HIV-1 Vpu Antagonism of Tetherin Inhibits Antibody-Dependent Cellular Cytotoxic Responses by Natural Killer Cells

ABSTRACT

The type I interferon-inducible factor tetherin retains virus particles on the surfaces of cells infected with vpu-deficient human immunodeficiency virus type 1 (HIV-1). While this mechanism inhibits cell-free viral spread, the immunological implications of tethered virus have not been investigated. We found that surface tetherin expression increased the antibody opsonization of vpu-deficient HIV-infected cells. The absence of Vpu also stimulated NK cell-activating FcγRIIIa signaling and enhanced NK cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). The deletion of vpu in HIV-1-infected primary CD4⁺ T cells enhanced the levels of antibody binding and Fc receptor signaling mediated by HIV-positive-patient-derived antibodies. The magnitudes of antibody binding and Fc signaling were both highly correlated to the levels of tetherin on the surfaces of infected primary CD4 T cells. The affinity of antibody binding to FcγRIIIa was also found to be critical in mediating efficient Fc activation. These studies implicate Vpu antagonism of tetherin as an ADCC evasion mechanism that prevents antibody-mediated clearance of virally infected cells.

IMPORTANCE The ability of the HIV-1 accessory factor to antagonize tetherin has been considered to primarily function by limiting the spread of virus by preventing the release of cell-free virus. This study supports the hypothesis that a major function of Vpu is to decrease the recognition of infected cells by anti-HIV antibodies at the cell surface, thereby reducing recognition by antibody-dependent clearance by natural killer cells.

INTRODUCTION

Human immunodeficiency virus type 1 (HIV-1) infection activates the innate immune system, but this activation fails to yield viral clearance or sterilizing immunity (1, 2). During the initial phases of acute HIV infection, the induction of type I interferon (IFN) upregulates a host of antiviral factors, including the HIV-1 restriction factor tetherin (BST-2/CD317) (3). Tetherin was found to be responsible for the retention of virus particles on the surfaces of cells infected with HIV-1 that lack the accessory protein Vpu (4, 5). Tetherin is constitutively expressed on several cell types, including mature B cells, plasma cells, and plasmacytoid dendritic cells, and tetherin is further upregulated by type I IFN in both macrophages and lymphocytes (6, 7). Within cells, tetherin is predominantly localized to the trans-Golgi network (TGN), as well as the plasma membrane and endosomal compartments (7–10). Tetherin inhibits the cell-free release of a diverse range of enveloped viruses, including nearly all retroviruses tested (11). The ability of tetherin to retain viruses on the cell surface requires a unique structural topology which features two membrane-interacting domains linked by a coiled coil domain and the ability of these molecules to form stable disulfide-linked dimers (12). Studies using fluorescence microscopy and transmission electron microscopy have shown that tetherin accumulates at sites of virus assembly and directly links nascent viruses to the plasma membrane (8, 12–14).

To counteract the retention of virus particles, HIV-1 has evolved the accessory protein Vpu, whose expression in infected cells leads to the downmodulation of tetherin from the plasma membrane (15). Vpu antagonism of tetherin occurs through ubiquitin-mediated degradation, as well as TGN sequestration, both of which lead to a decrease in tetherin surface expression (16–18).

While tetherin was proposed to inhibit viral spread, recent studies find that tethered virus may enhance or diminish viral dissemination through cell-cell contact (19–21). Several studies have shown that Vpu expression can be rapidly lost during in vitro culture (22, 23). The deletion of vpu from HIV-1 does not greatly diminish the spread of virus in infected T cell lines, implying that viral release is not essential for spread of HIV in cell culture (24). The lack of a strict requirement for Vpu for virus replication in vitro has led some to consider tetherin to be a modulator of the mode of infection (inhibiting cell-free infection but not cell-cell infection) rather than a strict inhibitor of viral growth (25). In vivo there may be additional reasons to enhance release of cell-free virus particles from the infected cell.

A potential advantage to preventing virus retention on the surfaces of infected cells is for immune evasion. Tethered virus particles may be recognized by circulating antibodies, resulting in the opsonization of infected cells. Antibody-opsonized cells can be cleared through complement-mediated lysis, Fc receptor-mediated phagocytosis via macrophages or antibody-dependent cellular cytotoxicity (ADCC)-mediated killing initiated through FcγRIIIa stimulation on the surfaces of granulocytes, macrophages, or natural killer (NK) cells (26–28). NK cells can also mediate the noncytolytic suppression of viral replication through the secretion of the inhibitory chemokines CCL3, CCL4, and CCL5 (29–31).

Nonneutralizing activities of antibodies have been proposed to play an important role in protective immune responses (32). The RV144 HIV vaccine trial found a correlation between the induction of nonneutralizing antibodies and protective immunity (33, 34). Other studies have detected greater ADCC responses in HIV-infected patients with slower disease progression and lower viral loads (35, 36). The primary population that mediates ADCC against virus-infected cells is NK cells, whose functions have also been correlated with slower disease progression and greater immune protection in exposed but uninfected individuals (37). Finally, several studies have demonstrated that elite controllers and long-term nonprogressors (LTNP) have higher ADCC and antibody-dependent cytotoxic viral inhibition antibody titers compared to viremic individuals (38–40).

In light of the potential contribution of ADCC in conferring protective immune responses against HIV infection, we examined whether the retention of viral particles by tetherin on the surfaces of infected cells enhances the detection and clearance of infected lymphocytes via ADCC. We hypothesized that tetherin surface expression enhances anti-HIV antibody binding and modulates the susceptibility of infected CD4⁺ T cells to ADCC by NK cells.

MATERIALS AND METHODS

Tetherin^low and tetherin^high CD4⁺ Jurkat cells.

The Jurkat E6 cell line was obtained from Arthur Weiss from the National Institutes of Health (NIH) AIDS Reagent Program. The cells were stained with tetherin antibody, clone RS38E (BioLegend), and flow sorted into tetherin^low and tetherin^high populations. These sorted clones maintained a stable tetherin phenotype in culture. To facilitate cell discrimination in cell mixing experiments, these cells were transduced to stably express green fluorescent protein (GFP; MSCV-GFP Puro retroviral vector). Cells were maintained in RPMI 1640 containing 10% fetal calf serum (FCS), penicillin-streptomycin, glutamine, and puromycin (2 μg/ml). To minimize nonspecific killing by primary NK cells, the tetherin^low and tetherin^high Jurkat cells were cocultured for 3 days with primary NK cells from three different donors.

Viruses and infection of CD4⁺ lymphocytes.

The HIV-1 reporter viruses used were replication-competent, full-length, infectious molecular clones derived from a pNL4.3 background. These constructs contain mCherry in place of the HIV-1 accessory protein Nef, and functional Nef expression is restored by the insertion of an internal ribosome entry site (41, 42). The Δvpu mutant has a stop codon inserted into the start of the vpu gene, and the A14L vpu mutant was generated by overlap-extension PCR mutagenesis. Viral stocks were generated by transfection of these molecular clones into 293T cells using calcium phosphate methods (43). Viral supernatants were harvested at 48 h posttransfection, passed through 0.45-μm-pore-size filters, and stored at −80°C.

