HIV transmission across synapses formed by CD4 T cells and dendritic cells appears to be resistant to neutralization by antigp41 antibodies . More controversial data exist on the effect of antigp41 antibodies on HIV spread between T cells, through contacts between productively infected and target CD4 T cells . In this case, cell-to-cell fusion is sensitive , whereas HIV transfer to target cells is resistant to neutralization . Moreover, little is known on the effect of neutralizing antibodies (nAbs) on trogocytosis, a mechanism of cellular communication that involves antigen and membrane exchange between the synaptic partners .
A reduced number of broadly nAbs, effective against a wide spectrum of HIV strains, have been isolated. Among them, IgGb12, 2G12, 4E10 and 2F5 are the best characterized . IgGb12 and 2G12 are directed against the CD4 binding site and a carbohydrate epitope in gp120, respectively. 2F5 and 4E10 bind adjacent epitopes located on the membrane proximal external region of gp41. The infusion of 2G12, 4E10 and 2F5 to HIV-infected patients selected HIV escape mutants to 2G12  but raised concerns about the in-vivo function of the antigp41 nAbs . The pre-eminent role of virological synapses in HIV spread in vivo might account for this lack of activity [2,9].
By analyzing the effect of nAbs on virological synapses, we show that IgGb12, but not antigp41 nAbs, block cellular contacts, endocytic virus transmission and trogocytic antigen exchange between synaptic partners. Despite these mechanistic differences, all nAbs efficiently blocked infectious HIV spread between T cells.
Materials and methods
The antibodies 2F5 and 4E10 from Dr H Katinger and IgGb12 from Dr D Burton and C Barbas; and the gp41-derived C34 peptide from DAIDS, NIAID were obtained through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program. C34 is a fusion inhibitor covering the 628-661 amino-acid sequence of gp41, similar to T-1249 (residues 628–663) and to T-20 (residues 638–673). The anti-CD4 monoclonal antibody (mAb) Leu3a was from Becton Dickinson (Franklin Lakes, New Jersey, USA).
Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient of blood cells provided by the local blood bank and immediately used to purify CD4 T cells (>95%) by immunomagnetic negative selection (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). MOLT-4/chemokine receptor 5 (MOLT/CCR5) cells uninfected or chronically infected with the NL4-3 and BaL HIV isolates (>90% producing HIV particles) have been previously described [10–12]. Cells were maintained in RPMI1640 (Gibco; Gibco-BRL, Grand Island, New York, USA) supplemented with 10% fetal calf serum (FCS; Gibco) and used without stimulation. 293T cells were maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM)/10% FCS (Gibco).
Cocultures of MOLT/CCR5 cells with primary CD4 T cells
Chronically infected MOLT/CCR5 cells (200 000 cells/well) were preincubated in 96-well plates either alone (control), with nAbs (30 μg/ml for IgGb12 and 100 μg/ml for both 4E10 and 2F5) or with the inhibitors Leu3a (0.25 μg/ml) or C34 (5 μg/ml) for 1 h at 37°C, before adding 200 000 CD4 T cells. Inhibitor concentrations were selected in bases of complete inhibition of cell-to-cell fusion. Uninfected MOLT/CCR5 cells were used as negative control.
Cellular contacts were measured by flow cytometry after 2 h of incubation as described previously using 5-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMRA)-labeled CD4 T cells . MOLT cells staining for CMRA were considered to be cellular conjugates. Percentage was calculated as follows:
HIV transfer: HIV particles associated with CD4 T, which corresponds to incoming virions specifically captured during cellular contacts , were analyzed by flow cytometry after 24 h of coculture. Fixed and permeabilized cells (Fix & Perm; Caltag Laboratories, Burlingame, California, USA) were stained with the PE-labeled KC57 anti-HIV-p24 antigen mAb (Beckman & Coulter, Fullerton, California, USA). Staining was analyzed in living CD4 T cells.
