JAIDS Journal of Acquired Immune Deficiency Syndromes:
Interleukin-7 Receptor Expression on CD8 T-Cells Is Downregulated by the HIV Tat Protein
Faller, Elliott M MSc*; McVey, Mark J MSc*; Kakal, Juzer A BSc*; MacPherson, Paul A MD, PhD*†‡
Received for publication February 8, 2006; accepted May 22, 2006.
From the *Ottawa Health Research Institute, Ottawa, Ontario, Canada; †Division of Infectious Diseases, Ottawa Hospital General Campus, Ottawa, Ontario, Canada; and ‡Departments of Medicine, and Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada.
Supported by grants from the Canadian Institutes for Health Research-Pilot Project Grants for New Investigators, the Canadian Foundation for AIDS Research, and the J.P. Bickell Foundation for Medical Research.
Reprints: Paul MacPherson, MD, PhD, Division of Infectious Diseases, The Ottawa Hospital, General Campus, 501 Smyth Road, Ottawa, Ontario, Canada, K1H 8L6 (e-mail: email@example.com).
We have previously shown decreased expression of the interleukin (IL)-7 receptor α-chain (CD127) on CD8 T-cells in HIV-infected patients and an apparent recovery of this receptor in those receiving antiretroviral therapy with sustained viral suppression. Here, we demonstrate that the HIV Tat protein specifically downregulates cell surface expression of CD127 on human CD8 T-cells in a dose- and time-dependent manner. The effects of Tat on CD127 expression could be blocked with anti-Tat monoclonal antibodies or by preincubating Tat with heparin. Tat had no effect on the expression of other cell surface proteins examined, including CD132, or on cell viability over 72 hours. Further, CD127 expression was not altered by other HIV proteins, including gp160 or Nef. Preincubation of purified CD8 T-cells with Tat protein inhibited CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Because IL-7 signaling is essential for optimal CD8 T-cell proliferation and function, the downregulation of CD127 and apparent inhibition of cytotoxic activity by Tat may play an important role in HIV-induced immune dysregulation and impaired cell-mediated immunity.
It is well established that HIV infection results in a loss of CD8 T-cell activity. Impaired cell-mediated immunity is, in fact, the clinical hallmark of HIV infection. In vitro studies have confirmed functional deficits in cytotoxic T-cells isolated from HIV-positive patients, including reduced proliferation and impaired cytolytic activity in response to mitogens and alloantigens.1-4 More recently, it has been demonstrated that HIV- and Epstein-Barr virus (EBV)-specific CD8 T-cells can be found in the circulation at relatively normal frequencies in HIV-infected patients with advanced disease,5-9 yet these cells respond poorly to their respective antigens and fail to express perforin and interferon (IFN)-γ or to demonstrate cytolytic activity in standard chromium release assays.6,9-13
Interleukin (IL)-7 is essential for normal T-cell development and function. Signaling occurs via the IL-7 receptor (R), a heterodimer composed of a unique α-chain (CD127),14 and a common γ-chain (CD132)15 that is shared with the receptors for IL-2, IL-4, IL-9, IL-15, and IL-21.16,17 IL-7 promotes the survival and differentiation of immature T-cells within the thymus18,19 and is critical for immune homeostasis, both for the maintenance of naive T-cells20-24 and for the establishment of memory T-cells in mice.25-28 IL-7 also plays an important role in the activation and proliferation of cytotoxic CD8 T-cells. IL-7 has been shown to stimulate proliferation of CD8 T-cells in a time- and dose-dependent manner in humans and in murine models.29-32 Consistent with this, CD8 T-cells from IL-7R−/− mice proliferate poorly, with approximately half undergoing apoptosis.33,34 IL-7 has also been shown to upregulate telomerase activity in CD45RA+ T-cells isolated from human cord blood,32 an activity necessary for clonal expansion of activated T-cells. In addition to its effects on proliferation, IL-7 also enhances CD8 T-cell antiviral and antitumor cytolytic activity35-41 and upregulates perforin expression, a protein used by CD8 T-cells to lyse their targets.42 Perforin regulation is thought to occur via activation of the transcription factor STAT5.43 Thus, IL-7 plays an essential role in the activation of cell-mediated immunity by enhancing the proliferation and cytolytic potential of CD8 T-cells.
Given the importance of IL-7 in CD8 T-cell function, decreased IL-7 signaling could explain, in part, the impaired CD8 T-cell activity characteristic of progressive HIV disease. In a cross-sectional study comparing CD127, we, in fact, reported a 67% reduction in the proportion of CD8 T-cells expressing the IL-7R α-chain in HIV-positive patients with uncontrolled viral replication (mean CD4 cell count = 232 cells/μL) compared with healthy HIV-seronegative controls.44 When HIV-positive patients on antiretroviral therapy with sustained viral suppression were examined, CD127 expression approached normal levels to 73% of that seen in controls. The duration of viral suppression was the only parameter that correlated with the apparent recovery of CD127 expression in effectively treated patients. Several other groups have since reported similar findings.45-49 Vingerhoets et al50 also observed lower CD127 expression on CD8 T-cells from HIV-infected individuals and found that these cells were less able to form blasts and upregulate CD25 in response to IL-7 compared with controls. Consistent with this, Ferrari et al51 demonstrated that anti-HIV cytotoxic T lymphocytes (CTLs) from patients with advanced disease could not be expanded in vitro after stimulation with HIV antigens and IL-7. Thus, it seems that HIV infection is associated with downregulation of the IL-7R, which may, in turn, lead to impaired CD8 T-cell activity.
