Human Endothelial Cells Enhance Human Immunodefici
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病菌学杂志 2005年第1期
Section of Immunobiology
Department of Genetics
Department of Pathology
Department of Dermatology
Interdepartmental Program in Vascular Biology and Transplantation
Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut
Department of Pathophysiology, Kazan State Medical University, Kazan, Russia
ABSTRACT
Infected CD4+ T cells are the primary sites of human immunodeficiency virus type 1 (HIV-1) replication in vivo. However, signals from professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, greatly enhance HIV-1 replication in T cells. Here, we report that in cocultures, vascular endothelial cells (ECs), which in humans can also serve as APCs, can enhance HIV-1 production of both CCR5- and CXCR4-utilizing strains approximately 50,000-fold. The observed HIV-1 replication enhancement conferred by ECs occurred only in memory CD4+ T cells, required expression of major histocompatibility complex class II (MHC-II) molecules by the ECs, and could not be conferred by fixed ECs, all of which are consistent with a requirement for EC-mediated T-cell activation via T-cell receptor (TCR) signaling. Deletion of nef (Nef–) decreased HIV-1 production by approximately 100-fold in T cells cocultured with ECs but had no effect on virus production in T cells cocultured with professional APCs or fibroblasts induced to express MHC-II. Human ECs do not express B7 costimulators, but Nef– replication in CD4+-T-cell and EC cocultures could not be rescued by anti-CD28 antibody. ECs act in trans to enhance wild-type but not Nef– replication and facilitate enhanced wild-type replication in na?ve T cells when added to T-cell or B-lymphoblastoid cell cocultures, suggesting that ECs also provide a TCR-independent signal to infected T cells. Consistent with these in vitro observations, wild-type HIV-1 replicated 30- to 50-fold more than Nef– in human T cells infiltrating allogeneic human skin grafts on human huPBL-SCID/bg mice, an in vivo model of T-cell activation by ECs. Our studies suggest that ECs, which line the entire cardiovascular system and are, per force, in frequent contact with memory CD4+ T cells, provide signals to HIV-1-infected CD4+ T cells to greatly enhance HIV-1 production in a Nef-dependent manner, a mechanism that could contribute to the development of AIDS.
INTRODUCTION
Although CD4+ T cells are the primary target in progressive human immunodeficiency virus type 1 (HIV-1) infection, antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages, and B cells play an important role in infection by enhancing viral replication within an infected T cell (57, 61). The role for APCs is underscored by the fact that productive HIV-1 infections occur in discrete, multicellular microenvironments where T cells respond to antigen. It is thought that HIV-1 exploits the complex paracrine and cell contact-dependent interactions that occur between APCs and T cells within these lymphoid microenvironments to enhance both replication and the spread of infection (9, 13, 21, 47).
In humans, microvascular endothelial cells (ECs) appear to be important APCs for the activation of circulating CD4+ memory T cells and function to initiate recall responses to antigens in peripheral tissues (11). Microvascular ECs in humans and other large mammals express both class I and class II major histocompatibility complex (MHC-I and MHC-II, respectively) molecules on their surfaces in vivo (64) and regularly come into contact with circulating lymphocytes within the microvasculature, especially within capillaries and venules (49). Cultured ECs provide antigenic stimulation and costimulation to resting memory T cells, resulting in proliferation and cytokine elaboration (28, 31, 37). Cultured human ECs, left untreated, lose MHC-II expression; however, gamma interferon (IFN-) restores this expression so that ECs can acquire, process, and present antigen in a manner that effectively activates resting peripheral blood CD4+ T cells and T-cell clones (40, 52, 53, 56). Unlike professional APCs, human ECs do not generally express B7 molecules (CD80 or CD86), which may contribute to their inability to induce activation of na?ve T cells. They do, however, express other costimulators, for example, LFA-3 and CD40 (11, 18, 19, 60). The ability of cultured ECs to activate allogeneic resting memory T cells distinguishes them from fibroblasts or smooth muscle cells, which can also express MHC-II following pretreatment with IFN- in culture but cannot induce allogeneic T-cell activation (44, 51). Allogeneic stimulation operates through the same T-cell receptor (TCR)-dependent mechanisms as stimulation through non-self peptides and more closely mimics physiological activation than anti-CD3 or phytohemagglutinin (PHA) treatment (11).
A difficulty in determining the contribution of host cells that may act in peripheral tissue (such as ECs) to enhance HIV-1 replication is the lack of animal models. One small animal model that could provide such information is huPBL-SCID/bg mice. Human lymphocytes can be adoptively transferred into SCID mice with a beige mutation (SCID/bg), which compromise NK cell function as well as B-cell and T-cell development (29). These animals can acquire significant numbers of circulating human T cells but no other human leukocyte type 7 to 14 days following intraperitoneal (i.p.) inoculation of human peripheral blood mononucleocytes (PBMCs) (25, 44). When PBMCs from one donor are introduced into SCID/bg mice bearing a human skin graft from a second donor, the circulating T cells in the huPBL-SCID/bg mice infiltrate and reject the allogeneic (to the T cells) skin (44, 45). In this model, human graft ECs are the principal APC population and the principal targets of effector T cells in the dermis. Thus, this model provides a means of examining T-cell and EC interactions in vivo.
Nef is an important contributor to HIV-1 pathogenesis in vivo. In several independent studies, HIV-1-infected human patients who have exhibited slow progression or nonprogression to AIDS have been found to harbor virus with large deletions in the nef gene (16, 35, 36). These patients maintain more stable CD4+-T-cell counts as well as lower viral loads in the peripheral blood than do typical progressors. Similarly, the introduction of a 182-base pair deletion into the simian immunodeficiency SIVmac239 nef gene results in a recombinant that replicates at low or undetectable levels and does not lead to progressive disease in approximately 95% of infected rhesus macaques (4, 33, 66). The rare macaques that do progress to AIDS do so in a much longer time span than do their counterparts infected with the wild-type virus. The most commonly studied activities of Nef have been CD4 and MHC-I down-regulation, infectivity enhancement, the regulation of apoptosis, and the increase of cellular activation status (3, 12, 22, 55, 58). These activities are conserved in HIV-1 and SIV Nef, and, in fact, HIV-1 Nef can substitute functionally for SIV Nef in a pathogenic simian-human immunodeficiency virus-Nef recombinant (2).
Here we show that cultured human vascular ECs provide Nef-mediated signals that substantially enhance HIV-1 replication. We also report that in huPBL-SCID/bg mice engrafted with human skin, Nef greatly increases HIV-1 replication in allogeneic T cells retained within the graft. We show that the level of dependence of the EC effect on HIV-1 Nef, its capacity to induce virus release in the absence of exogenous signals, its independence of envelope second receptor usage, and the specificity of Nef's effect to infected T cells is, in one way or another, distinct from that observed for other APC or T-cell coculture systems. Collectively, these observations suggest that ECs provide unique signals to CD4+ T cells that result in greatly enhanced HIV-1 production.
MATERIALS AND METHODS
Isolation and culture of human cells. All human cells and tissues were obtained under protocols approved by the Yale Human Investigations Committee. PBMCs were isolated from HIV-1 seronegative donors by density gradient centrifugation of leukapharesis products by using lymphocyte separation medium (Gibco BRL, Grand Island, N.Y.). CD4+ T cells were isolated from PBMCs by positive selection by using Dynabeads (Dynal, Lake Success, N.Y.). The selected population obtained by this procedure was routinely >97% CD4+ CD3+ by flow cytometry (data not shown). Cells were subsequently purified by using further negative selection as described previously (17). Activated CD4+ T cells were removed by incubating with an anti-HLA-DR antibody at a concentration of 5 μg/ml (LB3.1; gift of J. Strominger, Harvard University, Cambridge, Mass.) for 20 min, washing twice, and depleting by using magnetic beads conjugated to goat anti-mouse antibody (Dynal). Na?ve and memory subsets of T cells were isolated from the CD4-selected population by further negative selection by using anti-CD45RA or anti-CD45RO antibodies at a concentration of 2 μg/ml (Biosource, Camarillo, Calif.). For proliferation assays, the cells were stained with 250 nM carboxy-fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oreg.) for 15 min prior to coculture with ECs.
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords by collagenase digestion and maintained in culture by using conditions and reagents as previously described (50). When indicated, the cells were treated with 50 ng of IFN- (Biosource) per ml for 3 days prior to cocultivation. After pretreatment with IFN-, the ECs were uniformly HLA-DR positive (data not shown). B lymphoblastoid cells (BLCs) from the same donors as the HUVECs were generated by Epstein-Barr virus immortalization of cord blood mononuclear cells as previously described (7). Primary fibroblasts were isolated from cadaveric human skin via explant outgrowth (63).
To isolate blood monocytes, PBMCs were incubated at 37°C on human plasma-derived fibronectin-coated tissue culture plastic overnight (Falcon, Bedford, Mass.). Nonadherent cells were vigorously washed off with phosphate-buffered saline (PBS; Gibco BRL). Fluorescence-activated cell sorting (FACS) revealed that the majority of the remaining cells were positive for the monocyte/macrophage-specific marker CD14 (data not shown). Human DCs were derived from monocyte populations by standard methods. In brief, CD14+ cells were isolated by negative selection by using Dynabeads (Dynal). The cells were then cultured in 500 U of interleukin-4 (IL-4; Sigma-Aldrich, St. Louis, Mo.) per ml and 800 U of granulocyte-macrophage colony-stimulating factor (Sigma-Aldrich) per ml. On day 5, tumor necrosis factor was added to the medium at a concentration of 100 U/ml to mature the cells. The remaining cells were predominantly CD14–, CD11c+, CD80+, CD83+, and CD86+. The cells were used on day 8 or day 9 postdifferentiation.
