Nef Is Required for Efficient HIV-1 Replication in Cocultures of Dendritic Cells and Lymphocytes

Virology 286, 225–236 (2001) doi:10.1006/viro.2001.0984, available online at http://www.idealibrary.com on

Nef Is Required for Efficient HIV-1 Replication in Cocultures of Dendritic Cells and Lymphocytes Caroline Petit,* Florence Buseyne,† Claire Boccaccio,‡ Jean-Pierre Abastado,‡ Jean-Michel Heard,* and Olivier Schwartz* ,1 *Unite´ Re´trovirus et Transfert Ge´ne´tique, †Laboratoire d’Immunopathologie Virale, URA CNRS 1930, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; and ‡ImmunoDesigned Molecules Research Laboratories, Institut des Cordeliers, 15 rue de l’Ecole de Me´decine, 75006 Paris, France Received March 6, 2001; returned to author for revision April 16, 2001; accepted April 30, 2001 Dendritic cells (DCs) are thought to play a crucial role in the pathogenesis of HIV-1 infection. DCs are believed to transport virus particles to lymph nodes before transfer to CD4 ⫹ lymphocytes. We have investigated the role of Nef in these processes. HIV-1 replication was examined in human immature DC-lymphocyte cocultures and in DCs or lymphocytes separately. Using various R5-tropic and X4-tropic HIV-1 strains and their nef-deleted (⌬nef) counterparts, we show that Nef is required for optimal viral replication in immature DC-T cells clusters and in T lymphocytes. Nef exerts only a marginal role on viral replication in immature DCs alone as well as on virion capture by DCs, long-term intracellular accumulation and transmission of X4 strains to lymphocytes. We also show that wild-type and ⌬nef virions are similarly processed for MHC-I restricted exogenous presentation by DCs. Taken together, these results help explain how HIV-1 Nef may affect viral spread and immune responses in the infected host. © 2001 Academic Press Key Words: HIV-1; Nef; dendritic cells; lymphocytes; virus transmission; MHC-I exogenous presentation.

INTRODUCTION

et al., 1993). Nef also down-regulates the surface expression of major histocompatibility complex class I molecules (MHC-I) (Le Gall et al., 1998; Schwartz et al., 1996), preventing the recognition and lysis of infected cells by both CD8 ⫹ cytotoxic T lymphocytes (CTLs) and NK cells (Cohen et al., 1999; Collins et al., 1998). In addition, Nef exerts direct positive effects on the virus life cycle in primary human lymphocytes and in macrophages (Aiken and Trono, 1995; de Ronde et al., 1992; Glushakova et al., 1999; Miller et al., 1994; Spina et al., 1994), probably by facilitating early (entry or postentry) steps of the replicative cycle (Aiken and Trono, 1995; Miller et al., 1994; Schwartz et al., 1995). Finally, Nef mediates lymphocyte chemotaxis and activation when expressed in macrophages, indicating that the viral protein may also play a role in lymphocyte recruitment (Swingler et al., 1999). Dendritic cells (DCs) are also natural targets of HIV-1 replication. However, the importance of Nef for viral replication in these cells remains poorly documented. DCs are the most potent antigen presenting cells that can stimulate resting naive T lymphocytes and initiate CTL responses (Banchereau and Steinman, 1998). Immature DCs residing in peripheral tissues, including the skin and several mucosal surfaces, are specialized in capturing and processing antigens. Immature DCs do not express high levels of surface molecules required for potent T-cell stimulation. After antigen capture, DCs mature and travel toward secondary lymphoid organs, processing antigens for presentation and acquiring the ca-

The nef gene is required for efficient in vivo replication and pathogenicity of human and simian immunodeficiency viruses (Cullen, 1998; Harris, 1996). Experimental infection of macaques with SIVmac⌬nef is characterized by a low level of viral replication (Chakrabarti et al., 1995; Kestler et al., 1991). In humans, several long-term nonprogressors have been identified, in which only nefdeleted proviruses were detected (Deacon et al., 1995; Kirchoff et al., 1995). These individuals display low viral loads and remained healthy for more than a decade, although some HIV⌬nef-infected patients show signs of immune defects (Greenough et al., 1999). The cellular and virological mechanisms responsible for this role in vivo remain unclear. Multiple activities of Nef have been described in vitro: Nef interferes with cellular signal transduction pathways and interacts with a number of kinases (Saksela, 1997). Nef down-regulates the cell surface expression of CD4, the primary receptor for HIV and SIV (Guy et al., 1987; Piguet et al., 1999). Reduced CD4 surface levels could prevent superinfections, increase the life span of viral envelope-expressing cells, enhance envelope incorporation into virions, and alter activity of CD4 ⫹ T cells (Benson et al., 1993; Lama et al., 1999; Ross et al., 1999; Schwartz et al., 1993; Skowronski