It has recently been demonstrated that IL-7 downregulates expression of its own receptor, including surface protein and mRNA transcripts.52 Because HIV-infected individuals have increased plasma concentrations of IL-7 compared with uninfected controls,53,54 it is possible that elevated plasma IL-7 causes reduced expression of CD127 on CD8 T-cells in HIV-positive patients. We suspect, however, that the IL-7 concentrations found in HIV-positive patients are alone insufficient to induce CD127 downregulation in vivo. First, IL-7 concentrations in the range of 500 to 10,000 pg/mL are required in vitro to downregulate CD127 on purified CD8 T-cells isolated from healthy volunteers49,52 (our unpublished observations). In HIV-seronegative individuals, plasma IL-7 concentrations average 2.2 pg/mL and increase in HIV-infected individuals to an average of 18 pg/mL, with a reported maximum of 55 pg/mL.20,48,54,55 It is thus questionable whether the concentrations of IL-7 required to downregulate CD127 in vitro can be achieved in vivo. Similar results have been noted in simian immunodeficiency virus (SIV)-infected rhesus macaques,23 in which subcutaneous administration of IL-7 led to a significant decline in CD127 expression on CD8 T-cells. Here, again, supraphysiologic concentrations of IL-7 exceeding 1000 pg/mL in plasma were achieved, although lower concentrations were not tested. These findings are consistent with 2 recent studies45,47 demonstrating no correlation between plasma IL-7 levels (maximum levels: 40 and 60 pg/mL) and expression of CD127 on CD8 T-cells in HIV-positive individuals. In addition, Mussini et al56 noted that patients on antiretroviral therapy who experience a rapid increase in CD4 T-cells have plasma IL-7 levels some 10-fold higher than controls yet express CD127 on CD8 T-cells at near-normal levels. Perhaps most convincing is that although T-cells isolated from HIV-negative individuals downregulate CD127 in response to high concentrations of IL-7 and recover the receptor once IL-7 is removed from the medium, T-cells isolated from HIV-positive patients remain low or negative for CD127 when cultured in the absence of IL-7.48 Although direct comparisons between in vitro studies using recombinant protein and in vivo observations can be difficult, it seems that IL-7 alone at concentrations typically found in HIV-positive patients is unlikely to explain the downregulation of CD127 on CD8 T-cells in these individuals.
Several HIV gene products have been shown to alter the expression of cellular proteins. The HIV Tat protein, a 14-kd polypeptide produced from multiply spliced viral transcripts, is a well-known activator of viral gene transcription. Tat binds to a secondary stem-loop structure called the transactivation response region (TAR) located at the 5′ end of all viral RNA transcripts and both alters histone acetylation around the viral long terminal repeat (LTR) and directly enhances the processivity of RNA polymerase II.57,58 In this way, Tat causes an increased accumulation of full-length viral transcripts. Depending on cell type, Tat has also been shown to alter the expression of a number of cellular genes. Tat upregulates the expression of IL-2, IL-8, and IL-10 in T-lymphoblastic cell lines,59-62 IL-6 in HeLa and B-lymphoblastoid cells,63,64 and tumor necrosis factor-α (TNFα) in monocytes.65 Tat also upregulates the IL-4R on Raji cells, a B-lymphoblastoid cell line.66 Although Tat has been shown to suppress IL-2R α-chain (CD25) expression on the H9 T-lymphoid cell line,67 it seems to have no effect on the expression of this receptor on primary T-cells isolated from healthy donors.68 Finally, Tat also downregulates major histocompatibility complex (MHC) class I gene expression on HeLa cells.69,70 Tat seems to mediate these pleiotropic effects by interacting with cellular acetyltransferases. The reversible acetylation of lysine residues on histones and transcription factors, regulated by a dynamic equilibrium between acetyltransferases and deacetylases, governs the assembly and activity of transcription complexes bound to cellular gene promoters.71-73 Tat, itself acetylated,74-76 binds to a number of lysine acetyltransferases (LATs), including p300/CBP, Tip60, TAFII250, and p300/CBP associated factor (PCAF), and enhances or inhibits LAT enzymatic activity.72 As a result, Tat is able to influence the acetylation of transcription factors and histones and thus upregulate or repress the expression of cellular genes. Tat has, in fact, been shown to alter the activity of nuclear factor-κB (NF-κB) and activator protein (AP)-1 in T-cells.60,77-82
Interestingly, Tat seems to function in an autocrine and/or paracrine fashion. Full-length Tat protein is secreted by HIV-infected cells and can be found in culture media during peak infection.83,84 Secreted Tat is also rapidly internalized by a variety of cell types in culture, including lymphocytes,84-86 and it is now well established that the arginine-rich basic domain of Tat (amino acids 48-60) is responsible for uptake across the cell membrane.87,88 Specifically, Tat binds via its basic domain to heparan sulfate glycosaminoglycans on the cell surface and is then internalized by caveolar endocytosis.86,89-91 Preincubating Tat protein with heparin blocks Tat binding to the cell membrane and entry into the cytoplasm.89-91 Several studies have demonstrated that Tat is taken up by cells within 90 minutes of being added to the medium and colocalizes with caveolin-1-containing cytoplasmic vesicles.91 Within 4 hours, Tat translocates to the nucleus where it upregulates transcription from the HIV LTR.84,87-89,91 Several authors have suggested that secreted Tat may act in a paracrine fashion to enhance HIV replication in unstimulated latently infected CD4+ cells.86
In view of the established paracrine activity of HIV Tat protein and its effects on cytokine and cytokine receptor expression, we hypothesized that this viral factor may also affect expression of the IL-7R α-chain. We demonstrate here that, indeed, soluble HIV Tat protein specifically downregulates surface expression of CD127 on purified CD8 T-cells and that this downregulation results in impaired CD8 T-cell proliferation and perforin synthesis after stimulation with IL-7. Decreased CD127 expression on CD8 T-cells by Tat may then explain, at least in part, the impaired cell-mediated immunity and ineffective immunologic control of viral replication evident in HIV-positive patients with progressive disease.