Primary CD4⁺ T cells were purified from peripheral blood mononuclear cells (PBMCs) obtained from deidentifed HIV-negative blood donors (New York Blood Center) using a Miltenyi Biosciences negative isolation kit according to manufacturer's instructions and stored in liquid nitrogen prior to use. Primary CD4⁺ T cells were thawed and activated using irradiated, allogeneic PBMCs cocultured at a 2:1 ratio in RPMI 1640 containing 10% FCS, 5% human serum, interleukin-2 (50 IU), and phytohemagglutinin (PHA; 4 μg/ml).

CD4⁺ lymphocytes were infected (multiplicity of infection of 0.5) by spinoculation methods at a density of 2.5 × 10⁵ cells/well in a flat-bottom, 96-well plate. Cells were centrifuged at 1,200 × g for 90 min at 25°C and then returned to 37°C (44). Amaxa nucleofection was used to transfect HIV-1 constructs in tetherin^low and tetherin^high Jurkat cells. A total of 7 × 10⁶ cells were resuspended with proviral expression plasmid in 120 μl of Amaxa T-cell line solution V (Lonza). Nucleofections were performed using program S18 on a Nucleofector 2b (Lonza). Viable cells were purified from transfected populations via Ficoll density gradient purification at 24 h posttransfection and subsequently used as target cells. Overexpression of tetherin in tetherin^low cells was performed by cotransfection of a mammalian tetherin expression construct, IRAT (Thermo Scientific), along with specified HIV-1 reporter virus constructs using the nucleofection technique described above.

Flow cytometry analysis.

Recombinant 4E10 and HIV Ig pooled polyclonal patient sera were obtained from the NIH AIDS Reagent Program. The b12 antibody was purchased from Polymun Scientific (Vienna, Austria). b12 mutants with enhanced ADCC capacity (b12 double [S239D/I332E] and triple [S239D/I332E/A330L] mutants), along with a control LALA b12 mutant that has a diminished capacity to signal via the FcγRIIIa, were expressed as previously described (45). Indirect surface antibody staining with all anti-HIV antibodies was performed using the primary antibodies at 2 μg/ml in phosphate-buffered saline (PBS)–2% FCS for 30 min at 4°C. A secondary antibody, anti-human IgG-Alexa Fluor 647 (Invitrogen), was used at 2 μg/ml. The cells were fixed in 2% paraformaldehyde-PBS prior to analysis by flow cytometry. For tetherin surface staining, an anti-tetherin allophycocyanin-conjugated (APC) antibody (BioLegend) was used at a concentration of 5 μg/ml. Flow cytometry was performed on a BD Fortessa (Becton Dickinson, San Jose CA), and analysis was performed using FlowJo v8.7.3 (TreeStar, Ashland, OR).

FcγRIIIa stimulation assay.

Activation of FcγRIIIa signaling was measured by using a Jurkat NFAT-luc+FcγRIIIa cell line (Jur-γRIIIa; Promega). FcγRIIIa signaling activates the NFAT transcription factor, inducing expression of firefly luciferase driven by an NFAT-responsive promoter (46). Tetherin^low and tetherin^high CD4⁺ lymphocytes infected with wild-type (WT) or Δvpu HIV-1 were purified by using a Ficoll-Hypaque gradient, normalized to 15 to 20% infection, and preincubated for 15 min with the indicated concentration of anti-HIV antibodies. Target cells were cocultured with the Jur-γRIIIa cells at a 5:1 effector/target ratio for 16 h. The cells were lysed, and the firefly luciferase activity was determined with a luciferase assay kit (Promega). Jur-γRIIIa cells cocultured with the infected target populations in the absence of antibody provided background (antibody-independent) luciferase production, and these levels were subtracted from the signal to yield antibody-specific activation in relative light units.

CD107a degranulation.

The degranulation of primary NK cells was measured as previously described (47). Briefly, HIV-transduced cells were purified by using a Ficoll-Hypaque gradient to remove cellular debris, normalized to 15 to 20% infection, and dye labeled with the CellTrace violet indicator dye (Invitrogen). Infected targets were preincubated with the indicated concentrations of antibody for 15 min and cocultured with primary NK cells at a 10:1 effector/target ratio at 37°C for 2 h. After 1 h, Golgi Stop (monensin; BD Biosciences) was added to the coculture. The cells were then washed and stained for CD3 (OKT3; BioLegend), CD56 (MEM-188; BioLegend), and CD107a (H4A3; BioLegend) surface expression and analyzed by flow cytometry.

ADCC assay.

Primary NK cells were purified from PBMCs from HIV-negative blood donors (New York Blood Center) using a Miltenyi negative selection kit for CD56⁺ CD3⁻ NK cells according to the manufacturer's protocol and stored in liquid nitrogen until needed. On the day of the ADCC assays, NK cells were thawed and allowed to recover for 4 h in RPMI medium containing 10% IgG-low FCS, 20 IU of IL-2, penicillin-streptomycin, and glutamine. CD4⁺ lymphocytes were infected with WT or Δvpu HIV-1 purified by using a Ficoll-Hypaque gradient to eliminate cellular debris, normalized to 15 to 20% infection, and dye labeled with 1 mM CellTrace violet indicator dye (Invitrogen). The cells were then resuspended in assay medium (RPMI, 10% IgG-low FCS) and plated at a density of 5 × 10⁴ cells per well in a 96-well, round-bottom plate. After preincubation with the indicated concentration of antibodies for 15 min, the target cells were cocultured with primary NK cells at 10:1 effector/target ratio at 37°C for 6 h. After coculture, the cells were washed in PBS, fixed in PBS with 2% paraformaldehyde, and analyzed by flow cytometry. The specific killing of HIV⁺ cells was assessed by the loss of mCherry^high cells as a proportion of the input target cells. Cells cocultured in the absence of anti-HIV antibodies defined the level of nonspecific or background killing (∼30 to 40%).

IgG antibody isolation from sera obtained from HIV-positive and HIV-negative patients.

HIV-positive and HIV-negative plasma samples were collected from patients in the Jack Martin Clinic at Mount Sinai Hospital or from healthy volunteers in New York, NY, according to an institutional review board (IRB)-approved protocol (IRB no. 08-0464). Samples were provided deidentified with basic clinical profiles. Polyclonal IgG was isolated from 500 μl of serum using a NAb spin kit (Thermo Scientific) for IgG antibody isolation, according to the manufacturer's protocol. After elution, polyclonal IgG was desalted using Zeba spin desalting columns (Thermo Scientific) according to the manufacturer's instructions, and the IgG yields were quantified using an Easy-Titer IgG assay kit (Thermo Scientific). Isolated IgGs were then divided into aliquots and stored at −80°C until used in binding and Fc receptor stimulation assays.

Statistical analysis.