1 To whom correspondence and reprint requests should be addressed. Fax: ⫹33 1 45 68 89 40. E-mail: [email protected].

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pacity to attract and activate resting CD8 ⫹ CTLs during that journey. DCs are believed to play a crucial role in the pathogenesis of HIV-1 infection (Banchereau and Steinman, 1998; Blauvelt, 1997; Knight and Patterson, 1997; Rowland-Jones, 1999). Immature DCs in mucosal tissues may represent the initial targets for HIV during primary infection, and these cells are suspected to be responsible for the transport of virus to lymph nodes and transfer to CD4 ⫹ lymphocytes (Banchereau and Steinman, 1998; Blauvelt, 1997; Knight and Patterson, 1997; RowlandJones, 1999). DCs express low levels of the HIV receptors CD4 and CCR5 (Ayehunie et al., 1997; Granelli-Piperno et al., 1996; Klagge and Schneider-Schaulies, 1999; O’Doherty et al., 1993; Rubbert et al., 1998). DCs are considered either negative or weakly positive for CXCR4 expression, depending on the study (Ayehunie et al., 1997; GranelliPiperno et al., 1996; Klagge and Schneider-Schaulies, 1999; Rubbert et al., 1998). These contradictory results may be due to different maturation states of DCs, which regulate chemokine receptor surface expression (Lin et al., 1998). HIV-1 replicates rather inefficiently in DCs. R5-tropic, but not X4-tropic, HIV-1 strains induce chemotaxis and replicate in immature DCs (Granelli-Piperno et al., 1998; Lin et al., 2000). However, both R5- and X4tropic HIV-1 readily bind and enter DCs (Ayehunie et al., 1997; Granelli-Piperno et al., 1999, 1996; Klagge and Schneider-Schaulies, 1999). HIV-1 transmission from DCs to T cells involves a DC-specific receptor, DC-SIGN, which binds viral envelope glycoproteins and retains the attached virus in an infectious state (Geijtenbeek et al., 2000). Interestingly, DC-T cell clusters are the sites of extremely efficient HIV-1 (Cameron et al., 1992; GranelliPiperno et al., 1998, 1999; Pope et al., 1994) and SIV (Ignatius et al., 1998; Messmer et al., 2000; Pope et al., 1997) replication in culture, and evidence exists for an active HIV-1 replication within DC-T clusters in lymphoid tissues (Frankel et al., 1996). The observation that Nef affects viral loads as soon as the early stages of infection in humans and monkeys (Chakrabarti et al., 1995; Deacon et al., 1995; Kestler et al., 1991; Kirchoff et al., 1995) suggests that this factor may play an important role in the interactions between HIV-1, DCs, and T cells. Recently, Messmer et al. reported that SIVmac239⌬nef displayed decreased replicative capacity in cocultures of simian immature DCs and T cells (Messmer et al., 2000). However, because immature DCs are not susceptible to SIVmac239 replication, whether SIV Nef is required for viral replication in DCs or for viral transmission from DCs to T cells could not be determined in this study (Messmer et al., 2000). We have investigated here the role of Nef on HIV-1 replication in human immature DCs-T cell cocultures and in separate cultures of DCs or lymphocytes. Using various R5 and X4 HIV-1 strains and their nef-deleted counterparts, we report that in mixtures of immature DCs and T cells, the

level of replication of ⌬nef was significantly lower than that of the wild-type. Altogether, our results indicate that Nef is required for optimal viral replication in immature DC-T cell clusters and in T lymphocytes, but exerts only a marginal role in immature DCs. We also show that wild-type and ⌬nef virions are similarly captured and processed for MHC-I restricted exogenous presentation by DCs. RESULTS Role of Nef on HIV-1 replication in immature DC-T cell cultures To investigate the role of Nef on HIV-1 replication in primary immature DC-T cell cultures, we compared the kinetics of viral growth of WT and ⌬nef isogenic viruses. Immature DCs have been reported to express CD4 and CCR5, albeit at lower levels than on T cells (Ayehunie et al., 1997; Granelli-Piperno et al., 1996; O’Doherty et al., 1993). Flow cytometry analysis indicated that monocytederived immature DCs used in this study expressed CD4, low levels of CCR5, and virtually no CXCR4 at the cell surface (Fig. 1A). Since HIV replication in DCs depends on the tropism of the virus (Granelli-Piperno et al., 1998), we analyzed the replication of two R5 isolates, NLAD8 and YU-2, and one X4 isolate, NL43. Immature DCs were exposed to low virus doses (7 ng of p24 per 2 ⫻ 10 5 cells) in an overnight incubation; cells were then washed to remove extracellular virus and mixed with autologous T lymphoblasts that had been activated 3 days before coculture. Viral replication was assessed by measuring p24 production in cell supernatants. All three WT viruses replicated in these mixed cultures (Fig. 1B). With NLAD8 and YU-2, the levels of p24 production reached 100–200 ng/ml at days 8–12 postinfection (pi). They were lower with NL43 (peak at 20 ng/ml), suggesting that R5 strains replicate more efficiently than X4 strains in DC-T cell cultures. Interestingly, ⌬nef replication was significantly impaired when compared to WT viruses (Fig. 1B). Moreover, the defect of ⌬nef viruses varied with the viral strain. NLAD8⌬nef replication was moderately affected, with about a threefold reduction in p24 production when compared to WT virus (Fig. 1B). With YU-2 and NL43 isolates, ⌬nef replication was more dramatically impaired, reaching only 5–10 ng p24/ml at day 12 pi. This corresponded to about a 10-fold reduction in p24 production when compared with WT viruses. Therefore, HIV replication is significantly affected in primary DC-T cell cultures in the absence of Nef. The replicative defect of ⌬nef viruses is observed with both R5 and X4 strains and is thus independent of viral tropism. Of note, a similar defect of ⌬nef viruses was observed when cells were infected with a 10-fold higher viral inoculum (not shown) or when immature DCs were mixed with heterologous activated T cells (not shown). One attractive feature of the DC-T coculture model is

