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. Author manuscript; available in PMC: 2008 Jul 7.
Published in final edited form as: Virology. 2007 Sep 4;369(1):214–225. doi: 10.1016/j.virol.2007.07.031

Antigen stimulation induces HIV envelope gp120-specific CD4+ T cells to secrete CCR5 ligands and suppress HIV infection

Gurvinder Kaur a,b, Michael Tuen a, Diana Virland a, Sandra Cohen a, Narinder K Mehra b, Christian Münz c, Sayed Abdelwahab d,1, Alfredo Garzino-Demo d, Catarina E Hioe a,*
PMCID: PMC2443714  NIHMSID: NIHMS34302  PMID: 17765942

Abstract

CD4+ T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4+ T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4+ T cells elicited production of high amounts of CCR5 chemokines MIP-1α (CCL3), MIP-1β (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-γ or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4+ T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1β and IFN-γ rapidly declined. Thus, vaccines that trigger HIV-specific CD4+ T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4+ T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.

Introduction

Virus-specific CD4+ T cells are a critical component of immune responses against HIV, but these cells are also the prime targets for HIV infection. Indeed, HIV-specific CD4+ T cells have been reported to be preferentially infected with the virus as compared to CD4+ T cells specific for other antigens (Douek et al., 2002), and they were depleted early during the primary HIV infection due to apoptosis and/or cytopathic effects of HIV infection (Brenchley et al., 2006; Malhotra et al., 2003; Yue et al., 2005; Zaunders et al., 2005). In the SIV model, Staprans et al. (Staprans et al., 2004) showed that increased CD4+ T cell proliferation was associated with enhanced levels of virus replication and pathogenesis in rhesus macaques that were vaccinated prior to SIV challenge. Maier et al. (Maier et al., 2000) also presented evidence that activation of CD4+ T cells by superantigens leads to upregulation of the surface expression of the chemokine receptors CCR5 and CXCR4, rendering the cells highly susceptible to HIV infection. Hence, there are concerns that stimulating and expanding HIV-specific CD4+ T cells, by vaccination for example, may supply more target cells for the virus and increase the host susceptibility to HIV infection. Nevertheless, not all of virus-specific CD4+ T cells in HIV infected subjects are depleted and only <10% are infected by the virus (Douek et al., 2002; Mattapallil et al., 2005). Our earlier study demonstrated that CD4+ T cells specific for HIV envelope gp120 were present at low frequencies in the peripheral blood of each of the HIV-infected subjects studied (Cohen et al., 2003). Notably, a proportion of these cells remained uninfected and could be propagated readily by antigen stimulation in vitro. It is clear that a fraction of HIV-specific CD4+ T cells are able to escape from or resist HIV infection, although the mechanisms contributing to their virus-free condition are yet not known.

Previous studies have shown that in addition to their traditional roles of providing help to virus-specific CD8+ T cells and B cells, CD4+ T cells can also mediate direct anti-viral functions through secretion of cytokines and chemokines that block or suppress HIV infection (Abdelwahab et al., 2003; Norris et al., 2004; Robbins et al., 1998; Saha et al., 1998). Some CD4+ T cells have also been shown to mediate cytolysis of virus-infected cells (Norris et al., 2004; Norris et al., 2001). Analyses of immortalized T cell clones generated from HIV-infected patients with different clinical profiles demonstrated that CD4+ T cells from non-progressors secreted high levels of RANTES, MIP-1α and MIP-1β, and were resistant to infection by CCR5-tropic HIV-1, while CD4+ T cells from AIDS patients were deficient in the production of these CC chemokines and were readily infected by the virus (Saha et al., 1998). HIV-specific CD4+ T cells from exposed uninfected subjects expressing the wild type CCR5 allele also secreted high levels of CC-chemokines with potent anti-viral activities upon antigen stimulation (Furci et al., 1997). Nevertheless, it is not known to what extent the CC chemokine production by these CD4+ T cells confers protection against HIV.

While many studies have demonstrated that CD8+ T cells, natural killer cells and macrophages are among the different immune cells that can be stimulated to produce high levels of the CC chemokines RANTES, MIP-1α and MIP-1β (Bernstein et al., 2004; Cocchi et al., 1995; Fehniger et al., 1998; Oliva et al., 1998; Verani et al., 1997), limited information is available regarding the capacity of antigen-specific CD4+ T cells to produce anti-HIV chemokines and protect themselves against the virus. A previous study of an influenza virus-specific CD4+ cytolytic T cell line demonstrated that antigen stimulation led to suppression of CCR5-tropic HIV infection in these CD4+ T cells and that the suppressive activity was mediated primarily by CC chemokines produced in the culture (Robbins et al., 1998). These chemokines inhibited HIV-1 entry not only by sterically blocking the CCR5 receptors but also by downregulating CCR5 cell surface expression. Whether HIV-specific CD4+ T cells generated during infection can exhibit a similar anti-HIV function has not been determined. Since the numbers of HIV-specific CD4 T cells are usually very low in the peripheral blood ex vivo from HIV-infected subjects, we had previously expanded these cells by 3 to 4 rounds of antigen stimulation and generated short-term primary lines (Cohen et al., 2003). In this study, two CD4+ T cell lines recognizing epitopes in the C1 and C2 regions of HIV envelope gp120 (PS01 and PS02) were examined. These cell lines were generated previously from PBMC of HIV-infected subjects who are CCR5 Δ32 heterozygotes and maintain their virus loads below 5000 copies/ml without anti-retroviral therapy. The cells have been shown to produce IFN-γ upon stimulation with their cognate antigens (Cohen et al., 2003), but their anti-viral activities have not been investigated. The present study reports the capacity of these gp120-specific CD4+ T cells to secrete high amounts of CCR5 chemokines in response to stimulation with their cognate antigens, the role of these chemokines in in vitro protection against HIV challenge, and the effect of HIV infection on production of IFN-γ and the CCR5 ligand MIP-1β.

