JOURNAL OF VIROLOGY, May 2004, p. 5223–5232 0022-538X/04/$08.00⫹0 DOI: 10.1128/JVI.78.10.5223–5232.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 10
Human Immunodeficiency Virus Type 1 Activates Plasmacytoid Dendritic Cells and Concomitantly Induces the Bystander Maturation of Myeloid Dendritic Cells Jean-Franc¸ois Fonteneau,1† Marie Larsson,2† Anne-Sophie Beignon,2 Kelli McKenna,2 Ida Dasilva,2 Ali Amara,3 Yong-Jun Liu,4 Jeffrey D. Lifson,5 Dan R. Littman,3 and Nina Bhardwaj2* Institut de Biologie, INSERM U463, Nantes, France,1 and Departments of Medicine and Pathology2 and the Howard Hughes Medical Institute and Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine,3 New York University School of Medicine, New York, New York; Department of Immunology and Center for Cancer Immunology Research, MD Anderson Cancer Center, University of Texas, Houston, Texas4; and AIDS Vaccine Program, SAIC/Frederick, Inc., National Cancer Institute, Frederick, Maryland5 Received 13 November 2003/Accepted 21 January 2004
In this study, we analyzed the phenotypic and physiological consequences of the interaction of plasmacytoid dendritic cells (pDCs) with human immunodeficiency virus type 1 (HIV-1). pDCs are one cellular target of HIV-1 and respond to the virus by producing alpha/beta interferon (IFN-␣/) and chemokines. The outcome of this interaction, notably on the function of bystander myeloid DC (CD11cⴙ DCs), remains unclear. We therefore evaluated the effects of HIV-1 exposure on these two DC subsets under various conditions. Bloodpurified pDCs and CD11cⴙ DCs were exposed in vitro to HIV-1, after which maturation markers, cytokine production, migratory capacity, and CD4 T-cell stimulatory capacity were analyzed. pDCs exposed to different strains of infectious or even chemically inactivated, nonreplicating HIV-1 strongly upregulated the expression of maturation markers, such as CD83 and functional CCR7, analogous to exposure to R-848, a synthetic agonist of toll-like receptor-7 and -8. In addition, HIV-1-activated pDCs produced cytokines (IFN-␣ and tumor necrosis factor alpha), migrated in response to CCL19 and, in coculture, matured CD11cⴙ DCs, which are not directly activated by HIV. pDCs also acquired the ability to stimulate naïve CD4ⴙ T cells, albeit less efficiently than CD11cⴙ DCs. This HIV-1-induced maturation of both DC subsets may explain their disappearance from the blood of patients with high viral loads and may have important consequences on HIV-1 cellular transmission and HIV-1-specific T-cell responses. they probably play a role in the initiation of T-cell responses in a manner similar to that of the myeloid DCs (12, 24). Several studies have shown that, during HIV infection, both DC subsets are substantially reduced in patients’ blood (5, 13, 17, 19, 20, 30, 39, 46, 48). In some of these studies, this decrease correlated with the plasma viral load and was partially reversed following highly antiretroviral therapy (HAART) (5, 19, 46, 48). Several hypotheses have been proposed to explain their disappearance from the peripheral blood of HIV-1⫹ patients, such as the failure of DC precursors to differentiate, the death of DCs due to HIV infection, or the relocalization of DCs in secondary lymphoid tissues as a consequences of DC maturation induced by the virus (45). This last hypothesis is supported by the observations of Yonezawa et al. that HIV induces activation of pDCs, a process characterized by production of IFN-␣ and increased expression of costimulatory molecules CD80 and CD86 (51). However, the fact that CD11c⫹ DCs do not mature when exposed to HIV in vitro is not in favor of this hypothesis (32). In this study we analyzed the effects of HIV-1 exposure on the maturation status of highly purified blood pDCs and CD11c⫹ DCs. We confirm that CD11c⫹ DCs exposed to HIV-1 in vitro do not mature, contrary to pDCs which mature rapidly following contact with infectious HIV-1, and interestingly with aldrithiol-2 (AT-2) chemically inactivated HIV-1