Generation of virus stocks. To generate infectious pNL4-3 (Nef+) or derivative HIV-1 (Nef–), p83-10 plasmid DNA (containing 3' NL4-3 or derivative sequences) was digested with EcoRI (New England Biolabs, Beverly, Mass.), column purified (QIAGEN, Valencia, Calif.), and combined with an equal amount of similarly prepared EcoRI-digested p83-2 plasmid DNA (containing 5' NL4-3 sequences). The two plasmids were fused by using T4 DNA ligase (New England Biolabs). This mixture was then introduced into the permissive cell line CEMx174 by DEAE dextran transfection (46). The transfected cells were cultured in complete medium consisting of RPMI 1640 (Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gibco BRL), penicillin-streptomycin, and L-glutamine (Gibco BRL). For the generation of HIV-1 stocks, culture supernatants containing virus were harvested daily beginning 48 h after the first observation of cytopathic effect and stored at –70°C.
Cell free JR-CSF HIV was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS. National Institute of Allergy and Infectious Diseases, National Institutes of Health catalog number 394). PBMC-derived viral stocks of JR-CSF were produced by infecting freshly isolated PBMCs isolated from an HIV-1-negative donor that had been pretreated with PHA (5 μg/ml) for 3 days and maintained in RPMI medium supplemented with 20% FBS and 50 IU of recombinant human IL-2 (Biological Resources Branch, National Cancer Institute) per ml.
Virus infections in vitro. HUVECs, BLCs, adherent PBMCs (monocytes), DCs, or dermal fibroblasts were used as stimulator cells in cocultures. BLCs in cocultures were pretreated with mitomycin C for 30 min (at a concentration of 50 μg/ml in PBS) (Sigma-Aldrich). In coculture infection experiments, approximately 750,000 HLA-DR-negative, CD4+ T cells and approximately 150,000 to 250,000 adherent PBMCs, BLCs, DCs, or fibroblasts were used in 24-well microculture plates (Falcon). The cells were maintained in 1.0 ml of complete medium.
Primary quiescent CD4+ T cells were cultured alone or in coculture with ECs, BLCs, adherent PBMCs, or DCs. These cultures were infected within 1 h of coculture with HIV-1 at a multiplicity of infection of 0.05. In each case the infections were carried out in triplicate. After overnight incubation with HIV-1, nonadherent cells and culture supernatant were subjected to centrifugation. The supernatant was then removed, the cells were washed with PBS to remove residual virus, and the cells were resuspended in fresh complete medium. At 3-day intervals, the media were changed; supernatants were aliquoted and stored at –70°C.
The growth kinetics of HIV-1 under various experimental conditions was assayed by the concentration of HIV-1 p24Gag in the culture supernatants as assessed by enzyme-linked immunosorbent assay (ELISA; Coulter, Hialeah, Fla.) or by real-time reverse transcription-PCR (RT-PCR) as described below.
CIITA transduction of HUVECs. The plasmid pCD/CIITA, containing the complete human class II transactivator (CIITA) cDNA, was a gift of D. Johnson (Yale University, New Haven, Conn.). A 3.4-kb fragment comprising the coding sequence, with an additional six amino acids encoding a hemagglutinin epitope following the initial methionine, was amplified by PCR, and the resultant fragment was subcloned into the EcoRI site of the retroviral vector LZRSpBMN-Z, a gift of G. P. Nolan (Stanford University, Palo Alto, Calif.). This construct was transfected into the Phoenix-Ampho packaging cell line by using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Puromycin-resistant cells were selected and used to condition the medium. The collection of retrovirus-conditioned medium and transduction of primary HUVEC cultures were performed as described previously (38). Cells transduced with the original cloning vector encoding a LacZ protein absent CIITA sequences served as the control for these experiments.
Stimulating, blocking, and conjugated antibodies. For blocking experiments, the monoclonal antibodies (MAbs) used were TS2/18 (anti-CD2; a gift from T. Springer, Center for Blood Research, Boston, Mass.) and LB3.1, as well as an immunoglobulin G (IgG) control (Calbiochem, San Diego, Calif.) at a concentration of 10 μg/ml. For stimulation experiments, anti-CD28 antibody (BD Pharmingen, San Jose, Calif.) was used at a concentration of 1.0 μg/ml. The antibodies were added at the time of infection, were included in every medium change (every 3 days), and were therefore present throughout the duration of the cocultures. For FACS analyses, all the fluorescently conjugated antibodies -CD3, -CD4, -CD11c, -CD14, -CD19, -CD83, -HLA-DR, and -ICAM-1 (BD Biosciences, Palo Alto, Calif.) were used at a 1:50 to 1:100 dilution. For immunohistochemistry, the -CD3 clone, PS1, was used undiluted per the manufacturer's instructions (BD Biosciences).
Fixation of ECs with paraformaldehyde. Where indicated, ECs were lightly fixed as described previously (54). In brief, the cells were washed three times in Hanks balanced salt solution (Gibco BRL). They were then fixed for 15 min in PBS containing 1% freshly prepared paraformaldehyde-2% FBS. They were subsequently washed three times with RPMI medium and left in complete medium overnight at 37°C. The next day, the cells were washed again with Hanks balanced salt solution three times before use.
Quantification of intracellular HIV-1 p24Gag production. The production of intracellular p24Gag was assessed by using the fluorescein isothiocyanate (FITC)-conjugated anti-p24Gag MAb KC-57-FITC (Coulter). For these experiments, the CD4+ cells were removed from the cultures on days 6, 9, and 12 postinfection and washed twice with cold PBS containing 1% FBS. The cells were then permeabilized by using cytofix-cytoperm solution (BD Biosciences) for 20 min at 4°C. The permeabilzed cells were washed twice with Perm/Wash buffer (BD Biosciences) following staining with KC-57 or the IgG-FITC isotype control MAb (Coulter) for 30 min at room temperature. The cells were then washed twice with staining buffer (BD Biosciences) containing 1% FBS and were analyzed by using a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed by using CellQuest software (BD Biosciences).
HIV-1 infection of huPBL-SCID/bg mice. By using a protocol approved by the Yale School of Medicine Animal Care and Use Committee as well as the Human Investigation Committee, cadaveric human skin was engrafted onto C.B-17 SCID/beige mice (Taconic Farms, Germantown, N.Y.) as previously described (44). The grafts were allowed to heal for 4 to 6 weeks prior to use in experimental infections. For these experiments the engrafted animals were inoculated by i.p. injection with 3 x108 human PBMCs. Six days later the mice were infected with Nef+ or Nef– diluted to contain 100 ng of p24Gag, again by i.p. injection. On day 11 after the introduction of the PBMCs, the skin was harvested from the mice and cut into halves. One half was fixed in formalin overnight and subsequently paraffin embedded. The samples were deparaffinized and prepared for antigen retrieval as previously described (44). The samples were stained with primary antibody overnight at 4°C and were sequentially incubated with biotinylated goat anti-mouse secondary antibody, streptavidin-horseradish peroxidase-conjugated ABC reagent, and Nova Red substrate (Vector Laboratories, Burlingame, Calif.). The samples were counterstained with hematoxylin for 2 to 3 min. The other half was placed immediately in Trizol and processed according to the manufacturer's instructions (Invitrogen) by using a tissue homogenizer (Brinkmann, Westbury, N.Y.). The RNA was extracted from tissue homogenates by using a phenol-chloroform separation, precipitated by isopropanol, and washed with 70% ethanol. The RNA was further purified by using the RNeasy Protect Kit (QIAGEN) and served as the template for quantitative real-time RT-PCR.
Quantitative real-time RT-PCR. Total RNA from human skin samples or from PBMCs of HIV-1-infected mice or viral RNA from cultured cells (isolated by using a QIAmp virus RNA mini kit [QIAGEN]) was quantified by using a RiboGreen RNA quantitation assay (Molecular Probes). Equal amounts of total RNA were used in the subsequent quantification of HIV-1 RNA by quantitative real-time RT-PCR by using the primers 5'-TTAGAGCCTTTTAGAAAACAAAATCCA-3'-forward and 5'-TCTATTTTTGTTCTATGCTGCCCTATT-3'-reverse (Invitrogen) and the 3' VIC-labeled oligonucleotide probe 5'-ATCCTACATACAAATCAT (ABI, Foster City, Calif.). We have demonstrated previously that this primer-probe set can quantify HIV-1 over a very wide linear dynamic range (14). Quantification of human ?-actin gene transcription served as the control for these experiments and utilized a specific primer-probe set (Assays-on-Demand; ABI). RT-PCRs were performed on an ABI Prism 7000 sequence detection system by using reagents from the Taqman Gold RT-PCR kit (ABI). Reaction conditions were as follows: 2 min at 50°C, 10 min at 95°C, and 55 cycles of 15 s at 95°C and 1 min at 60°C.
RESULTS
The enhancement of HIV-1 replication in T cells cocultured with ECs. Human ECs serve as APCs to circulating memory T cells, facilitating immune surveillance under physiological conditions. Since other APCs influence HIV-1 replication in infected CD4+ T cells, we wondered if ECs would have a similar effect. To test this possibility, we initially used an in vitro coculture system of allogeneic human vascular ECs and resting peripheral blood CD4+ T cells. In this system the signals provided by allogeneic MHC molecules mimic those provided by antigenic peptide presented on autologous MHC molecules, and alloreactive T cells in both the na?ve and memory subsets are sufficiently numerous to measure the activation of resting human T cells without specific in vivo priming (11).
First, we examined the activation status of purified T cells to be used in EC and T-cell infection experiments. FACS analyses revealed that these cells expressed high levels of CD4 but lacked activation marker expression, i.e., HLA-DR, CD69, and CD25 (Fig. 1A). In addition, they were activated in response to IFN--pretreated ECs. By 24 h of coculture, these cells will produce cytokines, including IL-2, IL-4, IL-10, and IFN-. Within a few days, these cells will acquire activation markers, including CD25 and CD69; furthermore, by day 9, these cells will proliferate in response to IFN--pretreated ECs but not in response to untreated ECs lacking HLA-DR expression (Fig. 1B), and they will not proliferate when cultured alone (data not shown). The proliferating cells were CD45RO+. Based on these observations, we consider CD4+ T cells isolated under these conditions to be resting but not anergic and capable of being activated by allogeneic HLA-DR molecules expressed by ECs.