Purified HIV-1 Tat protein (86 amino acids) was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH, Bethesda, MD), or was purchased from Advanced Bioscience Laboratories, Inc. (Kensington, MD). Protein was received lyophilized and was resuspended to 1 mg/mL in phosphate-buffered saline (PBS) containing 1 mg/mL of bovine serum albumin (BSA) and 0.1 mM of dithiothreitol. Tat protein is reportedly >95% pure by heparin affinity chromatography and reverse phase high-performance liquid chromatography (HPLC). Purified HIV-1 gp160 and Nef protein were obtained from Immunodiagnostics, Inc. (Woburn, MA). Whole-killed Candida was from Greer Laboratories, Inc. (Lenoir, NC). The following fluorochrome-labeled monoclonal antibodies were purchased from Immunotech Beckman Coulter (Marseille, France): anti-CD2-phycoerythrin (PE) (39C1.5), anti-CD3-fluorescein isothiocyanate (FITC) (UCHT1), anti-CD8-PC5 (B9.11), anti-CD16-PE (3G8), anti-CD25-FITC (B1.49.9), anti-CD28-phycoerythrin-Texas Red (ECD) (CD28.2), anti-CD38-PE (T16), anti-CD45RA-ECD (2H4), anti-CD45RA-FITC (ALB11), anti-CD45RO-FITC (UCHL1), anti-CD56-PC5 (NKH-1), anti-CD62L-ECD (DREG56), anti-CD127-PE (R34.34), anti-human leukocyte antigen-D-related (HLA-DR)-PE (Immu-357), and antiperforin-FITC (δG9). Anti-CD132-PE (AG184) plus anti-CD3 (HIT3a) and anti-CD28 (CD28.2) monoclonal antibodies were purchased from BD Biosciences: Pharmingen: (Mississauga, ON, Canada). All fluorochrome-labeled antibodies were titrated and used at saturating concentrations. Anti-Tat monoclonal antibody (IgG1) and IgG1 isotype control (107.3) were purchased from Immunodiagnostics, Inc., and BD Biosciences: Pharmingen, respectively. IL-7 was obtained from R&D Systems (Minneapolis, MN), resuspended in PBS, and stored at −20°C. The Annexin V staining kit and propidium iodide were purchased from BD Biosciences: Pharmingen. Heparin was from Organon (Toronto, Ontario, Canada), and lipopolysaccharide (LPS) was from Sigma-Aldrich (Oakville, ON, Canada). 3H-thymidine was purchased from Amersham Biosciences (Baie d’Urfe, QC, Canada).
Cell Purification and Culture
Blood from healthy HIV-seronegative donors was drawn into tubes containing sodium heparin, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density centrifugation. Cells were washed with PBS and resuspended at 1 × 106 cells/mL. CD8 T-cells were then purified from PBMCs using the MACS Microbead CD8+ Cell AutoMACS Isolation System (Miltenyi Biotec, Auburn, CA) according to the manufacturer's directions. Cell purity was consistently >95% CD8+ by flow cytometric analysis, with only 2.6% ± 0.99% (mean ± SEM) CD8+, CD3−, CD16+, and CD56+ natural killer (NK) cells. Our cultures were thus composed of >95% purified CD8 T-cells.
After isolation, purified CD8 T-cells were generally allowed to recover overnight at 1 × 106 cells/mL in media composed of RPMI 1640 (Hyclone, Logan, UT) supplemented with 20% fetal calf serum (FCS; Cansera, Rexdale, Ontario, Canada) plus penicillin and streptomycin (RPMI-20). After overnight culture, CD8 T-cells were incubated in media alone (RPMI-20), with purified HIV Tat (10 μg/mL unless otherwise specified), or with other proteins or reagents as indicated. All cultures were maintained in a humidified incubator at 37°C in the presence of 5% CO2.
This work was reviewed and approved by the Ottawa Health Research Institute Research Ethics Board.
At the times indicated, cells were incubated with the appropriate fluorochrome-labeled antibodies for 30 minutes in the dark at room temperature and then analyzed by flow cytometry using a Coulter Epics ALTRA flow cytometer (Fullerton, CA). The IL-7R α-chain was detected using anti-CD127-PE (R34.34) from Immunotech Beckman Coulter. Live cells were gated on the basis of side and forward scatter. At least 10,000 events were recorded for each sample. Isotype controls were performed for each fluorochrome-conjugated antibody. Resulting profiles were analyzed with the EXPO version 2.0 software package.
Cell Viability Assays
Purified CD8 T-cells were incubated in media alone or with Tat protein (10 μg/mL) in a humidified incubator at 37°C. At 24 and 72 hours, cells were stained with Annexin V-FITC and propidium iodide according to the manufacturer's directions using the Apoptosis Detection Kit I from BD Biosciences: Pharmingen. As a positive control for apoptosis, purified CD8 T-cells were incubated in parallel with camptothecin (Sigma-Aldrich) at a final concentration of 10 μM.