Statistical analysis of data was performed using Prism software (GraphPad, San Diego, CA). Significance levels between infected populations were calculated by using an unpaired two-tailed t test. Correlations between Fc receptor signaling and tetherin surface expression were calculated by using a Pearson correlation test, and the significance of the change in signaling comparing WT and Δvpu HIV-1-infected cells was calculated with a Wilcoxon matched-pair test. P values of ≤0.05 were considered significant.

RESULTS

Tetherin model system.

To examine how the expression of tetherin affects the susceptibility of CD4⁺ T lymphocytes to ADCC, we exploited the heterogeneous surface expression of tetherin on the CD4⁺ Jurkat E6 T cell line. We used flow cytometry to sort these cells into populations expressing high or low levels of tetherin, delineated as tetherin^high and tetherin^low cells, which maintained stable surface expression when propagated in culture (Fig. 1A). We further characterized these populations for surface CD4 and CXCR4 expression and found no significant differences in the expression of either molecule on the two cell lines (data not shown). We infected these cells with an mCherry-expressing wild-type (WT) molecular clone of HIV-1 or an isogenic clone that was deficient for Vpu expression (Δvpu). After infection with WT or Δvpu HIV-1, no changes in tetherin surface expression were detected in the tetherin^low cells (Fig. 1B). In contrast, tetherin surface expression was downmodulated in the tetherin^high cells infected with WT virus, whereas high tetherin expression remained unaltered after infection with Δvpu HIV-1 (Fig. 1B). The HIV constructs used in the present study encode the mCherry reporter in the nef position, and the accumulation of high levels of mCherry fluorescence demarcates cells in the late stages of productive infection. Consistent with vpu being a Rev-dependent gene expressed in the late stages of infection, we observed tetherin downmodulation predominantly in the mCherry^high cells infected with WT HIV-1 but not Δvpu HIV-1 (Fig. 1C). HIV-1 Vpu and Env are expressed from the same bicistronic mRNA, so the deletion of vpu could lead to increased amounts of total Env expression. To examine the total Gag and Env expression levels in the tetherin^low and tetherin^high cells infected with WT and Δvpu HIV-1, we performed Western blot analysis. The total level of HIV Env expression in the Δvpu HIV-1-infected versus WT HIV-1-infected cells was slightly reduced in both tetherin^low and tetherin^high cells (Fig. 1D). The levels of HIV Gag were similar in Δvpu HIV-1- versus WT HIV-1-infected tetherin^low and tetherin^high cells (Fig. 1D).

FIG 1.

Tetherin CD4⁺ T cell model system. (A) Heterogeneous expression of surface tetherin on unsorted CD4⁺ Jurkat E6 cells (left). The Jurkat E6 cells were stained for tetherin expression (red line) and then flow sorted into tetherin^low (center) and tetherin^high (right) populations, which remained stable in culture. (B) Tetherin^low and tetherin^high cells were infected with WT (blue line) or Δvpu (red line) mCherry fluorescent protein-expressing HIV-1, and surface tetherin on the mCherry-positive infected cells was assessed by flow cytometry. The histogram plots show the modulation of surface tetherin on the HIV-infected tetherin^low (left) and tetherin^high (right) cells 48 h after infection. (C) Mean fluorescence intensity (MFI) of surface tetherin in HIV-infected tetherin^low and tetherin^high cells. (D) Western blot showing the levels of HIV-1 protein expression in the tetherin^low and tetherin^high cells infected with WT or Δvpu HIV-1. Samples were normalized to 20% infection, as indicated by mCherry expression. Cellular lysates were probed with polyclonal anti-HIV antisera, an anti-HIV Env, and an anti-mCherry antibody. (E) Tetherin^low and tetherin^high cells (GFP⁺) were infected with WT and Δvpu HIV-1 (mCherry⁺) for 48 h, and the surface tetherin (cyan) localization was assessed by spinning disk confocal microscopy. Maximum intensity projections are displayed.

When examined by confocal microscopy, tetherin surface expression was not detected in the tetherin^low population infected with WT or Δvpu HIV-1 (Fig. 1E). However, reduced levels of tetherin surface expression were observed in tetherin^high cells infected with WT virus compared to uninfected cells. Moreover, tetherin^high cells infected with Δvpu HIV-1 maintained similar levels of tetherin expression compared to uninfected tetherin^high cells. In agreement with previous studies, tetherin was observed to accumulate in large puncta on the surfaces of uninfected and Δvpu HIV-1-infected tetherin^high cells (19).

Binding of anti-HIV antibodies to infected T lymphocytes correlates with tetherin surface expression.

We next examined the binding of a panel of anti-HIV Env antibodies to the surfaces of infected cells. The monoclonal anti-HIV antibodies b12, 2G12 and 4E10 are broadly neutralizing antibodies (48, 49). In addition to the monoclonal antibodies, a patient polyclonal anti-HIV immunoglobulin was also used to examine the level of antibody binding to the surfaces of tetherin^low and tetherin^high cells infected with WT or Δvpu HIV-1 (Fig. 2A). In the tetherin^low cells, no significant differences in the percentages of antibody binding were noted (Fig. 2A and B). The binding of monoclonal antibodies may represent the enhanced levels of Env present on the cell surface and/or the exposure of particular conformations of Env. The weak staining of the 2G12, which binds a glycan moiety and is not conformationally dependent, and the pooled patient IgG, generally indicate that the surface Env levels are not strongly altered by the presence or absence of Vpu in tetherin^low cells. Infection of the tetherin^high cells with WT or Δvpu HIV-1 induced higher levels of anti-HIV antibody binding in comparison to the tetherin^low cells, with the highest levels of antibody binding to the tetherin^high cells infected with Δvpu HIV-1. Similar to the modulation of surface tetherin (Fig. 1B and C), strong anti-HIV antibody binding was only observed in mCherry^high HIV-infected cells. We previously observed that the total level of HIV Env expression in Δvpu HIV-1-infected cells was slightly reduced compared to infection with WT HIV-1 (Fig. 1D); this suggests that the increase in surface Env detected in Δvpu HIV-1-infected cells is not likely attributable to an increase in total Env expression but rather to the tetherin-mediated retention of virus particles on the surfaces of Δvpu HIV-1-infected cells.

FIG 2.

High tetherin expression correlates with anti-HIV antibody binding on HIV-infected lymphocytes. Tetherin^low and tetherin^high cells were infected with WT or Δvpu HIV-1, and the levels of surface antibody binding on HIV-infected cells were assessed by using a panel of broadly neutralizing anti-HIV antibodies or polyclonal patient sera. (A) Surface binding of anti-Env neutralizing antibodies b12, 2G12, and 4E10 and polyclonal anti-HIV IgG sera, as measured by flow cytometry. The percentages of HIV-infected cells binding antibody are shown in boldface. (B) Mean percentages of Env-positive cells from five biological replicates. (C) The mean fluorescence of the Env-positive cells was plotted (average of five independent experiments). (D) The fluorescence index was calculated by multiplying the percentage of infected cells by the MFI index. The graph shows the fluorescent index values of anti-HIV antibody binding to the surfaces of tetherin^high and tetherin^low HIV-1-infected populations. The levels of antibody binding in panels B to D were calculated from five independent staining experiments.