To determine if ECs supported HIV-1 production in these resting T cells, T cells were infected with HIV-1 strain NL4-3 (the stock was derived from CEMx174 cells) either alone or in cocultures with untreated and IFN--pretreated ECs. IFN--pretreated ECs infected with the same stock served as a control for these experiments. When cultures were assayed by ELISA, we were unable to consistently detect HIV-1 in the culture supernatants in wells containing T cells or ECs alone (data not shown). However, in wells containing T cells and ECs, we observed levels of HIV-1 in culture supernatants that were 1,000-fold greater than the detection limit of the assay (data not shown). To more accurately quantify the level of HIV-1 replication enhancement in T cells conferred by IFN--treated ECs over T cells alone, we used real-time RT-PCR. In these experiments we quantified levels of HIV-1 RNA in cultures with T cells alone or IFN--pretreated ECs alone or cocultures of these two cell types. The level of HIV-1 produced in treated EC and T-cell cocultures was substantially enhanced over T cells or ECs alone, producing 50,000-fold more viral RNA (Fig. 2A). To determine if the capacity of ECs to enhance HIV-1 replication in T cells is linked to the capacity of ECs to activate T cells, we also compared the effects of ECs pretreated or not with IFN-. In cultures with untreated ECs that lacked HLA-DR, we observed very low levels of NL4-3 production that were substantially lower than levels observed for cultures that contained IFN--pretreated ECs (Fig. 2A).
To ensure that the observed HIV-1 growth phenotype was not dependent on the particular stock or the cell type host used to produce the virus stock, HIV-1 derived from cultured PBMCs from a donor not infected with HIV-1 were tested in this assay. In these experiments we observed levels of HIV-1 production that were comparable to levels in experiments with the CEMx174-derived stock (data not shown). NL4-3 utilizes CXCR4 as a second receptor to gain entry into T cells. HIV-1 strains that utilize this second receptor are usually observed in the later stages of infection (41). During the long asymptomatic period of infection, viruses that utilize CCR5 predominate (41). To assess if second receptor usage substantially affected the capacity of ECs to enhance HIV-1 replication in infected T cells, we infected EC and T-cell cocultures with the strain JR-CSF, which utilizes CCR5 for cell entry. In these experiments, we observed levels of JR-CSF replication that were comparable to those observed for NL4-3 (data not shown).
EC MHC-II expression contributes to enhanced HIV-1 replication in T cells. To more specifically test whether HLA-DR expression is a relevant change induced by IFN-, we used transduction of CIITA to upregulate MHC-II surface expression on ECs to levels comparable to those induced by IFN- without affecting additional IFN- gene targets such as ICAM-1 (Fig. 2B). The levels of the costimulatory molecule LFA-3 are unaffected by IFN- pretreatment. CIITA-transduced ECs (with or without IFN- pretreatment) were cultured with resting T cells and infected with NL4-3. In these experiments we observed similar levels of peak HIV-1 replication in cultures with either IFN--prestimulated ECs or nonstimulated ECs, although the IFN--pretreated cells did produce higher levels of virus on day 6 postinfection (Fig. 2C). Thus, the up-regulation of MHC-II on ECs was largely sufficient to replace pretreatment with IFN-.
We also directly investigated the roles (in enhanced HIV-1 production) of HLA-DR as well as LFA-3 to EC-mediated enhancement of HIV-1 production by using inhibitory antibodies. For these studies HIV-1-infected EC and T-cell cocultures were incubated with blocking antibodies to HLA-DR or CD2 (the LFA-3 receptor on T cells). In these cultures the HLA-DR and CD2 antibodies inhibited HIV-1 replication substantially (Fig. 3A). During HIV-1 egress, HLA-DR and CD2 can be incorporated into the viral envelope (5). It has been shown that preincubation of HIV-1 with antibodies to HLA-DR can lower viral infectivity (10). Thus, it was necessary to establish if the inhibition of HIV-1 replication by the blocking antibodies was due to a disruption of signals between ECs and CD4+ T cells or to neutralization of virus infectivity due to the incorporation of antibody targets in the viral envelope. For these experiments the same HLA-DR and CD2 blocking antibodies were added to HeLa/CD4-?gal cells at the same time that HIV-1 was added, and their effect on HIV-1 infectivity was measured (34). These experiments demonstrated that the blocking antibodies did not directly reduce the infectivity of HIV-1 (data not shown). Thus, the repression of HIV-1 production in the presence of the blocking antibodies was likely due to the interference of EC and T-cell interactions and/or activation signals.
Lightly fixed ECs can provide costimulation to PHA-activated T cells but will no longer activate T cells through TCR signals (54). To confirm that class II signaling through the TCR was necessary for the EC and T-cell effect, we investigated whether fixation of the ECs would affect their capacity to enhance NL4-3 replication. For these experiments CIITA-transduced ECs were treated with paraformaldehyde prior to cocultivation with CD4+ T cells. In these conditions we observed very low levels of virus replication, over 100-fold lower than was observed than in EC and T-cell cocultures containing living ECs (Fig. 3B). Paraformaldehyde fixation of IFN--treated ECs gave similar results (data not shown).
Characterization of the EC effect on HIV-1 replication in CD4+ T cells. To examine whether sustained EC contact was necessary for enhanced HIV-1 replication, T cells were separated from the EC monolayer at 1, 3, and 6 days after HIV-1 was introduced in the EC and T-cell cocultures. At the time of separation, very little virus had been released into the coculture supernatant (Fig. 3C). We monitored HIV-1 production from the T cells at days 9 and 12 postinfection. When ECs and T cells were separated after 6 days of coculture, the isolated T cells produced virus levels that were comparable to T cells that remained in contact with ECs (Fig. 3C). If the T cells and ECs were separated after 1 or 3 days, there was little detectable virus in the supernatant (data not shown). While this may suggest that sustained EC contact is required for virus replication at early times, this result may also be explained by the inability of cultured T cells to survive without homeostatic signals provided by APCs or cytokines. However, once the response is activated, EC contact appears unnecessary.
We next investigated whether the EC effect on HIV-1 replication occurred in all CD4+ T cells. Unlike professional APCs, ECs effectively activate resting memory but not na?ve CD4+ T cells (39). We suspected, therefore, that the primary cells in which HIV-1 replication would be affected would be memory T cells and examined if NL4-3 replication was restricted to memory T cells in EC and T-cell cocultures. The CXCR4 utilizing strain was appropriate for these experiments since both memory and na?ve T cells express this secondary HIV-1 receptor (42). For these experiments CD4+ cells were separated by magnetic bead-mediated negative selection into CD45RO+ (memory) and CD45RA+ (na?ve) subpopulations. FACS confirmed the purity of these isolations (Fig. 4A). The purified cells were then incubated with IFN--pretreated ECs and infected with NL4-3. In cocultures containing CD45RO+ T cells, we observed substantially higher levels of HIV-1 production than in cocultures containing CD45RA+ T cells (Fig. 4B).
A role for Nef in the enhanced HIV-1 production in EC and T-cell cocultures. It has been demonstrated that HIV-1 Nef can increase the activation status of HIV-1 infected T cells (20). We examined whether the EC effect on HIV-1 production could operate by providing activation signals to T cells, which could be modulated by Nef. To test this hypothesis, equivalent infectious doses of wild-type NL4-3 (hereafter termed Nef+) or a derivative strain that contained a large deletion in the nef gene (Nef–) were used to infect EC and T-cell cocultures. In these studies Nef+ replicated to approximately 100-fold higher levels than did Nef– (Fig. 5A). To ensure that the poor growth of Nef– was not inherent for this recombinant, Nef+ and Nef– were used to infect PBMCs pretreated with PHA and IL-2. Under these conditions Nef– replicated at a level similar to that of Nef+ (Fig. 5B), indicating that in fully stimulated cells, the deletion of Nef did not substantially influence virus replication.
We also examined whether the Nef+ growth advantage in EC and T-cell cocultures was EC specific or if the activation of allogeneic CD4+ T cells by other MHC-II-expressing cell types could substitute functionally for ECs. For these studies nonstimulated CD4+ T cells were cocultured with professional APCs (represented by BLCs, adherent PBMCs, or DCs), or with non-APCs (represented by IFN--pretreated dermal fibroblasts). The cultures were then infected with Nef+ or Nef–. Although there was significant virus production from T cells cocultivated with BLCs, adherent PBMCs, or DCs, the levels were independent of an intact nef gene (Fig. 5C to E). Conversely, cocultivation with fibroblasts induced very low levels of either Nef+ or Nef– replication in CD4+ T cells (Fig. 5F). BLCs, adherent PBMCs, and DCs alone produced very low levels of the virus, and fibroblasts did not produce detectable levels of virus (data not shown).
The enhanced growth of Nef+ in EC and T-cell cocultures could be due to an increase in the number of Nef+ producing cells or to an increase in the number of virions produced by each cell. To distinguish between these two possibilities, we assayed for the levels of intracellular HIV-1 p24Gag in T cells infected with Nef+ or Nef– by using indirect immunofluorescence and FACS analyses. In these experiments we observed that Nef+-infected cultures contained a subpopulation of T cells that harbored a high concentration of intracellular p24Gag that was not observed in Nef– cultures (Fig. 6). The increase in this population correlated temporally with the increase in p24Gag in the supernatant (data not shown).
Characterization of the EC effect on Nef-dependent HIV-1 replication. The Nef-dependent effect of ECs could be due to an EC-specific positive signal mediated via Nef or to the absence of a signal provided by professional APCs that renders Nef superfluous. HUVECs do not express B7 molecules (18, 19, 60) and, hence, unlike professional APCs, ECs cannot provide costimulation to T cells through CD28. To test if the absence of B7 molecules on the EC surface could account for their inability to support Nef– replication, we examined whether Nef– replication in EC and T-cell cocultures could be rescued by exogenous costimulation through CD28. For these studies EC and T-cell cocultures infected with Nef+ or Nef– were incubated either with a stimulatory anti-CD28 antibody or an isotype-matched control. Although CD28 costimulation enhanced Nef+ replication to a small extent, it did not appreciably increase Nef– replication (Fig. 7A). In these same experiments, anti-CD28 antibody markedly augmented IL-2 production in Nef+- or Nef–-infected cultures or in uninfected cultures (Fig. 7B). These experiments suggest that Nef was not simply substituting for costimulation to increase the number of T cells that become fully activated in the presence of an inefficient APC.
