Abstract
We previously showed that expression of human FcγRI on TZM-bl cells potentiates neutralization by gp41 membrane-proximal external region (MPER)-specific antibodies. Here we show that lysosomotropic reagents known to block phagocytosis do not diminish this effect. We also show that FcγRI occasionally potentiates neutralization by antibodies against the V3 loop of gp120 and cluster I of gp41. We conclude that FcγRI provides a kinetic advantage for neutralizing antibodies against partially cryptic epitopes independent of phagocytosis.
TEXT
Many cells of the immune system express Fc receptors that bind immune complexes through the constant region of immunoglobulins to mediate a variety of innate and acquired immune defense mechanisms (1, 2). In the case of cell-free HIV-1, FcγRs have been implicated in both infection enhancement (3) and virus destruction by phagocytosis (4, 5, 6). To study the impact of the four major Fcγ receptors on cell-free HIV-1 in greater detail, we stably expressed each of the cDNAs for the human FcγRs on TZM-bl cells. Using these new cell lines, we assayed a panel of HIV-1-positive sera and monoclonal antibodies (MAbs) for neutralization potency against several strains of HIV-1. We showed that expression of FcγRI/CD64 had a profound effect on HIV-1 neutralization by the gp41 membrane-proximal external region (MPER)-specific MAbs 2F5 and 4E10 (7). In the presence of FcγRI expression, neutralization titers for 4E10 and 2F5 were increased as much as 5,000-fold depending on the virus used. Given that both 2F5 and 4E10 have been shown to prevent viral infection in a macaque model (8) and that both MAbs are broadly neutralizing, an understanding of their neutralizing mechanism may be beneficial for vaccine development.
As we proposed previously, at least two mechanisms might account for the observed positive effect of FcγRI on HIV-1 neutralization. One would involve the pre-positioning of Abs at the cell surface, thereby giving them a kinetic advantage in accessing epitopes that are more-readily exposed after virus attachment. This mechanism is supported by the finding that cell-free HIV-1 immune complex formation was not required prior to Ab-FcγRI engagement in order for FcγRI to augment the neutralization activities of 2F5 and 4E10 (7).
A second mechanism that may potentially facilitate HIV-1 neutralization is phagocytosis. HeLa cells, from which the TZM-bl cell line was developed, are known to exhibit properties of nonprofessional phagocytes (9, 10, 11). Several mammalian cells have been shown to acquire phagocytic properties upon expression of FcγRs (12, 13, 14). Thus, surface expression of FcγRs may, in principle, turn TZM-bl cells into professional phagocytes.
Macrophages, dendritic cells, and neutrophils use phagocytosis to internalize particles larger than 0.5 μm. This process can be triggered by diverse membrane components, such as scavenger receptors, complement receptors, and Fc receptors (13, 14). During phagocytosis, internalized particles are trafficked into a complex of membrane-bound structures known as phagosomes (15). At the end of the phagocytic process, particle degradation requires phagosome maturation, a process by which these vesicles become increasingly acidified by acquiring different proteins and eventually fuse with acidic lysosome structures (15).
The antibiotic bafilomycin A1 and the weak base chloroquine are well-characterized compounds known to block acidification and protein degradation in lysosomes (16, 17, 18). We used these drugs to test whether phagocytosis facilitates HIV-1 neutralization in cells expressing FcγRI. The drugs were used at the highest concentrations that were nontoxic to TZM-bl and TZM-bl/FcγRI cells. To verify that the drugs were capable of blocking lysosomal acidification, we tested the ingestion of pHrodo Escherichia coli BioParticles (Invitrogen, Carlsbad, CA) by TZM-bl/FcγRI cells in the presence and absence of each drug. In this phagocytosis assay, the fluorescence of the pHrodo dye dramatically increases as the pH of the surroundings becomes acidic (15). Cells were treated for 4 h with either 1 nM bafilomycin A1 or 50 μM chloroquine at 37°C (Sigma-Aldrich, St. Louis, MO), trypsinized, washed with phosphate-buffered saline, and incubated with pHrodo E. coli at 37°C for 15 min. Cells were then washed, fixed with 1% formaldehyde, and analyzed by flow cytometry using a BD FACSCalibur analyzer (BD Biosciences). Compensation and analysis were performed by using FlowJo software (Tree Star). Approximately 50% of the TZM-bl/FcγRI cells were positive in the absence of drug, whereas only 25% were positive in the presence of drug, demonstrating the capacity of these lysosomotropic agents to block phagocytosis in this cell line.