The efficiency of ADCC is dependent on the concentration and stability of antigen expressed on the surfaces of target cells (28). Therefore, the clearance of infected cells depends not only on the frequency of opsonized target cells but also on the density of antibody bound to their surface. We therefore calculated a combined measure of anti-HIV antibody binding to the infected populations by multiplying the percentage of infected cells that bound antibody by their mean fluorescence intensity (MFI). The total amount of anti-HIV IgG, b12, and 4E10 antibody bound to the surfaces of tetherin^high cells infected with Δvpu HIV-1 was approximately 2 orders of magnitude higher than the level of binding to the tetherin^low cells infected with WT and/or Δvpu HIV-1 (Fig. 2D). Moreover, in examining the magnitude of antibody binding, we detected significant differences among all of the infected populations with all the neutralizing antibodies tested. Previously, the monoclonal antibody b12 has been shown to be more efficient than other anti-HIV antibodies (i.e., 2F5 and 4E10) at mediating antibody-dependent cell-mediated viral inhibition (50). For this reason, we utilized the b12 antibody to examine whether Vpu modulates the efficiency of ADCC against HIV-infected cells.

The abundance of surface tetherin expression correlates with Fc receptor signaling, primary NK degranulation, and ADCC.

The FcγRIIIa serves as the primary receptor on NK cells, granulocytes, and macrophages to detect antibody-opsonized targets, and it initiates the signaling cascade that leads to ADCC (28). During viral infections, ADCC is mostly mediated via FcγRIIIa stimulation on NK cells. The Jurkat NFAT-luc⁺ FcγRIIIa (Jur-γRIIIa) effector cell line expresses the FcγRIIIa and an NFAT-sensitive luciferase-reporter that is activated by FcγRIIIa stimulation (Fig. 3A) (46).

FIG 3.

Tetherin surface expression increases FcγRIIIa stimulation. (A) Diagram depicting the FcγRIIIa assay. A Jurkat E6-derived indicator cell line expresses FcγRIIIa. Upon FcγRIIIa stimulation, activation of the NFAT transcription factor induces luciferase expression. Tetherin^low and tetherin^high cells were infected with WT or Δvpu HIV-1 for 48 h and normalized to 15 to 20% infection, as indicated by mCherry expression. Infected populations were then cocultured in the presence or absence of either the WT b12 antibody (B) or a LALA b12 antibody with an altered Fc region that abrogates FcγRIIIa stimulation (C). These populations were cocultured at a 5:1 ratio of FcγRIIIa-expressing effector to HIV-infected target cells for 16 h. After 16 h, the luciferase activity in the lysed cells indicates FcγRIIIa activation. The graphs show the means of results of three independent experiments conducted in duplicate. *, P < 0.05; **, P, < 0.01; ***, P < 0.001.

The tetherin^low and tetherin^high cells were infected with WT or Δvpu HIV-1 and then cocultured with Jur-γRIIIa cells in the presence of increasing concentrations of the b12 antibody. The tetherin^high cells infected with Δvpu HIV-1 yielded 3- to 2-fold-higher levels of FcγRIIIa stimulation compared to the tetherin^low populations infected with WT or Δvpu HIV-1, respectively (Fig. 3B). The tetherin^high cells infected with Δvpu HIV-1 also yielded 70% higher levels of Fc receptor stimulation compared to the tetherin^high cells infected with WT HIV-1 (P < 0.01) (Fig. 3B). The lowest levels of Fc receptor stimulation were observed in response to the tetherin^low cells infected with WT HIV-1 at all b12 antibody concentrations tested (Fig. 3B). Fc receptor signaling stimulated by tetherin^low cells infected with Δvpu HIV-1 was greater than that stimulated by infection with WT HIV-1, at the highest concentration (10 μg/ml) of b12 antibody tested (Fig. 3B). Coculturing the infected populations with the Jur-γRIIIa cells in the absence of b12 did not produce luciferase levels above background (Jur-γRIIIa cells cultured alone).

To examine the specificity of the Fc receptor signaling, we utilized the LALA b12 antibody that contains a mutation in its constant region, which abrogates Fc receptor signaling while maintaining the ability to bind HIV-1 Env (45). Fc receptor signaling in response to the infected populations in both tetherin^low and tetherin^high cells was completely absent in the presence of the LALA b12 (Fig. 3C). This observation demonstrates that tetherin-mediated FcγIIIR stimulation is dependent on antibody Fc region engagement.

A consequence of FcγIIIR signaling is the mobilization and release of lytic granules into antibody-opsonized target cells. During degranulation, LAMP-1 (CD107a), which is normally contained within lytic granules, becomes expressed on the surfaces of NK cells (51). To examine the ability of vpu/tetherin to influence NK cell degranulation, we cocultured the infected cell populations with primary CD3-CD56⁺ NK cells and then measured the accumulation of CD107a on the surfaces of the NK cells (Fig. 4A). We observed a modest but specific 4-fold increase in the level of NK cell degranulation in response to the Δvpu HIV-1-infected tetherin^high cells compared to WT HIV-1-infected tetherin^high cells or WT or Δvpu HIV-infected tetherin^low cells (Fig. 4B and C).

FIG 4.

Tetherin surface expression increases primary NK cell degranulation and ADCC killing. Tetherin^low and tetherin^high cells infected with either WT or Δvpu HIV-1 were cocultured with primary CD56⁺ CD3⁻ NK cells at a 1:10 ratio of infected target to NK cells in the presence or absence of different concentrations of the b12 antibody. (A) Gating scheme of the assays uses CellTracker violet staining to discriminate HIV-infected target cells (Violet⁺) from NK cells (upper left panel). To assess the activation of NK cells, we measured surface CD107a in CD56⁺ cells (bottom panels). To assess infected cell killing, we measured the loss of HIV-infected cells (mCherry⁺) from the violet cells (upper right panel). (B) NK cell activation was measured after coculture with the HIV-infected CD4⁺ cells for 2 h in the presence or absence of b12 antibody. The levels of CD107a degranulation on NK cells indicate activation. (C) Graph representing the fold change in CD107a expression on the surfaces of NK cells in response to HIV-infected tetherin^low and tetherin^high cells. The fold difference was calculated by dividing the levels of CD107a degranulation in the presence of b12 by those observed in the absence of antibody. (D) A primary NK ADCC assay was conducted in which NK cells were cocultured with HIV-infected tetherin^high or tetherin^low cells for 6 h. The levels of specific killing within each HIV-infected population were assessed by flow cytometry. Dot plots depict the levels of NK cell-mediated ADCC in the absence of b12 (left panel) or in the presence of 10 μg of b12/ml (right panel). (E) Graphs represent the percentages of NK cell-mediated killing of tetherin^low and tetherin^high cells infected with WT or Δvpu HIV after exposure to 0.1, 1, or 10 μg of b12 antibody/ml. The primary NK cells used in these assays were derived from four or five different donors. Each dot on the scatter plot represents the mean of technical replicates from each donor. *, P < 0.05; **, P < 0.01.