Intracellular Perforin Expression
Purified CD8 T-cells were preincubated in RPMI-20 at 1 × 106 cells/mL for 72 hours in media alone or with purified Tat protein (10 μg/mL). Cells were then stimulated with IL-7 (20 ng/mL). At regular 24-hour intervals, cells were washed with PBS and then fixed and permeablized using the Fix and Perm Cell permeablization kit from Caltag Laboratories (Burlingame, CA). Cells were then incubated with anti-CD8-PC5 and antiperforin-FITC antibodies at room temperature for 30 minutes in the dark. Samples were analyzed by flow cytometry as described above.
Purified CD8 T-cells were preincubated in RPMI-20 at 1 × 106 cells/mL for 72 hours in media alone or with purified Tat protein (10 μg/mL). The cells were then transferred to a 96-well tissue culture plate at 5 × 105 cells/mL and stimulated in triplicate with anti-CD3 plus anti-CD28 monoclonal antibodies (3 μg/mL and 2 μg/mL, respectively) with or without IL-7 (5 ng/mL). After an additional 48 hours of incubation, cultures were pulsed with 1 μCi of 3H-thymidine for 18 hours. Cells were then harvested onto Filtermat paper (Perkin Elmer, Wellesley, MA), and β-radioactivity was measured using a 96-well liquid scintillation counter.
The Interleukin-7 Receptor Is Downregulated on CD8 T-Cells by Soluble HIV Tat Protein
We previously demonstrated decreased expression of CD127 on circulating CD8 T-cells in patients with active HIV replication.44 Because the HIV Tat protein has been shown to influence the expression of a number of cellular proteins in a paracrine fashion, we hypothesized that Tat might downregulate CD127 expression on CD8 T-cells. To examine this directly, purified CD8 T-cells from healthy donors were incubated in RPMI-20 with or without purified Tat protein (10 μg/mL) and analyzed by flow cytometry at 24-hour intervals (n = 15; Figs. 1A-C). While the level of CD127 expression on CD8 T-cells cultured in media alone varied little over time, purified Tat protein induced a 35% ± 3.3% reduction in the number of CD8 T-cells expressing CD127 over 72 hours compared with controls. Further, among those cells expressing CD127 at 72 hours, there was a 38% ± 2.9% decline in the mean channel fluorescence, indicating a reduction in the total number of receptors present on the cell surface (see Figs. 1B, D). These effects were time dependent, with the greatest decrease in CD127 expression occurring within the first 24 hours after exposure to Tat. The effects of Tat were also dose dependent. Increasing concentrations of purified Tat protein (1, 2.5, 5, and 10 μg/mL) induced incremental reductions in CD127 expression (Fig. 1E).
Downregulation of CD127 on CD8 T-Cells Is Mediated Specifically by the HIV Tat Protein
To determine if the reduction in CD127 expression on CD8 T-cells was mediated by HIV Tat protein and not a contaminant in the preparation, 10 μg of purified Tat was pretreated for 3 hours at 37°C with proteinase K, which, in turn, was inactivated at 95°C for 1 hour. Purified CD8 T-cells from healthy donors (n = 4) were then incubated with native or proteinase K-treated Tat protein. As shown in Figure 2A, prior treatment of Tat with proteinase K abolished its ability to downregulate CD127.
Although Tat is purified by HPLC and reported to be >95% pure, it is expressed in Escherichia coli; as a result, the preparation could contain LPS. Notably, CD8 T-cells lack CD14 and Toll-like receptor-4, and thus should not respond to LPS. However, Kaya et al92 demonstrated LPS-induced activation of murine CD4 and CD8 T-cells via the complement receptor types 1 and 2 (CR1 and CR2) present on both T-cell subsets. To rule out any effects of LPS definitively, we incubated purified CD8 T-cells with 1 and 5 μg/mL of LPS and followed CD127 expression by flow cytometry. As expected, LPS had no effect on CD127 expression (Fig. 2A).
To determine if the effects on CD127 expression were mediated specifically by HIV Tat, purified Tat protein was preincubated for 30 minutes with an equimolar concentration of anti-Tat IgG1 monoclonal antibody or with heparin (3.5 μg/mL). Heparin binds directly to Tat protein and prevents its uptake by cells in culture,89,91 and it has been used in several studies to block the effects of extracellular Tat, including Tat-induced transactivation of the HIV LTR.90 Neither heparin alone nor isotype control IgG1 antibody had any effect on CD127 expression when incubated with CD8 T-cells over 72 hours (101% and 102% CD127 expression, respectively) compared with media alone. As expected, purified Tat protein and Tat preincubated with nonspecific isotype control IgG1 antibody induced typical 36% and 35% reductions in CD127 expression, respectively, on CD8 T-cells after 72 hours. In contrast, preincubation of Tat with anti-Tat monoclonal IgG1 or heparin completely abrogated the effects of Tat on CD127 expression. After 72 hours of incubation, CD127 expression on CD8 T-cells was 98% in the presence of Tat plus anti-Tat monoclonal antibody and 102% in the presence of Tat plus heparin relative to the media control (Table 1). The ability of the anti-Tat monoclonal antibody and heparin to block the downregulation of CD127 on CD8 T-cells clearly indicates that the effect is mediated by Tat protein.
Downregulation of CD127 on CD8 T-Cells Is Not Mediated by Other HIV or Nonviral Proteins
Although CD127 is clearly downregulated by the HIV Tat protein, we considered the possibility that this is a nonspecific effect mediated by exposure of purified CD8 T-cells to foreign protein. To address this possibility, CD8 T-cells were isolated from healthy donors (n = 3) and incubated in RPMI-20 with 10 μg/mL of purified HIV gp160, HIV Nef protein, or whole-killed Candida. Among these proteins, Tat alone induced downregulation of CD127 on CD8 T-cells (Fig. 2B).