Materials and Methods

HIV envelope-specific CD4+ T cell lines

Primary CD4+ T cell lines recognizing epitopes in the C1 and C2 regions of gp120 were generated from PBMCs of chronically HIV-1 infected non-progressors PS01 and PS02. PS01 and PS02 have been HIV-seropositive since 1991 and 1985, respectively, and have maintained HIV RNA loads of <5000 copies/ml and CD4+ cell counts >600/μl essentially without anti-retroviral therapy. PS01 received only 8-months of anti-retroviral therapy prior to 2000, while PS02 never had any anti-retroviral therapy. Both subjects are heterozygous for the CCR5 Δ32 allele (unpublished data). All subjects gave informed consent as required. The study was reviewed and approved by the Veterans Affairs New York Harbor Healthcare System Institutional Review Board. The initial characterization of the cell lines has previously been reported (Cohen et al., 2003). These short-term cell lines were frozen after the initial 3 or 4 rounds of antigen stimulation, re-stimulated with gp120 after thawing, and maintained in cultures for approximately 2 months in complete RPMI-1640 media supplemented with 10% human AB serum and 20 U/ml of human recombinant IL-2 (Roche). The cells were stimulated with gp120 every 2-3 weeks and used in the experiments 7 or more days after antigen stimulation. Autologous PBMCs were used as antigen-presenting cells for routine expansion of the cultures. However, in order to measure chemokines produced specifically by the CD4 T cells and not by other cells in the cultures, experiments were set up such that the CD4 T cells were stimulated with gp120 peptides without additional antigen-presenting cells. These CD4+ T cells expressed MHC class II and were capable of presenting gp120 peptides to one another to elicit production of cytokines and chemokines as well as proliferation. When contaminating feeder cells (usually CD8+ cells) were still present in the cultures, we depleted these cells with antibody-coated magnetic beads (Dynabeads) to achieve nearly 100% pure population of CD4+ CD3+ cells. Cytolytic activity of the CD4+ T cells was assessed in the standard 51Cr release assay using autologous Epstein Barr virus-transformed B lymphoblastoid cells as target cells.

Intracellular staining

Intracellular staining to detect production of cytokines and chemokines in the CD4+ T cell lines was performed as previously described (Cohen et al., 2003) with minor modifications. Briefly, CD4+ T cell lines PS01 and PS02 were incubated with 1 μg/ml of HIV-1 envelope peptides pC1a (NFNMWKNNMVEQMHEDIISL), pC2 (PKISFEPIPIHYCAPAGFAI), an irrelevant envelope peptide (YTTKNIIGTIRQAHCNISRA), or no antigen for 8 hrs. Brefeldin A (10 μg/ml) was added in the last 6 hrs of incubation. At the end of the incubation, the cells were washed, fixed, and stored at -4°C. Prior to staining, the cells were permeabilized with BD FACSPerm solution, and stained with fluorescence-conjugated antibodies to CD3, CD4 or CD8, IL-2, and IFN-γ or MIP-1β (BD Bioscience; catalog #: 347344, 340443, 555369, 340448, 340512, 550078). In some experiments, surface CCR5 staining was also performed on the CD4+ T cell lines treated with the specific peptides, an irrelevant peptide control, or medium alone. After 6, 24, and 48 hrs of antigen stimulation, the cells were stained with fluorescence-conjugated antibodies to CD3, CD4, and CCR5 (BD Bioscience catalog #: 347344, 340133, 550856). All flow cytometric experiments were performed using a BD FACSCalibur flow cytometer and the data were analyzed using the FlowJo software.

Measurement of chemokines and anti-HIV activity in culture supernatants

To detect chemokines secreted in the supernatants, the CD4+ T cell lines were either stimulated with their specific peptides or left untreated, and the supernatants were then collected on the designated days, frozen at -80°C, and stored for analyses by ELISA. Commercial ELISA kits (R&D Systems, Minneapolis, MN, USA) were used to measure all chemokines.

Assessment of HIV-suppressive activity in supernatants of the CD4+ T cell lines was done as previously described (Sun et al., 2004). Briefly, phytohemagglutinin-stimulated PBMCs from healthy donors were infected with HIV-1BaL (100 TCID50) for 2 hrs, washed, and then treated with complete media containing 25% or 50% of the CD4+ T cell supernatants. For controls, we treated the infected PBMCs with 25% or 50% of media containing no T cell supernatant. The contribution of CC chemokines was evaluated by adding a cocktail of neutralizing polyclonal antibodies to RANTES, MIP-1α, MIP-1β and MDC (5 μg/ml each) or isotype-matched control antibodies (R&D Systems).

HIV-1 infection in antigen-stimulated CD4+ T cell lines

To assess the effects of antigen stimulation on virus infection, the cells were treated with the specific peptides or medium alone for 6 hrs and then infected with varying doses of HIV-1BX-08 isolate. The virus stock was propagated in phytohemagglutinin-stimulated PBMCs to yield at least 100 ng p24/ml. Prior to use in the experiments, the virus particles were centrifuged using the TLA100.3 rotor in the Beckman Optima MAX ultracentrifuge (43,000 rpm for 1 hr at 4°C) to remove mitogen and other contaminants present in the virus preparations. The virus pellets were suspended in fresh medium and used to infect the CD4+ T cells. The numbers of HIV RNA copies present in the pelleted virus samples were measured by the branched DNA test (HIV RNA 3.0; Bayer). One day after infection, the cells were washed and cultured with fresh RPMI-1640 medium supplemented with 10% human AB serum and 20 U/ml of IL-2 (Roche). Culture supernatants were collected on the designated days and stored at -80°C. The concentrations of HIV-1 p24 antigens in the supernatants were measured by non-commercial ELISA.

To detect HIV-infected cells and IFN-γ– or MIP-1β-producing cells in the cultures, cells were harvested periodically, fixed, and stained. The previously described intracellular staining protocol was followed (Cohen et al., 2003), with the exception that fluorescence-conjugated antibodies to CD3 and CD8 were used to gate the cell population studied and fluorescence-conjugated anti-p24 monoclonal antibody (KC57; Coulter) was used to detect virus-infected cells.

Antigen stimulation of HIV-infected CD4+ T cell lines

To assess the effect of HIV infection on envelope-specific CD4+ T cell responses, the CD4+ T cell lines were first infected with pelleted stocks of HIV-1BX08 (100 ng p24 per 5 ×106 CD4+ T cells) or left untreated for 24 hrs. After washing to remove free virus, the cells were cultured in RPMI 1640 medium with 20% FCS and IL-2 (20 U/ml; Roche). The infection condition was optimized in order to infect the majority of the cells within 5 days while the cells remained viable. On the designated days, the cells were treated with the specific peptides or medium alone, and stained with fluorescence-conjugated antibodies to CD3, CD8, p24, and IFN-γ or MIP-1β as described above.

Results

HIV gp120-specific CD4+ T cell lines produce IFN-γ and CCR5 ligands upon antigen stimulation

This study evaluated whether HIV-specific CD4+ T cells could produce anti-viral factors and protect themselves from HIV infection. Considering that the vast majority of primary HIV-1 isolates worldwide are CCR5-tropic and the viruses transmitted from one subject to another are mainly CCR5-tropic (Hladik et al., 2007; Li et al., 2005a; Li et al., 2006), we focused our analyses on the CC chemokines that are ligands forf the CCR5 receptor and that are potent inhibitors of CCR5-tropic viruses, i.e. MIP-1α (CCL3), MIP-1β (CCL4), and RANTES (CCL5). In addition, we measured production of IFN-γ and IL-2, two cytokines commonly tested to measure antigen-specific T cell responses. Two primary CD4+ T cell lines (PS01 and PS02) recognizing distinct epitopes in the C1 and C2 regions of HIV envelope gp120 were tested (Cohen et al., 2003). These lines were expanded by repeated stimulation with gp120-pulsed antigen-presenting cells and maintained as short-term cultures (for <10 weeks) in the presence of IL-2.