At least two dendritic cell (DC) subsets have been described in humans: the CD11c⫹ myeloid DCs, which include Langerhans cells, dermal and interstitial DCs, and the CD11c⫺/ CD123⫹/CD4⫹ plasmacytoid DCs (pDCs) (36). CD11c⫹ DCs play an important role in antiviral immune responses by acquiring and processing viral antigens into peptides for major histocompatibility complex presentation to T cells in secondary lymphoid organs (4). pDCs, also known as alpha/beta interferon (IFN-␣/)-producing cells (IPCs), are located in blood and in secondary lymphoid organs (25, 47). These cells express CD4 and CD123 (the interleukin-3 [IL-3] receptor alpha chain) but lack expression of myeloid-related markers, such as CD11b, CD11c, CD13, and CD33. pDCs participate in innate immune responses to different types of viruses (influenza virus, herpes simplex virus, HIV-1) by producing large amounts of IFN-␣, - and, - (21, 22, 34, 49). This activation of pDCs by viruses induces their differentiation from their precursor form, characterized by a plasmacytoid morphology, to their differentiated form where they adopt a DC morphology. In addition,
* Corresponding author. Mailing address: The NYU School of Medicine, Departments of Pathology and Medicine, MSB507, 550 First Ave., New York, NY 10016. Phone: (212) 263-5814. Fax: (212) 2636729. E-mail:
[email protected]. † J.-F.F. and M.L. contributed equally to this work. 5223
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FIG. 1. HIV-1 activates pDCs but not CD11c⫹ DCs. pDCs and CD11c⫹ DCs were obtained by magnetic bead depletion and fluorescenceactivated cell sorting. (A) pDCs were cultured with Mv, 300 ng of HIV-1MN/ml, or 20 ng of IL-3/ml. All HIV-1 amounts are based on p24 equivalents per milliliter. After 16 h, pictures of cultures were taken by using an inverted microscope. (B) pDCs were cultured with 20 ng of IL-3/ml, 300 ng of HIV-1 (right panel, MN, left panel, JR-CSF)/ml, AT-2 HIV-1 (right panel, MN, left panel, JR-CSF), Mv, or 10 M R-848 while CD11c⫹ DCs were cultured alone, with HIV-1, AT-2 HIV-1, Mv, or R-848. After 16 h, expression of CCR7, HLA-DR, CD80, CD83, and CD86 was measured. CCR7 expression was not assessable on CD11c⫹ DCs because the FL2 channel was saturated by PE-conjugated anti-CD11c MAb used to sort these cells. DC survival can be estimated by the percentage of gated cells on the forward scatter/side scatter dot plot. Numbers on histogram plots represent means of fluorescence intensity. (C) pDCs were cultured with Mv, 300 ng of HIV-1MN/ml, 500 ng of HIV-1NL4-3/ml, or HIV-1⌬env. After 16 h, expression of CD83 was measured. (D) Presence of IFN-␣ and TNF-␣ in culture supernatants as measured by ELISA. Each figure is representative of at least three different experiments demonstrating similar trends.
(AT-2 HIV-1, which does not replicate). The activated pDCs produce IFN-␣ and tumor necrosis factor alpha (TNF-␣), strongly upregulate some maturation markers (CD83 and CCR7), and slightly upregulate costimulatory molecules
(CD80 and CD86). They acquire the capacity to migrate toward CCL19, the ligand of CCR7 expressed in lymphoid organs. In addition, we show that pDC secretion in response to HIV-1 induces the maturation of bystander CD11c⫹ DCs,
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characterized by an induction of CD83 and CCR7 expression and the upregulation of CD80 and CD86. The direct maturation of pDCs and indirect maturation of CD11c⫹ DCs by HIV-1 observed in our in vitro study are in support of the hypothesis that the disappearance of both DC subsets from the patient blood may be due to their CCL19-driven relocalization into secondary lymphoid organs. This phenomenon may have important consequences for the development of HIV infection by furnishing accelerated cellular transport for the virus from the site of infection to the lymphoid compartment where viral spread and replication are most efficient. MATERIALS AND METHODS pDC and CD11cⴙ DC purifications. Leukocyte-enriched buffy coats were obtained from the New York Blood Center (New York, N.Y.). Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation on Ficoll-Hypaque (Amersham Pharmacia Biotech). pDCs and CD11c⫹ DCs were initially enriched from PBMCs by magnetic bead depletion of T and B lymphocytes, monocytes, NK cells, and erythrocytes by using monoclonal antibodies (MAbs) against CD3, CD19, CD14, CD56, and glycophorin A. pDCs (CD3⫺, CD11c⫺, CD14⫺, CD16⫺, CD20⫺, CD4⫹ cells) and CD11c⫹ DCs (CD3⫺, CD11c⫹ High, CD14⫺, CD16⫺, CD20⫺, CD4⫺/⫹ cells) were then purified from the enriched population by fluorescence-activated cell sorting (FACS) (FACs Vantage; Becton Dickinson (BD), Franklin Lakes, N.J.) using fluorescein isothiocyanate (FITC)-conjugated MAbs against CD3, CD14, CD16, and CD20 (BD), phycoerythrin (PE)-conjugated MAb against CD11c (BD), and PE-cy5-conjugated MAb against CD4 (Caltag, Burlingame, Calif.) or CD4V4 (BD). pDCs and CD11c⫹ DCs were more than 95 to 98% pure. Infectious