC based on CD4⁺, CD25⁺, and CD127⁻ expression. Greater than 90% of the sorted cells expressed FoxP3 (Fig. 5⇓A).

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FIGURE 5.

Endogenously present or activation induced (plus FoxP3⁺,CD25⁻) FoxP3⁺ Treg cell suppressive activity is not modulated by Env. A, Isolation of FoxP3⁺ Treg cells from PBMC using CD4, CD25, and CD127. CD4⁺ live lymphocyte sized cells (gate in top panel of A) were sorted for high CD25 and no CD127 expression (gate in middle panel of A). The sorted cells were stained for FoxP3 (A, lower panel) and demonstrated greater than 90% positivity. B, As a source of suppressors, PBMC were cultured with Env expressing or control 293T cells, followed by isolation of CD4⁺, CD25⁺, CD127⁻ T cells by flow cytometric sorting (detailed in A) 48 h after Env exposure. CFSE-labeled responder cells (autologous PBMC depleted of CD25⁺ cells) were cocultured with graded numbers of sorted CD4⁺, CD25⁺, CD127⁻ suppressor cells in the presence of anti-CD3 and IL-2 stimulation. CFSE intensity of responder cells with CD4 staining was monitored by flow cytometry at day 4 to quantify responder cell proliferation. C, Percent of input CD4⁺ T cells that proliferated was calculated for each condition. ¹p < 0.007. p values calculated using 2-tailed homoscedastic student’s t test comparing 293T Env-treated CD4⁺ T cell proliferation to no Env-treated. D, As a source of suppressors, CD25-depleted PBMC were cultured with Env expressing or control 293T cells in the presence of anti-CD3 and IL-2 stimulation, followed by isolation of CD4⁺, CD25⁺, CD127⁻ T cells 48 h after stimulation, as described in A. CFSE-labeled responder cells (autologous PBMC depleted of CD25⁺ cells) were cocultured with graded numbers of sorted CD4⁺, CD25⁺, CD127⁻ suppressor cells in the presence of anti-CD3 and IL-2 stimulation. CFSE intensity of responder cells with CD4 staining was monitored by flow cytometry at day 4 to quantify responder cell proliferation. E, Percent of input CD4⁺ T cells that proliferated was calculated for each condition. ¹p < 0.009. p values calculated using 2-tailed homoscedastic student’s t test comparing 293T-Env-treated CD4⁺ T cell proliferation to no Env-treated. Data are representative of at least three repetitions.

We observed a population of FoxP3⁺ cells that did not express CD25 after CD25-depletion of PBMC that accounted for ∼40% of the FoxP3⁺, CD4⁺ cells. In addition to natively expressed FoxP3, an additional population of FoxP3⁺ cells also appears after activation. These cells are derived from FoxP3⁻, CD4⁺ cells and their ability to suppress immune responses is controversial (25). We attempted to use the above sorting strategy to isolate FoxP3⁺ Treg cells after Env exposure and anti-CD3 stimulation. It should be noted that activation induces up-regulation of CD25 as well as down-regulation of CD127 on non-Treg CD4⁺ T cells, making the isolation of FoxP3⁺ cells less accurate. However, approximately 30–50% of the sorted cells from stimulated PBMCs expressed FoxP3.

Sorted CD4⁺, CD25⁺, CD127⁻ cells (90% FoxP3⁺) inhibited responder CD4⁺ T cell proliferation in a dose-dependent manner, where in the absence of added Tregs, 56.2% of the input cells proliferated after stimulation and the addition of one Treg per three responder cells (CD25-depleted PBMC) decreased the percent of proliferating CD4⁺ T cells to 29.1 and at one Treg per 6 responder cells, 46.4% of the CD4⁺ T cells proliferated (Fig. 5⇑, B and C). However, Env-exposed and control CD4⁺, CD25⁺, CD127⁻ T cells were equally suppressive, because no significant difference in the proportion of dividing responder cells was found between them (Fig. 5⇑, B and C).

We then determined whether Env induces a more suppressive activity of activation induced FoxP3⁺ cells, which also contained the FoxP3⁺, CD25⁻ population, by sorting CD4⁺, CD25⁺, CD127⁻ T cells from PBMC, which were first depleted of CD25⁺ cells, then cocultured with 293T cells (with and without Env), and finally activated by anti-CD3. Activation induced CD4⁺, CD25⁺, CD127⁻ T cells (∼30–50% FoxP3⁺), although slightly less suppressive than natural FoxP3⁺ cells isolated from unstimulated PBMC, inhibited CD4⁺ T cell proliferation in a dose-dependent manner (Fig. 5⇑, D and E). Of importance, no difference in suppressive ability between Env exposed and control activation-induced Treg cells was observed (Fig. 5⇑, D and E). Taken together, these data indicate that Env exposure does not enhance the functional suppressive activity of either pre-existing Treg or activation induced FoxP3⁺ cells.