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Blood, 1 June 2006, Vol. 107, No. 11, pp. 4466-4474. Prepublished online as a Blood First Edition Paper on February 9, 2006; DOI 10.1182/blood-2005-08-3490.
IMMUNOBIOLOGY Efficient inhibition of HIV-1 replication in human immature monocyte-derived dendritic cells by purified anti–HIV-1 IgG without induction of maturationFrom the Institut de Virologie, Strasbourg, France; and Veterans Affairs and New York University Medical Centers, NY.
During mucosal HIV transmission, immature dendritic cells (DCs) present in the mucosa are among the first cellular targets of the virus. Previous studies have analyzed the inhibition of HIV-1 transfer from human mature DCs to T lymphocytes by neutralizing IgG, but so far no in vitro data regarding the capacity of antibodies to inhibit HIV-1 infection of immature DCs have been reported. Here, we found an increased HIV-inhibitory activity of monoclonal IgG and purified polyclonal IgG when immature monocyte-derived dendritic cells (iMDDCs) were used as target cells instead of autologous blood lymphocytes. We showed that Fc  RII is involved in the mechanism for inhibiting HIV-1 infection of iMDDCs by IgG, whereas no induction of maturation was detected at concentrations of IgG that result in a 90% reduction of HIV replication. After induction of FcRI expression on iMDDCs by IFN-, an augmentation of the HIV-inhibitory activity of IgG, related to the expression of FcRI, was observed. Taken together, our results demonstrate the participation of FcRs in HIV-1 inhibition by IgG when iMDDCs are the targets. We propose that IgG is able to efficiently inhibit HIV-1 replication in iMDDCs and should be one of the components to be induced by vaccination.
Dendritic cells (DCs) constitute an essential component of the immune system.1 These cells, present at trace level in all organs,2 play a crucial role in bridging innate and acquired immune responses to pathogens.3 Mucosal HIV-1 transmission is the major mode of infection, and immature myeloid DCs (MDCs) present at mucosal sites are among the first cells targeted by the virus. DCs play an important role in virus transmission, dissemination, and persistence of HIV-1 infection and are considered as reservoirs for the virus in lymphoid tissues where they may contribute to the infection of newly recruited T lymphocytes.3-6 Different subsets of DCs have been found to be infected in vivo and in vitro,5-11 although the frequency of HIV-infected DCs is often 10 to 100 times lower compared with CD4+ T lymphocytes.12 It has been reported that HIV-1 proteins, such as gp-120, Nef, and Tat, can each induce maturation of MDCs, but maturation induced by whole HIV infectious particles is more controversial.5,13 Some authors have shown that plasmacytoid DCs (PDCs) can mature after in vitro infection,14 whereas maturation of MDCs seems to occur as a bystander effect due to cytokines produced by PDCs after HIV-1 exposure.15 On the other hand, once MDDCs are infected by HIV-1, their maturation induced by TLR4 or CD40L ligation was impaired.13,16 Moreover, abnormal maturation induced by LPS has been measured after exposure of iMDDCs to HIV-1 gp-120.17,18 Recently, it has been shown that LPS-induced maturation could be prevented by addition of recombinant Vpr.19 Thus, new evidence has shown that HIV-1 could interfere with DC immune responses by impairing their maturation process, their cytokine production, and their allogenic T-cell stimulatory function; this could contribute to immune dysfunction in AIDS patients.5
Apart from the classic infectious process,20 binding and uptake of viruses by DCs will induce iDCs to respond rapidly to virus exposure by several antigen-internalization pathways such as phagocytosis, receptor-mediated endocytosis, and macropinocytosis.3,21-24 These cells bind immune complexes (ICs) via Fc Few studies have analyzed inhibition of HIV transmission from mature DCs to T lymphocytes by antibodies.4,13,32 Frankel et al have found an increased inhibitory activity of neutralizing mAbs when a virus/antibody mixture is added to mature DCs before transfer to T lymphocytes versus direct infection of T lymphocytes.32 Similarly, Ganesh et al have observed that mAb 2F5 is able to prevent transfer of HIV from mature MDDCs to T lymphocytes during the first 48 hours, whereas protection of T lymphocyte infection is no longer recorded after 4 days of culture.13 The authors concluded that antibodies cannot protect HIV-1 R5 strain transfer to autologous T lymphocytes during infectious synapse formation with mature MDDCs.13 However, these studies did not analyze the effects of anti–HIV-1 antibodies on infection of iMDDCs.