Intracellular staining assays showed that both the PS01 and the PS02 cell lines produced IFN-γ, IL-2, and MIP-1β in response to their respective gp120 peptides, pC1a and pC2. Dot plots from one representative experiment are shown in Fig. 1. In both cell lines, MIP1β responses were consistently more robust than IFN-γ or IL-2 responses. From multiple experiments with different stocks of the cell lines, the percentages of MIP-1β producers reached 8-25% (mean 16%) for PS01 and 44-97% (mean 71%) for PS02, while the percentages of IFN-γ producers were 5-14% (mean 12%) for PS01 and 3-60% (mean 24%) for PS02. These responses were specific, as no response was detected when the cells were treated with an irrelevant gp120 peptide control. By comparison, the IL-2 responses were barely detectable; 1.6-2.9% (mean 2.3%) of PS01 cells and only 0.06-0.4% (mean 0.25%) of PS02 cells produced this cytokine. The low IL-2 production may be due to the culturing conditions which include exogenous IL-2. Nevertheless, these data are consistent with a previous report showing that in PBMCs from healthy subjects with recall responses to tetanus toxoid, the production of CCR5 ligands was much more readily detectable than that of IFN-γ while IL-2 secretion was the poorest (Sun et al., 2004).

Fig 1. HIV gp120-specific CD4+ T cells produce IFN-γ and MIP-1β after antigen stimulation.

Fig 1

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CD4+ T cell lines PS01 (A) and PS02 (B), which are specific for epitopes in the C1 and C2 regions of gp120, were stimulated for 6 hrs with the relevant peptides (pC1a or pC2), control peptide, or no antigen. MAb staining was then performed to detect CD4 (not shown), CD3, IL-2, and IFN-γ or MIP-1β. The frequencies of responding CD4+ T cells were determined by flow cytometry. Top panels are dot plots showing CD3 and MIP-1β; the bottom panels are dot plots showing IL-2 and IFN-γ. Background surface fluorescence was determined from isotype-matched control mAb staining, while cut-off for IL-2, IFN-γ, and MIP-1β was based on specific mAb staining of untreated cultures (left panels). Plots are representative of five independent experiments.

When the supernatants from these T cell cultures were tested by ELISA, we readily detected the presence of the CCR5 ligands RANTES, MIP-1α, and MIP-1β, but not of MDC (macrophage-derived chemokine or CCL22), which inhibits both CCR5- and CXCR4-tropic viruses (Fig. 2). Notably, secretion of these chemokines was sustained in the PS01 cells for > 20 days, while the PS02 cells secreted high levels of these CCR5 ligands only within the first 1-3 days after antigen stimulation. These data clearly show distinct patterns of chemokine production by these two cell lines. While the reason for this difference is not known, the PS01 line contained a small, but consistently detectable, fraction of antigen-specific CD4+ T cells that produced both IFN-γ and IL-2. These multifunctional CD4+ T cells might produce additional cytokines such as TNF-α that upregulate and sustain production of CCR5 ligands (Brice et al., 2000; Hornung et al., 2000). The PS02 cell line, on the other hand, appeared to contain mostly MIP-1β-producing cells, with few IFN-γ-producing cells and no IL-2-producing cells (Fig. 1).

Fig 2. Secretion of CCR5 ligands by CD4+ T cells after antigen stimulation.

Fig 2

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CD4+ T cell lines PS01 and PS02 were stimulated with peptides pC1a and pC2, respectively, and cultured for up to 1 month in the presence of IL-2. Culture supernatants were collected and replaced with fresh medium on the designated days. The levels of RANTES, MIP1α, MIP1β, and MDC in the supernatants were determined by ELISA. Representative data from one of two experiments are shown.

CC-chemokine production is associated with HIV-suppressive activity

To directly evaluate anti-HIV activities of the gp120-specific CD4+ T cells, we tested the CD4+ T cell line PS02 that consistently had a high number of CC-chemokine producers upon antigen stimulation but could also be cultured without detectable chemokine secretion in the absence of antigen. PS02 cells were stimulated with the specific gp120 peptide for 6 hrs and then infected with a CCR5-tropic primary HIV-1 isolate BX08. For comparison, a parallel culture of unstimulated cells was also infected with the same virus. Both cultures were maintained in medium containing IL-2 for up to 10 days. Virus infection was monitored by measuring p24 in the supernatants with ELISA. Fig. 3A shows that antigen stimulation suppressed infection with BX08 virus at 7.5×106 v-RNA copies/ml (equivalent to 100 ng p24/ml), especially at the earlier time points. When one or two log lower doses of virus were used, the suppression was nearly complete throughout the observation period (Fig. 3B-C). Similar results were observed when p24+ cells were detected in the cultures by flow cytometry (Fig. 4A). During this observation period (6 days), the cell viability was similar in both the antigen-stimulated and the unstimulated cultures. The PS02 cell line had no antigen-specific cytolytic activity (Fig. 4D). Rather, the virus-suppressive activity was associated with induction of IFN-γ and MIP-1β in the CD4+ T cells within 6 hrs after gp120 peptide stimulation (Fig. 4B and 4C). Interestingly, IFN-γ production waned quickly, such that no IFN-γ+ cells were detected above background by day 6. In contrast, MIP-1β production was stable through day 4 and started to drop only by day 6 by ∼50%. These results were consistent with the data in Fig. 2 showing that CC chemokines were secreted by PS02 cells for 1-3 days after antigen stimulation.

Fig 3. Antigen stimulation renders gp120-specific CD4+ T cells more resistant to HIV infection.

Fig 3

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T cells were treated with peptide pC2 or untreated for 6 hrs, and then infected with varying doses of BX08: A. 7.5 × 106 v-RNA copies/ml, B: 7.5 × 105 v-RNA copies/ml, and C: 7.5 × 104 v-RNA copies/ml. The levels of p24 in the culture supernatants on the designated days were measured by ELISA.

Fig. 4. Suppression of HIV replication in gp120 peptide-stimulated CD4+ T cells correlates with MIP-1β production.

Fig. 4

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A: PS02 CD4+ T cells were stimulated for 6 hrs with peptide pC2 or left untreated, and exposed 24 hrs to infectious BX08 (100 ng p24/ml). The cells were then washed and incubated for up to 6 days. On the designated days, the cells were harvested and fixed. MAb staining and flow cytometric analyses were done on all cells concurrently to detect the fractions of CD4+ T cells bearing intracellular p24. PS02 cells that were not infected with virus were used as negative controls. Mean and standard deviation from duplicate wells are presented. B and C: Parallel cultures of uninfected PS02 cells were also set up in order to monitor IFN-γ and MIP-1β production during the observation period. The cells were treated for 6 hrs with peptide pC2 or with medium alone. A fraction of the cells were harvested and fixed immediately (day 0), while the rest were cultured for up to 6 days. On days 2, 4 and 6, additional fractions of the cells were harvested and fixed. Brefeldin A was added 4 hrs prior to fixing. Intracellular cytokine staining was done on all cells at the same time. The percentages of CD4+ T cells producing IFN-γ or MIP-1β were determined by flow cytometry. Data are representative of three independent experiments. D: Cytolytic activity of PS02 cells was assessed in the standard 51Cr-release assay. Autologous B lymphoblastoid cells treated with peptide pC2, control peptide, or no peptide were used as target cells and incubated for 18 hrs with PS02 cells at the designated effector/target (E/T) ratios.