Downregulation of CD127 on CD8 T-Cells by Tat Is Not the Result of Cell Death or Apoptosis
There are conflicting reports of exogenous Tat protein inducing apoptosis in cultured T-cells.93-96 To ensure that the downregulation of CD127 was, in fact, occurring on viable cells, purified CD8 T-cells isolated from healthy donors (n = 4) were incubated with or without Tat (10 μg/mL) for up to 72 hours. In the presence and absence of Tat, the viability of gated cells was 100%, as indicated by propidium iodide exclusion (Fig. 3A). Forward and side scatter profiles of the entire cell population did not change significantly over 72 hours of incubation, and the viability of the entire population was maintained at >90%, irrespective of the presence of Tat. Apoptotic cells, as identified by annexin V staining, averaged 10.3% of gated cells, which, again, was not different between CD8 T-cells exposed to Tat and media controls (Fig. 3B). In fact, cells incubated with Tat protein and analyzed within the lymphocyte gate consistently showed lower annexin V staining, although this difference never reached statistical significance. Because the greatest decrease in CD127 expression on purified CD8 T-cells occurred within the first 24 hours of exposure to Tat and because these cells remained viable for at least 72 hours in culture, we conclude that the changes in CD127 expression were not the result of cell death or apoptosis.
HIV Tat Protein Specifically Downregulates CD127
Because the HIV Tat protein is internalized by caveolar-mediated endocytosis, we next questioned whether exposure to Tat induces a generalized and nonspecific reduction in cell surface proteins on CD8 T-cells. To investigate this possibility, purified CD8 T-cells isolated from healthy donors (n = 4 to n = 8) were incubated with 10 μg/mL of Tat protein, and expression of several cell surface proteins was followed over 72 hours by flow cytometry. As shown in Figure 4A, Tat induced downregulation of CD127 on CD8 T-cells but had no effect on the expression of CD2, CD3, CD8, CD25, CD28, CD45RA, CD45RO, CD56, or CD62L. Expression of these cell surface proteins was unaffected in terms of the number of cells positive and in the extent of surface expression, as indicated by mean channel fluorescence. It is notable that Tat did not affect the surface expression of CD132, the common γ-chain that associates with CD127 to form the heterodimeric IL-7R (Fig. 4B). Given that the expression of CD25 and CD132 was unaffected, it is unlikely that the effects of Tat on CD8 T-cells can be generalized to the family of common γ-chain cytokine receptors. These data then indicate that the HIV Tat protein specifically targets CD127 on CD8 T-cells.
CD127 Is Downregulated by the HIV Tat Protein on Naive and Memory CD8 T-Cells
Among T-cells, CD127 expression is primarily limited to the naive and memory subsets.28,97 We then questioned whether Tat downregulated CD127 on either or both of these subpopulations. CD8 T-cells were purified from healthy donors (n = 4), incubated with 10 μg/mL of Tat protein, and then analyzed for CD127 expression on naive (CD45RA+CD62L+) and memory (CD45RO+) cells. Both subsets were affected equally and mirrored the total CD8 T-cell population in terms of degree and rate of CD127 downregulation (Fig. 5).
Downregulation of CD127 by Tat Is Not the Result of Nonspecific CD8 T-Cell Stimulation
CD8 T-cell stimulation induces a number of phenotypic changes, including upregulation of CD25, HLA-DR, and perforin as well as downregulation of CD127.28 We questioned therefore whether exogenous Tat could induce nonspecific activation of purified CD8 T-cells resulting in the downregulation of CD127. To investigate this possibility, CD8 T-cells from healthy HIV-seronegative donors were incubated in RPMI-20 with purified Tat protein (10 μg/mL) or with anti-CD3 and anti-CD28 monoclonal antibodies. Cells stimulated through CD3 and CD28 ligation and Tat-treated cells both downregulated CD127 on the cell surface as anticipated, although the decrease in expression was greater with anti-CD3 and anti-CD28 compared with Tat alone (25% ± 4% vs. 51% ± 2% CD127+, respectively, at 24 hours; Fig. 6A). As expected, stimulation of CD8 T-cells with anti-CD3 plus anti-CD28 monoclonal antibodies caused an increase in CD25 expression (1.7-fold) within 24 hours. CD8 T-cells incubated with purified Tat showed no change in CD25 over 72 hours (Fig. 6B). Surface expression of HLA-DR and CD38 also appropriately increased after stimulation with anti-CD3 and anti-CD28 monoclonal antibodies but did not change at all after exposure to purified Tat protein (data not shown). This is in agreement with the data presented in Figure 4A showing no change in the expression of other cell surface proteins in response to Tat, including CD28 and CD62L, which are normally downregulated after CD8 T-cell stimulation, and CD56, which is normally upregulated after CD8 T-cell stimulation. Consistent with cell phenotype, exogenous Tat protein also did not stimulate CD8 T-cell proliferation. In the presence of Tat, incorporation of 3H-thymidine remained at background levels, whereas cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies showed marked proliferation (Table 2).
Once stimulated, CD8 T-cells mature into cytolytic effector cells and upregulate perforin. Smyth et al42 demonstrated several years ago that IL-7 causes an increased accumulation of perforin gene transcripts in human CD8 T-cells. Similarly, we have found that IL-7 induces an increase in intracellular perforin expression in CD8 T-cells and that accumulation of perforin is time dependent. Exposure to Tat protein, however, did not induce perforin synthesis in CD8 T-cells (Fig. 6C). Given the stable phenotype, absence of perforin induction, and lack of proliferation, we conclude that the decrease in CD127 expression induced by exogenous purified Tat protein is not attributable to nonspecific stimulation of CD8 T-cells.