Here, we have analyzed the capacity of monoclonal IgG or polyclonal IgG purified from sera of HIV-1–infected individuals to inhibit HIV-1 replication in iMDDCs and to induce cell maturation. We have shown for the first time that anti–HIV-1 IgGs are able to efficiently inhibit HIV-1 infection of human iMDDCs without induction of maturation and demonstrate that Fc
Antibodies and reagents
mAbs HLA-DR–PE (G46-6), HLA-ABC–PE (G46-2.6), CD1a-PE (HI149), CD11c-PE (HL3), CD16-PE (3G8), CD40-PE (5C3), CD80-PE (L307.4), CD83-PE (HB15e), CD86-PE (FUN-1), CD89-PE (A59), CD206-PE (19.2), and DC-SIGN–PE (DCN46) were purchased from BD Pharmingen (San Diego, CA). mAbs CD4-PC5 (13B8.2), CD8-PE (B9.11), CD14-PC5 (RMO52), CD32-PE (2E1), CD64-PC5 (22), CD45RO-ECD (UCHL1), DC-LAMP–PE (104.G4), and p24-RD1 or -FITC (KC57) were purchased from Beckman-Coulter (Roissy, France). Purified mAbs anti–human Fc Preparation of human iMDDCs
Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were obtained by Ficoll-Hypaque sedimentation. PBMCs were either stimulated by PHA for 3 days or used for isolation of blood monocytes with a one-step immunomagnetic separation procedure of CD14+ cells according to the manufacturer's instructions (EasyStep; Stem cells biotechnologies, Vancouver, BC, Canada). This purified cell fraction contained more than 99% of CD14+ monocytes as determined by flow cytometry analysis. The purified CD14+ monocytes were cultured at 2 x 106 cells/mL in RPMI 5% FCS supplemented with 10 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF; R&D systems, Minneapolis, MN) plus 20 ng/mL IL-4 (R&D systems) at 37°C and 5% CO2 in air. To analyze HIV neutralization by antibodies in both iMDDCs and autologous lymphocytes, 15% of unstimulated autologous peripheral blood lymphocytes (PBLs) were added to CD14+ monocyte culture for 6 days. Every 3 days, GM-CSF and IL-4 were newly added to cultures. Fc Virus stocks R5 HIV-1 primary isolates Bx08 and BaL (subtype B) were provided by Prof H. Fleury (Bordeaux, France) and through the National Institutes of Health (NIH; Bethesda, MD) from Drs S. Gartner, M. Popovic, and R. Gallo. Virus stocks were obtained as previously described.38,39 To eliminate the presence of bystander activation factors in virus preparation that could induce MDDC maturation, viruses were purified on sephacryl S-1000 superfin column as described.40 Purified virus preparations were aliquoted and frozen at the concentration of 10 to 15 µg/mL p24. For each iMDDC virus titration was performed at day 4 in conditions similar to neutralization. Antibody neutralization assays Neutralization was assessed by flow cytometry detection of intracellular p24 viral antigen after infection of PHA-stimulated PBMCs or iMDDCs, using experimental conditions similar to those previously described.37 Briefly, 25 µL purified HIV-1 (concentration ranging from 2 to 4 µg/mL p24 depending on the strain, leading to 5% infected cells at 64 hours) was preincubated for 1 hour with 25 µL mAb or polyclonal IgG or IgA purified from HIV-1 patients before the addition of 25 µL iMDDCs at 15 x 106 cells/mL (in RPMI 1640 medium with 5% FCS, supplemented with human recombinant IL-4 plus GM-CSF) or of 25 µL PHA-stimulated PBMCs at 20 x 106 cells/mL (in RPMI medium with 10% FCS plus IL-2). The numbers of infected cells were determined by the detection of intracellular p24 antigen by flow cytometry, 24 or 64 hours after the infection of PHA-stimulated PBMCs or iMDDCs, respectively. Neutralizing titers were determined as the concentration of antibodies, which results in 90% reduction of infected cells. Flow cytometry analysis To assess the immunophenotypic profile of iMDDCs, cells were washed once with PBS plus 3% FBS and incubated for 15 minutes at 4°C with antibodies. After washing, the stained cells were either fixed in 1.5% paraformaldehyde (Sigma, St Louis, MO) and/or further monitored for virus replication by intracellular p24 detection.37,41 Cells were characterized by flow cytometry (FACScan; Becton Dickinson, San Jose, CA).