and chemically inactivated HIV-1. Virions of HIV-1MN (X4) and HIV-1JR-CSF (R5) were produced from the HIV-1 CEMX174 (T1) cell line as previously described (3, 14, 40). Briefly, virions were purified by sucrose gradient ultracentrifugation after cells were removed from harvested cell cultures by tangential flow filtration or by clarification. The virus-containing fractions, as determined by the UV profile and sucrose density, were pooled and centrifuged at 30,000 rpm (Beckman continuous flow rotor model CF32Ti) for 1 h to pellet the virus. The resulting viral pellet was resuspended in TNE (0.01 M Tris-HCl [pH 7.2], 0.1 M NaCl, and 0.1 mM EDTA in Milli-Q water) buffer to a final 1,000-fold concentration relative to the original volume of cell culture fluid. Aliquots were stored in liquid nitrogen vapor. Samples were titrated for the presence of infectious virus by using AA2CL.1 cells and HIV-1 p24 antigen capture kits (AVP; National Cancer Institute, Frederick, Md.). For preparation
of noninfectious virus (AT-2 HIV-1) with functionally intact envelope glycoproteins, virus was inactivated by treatment with 1 mM 2,2⬘-dithiodipyridine (AT-2; Sigma) and purified and concentrated like nontreated HIV-1 as previously described (3, 14, 15, 43). Microvesicles (Mv), used as a negative control, were prepared from supernatants of uninfected cell cultures in a manner identical to that used for virus preparation from infected cells (8). HIV-1NL4-3 (X4) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health; pNL4-3 was from Malcolm Martin (1), and HIV-1⌬-Env was a kind gift of Dan Littman. pDC and CD11cⴙ DC culture. pDCs and CD11c⫹ DCs were cultured at 106 cells/ml with at least 105 DCs per well in flat-bottom 96-well plates in RPMI 1640 medium with 20 g of gentamicin (Gibco BRL, Grand Island, N.Y.)/ml and 5% pooled human serum (PHS; Cellgro, Herndon, Va.). pDCs were cultured with 20 ng of IL-3 (R&D, Minneapolis, Minn.)/ml, 300 ng of HIV-1 (MN, JR)/ml, 300 ng of AT-2 HIV-1 (MN, JR)/ml, 500 ng of HIV-1 (NL4-3 or ⌬-Env)/ml, Mv (at equivalent total protein dilutions used for intact HIV), or 10 M R-848 (Resiquimod, S-28463; GLSynthesis Inc., Worcester, Mass.). All HIV-1 amounts are based on p24 equivalents per milliliter. CD11c⫹ DCs were cultured alone or with 300 ng of HIV-1 (MN, JR)/ml, 300 ng of AT-2 HIV-1 (MN, JR)/ml, Mv, or 10 M R-848. After 16 h at 37°C, DCs were collected, washed twice, counted for viable cells, and used for experiments. DC culture supernatants were stored at ⫺80°C for subsequent evaluation in IFN-␣, TNF-␣, IL-12 enzyme-linked immunosorbent assays (Endogen, Rockford, Ill.) and for myeloid DC maturation experiments. Microscopic pictures of culture were performed by using an inverted microscope Zeiss Axioplan (Thornwood, N.Y.) equipped with a Hamamatsu digital camera (CA-742-96-12NR; Bridgewater, N.J.) and analyzed by using Photoshop 6.0 (Adobe Systems Incorporated, San Jose, Calif.). For coculture experiments, 1.5 ⫻ 105 pDCs were cultured with 1.5 ⫻ 105 CD11c⫹ DCs at final concentrations of 106 cells/ml as described above. Phenotype. DCs were stained with FITC-conjugated MAbs against HLA-DR (Iotest; Beckman Coulter, Miami, Fla.), CD80, CD86 (Pharmingen, Franklin Lakes, N.J.), CD83 (BD or Iotest), PE-conjugated MAbs against CD83 (Iotest), CCR7 (Pharmingen), or the respective FITC- or PE-conjugated isotype control MAbs (Pharmingen). DCs were fixed with phosphate-buffered saline containing 1% formaldehyde (Sigma-Aldrich, St. Louis, Mo.) prior to analysis. Fluorescence was analyzed by flow cytometry on a FACScan using Cell Quest software (BD). CCL19 migration assay. Differentially treated DCs (5 ⫻ 104) were incubated in 100 l of RPMI medium containing 5% PHS in the upper chamber of a transwell 24-well plate with 5.0-m pore sizes (Corning, Somerset, N.J.). The lower chamber contained 500 l of RPMI medium containing 5% PHS with or without 25 ng of CCL19 chemokine (R&D)/ml. After 2 h, cells in the lower chamber were harvested and counted. Maturation of monocyte-derived DCs by supernatants of HIV-1 exposed blood
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FIG. 2. pDCs activated by HIV-1 migrates toward CCL19. pDCs were cultured with 20 ng of IL-3/ml, 300 ng of HIV-1 (MN or JRCSF)/ml, 300 ng of AT-2 HIV-1 (MN or JR-CSF)/ml, or 10 M R-848. After 16 h, chemotaxis toward 25 ng of CCL19/ml was measured by using a transwell assay. Shown are representative results from four different experiments demonstrating similar trends.