Subsequently, we examined whether suppression of HIV infection was mediated by the CC-chemokines secreted by the CD4+ T cells. PHA-treated PBMCs from an HIV-seronegative donor were infected with CCR5-tropic HIV-1 BaL, in the presence of the CD4+ T cell supernatants which were pre-treated with either a mixture of blocking Abs to RANTES, MIP-1α, MIP-1β, and MDC, or with irrelevant control Abs (Fig. 5). Addition of the cell supernatant pretreated with control Abs suppressed virus infection, and the suppressive effects were eliminated by pretreatment with anti-chemokine Abs. Medium that contained no T cell supernatant and were treated with Abs was included as negative controls and showed no virus-suppressive activity. Since the CD4+ T cell supernatant contained RANTES, MIP-1α and MIP-1β (Fig. 2), these results indicate that the suppression of HIV infection observed here was primarily due to these CC-chemokines. Of note, the addition of anti-chemokine Abs enhanced infection above that seen in PBMCs cultured with virus alone. This enhancing effect is most likely due to the blocking of CC-chemokines produced by mitogen-stimulated PBMCs by the Abs (Furci et al., 1997; Kinter et al., 1996; Sun et al., 2004). The same supernatants were also tested for suppressive activity against CXCR4-tropic HIV-1IIIB, but the levels of suppression detected were low and inconsistent (data not shown).

Fig 5. Chemokines secreted by gp120-specific CD4+ T cells mediate suppression of HIV infection.

Fig 5

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Supernatant collected from the culture of gp120-specific CD4+ T cells after antigen stimulation (left graph) or medium containing no supernatant (right graph) were treated with a combination of antibodies against MIP-1α, MIP-1β, RANTES, and MDC (anti-CK Abs) or with control antibodies. The treated supernatants or medium were added to mitogen-blasted PBMCs infected with HIV-1 BaL at a final concentration of 25% (a 1:4 dilution). Comparable results were obtained with T cell supernatant diluted 1:2 (data not shown). The levels of p24 in the cultures were monitored by ELISA. p24 levels in virus-infected PBMCs that were left untreated (medium only) were also assessed for comparison.

CC-chemokine production is not accompanied by CCR5 downregulation

The binding of chemokines to their G protein-coupled receptors causes receptor internalization that can down-regulate the surface expression of these receptors (Fernandis et al., 2002; Mack et al., 1998; Signoret et al., 2005). To determine whether the secretion of CC-chemokines upon antigen stimulation of gp120-specific CD4+ T cells is accompanied by down-regulation of CCR5 from the cell surface, we measured the levels of CCR5 expression on the surface of PS02 cells following treatment with the relevant gp120 peptide, control peptide, or medium alone. The flow cytometry data demonstrate that after a 6 hr treatment with the antigenic gp120 peptide, the CCR5 levels on the CD4+ T cell surface did not change (Fig. 6A). Surface levels of CD4+ also remained unchanged (data not shown). At this time point, significant percentages of the cells in the culture produced MIP-1β and IFN-γ (Fig. 6B). These data indicate that while antigen activation of gp120-specific CD4+ T cells elicits production of CC-chemokines that suppress HIV infection, the suppressive activity is not likely due to down-regulation of the chemokine receptor CCR5.

Fig 6. Upon peptide stimulation, gp120-specific CD4+ T cells express IFN-γ and MIP-1β but chemokine receptors CCR5 are not down-regulated.

Fig 6

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A: Gp120-specific CD4+ T cell line PS02 was treated for 6 hrs with specific peptide pC2 (solid black line), irrelevant peptide control (dotted black line), or no peptide (solid gray line), and assessed for surface expression of CCR5 by CCR5-specific mAb staining and flow cytometric analysis. Cells staining with isotype-matched irrelevant mAb were used to determine background fluorescence (dotted gray line). B: Parallel cultures of cells were similarly treated with peptide pC2, control peptide, or no peptide, and then stained intracellularly with mAbs to IFN-γ (left panel) or MIP-1β (right panel). The percentages of positive cells were determined by flow cytometry. Data are representative of two separate experiments.

Production of IFN-γ and MIP-1β is impaired by HIV infection

Gp120-specific CD4+ T cells were capable of suppressing HIV infection, but this anti-viral activity was only achieved if the cells were first stimulated by their specific gp120 peptides to produce CC-chemokines. The resistance to infection was however incomplete; when the cells were exposed to relatively high doses of virus, virus replication still occurred at low levels. To assess if the gp120-specific CD4+ T cells infected with HIV were able to respond to their specific antigens, PS02 cells were first infected with CCR5-tropic HIV-1 BX-08 for 0 to 5 days, and then stimulated with the specific gp120 peptide at different time points. Intracellular staining assays were performed to detect cells expressing p24 and IFN-γ or MIP-1β. As expected, very little infection (<5%) was observed 24 hrs after infection, but almost all of the cells were p24+ by days 4-5 (Fig. 7). On day 1, when a few cells were positive for p24, the CD4+ T cells were responsive to gp120 peptides, and the frequency of CD4+ T cells producing IFN-γ or MIP-1β in the virus-infected culture was comparable to the frequency of CD4+ responders in the uninfected cultures. However, from day 2 to day 5, as the number of p24+ cells and intensity of p24 staining in all cells rose, the frequency of cells responsive to gp120 peptides declined, almost to the background level by day 5. In a parallel uninfected culture, the numbers of cells that produced IFN-γ or MIP-1β in response to gp120 peptide were relatively stable from day 0 to day 5. These results demonstrate deleterious effects of HIV infection on the virus-specific CD4+ T cells and particularly on the anti-viral functions of these cells.

Fig 7. HIV infection impairs the production of IFN-γ and MIP-1β by gp120-specific CD4 T cells.

Fig 7

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Uninfected and BX08-infected PS02 cells were cultured in parallel for 4 or 5 days. On the designated days, a fraction of the cells in each culture were treated with peptide pC2 for 6 hrs and stained with mAbs to p24, IFNγ or MIP-1β. The background fluorescence for p24 was based on cells collected on day 0 and on uninfected cells collected on days 1-5. Cells treated with no peptide were also stained for IFN-γ or MIP-1β and used to determine the cut-off for the respective cytokine and chemokine. Flow cytometric analyses were performed to measure the percentages of CD4 T cells positive for p24 and IFN-γ or MIP-1β. A: Dot plots of HIV-infected and uninfected PS02 cells stained with mAbs to p24 and IFN-γ. B: Dot plots of HIV-infected and uninfected PS02 cells stained with mAbs to p24 and MIP-1β. C: Changes in percentages of CD4 T cells positive for p24 and IFN-γ (top graphs) or p24 and MIP-1β (bottom graphs) over time in HIV-infected and uninfected PS02 cultures.