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FIG. 1. Surface expression of HIV receptors in immature DCs and replication of HIV-1 WT and ⌬nef in immature DCs-T cell cocultures. (A) Surface levels of HIV receptors CD4, CXCR4, and CCR5 in immature DCs. Cells were stained with Leu3A (anti-CD4), 12G5 (anti-CXCR4), or 2D7 (anti-CCR5) mAbs and revealed with a phycoerythrin-labeled secondary antibody. Cells were analyzed by flow cytometry. CTRL, cells stained with an irrelevant isotypic mAb. (B) Replication of isogenic WT and ⌬nef isogenic HIV-1 in immature DCs-T cell cocultures. DCs (2 ⫻ 10 5) were exposed to the indicated viruses (7 ng of p24). After overnight incubation, cells were washed to remove unbound viruses and mixed with 8 ⫻ 10 5 autologous lymphoblasts that had been activated 3 days before (top) or with 8 ⫻ 10 5 autologous nonactivated lymphocytes. Viral replication was assessed by measuring p24 production in the culture supernatants over a 12-day period. Days pi: days postinfection. Data are representative of three experiments.

that it promotes the growth of HIV without prior activation of T cells. We therefore examined the effects of Nef deletion on viral replication in cocultures of DCs and

autologous nonactivated PBLs. All three WT viruses replicated in these mixed cultures (Fig. 1B). Levels of p24 production were much lower than when PBMCs were

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activated before coculture. With NLAD8 and YU-2, p24 production reached 10 ng/ml at days 12–14 pi and was even lower with NL43 (peak below 5 ng/ml). Of note, none of the three viral strains replicated in nonactivated PBMCs alone (not shown), confirming that viral replication in this experimental system requires the presence of DCs. As for DC-activated T-cell cocultures, ⌬nef replication was significantly impaired when compared to WT viruses, and the ⌬nef defect varied with the viral strain (Fig. 1B). When compared to WT, NLAD8⌬nef replication was moderately affected, with about a twofold reduction in p24 production virus. With YU-2 and NL43 isolates, ⌬nef defect was more marked (Fig. 1B). Altogether, these experiments indicate that Nef is required for efficient HIV-1 replication in DC-T cell cultures irrespective of the activation state of T lymphocytes. Influence of Nef on HIV-1 replication in immature DCs We then characterized the replicative defect of ⌬nef viruses in immature DC-T cell cocultures. More precisely, we examined the role of Nef on viral replication in immature DCs and in T cells separately, and on viral transmission from immature DCs to T lymphocytes. To study the role of Nef on HIV-1 replication in immature DCs, we compared the viral growth of WT and ⌬nef isogenic NLAD8, YU-2, or NL43 strains. We used two viral inputs (7 and 50 ng of p24 per 2 ⫻ 10 5 target cells, respectively), since the effect of Nef may be more pronounced at low viral doses (Miller et al., 1994; Spina et al., 1994). Whatever the viral input, both NLAD8 and YU-2 strains replicated in immature DCs, whereas NL43 was unable to grow in these cells (Fig. 2A). This confirmed the finding that immature DCs selectively replicate R5-tropic HIV-1 (Granelli-Piperno et al., 1998). At the lower viral input, which was the same as that used in immature DCs-T cell cocultures, replication of WT NLAD8 or YU-2 reached 2–3 ng p24/ml at day 12 pi (Fig. 2). Therefore, as previously reported (Cameron et al., 1992; Frank et al., 1999; Granelli-Piperno et al., 1998; Pope et al., 1995), viral replication is much less efficient in immature DCs than in DCs-T cell cocultures (compare p24 levels in Figs. 2A and 1B). At the higher viral input, virus production reached 30 and 80 ng of p24 for NLAD8 and YU-2, respectively (Fig. 2A). Interestingly, the replication of ⌬nef viruses was not, or only slightly, affected in DCs. NLAD8 WT and ⌬nef yielded similar p24 production levels at high viral input. Even at the lower viral input, replication of NLAD8⌬nef was not significantly affected (Fig. 2A). Similarly, only a slight difference (less than twofold) was observed between YU-2 WT and ⌬nef viruses (Fig. 2A). Altogether, these results indicate that R5 HIV-1 strains are able to replicate in immature DCs, albeit with a low efficiency. Moreover, Nef is only marginally necessary for HIV-1 replication in immature DCs.