Next, we examined whether higher levels of Fc receptor signaling and degranulation in response to tetherin^high cells infected with Δvpu HIV-1 rendered these targets more susceptible to NK cell-mediated ADCC. Cell death was measured using a flow cytometry-based killing assay, in which primary NK cells were cocultured with dye-labeled, HIV-infected target cells (Fig. 4A). The levels of killing were assessed by quantifying the specific loss of mCherry^high HIV-infected cells as a proportion of the total target cell population (Fig. 4D). The loss of mCherry^high cells was measured since late viral gene expression and anti-HIV antibody binding predominantly occur in the mCherry^high cells (Fig. 1A). This approach allowed us to detect preferential killing of the HIV-1-infected cells over a background of 30 to 40% killing.

The tetherin^low and tetherin^high cells infected with WT or Δvpu HIV-1 were cocultured with primary NK cells from seven different donors in the absence or presence of the b12 antibody (10, 1, and 0.1 μg/ml). At all of the b12 antibody concentrations tested, the tetherin^high cells infected with Δvpu HIV-1 were more susceptible to primary NK cell-mediated ADCC than either tetherin^high or tetherin^low cells infected with WT HIV-1 (Fig. 4E). At the lowest concentration, tetherin^high cells infected with Δvpu HIV-1 were 8-fold more susceptible to primary NK cell-mediated ADCC than tetherin^high cells infected with WT HIV-1 and ∼20-fold more susceptible than the tetherin^low cells infected with WT HIV-1 (Fig. 4E). These data demonstrate that high tetherin surface expression in conjunction with Δvpu HIV-1 infection induces primary NK degranulation and enhanced ADCC responses against HIV-infected lymphocytes. Thus far, we observed that Fc receptor stimulation correlated well with the levels of ADCC mediated by primary NK cells. Below, we exploited the FcγIIIR assay system to further examine whether tetherin is required to increase the susceptibility of Δvpu HIV-1-infected lymphocytes to ADCC.

The complementation of surface tetherin in an HIV-infected T-cell line increases their capacity to signal through the FcγRIIIa.

Because tetherin^low cells represent a common subpopulation of the Jurkat E6 cell line, we wanted to test whether the phenotypes in this population could be complemented by overexpression of exogenous tetherin. To examine whether the lack of tetherin was the primary reason that the tetherin^low cells fail to activate FcγRIIIa signaling, we cotransfected them with a tetherin expression construct and WT or Δvpu HIV-1. Transfection with WT or Δvpu HIV-1 alone did not induce tetherin surface expression; however, cotransfection with tetherin yielded 4- and 15-fold increases, respectively, in surface tetherin expression on cell populations in which the WT and Δvpu HIV-1 were cotransfected (Fig. 5A). The lower levels of surface tetherin in the WT population reflect Vpu antagonism, whereas the higher levels observed in the Δvpu HIV-1-infected cells represent baseline levels of tetherin expression.

FIG 5.

Impact of tetherin overexpression or mutation of a residue in Vpu required for tetherin downmodulation on infected-cell opsonization and FcγRIIIa signaling. (A) Tetherin^low cells transfected with the tetherin expression construct IRAT show higher levels of tetherin staining. Cells were stained with 5 μg of anti-tetherin APC antibody/ml. Histogram flow cytometry plots depict the level of tetherin in the tetherin^low cells that were nucleofected with WT HIV-1 (upper left), WT HIV-1 plus IRAT (upper right), Δvpu HIV-1 (lower left), or Δvpu HIV-1 plus IRAT (lower right). (B) Dot plots show the levels of surface b12 antibody binding to the surfaces of tetherin^low cells nucleofected with WT HIV-1 (upper left), WT HIV-1 plus IRAT (upper right), Δvpu HIV-1 (lower left), or Δvpu HIV-1 or IRAT (lower right). (C) The nucleofected populations were cocultured at a 5:1 ratio of Jur-γRIIIa effector cells to infected target cells for 16 h. After coculture, the total samples were lysed, and the levels of luciferase activity were measured. The graph depicts the relative light units produced by FcγIIIR-expressing cells in response to HIV-infected and b12-opsonized CD4⁺ populations. (D) The levels of surface NTB-A, PVR and tetherin expression, along with the levels of b12 binding to tetherin^high cells infected with WT, Δvpu, or vpu(A14L) HIV-1 were assessed using surface antibody staining, followed by flow cytometry. Human anti-NTB-A, anti-PVR, and anti-tetherin antibodies were used at 5 μg/ml. (E) Graph showing the levels of FcγRIIIa stimulation in response to WT, Δvpu, or vpu(A14L) HIV-infected tetherin^high cells. The data are from a representative experiment performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Anti-HIV antibody binding to these tetherin^low cells cotransfected with HIV WT and tetherin expression vector revealed a 1.7-fold higher percentage of b12 binding to cotransfected cells compared to cells transfected only with WT HIV-1 (Fig. 5B). The percentage of tetherin-positive cells was 2.8-fold higher in the tetherin^low cells cotransfected with tetherin and Δvpu HIV-1 compared to cells transfected with Δvpu HIV-1 alone (Fig. 5B). The fluorescence index of the antibody-binding cells, calculated as the product of the percentage of HIV antibody-binding cells and the mean fluorescence signal in these cells, showed that the greatest antibody staining was present in cells cotransfected with the tetherin expression vector and the HIV Δvpu viral construct (Fig. 5C). When these populations were cocultured with the Jur-γRIIIa cells, only the tetherin and Δvpu HIV-1-cotransfected cells stimulated FcγRIIIa signaling in a b12 antibody dose-dependent manner (Fig. 5D). These results suggest that the expression of tetherin alone was sufficient to restore greater anti-HIV antibody binding and the Fc-stimulating capacity of tetherin^low cells transduced with Δvpu HIV-1.

The specific loss of vpu antagonism for tetherin increases the capacity of HIV-1-infected CD4⁺ T cells to signal through the FcγRIIIa (CD16).

In addition to downmodulating tetherin, vpu has previously been shown to have effects on regulating cell surface CD4, CD1d, NK T and B cell antigen (NTB-A), and poliovirus receptor (PVR) (52–54). Therefore, we investigated whether a mutation in vpu that specifically affects the ability to antagonize tetherin would enhance the ability of HIV-infected lymphocytes to stimulate antibody-dependent FcγRIIIa signaling.

It has been reported that an alanine-to-leucine point mutation in Vpu at amino acid 14 (A14L) causes a loss of tetherin antagonism while maintaining its ability to downmodulate CD4 (55). We first examined the effect of this mutation on tetherin and b12 antibody binding. Infection of the tetherin^high cells with the HIV vpu mutation A14L [vpu(A14L)] abrogated the ability of Vpu to antagonize tetherin surface expression to the same level as infection with Δvpu HIV-1 (Fig. 5E). The level of anti-HIV antibody binding to the tetherin^high cells infected with vpu(A14L) HIV-1 was significantly higher than that of WT HIV-1 (Fig. 5E). The magnitude of b12 antibody binding was similar to that observed with Δvpu infections (Fig. 5E). In addition, we examined the effect of the A14L mutation on NTB-A and PVR expression. Since CD1d is primarily expressed on dendritic cells and only affects NKT cells, we focused on NTB-A and PVR. Infection of tetherin^high cells with the vpu(A14L) HIV-1 mutant did not affect either NTB-A or PVR surface expression compared to infection with WT or Δvpu HIV-1 (Fig. 5E).