To test for an effect on neutralization, TZM-bl cells and TZM-bl/FcγRI cells were pretreated for 4 h with either 1 nM bafilomycin A1 or 50 μM chloroquine at 37°C and used in standard neutralization assays (7) in the continued presence of the drugs. Two HIV-1 subtype B Env-pseudotyped viruses (6535.3 and QH0692.42) were assayed with 2F5, 4E10, and IgG1b12. As shown in Fig. 1, expression of FcγRI dramatically improved the neutralizing activity of 2F5 and 4E10 against both viruses in the absence of lysosomotropic agents. Moreover, neither lysosomotropic agent showed any evidence of reversing the FcγRI-mediated effect on 2F5 and 4E10. As reported previously, FcγRI had no effect on the neutralizing activity of IgG1b12 (7), and the activity of this MAb was unaltered by the lysosomotropic agents.
Fig 1.
Effect of lysosomotropic agents on HIV-1 neutralization in TZM-bl and TZM-bl/FcγRI cells. Cells were pretreated with either 1 nM bafilomycin A1 or 50 μM chloroquine as described in the text and then used to test neutralization of HIV-1 subtype B Env-pseudotyped viruses 6535.3 and QH0692.42 (both produced in 293T cells) by 2F5, 4E10, and IgG1b12 in the continued presence of the lysosomotropic agents. TZM-bl/FcγRI cells that were untreated or treated with either chloroquine or bafilomycin are shown as closed circles, closed squares, or closed triangles, respectively. Corresponding conditions with TZM-bl cells are shown as open circles, open squares, and open triangles, respectively.
The results presented here indicate that phagocytosis is not a mechanism responsible for the enhanced neutralizing activity of MPER-specific antibodies in TZM-bl/FcγRI cells. Though the lysosomotropic agents inhibited the phagocytosis of pHrodo E. coli BioParticles by only 50% under the conditions used (conditions that were clearly free of toxic effects to the cells), we saw no reduction in HIV-1-neutralizing activity in their presence. It remains possible that immune complexes may be trapped at the cell membrane in partial phagocytic cups induced by the incomplete activation of the FcγRs (19).
A recent study showed that activation of FcRs by immune complexes blocked the replication of HIV-1 and related primate lentiviruses in human macrophages (20). This activation restricted HIV-1 reverse transcription and integration but not viral entry, nuclear import, or gene expression from integrated proviruses and was not due to enhanced degradation of incoming viral proteins. This FcR-mediated restriction of lentiviruses also appeared to involve the induction of the cyclin-dependent kinase inhibitor p21Cip1/WAF1. It is unlikely that a similar mechanism may be responsible for the efficient neutralization of HIV by MPER MAbs because this phenomenon does not occur with every immune complex, as was observed in the FcR-mediated restriction of lentiviruses in macrophages (20).
A more likely mechanism for enhanced Ab-mediated neutralization of HIV-1 in TZM-bl/FcγRI cells is that the Fc receptor pre-positions Abs at the virus surface, thereby breaching the steric barrier at the virus-cell interface (21) for Abs whose epitopes are not fully revealed until the Env glycoprotein spike changes conformation upon receptor and coreceptor binding (22–25). We note that CD89 (FcαR1) expression on TZM-bl cells does not potentiate the neutralizing capacity of the IgA version of 2F5 (our unpublished results). This finding suggests that the structure of IgG and the characteristics of its interaction with FcγRI are most favorable for the pre-positioning effect. The unusual very long third complementarity-determining region of the heavy chain, CDR H3, of these MAbs may contribute to this property (26).
The FcγRI effect on 2F5 and 4E10 has been seen with every HIV-1 isolate we have tested that is sensitive to these MAbs, and we have seen a similar effect with other MPER MAbs, including CAP206-CH12 (27) and m66 (28). Previously, however, we had not seen an effect with Abs against other epitopes (7). We have now tested a larger number of MAbs against four additional tier 2 viruses and show that FcγRI can occasionally augment the neutralizing activity of MAbs against the V3 loop of gp120 and against cluster I epitopes in gp41 (Table 1), although not with the same intensity and frequency seen with MPER MAbs. The V3-specific MAb 447-52D and the gp41 cluster I-specific MAbs 240-D, 256-D, and 50-69 have been shown to inhibit replication of HIV-1 strains BaL, Bx08, and TV1 in human macrophages and immature dendritic cells expressing FcγRs but not in human peripheral blood mononuclear cells (5, 6).