EC and Nef effects on T cells activated by professional APCs. In the preceding experiments ECs served both as APCs, presenting allogeneic HLA-DR to T cells, and as enhancers of Nef+ replication. To examine whether these properties could be uncoupled, we investigated whether ECs would be able to provide their Nef-dependent signal in trans to T cells activated by other APCs. To test this possibility we utilized a three-cell coculture system that included BLCs, T cells, and ECs. As mentioned previously, cocultivation of T cells with BLCs produced moderate levels of both Nef+ and Nef–. The addition of ECs to BLC and T-cell cocultures selectively augmented Nef+ replication, while Nef– replication continued only at moderate levels (Table 1). Surprisingly, despite the 50-fold increase of HIV-1 production with the addition of ECs, levels of IL-2 production were similar in the BLC and T-cell cocultures and the cocultures of BLCs, ECs, and T cells (Table 1). These experiments suggest that ECs are able to provide an additional non-TCR-mediated signal that confers a growth advantage to Nef+ virus.
As mentioned previously, the EC effect on HIV-1 replication was confined to memory T cells. We examined if EC signals to na?ve T cells activated by BLCs would enhance HIV-1 production. In these experiments we again observed that na?ve T cells supported substantially lower levels of Nef+ production than memory T cells in EC and T-cell cocultures (Fig. 8). However, in cocultures of ECs, BLCs, and T cells, high levels of Nef+ were produced both in memory and na?ve T cells, and the level was actually higher than in EC and T-cell cocultures containing memory T cells (Fig. 8). These data confirm that EC-derived signals affect activated T cells and do not require that the EC be the APC responsible for T-cell activation.
The effect of Nef on extranodal HIV-1 replication in vivo. Having established that ECs support Nef-dependent HIV-1 production in cultured CD4+ T cells, we next determined if Nef could also enhance HIV-1 replication in CD4+ T cells in an in vivo model in which ECs served as the primary APC. For these studies we utilized mechanisms of T-cell-mediated allograft rejection in huPBL-SCID/bg mice bearing a vascularized human skin graft. In these grafts the dermis contains few, if any, resident professional APCs, and the T-cell response appears to be activated by graft ECs. Mice with well-healed skin grafts, which were perfused through retained human microvessels, were reconstituted with human PBMCs via i.p. injection. Human APCs do not seem to be adaptively transferred via the i.p. route, rendering ECs as the primary APCs in this system (44). Six days after the introduction of the PBMCs, these mice were infected with Nef+ or Nef– (23), again by i.p. injection. On day 11 after the introduction of the PBMCs, the mice were sacrificed, and the skin grafts were harvested. Quantitative real-time RT-PCR of RNA derived from these grafts revealed that, despite comparable levels of infiltration by human T cells (Fig. 9A and B), the samples from Nef+-infected animals harbored 30- to 50-fold higher levels of viral RNA than did the Nef–-infected animals (Fig. 9C). Immunohistochemical staining with an anti-p24Gag antibody was sufficiently sensitive to detect the presence of virus within infiltrating T cells following Nef+ but not Nef– viral infection (Fig. 9D). Since there is no evidence of infiltrating monocytes or macrophages in these grafts (44), HIV-1 replication within the rejecting skin appears to occur exclusively within T cells.
DISCUSSION
In recent years the role of professional APCs in facilitating efficient HIV-1 replication in infected CD4+ T cells has become increasingly apparent. APCs induce the development of HIV-1-favorable microenvironments through the release of soluble factors and through direct cellular contacts. Here, we report the discovery that an additional APC type, namely, vascular ECs, also play a role in supporting HIV-1 production from CD4+ T cells, likely through a unique mechanism.
Previous reports have demonstrated a role for Nef in other APC and T-cell coculture systems. The presence of Nef increased viral replication 4- to 10-fold in T cells cocultured with immature DCs (48). We observed a much larger difference in Nef+ versus Nef– replication, i.e., 100-fold in EC and T-cell cocultures, suggesting a mode for Nef action that is distinct in these two different culture systems. Furthermore, a recent study has also revealed that Nef expression in infected macrophages facilitates infection of resting T cells (62). In that study Nef is thought to exert its effect within the APCs and not within the infected T cells. In our studies we believe that the Nef effect is specific for the infected T cells both in vitro and in vivo since ECs are associated with extremely low levels of viral RNA in our hands. Moreover, in vivo, there is no evidence of human or mouse monocytes or macrophages in human skin engrafted onto SCID/bg mice (42). Consequently, HIV-1 replication in the graft appears to be exclusively within T cells (44).
ECs have distinct attributes that distinguish them from other APCs, which may underlie their seemingly unique signaling capacity to modulate HIV-1-infected T cells. For example, while CD8+ T cells stimulated by professional APCs express early activation markers only transiently, EC-stimulated CD8+ T cells maintain prolonged expression of CD25 and CD69 (17). In addition, there is evidence that ECs activate a unique subset of allogeneic T cells. B-cell-stimulated T-cell lines can often be reactivated by both the BLCs and ECs (8), whereas EC-activated T cells are often EC specific. Furthermore, ECs can render CD4+ T cells resistant to cyclosporine (32). EC-stimulated CD4+ T cells also experience rapid and sustained expression of CD154 (CD40L), whereas monocyte or B-cell-stimulated T cells do not (43). Prolonged CD40L-CD40 interactions between B cells and EC-experienced T cells could contribute to the enhanced Nef+ replication in cocultures of ECs, B cells, and T cells and could underlie the capacity of na?ve T cells to support enhanced Nef+ replication under these conditions. We postulate that the distinctive properties of ECs that facilitate HIV-1 production in T cells may be a function of the array of costimulator and adhesion molecules that are either enriched or exclusively expressed on the EC surface. These molecules, which include members of the tumor necrosis factor superfamily and members of the B7 superfamily (11), represent potential candidates for the EC-derived signal(s) that facilitate HIV-1 replication in T cells.
We argue that the Nef dependence of the EC effect cannot be explained as simply augmenting TCR-based signaling pathways. If Nef were merely augmenting TCR-based signaling in our system, we would have anticipated that augmenting TCR-based signals by other means, i.e., via the addition of a stimulating anti-CD28 antibody, would rescue Nef– replication. In addition, we observed that the addition of ECs to BLC and T-cell cocultures greatly enhanced Nef+ production without greatly changing IL-2 elaboration, and the addition of BLCs to EC and T-cell cocultures greatly enhanced HIV-1 production in na?ve T cells. Furthermore, we do not believe that full TCR-based activation is even required for viral replication in our system. The frequency of p24Gag/high cells consisted of 5 to 10% of the total T-cell population; this is greater than the numbers of T cells that are fully activated by ECs. By limiting dilution analysis, precursor frequencies of T cells that respond to allogeneic EC have been routinely measured at between 1/1,000 and 1/5,000 (44). There are several explanations for these findings. For example, HIV-1 replication may require activation within the population of T cells but may be occurring in cells that are not directly recognizing MHC molecules on the EC surface, i.e., bystander cells. Alternatively, HIV-1 replication in this system may only require a low level of TCR-derived signals that is insufficient for complete activation. Weak signals may be produced by low-affinity cross-reactivity with foreign MHC molecules similar to those required by T cells for homeostatic TCR tickling by self MHC molecules (30). The low-level cross-reactivity has been shown to have a selective stimulatory function (59), which may be altered by Nef to facilitate productive infection within, and HIV-1 production from, incompletely stimulated T cells.
We observed that light fixation of cells abolished the capacity of ECs to enhance HIV-1 replication in T cells. Light fixation of the ECs preserves some aspects of their APC function and compromises others. Fixed ECs are equal to living ECs at providing costimulation to PHA-activated CD4+ T cells, which as with living ECs, is mediated via surface-bound molecules, i.e., LFA-3 (54). However, they lose their ability to fully stimulate alloreactive CD4+ T cells as well as the ability to confer cyclosporine resistance to T cells (J. S. Pober, unpublished observations). The inability of fixed ECs to support virus production may be a result of their inability to adequately stimulate resting CD4+ T cells, but it may also indicate the requirement for active EC metabolism. It is also possible that signals derived from HIV-1-infected T cells affect EC metabolism in ways that result in enhanced HIV-1 replication in EC and T cell cocultures. For example, HIV-1 Tat can be secreted from HIV-1-infected T cells, and this soluble form can enter ECs through specific receptors (6, 15, 24, 65). Soluble Tat acts as a promiscuous transcription factor that can promote transcription from many genes in ECs including IL-6, E-selectin, and IL-8 (1, 26, 27). The result of this aberrant transcription control is that ECs can behave similarly to they would in response to inflammation (27), which could influence HIV-1 replication in T cells. Interestingly, we observed that after 6 days of cocultivation, T cells could be separated from the ECs without adversely affecting HIV-1 production, suggesting that once signals have been exchanged between the two cell types, previously resting T cells are rendered competent for virus production.
In summary, we report that EC and T-cell coculture reveals a novel APC and T-cell interaction that results in greatly increased HIV-1 production from previously resting T cells. The specific characteristics of this interaction suggest that the mechanism of EC support of HIV-1 production in infected T cells is distinct from the mechanisms of other documented APC and T-cell interactions and could be determined by an undefined function of Nef.
ACKNOWLEDGMENTS
We thank Louise Benson, Gwendoline Davis, and Lisa Gras for excellent assistance in cell culture and animal handling.
This work was support by NIH grant R01 HL51014 and American Cancer Society institutional research grant IRG 58-012-42. J.C. was supported by NIH training grant GM07205; J.W. was supported by a National Science Foundation graduate research fellowship.