Discussion

This study provides the first evidence that antigen stimulation of HIV-specific CD4+ T cells protected these cells against the subsequent challenge by CCR5-tropic HIV-1, and that the virus-inhibitory activity was associated with the secretion of CC chemokines. Prior to antigen stimulation, the cells produced undetectable or low amounts of these chemokines and were readily infected by HIV. Within hours after antigen stimulation, these cells secreted high levels of CC chemokines with potent suppressive activities against CCR5-tropic HIV-1 that could be blocked by anti-chemokine antibodies. In contrast, MDC, which is effective against both CCR5- and CXCR4-tropic HIV, was not produced. Since little and inconsistent suppression was observed against CXCR4-tropic HIV-1 (data not shown), we did not analyze other soluble factors known to inhibit CXCR4-tropic isolates, such as SDF-1 (stromal derived factor 1) and α-defensins. These observations are corroborated by published reports that CD4+ T cells specific for other antigens secrete mainly CCR5 ligands and do not suppress CXCR4-tropic isolates (Lotti et al., 2002; Norris et al., 2004; Robbins et al., 1998). Interestingly, production of CC-chemokines in the gp120-specific CD4+ T cells was not accompanied by down-regulation of cell surface CCR5. Rather, the CCR5 ligands most likely inhibited virus infection by directly interfering with virus entry. Studies are now underway to further test this hypothesis and examine other steps in the virus life cycle that are affected.

The gp120-specific CD4+ T cell lines PS01 and PS02 also secreted IFN-γ in response to antigen stimulation, but as compared to the robust and durable CCR5 ligand response, IFN-γ production was very transient and was observed only in a small fraction of the cells. Moreover, there is no clear evidence that IFN-γ mediates direct anti-HIV activities (Creery et al., 2004; Mackewicz et al., 1994). It should also be noted, that unlike many of the CD4+ T cell lines or clones reported in the literature (Lotti et al., 2002; Misko et al., 1984; Norris et al., 2004; Paludan et al., 2002; Robbins et al., 1998), the gp120-specific CD4+ T cell lines studied here had no cytolytic activity (Fig. 4D and data not shown). Nevertheless, factors other than CCR5 ligands might contribute to virus resistance and suppression in these CD4+ T cells. Of note, decreased HIV replication has been observed in heterozygous CCR5 Δ32 cells (Liu et al., 1996). Reduced CCR5 expression due to CCR5/CCR5 Δ32 heterodimerization has also been reported in T cells of CCR5 Δ32 heterozygotes and correlated with lower levels of HIV replication and delayed onset of disease progression in these subjects (Benkirane et al., 1997; Cohen et al., 1997; Gorry et al., 2007). In view of the lower levels of CCR5 expression and HIV replication in the heterozygous CCR5 Δ32 cells, HIV infection in these cells may be relatively sensitive to inhibitory effects of the CCR5 ligands as compared with wild type cells, but further investigation is necessary to examine this issue.

The capacity of the gp120-specific CD4+ T cells to produce CCR5 ligands and suppress HIV infection depended on antigen stimulation prior to or soon after exposure of the cells to the virus (Figs. 3 and 7). After the cells become infected, the capacity of these cells to respond to antigen stimulation and produce chemokines was rapidly abrogated (Fig. 7). These results are consistent with earlier reports demonstrating that HIV infection decreases cytotoxicity and cytokine-secreting capacity of CD4+ T cells specific for other pathogens, such as influenza virus and M. tuberculosis (Robbins et al., 1998; Sutherland et al., 2006). In our experimental design, there appeared to be a narrow window of ≤ 1 day, where the cells were responsive to antigen stimulation after virus exposure and almost all cells were still negative for intracellular p24. However, in this in vitro experiment, a relatively high dose of virus was used in order to infect the majority of the cells within 4-5 days. The rates of infection and spread to virus-specific CD4+ T cells in vivo after human subjects are exposed to the virus may not necessarily be as rapid. When rhesus macaques were infected intravaginally with SIVmac251, SIV RNA-bearing cells rose up only after 8 days of infection (Li et al., 2005b). We postulate that if virus-specific CD4+ T cells that produce CCR5 ligands are recruited within the initial week to the mucosal sites of primary infection, they will protect themselves and other CD4+ T cells which would otherwise succumb to virus infection and become non-responsive. Therefore, HIV vaccines should include components that trigger virus-specific CD4+ T cells to secrete high levels of these chemokines. Without vaccination, CD4+ T cells specific for gag and envelope were found in the peripheral blood of subjects with very early primary HIV infection (< 30 days after infection or onset of symptoms), but the cells were rapidly lost thereafter (Malhotra et al., 2003; Zaunders et al., 2005). These early responses were reported to include CD4+ cytotoxic effector cells, but the capacity of the cells to secrete CC chemokines that block entry and infection of the transmitted viruses was not examined. Nevertheless, it is also possible that upon virus challenge, production of CC chemokines by HIV-specific CD4+ T cells may lead to the recruitment of more inflammatory cells to the mucosal sites of infection, including dendritic cells that capture HIV via DC-SIGN, independent of CCR5, thereby facilitating the spread of the virus to the lymph nodes (Kwon et al., 2002; Sozzani, 2005; van Montfort et al., 2007).

This study, together with previous reports, demonstrate that CD4+ T cells specific for different antigens which can be stimulated to produce CCR5 chemokines and resist HIV infection are present in various HIV-infected and uninfected subjects. Hence, in addition to the CD4+ T cell lines PS01 and PS02 that were derived from HIV+ subjects who were able to control their virus infection without anti-retroviral therapy, envelope-specific CD4+ T cell clones secreting very high levels of CCR5 chemokines were also generated from peripheral blood cells of HIV-exposed uninfected individuals and of HIV-seronegative subjects immunized with recombinant gp120W61D protein in QS21/MPL adjuvant (Furci et al., 1997; Jones et al., 1999). A number of gag-specific CD4+ T cell clones and lines have been generated from acutely and chronically HIV-infected patients who have received HAART, and these cells produce CCR5 ligands (Lotti et al., 2002; Norris et al., 2004; Norris et al., 2001). CD4+ T cells specific for other antigens, such as tetanus toxoid and influenza virus hemagglutinin, from HIV-seronegative subjects also showed the capacity to secrete CCR5 ligands with rapid kinetics, that would allow for the blocking of HIV entry (Robbins et al., 1998; Sun et al., 2004). However, one important question that remains unaddressed is whether HIV-specific CD4 T cells capable of producing CCR5 ligands constitute a significant proportion of the CD4 T cell population. Only a subset of the CD4+ T cell population may produce CCR5 ligands, and these cells may secrete the chemokines with different kinetics, as indicated by the observed differences between PS01 and PS02 lines (Fig. 2). Lotti et al. (Lotti et al., 2002) also demonstrated that only a fraction of gag-specific CD4 T cell clones isolated from the HIV-seropositive subject with the strongest gag-specific proliferative response in their cohort produced high levels of MIP-1α and MIP-1β. Hence, one possibility is that a large proportion of HIV-specific CD4 T cells activated in response to the infection do not produce CCR5 ligands to a sufficiently high level and/or with a fast enough rate, and these cells become exceedingly prone to infection. This would explain the loss of significant percentages of virus-specific CD4 T cells and the small numbers that remain uninfected in most HIV-infected subjects.