Coculturing vpu(A14L) HIV-1-infected tetherin^high cells with Jur-γRIIIa cells resulted in a 2-fold increase in the level of FcγRIIIa stimulation compared to WT (Fig. 5F). The level of FcγRIIIa signaling was comparable to that induced by tetherin^high cells infected with Δvpu HIV-1 (Fig. 5F). These results demonstrate that the ability of Vpu to antagonize tetherin is important for the antibody opsonization of HIV-infected cells, which in turn increases FcγRIIIa signaling.

b12 antibodies with stronger binding to FcγRIIIa (CD16) mediate stronger Fc signaling by HIV-infected cells.

Another important factor in mediating the efficiency of ADCC is the capacity of the Fc portion of an antibody to signal through the FcγRIIIa. In particular, b12 antibodies with specific amino acid substitutions in their Fc regions have previously been shown to influence the levels of FcγRIIIa binding and ADCC (45). b12 antibodies with amino acid substitutions at residues 239 and 332 (double mutant) or residues 239, 332, and 330 (triple mutant) can mediate greater binding to the FcγIIIR and higher levels of ADCC compared to WT b12 antibody (45). Tetherin^high cells infected with either WT or Δvpu HIV-1 were cocultured with FcγRIIIa-expressing cells in the presence of WT b12, the b12 double mutant (S239D/I332E), or the b12 triple mutant (S239D/I332E/A330L). Titrations with these b12 antibodies resulted in higher levels of Fc receptor stimulation in response to the tetherin^high cells infected with Δvpu HIV-1 compared to WT HIV-1 (Fig. 6A, B, and C). Moreover, higher levels of FcγRIIIa stimulation were detected in response to the b12 double and triple mutants compared to the WT b12 antibody (Fig. 6A, B, and C). The highest levels of FcγRIIIa signaling were observed in response to Δvpu HIV-1-infected cells incubated with the b12 triple mutant, with an 18-fold higher level of FcγRIIIa stimulation in response to Δvpu HIV-1-infected cells compared to infection with WT HIV-1 (Fig. 6C). Interestingly, an ∼2-fold difference in FcγRIIIa signaling observed between tetherin^high cells infected with Δvpu HIV-1 versus WT HIV-1 at a 1-μg/ml concentration of WT b12 antibody was amplified to a 6.5-fold difference at the same concentration of the b12 triple mutant (Fig. 6A and C). Moreover, differences in FcγRIIIa signaling were maintained with the triple mutant at lower antibody concentrations (i.e., 0.01 μg/ml) in contrast to WT b12 antibody (Fig. 6C). Therefore, we observed that the tetherin-mediated activation of FcγRIIIa signaling was increased by mutations that are known to enhance the binding of antibodies to FcγRIIIa.

FIG 6.

The magnitude of tetherin-enhanced FcγRIIIa stimulation is modulated by changes in antibody Fc regions that affect FcγRIIIa binding. Tetherin^high CD4⁺ cells were infected with WT or Δvpu HIV-1 and 48 h later normalized to 20% infection. The infected populations were then incubated in the presence of WT b12 or b12 double (S239D/I332E) or triple (S239D/I332E/A330L) Fc mutants that confer enhanced binding and signaling through the FcγRIIIa (A to C). Graphs depict the levels of FcγRIIIa signaling in response to tetherin^high cells infected with WT or Δvpu HIV incubated with a titration of either WT b12 (A) b12 double-mutant (B), or b12 triple-mutant (C) antibodies. (D) Primary CD4⁺ T cells were activated for 3 days and then infected with WT or Δvpu HIV-1. At 4 days after infection, the populations were normalized to 10% infection and cocultured with the Jur-γRIIIa cells at a 5:1 effector/target ratio for 16 h. The graph depicts the results from a representative experiment showing the levels of FcγIIIR stimulation in response to primary CD4⁺ lymphocytes cultured with either WT or Δvpu HIV-1. The data are of representative experiments conducted in triplicate.

We next examined the b12 triple mutant for its ability to stimulate FcγRIIIa in response to infection of primary CD4⁺ T cells with Δvpu or WT HIV-1. Activated primary CD4⁺ T cells that expressed uniformly high tetherin levels were infected with either Δvpu or WT HIV-1. Infected cells were then cocultured with the Jur-γRIIIa cells in the absence or presence of the b12 triple mutant antibody. At all of the antibody concentrations tested, infection with Δvpu HIV-1 yielded 5- to 23-fold higher levels of FcγRIIIa stimulation compared to WT HIV-1-infected primary CD4⁺ cells (Fig. 6D).

Higher surface tetherin levels correlate with enhanced patient antibody binding to infected lymphocytes and FcγRIIIa signaling.

We next examined the extent to which the binding of HIV-positive-patient-derived antibodies to the surfaces of HIV-1-infected lymphocytes was influenced by tetherin surface expression. Polyclonal IgG was isolated from the sera of three HIV-positive and two HIV-negative donors and incubated with tetherin^low and tetherin^high cells infected with WT or Δvpu HIV-1 (Fig. 7A). In the tetherin^low cells, no significant differences in either the percentage or mean fluorescence intensity (MFI) of antibody binding were detected with either HIV-negative- or HIV-positive-patient-derived IgG antibodies. In the tetherin^high cells, higher levels of antibody binding (% and MFI) were detected in Δvpu HIV-1-infected cells compared to cells infected with HIV-1 WT. This effect was observed with all three HIV-positive-patient-derived polyclonal IgGs but not from the IgGs derived from the two HIV-negative donors (Fig. 7A).

FIG 7.

Higher tetherin surface expression correlates with enhanced HIV-positive-patient-derived IgG opsonization of HIV-infected T cells and enhanced Fc receptor stimulation. Tetherin^low and tetherin^high cells were infected WT or Δvpu HIV-1, and the levels of antibody binding were quantified by using purified polyclonal IgG antibodies derived from two HIV-negative and three HIV-positive donors. (A) Overlapping histograms show the levels of donor-derived IgG surface binding, as measured by flow cytometry in tetherin^low and tetherin^high cells that were infected with WT or Δvpu HIV-1. (B) Graphs depicting the levels of FcγRIIIa signaling in response to tetherin^low and tetherin^high cells infected with WT or Δvpu HIV-1 and incubated with a titration of different HIV-negative (left) or HIV-positive (right) donor-derived IgGs.