Role of Nef on HIV-1 replication in primary lymphocytes Since HIV-1 replication in immature DCs is not significantly affected by the absence of Nef, we next studied the growth of WT and ⌬nef isogenic NLAD8, YU-2, or NL43 strains in primary lymphocytes. The importance of Nef on HIV replication in PBMCs has been demonstrated (Aiken and Trono, 1995; de Ronde et al., 1992; Glushakova et al., 1999; Miller et al., 1994; Spina et al., 1994). In some studies, it has been reported that HIV⌬nef replicated poorly only in CD4⫹ cells stimulated after infection, when virus inoculum was low (Miller et al., 1994; Spina et al., 1994). In others, the replicative defect was also manifest in fully activated PBMCs and was independent of the multiplicity of infection (Aiken and Trono, 1995; de Ronde et al., 1992). In the immature DC-T cell cultures in which HIV⌬nef replication was impaired (Fig. 1), we used lymphocytes which had been activated before incubation with virus-exposed DCs. We therefore used the same protocol and analyzed viral replication in PBLs that had been activated with PHA 3 days before infection. As for immature DCs, we used two viral inputs (7 and 50 ng of p24 per 8 ⫻ 10 5 target cells, respectively). Wild-type NLAD8, YU-2, and NL43 induced p24 production in supernatants of infected cells (Fig. 2B), confirming that R5 and X4 strains replicate in PBLs. Both strains efficiently replicate and reached equivalent peaks of p24 production at each viral inoculum (100–150 ng p24/ml for NLAD8, 25–50 ng p24/ml for YU-2 and NL43). In contrast, the replicative capacity of ⌬nef mutants was dramatically affected. As in immature DC-T cell cultures, the defect of ⌬nef viruses varied with the viral strain. NLAD8⌬nef replication was only two- to threefold lower than WT virus (Fig. 2B). With YU-2 and NL43 isolates, ⌬nef replication was more significantly impaired (Fig. 2B). Nef-mutated viruses replicated very poorly, even at the higher viral inoculum, plateauing at only 5 ng/ml p24. Taken together, these results demonstrated a consistently positive influence of Nef on HIV-1 replication in primary lymphocytes. The absolute levels of viral replication, as well as the defects of nef-deleted viruses in primary cells, may vary according to donors. To obtain a better insight of the influence of Nef on viral replication, results from three to five independent experiments are shown in Fig. 3. Differences between WT and ⌬nef replication levels were expressed as ratios of p24 concentrations at the peak of viral production, with 100% corresponding to WT values. Calculations were performed for each viral strain and for each culture condition, including DCs and activated T cells separately and DC-activated T cell cocultures. Irrespective of cell culture conditions, ⌬nef virus defect was minimal with NLAD8 strain, whereas it was much more pronounced with YU-2 and NL43 strains (Fig. 3). Therefore, the replicative defect associated with the absence of Nef is strain-dependent. Since NLAD8 and NL43 are isogenic viruses differing only in the env gene, our result

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FIG. 2. HIV-1 WT and ⌬nef replication in separate cultures of immature DCs and activated PBLs. DCs (2 ⫻ 10 5 cells, A) and PHA-activated PBLs (8 ⫻ 10 5 cells, B) were exposed to either 50 ng (high inoculum panel) or 7 ng (low inoculum panel) p24 of the indicated viral strains. After overnight incubation, cells were washed to remove unbound virus. Viral replication was assessed by measuring p24 production in the culture supernatants. Data are representative of three experiments.

indicates that the Nef defect may be modulated by Env proteins. Data also indicates that Nef requirement for optimal viral growth varied with cell culture conditions. In immature DCs-T cell cocultures, ⌬nef replicated less efficiently than WT (2-fold, 10-fold, and 5-fold reduction of p24 peak levels for NLAD8, YU-2, and NL43, respectively). In immature DCs alone, the effect of Nef was less obvious

(⌬nef p24 levels reduced to 75 and 60% of those of WT for NLAD8 and YU-2, respectively). In contrast, Nef was required for efficient virus production in lymphocytes, as p24 levels reached by ⌬nef viruses were 4-, 10-, and 20-fold lower than for WT NLAD8, NL43, and YU-2, respectively (Fig. 3). Taken together, these experiments indicate a more prominent role of Nef in lymphocytes than in DCs.

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FIG. 3. Comparison of HIV-1 WT and ⌬nef replication in immature DC-activated PBLs cocultures, in immature DCs, and in activated PBLs. Cells were infected as described in Figs. 1–2 with the indicated viral strains. Viral replication was assessed by measuring p24 production in the culture supernatants. WT and ⌬nef replication levels were compared by calculating the ratio of p24 released at the peak of viral production with 100% corresponding to WT values. Data are mean ⫾ SD of three to five independent experiments. N.A., not applicable since NL43 does not replicate in immature DCs.

Role of Nef during transmission of X4-tropic HIV-1 from DC to T cells Since Nef is required for efficient HIV-1 replication in DCs-T cell cocultures, the transmission step between DCs and T cells may be impaired with ⌬nef viruses. We therefore developed an experimental system allowing the study of the capacity of DCs to transmit infectious viral particles. Since R5 strains replicate in immature DCs even in the absence of T cells (see Fig. 2A), it was important to use an X4 strain (NL43) for this analysis.

With the aim to overcome possible artifacts that could result from the altered replication of NL43⌬nef in PBLs (see Fig. 2B), DCs were cocultivated with immortalized T cells, in which Nef is dispensable for viral replication (Aiken and Trono, 1995; Miller et al., 1994; Schwartz et al., 1995; Spina et al., 1994). As we previously reported (Le Gall et al., 1997; Schwartz et al., 1995), cell-free infection of the CD4 ⫹ lymphoid line MT4 with NL43 WT or ⌬nef virions (7 ng of p24 per 8 ⫻ 10 5 cells) produced equivalent amounts of p24 with similar kinetics (Fig. 4C). To

FIG. 4. Viral transmission and stability of HIV-1 WT and ⌬nef viruses after capture by immature DCs. Immature DCs (2 ⫻ 10 5) were exposed to NL43 WT or ⌬nef (7 ng of p24). After overnight incubation, cells were washed to remove unbound virus. Cells were then mixed with 8 ⫻ 10 5 MT4 cells (A) or incubated 5 days before addition of 8 ⫻ 10 5 MT4 cells (B). As a control, MT4 cells (8 ⫻ 10 5) were directly exposed to cell-free NL43 WT or ⌬nef (7 ng of p24) (C). A 5-day incubation period at 37°C of cell-free NL43 WT or ⌬nef fully abrogates viral infectivity (D). Viral replication was assessed by measuring p24 production in the culture supernatants over a 14-day period. Data are representative of two experiments.