Table 1.
Occasional effect of FcγRI expression on the neutralizing activity of MAbs against the V3 loop of gp120 and cluster I of gp41 in TZM-bl cells
| MAb | Epitope | Reference | IC50 (μg/ml) in TZM-bl and TZM-bl/FcγRI cells of virusa: |
|||
|---|---|---|---|---|---|---|
| 6535.3 | QH0692.42 | SC22.3C2.LucR | RHPA.LucR | |||
| 1361 | V2 | 29 | >25/13.8 | >25/>25 | >25/>25 | >25/>25 |
| 1393A | V2 | 30 | 16.8/16.3 | >25/>25 | >25/>25 | >25/>25 |
| 1357D | V2 | 29 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 697-30D | V2 | 31 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 830A | V2 | 30 | >23/>23 | >23/>23 | >23/>23 | >23/>25 |
| 2297 | V2 | 32 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 2219 | V3 | 33 | 1.1/0.07 | 13.2/0.45 | >25/>25 | >25/0.4 |
| 3869 | V3 | 34 | 2.0/0.3 | 21.9/6.2 | >25/>25 | >25/0.9 |
| 447-52D | V3 | 35 | 0.3/0.05 | 7.8/0.9 | >25/>25 | >25/11.8 |
| 3074 | V3 | 36 | >25/10.6 | >25/17.7 | >25/>25 | >25/>25 |
| 654-30D | CD4bs | 37 | >25/22.3 | >25/>25 | >25/>25 | >25/>25 |
| 1008-30D | CD4bs | 38 | 22.0/>25 | >25/>25 | >25/>25 | >25/>25 |
| 729-30D | CD4bs | 39 | 16.8/14.6 | >25/>25 | >25/>25 | >25/>25 |
| 1331-160A | C5 | 29 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 670-30D | C5 | 39 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 858-30D | C5 | 39 | >22.5/>22.5 | >22.5/>22.5 | >22.5/>22.5 | >22.5/>22.5 |
| 240-D | gp41 cluster I | 40 | >25/0.27 | >25/>25 | >25/1.0 | >25/23.2 |
| 246-D | gp41 cluster I | 40 | >25/>25 | >25/>25 | >25/11.9 | >25/>25 |
| 50-69D | gp41 cluster I | 41 | >25/0.78 | >25/>25 | >25/6.4 | >25/>25 |
| 181D | gp41 cluster I | 40 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 126-7D | gp41 cluster II | 40 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
| 167D | gp41 cluster II | 40 | >25/>25 | >25/>25 | >25/>25 | >25/>25 |
Results are shown as IC50 for TZM-bl cells/IC50 for TZM-bl/FcγRI cells. Positive neutralization is shown in boldface type. Differences that were >3-fold between TZM-bl and TZM-bl/FcγRI cells are underlined (3-fold differences exceed the normal variation of the assay). Viruses 6535.3 and QH0692.42 were used as Env-pseudotyped viruses. Viruses SC22.3C2 and RHPA were used as infectious molecular clones that expressed the entire Env ectodomain of the designated HIV-1 strain (Env.IMC.LucR viruses) (42).
Given these results, we believe that FcγRI can augment the conventional entry-inhibiting neutralizing activity of Abs against multiple epitopes on gp120 and gp41 that tend to be poorly exposed prior to virus attachment. In this regard, the TZM-bl/FcγRI cell line appears to be an interesting tool for mechanistic studies of HIV-1 neutralization as well as for monitoring vaccine-elicited neutralizing Ab responses. It remains to be determined whether FcγRI-augmented neutralizing activity of HIV-1-specific Abs in vitro has potential value for vaccine protection.
ACKNOWLEDGMENTS
We thank the Duke Center for AIDS Research (CFAR) Flow Cytometry Facility for cell analyses.
This work was supported by the National Institutes of Health (HL59725 to S.Z.-P.), the Center for HIV/AIDS Vaccine Immunology (CHAVI), The Bill and Melinda Gates Foundation's Collaboration for AIDS Vaccine Discovery, and the Duke Center for AIDS Research.
Footnotes
Published ahead of print 13 February 2013
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