DCs. Monocytes were purified from PBMCs by 2 h of adherence in 6-well plates and were cultured with 300 U of IL-4 (Immunex, Seattle, Wash.)/ml and 100 IU of granulocyte-macrophage colony-stimulating factor (Schering-Plough, Kenilworth, N.J.)/ml for 5 days to obtain monocyte-derived immature DCs as previously described (7). Immature DCs were collected and cultured at 5 ⫻ 104 DCs per well of flat-bottom 96-well plates in 100 l of DC culture medium and exposed to 50 l of pDCs or CD11c⫹ DCs supernatants, 10 M R-848, or 300 ng of HIV-1MN/ml. Expression of CD83 and CCR7 on surfaces of monocyte-derived DCs was measured after 16 and 40 h, respectively, by flow cytometry. MAbs against TNF-␣ (BD), IFN-␣ and - receptor (Research Diagnostics, Inc., Flanders, N.J.), and polyclonal antibodies against IFN-␣ and IFN- (Research Diagnostics) were added in some experiments to block CD11c⫹ DC maturation induced by supernatants of HIV-1MN-activated pDC. Proliferation and polarization of allogeneic naïve CD4ⴙ T cells by DCs. Naïve CD4⫹ T cells were purified by negative magnetic depletion by using a CD4⫹ T-cell isolation kit (Miltenyi, Auburn, Calif.) and CD45RO MicroBeads (Miltenyi). Naïve CD4⫹ T cells (105) were cultured with different quantities of differentially treated allogeneic DCs in U-bottom 96-well plates in 200 l of RPMI 1640 medium with 20 g of gentamicin/ml and 5% PHS. After 4 days, 4 Ci of [3H]thymidine/well was added for 12 h. Cells were then harvested to assess proliferation. To analyze cytokine polarization of stimulated CD4⫹ T cells, they were restimulated for 12 h with 50 ng of phorbol myristate acetate (PMA; Sigma-Aldrich)/ml and 2 g of ionomycin (Sigma-Aldrich)/ml on day 6 of DC/ T-cell coculture. Brefeldin A (10 g/ml; Sigma-Aldrich) was added for the last 6 h. Cells were fixed, permeabilized, and stained for intracellular cytokines with PE-conjugated anti-IL-4 MAb and FITC-conjugated anti-IFN-␥ MAb (BD) and analyzed by flow cytometry on a FACScan.
RESULTS HIV-1 induces the maturation of pDCs but not CD11cⴙ DCs. pDCs and CD11c⫹ DCs were purified from PBMCs of healthy donors by a magnetic bead depletion of T and B lymphocytes, monocytes, NK cells, and erythrocytes followed by FACS as described previously (34). After purification, approximately 0.12 and 0.22% of the starting population of PBMCs consisted of pDCs and CD11c⫹ DCs, respectively, with purities greater than 95%, often reaching 98%. We and others have reported that pDCs exposed to viruses like influenza A or herpes simplex virus secrete IFN-␣ and TNF-␣, which promote their viability and induce their maturation (24, 34). Because HIV induces the secretion of IFN-␣ by pDCs (21), we tested whether pDCs undergo maturation after encountering HIV-1. After 16 h of coculture with HIV-1MN (X4), purified pDCs developed dendrites, clustered together, and survived for more than 2 days (Fig. 1A), similar to what was previously observed in response to influenza A virus (24).
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This effect was due to HIV-1 as the corresponding control cultures of pDCs and Mv (isolated in a manner analogous to that of HIV but from uninfected lines) failed to induce morphological changes or to prolong viability. Interestingly, similar observations were made after pDCs were exposed to HIV-1 chemically inactivated with AT-2 (AT-2 HIV-1MN, data not shown). AT-2 covalently modifies free sulfhydryl moieties on the cysteines of internal retroviral proteins, including the retroviral zinc finger motifs of the nucleocapsid, without altering the conformation of the envelope glycoproteins (3). Therefore, HIV need not be replication competent in order to promote pDC viability. pDCs cultured with IL-3, a known survival factor for these cells, resulted in formation of several big clusters with rare isolated cells. The morphological changes observed in pDCs exposed to HIV-1 were accompanied by phenotypic changes (Fig. 1B, upper panel). pDCs cultured either with infectious virus (HIV1MN) or with AT-2 HIV-1MN mature and upregulate CD83, CD80, CD86, HLA-DR, and CCR7 molecules more so than to pDCs exposed to Mv or IL-3. However, the levels of costimulatory molecules (CD80 and CD86) on HIV-1 activated pDCs were consistently lower than levels induced by R-848, a small synthetic imidazoquinoline agonist of toll-like receptor-7 (TLR-7) and TLR-8 (2, 28). Maturation and prolonged survival of pDCs were also observed in response to different R5 HIV-1 strains: HIV-1JR-CSF, AT-2 HIV-1JR-CSF (Fig. 1B, upper panel), HIV-1ADA, and AT-2 HIV-1ADA (data not shown). Thus, the activating effects of HIV on pDCs occur