Earlier studies of T cell responses in HIV-infected and uninfected subjects typically measured IFN-γ, IL-2, and/or TNF-α, but not CCR5 chemokines. Only in late 2006, with the use of polychromatic flow cytometry, production of MIP-1β was monitored along with IFN-γ, TNF-α, and IL-2, in CD4+ and CD8+ T cell responses to CMV and HIV, respectively (Betts et al., 2006; Casazza et al., 2006). It is of interest to note that MIP-1β production dominated CD8+ T cell responses to different HIV antigens and was one of the CD8+ T cell functions that discriminate HIV-infected non-progressors from progressors (Betts et al., 2006). Whether vigorous production of CCR5 ligands also characterizes HIV-specific CD4+ T cell responses in non-progressors as compared to progressors remains to be determined. It is also not known if high frequencies of CCR5-ligand-producing T cells are induced by candidate HIV vaccines that have been tested in pre-clinical and clinical trials. The parameters used to evaluate T cell responses to these vaccines typically do not include CCR5 ligands, even though these chemokines directly mediate potent anti-HIV functions.

The present study shows that CCR5 ligands were secreted by gp120-specific CD4+ T cells after antigen stimulation. CCR5 chemokine production was much robust and durable as compared to IFN-γ and IL-2, and importantly, production of these chemokines led to the suppression of virus infection in these cells. These results imply that antigen-driven induction of HIV-specific CD4+ T cell responses that are accompanied by secretion of high amounts of CCR5 ligands will provide these cells some degree of protection from virus infection. Taken together with the published reports mentioned above, the data also strengthen the case for including CCR5 ligand production among the CD4+ T cell responses evaluated in HIV vaccine trials and in HIV-infected subjects.

Acknowledgments

This work was supported by funds from a Merit Review Award and the Research Enhancement Award Program of the US Department of Veterans Affairs, the New York University Center for AIDS Research Immunology Core (AI-27742), the Training Program in TB and HIV Prevention and Treatment (D43 TWO1409), and by NIH grant AI-48371.

The authors would like to thank Dr. Jennifer Fuller for reviewing and editing the manuscript and the volunteers for their dedication and efforts to make this study possible.