Effect of Tat on CD127 Expression Is Reversible
We questioned whether CD127 is irreversibly downregulated on CD8 T-cells by Tat or whether Tat is continuously required to maintain suppression. To investigate this, purified CD8 T-cells were cultured with Tat protein, and at 24-hour intervals, aliquots were removed, centrifuged at 1600 rpm, and resuspended in fresh RPMI-20. As shown in Figure 7, once Tat was removed from the culture media, CD127 surface expression recovered back to baseline within 24 hours and remained stable thereafter. Full recovery of CD127 was evident even on cells incubated with Tat for up to 72 hours. This suggests that Tat does not irreversibly affect CD8 T-cells but, instead, is continually required to maintain suppression of CD127. Of note, as can be seen in Figure 7, cells cultured in the presence of Tat for up to 96 hours tended to show a slight rebound in CD127 expression. We attribute this to depletion of soluble Tat in our cultures. Indeed, if supplemental Tat protein was added at 48 or 72 hours, this rebound was not evident (data not shown).
The Concentration of Tat Required to Downregulate CD127 Is Dependent on Cell Concentration
Relatively high concentrations of Tat protein (up to 10 μg/mL) were required to demonstrate significant downregulation of CD127 expression on CD8 T-cells. This is likely attributable to the lack of posttranslational modification of Tat produced in E. coli and to the relative biologic activity of a purified protein preparation. Because Tat is taken up by individual cells in culture and so removed from the medium, one would also predict a stoichiometric relation between the cell concentration and the concentration of Tat protein required in vitro to induce a decline in CD127. To investigate this possibility, CD8 T-cells were isolated from healthy donors and incubated in RPMI-20 at 1.0, 0.5, and 0.25 million cells/mL in the presence of decreasing concentrations of Tat protein. As predicted, less Tat was required to induce the same degree of CD127 downregulation when fewer cells were present in the culture (Fig. 8). Indeed, at 72 hours, 2.5 μg/mL of Tat downregulated CD127 on CD8 T-cells by 11.5% ± 4.5% in cultures of 1.0 million cells/mL, by 24.8% ± 4.0% in cultures of 0.5 million cells/mL, and by 39.2% ± 2.5% in cultures of 0.25 million cells/mL. Thus, the concentration of Tat required to induce significant downregulation of CD127 on CD8 T-cells is determined, at least in part, by the concentration of cells present in the culture.
The Presence of Exogenous Tat Protein Impairs CD8 T-Cell Response to Stimulation
IL-7 plays an important role in the activation of cytotoxic CD8 T-cells. Signaling via the IL-7R stimulates proliferation of CD8 T-cells and induces upregulation of perforin. Because the HIV Tat protein downregulates CD127 on the surface of these cells, we questioned whether this effect was functionally significant. To examine this, purified CD8 T-cells (1.0 × 106 cells/mL) were preincubated in medium alone or with Tat (10 μg/mL) for 72 hours and then stimulated with anti-CD3 and anti-CD28 monoclonal antibodies ± IL-7 (5 ng/mL). As shown in Figure 9A, addition of IL-7 enhances CD8 T-cell proliferation, with a proliferation index more than 2-fold greater than stimulation with anti-CD3 and anti-CD28 monoclonal antibodies alone. Preincubation with Tat, however, completely blocked the effects of IL-7. When stimulated with anti-CD3 and anti-CD28 monoclonal antibodies plus IL-7 (5 ng/mL), CD8 T-cells pretreated with Tat proliferated to the same extent as untreated cells stimulated with only anti-CD3 and anti-CD28 antibodies. It would seem that by downregulating CD127, Tat is able to block stimulation with IL-7 and to impair CD8 T-cell proliferation. Tat also inhibited CD8 T-cell proliferation to some extent in the absence of IL-7, suggesting that additional pathways may be affected by this protein (data not shown). These additional effects may explain why the inhibition in cell proliferation was more complete than may have been anticipated, given the fact that CD127 is not fully downregulated on all Tat-treated cells in our cultures.
We also examined CD8 T-cells for their ability to synthesize perforin in the presence of exogenous Tat protein. While IL-7 (20 ng/mL) alone upregulates accumulation of intracellular perforin in CD8 T-cells, preincubation with Tat completely blocked this effect. As predicted, CD8 T-cells pretreated with Tat showed no accumulation of perforin after addition of IL-7 to the culture (Fig. 9B). These data suggest that by downregulating CD127, Tat inhibits IL-7-induced accumulation of perforin in CD8 T-cells, and thus likely impairs cytolytic activity.
Impaired CD8 T-cell proliferation and function are well described during HIV infection. Although declining CD4 T-cell help likely plays a role in reduced cell-mediated immunity, direct effects on CD8 T-cells are also evident. We previously demonstrated reduced expression of the IL-7R α-chain on CD8 T-cells from HIV-infected patients with active viral replication.44 Here, we show that the HIV Tat protein is likely responsible, at least in part, for this downregulation and that this downregulation results in impaired CD8 T-cell function.