Nef AND HIV REPLICATION IN DENDRITIC T-CELL COCULTURES

monitor the transmission of viral particles from DCs to MT4 cells, DCs were pulsed overnight with NL43 WT or ⌬nef virions (7 ng of p24 per 2 ⫻ 10 5 cells). Cells were then washed extensively to remove extracellular virions and mixed with MT4 cells. Efficient replication occurred in recipient MT4 cells (Fig. 4A). The same kinetics were observed for WT and ⌬nef viruses (Fig. 4A), indicating that Nef is dispensable for transmitting viral particles from DCs to MT4 cells. It has been reported recently that transmission of HIV-1 from DCs to T cells involves a specific DC receptor, DC-SIGN, which binds the viral envelope protein and retains the captured virus in an infectious state (Geijtenbeek et al., 2000). Using THP1 monocytoid cell lines stably expressing DC-SIGN, it was demonstrated that this molecule is able to capture and retain HIV-1 for more than 4 days, after which the virus can still infect permissive target cells (Geijtenbeek et al., 2000). We thus examined whether immature DCs are able to capture viral particles for long period of times and the influence of Nef in this process. DCs were pulsed overnight with NL43 WT and ⌬nef virions (7 ng of p24 per 2 ⫻ 10 5 cells). Cells were then washed extensively and 5 days later they were mixed with MT4 cells. Efficient viral replication occurred only after addition of recipient MT4 cells (Fig. 4B). Again, similar kinetics of viral production were observed for WT and ⌬nef viruses (Fig. 4B). Of note, a 5-day incubation at 37°C in culture medium of cell-free NL43 WT and ⌬nef abolished viral infectivity (Fig. 4D). Therefore, primary immature DCs are able to capture and retain HIV-1 in an infectious state for at least 5 days. NL43⌬nef particles were captured and transmitted to target cells as efficiently as their WT counterparts, even after 5 days. Altogether, these experiments strongly suggest that Nef does not affect virion capture, long-term intracellular accumulation, and transmission of X4 strains to T lymphocytes by DCs. Role of Nef on MHC-I restricted presentation of HIV-1 virion antigens by DCs A major function of DCs is to stimulate resting naive T lymphocytes and to initiate primary CTL responses (Banchereau and Steinman, 1998). We have recently shown that immature DCs capture and process incoming HIV-1 virions through the MHC-I-restricted presentation pathway, leading to CTL activation in the absence of viral protein neosynthesis (Buseyne et al., 2001). Presentation of exogenous HIV-1 antigens required adequate virusreceptor interactions and fusion of viral and cellular membranes. We compared the ability of WT and ⌬nef virions to be processed for presentation by this exogenous MHC-I-restricted pathway. To this aim, immature HLA-A2⫹ DCs were exposed to various HIV-1 virions in the presence of AZT, to inhibit reverse transcription and subsequent synthesis of viral proteins. After overnight

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incubation, extracellular particles were washed away and DCs were cocultivated with the HLA-A2-restricted CD8 ⫹ CTL cell line EM71-1. EM71-1 cells were derived from an HIV-infected patient and recognize a well-characterized immunodominant epitope of the Gag p17 protein (Buseyne et al., 2001). DCs pulsed with the synthetic Gag epitope (SL9) activated EM71-1 cells specifically, as measured by IFN-␥ production in an Elispot assay (Fig. 5A). This epitope is present in BRU (which is a X4-tropic strain) and YU-2, but not in NL43 or NLAD8 isolates (Buseyne et al., 2001). We first tested the ability of immature DCs to present the Gag epitope after exposure to the BRU isolate. We observed that DCs pulsed with either WT or ⌬nef viruses efficiently activated EM71-1 cells (Fig. 5A). Immature DCs also efficiently activated effector cells upon exposure to the YU-2 strain (Fig. 5B). Again, no significant difference was noted between WT and ⌬nef viruses, at either high or low viral inoculum (Fig. 5B). Of note, HLA-A2⫺ DCs exposed to various HIV strains were not recognized by EM71-1 cells (not shown), indicating that the exogenous presentation of HIV-1 epitopes is appropriately MHC-I restricted. Altogether, these experiments indicate that DCs present a Gag epitope upon exposure to incoming virions in the absence of viral protein neosynthesis. The exogenous presentation is observed with similar efficiencies with WT and nef-deleted virions. Therefore, the presence of Nef in virus producer cells does not significantly influence the ability of progeny virions entering DCs to be processed for exogenous MHC-I presentation. DISCUSSION We demonstrate here deficient replication of various nef-deleted HIV strains in immature DC-T cell cocultures. DCs are thought to play a crucial role in HIV-1 transmission and propagation. Immature DCs residing in the skin and the mucosa might be the first cell targets of HIV-1. They are believed to transport virus particles and transmit a vigorous infection to T cells in lymph nodes (Cameron et al., 1996; Knight and Patterson, 1997). Although there is considerable evidence that DCs from blood, skin, or lymphoid organs harboring HIV-1 can be found in patients, the contribution of infected DCs to viral burden is probably marginal (Cameron et al., 1996). In contrast, HIV-1 replication is intense in conjugates of DCs and T cells (Cameron et al., 1996) and active viral production has been demonstrated in lymphoid tissues in DC–lymphocyte clusters (Frankel et al., 1996). In vivo, the Nef gene product confers a replicative advantage as early as the acute phase of infection (Chakrabarti et al., 1995; Kestler et al., 1991). The main conclusion of our study is that high levels of viral replication would occur when DCs exposed to wild-type virus encounter CD4 ⫹ T cells, whereas ⌬nef virus would disseminate much less efficiently in this environment.