Footnotes

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References

  1. Abdelwahab SF, Cocchi F, Bagley KC, Kamin-Lewis R, Gallo RC, DeVico A, Lewis GK. HIV-1-suppressive factors are secreted by CD4+ T cells during primary immune responses. Proc Natl Acad Sci U S A. 2003;100(25):15006–10. doi: 10.1073/pnas.2035075100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Benkirane M, Jin DY, Chun RF, Koup RA, Jeang KT. Mechanism of transdominant inhibition of CCR5-mediated HIV-1 infection by ccr5delta32. J Biol Chem. 1997;272(49):30603–6. doi: 10.1074/jbc.272.49.30603. [DOI] [PubMed] [Google Scholar]
  3. Bernstein HB, Kinter AL, Jackson R, Fauci AS. Neonatal natural killer cells produce chemokines and suppress HIV replication in vitro. AIDS Res Hum Retroviruses. 2004;20(11):1189–95. doi: 10.1089/aid.2004.20.1189. [DOI] [PubMed] [Google Scholar]
  4. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, Lederman MM, Benito JM, Goepfert PA, Connors M, Roederer M, Koup RA. HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood. 2006;107(12):4781–9. doi: 10.1182/blood-2005-12-4818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brenchley JM, Ruff LE, Casazza JP, Koup RA, Price DA, Douek DC. Preferential infection shortens the life span of human immunodeficiency virus-specific CD4+ T cells in vivo. J Virol. 2006;80(14):6801–9. doi: 10.1128/JVI.00070-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brice GT, Mayne AE, Villinger F, Ansari AA. A novel role for tumor necrosis factor-alpha in regulating susceptibility of activated CD4+ T cells from human and nonhuman primates for distinct coreceptor using lentiviruses. J Acquir Immune Defic Syndr. 2000;24(1):10–22. doi: 10.1097/00126334-200005010-00003. [DOI] [PubMed] [Google Scholar]
  7. Casazza JP, Betts MR, Price DA, Precopio ML, Ruff LE, Brenchley JM, Hill BJ, Roederer M, Douek DC, Koup RA. Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J Exp Med. 2006;203(13):2865–77. doi: 10.1084/jem.20052246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cocchi F, DeVico AL, Garzino-Demo A, Arya SK, Gallo RC, Lusso P. Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells. Science. 1995;270(5243):1811–5. doi: 10.1126/science.270.5243.1811. [DOI] [PubMed] [Google Scholar]
  9. Cohen OJ, Vaccarezza M, Lam GK, Baird BF, Wildt K, Murphy PM, Zimmerman PA, Nutman TB, Fox CH, Hoover S, Adelsberger J, Baseler M, Arthos J, Davey RT, Jr, Dewar RL, Metcalf J, Schwartzentruber DJ, Orenstein JM, Buchbinder S, Saah AJ, Detels R, Phair J, Rinaldo C, Margolick JB, Pantaleo G, Fauci AS. Heterozygosity for a defective gene for CC chemokine receptor 5 is not the sole determinant for the immunologic and virologic phenotype of HIV-infected long-term nonprogressors. J Clin Invest. 1997;100(6):1581–9. doi: 10.1172/JCI119682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen S, Tuen M, Hioe CE. Propagation of CD4+ T cells specific for HIV type 1 envelope gp120 from chronically HIV type 1-infected subjects. AIDS Res Hum Retroviruses. 2003;19(9):793–806. doi: 10.1089/088922203769232593. [DOI] [PubMed] [Google Scholar]
  11. Creery D, Weiss W, Lim WT, Aziz Z, Angel JB, Kumar A. Down-regulation of CXCR-4 and CCR-5 expression by interferon-gamma is associated with inhibition of chemotaxis and human immunodeficiency virus (HIV) replication but not HIV entry into human monocytes. Clin Exp Immunol. 2004;137(1):156–65. doi: 10.1111/j.1365-2249.2004.02495.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Douek DC, Brenchley JM, Betts MR, Ambrozak DR, Hill BJ, Okamoto Y, Casazza JP, Kuruppu J, Kunstman K, Wolinsky S, Grossman Z, Dybul M, Oxenius A, Price DA, Connors M, Koup RA. HIV preferentially infects HIV-specific CD4+ T cells. Nature. 2002;417(6884):95–8. doi: 10.1038/417095a. [DOI] [PubMed] [Google Scholar]
  13. Fehniger TA, Herbein G, Yu H, Para MI, Bernstein ZP, O'Brien WA, Caligiuri MA. Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection. J Immunol. 1998;161(11):6433–8. [PubMed] [Google Scholar]
  14. Fernandis AZ, Cherla RP, Chernock RD, Ganju RK. CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway. J Biol Chem. 2002;277(20):18111–7. doi: 10.1074/jbc.M200750200. [DOI] [PubMed] [Google Scholar]
  15. Furci L, Scarlatti G, Burastero S, Tambussi G, Colognesi C, Quillent C, Longhi R, Loverro P, Borgonovo B, Gaffi D, Carrow E, Malnati M, Lusso P, Siccardi AG, Lazzarin A, Beretta A. Antigen-driven C-C chemokine-mediated HIV-1 suppression by CD4(+) T cells from exposed uninfected individuals expressing the wild-type CCR-5 allele. J Exp Med. 1997;186(3):455–60. doi: 10.1084/jem.186.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gorry PR, Dunfee RL, Mefford ME, Kunstman K, Morgan T, Moore JP, Mascola JR, Agopian K, Holm GH, Mehle A, Taylor J, Farzan M, Wang H, Ellery P, Willey SJ, Clapham PR, Wolinsky SM, Crowe SM, Gabuzda D. Changes in the V3 region of gp120 contribute to unusually broad coreceptor usage of an HIV-1 isolate from a CCR5 Delta32 heterozygote. Virology. 2007 doi: 10.1016/j.virol.2006.11.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hladik F, Sakchalathorn P, Ballweber L, Lentz G, Fialkow M, Eschenbach D, McElrath MJ. Initial Events in Establishing Vaginal Entry and Infection by Human Immunodeficiency Virus Type-1. Immunity. 2007;26(2):257–270. doi: 10.1016/j.immuni.2007.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hornung F, Scala G, Lenardo MJ. TNF-alpha-induced secretion of C-C chemokines modulates C-C chemokine receptor 5 expression on peripheral blood lymphocytes. J Immunol. 2000;164(12):6180–7. doi: 10.4049/jimmunol.164.12.6180. [DOI] [PubMed] [Google Scholar]
  19. Jones GJ, von Hoegen P, Weber J, Rees AD. Immunization with human immunodeficiency virus type 1 rgp120W61D in QS21/MPL adjuvant primes T cell proliferation and C-C chemokine production to multiple epitopes within variable and conserved domains of gp120W61D. J Infect Dis. 1999;179(3):558–66. doi: 10.1086/314626. [DOI] [PubMed] [Google Scholar]
  20. Kinter AL, Ostrowski M, Goletti D, Oliva A, Weissman D, Gantt K, Hardy E, Jackson R, Ehler L, Fauci AS. HIV replication in CD4+ T cells of HIV-infected individuals is regulated by a balance between the viral suppressive effects of endogenous beta-chemokines and the viral inductive effects of other endogenous cytokines. Proc Natl Acad Sci U S A. 1996;93(24):14076–81. doi: 10.1073/pnas.93.24.14076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kwon DS, Gregorio G, Bitton N, Hendrickson WA, Littman DR. DC-SIGN-mediated internalization of HIV is required for trans-enhancement of T cell infection. Immunity. 2002;16(1):135–44. doi: 10.1016/s1074-7613(02)00259-5. [DOI] [PubMed] [Google Scholar]
  22. Li M, Gao F, Mascola JR, Stamatatos L, Polonis VR, Koutsoukos M, Voss G, Goepfert P, Gilbert P, Greene KM, Bilska M, Kothe DL, Salazar-Gonzalez JF, Wei X, Decker JM, Hahn BH, Montefiori DC. Human immunodeficiency virus type 1 env clones from acute and early subtype B infections for standardized assessments of vaccine-elicited neutralizing antibodies. J Virol. 2005a;79(16):10108–25. doi: 10.1128/JVI.79.16.10108-10125.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li M, Salazar-Gonzalez JF, Derdeyn CA, Morris L, Williamson C, Robinson JE, Decker JM, Li Y, Salazar MG, Polonis VR, Mlisana K, Karim SA, Hong K, Greene KM, Bilska M, Zhou J, Allen S, Chomba E, Mulenga J, Vwalika C, Gao F, Zhang M, Korber BT, Hunter E, Hahn BH, Montefiori DC. Genetic and neutralization properties of subtype C human immunodeficiency virus type 1 molecular env clones from acute and early heterosexually acquired infections in Southern Africa. J Virol. 2006;80(23):11776–90. doi: 10.1128/JVI.01730-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li Q, Duan L, Estes JD, Ma ZM, Rourke T, Wang Y, Reilly C, Carlis J, Miller CJ, Haase AT. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature. 2005b;434(7037):1148–52. doi: 10.1038/nature03513. [DOI] [PubMed] [Google Scholar]