Because Tat protein is secreted from infected cells during active HIV replication83,84 and can alter gene expression in uninfected cells60,77-79,81 in a paracrine fashion,86,91 we questioned whether this protein exerted an effect on the expression of the IL-7R α-chain on CD8 T-cells. Because infection of CD8 T-cells by HIV is likely a rare event, it seemed more biologically relevant to examine the effects of extracellular Tat on these cells. As predicted, purified Tat protein induced a significant decline in the expression of CD127 on CD8 T-cells isolated from healthy donors compared with cells maintained in medium alone. The effect was dose and time dependent and required the continual presence of Tat. Removal of Tat protein after up to 72 hours of incubation allowed full recovery of CD127 over the ensuing 24 hours. Interestingly, Tat downregulated CD127 expression equally on naive (CD45RA+CD62L+) and memory (CD45RO+) CD8 T-cells, mirroring the bulk CD8 T-cell population in kinetics and extent of decline. Prior treatment with proteinase K and anti-Tat monoclonal antibodies and preincubation with heparin abolished the ability of Tat to downregulate CD127. Further, purified HIV gp160, HIV Nef, and whole-killed Candida had no effect on the expression of CD127. Taken together, these data demonstrate that exogenous HIV Tat protein downregulates the IL-7R α-chain on CD8 T-cells.
In view of the important role that IL-7 plays in CD8 T-cell function, we questioned whether the reduction in CD127 expression induced by Tat was functionally significant. As expected, IL-7 significantly enhanced CD8 T-cell proliferation over stimulation with anti-CD3 plus anti-CD28 monoclonal antibodies. Preincubation with Tat, however, completely eliminated the effects of IL-7 and reduced proliferation back to levels similar to stimulation with anti-CD3 plus anti-CD28 antibodies alone. The inhibition of proliferation was more complete than anticipated, suggesting that Tat may have additional effects on cell cycle regulation. IL-7 also plays an important role in upregulating CD8 T-cell cytolytic activity, specifically by increasing expression of perforin. Here, again, preincubating CD8 T-cells with Tat inhibited accumulation of intracellular perforin in response to IL-7. Thus, by downregulating CD127 expression on CD8 T-cells, HIV Tat is able to block the stimulatory effects of IL-7 and to impair CD8 T-cell proliferation and cytolytic capacity.
Interestingly, this is not the first time that Tat has been shown to inhibit T-cell proliferation. Years ago, Viscidi et al98 demonstrated that purified Tat protein inhibited the proliferation of PBMCs from healthy donors after stimulation with tetanus toxoid. The inhibitory effect was concentration dependent, with 10 μg/mL of Tat inhibiting proliferation by 81%. Similarly, Chirmule et al68 later demonstrated that purified Tat protein (1-3 μg/mL) inhibited proliferation of CD8 T-cells cultured in the presence of autologous irradiated non-T-cells and stimulated with anti-CD3 monoclonal antibodies. As in our study, Tat did not affect cell viability or the surface expression of CD25. Interestingly, these authors also showed that Tat did not disrupt intracellular signaling after stimulation of the T-cell receptor (TCR), indicating that the inhibitory effects of Tat were mediated through a different pathway. We suggest that the inhibition observed in these studies was the result of Tat-induced downregulation of CD127 and the loss of IL-7 signaling in these mixed cell cultures.
While Tat downregulated CD127 on the surface of healthy CD8 T-cells, we found no effect on the overall phenotype of these cells or on cell viability. Several authors have suggested that during chronic viral infection, persistent antigen leads to continuous CD8 T-cell activation resulting in decreased CD127 expression and cellular exhaustion. Indeed, in mice previously infected with a weakly replicating strain of lymphocytic choriomeningitis virus (LCMV), persistent gp33 antigen caused a decrease in CD127 expression on LCMV gp33-specific CD8 T-cells.99 Consistent with immune activation, these cells exhibited an effector phenotype with increased granzyme B expression, increased annexin V staining, and a gradual decline in number. In contrast, Tat does not seem to induce CD127 downregulation by activating CD8 T-cells. Exposure to Tat did not result in upregulation of the activation markers CD25, CD38, or HLA-DR, and it did not stimulate cell proliferation or the accumulation of perforin. In fact, no changes in the expression of a number of cell surface markers were detected, suggesting a stable CD8 T-cell phenotype in the presence of purified Tat protein. Thus, Tat does not seem to induce downregulation of CD127 through nonspecific stimulation of CD8 T-cells.
The mechanism by which Tat is able to downregulate CD127 on CD8 T-cells is currently under investigation in our laboratory. We have recently detected a 4-fold decrease in CD127 mRNA transcripts in CD8 T-cells that shift from CD127hi to CD127lo after exposure to Tat (manuscript in preparation). This is consistent with 2 reports in the literature demonstrating lower levels of CD127 mRNA in T-cells isolated from HIV-infected patients compared with cells from healthy controls.45,48 Downregulation of CD127 gene transcription by Tat is also consistent with Tat's known effects on transcription initiation at various cellular gene promoters.
