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HIV-1 Tat protein induces PD-L1 (B7-H1) expression on dendritic cells through tumor necrosis factor alpha- and toll-like receptor 4-mediated mechanisms - PubMed

HIV-1 Tat protein induces PD-L1 (B7-H1) expression on dendritic cells through tumor necrosis factor alpha- and toll-like receptor 4-mediated mechanisms

Rémi Planès et al. J Virol. 2014 Jun.

Abstract

Chronic human immunodeficiency virus type 1 (HIV-1) infection is associated with induction of T-cell coinhibitory pathways. However, the mechanisms by which HIV-1 induces upregulation of coinhibitory molecules remain to be fully elucidated. The aim of the present study was to determine whether and how HIV-1 Tat protein, an immunosuppressive viral factor, induces the PD-1/PD-L1 coinhibitory pathway on human dendritic cells (DCs). We found that treatment of DCs with whole HIV-1 Tat protein significantly upregulated the level of expression of PD-L1. This PD-L1 upregulation was observed in monocyte-derived dendritic cells (MoDCs) obtained from either uninfected or HIV-1-infected patients as well as in primary myeloid DCs from HIV-negative donors. In contrast, no effect on the expression of PD-L2 or PD-1 molecules was detected. The induction of PD-L1 on MoDCs by HIV-1 Tat (i) occurred in dose- and time-dependent manners, (ii) was mediated by the N-terminal 1-45 fragment of Tat, (iii) did not require direct cell-cell contact but appeared rather to be mediated by soluble factor(s), (iv) was abrogated following neutralization of tumor necrosis factor alpha (TNF-α) or blocking of Toll-like receptor 4 (TLR4), (v) was absent in TLR4-knockoout (KO) mice but could be restored following incubation with Tat-conditioned medium from wild-type DCs, (vi) impaired the capacity of MoDCs to functionally stimulate T cells, and (vii) was not reversed functionally following PD-1/PD-L1 pathway blockade, suggesting the implication of other Tat-mediated coinhibitory pathways. Our results demonstrate that HIV-1 Tat protein upregulates PD-L1 expression on MoDCs through TNF-α- and TLR4-mediated mechanisms, functionally compromising the ability of DCs to stimulate T cells. The findings offer a novel potential molecular target for the development of an anti-HIV-1 treatment.

Importance: The objective of this study was to investigate the effect of human immunodeficiency virus type 1 (HIV-1) Tat on the PD-1/PD-L1 coinhibitory pathway on human monocyte-derived dendritic cells (MoDCs). We found that treatment of MoDCs from either healthy or HIV-1-infected patients with HIV-1 Tat protein stimulated the expression of PD-L1. We demonstrate that this stimulation was mediated through an indirect mechanism, involving tumor necrosis factor alpha (TNF-α) and Toll-like receptor 4 (TLR4) pathways, and resulted in compromised ability of Tat-treated MoDCs to functionally stimulate T-cell proliferation.

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Figures

FIG 1
FIG 1

HIV-1 Tat upregulates maturation markers on MoDCs. (a) Immature MoDCs were treated for 24 h with 50 nM recombinant GST-Tat 1–101 protein (Tat) or an equal amount of GST protein alone (GST). Untreated and LPS-treated (100 ng/ml) MoDCs were used as negative and positive controls, respectively. Tat and LPS, heat-inactivated for 20 min at 95°C, were also included as negative controls (Tat heated and LPS heated, respectively). After 24 h of treatment, MoDCs were harvested and analyzed for CD80, CD83, and CD86 cell surface expression by flow cytometry. The gray lines on the top 3 histograms represent labeling with the IgG isotype control. The filled histograms correspond to the phenotypes of untreated MoDCs, and the unfilled histograms represent the effect of the indicated treatment on the expression of CD83, CD80, and CD86 maturation markers. (b and c) MoDCs were treated with increasing amounts of chemically synthesized Tat protein (10 nM, 100 nM, or 500 nM) in either reduced or oxidized forms. Twenty-four hours later, CD86 cell surface expression was determined by flow cytometry. One representative experiment and a graphical representation of three independent experiments with statistical analysis are depicted in panels b and c, respectively. (d to f) Mean fluorescence intensity (MFI) of CD80, CD83, and CD86 expression on MoDCs derived from 10 different donors analyzed after 24 h of incubation in the absence or in the presence of 50 nM GST (GST) or GST-Tat 1–101 (Tat). (g to i) Increase of the MFI of CD80, CD83, and CD86 on Tat-treated compared to untreated MoDCs of each donor. The results are representative of at least 3 independent experiments. Data were compared by Mann-Whitney and Student's t statistical tests, and the results were considered to be statistically significant when P values were ≤0.05. Asterisks represent P values: *, P < 0.05; *** P < 0.001. (j) Characterization of monocyte-derived dendritic cell phenotype. Monocytes were differentiated into DCs during 5 days of culture in the presence of GM-CSF and IL-4. Differentiation was checked by monitoring the specific MoDC surface markers, including CD1a+, CD14−, CD80+, CD86+, and HLA-DR+ by flow cytometry. The immature status of MoDCs was verified by the weak expression of surface markers CD83, CD80, and CD86.