Immunophenotyping of iMDDCs
The phenotypic profile of human MDDCs was determined after 6 days of culture. After this period, cells had acquired the immunophenotype of iDCs (Figure 1). The mean percentages of MDDCs expressing the costimulatory molecules CD86 and CD80 were 40% and 47%, respectively, and only a very low percentage of these MDDCs was found positive for the maturation marker CD83. The loss of CD14 expression and the slow up-regulation of CD1a and Fc
Maturation of these cells was obtained after addition of LPS (Figure 1B) or TNF-
HIV-1 infection of iMDDCs The percentage of HIV-infected MDDCs was assessed by the detection of intracellular viral p24 Gag. Kinetics of HIV replication in iMDDCs were first determined. A few iMDDCs positive for p24 were detected at 48 hours, and the percentage of p24-positive cells reached 3.5 and 18 at 64 and 96 hours, respectively (Figure 2A). This p24 corresponds to new viral p24 proteins produced, as no intracellular p24 antigen was detected when 10 µM AZT was added together with or 24 hours after HIV-1 addition (Figure 2A). When AZT was added 48 hours later, the percentage of infected cells at 64 hours was identical to that measured in the absence of AZT, suggesting that the infected iMDDCs detected at 64 hours correspond to the first round of infection. This production of viral p24 in iMDDCs was delayed compared with PHA-stimulated PBMCs.41
Next, the relationship between virus input and the number of HIV-infected iMDDCs was assessed. Using serial dilutions of 2 purified HIV-1 primary isolates, we found a linear dose-response curve between the concentrations of virus inoculum ranging from 0.1 to 4 µg/mL p24 and the percentage of infected cells ranging from 1% to 6% (Figure 2B). The concentration of HIV-1 allowing 5% of HIV-infected iMDDCs after 64 hours was chosen for antibody neutralization experiments to be close to the experimental conditions used for single-cycle neutralization assay performed on PHA-stimulated PBMCs.41 The detection of HIV-infected cells by intracellular p24 staining used in this neutralization assay has the advantage to discriminate the infection of iMDDCs from the infection of lymphocytes. Infection of iMDDCs in the presence of autologous PBLs could thus be assessed and compared with the infection of highly purified iMDDCs. In the absence of added PBLs, 5.2% of iMDDCs were infected at 64 hours, whereas in the presence of 15% of autologous PBLs, 5.5% of iMDDCs (corresponding to 6.4% of the MDDC population) and 1.8% of PBLs (corresponding to 12% of the PBL population) were found infected (Figure 2C). After addition of neutralizing mAb 2F5 at 5 µg/mL, a strong reduction of the percentage of HIV-1BaL–infected iMDDCs was detectable (Figure 2C), whereas the percentage of HIV-1BaL–infected autologous PBLs only slightly decreased. These results showed that neutralizing mAb 2F5 was more efficient for inhibiting HIV infection of iMDDCs than blood lymphocytes. Increased HIV-neutralizing activities of anti–HIV IgG We extended this study to various polyclonal and monoclonal Abs. Four- to 10-fold lower neutralizing concentrations of polyclonal IgG were observed on iMDDCs compared with cocultured autologous PBLs (Table 1). These lower neutralizing concentrations were observed whether iMDDCs were purified or cultured in the presence of 15% of autologous PBLs (Table 1). The HIV-inhibitory activity of antibodies on autologous PBLs was comparable with that observed when PHA-stimulated PBMCs were used in a more "classical" neutralization assay.36 When using PHA-stimulated PBMCs, the HIV-1–infected cells were found to be essentially CD4+CD45RO+ T lymphocytes (not shown). Purified polyclonal IgG samples nos. 3 and 11 that had undetectable neutralizing activity on PHA-stimulated PBMCs decreased the percentage of HIV-infected iMDDCs by 90% at concentrations of 350 and 130 µg/mL, respectively, for HIV-1BaL and at concentrations of 275 and 165 µg/mL, respectively, for HIV-1Bx08 (Table 1). The 90% inhibitory concentrations (IC90) of 5 well-characterized neutralizing mAbs ranged between 1 and 5 µg/mL when iMDDCs were used, whereas their neutralizing concentrations were 10- to 50-fold higher on autologous PBLs or PHA-stimulated PBMCs (Tables 1, 2). Moreover, when a multiple cycle HIV-1BaL neutralization experiment was performed on highly purified iMDDCs, the concentration of mAb 2F5 resulting in 90% decrease of p24 production in the supernatant after 7 days was 1 µg/mL, a concentration similar to that determined by flow cytometry at 64 hours. On the contrary, the IC90 of polyclonal IgA samples nos. 6 and 8 were similar whether iMDDCs, autologous blood lymphocytes, or PHA-stimulated PBMCs were the targets of HIV-1 (Tables 1, 2). Polyclonal IgG or IgA purified from HIV-seronegative donors did not inhibit infection of these cells. Overall, monoclonal and polyclonal IgG exhibited more potent HIV-1–inhibitory activity when iMDDCs instead of PHA-stimulated PBMCs were used as target cells.