independently of virus tropism or viral replication. pDCs cultured with Mv alone underwent significant cell death as shown on the forward scatter/side scatter dot plot, indicating that pDC activation, maturation, and extended survival were not due to cellular components present in the virus preparations. The loss of viability in the presence of Mv was not due to a potentially toxic effect, as pDCs cultured in their absence underwent the same degree of death. Furthermore, Mv did not exert any notable toxicity on CD11c⫹ DCs (Fig. 1B, lower panel). In contrast to pDCs, the maturation status of blood CD11c⫹ DCs was not affected by exposure to HIV-1. Similar levels of spontaneous background maturation, ranging from 25 to 75% of CD83⫹ cells from one experiment to others, were observed on the CD11c⫹ DCs whether they were cultured alone or in the presence of HIV-1, AT-2 HIV-1, or Mv (Fig. 1B, lower panel). These results also show that the virion preparations we used are not contaminated with lipopolysaccharide, as we would otherwise have seen significant CD11c⫹ DC maturation. Likewise, the control Mv fractions, prepared in a similar fashion, did not induce maturation of either CD11c⫹ DCs or pDCs. However, maturation of 100% of the CD11c⫹ DCs was achieved by addition of R-848 to the culture medium, as seen by upregulation of CD83, CD80, and CD86 expression on the surface of these cells. Increased maturation of CD11c⫹ DCs was also obtained in response to influenza A virus as was previously described (24). This result confirms a previous report indicating that HIV does not mature myeloid DCs (32). To further define the HIV-1 molecules responsible for activation of pDCs, we cultured pDCs with Mv, HIV-1MN, HIV-1NL4-3, or envelope-deficient HIV (HIV-1⌬env). After 16 h we observed maturation of pDCs in response to HIV-
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1MN and HIV-1NL4-3, whereas no CD83 expression was observed on pDCs after culture with Mv and HIV-1⌬env (Fig. 1C), suggesting that surface gp120 of HIV-1 is involved in the activation of pDCs. This result further confirms that the maturation of pDCs by HIV-1 is due to the virus and not to eventual contaminants in the viral preparations. We next confirmed that pDCs exposed to HIV-1 produce IFN-␣, and we also observed production of TNF-␣ in response to the virus (Fig. 1D). The levels of IFN-␣ produced by pDCs in response to HIV-1 ranged from 1,480 to 13,070 pg/ml in the different experiments and were of similar magnitude to those observed in response to influenza virus (data not shown). In contrast, production of IFN-␣ and TNF-␣ by CD11c⫹ DCs was not observed in response to HIV-1. In some experiments, background TNF-␣ production by CD11c⫹ DC was detected in the cultures’ supernatants where the highest spontaneous maturation backgrounds were observed, but these levels never increased in the presence of HIV-1 (data not shown). We also examined IL-12 p70 production but failed to detect this cytokine in supernatants of pDCs (data not shown), as reported by others (37). IL-12 production was only detected in culture supernatants of CD11c⫹ DCs in response to R-848. pDCs matured by HIV-1 acquire chemotactic ability toward CCL19. Since pDCs express CCR7 following exposure to HIV-1, we tested their capacity to migrate toward CCL19 chemokine, the ligand of CCR7, which is secreted in the lymphoid compartment. After a 16-h culture either with IL-3, HIV-1 (MN or JR), AT-2 HIV-1 (MN or JR), or R-848, the chemotactic activity of pDC toward CCL19 was measured in a 2-h transwell assay. Migration of pDCs toward CCL19 was observed after stimulation with infectious HIV-1, inactivated AT-2 HIV-1, and R-848, whereas no chemotactic activity was observed after exposure to IL-3 (Fig. 2). These results were in accordance with the observed CCR7 expression on pDCs (Fig. 1B, upper panel). Secreted products of HIV-1-activated pDCs induce maturation of CD11cⴙ DCs. Because we detected TNF-␣ and IFN-␣ in the supernatants of HIV-1-activated pDCs and these cytokine are known maturation stimuli for CD11c⫹ DCs (44), we tested whether HIV-1-activated pDCs could induce the maturation of blood CD11c⫹ DCs in a bystander fashion. In a first set of experiments we cultured pDC and CD11c⫹ DCs alone or together and exposed them either to Mv or HIV-1MN. After 16 h, activation of pDCs was observed only in cultures containing HIV-1, independent of the presence of CD11c⫹ DCs, and was characterized by the upregulation of CD83, CD80, and CD86 (Fig. 3A). Maturation of CD11c⫹ DCs (i.e., upregulation of CD83, CD80, and CD86) was observed only in those cultures where pDCs and HIV-1 were also present, whereas no increase of CD11c⫹ DC maturation was evident in cultures where CD11c⫹ DCs were cultured alone, with Mv, or with HIV-1. To further address the nature of the CD11c⫹ DC maturation stimulus produced by HIV-1-activated pDCs, we cultured CD11c⫹ DCs with supernatants from pDCs or CD11c⫹ DCs exposed either to Mv or to HIV-1. As a control, CD11c⫹ DCs were exposed directly to HIV-1 or R-848. Maturation of CD11c⫹ as seen by CD83 staining was induced only by R-848 or by supernatants from HIV-1-exposed pDC (Fig. 3B), con-