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FIG. 5. MHC-I presentation of a Gag epitope derived from incoming HIV-1 virions. Immature DCs prepared from HLA-A2⫹ HIV-seronegative individuals were used as stimulator cells in an IFN-␥-Elispot assay. The effector was the CD8 ⫹ CTL line EM71-1, which recognizes an HLA-A2restricted epitope (SL9) from the Gag p17 protein. Stimulating cells were pretreated with AZT (5 ␮M), exposed to BRU WT and ⌬nef (250 ng p24) (A), or in an independent experiment, to YU-2 WT and ⌬nef at either low or high inoculum (25 or 250 ng p24, respectively) (B). Cells were then incubated with EM71-1 cells. Activity of EM71-1 cells is depicted as the number of IFN-␥ positive cells for 500 or 2500 effectors. As a positive control, stimulating cells were pulsed with the cognate peptide (SL9, 1 ␮g/ml). In the experiment depicted in B, the IFN-␥ positive cells were too numerous to be counted when the cognate peptide was used (not shown). Data are mean ⫾ SD of duplicates and are representative of two independent experiments.

In vitro, HIV-1 produced in DC–lymphocyte cocultures probably originates from different sources. A high viral production from DC-T cell syncytia, due to the coexpression of transcription factors, has been reported (GranelliPiperno et al., 1995). However, analysis of the cellular components of virions indicated that virus produced from immature DC-T cell cocultures primarily originates from T cells (Frank et al., 1999). We have dissected the replicative defect of ⌬nef HIV-1 in immature DC-T cocultures. The replication rates of HIV-1 in immature DCs alone were equivalent for wild-type and ⌬nef, whereas the most dramatic replicative defect of ⌬nef HIV-1 was observed in lymphocytes. Therefore, the compromised replication of ⌬nef in DCs-T cells mixture is most likely due to the lymphocytic component of the coculture, rather than to immature DCs. Although initially controversial, it is currently believed that purified DCs do not replicate HIV-1 with high efficiency (Cameron et al., 1996; GranelliPiperno et al., 1998, 1999). Accordingly, we report here that the X4 strain NL43 does not grow in immature DCs, whereas levels of viral production of the R5 stains YU-2 and NLAD8 were about 10–100 times lower in DCs alone than in T cells or in DCs-T cell cocultures. Furthermore, this low virus production was almost unchanged with ⌬nef viruses. This marginal effect of Nef in immature DCs could be due to a reduced number of replicative cycles of HIV-1, which will be consistent with the low levels of viral production in these cells. Alternatively, immature DCs might be “permissive” for ⌬nef replication, in a situation reminiscent of that of Vif (Madani and Kabat, 1998; Simon et al., 1998). On the other hand, our observation that Nef is not required for viral replication in immature DCs provides indirect evidence for the limited contribution of these cells to the viral burden in vivo.

DCs bind and capture HIV-1 through the DC-specific type C lectin, DC-SIGN, which is the receptor for ICAM-3 (Geijtenbeek et al., 2000). Transmission of DC-associated virus to T cells involves the interaction of other adhesion molecules, including LFA1/ICAM-1 and LFA3/ CD2 (Tsunetsugu-Yokota et al., 1997), indicating that close contacts between the two cell types are required for this process. We have investigated the role of Nef during viral transmission from DCs to T cells. However, R5 HIV-1 strains replicate in immature DCs in the absence of T cells, whereas ⌬nef viruses are altered in their replication in primary lymphocytes. These considerations precluded the study of the transmission of R5 strains and the use of primary lymphocytes for this part of our study. We therefore followed the transmission of the X4 strain NL43 from DCs to MT4 lymphoid cells, in which Nef is dispensable for viral replication. We observed similar replication kinetics for WT and ⌬nef viruses, strongly suggesting that Nef is not required for X4-tropic HIV-1 transmission from DCs to T cells. The productive infection of DCs by HIV-1 and their ability to capture virus are thought to be mediated through separate pathways (Blauvelt et al., 1997). Captured particles accumulate in a trypsin-resistant compartment, in which they retain their infectivity for several days (Blauvelt et al., 1997; Geijtenbeek et al., 2000). We show here that immature DCs similarly retain WT and ⌬nef HIV-1 strain NL43 in an infectious state for at least 5 days. Altogether, our experiments strongly suggest that Nef is not required for virion capture, long-term intracellular accumulation, and transmission of X4 strains to T lymphocytes by DCs. Replication of HIV-1 in vivo is highly productive and continuous. Productively infected lymphocytes have a half-life of less than 2 days and contribute to approxi-