  25. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA, Landau NR. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell. 1996;86(3):367–77. doi: 10.1016/s0092-8674(00)80110-5. [DOI] [PubMed] [Google Scholar]
  26. Lotti B, Wendland T, Furrer H, Yawalkar N, von Greyerz S, Schnyder K, Brandes M, Vernazza P, Wagner R, Nguyen T, Rosenberg E, Pichler WJ, Brander C. Cytotoxic HIV-1 p55gag-specific CD4+ T cells produce HIV-inhibitory cytokines and chemokines. J Clin Immunol. 2002;22(5):253–62. doi: 10.1023/a:1020066404226. [DOI] [PubMed] [Google Scholar]
  27. Mack M, Luckow B, Nelson PJ, Cihak J, Simmons G, Clapham PR, Signoret N, Marsh M, Stangassinger M, Borlat F, Wells TN, Schlondorff D, Proudfoot AE. Aminooxypentane-RANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. J Exp Med. 1998;187(8):1215–24. doi: 10.1084/jem.187.8.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mackewicz CE, Ortega H, Levy JA. Effect of cytokines on HIV replication in CD4+ lymphocytes: lack of identity with the CD8+ cell antiviral factor. Cell Immunol. 1994;153(2):329–43. doi: 10.1006/cimm.1994.1032. [DOI] [PubMed] [Google Scholar]
  29. Maier R, Bartolome-Rodriguez MM, Moulon C, Weltzien HU, Meyerhans A. Kinetics of CXCR4 and CCR5 up-regulation and human immunodeficiency virus expansion after antigenic stimulation of primary CD4(+) T lymphocytes. Blood. 2000;96(5):1853–6. [PubMed] [Google Scholar]
  30. Malhotra U, Holte S, Zhu T, Delpit E, Huntsberry C, Sette A, Shankarappa R, Maenza J, Corey L, McElrath MJ. Early induction and maintenance of Env-specific T-helper cells following human immunodeficiency virus type 1 infection. J Virol. 2003;77(4):2663–74. doi: 10.1128/JVI.77.4.2663-2674.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Mattapallil JJ, Douek DC, Hill B, Nishimura Y, Martin M, Roederer M. Massive infection and loss of memory CD4+ T cells in multiple tissues during acute SIV infection. Nature. 2005;434(7037):1093–7. doi: 10.1038/nature03501. [DOI] [PubMed] [Google Scholar]
  32. Misko IS, Pope JH, Hutter R, Soszynski TD, Kane RG. HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies. Int J Cancer. 1984;33(2):239–43. doi: 10.1002/ijc.2910330212. [DOI] [PubMed] [Google Scholar]
  33. Norris PJ, Moffett HF, Yang OO, Kaufmann DE, Clark MJ, Addo MM, Rosenberg ES. Beyond help: direct effector functions of human immunodeficiency virus type 1-specific CD4(+) T cells. J Virol. 2004;78(16):8844–51. doi: 10.1128/JVI.78.16.8844-8851.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Norris PJ, Sumaroka M, Brander C, Moffett HF, Boswell SL, Nguyen T, Sykulev Y, Walker BD, Rosenberg ES. Multiple effector functions mediated by human immunodeficiency virus-specific CD4(+) T-cell clones. J Virol. 2001;75(20):9771–9. doi: 10.1128/JVI.75.20.9771-9779.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oliva A, Kinter AL, Vaccarezza M, Rubbert A, Catanzaro A, Moir S, Monaco J, Ehler L, Mizell S, Jackson R, Li Y, Romano JW, Fauci AS. Natural killer cells from human immunodeficiency virus (HIV)-infected individuals are an important source of CC-chemokines and suppress HIV-1 entry and replication in vitro. J Clin Invest. 1998;102(1):223–31. doi: 10.1172/JCI2323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Paludan C, Bickham K, Nikiforow S, Tsang ML, Goodman K, Hanekom WA, Fonteneau JF, Stevanovic S, Munz C. Epstein-Barr nuclear antigen 1-specific CD4(+) Th1 cells kill Burkitt's lymphoma cells. J Immunol. 2002;169(3):1593–603. doi: 10.4049/jimmunol.169.3.1593. [DOI] [PubMed] [Google Scholar]
  37. Robbins PA, Roderiquez GL, Peden KW, Norcross MA. Human immunodeficiency virus type 1 infection of antigen-specific CD4 cytotoxic T lymphocytes. AIDS Res Hum Retroviruses. 1998;14(16):1397–406. doi: 10.1089/aid.1998.14.1397. [DOI] [PubMed] [Google Scholar]
  38. Saha K, Bentsman G, Chess L, Volsky DJ. Endogenous production of beta-chemokines by CD4+, but not CD8+, T-cell clones correlates with the clinical state of human immunodeficiency virus type 1 (HIV-1)-infected individuals and may be responsible for blocking infection with non-syncytium-inducing HIV-1 in vitro. J Virol. 1998;72(1):876–81. doi: 10.1128/jvi.72.1.876-881.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Signoret N, Hewlett L, Wavre S, Pelchen-Matthews A, Oppermann M, Marsh M. Agonist-induced endocytosis of CC chemokine receptor 5 is clathrin dependent. Mol Biol Cell. 2005;16(2):902–17. doi: 10.1091/mbc.E04-08-0687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sozzani S. Dendritic cell trafficking: more than just chemokines. Cytokine Growth Factor Rev. 2005;16(6):581–92. doi: 10.1016/j.cytogfr.2005.04.008. [DOI] [PubMed] [Google Scholar]
  41. Staprans SI, Barry AP, Silvestri G, Safrit JT, Kozyr N, Sumpter B, Nguyen H, McClure H, Montefiori D, Cohen JI, Feinberg MB. Enhanced SIV replication and accelerated progression to AIDS in macaques primed to mount a CD4 T cell response to the SIV envelope protein. Proc Natl Acad Sci U S A. 2004;101(35):13026–31. doi: 10.1073/pnas.0404739101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sun L, Abdelwahab SF, Lewis GK, Garzino-Demo A. Recall antigen activation induces prompt release of CCR5 ligands from PBMC: implication in memory responses and immunization. Int Immunol. 2004;16(11):1623–31. doi: 10.1093/intimm/dxh164. [DOI] [PubMed] [Google Scholar]
  43. Sutherland R, Yang H, Scriba TJ, Ondondo B, Robinson N, Conlon C, Suttill A, McShane H, Fidler S, McMichael A, Dorrell L. Impaired IFN-gamma-secreting capacity in mycobacterial antigen-specific CD4 T cells during chronic HIV-1 infection despite long-term HAART. Aids. 2006;20(6):821–9. doi: 10.1097/01.aids.0000218545.31716.a4. [DOI] [PubMed] [Google Scholar]
  44. van Montfort T, Nabatov AA, Geijtenbeek TB, Pollakis G, Paxton WA. Efficient Capture of Antibody Neutralized HIV-1 by Cells Expressing DC-SIGN and Transfer to CD4+ T Lymphocytes. J Immunol. 2007;178(5):3177–85. doi: 10.4049/jimmunol.178.5.3177. [DOI] [PubMed] [Google Scholar]
  45. Verani A, Scarlatti G, Comar M, Tresoldi E, Polo S, Giacca M, Lusso P, Siccardi AG, Vercelli D. C-C chemokines released by lipopolysaccharide (LPS)-stimulated human macrophages suppress HIV-1 infection in both macrophages and T cells. J Exp Med. 1997;185(5):805–16. doi: 10.1084/jem.185.5.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Yue FY, Kovacs CM, Dimayuga RC, Gu XX, Parks P, Kaul R, Ostrowski MA. Preferential apoptosis of HIV-1-specific CD4+ T cells. J Immunol. 2005;174(4):2196–204. doi: 10.4049/jimmunol.174.4.2196. [DOI] [PubMed] [Google Scholar]
  47. Zaunders JJ, Munier ML, Kaufmann DE, Ip S, Grey P, Smith D, Ramacciotti T, Quan D, Finlayson R, Kaldor J, Rosenberg ES, Walker BD, Cooper DA, Kelleher AD. Early proliferation of CCR5(+) CD38(+++) antigen-specific CD4(+) Th1 effector cells during primary HIV-1 infection. Blood. 2005;106(5):1660–7. doi: 10.1182/blood-2005-01-0206. [DOI] [PubMed] [Google Scholar]

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