HIV spread was quantified by PCR as described  with some modifications. Briefly, 500 000 CD4 T cells were cultured in 48-well plate with 500 000 NL4-3 chronically infected MOLT/CCR5 cells. Additional controls [zidovudine (ZDV) at 1 μg/ml, and mixtures of MOLT/CCR5 cells and CD4 T cells cultured in separate wells] were included. After 24 h, DNA was extracted using the Qiamp DNA Blood mini kit (Qiagen, Hilden, Germany) and amplified in triplicate using a Taqman universal PCR Master Mix, primers and probes for HIV and CCR5 (Applied Biosystems, Foster City, California, USA). Reactions (40 cycles of 15 s at 95°C for melting and 1 min at 60°C for annealing/extending) were conducted in an ABI 7000 Sequence Detection System (Applied Biosystems). Relative proviral DNA synthesis was calculated using the 2-ΔΔCT method . ΔΔCT = (CTHIV − CTCCR5)Test − (CTHIV − CTCCR5)Control, where CT is the fractional cycle number that reaches a fixed threshold.
Antigen transfer from uninfected to infected cells was assessed in cocultures of 50 000 293T cells expressing a CD4-green fluorescent protein (GFP) fusion protein  with 50 000 MOLT/CCR5 cells (uninfected or infected) labeled with the cell tracker dimethylacridinone (DDAO; Molecular Probes, Eugene, Oregon, USA). After 2 h, cellular conjugates were disrupted by vigorous shaking, cells were fixed and the GFP content of DDAO+ cells was analyzed by flow cytometry.
Gag-independent and gag-dependent antigen/membrane transfer from effector to target cells was assessed in cocultures of 50 000 CD4 T cells with 100 000 293T cells transfected (Calphos; BD Bioscience Clontech, Palo Alto, California, USA) with HIV Env alone or in combination with the ΔEnv plasmid PSG3, respectively. Gag-dependent transfer was assessed by intracellular p24 staining as described above, whereas gag-independent transfer was assessed by staining 293T cells with DiI (Molecular Probes)  and determining DiI content in CD4 T cells. Envelope plasmids used were NL4-3 and the fusion defective mutant 41.2 cloned in pCDNA3 .
Statistical analyses were performed using two-sided Student's t-test, including the Bonferroni correction. P values less than 0.05/n were considered to indicate statistical significance (n = number of comparisons).
Effect of neutralizing antibodies on early events of virological synapses
We have analyzed the effect of nAbs on cell-to-cell HIV transmission in a well defined model of coculture of HIV-infected MOLT/CCR5 cells with primary CD4 T cells [10,11]. First, we confirmed that all nAbs, as well as Leu3a and C34, inhibited NL4-3 and BaL infection in a standard neutralization assay (not shown) and syncytium formation in cocultures of MOLT/CCR5 cells with CD4 T cells . Next, we dissected the mechanism of action of different nAbs. The first step of synaptic HIV transmission, the formation of conjugates between CD4 T cells and MOLT/CCR5 cells infected with NL4-3 or BaL isolates, was significantly inhibited by Leu3a or IgGb12. In contrast, 4E10 and 2F5 (and C34) did not show inhibitory effects at concentrations that completely blocked fusion (Fig. 1a). Similar effects were observed on HIV NL4-3 or BaL transfer to CD4 T cells, which was exclusively inhibited by Leu3a and IgGb12 (Fig. 1b), whereas antigp41 nAbs failed to inhibit BaL transfer or induced a significant (P < 0.05) increase in viral NL4-3 particles captured by CD4 T cells (Fig. 1b), due to inhibition of death and syncytium formation. These data confirm the paradoxical effect of antigp41 nAbs on virus transmission.
Mechanisms of HIV transfer and antigen exchange by trogocytosis
To evaluate the relevance of the synapses formed in the presence of antigp41 antibodies, we analyzed the occurrence of trogocytic events. First, we determined the transfer of membranes from 293T cells expressing CD4-GFP to infected MOLT/CCR5 cells. Specific trogocytic transfer of CD4-GFP to NL4-3 and BaL-infected cells was observed (Fig. 2a). Trogocytosis was blocked by Leu3a or IgGb12, but not by C34 or nAbs 4E10 and 2F5, suggesting that gp120-mediated contacts are sufficient for trogocytic events to occur at the virological synapse.