FIG 2
FIG 2

HIV-1 Tat induced PD-L1 expression on MoDCs. (a) The effect of HIV-1 Tat protein on the expression of PD-L1 on MoDCs was analyzed by flow cytometry. MoDCs were treated for 24 h with 50 nM either recombinant GST-Tat 1–101 protein (GST-Tat) or GST alone (GST). Untreated cells were used as negative controls (Untreated). After 24 h, the levels of PD-1, PD-L1, and PD-L2 expression were determined by flow cytometry. (b) Dose-response effect of Tat. MoDCs were treated for 24 h with increasing amounts of recombinant GST-Tat 1–101 protein (10 nM, 50 nM, or 100 nM). The level of expression of PD-L1 was determined by flow cytometry. (c) Specificity of Tat action. MoDCs were treated for 24 h with 10 nM 1–86 Tat protein (Tat) or with 50 nM GST-Tat 1–101 protein (GST-Tat). Untreated cells were used as a negative control (Untreated). LPS-stimulated cells (100 ng/ml) were used as a positive control (LPS). To evaluate the specificity of the Tat effect on PD-L1 expression, additional controls were included: stimulation with 50 nM GST alone (GST), Tat preincubated for 30 min at 37°C with 3 μg/ml of anti-Tat antibodies (Tat + αTat), Tat heat inactivated for 20 min at 95°C (Tat heated), and LPS heat inactivated in the same conditions (LPS heated). (d) MoDCs were treated with increasing amounts of chemically synthesized Tat protein (10 to 500 nM) in either reduced or oxidized forms. Twenty-four hours later, PD-L1 cell surface expression was determined by flow cytometry. One representative experiment and graphical representation of three independent experiments with statistical analysis are depicted in panels d and e, respectively. (f) Mean fluorescence intensity (MFI) of PD-L1 on MoDCs derived from 10 different donors analyzed after 24 h of incubation in the absence or in the presence of 50 nM GST (GST) or GST-Tat 1–101 (Tat). (g) Increase of the MFI of PD-L1 on Tat-treated compared to untreated MoDCs of each donor. The results are representative of at least 3 independent experiments. Data were compared by Mann-Whitney and Student's t statistical tests, and the results were considered to be statistically significant when P values were ≤0.05. Asterisks represent P values: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3
FIG 3

Tat upregulates PD-L1 expression on DCs from HIV-1-infected and uninfected patients. (a to c) Effect of Tat on PD-L1 expression in primary myeloid DCs. Myeloid DCs from healthy donors were treated for 24 h with 100 nM GST-Tat 1–101 and analyzed by flow cytometry for PD-L1 expression in CD1c-positive cells. The data show one representative experiment (a), a graphical representation of four independent experiments with statistical analysis (b), and the increase of the MFI of PD-L1 on Tat-treated compared to untreated DCs of each donor (c). (d to f) Effect of Tat on PD-L1 expression in MoDCs derived from HIV-1-infected patients. MoDCs from HIV-1-infected patients were treated as described above and analyzed by flow cytometry for PD-L1 expression by gating on CD1a-positive cells. The data show one representative experiment (d), graphical representation of three independent experiments with statistical analysis (e), and the increase of PD-L1 expression on Tat-treated compared to untreated MoDCs of each HIV-1-infected donor (f). The results are representative of at least 3 independent experiments. Data of all the figures were compared by Student's t statistical tests, and the results were considered to be statistically significant when P values were ≤0.05. Asterisks represent P values: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4
FIG 4