Nef AND HIV REPLICATION IN DENDRITIC T-CELL COCULTURES

mately 99% of the viremia (Perelson et al., 1996). We show here that ⌬nef replication is severely compromised in activated lymphocytes, confirming the importance of the viral protein on HIV replication in PBMCs (Aiken and Trono, 1995; Brown et al., 1999; de Ronde et al., 1992; Glushakova et al., 1999; Miller et al., 1994; Spina et al., 1994). Thus, the low viral loads observed in SIV⌬nef or in HIV⌬nef-infected hosts (Deacon et al., 1995; Kestler et al., 1991; Kirchoff et al., 1995) are most likely the consequence of the replicative defect of mutated viruses in lymphocytes. In unstimulated lymphocytes, it has been reported that Nef may provide an activating signal necessary for reaching a high level of productive infection (Alexander et al., 1997; Brown et al., 1999; Glushakova et al., 1999; Wang et al., 2000). That Nef is also required in activated T cells suggests that other mechanisms are involved in the positive effects of this viral protein. Interestingly, we observed strain-specific differences in the dependence on Nef for replication in activated PBMCs. The defect of ⌬nef virus was minimal with NLAD8, whereas it was much more pronounced with YU-2 and NL43. Coreceptor usage apparently does not account for these differences, because both YU-2 and NLAD8 uses CCR5 for entry. In primary macrophages, opposite results concerning the requirement of Nef for HIV-1 replication have been reported. Whereas ⌬nef replication of YU-2 or SF162 strains is severely compromised in macrophages (Brown et al., 1999; Miller et al., 1994), similar replication efficiencies were observed with ⌬nef and wild-type viruses for the ADA isolate or for a NL43/BAL chimera (Meylan et al., 1998; Swingler et al., 1999). Consistent with our observation in lymphocytes, these differences may also be virus strain-dependent. Molecular and cellular mechanisms underlying these differences are not fully understood. The NLAD8 and NL43 viruses that we used here are isogenic strains differing only in the env gene. This indicates that the Nef defect may be modulated by Env proteins. We previously reported that Nef modulates the surface expression and cytopathic effects of gp120/gp41 complexes (Schwartz et al., 1993). Nef also enhances envelope glycoprotein incorporation into virions (Lama et al., 1999; Ross et al., 1999). Therefore, it will be of interest to compare the effects of Nef on gp120/gp41 function using different isogenics strains. Messmer et al. recently reported a positive role of SIV Nef on SIVmac239 replication in cocultures of simian immature DCs and T cells (Messmer et al., 2000). Our results indicate that SIV and HIV Nef share functional homologies in supporting viral replication in immature DC-T cells mixtures. However, our study differs from that of Messmer et al. in several respects. First, the effects of SIV Nef on viral replication in DCs alone or on viral transmission from DCs to T cells were not examined by Messmer et al. because immature DCs are not susceptible to SIVmac239 replication (Messmer et al., 2000). We show here that the prominent role of HIV-1 Nef on viral

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replication in this experimental system occurs in lymphocytes. Second, we observed that Nef is required for efficient HIV-1 replication in DC-T cell cultures irrespective of the activation state of T lymphocytes. In contrast, Messmer et al. reported that when DC-T cell cultures were activated with the superantigen SEB, both wt and ⌬nef SIVmac239 replicated with identical levels and kinetics (Messmer et al., 2000). This discrepancy could be due to experimental differences, or to inherent differences between human and monkey cells or virus strains used. On the other hand, the functional organization of SIV and HIV-1 Nef is different and includes variations in the ability to bind the TCR (Howe et al., 1998; Schaefer et al., 2000). This could influence the respective abilities of SIV and HIV-1 Nef to stimulate viral replication in activated or inactivated lymphocytes. Moreover, by using three HIV-1 isolates, we point out that the requirement for Nef in DC-T cell cocultures is viral strain-specific and depends on Env proteins. DCs can stimulate resting, naive T lymphocytes and, as a consequence, initiate CTL immune responses in vivo (Banchereau and Steinman, 1998). Following antigen capture in peripheral tissues, immature DCs migrate to secondary lymphoid organs. As cells travel, they mature, process antigens for presentation, and acquire the ability to attract and activate resting CD8 ⫹ cytotoxic T lymphocytes. In most cells, MHC-I molecules associate exclusively with peptides derived from endogenous cytosolic proteins. However, stimulation of CTLs against transplants, tumors, bacteria, or viruses that do not infect APCs requires presentation of exogenous antigens by DCs (Albert et al., 1998; Sigal et al., 1999; Yewdell et al., 1999). We have shown that immature DCs process antigens from incoming HIV-1 virions through the MHC-Irestricted presentation pathway, leading to CTL activation in the absence of viral protein neosynthesis (Buseyne et al., 2001). Presentation of exogenous HIV-1 antigens required adequate virus-receptor interactions and fusion of viral and cellular membranes (Buseyne et al., 2001). Thus, the only step of the viral cycle that is required for CTL stimulation by DCs is virus entry. Presentation of epitopes before the synthesis of viral proteins may be essential for CTL activation, since Nef down-regulates MHC-I expression and decreases immune recognition of cells that become productively infected (Collins et al., 1998; Schwartz et al., 1996). We show here that presentation of a Gag epitope derived from incoming virions is observed with similar efficiencies for WT and nef-deleted viruses. Our results strongly suggest that Nef does not influence the ability of incoming virions to be processed for MHC-I exogenous presentation. Moreover, they provide a likely explanation of how anti-HIV CTLs may be induced despite the downmodulating activity of Nef on MHC-I. In summary, we have shown here that Nef is required for optimal HIV-1 replication in mixtures of immature DCs