Trogocytosis from the infected to uninfected cells was studied coculturing Env-expressing 293T cells and primary CD4 T cells. To avoid membrane exchange induced by gp41-mediated fusion, we used an Env mutant (41.2) devoid of fusogenic activity . This Env mutant, which binds to CD4, promoted similar level of HIV transfer than the wild-type NL4-3 Env, when cotransfected with a ΔEnv HIV backbone (Fig. 2b). However, when transfected alone, the 41.2 mutant promoted background levels of membrane transfer to target cells, which were not significantly modified by Leu3a or C34. Consistently, wild-type Env induced membrane exchange exclusively by fusion-mediated mechanisms, as it was blocked to the same extent by Leu3a and C34 (Fig. 2c). Thus, detectable trogocytosis operates solely on the uninfected-to-infected cell direction in virological synapses.
Productive infection of target cells
To definitively ascertain whether endocytosis or trogocytosis contributes to the infection of target cells, we have quantified the synthesis of proviral DNA after 24 h of coculture of NL4-3-infected MOLT/CCR5 cells and primary CD4 T cells . We chose this time instead of shorter incubation times to maximize the possibilities of captured viruses to reach the cytoplasm of target cells and to start reverse transcription. Cocultures processed immediately after mixing cells (t = 0) showed levels of proviral DNA comparable with those of infected cells alone. After 24 h of coculture, the amount of proviral DNA increased 7.6 ± 3.5-fold and was inhibited by IgGb12, 4E10 and 2F5 antibodies to background levels, as observed in the presence of Leu3a, C34 or ZDV (Fig. 2d). Thus, infectious HIV spread is fully sensitive to high concentrations of nAbs, irrespective of the subunit targeted.
In our cell-to-cell HIV transmission model, antigp41 antibodies failed to block the formation of virological synapses, whereas the anti-CD4 binding site nAb IgGb12 completely blocked this process and the transmission of HIV particles. These results support the key role of gp120 and CD4 in cellular contacts  and might be relevant for neutralization efficiency. First, the ability of antigp41 antibodies to block fusion and death arrests the HIV transmission process after the formation of cellular conjugates increasing the amount of HIV particles transferred to CD4 T cells by an endocytic fusion-independent mechanism [4,11]. Second, cellular conjugates may be sufficient to activate cell communication by trogocytosis, defined as the unidirectional  or bidirectional  exchange of membrane patches between cells forming a synapse .
Although trogocytosis has been associated with little cytoplasm transfer compared to plasma membrane transfer , this mechanism could be sufficient to allow viral proteins and genomic RNA, both polarized to synaptic areas , reach target cells. Our data show trogocytosis in the uninfected toward the infected cell direction but not on the HIV transmission direction (Fig. 2). The observed trogocytosis is insensitive to antigp41 nAbs and may be explained by the reported elongation of CD4 T cell membranes (nanotubes or filopodia) [20,21] that are endocytosed by infected cells . The lack of trogocytosis in the direction of viral transmission supports the idea that gag-driven HIV budding is necessary for viral transfer  and is also consistent with the electron microscopy images of virological synapses (data not shown).
In addition to the lack of trogocytic HIV transfer, the fact that all nAbs block infectious viral transmission suggests that endocytosed virions are not able to reach the cytoplasm of target cells, at least at detectable levels, following 24 h of cellular contacts. Therefore, in contrast to other mechanisms of HIV spread through cellular contacts (transcytosis or dendritic cell-mediated transinfection), which are completely or partially resistant to neutralization [1,22], HIV spread between T cells is fully sensitive to humoral responses.
The present work was supported by the HIVACAT Program, the FIS project 05/1504, the FIPSE project 36600/06 and the Spanish AIDS network ‘Red Temática Cooperativa de Investigación en SIDA (RD06/0006)’. D.H. is supported by a grant from Sidaction (AI18-2/01284). J.B. and C.C. are researchers from Fundació Institut de Recerca en Ciències de la Salut Germans Trias i Pujol supported by the ISCIII and the Health Department of the Catalan Government (Generalitat de Catalunya). I.P is supported by a predoctoral grant from Generalitat de Catalunya and European Social Fund.
M.M. and I.P. performed neutralization and trogocytosis experiments. E.G. characterized envelope mutants. M.T.F.-F. performed electron microscopy. C.C. designed and analyzed quantitative PCR data; D.H., A.A. and G.G. designed and interpreted trogocytosis data; and M.B. and B.C. designed and interpreted neutralization data. J.B. coordinated the work and wrote the manuscript. All authors have read and approved the final version of the manuscript.
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