HIV-1 Tat protein induces PD-L1 expression on MoDCs via the N-terminal 1–45 fragment. (a) Equal amounts of GST, GST-Tat 1–45, and GST-Tat 1–101 recombinant proteins were analyzed by SDS-PAGE electrophoresis and immunoblotting using a monoclonal anti-Tat antibody targeting the N-terminal epitope (amino acids 1 to 15) or anti-GST antibodies. In panel b, the three recombinant proteins were tested for trans activation activity. HeLa cells stably transfected with a plasmid encoding the β-galactosidase protein under the control of the LTR promoter of HIV-1 were incubated for 24 h with 1 μM GST, GST-Tat 1–101, and GST-Tat 1–45 proteins. The HeLa cells were then washed with PBS, fixed with PBS-0.5% glutaraldehyde, and incubated for an additional 24 h with X-Gal as β-galactosidase substrate (0.4 mg/ml X-Gal, 5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2). The number of blue-dyed cells corresponding to trans-activated cells was counted under an optical microscope (at a ×400 magnification). The results are represented as numbers of blue cells per field. (c and d) MoDCs were treated for 24 h with 100 nM of either the full-length GST-Tat 1–101 protein or the truncated form, GST-Tat 1–45. Untreated and LPS-stimulated cells (100 ng/ml) were used as negative and positive controls, respectively. After 24 h, the expression of PD-L1 and PD-L2 was analyzed by flow cytometry. (c) Corresponds to one representative experiment; (d) corresponds to graphical representation of three independent experiments with statistical analysis. The results are representative of at least 3 independent experiments. Asterisks represent P values: *, P < 0.05; **, P < 0.01.

FIG 5
FIG 5

Tat upregulates PD-L1 expression on MoDCs through an indirect mechanism. MoDCs were treated with increasing amounts of Tat protein (50 nM, 100 nM) for 1 h and then washed once with PBS to remove unbound Tat protein. Tat-treated MoDCs were then cocultured with autologous Tat-untreated MoDCs at a 1:1 ratio to allow cell-cell contact. To discriminate between the untreated and Tat-treated MoDCs in the coculture, untreated cells were prelabeled with 1 μM CFSE [CFSE(+)], whereas Tat-treated cells were kept unlabeled [CFSE(−)]. (a) Corresponds to one representative experiment. The upper histogram shows CFSE+ and CFSE− MoDC coculture. After 24 h of coculture, the level of PD-L1 was determined by flow cytometry by gating on CFSE-positive cells (left histograms) and CFSE-negative cells (right histograms). The filled histograms correspond to the phenotypes of untreated MoDCs, and the unfilled histograms represent PD-L1 expression after the indicated treatment. (b) Corresponds to graphical representation of three independent experiments with statistical analysis. The results are representative of at least 3 independent experiments. Asterisks represent P values: **, P < 0.01; ***, P < 0.001.

FIG 6
FIG 6

Role of soluble factors on the induction of PD-L1 expression by HIV-1 Tat. (a and b) Tat-treated MoDCs (Tat) and autologous untreated MoDCs (Untreated) were cocultured for 24 h in two different compartments separated by a 1-μm microporous membrane to avoid cell-cell contact. MoDCs that had been pretreated for 1 h with 100 nM Tat protein and washed three times with sterile PBS, to remove any residual Tat protein, were placed in the upper chamber of the transwell (Up). The untreated MoDCs were placed in the bottom chamber of the transwell (Down). Untreated MoDCs alone (Untreated) and MoDCs directly treated for 24 h with 100 nM Tat protein (Tat) were used as negative and positive controls, respectively. Following a 24-h coculture, the level of expression of PD-L1 was determined by flow cytometry in each compartment. (b) A graphical representation of three independent experiments showing PD-L1 MFI ± standard deviation with statistical analysis. Asterisks represent P values: *, P < 0.05; **, P < 0.01. (c) MoDCs were stimulated with 100 nM HIV-1 Tat protein for various times. As a negative control, MoDCs were kept untreated (filled histograms). After the indicated time (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h), the level of PD-L1 expression was determined by flow cytometry, as described above. Filled histograms indicate the basal level of PD-L1 expression on untreated MoDCs, and the unfilled histograms represent the level of PD-L1 expression following treatment with the GST-Tat 1–101 protein. Data are representative of three independent experiments. (d to i) Cell supernatants were harvested at the indicated time (0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h) in untreated MoDCs (white bars) and in GST-Tat 1–101-treated MoDCs (black bars). The levels of TNF-α, IL-12p70, IL-6, IFN-α, IL-10, and IFN-γ cytokines were determined by ELISA, as detailed in Materials and Methods. The results are representative of at least 3 independent experiments. Data are the means from triplicates wells ± standard deviations.