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Department of Genetics
Department of Pathology
Department of Dermatology
Interdepartmental Program in Vascular Biology and Transplantation
Department of Epidemiology and Public Health, Yale University School of Medicine, New Haven, Connecticut
Department of Pathophysiology, Kazan State Medical University, Kazan, Russia
ABSTRACT
Infected CD4+ T cells are the primary sites of human immunodeficiency virus type 1 (HIV-1) replication in vivo. However, signals from professional antigen-presenting cells (APCs), such as dendritic cells and macrophages, greatly enhance HIV-1 replication in T cells. Here, we report that in cocultures, vascular endothelial cells (ECs), which in humans can also serve as APCs, can enhance HIV-1 production of both CCR5- and CXCR4-utilizing strains approximately 50,000-fold. The observed HIV-1 replication enhancement conferred by ECs occurred only in memory CD4+ T cells, required expression of major histocompatibility complex class II (MHC-II) molecules by the ECs, and could not be conferred by fixed ECs, all of which are consistent with a requirement for EC-mediated T-cell activation via T-cell receptor (TCR) signaling. Deletion of nef (Nef–) decreased HIV-1 production by approximately 100-fold in T cells cocultured with ECs but had no effect on virus production in T cells cocultured with professional APCs or fibroblasts induced to express MHC-II. Human ECs do not express B7 costimulators, but Nef– replication in CD4+-T-cell and EC cocultures could not be rescued by anti-CD28 antibody. ECs act in trans to enhance wild-type but not Nef– replication and facilitate enhanced wild-type replication in na?ve T cells when added to T-cell or B-lymphoblastoid cell cocultures, suggesting that ECs also provide a TCR-independent signal to infected T cells. Consistent with these in vitro observations, wild-type HIV-1 replicated 30- to 50-fold more than Nef– in human T cells infiltrating allogeneic human skin grafts on human huPBL-SCID/bg mice, an in vivo model of T-cell activation by ECs. Our studies suggest that ECs, which line the entire cardiovascular system and are, per force, in frequent contact with memory CD4+ T cells, provide signals to HIV-1-infected CD4+ T cells to greatly enhance HIV-1 production in a Nef-dependent manner, a mechanism that could contribute to the development of AIDS.
INTRODUCTION
Although CD4+ T cells are the primary target in progressive human immunodeficiency virus type 1 (HIV-1) infection, antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages, and B cells play an important role in infection by enhancing viral replication within an infected T cell (57, 61). The role for APCs is underscored by the fact that productive HIV-1 infections occur in discrete, multicellular microenvironments where T cells respond to antigen. It is thought that HIV-1 exploits the complex paracrine and cell contact-dependent interactions that occur between APCs and T cells within these lymphoid microenvironments to enhance both replication and the spread of infection (9, 13, 21, 47).
In humans, microvascular endothelial cells (ECs) appear to be important APCs for the activation of circulating CD4+ memory T cells and function to initiate recall responses to antigens in peripheral tissues (11). Microvascular ECs in humans and other large mammals express both class I and class II major histocompatibility complex (MHC-I and MHC-II, respectively) molecules on their surfaces in vivo (64) and regularly come into contact with circulating lymphocytes within the microvasculature, especially within capillaries and venules (49). Cultured ECs provide antigenic stimulation and costimulation to resting memory T cells, resulting in proliferation and cytokine elaboration (28, 31, 37). Cultured human ECs, left untreated, lose MHC-II expression; however, gamma interferon (IFN-) restores this expression so that ECs can acquire, process, and present antigen in a manner that effectively activates resting peripheral blood CD4+ T cells and T-cell clones (40, 52, 53, 56). Unlike professional APCs, human ECs do not generally express B7 molecules (CD80 or CD86), which may contribute to their inability to induce activation of na?ve T cells. They do, however, express other costimulators, for example, LFA-3 and CD40 (11, 18, 19, 60). The ability of cultured ECs to activate allogeneic resting memory T cells distinguishes them from fibroblasts or smooth muscle cells, which can also express MHC-II following pretreatment with IFN- in culture but cannot induce allogeneic T-cell activation (44, 51). Allogeneic stimulation operates through the same T-cell receptor (TCR)-dependent mechanisms as stimulation through non-self peptides and more closely mimics physiological activation than anti-CD3 or phytohemagglutinin (PHA) treatment (11).
A difficulty in determining the contribution of host cells that may act in peripheral tissue (such as ECs) to enhance HIV-1 replication is the lack of animal models. One small animal model that could provide such information is huPBL-SCID/bg mice. Human lymphocytes can be adoptively transferred into SCID mice with a beige mutation (SCID/bg), which compromise NK cell function as well as B-cell and T-cell development (29). These animals can acquire significant numbers of circulating human T cells but no other human leukocyte type 7 to 14 days following intraperitoneal (i.p.) inoculation of human peripheral blood mononucleocytes (PBMCs) (25, 44). When PBMCs from one donor are introduced into SCID/bg mice bearing a human skin graft from a second donor, the circulating T cells in the huPBL-SCID/bg mice infiltrate and reject the allogeneic (to the T cells) skin (44, 45). In this model, human graft ECs are the principal APC population and the principal targets of effector T cells in the dermis. Thus, this model provides a means of examining T-cell and EC interactions in vivo.
Nef is an important contributor to HIV-1 pathogenesis in vivo. In several independent studies, HIV-1-infected human patients who have exhibited slow progression or nonprogression to AIDS have been found to harbor virus with large deletions in the nef gene (16, 35, 36). These patients maintain more stable CD4+-T-cell counts as well as lower viral loads in the peripheral blood than do typical progressors. Similarly, the introduction of a 182-base pair deletion into the simian immunodeficiency SIVmac239 nef gene results in a recombinant that replicates at low or undetectable levels and does not lead to progressive disease in approximately 95% of infected rhesus macaques (4, 33, 66). The rare macaques that do progress to AIDS do so in a much longer time span than do their counterparts infected with the wild-type virus. The most commonly studied activities of Nef have been CD4 and MHC-I down-regulation, infectivity enhancement, the regulation of apoptosis, and the increase of cellular activation status (3, 12, 22, 55, 58). These activities are conserved in HIV-1 and SIV Nef, and, in fact, HIV-1 Nef can substitute functionally for SIV Nef in a pathogenic simian-human immunodeficiency virus-Nef recombinant (2).
Here we show that cultured human vascular ECs provide Nef-mediated signals that substantially enhance HIV-1 replication. We also report that in huPBL-SCID/bg mice engrafted with human skin, Nef greatly increases HIV-1 replication in allogeneic T cells retained within the graft. We show that the level of dependence of the EC effect on HIV-1 Nef, its capacity to induce virus release in the absence of exogenous signals, its independence of envelope second receptor usage, and the specificity of Nef's effect to infected T cells is, in one way or another, distinct from that observed for other APC or T-cell coculture systems. Collectively, these observations suggest that ECs provide unique signals to CD4+ T cells that result in greatly enhanced HIV-1 production.
MATERIALS AND METHODS
Isolation and culture of human cells. All human cells and tissues were obtained under protocols approved by the Yale Human Investigations Committee. PBMCs were isolated from HIV-1 seronegative donors by density gradient centrifugation of leukapharesis products by using lymphocyte separation medium (Gibco BRL, Grand Island, N.Y.). CD4+ T cells were isolated from PBMCs by positive selection by using Dynabeads (Dynal, Lake Success, N.Y.). The selected population obtained by this procedure was routinely >97% CD4+ CD3+ by flow cytometry (data not shown). Cells were subsequently purified by using further negative selection as described previously (17). Activated CD4+ T cells were removed by incubating with an anti-HLA-DR antibody at a concentration of 5 μg/ml (LB3.1; gift of J. Strominger, Harvard University, Cambridge, Mass.) for 20 min, washing twice, and depleting by using magnetic beads conjugated to goat anti-mouse antibody (Dynal). Na?ve and memory subsets of T cells were isolated from the CD4-selected population by further negative selection by using anti-CD45RA or anti-CD45RO antibodies at a concentration of 2 μg/ml (Biosource, Camarillo, Calif.). For proliferation assays, the cells were stained with 250 nM carboxy-fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, Oreg.) for 15 min prior to coculture with ECs.
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords by collagenase digestion and maintained in culture by using conditions and reagents as previously described (50). When indicated, the cells were treated with 50 ng of IFN- (Biosource) per ml for 3 days prior to cocultivation. After pretreatment with IFN-, the ECs were uniformly HLA-DR positive (data not shown). B lymphoblastoid cells (BLCs) from the same donors as the HUVECs were generated by Epstein-Barr virus immortalization of cord blood mononuclear cells as previously described (7). Primary fibroblasts were isolated from cadaveric human skin via explant outgrowth (63).
To isolate blood monocytes, PBMCs were incubated at 37°C on human plasma-derived fibronectin-coated tissue culture plastic overnight (Falcon, Bedford, Mass.). Nonadherent cells were vigorously washed off with phosphate-buffered saline (PBS; Gibco BRL). Fluorescence-activated cell sorting (FACS) revealed that the majority of the remaining cells were positive for the monocyte/macrophage-specific marker CD14 (data not shown). Human DCs were derived from monocyte populations by standard methods. In brief, CD14+ cells were isolated by negative selection by using Dynabeads (Dynal). The cells were then cultured in 500 U of interleukin-4 (IL-4; Sigma-Aldrich, St. Louis, Mo.) per ml and 800 U of granulocyte-macrophage colony-stimulating factor (Sigma-Aldrich) per ml. On day 5, tumor necrosis factor was added to the medium at a concentration of 100 U/ml to mature the cells. The remaining cells were predominantly CD14–, CD11c+, CD80+, CD83+, and CD86+. The cells were used on day 8 or day 9 postdifferentiation.