As HIV-inhibitory activity of recombinant soluble CD4 (Tables 1, 2) or of T20 fusion inhibitor (not shown and Herrera et al43) was similar for the different cell types used in this neutralization study, a step distinct from the binding of HIV-1 to CD4 or the fusion of HIV-1 with the cellular membrane may be involved in the increased HIV-1–inhibitory activity of IgG on iMDDCs. Kinetics of antibody addition showed that if neutralizing monoclonal antibody 2F5 or polyclonal IgG no. 11 was added 3 hours after addition of HIV to iMDDCs, the potent HIV-inhibitory activity was lost (Figure 3), suggesting that antibodies interfered with the first events of HIV-1 entry in iMDDCs. HIV-1 inhibition by IgG is not due to iMDDC maturation
It has been previously shown that maturation of bone marrow–derived mouse DCs can be induced by antigen-IgG ICs.31 As others have indicated that HIV-1 production is lower in mature DCs than in iDCs, which may be due to a postentry block44,45 and/or to variations in coreceptor expression,8 maturation of MDDCs in the presence of HIV-1 antibody ICs may explain the increased inhibitory activities of these antibodies on iMDDCs. MDDC maturation can be induced by cellular or viral factors present in crude virus suspension; therefore, they were eliminated by gel filtration. After infection with the purified HIV-1BaL, the infected MDDCs have similar phenotype as the total population (mean ± SD of 3 different cell preparations): 40% ± 5% of MDDCs were positive for CD80; 49% ± 7% for CD86; 2% ± 1% for CD83; 84% ± 8% for DC-SIGN; 4% ± 1% for Fc
Next, we analyzed the number of MDDCs positive for CD83 in the presence of HIV-Ig ICs. After addition of monoclonal IgG as well as polyclonal IgA or IgG at concentrations equal or below the IC90, no significant change in the percentage of MDDCs positive for CD83 was detected (Table 3; Figure 4A). We also failed to observe any change in the percentage of MDDCs positive for CD86bright and for intracellular lysosomal glycoprotein DC-LAMP after addition of HIV-1BaL complexed with antibodies (not shown), whereas LPS significantly increased the percentage of MDDCs positive for CD83 (Table 3). Thus, a 90% reduction of HIV infection by antibodies was observed in the absence of induction of MDDC maturation.
Of special interest, at high concentrations of mAb 2F5 (100 µg/mL) or polyclonal IgG (650 µg/mL), a maturation signal resulting in an increased expression of CD83 (Figure 4B) and of intracellular DC-LAMP (not shown) was triggered. This maturation was observed in the presence or in the absence of infection and also with HIV-seronegative polyclonal IgG samples, demonstrating that this maturation was not related to HIV infection and was not specific to IgG directed against HIV-1. Of interest, 650 µg/mL polyclonal IgA sample no. 8 did not induce the expression of CD83 (Figure 4B) or DC-LAMP (not shown) in these cells, suggesting that this mechanism of maturation is specific to Fc Rs.