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firming our hypothesis that TNF-␣ alone or with other soluble factors produced by pDCs is responsible for the maturation of CD11c⫹ DCs. Indeed, we were able to block bystander maturation of CD11c⫹ DC by the addition of a mixture of neutralizing antibodies against IFN-␣, IFN-, TNF-␣, and IFN-␣ and - receptor (Fig. 3C). Significantly, we could not induce the maturation of CD11c⫹ DCs with recombinant IFN-␣ alone but could do so with recombinant TNF-␣, as expected (data not shown). Given that the number of blood CD11c⫹ DCs obtained after each purification is very low, we further characterized the effects of supernatants from HIV-1-exposed pDCs and CD11c⫹ DCs on monocyte-derived immature DCs, which can be obtained in large quantities. We observed that maturation of monocyte-derived DC was induced by supernatants of pDCs activated by either strain of HIV-1 (MN or JR-CSF) and also by the corresponding chemically inactivated AT-2 virus, whereas CD11c⫹ DC supernatants did not induce maturation of monocyte-derived DCs (Fig. 3D). As soluble products from HIV-1-activated pDCs upregulate CD80, CD83, and CD86 on the surface of CD11c⫹ DCs (Fig. 3A), we next tested if CCR7 is also upregulated on myeloid DCs. It was technically not possible to address this question on blood-purified CD11c⫹, because the anti-CCR7 MAb used in this study is conjugated to PE and the FL-2 fluorescence of CD11c⫹ DCs is saturated by the staining with PE-conjugated anti-CD11c MAb used to purify them. Therefore, we chose to use monocyte-derived DCs to test this hypothesis. After 40 h of culture, CCR7 expression on monocyte-derived DCs was only observed in response to R-848 and to supernatants of HIV-1activated pDCs (Fig. 3E). Altogether, these results suggest that CD11c⫹ DCs stimulated by HIV-1-activated pDCs mature and acquire the capacity to migrate to secondary lymphoid organs. T-cell-stimulatory ability of HIV-1-activated pDCs. Because pDCs are activated following exposure to HIV-1, we tested their ability to stimulate allogeneic naïve CD4⫹ T cells. HIV-1-exposed pDCs were similar to or somewhat less efficient than IL-3 or Mv/IL-3 treated pDCs in inducing T-cell proliferation depending on the experiments (Fig. 4A). Furthermore, the responses induced by HIV-activated pDCs were consistently lower than the proliferation induced by CD11c⫹ DCs or R-848-treated pDCs. Overall, the differential stimulatory activities observed in these experiments correlated with the levels of costimulatory molecules (CD80 and CD86) seen on the surface of these cells (Fig. 1B and 3A), suggesting that the HIV-1-induced activation of pDCs does not lead to their complete maturation. However, one should bear in mind that HIV confers sufficient signals to pDCs to render them viable, sensitive to CCL19-mediated chemotactic effect, and capable of stimulating T cells (albeit at levels lower than those of potent stimuli such as R-848), whereas Mv-stimulated pDCs die rapidly (Fig. 1) and do not substantially support T-cell activation (data not shown). This T-cell-activating ability was verified in subsequent experiments where we tested the capacity of T cells stimulated by HIV-activated pDCs to produce IFN-␥ and IL-4. Restimulated CD4⫹ T cells were polarized to preferentially secrete IFN-␥ instead of IL-4 after stimulation with R-848-matured CD11c⫹ DCs, Mv/IL-3- or HIV-exposed pDCs and especially with R-848 pDCs (Fig. 4B). In contrast, more IL-4-
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producing CD4 T cells were observed in populations stimulated by immature CD11c⫹ DCs (MV/IL-3- and HIV-1exposed CD11c⫹ DC). Therefore, HIV-1 activation is similar to that of other viruses (e.g., influenza) in programming pDCs to prime Th1 responses. HIV-1 thus activates pDCs at several levels: enhanced viability, upregulation of maturation markers, secretion of antiviral and proinflammatory cytokines, enhanced migration towards nodal cytokines, T-cell-activating potential, and, finally, concomitant bystander maturation of CD11c⫹ DCs.