Generation of virus stocks. To generate infectious pNL4-3 (Nef+) or derivative HIV-1 (Nef–), p83-10 plasmid DNA (containing 3' NL4-3 or derivative sequences) was digested with EcoRI (New England Biolabs, Beverly, Mass.), column purified (QIAGEN, Valencia, Calif.), and combined with an equal amount of similarly prepared EcoRI-digested p83-2 plasmid DNA (containing 5' NL4-3 sequences). The two plasmids were fused by using T4 DNA ligase (New England Biolabs). This mixture was then introduced into the permissive cell line CEMx174 by DEAE dextran transfection (46). The transfected cells were cultured in complete medium consisting of RPMI 1640 (Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gibco BRL), penicillin-streptomycin, and L-glutamine (Gibco BRL). For the generation of HIV-1 stocks, culture supernatants containing virus were harvested daily beginning 48 h after the first observation of cytopathic effect and stored at –70°C.
Cell free JR-CSF HIV was obtained through the AIDS Research and Reference Reagent Program (Division of AIDS. National Institute of Allergy and Infectious Diseases, National Institutes of Health catalog number 394). PBMC-derived viral stocks of JR-CSF were produced by infecting freshly isolated PBMCs isolated from an HIV-1-negative donor that had been pretreated with PHA (5 μg/ml) for 3 days and maintained in RPMI medium supplemented with 20% FBS and 50 IU of recombinant human IL-2 (Biological Resources Branch, National Cancer Institute) per ml.
Virus infections in vitro. HUVECs, BLCs, adherent PBMCs (monocytes), DCs, or dermal fibroblasts were used as stimulator cells in cocultures. BLCs in cocultures were pretreated with mitomycin C for 30 min (at a concentration of 50 μg/ml in PBS) (Sigma-Aldrich). In coculture infection experiments, approximately 750,000 HLA-DR-negative, CD4+ T cells and approximately 150,000 to 250,000 adherent PBMCs, BLCs, DCs, or fibroblasts were used in 24-well microculture plates (Falcon). The cells were maintained in 1.0 ml of complete medium.
Primary quiescent CD4+ T cells were cultured alone or in coculture with ECs, BLCs, adherent PBMCs, or DCs. These cultures were infected within 1 h of coculture with HIV-1 at a multiplicity of infection of 0.05. In each case the infections were carried out in triplicate. After overnight incubation with HIV-1, nonadherent cells and culture supernatant were subjected to centrifugation. The supernatant was then removed, the cells were washed with PBS to remove residual virus, and the cells were resuspended in fresh complete medium. At 3-day intervals, the media were changed; supernatants were aliquoted and stored at –70°C.
The growth kinetics of HIV-1 under various experimental conditions was assayed by the concentration of HIV-1 p24Gag in the culture supernatants as assessed by enzyme-linked immunosorbent assay (ELISA; Coulter, Hialeah, Fla.) or by real-time reverse transcription-PCR (RT-PCR) as described below.
CIITA transduction of HUVECs. The plasmid pCD/CIITA, containing the complete human class II transactivator (CIITA) cDNA, was a gift of D. Johnson (Yale University, New Haven, Conn.). A 3.4-kb fragment comprising the coding sequence, with an additional six amino acids encoding a hemagglutinin epitope following the initial methionine, was amplified by PCR, and the resultant fragment was subcloned into the EcoRI site of the retroviral vector LZRSpBMN-Z, a gift of G. P. Nolan (Stanford University, Palo Alto, Calif.). This construct was transfected into the Phoenix-Ampho packaging cell line by using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Puromycin-resistant cells were selected and used to condition the medium. The collection of retrovirus-conditioned medium and transduction of primary HUVEC cultures were performed as described previously (38). Cells transduced with the original cloning vector encoding a LacZ protein absent CIITA sequences served as the control for these experiments.
Stimulating, blocking, and conjugated antibodies. For blocking experiments, the monoclonal antibodies (MAbs) used were TS2/18 (anti-CD2; a gift from T. Springer, Center for Blood Research, Boston, Mass.) and LB3.1, as well as an immunoglobulin G (IgG) control (Calbiochem, San Diego, Calif.) at a concentration of 10 μg/ml. For stimulation experiments, anti-CD28 antibody (BD Pharmingen, San Jose, Calif.) was used at a concentration of 1.0 μg/ml. The antibodies were added at the time of infection, were included in every medium change (every 3 days), and were therefore present throughout the duration of the cocultures. For FACS analyses, all the fluorescently conjugated antibodies -CD3, -CD4, -CD11c, -CD14, -CD19, -CD83, -HLA-DR, and -ICAM-1 (BD Biosciences, Palo Alto, Calif.) were used at a 1:50 to 1:100 dilution. For immunohistochemistry, the -CD3 clone, PS1, was used undiluted per the manufacturer's instructions (BD Biosciences).
Fixation of ECs with paraformaldehyde. Where indicated, ECs were lightly fixed as described previously (54). In brief, the cells were washed three times in Hanks balanced salt solution (Gibco BRL). They were then fixed for 15 min in PBS containing 1% freshly prepared paraformaldehyde-2% FBS. They were subsequently washed three times with RPMI medium and left in complete medium overnight at 37°C. The next day, the cells were washed again with Hanks balanced salt solution three times before use.
Quantification of intracellular HIV-1 p24Gag production. The production of intracellular p24Gag was assessed by using the fluorescein isothiocyanate (FITC)-conjugated anti-p24Gag MAb KC-57-FITC (Coulter). For these experiments, the CD4+ cells were removed from the cultures on days 6, 9, and 12 postinfection and washed twice with cold PBS containing 1% FBS. The cells were then permeabilized by using cytofix-cytoperm solution (BD Biosciences) for 20 min at 4°C. The permeabilzed cells were washed twice with Perm/Wash buffer (BD Biosciences) following staining with KC-57 or the IgG-FITC isotype control MAb (Coulter) for 30 min at room temperature. The cells were then washed twice with staining buffer (BD Biosciences) containing 1% FBS and were analyzed by using a FACSCalibur flow cytometer (BD Biosciences). The data were analyzed by using CellQuest software (BD Biosciences).
HIV-1 infection of huPBL-SCID/bg mice. By using a protocol approved by the Yale School of Medicine Animal Care and Use Committee as well as the Human Investigation Committee, cadaveric human skin was engrafted onto C.B-17 SCID/beige mice (Taconic Farms, Germantown, N.Y.) as previously described (44). The grafts were allowed to heal for 4 to 6 weeks prior to use in experimental infections. For these experiments the engrafted animals were inoculated by i.p. injection with 3 x108 human PBMCs. Six days later the mice were infected with Nef+ or Nef– diluted to contain 100 ng of p24Gag, again by i.p. injection. On day 11 after the introduction of the PBMCs, the skin was harvested from the mice and cut into halves. One half was fixed in formalin overnight and subsequently paraffin embedded. The samples were deparaffinized and prepared for antigen retrieval as previously described (44). The samples were stained with primary antibody overnight at 4°C and were sequentially incubated with biotinylated goat anti-mouse secondary antibody, streptavidin-horseradish peroxidase-conjugated ABC reagent, and Nova Red substrate (Vector Laboratories, Burlingame, Calif.). The samples were counterstained with hematoxylin for 2 to 3 min. The other half was placed immediately in Trizol and processed according to the manufacturer's instructions (Invitrogen) by using a tissue homogenizer (Brinkmann, Westbury, N.Y.). The RNA was extracted from tissue homogenates by using a phenol-chloroform separation, precipitated by isopropanol, and washed with 70% ethanol. The RNA was further purified by using the RNeasy Protect Kit (QIAGEN) and served as the template for quantitative real-time RT-PCR.
Quantitative real-time RT-PCR. Total RNA from human skin samples or from PBMCs of HIV-1-infected mice or viral RNA from cultured cells (isolated by using a QIAmp virus RNA mini kit [QIAGEN]) was quantified by using a RiboGreen RNA quantitation assay (Molecular Probes). Equal amounts of total RNA were used in the subsequent quantification of HIV-1 RNA by quantitative real-time RT-PCR by using the primers 5'-TTAGAGCCTTTTAGAAAACAAAATCCA-3'-forward and 5'-TCTATTTTTGTTCTATGCTGCCCTATT-3'-reverse (Invitrogen) and the 3' VIC-labeled oligonucleotide probe 5'-ATCCTACATACAAATCAT (ABI, Foster City, Calif.). We have demonstrated previously that this primer-probe set can quantify HIV-1 over a very wide linear dynamic range (14). Quantification of human ?-actin gene transcription served as the control for these experiments and utilized a specific primer-probe set (Assays-on-Demand; ABI). RT-PCRs were performed on an ABI Prism 7000 sequence detection system by using reagents from the Taqman Gold RT-PCR kit (ABI). Reaction conditions were as follows: 2 min at 50°C, 10 min at 95°C, and 55 cycles of 15 s at 95°C and 1 min at 60°C.
RESULTS
The enhancement of HIV-1 replication in T cells cocultured with ECs. Human ECs serve as APCs to circulating memory T cells, facilitating immune surveillance under physiological conditions. Since other APCs influence HIV-1 replication in infected CD4+ T cells, we wondered if ECs would have a similar effect. To test this possibility, we initially used an in vitro coculture system of allogeneic human vascular ECs and resting peripheral blood CD4+ T cells. In this system the signals provided by allogeneic MHC molecules mimic those provided by antigenic peptide presented on autologous MHC molecules, and alloreactive T cells in both the na?ve and memory subsets are sufficiently numerous to measure the activation of resting human T cells without specific in vivo priming (11).
First, we examined the activation status of purified T cells to be used in EC and T-cell infection experiments. FACS analyses revealed that these cells expressed high levels of CD4 but lacked activation marker expression, i.e., HLA-DR, CD69, and CD25 (Fig. 1A). In addition, they were activated in response to IFN--pretreated ECs. By 24 h of coculture, these cells will produce cytokines, including IL-2, IL-4, IL-10, and IFN-. Within a few days, these cells will acquire activation markers, including CD25 and CD69; furthermore, by day 9, these cells will proliferate in response to IFN--pretreated ECs but not in response to untreated ECs lacking HLA-DR expression (Fig. 1B), and they will not proliferate when cultured alone (data not shown). The proliferating cells were CD45RO+. Based on these observations, we consider CD4+ T cells isolated under these conditions to be resting but not anergic and capable of being activated by allogeneic HLA-DR molecules expressed by ECs.