FIG 7
FIG 7

Tat upregulates PD-L1 expression on MoDCs through a TNF-α-dependent mechanism. (a) MoDCs from three different donors were incubated in Tat-conditioned (unfilled histograms) or unconditioned (filled histograms) medium. In a parallel experiment, to determine the implication of TNF-α, IL-10, and IFN-γ cytokines in the PD-L1 upregulation induced by Tat protein, those cytokines were first selectively neutralized in Tat-conditioned medium by preincubation for 90 min with 20 μg/ml of anticytokine-specific antibodies (+anti-TNF-α, +anti-IL-10, and +anti-IFN-γ). After 24 h of coculture, the level of PD-L1 expression was determined by flow cytometry. The data show the results from three independent experiments. (b) MoDCs were treated by Tat protein for 1 h, washed, and cocultured with autologous untreated MoDCs at a 1:1 ratio as detailed in the Fig. 5 legend. To determine the implication of TNF-α, similar experiments were performed in the presence of 20 μg/ml of anti-TNF-α-specific antibody (+anti-TNF-α) or the corresponding isotype Ig (+Isotype). After 24 h of coculture, the level of PD-L1 expression was determined by flow cytometry on both CFSE positive (+) and CFSE negative (−) MoDCs. (c) Corresponds to graphical representation of three independent experiments with statistical analysis. The results are representative of at least 3 independent experiments. Asterisks represent P values: **, P < 0.01; ***, P < 0.001. (d) MoDCs from three different healthy blood donors were treated with recombinant human TNF-α (50 ng/ml) or kept untreated. After 24 h, the expression of PD-L1 was analyzed by flow cytometry. The figure shows graphical representation of three independent experiments with statistical analysis. Asterisks represent P values: ***, P < 0.001.

FIG 8
FIG 8

Role of Tat-TLR4 interaction in PD-L1 upregulation. (a) MoDCs were treated with 10 nM Tat protein or kept untreated without (Mock) or with increasing amount of anti-TLR4 antibodies (1 μg/ml, 5 μg/ml, 10 μg/ml). After 24 h, the level of expression of PD-L1 was determined by flow cytometry. Flow plots show one representative result from three independent experiments. (b) The level of TNF-α present in the supernatants of Tat-treated and -untreated MoDCs with or without the anti-TLR4-blocking MAb was determined by ELISA. Data are the means of triplicates wells ± standard deviations. (c) BMDCs derived from either wt (dark-gray bars) or TLR4-KO mice (light-gray bars) were treated for 24 h with 100 nM GST-Tat protein, 1 μg/ml TLR1/2 ligand (Pam3CSK4), and 1 μg/ml LPS as a positive control. Tat-conditioned and unconditioned medium from wt BMDCs was also included as an internal control. After 24 h, the level of PD-L1 expression was determined by flow cytometry. Histograms show cumulative data obtained from three independent experiments with statistical analysis. Asterisks represent P values: *, P < 0.05; **, P < 0.01. (d) The level of TNF-α present at 24 h in the supernatants of each group of BMDCs treated, determined by ELISA, is indicated: untreated; GST, 100 nM; GST-Tat 1–101, 100 nM; LPS, 1 μg/ml; Pam3CSK4, 1 μg/ml. The results are representative of at least 3 independent experiments. Data are the means of triplicates wells ± standard deviations.

FIG 9
FIG 9

Effect of HIV-1 Tat protein on the ability of MoDCs to induce T-cell proliferation. Peripheral blood lymphocytes (PBLs) were labeled with CFSE (2 μM) and kept in culture for 5 days alone (a), in the presence of a suboptimal concentration (10 ng/ml) of anti-CD3 (b), or cocultured with autologous MoDCs (previously treated for 48 h, as indicated) in the presence of anti-CD3 (c to i). MoDCs were kept untreated (c, f) or treated with 50 nM synthetic oxidized Tat (d, g) or 100 nM recombinant GST-Tat (e, h) protein. Coculture of MoDCs (2 × 105 cells) and PBLs (4 × 105 cells) was performed in the absence (c to e) or presence (f to h) of anti-PD-L1 antibodies at 10 μg/ml or an equal amount of isotype antibodies (i). After 5 days of coculture, cells were harvested and CD3-positive cells were labeled firstly with a mouse anti-CD3 antibody and then with an anti-mouse IgG2a Alexa Fluor 633 antibody. Proliferation was then analyzed by flow cytometry in CD3+ cells. The results are representative of at least 3 independent experiments.

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