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and T cells, in a system relevant for the physiopathology of the infection. Furthermore, wild-type and ⌬nef virions are similarly captured and processed for MHC-I restricted exogenous presentation by DCs. Altogether, these results help explain how Nef affects viral spread and immune responses in vivo. MATERIALS AND METHODS Generation of mononuclear subsets DCs were prepared as described using a VacCell processor (IDM, Paris, France) (Goxe et al., 1998). Briefly, peripheral blood mononuclear cells (PBMCs) from leukapheresis were cultured 7 days in serum-free AIM-V medium (Gibco) supplemented with 500 U/ml GM-CSF (a kind gift from Novartis) and 50 ng/ml IL-13 (Sanofi), and DCs were isolated by elutriation. This isolation procedure yielded CD1a ⫹ MHC-I ⫹, MHC-II ⫹, CD64 ⫺, CD83 ⫺, CD80 low, CD86 low cells, a phenotype corresponding to immature DCs. DC purity was ⬎95%. PBMCs were activated with PHA and cultivated in the presence of IL-2 (50 U/ml, Chiron). Nonactivated PBMCs were allowed to adhere for 2 h at 37°C. Nonadherent cells were washed off and used for further studies. Flow cytometry analysis DCs were washed in PBS and incubated with monoclonal antibodies (mAbs) for 30 min at 4°C in PBA (1% bovine serum albumin, 0.1% sodium azide in PBS). After staining, cells were fixed in PBA containing 1% paraformaldehyde and analyzed with a FACScalibur cytofluorometer (Becton–Dickinson). Cells were stained with the following mAbs: anti-CD4: Leu 3A (IgG1, Becton– Dickinson), anti-CXCR4 (IgG2a, 12G5), and anti-CCR5 (IgG2a, 2D7), obtained through the NIH AIDS Research and Reference Reagent Program. As a control, cells were stained with an isotypic IgG2a mAb (Becton–Dickinson). Secondary antibody was a phycoerythrin-labeled goat anti-mouse antibody (Southern Biotechnology). Viruses and infections The use of X4-tropic NL43 WT and ⌬nef, and BRU WT and ⌬nef HIV-1, has been described elsewhere (Schwartz et al., 1995). The R5-tropic NLAD8 WT strain was a kind gift of Eric Freed (Englund et al., 1995). It carries a portion of the env gene from the R5-tropic clone AD8 on a pNL4–3 backbone (Englund et al., 1995). NLAD8 ⌬nef was generated by inserting a frameshift mutation at the unique XhoI site of the viral genome. The R5-tropic YU-2 WT and ⌬nef strains were a kind gift of Mark Feinberg (Miller et al., 1994). Viruses were produced by transient transfection and used for infections as described (Schwartz et al., 1998). For all strains, in single-cycle replication assays using HeLa-CD4 ⫹CCR5 ⫹ LTR–LacZ indicator cells (Mare´chal et al., 1998), ⌬nef

infectivity was reduced by 20 to 60% compared to that of WT (not shown). Infections were carried out as described (Schwartz et al., 1995). When stated, PBMCs were activated 3 days before infection with the indicated viral strains. PBMCs were maintained in the presence of IL-2 throughout the experiments. For coculture infections, DCs were first exposed to the indicated doses of viruses. After an overnight incubation at 37°C, cells were washed and target cells (PBMCs or MT4 cells) were then added at a ratio of four targets for one DC. After infection, DCs were cultivated in RPMI medium (Gibco) supplemented with 10% human AB⫹ serum. Similar viral replication curves were obtained when DCs were cultivated in serum-free AIM-V medium supplemented with GM-CSF and IL-13 (not shown). Viral replication was assessed by measuring p24 production by ELISA (Du Pont de Nemours-NEN) in culture supernatants. CTL line The CTL line EM71-1 has been described elsewhere (Buseyne et al., 2001). It was derived from a child perinatally infected with HIV-1 by repeated stimulations of PBMC with irradiated autologous B-EBV cells coated with the p17 Gag peptide SLYNTVATL (SL9, originally described by Tsomides et al., 1994) in the presence of allogeneic irradiated PBMC. Peptide recognition was HLA-A2 restricted, with a SD50 of 0.5 ng/ml in 51Cr release assays (not shown). This epitope is present in BRU and YU-2 HIV-1 strains. Ninety-seven percent of EM71-1 cells were CD8⫹ and CD3 ⫹, and 94% were stained with SL9-HLA-A2 tetramers (Buseyne et al., 2001). CTL culture conditions were as described (Buseyne et al., 1998). Elispot assay The reverse-transcriptase inhibitor AZT (5 ␮M, Sigma) was added to cells 3 to 5 h before exposure to viruses and maintained throughout the assay. DCs (2 ⫻ 10 6 cells) were exposed to the indicated viruses for 1 h in a volume of 1 ml and diluted twice in fresh medium before overnight incubation. Viral inoculum was 25 or 250 ng of p24/2 ⫻ 10 6 cells. Stimulator cells (DCs) were washed twice before incubation with effectors. IFN-␥ production was measured in a Elispot assay as described elsewhere (Buseyne et al., 2001). Briefly, targets and effectors were incubated overnight in nitrocellulose-bottomed 96well plates (Millipore) coated with anti-IFN-␥ mAb 1-D1K (15 ␮g/ml, Mabtech). IFN-␥ production was revealed by sequential incubations with biotinylated anti-IFN-␥ mAb 7-B6-1 (1 ␮g/ml, Mabtech), streptavidine-alkaline phosphatase (0.5 U/ml, Boehringer Mannheim), and BCIPNBT substrate (Promega). Positive spots were counted using a binocular microscope. ACKNOWLEDGMENTS We thank Susan Michelson and Nathalie Sol-Foulon for critical reading of the manuscript. We thank Eric Freed, Mark Feinberg, and the NIH

Nef AND HIV REPLICATION IN DENDRITIC T-CELL COCULTURES AIDS Research and Reference Reagent Program for the kind gift of reagents. This work was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), SIDACTION, and the Pasteur Institute. C.P. is a fellow of the ANRS.

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