Participation of Fc RII in HIV-1 inhibition by anti–HIV IgG
As iMDDCs are antigen-presenting cells, which express several Fc
As the Fc
Participation of Fc
It has been reported that Fc
The aim of this study was to analyze the mechanism of HIV inhibition by antibodies when iDCs were used as target cells. For this purpose, we used DCs differentiated from human blood CD14+ monocytes with a combination of IL-4 and GM-CSF or IL-4, GM-CSF, and IFN- , as they exhibit immunophenotypic and functional characteristics of human blood iDCs.47 In our culture conditions, we found that iMDDCs were infected by R5 primary isolates as recorded by intracellular p24 antigen detection. Indeed, various studies have previously reported that iDCs are susceptible to and support subsequent replication of R5 HIV-1 strains,13,20,32,47,48 although less efficiently than CD4+ T lymphocytes.5 We showed that addition of AZT after 24 hours of infection still completely abrogates p24 production, suggesting that HIV replication in iMDDCs was delayed compared with PHA-stimulated PBMCs41 or monocyte-derived macrophages (MDMs).37 It has been recently shown that HIV-1 virions were internalized in iDCs as well as in mature DCs over the first hours in the absence of viral replication.49,50 At that time, the virus retained could be efficiently transferred from DCs to CD4+ T lymphocytes,48-51 suggesting that virus was protected in specific DC compartments.48 Later on, the retained HIV-1 particles were mainly destroyed in iDCs and only some may lead to productive infection.48 Thus, DCs may transfer HIV-1 to CD4+ T lymphocytes in 2 distinct phases: the first one driven from an endosomal pathway (without induction of an acidic pH lysosomal degradation) to the DC–T cell synapse49 and the second one through de novo HIV-1 production that occurs later and may be responsible for the selective transmission of R5 strains in vivo.48 In this study, we analyzed the inhibition of R5 HIV replication by monoclonal and polyclonal IgGs when iMDDCs were used as target cells. The assay used allowed us to discriminate infected iMDDCs from infected PBLs. We found that anti–HIV IgG was able to inhibit HIV-1 replication more efficiently in iMDDCs than in PBLs, whereas no change of the neutralizing activity was observed for purified polyclonal IgA. Kinetics of IgG addition indicated that IgG inhibits an early event in HIV infection, which did not favor a mechanism of antibody-dependent cellular cytotoxicity (ADCC). Natural killer (NK) cells could mediate ADCC but they were not detected in our MDDC preparation. Previous reports have shown that human DCs were unable to mediate ADCC,46,52 but more recently, it has been described that a specific subset of native DCs in blood from humans and mice has the ability to lyse tumor cells through efficient ADCC.53,54
It is well known that IgG-Fc
DCs are antigen-presenting cells that have the capacity to phagocytose antigen-IgG ICs. To investigate the involvement of such a mechanism of HIV inhibition by IgG, the inhibitory activity of Fab or F(ab')2 fragments of IgG has been determined. We found a diminution of the inhibitory activity of Fab or F(ab')2 compared with whole IgG. By specific blockade of Fc
It is likely that the in vitro mechanism of HIV-1 inhibition by anti–HIV IgG in iMDDC is that HIV-IgG ICs are rerouted to a lysosomal degradation pathway after internalization through Fc
Submitted August 29, 2005; accepted January 26, 2006.
Prepublished online as Blood First Edition Paper, February 9, 2006; DOI 10.1182/blood-2005-08-3490.
Supported by grants from the European Union (QLK2-CT-1999-01321 "Eurovac"); Agence Nationale de Recherches sur le SIDA (ANRS); and National Institutes of Health (HL59725, AI36085, and AI27742).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: V. Holl, EA 3770, Université Louis Pasteur, Institut de Virologie, 3 rue Koeberlé, F-67000 Strasbourg, France; e-mail: vincent.holl{at}hemato-ulp.u-strasbg.fr.
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Abstract
Introduction
(not shown) for 24 hours. Addition of LPS induced a rapid up-regulation of the costimulatory molecules CD83, CD86bright, and CD40 together with a diminution of CD206+ MDDCs (
) or in the absence (
) of HIV-1BaL. Abs were added at concentrations that resulted in 90% inhibition of infection (IC90) (A) or at 5- to 25-fold higher concentrations (B). Values are from one representative experiment, repeated 4 times with iMDDCs from different HIV-negative donors. In A and B, 
) or cyclized V3 loop peptide (at 70 µg/mL; 
) to mAb 447-52D and purified HIV-1BaL for 1 hour before addition of iMDDCs (D). Values are mean ± SD of 3 independent wells from one representative experiment.
(10 µg/mL) directed against each human Fc