To determine if ECs supported HIV-1 production in these resting T cells, T cells were infected with HIV-1 strain NL4-3 (the stock was derived from CEMx174 cells) either alone or in cocultures with untreated and IFN--pretreated ECs. IFN--pretreated ECs infected with the same stock served as a control for these experiments. When cultures were assayed by ELISA, we were unable to consistently detect HIV-1 in the culture supernatants in wells containing T cells or ECs alone (data not shown). However, in wells containing T cells and ECs, we observed levels of HIV-1 in culture supernatants that were 1,000-fold greater than the detection limit of the assay (data not shown). To more accurately quantify the level of HIV-1 replication enhancement in T cells conferred by IFN--treated ECs over T cells alone, we used real-time RT-PCR. In these experiments we quantified levels of HIV-1 RNA in cultures with T cells alone or IFN--pretreated ECs alone or cocultures of these two cell types. The level of HIV-1 produced in treated EC and T-cell cocultures was substantially enhanced over T cells or ECs alone, producing 50,000-fold more viral RNA (Fig. 2A). To determine if the capacity of ECs to enhance HIV-1 replication in T cells is linked to the capacity of ECs to activate T cells, we also compared the effects of ECs pretreated or not with IFN-. In cultures with untreated ECs that lacked HLA-DR, we observed very low levels of NL4-3 production that were substantially lower than levels observed for cultures that contained IFN--pretreated ECs (Fig. 2A).
To ensure that the observed HIV-1 growth phenotype was not dependent on the particular stock or the cell type host used to produce the virus stock, HIV-1 derived from cultured PBMCs from a donor not infected with HIV-1 were tested in this assay. In these experiments we observed levels of HIV-1 production that were comparable to levels in experiments with the CEMx174-derived stock (data not shown). NL4-3 utilizes CXCR4 as a second receptor to gain entry into T cells. HIV-1 strains that utilize this second receptor are usually observed in the later stages of infection (41). During the long asymptomatic period of infection, viruses that utilize CCR5 predominate (41). To assess if second receptor usage substantially affected the capacity of ECs to enhance HIV-1 replication in infected T cells, we infected EC and T-cell cocultures with the strain JR-CSF, which utilizes CCR5 for cell entry. In these experiments, we observed levels of JR-CSF replication that were comparable to those observed for NL4-3 (data not shown).
EC MHC-II expression contributes to enhanced HIV-1 replication in T cells. To more specifically test whether HLA-DR expression is a relevant change induced by IFN-, we used transduction of CIITA to upregulate MHC-II surface expression on ECs to levels comparable to those induced by IFN- without affecting additional IFN- gene targets such as ICAM-1 (Fig. 2B). The levels of the costimulatory molecule LFA-3 are unaffected by IFN- pretreatment. CIITA-transduced ECs (with or without IFN- pretreatment) were cultured with resting T cells and infected with NL4-3. In these experiments we observed similar levels of peak HIV-1 replication in cultures with either IFN--prestimulated ECs or nonstimulated ECs, although the IFN--pretreated cells did produce higher levels of virus on day 6 postinfection (Fig. 2C). Thus, the up-regulation of MHC-II on ECs was largely sufficient to replace pretreatment with IFN-.
We also directly investigated the roles (in enhanced HIV-1 production) of HLA-DR as well as LFA-3 to EC-mediated enhancement of HIV-1 production by using inhibitory antibodies. For these studies HIV-1-infected EC and T-cell cocultures were incubated with blocking antibodies to HLA-DR or CD2 (the LFA-3 receptor on T cells). In these cultures the HLA-DR and CD2 antibodies inhibited HIV-1 replication substantially (Fig. 3A). During HIV-1 egress, HLA-DR and CD2 can be incorporated into the viral envelope (5). It has been shown that preincubation of HIV-1 with antibodies to HLA-DR can lower viral infectivity (10). Thus, it was necessary to establish if the inhibition of HIV-1 replication by the blocking antibodies was due to a disruption of signals between ECs and CD4+ T cells or to neutralization of virus infectivity due to the incorporation of antibody targets in the viral envelope. For these experiments the same HLA-DR and CD2 blocking antibodies were added to HeLa/CD4-?gal cells at the same time that HIV-1 was added, and their effect on HIV-1 infectivity was measured (34). These experiments demonstrated that the blocking antibodies did not directly reduce the infectivity of HIV-1 (data not shown). Thus, the repression of HIV-1 production in the presence of the blocking antibodies was likely due to the interference of EC and T-cell interactions and/or activation signals.
Lightly fixed ECs can provide costimulation to PHA-activated T cells but will no longer activate T cells through TCR signals (54). To confirm that class II signaling through the TCR was necessary for the EC and T-cell effect, we investigated whether fixation of the ECs would affect their capacity to enhance NL4-3 replication. For these experiments CIITA-transduced ECs were treated with paraformaldehyde prior to cocultivation with CD4+ T cells. In these conditions we observed very low levels of virus replication, over 100-fold lower than was observed than in EC and T-cell cocultures containing living ECs (Fig. 3B). Paraformaldehyde fixation of IFN--treated ECs gave similar results (data not shown).
Characterization of the EC effect on HIV-1 replication in CD4+ T cells. To examine whether sustained EC contact was necessary for enhanced HIV-1 replication, T cells were separated from the EC monolayer at 1, 3, and 6 days after HIV-1 was introduced in the EC and T-cell cocultures. At the time of separation, very little virus had been released into the coculture supernatant (Fig. 3C). We monitored HIV-1 production from the T cells at days 9 and 12 postinfection. When ECs and T cells were separated after 6 days of coculture, the isolated T cells produced virus levels that were comparable to T cells that remained in contact with ECs (Fig. 3C). If the T cells and ECs were separated after 1 or 3 days, there was little detectable virus in the supernatant (data not shown). While this may suggest that sustained EC contact is required for virus replication at early times, this result may also be explained by the inability of cultured T cells to survive without homeostatic signals provided by APCs or cytokines. However, once the response is activated, EC contact appears unnecessary.
We next investigated whether the EC effect on HIV-1 replication occurred in all CD4+ T cells. Unlike professional APCs, ECs effectively activate resting memory but not na?ve CD4+ T cells (39). We suspected, therefore, that the primary cells in which HIV-1 replication would be affected would be memory T cells and examined if NL4-3 replication was restricted to memory T cells in EC and T-cell cocultures. The CXCR4 utilizing strain was appropriate for these experiments since both memory and na?ve T cells express this secondary HIV-1 receptor (42). For these experiments CD4+ cells were separated by magnetic bead-mediated negative selection into CD45RO+ (memory) and CD45RA+ (na?ve) subpopulations. FACS confirmed the purity of these isolations (Fig. 4A). The purified cells were then incubated with IFN--pretreated ECs and infected with NL4-3. In cocultures containing CD45RO+ T cells, we observed substantially higher levels of HIV-1 production than in cocultures containing CD45RA+ T cells (Fig. 4B).
A role for Nef in the enhanced HIV-1 production in EC and T-cell cocultures. It has been demonstrated that HIV-1 Nef can increase the activation status of HIV-1 infected T cells (20). We examined whether the EC effect on HIV-1 production could operate by providing activation signals to T cells, which could be modulated by Nef. To test this hypothesis, equivalent infectious doses of wild-type NL4-3 (hereafter termed Nef+) or a derivative strain that contained a large deletion in the nef gene (Nef–) were used to infect EC and T-cell cocultures. In these studies Nef+ replicated to approximately 100-fold higher levels than did Nef– (Fig. 5A). To ensure that the poor growth of Nef– was not inherent for this recombinant, Nef+ and Nef– were used to infect PBMCs pretreated with PHA and IL-2. Under these conditions Nef– replicated at a level similar to that of Nef+ (Fig. 5B), indicating that in fully stimulated cells, the deletion of Nef did not substantially influence virus replication.
We also examined whether the Nef+ growth advantage in EC and T-cell cocultures was EC specific or if the activation of allogeneic CD4+ T cells by other MHC-II-expressing cell types could substitute functionally for ECs. For these studies nonstimulated CD4+ T cells were cocultured with professional APCs (represented by BLCs, adherent PBMCs, or DCs), or with non-APCs (represented by IFN--pretreated dermal fibroblasts). The cultures were then infected with Nef+ or Nef–. Although there was significant virus production from T cells cocultivated with BLCs, adherent PBMCs, or DCs, the levels were independent of an intact nef gene (Fig. 5C to E). Conversely, cocultivation with fibroblasts induced very low levels of either Nef+ or Nef– replication in CD4+ T cells (Fig. 5F). BLCs, adherent PBMCs, and DCs alone produced very low levels of the virus, and fibroblasts did not produce detectable levels of virus (data not shown).
The enhanced growth of Nef+ in EC and T-cell cocultures could be due to an increase in the number of Nef+ producing cells or to an increase in the number of virions produced by each cell. To distinguish between these two possibilities, we assayed for the levels of intracellular HIV-1 p24Gag in T cells infected with Nef+ or Nef– by using indirect immunofluorescence and FACS analyses. In these experiments we observed that Nef+-infected cultures contained a subpopulation of T cells that harbored a high concentration of intracellular p24Gag that was not observed in Nef– cultures (Fig. 6). The increase in this population correlated temporally with the increase in p24Gag in the supernatant (data not shown).
Characterization of the EC effect on Nef-dependent HIV-1 replication. The Nef-dependent effect of ECs could be due to an EC-specific positive signal mediated via Nef or to the absence of a signal provided by professional APCs that renders Nef superfluous. HUVECs do not express B7 molecules (18, 19, 60) and, hence, unlike professional APCs, ECs cannot provide costimulation to T cells through CD28. To test if the absence of B7 molecules on the EC surface could account for their inability to support Nef– replication, we examined whether Nef– replication in EC and T-cell cocultures could be rescued by exogenous costimulation through CD28. For these studies EC and T-cell cocultures infected with Nef+ or Nef– were incubated either with a stimulatory anti-CD28 antibody or an isotype-matched control. Although CD28 costimulation enhanced Nef+ replication to a small extent, it did not appreciably increase Nef– replication (Fig. 7A). In these same experiments, anti-CD28 antibody markedly augmented IL-2 production in Nef+- or Nef–-infected cultures or in uninfected cultures (Fig. 7B). These experiments suggest that Nef was not simply substituting for costimulation to increase the number of T cells that become fully activated in the presence of an inefficient APC.