To detect the level of FcγRIIIa stimulation induced by the different donor-derived antibodies, we cocultured the tetherin^low and tetherin^high cells infected with WT or Δvpu HIV-1 with Jur-γRIIIa cells in the presence of increasing concentrations of the donor-derived IgG antibodies. Significantly higher levels of FcγRIIIa stimulation were detected in the tetherin^high population infected with Δvpu HIV-1 compared to the tetherin^high cells infected with WT HIV-1 or tetherin^low populations infected with WT or Δvpu HIV-1, in response to all three HIV-positive patient-derived antibodies when tested at the 10-μg/ml concentration (Fig. 7B). Of note, significant stimulation of FcγRIIIa was not observed in response to the infected populations cultured in the presence of antibodies derived from HIV-negative donors (Fig. 7B).

Higher surface tetherin levels correlate with enhanced patient antibody binding to infected lymphocytes and FcγRIIIa signaling in primary HIV-infected lymphocytes.

We next sought to determine whether the tetherin expression on HIV-infected primary CD4⁺ T cells correlated with the ability of these cells to bind polyclonal anti-HIV antibodies and to stimulate Fc receptor signaling. Primary activated CD4⁺ T cells from three different HIV-negative donors were infected with either WT or Δvpu HIV-1, and the level of antibody binding was quantified. None of the control HIV-negative antibodies bound to the surfaces of the infected primary CD4⁺ T cells (Fig. 8A, left). All three HIV-positive patient-derived IgGs yielded significantly higher levels of antibody binding (% and MFI) to the surfaces of cells infected with Δvpu HIV-1 compared to WT HIV-1 (Fig. 8A, right). Interestingly, the differences in IgG antibody binding in both WT and Δvpu HIV-infected cells correlated well with the levels of tetherin surface expression that was retained on the different HIV-infected primary T cells (Fig. 8B). There was a robust, linear relationship between tetherin expression and HIV IgG binding on infected cells (Pearson correlation r > 0.94, R² > 0.9, and P < 0.005). The level of antibody binding appeared to be influenced both by the baseline expression of surface tetherin, and the magnitude of Vpu-mediated downmodulation of tetherin from the surfaces of primary CD4⁺ T cells.

FIG 8.

Tetherin surface expression correlates with enhanced HIV-positive-patient-derived IgG opsonization of HIV-infected T cells and enhanced Fc receptor stimulation in primary CD4⁺ T cells. (A) Primary CD4⁺ T cells isolated from three HIV-negative donors (D3890, D8800, and D7300) were activated for 2 days and infected with WT or Δvpu HIV-1. Two days after infection, the populations were normalized to 20% infection, and the levels of surface IgG binding were assessed. Overlapping histograms indicate the levels of surface IgG bound to primary CD4⁺ T cells infected with WT or Δvpu HIV-1 and incubated with a titration of different HIV-negative (left)- and HIV-positive (right)-donor-derived IgGs. (B) Graphs illustrate the correlation between the levels of antibody binding and the levels of tetherin surface expression in WT or Δvpu HIV-1-infected cells that were incubated with the HIV-positive-donor-derived IgG. The Spearman correlation was calculated, and a linear regression curve was plotted showing the r and P values for each graph. (C) Panels depict the levels of FcγRIIIa stimulation in primary CD4⁺ T cells infected with WT or Δvpu HIV-1 and incubated with a titration of HIV-positive-donor-derived IgGs from three different donors. Asterisks mark individual donors that yielded statistically significant differences between signaling stimulated by WT HIV-1 (open symbols)- and Δvpu HIV-1 (open symbols)-infected cells. (D) Cells from three different HIV-negative donor cells were treated with HIV-positive-patient-derived IgGs from three different patients, following infection with WT or Δvpu HIV-1. The enhancement of Fc receptor activation in the absence of Vpu is measured by the Wilcoxon matched-pair test at each concentration of antibody tested. (E) Panels show the positive correlation between the levels of FcγRIIIa signaling and the levels of tetherin surface expression on HIV-1-infected primary CD4⁺ T cells. WT and Δvpu HIV-infected cells are intermixed in the graph, showing a strong positive correlation between surface tetherin levels and Fc receptor signaling at 10, 1, or 0.1 μg of patient IgG/ml. A linear regression curve is plotted with the P values indicated (*, P < 0.05; ***, P < 0.001).

To examine the level of FcγRIIIa signaling induced by WT and Δvpu HIV-1-infected primary cells, the infected cells were cocultured with the Jur-γRIIIa cells in the presence of increasing concentrations of the control HIV-negative or HIV-positive donor-derived antibodies. Neither of the HIV-negative antibodies yielded different levels of FcγRIIIa stimulation in response to any of the infected populations (data not shown). All three HIV-positive patient IgGs (donors 1, 2, and 3) yielded higher levels of Fc receptor stimulation in response to Δvpu HIV-1 infection compared to the WT HIV-1-infected cells. Statistically significant differences for over half of the Δvpu HIV-1 versus WT HIV-1 comparisons were observed at the 1.0- and 10-μg/ml concentrations (Fig. 8C). We used a matched-pair analysis wherein the different CD4⁺ donor cells and donor HIV-positive IgGs were considered in aggregate, directly examining the impact of Vpu on Fc receptor signaling, and the lack of Vpu expression in the infected cells gave rise to a significant increase in Fc signaling at all of the concentrations tested (Fig. 8D).

Since the levels of antibody binding appeared to correlate with the level of surface tetherin expressed among the different infected populations, we plotted the absolute level of Fc receptor stimulation in relation to the level of tetherin surface expression in WT HIV-1- and Δvpu HIV-1-infected cells (Fig. 8E). Each of the three HIV-positive-patient-derived antibodies was tested on different primary cell target cells, and we observed a strong positive correlation between the level of Fc receptor stimulation and the level of tetherin surface expression in infected cells.

DISCUSSION

ADCC is postulated to be a critical component of protective immune responses against HIV-1/SHIV infections (34, 35, 38, 56–58). In the present study, we show that surface expression of the type I IFN-inducible factor tetherin enhances the presentation of viral antigen on the surfaces of HIV-infected lymphocytes, thereby increasing HIV-antibody binding. This enhanced opsonization rendered HIV-infected lymphocytes more potent stimulators of Fc signaling with greater susceptibility to NK cell-mediated killing via ADCC. The ability of Vpu to diminish tetherin expression on the surfaces of infected cells may protect infected cells from antibody-dependent, FcγR-mediated NK cell killing.

A critical component in mediating ADCC is the presentation of viral antigens on the surfaces of infected cells. During infection with WT HIV-1, Env glycoproteins are expressed at low density on the surfaces of infected cells due to endocytosis or gp120 shedding (59, 60). In the absence of Vpu, we found that tetherin was able to present higher levels of HIV-1 antigens on the surfaces of infected cells, which in turn led to enhanced antibody opsonization (Fig. 1A). This suggests that tetherin could play a major role in enhancing the otherwise limited expression of HIV Env on the surfaces of infected cells. In addition, we observed that surface tetherin induces the formation of large puncta of viral aggregates, which were not present in the absence of tetherin or when tetherin-expressing cells were infected with viruses expressing vpu. Since cross-linking of FcγRIIIa induces signaling (28), the formation of focal aggregates of antibody bound tethered virus may further enhance ADCC by acting as a concentrating mechanism for FcγRIIIa signaling.