In our experiments, Tat was required in nanomolar concentrations (2.5-10 μg/mL) to downregulate CD127 on CD8 T-cells. Although this concentration is similar to that used in most in vitro studies,68,83,98 it is approximately 10-fold higher than estimates of Tat concentration in patient sera (300-500 ng/mL).100 There are a number of reasons why more protein may be required in vitro. First, we have demonstrated here that the concentration of Tat required to downregulate CD127 is proportional to the concentration of cells in the culture. Second, the Tat protein used in our experiments is produced in bacteria, and thus is unlikely to be posttranslationally modified. Tat is known to associate with the eukaryotic acetyltransferases p300, PCAF, and hGCN5,75,76,101 and acetylation of Tat at Lys28 and Lys50 independently enhances Tat activity in transcriptional assays.75,76,101 It may be that lower concentrations of the more active acetylated form of Tat are required to downregulate CD127 on CD8 T-cells in vitro. This is currently being investigated in our laboratory. Finally, other factors in addition to Tat may be involved in the downregulation of CD127 in vivo. IL-2, IL-4, IL-6, IL-7, and IL-15 all downregulate CD127 on CD8 T-cells isolated from mice.52 We have recently found that Tat and IL-7, when added at suboptimal concentrations in which each alone has only a small effect on CD127 expression, cause a much greater decrease in CD127 on the surface of CD8 T-cells when added in combination (manuscript in preparation). Thus, it may be that Tat amplifies normal feedback mechanisms regulating IL-7R expression. Lower concentrations of Tat then may be required in vivo, where Tat is appropriately posttranslationally modified and is not studied in isolation.
Although CD127 expression is reduced on most CD8 T-cells in untreated HIV-infected individuals,44 we have consistently found that only 35% to 45% of CD8 T-cells from healthy donors significantly downregulate CD127 on exposure to purified Tat in vitro. There are a number of possible explanations for this observation. First, the half-life of Tat protein in solution is estimated to be approximately 24 hours.83 In our in vitro experiments, it is possible that Tat becomes limiting. We doubt that this is the case, however, because the addition of supplemental Tat to our cultures at 48 and 72 hours did not result in further suppression of CD127 expression (data not shown). It is also possible that specific subpopulations of CD8 T-cells are unaffected by Tat. These cells may be depleted in HIV-infected patients yet comprise a significant proportion of the total population in healthy individuals and remain CD127+ after exposure to Tat in culture. Although we have not examined all CD8 T-cell phenotypes, downregulation of CD127 on naive and memory cells mirrors the total CD8 T-cell population, with neither subset demonstrating a more limited effect. This possibility requires further investigation. A third possibility is that after the initial rapid decrease in CD127 on CD8 T-cells, Tat continues to induce a slow decline in CD127 expression beyond 72 hours. Certainly CD8 T-cells isolated from HIV-infected individuals have been exposed to Tat for a considerably longer period. Perhaps the most likely explanation is the synergistic effect demonstrated between Tat and IL-7 (unpublished data). Similar to what we have seen in vitro, the combined effects of Tat and IL-7 in HIV-infected patients may have a greater effect in reducing CD127 expression than either alone.
Several authors have suggested that the generalized decrease in CD127 expression on CD8 T-cells in HIV-infected patients is the result of chronic immune activation. This model is unsatisfying in several aspects. Consistent with our previously published data, several groups have now reported a significant decrease in CD127 expression on CD8 T-cells isolated from untreated HIV-infected individuals.45-48 Notably, all groups have documented reduced CD127 on the entire CD8 T-cell population. It remains unclear, however, how persistent HIV antigens can cause chronic stimulation of non-HIV-specific CD8 T-cells. Indeed, Paiardini et al45 have shown that in untreated HIV-positive patients, CD127 is downregulated not only on HIV-specific CD8 T-cells but on cytomegalovirus (CMV)- and EBV-specific CD8 T-cells compared with the respective cell populations in healthy HIV-seronegative controls. In this study, it is significant that the average CD4+ cell count within the untreated HIV-positive patient group was 367 cells/μL (range: 88-765 cells/μL). Because CMV reactivation rarely if ever occurs at CD4+ cell counts >50 cells/μL, it cannot be argued that the downregulation of CD127 on CMV-specific CD8 T-cells was the result of chronic stimulation with CMV antigen. Further, Boutboul et al46 have reported that most HIV-, CMV-, and EBV-specific CD8 T-cells in untreated HIV-positive patients are IL-7Rαlo/negPerforinlo, with very few IL-7Rαneg/loPerforinhi cells. This is in contrast to the LCMV model in mice, where chronic stimulation with persistent gp33 antigen caused a decrease in CD127 expression on LCMV gp33-specific CD8 T-cells but a concomitant increase in granzyme B.99 Finally, consistent with our previous report,44 2 groups have since demonstrated decreased CD127 expression on naive (CD45RA+CCR7+) CD8 T-cells isolated from untreated HIV-infected patients compared with seronegative controls.47,48 It is difficult to reconcile reduced expression of CD127 attributable to chronic stimulation with the naive phenotype. Thus, it seems that downregulation of CD127 on CD8 T-cells during active HIV replication may not be attributable to immune activation. We suggest instead that Tat protein secreted by infected CD4+ cells acts in a paracrine fashion to downregulate CD127 on neighboring CD8 T-cells, irrespective of their antigen specificity. In this case and consistent with the data, most of the CD8 T-cell population would be affected, including naive and memory cells. This may explain the generalized decrease in cell-mediated immunity, including the ineffective control of HIV replication by HIV-specific CD8 T-cells and the increased susceptibility to opportunistic pathogens that is readily seen in HIV-infected patients.
We have previously shown that the IL-7R α-chain is downregulated on naive and memory CD8 T-cells in patients during active HIV replication; here, we demonstrate that this downregulation is mediated, at least in part, by the HIV Tat protein. Because IL-7 enhances the proliferation of CD8 T-cells and upregulates the expression of perforin, decreased IL-7R expression would be expected to limit CD8 T-cell responses to antigen, leading to impaired cell-mediated immunity.
The authors thank Drs. Jonathan Angel, Ashok Kumar, and Angela Crawley for helpful discussion and critical review of the manuscript. They also thank Karl Parato for technical assistance with flow cytometry.
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