EC and Nef effects on T cells activated by professional APCs. In the preceding experiments ECs served both as APCs, presenting allogeneic HLA-DR to T cells, and as enhancers of Nef+ replication. To examine whether these properties could be uncoupled, we investigated whether ECs would be able to provide their Nef-dependent signal in trans to T cells activated by other APCs. To test this possibility we utilized a three-cell coculture system that included BLCs, T cells, and ECs. As mentioned previously, cocultivation of T cells with BLCs produced moderate levels of both Nef+ and Nef–. The addition of ECs to BLC and T-cell cocultures selectively augmented Nef+ replication, while Nef– replication continued only at moderate levels (Table 1). Surprisingly, despite the 50-fold increase of HIV-1 production with the addition of ECs, levels of IL-2 production were similar in the BLC and T-cell cocultures and the cocultures of BLCs, ECs, and T cells (Table 1). These experiments suggest that ECs are able to provide an additional non-TCR-mediated signal that confers a growth advantage to Nef+ virus.
As mentioned previously, the EC effect on HIV-1 replication was confined to memory T cells. We examined if EC signals to na?ve T cells activated by BLCs would enhance HIV-1 production. In these experiments we again observed that na?ve T cells supported substantially lower levels of Nef+ production than memory T cells in EC and T-cell cocultures (Fig. 8). However, in cocultures of ECs, BLCs, and T cells, high levels of Nef+ were produced both in memory and na?ve T cells, and the level was actually higher than in EC and T-cell cocultures containing memory T cells (Fig. 8). These data confirm that EC-derived signals affect activated T cells and do not require that the EC be the APC responsible for T-cell activation.
The effect of Nef on extranodal HIV-1 replication in vivo. Having established that ECs support Nef-dependent HIV-1 production in cultured CD4+ T cells, we next determined if Nef could also enhance HIV-1 replication in CD4+ T cells in an in vivo model in which ECs served as the primary APC. For these studies we utilized mechanisms of T-cell-mediated allograft rejection in huPBL-SCID/bg mice bearing a vascularized human skin graft. In these grafts the dermis contains few, if any, resident professional APCs, and the T-cell response appears to be activated by graft ECs. Mice with well-healed skin grafts, which were perfused through retained human microvessels, were reconstituted with human PBMCs via i.p. injection. Human APCs do not seem to be adaptively transferred via the i.p. route, rendering ECs as the primary APCs in this system (44). Six days after the introduction of the PBMCs, these mice were infected with Nef+ or Nef– (23), again by i.p. injection. On day 11 after the introduction of the PBMCs, the mice were sacrificed, and the skin grafts were harvested. Quantitative real-time RT-PCR of RNA derived from these grafts revealed that, despite comparable levels of infiltration by human T cells (Fig. 9A and B), the samples from Nef+-infected animals harbored 30- to 50-fold higher levels of viral RNA than did the Nef–-infected animals (Fig. 9C). Immunohistochemical staining with an anti-p24Gag antibody was sufficiently sensitive to detect the presence of virus within infiltrating T cells following Nef+ but not Nef– viral infection (Fig. 9D). Since there is no evidence of infiltrating monocytes or macrophages in these grafts (44), HIV-1 replication within the rejecting skin appears to occur exclusively within T cells.
DISCUSSION
In recent years the role of professional APCs in facilitating efficient HIV-1 replication in infected CD4+ T cells has become increasingly apparent. APCs induce the development of HIV-1-favorable microenvironments through the release of soluble factors and through direct cellular contacts. Here, we report the discovery that an additional APC type, namely, vascular ECs, also play a role in supporting HIV-1 production from CD4+ T cells, likely through a unique mechanism.
Previous reports have demonstrated a role for Nef in other APC and T-cell coculture systems. The presence of Nef increased viral replication 4- to 10-fold in T cells cocultured with immature DCs (48). We observed a much larger difference in Nef+ versus Nef– replication, i.e., 100-fold in EC and T-cell cocultures, suggesting a mode for Nef action that is distinct in these two different culture systems. Furthermore, a recent study has also revealed that Nef expression in infected macrophages facilitates infection of resting T cells (62). In that study Nef is thought to exert its effect within the APCs and not within the infected T cells. In our studies we believe that the Nef effect is specific for the infected T cells both in vitro and in vivo since ECs are associated with extremely low levels of viral RNA in our hands. Moreover, in vivo, there is no evidence of human or mouse monocytes or macrophages in human skin engrafted onto SCID/bg mice (42). Consequently, HIV-1 replication in the graft appears to be exclusively within T cells (44).
ECs have distinct attributes that distinguish them from other APCs, which may underlie their seemingly unique signaling capacity to modulate HIV-1-infected T cells. For example, while CD8+ T cells stimulated by professional APCs express early activation markers only transiently, EC-stimulated CD8+ T cells maintain prolonged expression of CD25 and CD69 (17). In addition, there is evidence that ECs activate a unique subset of allogeneic T cells. B-cell-stimulated T-cell lines can often be reactivated by both the BLCs and ECs (8), whereas EC-activated T cells are often EC specific. Furthermore, ECs can render CD4+ T cells resistant to cyclosporine (32). EC-stimulated CD4+ T cells also experience rapid and sustained expression of CD154 (CD40L), whereas monocyte or B-cell-stimulated T cells do not (43). Prolonged CD40L-CD40 interactions between B cells and EC-experienced T cells could contribute to the enhanced Nef+ replication in cocultures of ECs, B cells, and T cells and could underlie the capacity of na?ve T cells to support enhanced Nef+ replication under these conditions. We postulate that the distinctive properties of ECs that facilitate HIV-1 production in T cells may be a function of the array of costimulator and adhesion molecules that are either enriched or exclusively expressed on the EC surface. These molecules, which include members of the tumor necrosis factor superfamily and members of the B7 superfamily (11), represent potential candidates for the EC-derived signal(s) that facilitate HIV-1 replication in T cells.
We argue that the Nef dependence of the EC effect cannot be explained as simply augmenting TCR-based signaling pathways. If Nef were merely augmenting TCR-based signaling in our system, we would have anticipated that augmenting TCR-based signals by other means, i.e., via the addition of a stimulating anti-CD28 antibody, would rescue Nef– replication. In addition, we observed that the addition of ECs to BLC and T-cell cocultures greatly enhanced Nef+ production without greatly changing IL-2 elaboration, and the addition of BLCs to EC and T-cell cocultures greatly enhanced HIV-1 production in na?ve T cells. Furthermore, we do not believe that full TCR-based activation is even required for viral replication in our system. The frequency of p24Gag/high cells consisted of 5 to 10% of the total T-cell population; this is greater than the numbers of T cells that are fully activated by ECs. By limiting dilution analysis, precursor frequencies of T cells that respond to allogeneic EC have been routinely measured at between 1/1,000 and 1/5,000 (44). There are several explanations for these findings. For example, HIV-1 replication may require activation within the population of T cells but may be occurring in cells that are not directly recognizing MHC molecules on the EC surface, i.e., bystander cells. Alternatively, HIV-1 replication in this system may only require a low level of TCR-derived signals that is insufficient for complete activation. Weak signals may be produced by low-affinity cross-reactivity with foreign MHC molecules similar to those required by T cells for homeostatic TCR tickling by self MHC molecules (30). The low-level cross-reactivity has been shown to have a selective stimulatory function (59), which may be altered by Nef to facilitate productive infection within, and HIV-1 production from, incompletely stimulated T cells.
We observed that light fixation of cells abolished the capacity of ECs to enhance HIV-1 replication in T cells. Light fixation of the ECs preserves some aspects of their APC function and compromises others. Fixed ECs are equal to living ECs at providing costimulation to PHA-activated CD4+ T cells, which as with living ECs, is mediated via surface-bound molecules, i.e., LFA-3 (54). However, they lose their ability to fully stimulate alloreactive CD4+ T cells as well as the ability to confer cyclosporine resistance to T cells (J. S. Pober, unpublished observations). The inability of fixed ECs to support virus production may be a result of their inability to adequately stimulate resting CD4+ T cells, but it may also indicate the requirement for active EC metabolism. It is also possible that signals derived from HIV-1-infected T cells affect EC metabolism in ways that result in enhanced HIV-1 replication in EC and T cell cocultures. For example, HIV-1 Tat can be secreted from HIV-1-infected T cells, and this soluble form can enter ECs through specific receptors (6, 15, 24, 65). Soluble Tat acts as a promiscuous transcription factor that can promote transcription from many genes in ECs including IL-6, E-selectin, and IL-8 (1, 26, 27). The result of this aberrant transcription control is that ECs can behave similarly to they would in response to inflammation (27), which could influence HIV-1 replication in T cells. Interestingly, we observed that after 6 days of cocultivation, T cells could be separated from the ECs without adversely affecting HIV-1 production, suggesting that once signals have been exchanged between the two cell types, previously resting T cells are rendered competent for virus production.
In summary, we report that EC and T-cell coculture reveals a novel APC and T-cell interaction that results in greatly increased HIV-1 production from previously resting T cells. The specific characteristics of this interaction suggest that the mechanism of EC support of HIV-1 production in infected T cells is distinct from the mechanisms of other documented APC and T-cell interactions and could be determined by an undefined function of Nef.
ACKNOWLEDGMENTS
We thank Louise Benson, Gwendoline Davis, and Lisa Gras for excellent assistance in cell culture and animal handling.
This work was support by NIH grant R01 HL51014 and American Cancer Society institutional research grant IRG 58-012-42. J.C. was supported by NIH training grant GM07205; J.W. was supported by a National Science Foundation graduate research fellowship.
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