All three broadly neutralizing HIV antibodies tested bound to the greatest extent the tetherin^high cells infected with Δvpu HIV-1. Even though the monoclonal b12, 2G12, and 4E10 antibodies have broad and potent capacity to neutralize cell-free infection (48, 49, 61), these antibodies bound infected lymphocytes to various degrees, suggesting that the levels of antibody opsonization depend on the epitope recognized. 2G12 recognizes a largely conformation-insensitive glycan-based epitope, so it may more accurately reflect the total amount of Env expressed on the surfaces of infected cells. The b12 antibody binds the CD4 binding site, while the 4E10 recognizes a fusion-induced intermediate in the membrane-proximal external region, so variations in these antibodies may also reflect some degree of enhanced epitope exposure. The degree to which Env conformations vary between plasma membrane-embedded Env versus that on tethered virus particles is still unknown. Since the b12 antibody has previously been demonstrated to mediate efficient antibody-dependent cytotoxic viral inhibition responses (50), we further tested this antibody. We found that increased b12 antibody binding correlated with higher levels of FcγRIIIa signaling, NK cell degranulation, and NK cell-mediated ADCC.

In the tetherin^low cell line it is possible that deficiencies other than the low tetherin expression may reduce the levels of Env and/or virus particles on the surfaces of the cells. Overexpressing tetherin in the tetherin^low cells along with Δvpu HIV further increased antibody binding and FcγRIIIa signaling, suggesting that the low tetherin surface expression is a major determinant of the antibody phenotype in these cells. It was also important to determine whether the ability of Vpu to antagonize tetherin expression was required to evade ADCC responses. Vpu has many putative functions, but a leucine substitution mutation (A14L) within the transmembrane domain of Vpu specifically abrogates tetherin antagonism while maintaining its ability to antagonize CD4 (55). We show here that the vpu(A14L) mutant does not modulate NTB-A or PVR expression in the tetherin^high cells, where the levels of these proteins were low or undetectable, respectively. In contrast, we observed that high levels of tetherin surface expression were maintained after infection with vpu(A14L) HIV-1. Consequently, higher levels of b12 antibody binding were observed in the vpu(A14L) HIV-1-infected population compared to those infected with WT HIV-1. Furthermore, FcγRIIIa signaling induced by lymphocytes infected with vpu(A14L) HIV-1 was similar to that triggered by cells infected with Δvpu HIV-1. We conclude that in this system the ability of vpu to modulate NTB-A or PVR was not required to evade ADCC responses.

Previous studies have demonstrated the importance of antibody Fc regions in dictating the magnitude of ADCC activity. When bound to tetherin^high Δvpu HIV-1-infected cells, the b12 double and triple mutants exhibited enhanced FcγRIIIa stimulation relative to the WT b12 antibody. Furthermore, primary CD4⁺ T cells infected with Δvpu HIV-1 in the presence of the b12 triple mutant had a significantly higher capacity to mediate FcγRIIIa signaling compared to WT HIV-infected cells. These results indicate that the magnitude of tetherin-mediated FcγRIIIa stimulation can be enhanced by antibodies with greater affinity for the Fc receptor.

Importantly, the loss of Vpu increased anti-HIV antibody opsonization and Fc receptor signaling by primary infected CD4⁺ T cells using IgG antibodies isolated from HIV⁺ patients. Baseline tetherin surface expression on primary CD4⁺ T cells and the ability of Vpu to antagonize tetherin varied in different donor cells; however, the presence of vpu decreased surface tetherin expression in all HIV-infected CD4⁺ T cells. We observed that higher levels of tetherin expression correlated with enhanced Fc receptor stimulation, which further correlated with the level of HIV patient antibody binding to the surfaces of HIV-1-infected target cells. Interestingly, these correlations grew more significant as the concentration of antibodies increased. We conclude that Vpu antagonism of tetherin can inhibit the opsonization and detection of HIV-infected lymphocytes as the concentrations of HIV-specific antibodies increase during the course of infection.

Two very recent publications propose that CD4 downregulation by nef and vpu decreases the exposure of CD4 induced (CD4i) epitopes on HIV Env at the cell surface. These studies found that the lack of Nef or Vpu increased CD4i antibody binding to HIV-infected cells and ADCC (62, 63). Interestingly, Pham et al. reported that the deletion of vpu alone did not have a major impact on CD4 downmodulation, yet in experiments with HIV-positive patient antibodies, vpu deletion had a major impact on both antibody binding and ADCC. This may indicate that the lack of vpu, and likely its antagonism of tetherin, contribute to the majority of antibody binding and ADCC against HIV-infected lymphocytes.

The ability of tetherin to increase the surface density of Env by interfering with particle release appears to have variable effects on the cell-to-cell transmission of HIV-1. On the one hand, tetherin increases the amount of Env at the cell surface, which may increase cell-cell interactions and virological synapse formation, while on the other hand, the infectivity of the synapse formed may be decreased (19–21). Given the variable impact of vpu on cell to cell spread, it is compelling to consider other important functions of Vpu that relate to its impact on immune functions. We have demonstrated here that Vpu antagonism of tetherin can contribute to immune evasion from ADCC, suggesting that a central role for Vpu in vivo is the evasion of infected cell clearance. In line with this supposition, four of the five surface molecules, which now include tetherin, that are reported to be antagonized by Vpu naturally function to modulate the efficiency of NK cell responses during HIV infection (i.e., tetherin, NTB-A, CD1d, and PVR). Taken together, these findings suggest that the principal function of vpu in vivo is the evasion of antibody- and NK cell-mediated immune clearance mechanisms.

There is strong evidence indicating that ADCC responses can provide protective immunity against HIV-1 (64–66). Understanding that tetherin and Vpu can modulate antibody-dependent cellular responses allows us to consider approaches to enhance vaccines or antibody-based therapies. Because Vpu can limit the detection and killing of HIV-infected cells by ADCC, targeting Vpu in an ADCC-inducing vaccine strategy may increase viral antigen presentation by reducing the capacity of Vpu to antagonize tetherin. Intriguingly, ADCC responses in LTNP/elite controllers have already been observed to be broadly targeting and, in some cases, have been shown to target epitopes in Vpu (36, 47). It would be interesting to examine whether targeting Vpu in vaccines may exert immune pressure on the regions required to antagonize tetherin, thereby acting synergistically to improve the ADCC responses in these patients.

In the present study, we have demonstrated that tetherin can facilitate HIV-infected cell clearance through ADCC. The broader implication of these findings is that tetherin may also act on other viral infections to aid in the detection and clearance of infected cells through antibody-mediated mechanisms. In addition to ADCC, it is important to consider that the antibody Fc region can modulate various cellular effector functions, including cytokine release, phagocytosis, and the release of virus-inhibiting chemokines. Tetherin-mediated opsonization may also induce complement-dependent cytotoxicity. In addition, recent studies have implicated tetherin as an innate immune signaling receptor that functions through NF-κB pathways (67, 68). Thus, IFN-induced tetherin surface expression is likely to function as an important immune modulator during viral infections, acting as a bridge between the innate and adaptive immune responses.