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FIG. 3. pDCs activated by HIV-1 activate CD11c⫹ DCs. (A) CD11c⫹ DCs and pDCs were cultured alone or together in the presence of Mv or 300 ng of HIV-1MN/ml. After 16 h, surface expression of CD83, CD80, and CD86 was measured using CD11c⫹ as a marker to discriminate DC subsets. (B) CD11c⫹ DCs were cultured alone, with 300 ng of HIV-1MN/ml, 10 M R-848, or a 1:2 dilution of supernatants of pDCs or CD11c⫹ DCs cultured for 16 h with Mv or 300 ng of HIV-1MN/ml. After overnight culture, expression of CD83 by CD11c⫹ DCs was measured. (C) CD11c⫹ DCs were cultured in the absence or presence of neutralizing antibodies against IFN-␣, IFN-, TNF-␣, and IFN-␣/- receptor and with supernatants of pDCs previously cultured for 16 h with 300 ng of HIV-1MN/ml. After overnight culture, expression of CD83 by CD11c⫹ DCs was measured. (D) Immature monocyte-derived DCs were cultured alone, with 10 M R-848, or a 1:3 dilution of supernatants of pDCs or CD11c⫹ DCs cultured for 16 h with Mv, 300 ng of HIV-1 (right panel, MN; left panel, JR-CSF)/ml or 300 ng of AT-2 HIV-1 (right panel, MN; left panel, JR-CSF)/ml. After overnight culture, expression of CD83 by monocyte-derived DCs was measured. (E) Immature monocyte-derived DCs were cultured alone, with R-848, 300 ng of HIV-1MN/ml, or a 1:3 dilution of supernatants of pDCs or CD11c⫹ DCs cultured during 16 h with Mv or 300 ng of HIV-1MN/ml. After 40 h, expression of CD83 by monocyte-derived DCs was measured. Each image is representative of at least three different experiments.
DISCUSSION In this in vitro study we assessed the effects of HIV-1 exposure upon various DC subsets. We report for the first time that pDCs exposed to X4 or R5 HIV-1, either infectious or chemically inactivated (AT-2 HIV-1), produce cytokines such as
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FIG. 3—Continued.
IFN-␣ and TNF-␣, upregulate the DC maturation markers CD83 and CCR7, and to a lesser extent upregulate the costimulatory molecules CD80 and CD86. Furthermore, HIV-1activated pDCs gain migratory capacity toward CCL19, a chemokine produced in secondary lymphoid organs. We also confirm that, in contrast to pDCs, CD11c⫹ DCs do not mature upon contact with HIV-1 but that they do mature indirectly as a consequence of the production of cytokines by HIV-activated pDCs (TNF-␣, possibly in conjunction with IFN-␣ and IFN-). CD11c⫹ DCs matured by these factors upregulate CD80, CD83, CD86, and CCR7 molecules, suggesting that they acquire CCL19-driven chemotactic ability. Finally, we report that pDCs exposed to HIV-1 stimulate naïve CD4⫹ T cells to preferentially produce Th1 cytokines, although the effects are weaker than the stimulatory capacity of R-848-matured pDCs or CD11c⫹ DCs. The maturation of both DC subsets by HIV-1 may have important consequences on their trafficking, virus uptake, and cellular transmission in trans and on the HIV-1specific immune response as discussed below. In addition, the relocalization of both subsets of DCs into secondary lymphoid compartments may affect the capacity of the immune system to fight other pathogens in the periphery and may participate in the development of AIDS. The direct and indirect activation of lymphoid and myeloid DCs observed here may account for their decline in the blood
mononuclear cell fractions of seropositive patients, and in particular, untreated patients (5, 13, 17, 19, 20, 30, 39, 46, 48). Indeed, HIV-1-activated DCs express CCR7 and may relocate to the secondary lymphoid organs where HIV-1 replication is the most active. CD11c⫹ DCs have been shown to be one of the first mucosal cells targeted during oral and sexual transmission (9, 16, 31, 50). They capture HIV through C-type lectin (e.g., DC-SIGN, mannose receptor)-dependent and -independent pathways, thereby promoting the subsequent transmission of virus to CD4⫹ T cells (26, 27). Given that CD11c⫹ DCs do not mature directly in response to HIV, their migration from mucosa to secondary lymphoid organs and transmission of HIV-1 to CD4⫹ T is probably facilitated by the release of maturation stimuli in the local environment. This may be provided by bacteria or other pathogens accompanying HIV as it engages the mucosa and/or through cytokines produced by HIV-activated pDCs, for example, in the blood, where viral loads can approach high levels. pDCs in turn may be recruited to mucosal sites through chemokines produced by CD11c⫹ DCs following reprogramming by HIV tat (32). Indeed, pDCs accumulate at sites of chronic inflammation, for example, lupus skin lesions (18), and in nasal mucosa following the triggering of allergic rhinitis (33). However, a few studies have questioned whether pDCs routinely accumulate in mucosal sites. In colonic and rectal mucosa, Bell et al. failed to detect
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FIG. 4. pDCs activated by HIV-1 remain poor activators of naïve CD4⫹ T cells. (A) pDCs were cultured with Mv and IL-3, with 300 ng of HIV-1MN/ml, or with 10 M R-848, while CD11c⫹ DCs were cultured with Mv, HIV-1MN, or with R-848. After 16 h DCs were washed and cocultured with allogeneic naïve CD45RA⫹ CD4⫹ T cells at different T-cell/DC ratios. After 4 days, [3H]thymidine was added for 12 h. Cells were then harvested to assess proliferation. (B) pDCs and CD11c⫹ DCs were cultured with naïve CD4⫹ T cells at a ratio of 1:30. After 7 days the T cells were restimulated with PMA and ionomycin in presence of brefeldin A and were analyzed by intracellular flow cytometry for production of IFN-␥ or IL-4. The percentage of T cells producing cytokines is depicted in the FACS profiles. Each image is representative of at least three different experiments.
pDCs even in inflamed mucosa from Crohn’s disease patients (6). Further studies will be necessary to determine whether pDCs localize in common mucosal sites of HIV entry, e.g., oral and vaginal-cervical mucosa. In the case of intravenous HIV infections, pDCs may be one of the first cells to transport virus into lymph nodes, because pDCs are cellular targets of HIV (17, 23, 41) and because they rapidly acquire CCL19-driven migratory capacity after contact with this virus. By maturing bystander CD11c⫹ DCs, HIVactivated pDCs may be a major intermediary for ensuring the rapid trafficking of virus to lymph nodes. This phenomenon may also affect the development of anti-HIV-1 T-cell responses and may play a role in the transmission of HIV-1 to CD4⫹ T cells. It has been reported that pDCs exposed to influenza A virus are able to process and present viral antigens to stimulate anti-influenza CD4⫹ and CD8⫹ T-cell memory responses as efficiently as influenza virus-infected CD11c⫹ DCs (24). This result suggests that HIV-1-infected pDCs will also have the capacity to process and present HIV-1 antigens to naive CD4⫹ T cells, a phenomenon we are presently investigating. pDCs can present HIV- and cytomegalovirus-derived peptides to memory T cells following stimulation with R-848 and other TLR agonists (37), but the processing capacity of whole HIV virions remains to be shown. Other investigators have reported that pDCs support HIV-1 replication without
additional stimuli, although this remains controversial. Nevertheless, it is possible that pDCs may facilitate virus transfer while concomitantly processing and presenting viral antigens to T cells, a situation somewhat analogous to that of CD11c⫹ DCs. This hypothesis is further supported by the observation of Fong et al., who showed that pDCs support productive virus replication after engagement of CD40 by CD40L and are able to transmit HIV to PHA-activated CD4⫹ T cells (23). The fact that HIV antigens may be presented by pDCs to T cells in the lymph node may have an effect on the quality of the anti-HIV immune responses. pDCs used to stimulate allogeneic naïve T cells have been shown to induce Th1 and Th2 as well as suppressive T-cell responses depending on the method used to culture them and the nature of the stimulus (12, 24, 29, 34, 42). In a mouse model it has been shown that the quantity of Ag presented by pDCs may also affect the polarization of the T-cell response (10). However, in our hands, we observed mainly a Th1 polarization of allogeneic CD4⫹ T-cell response by HIV activated pDCs. The outcome of the continuous exposure of DCs to the maturation stimuli that HIV-1 provides now has to be characterized. It is possible that this continuous exposure may induce dysregulation of DC function, because it has been observed that the remaining DCs in blood of HAART-naïve patients
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have a defect in their capacity to stimulate allogeneic T-cell proliferation (17). Interestingly, we show that chemically inactivated virus (AT-2 HIV-1) is as efficient as the natural virus to directly mature the pDCs and indirectly mature the CD11c⫹ DCs. AT-2 HIV-1 is a candidate reagent for an HIV-1 vaccine, because these virions can fuse and enter targets cells, notably DCs, without productive infection, thereby allowing HIV antigens to be presented by DCs to T cells (11, 35). Indeed, inactivated simian immunodeficiency virus-pulsed DCs were effective therapeutic vaccines in a primate model (38). Our results add to the potency of these inactivated virions by showing that, in addition to being an effective immunogen, they have endogenous adjuvant capacity through stimulation of pDCs and bystander CD11c⫹ DC maturation. Our results offer a better understanding of the mechanisms influencing the trafficking of HIV and some of its cellular targets from the periphery to secondary lymphoid organs. These observations suggest unique ways to take advantage of this phenomenon to efficiently activate anti-HIV immune responses in patients.
12. 13.
14.
15.
16. 17. 18.
ACKNOWLEDGMENTS 19.
These studies were supported by the NIH grants AI 44628 and C.A. to N.B. and a Burroughs Wellcome Clinical Investigator Grant, a Doris Duke Foundation Award, and a Cancer Research Institute grants to N.B. N.B. is an Elizabeth Glaser scientist. This work was also supported in part with federal funds from the National Cancer Institute, the National Institutes of Health, under contract NO1-CO-124000 to J.D.L. Funding was also provided by the ARC (Association pour la Recherche sur le Cancer) to J.-F.F. and FRM (Fondation pour la Recherche Me´dicale) to A.-S.B.
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