Although T cells potentially represent key actors in the first line of defence during the early phases of HIV infection [1,2][1,2], T-cell dysfunction in infected HIV individuals is a common event associated to disease progression . An important factor associated to a defective pathogen clearance in chronic infection is T-cell exhaustion, defined as the progressive loss of functions by consumption in antigen-specific T cells [4,5][4,5].
It has been previously shown that during HIV infection, a down-modulation of CD3ζ is induced on αβ and γδ T cells [6,7][6,7], correlating with their functional inability. In other chronic inflammations and in high-load antigenic persistence, selective defects in global CD8+ T-cell function have been associated with down-regulation of CD3ζ expression. These include autoimmune disorders , malignancy , and microbial infections [10–12][10–12][10–12].
CD3ζ molecule is a 16-kDa transmembrane protein expressed as a disulfide-linked homodimer; it is indispensable for coupling antigen recognition by the T-cell receptor (TCR) and downstream T-cell response [13,14][13,14], and its down-modulation could represent a mechanism by which T-cell anergy is induced during HIV infection.
Different factors were described to induce an impairment of CD3ζ expression, such as arginine deprivation  or tryptophan catabolites accumulation . Both mechanisms are mediated by the activity of arginase I (ArgI) and indoleamine 2,3-dioxygenase (IDO), mostly produced by myeloid-derived suppressor cells (MDSC) . MDSC are a heterogeneous population comprising myeloid-cell progenitors . In pathological conditions such as cancer, various infectious diseases or some autoimmune disorders, a partial block in the differentiation of immature myeloid cells into mature myeloid cells results in an expansion of the MDSC population . Importantly, the activation of these cells in a pathological context results in the up-regulated expression of immune suppressive factors such as Arg1, IDO and inducible nitric oxide synthase (iNOS), and an increase in the production of NO (nitric oxide) and reactive oxygen species (ROS) . MDSC can be divided into two subsets: monocytic (M-MDSC) and granulocytic (Gr-MDSC). In humans, the monocytic subset contains CD14+ cells, whereas the granulocytic subset contains CD14– CD15+ cells [17,19][17,19].
Two recent papers show that levels of MDSC are elevated in HIV+ patients and correlate with disease progression [20,21][20,21]. However, the mechanisms used by MDSC to inhibit T-cell function against HIV-infected cells have not been clearly shown. In the present work we evaluated the role of Gr-MDSC in regulating CD3ζ expression during HIV infection. We found that in HIV+ patients, the presence of a high frequency of Gr-MDSC correlates with a lower expression of CD3ζ; moreover, the depletion of Gr-MDSC in vitro was able to restore CD3ζ level in T cells, thus enhancing the HIV-specific T-cell response. As for the relevant mechanism, we showed that CD3ζ down-modulation was due to the inhibition of its transcription factors ELF-1 (E74 like-factor 1), induced by Gr-MDSC throughout a cell contact-dependent mechanism.
Materials and methods
One hundred and five HIV+ patients afferent to the ‘Lazzaro Spallanzani’ National Institute for Infectious Diseases (INMI) (Rome, Italy) were recruited. Eighty-five HIV+ patients on cART with CD4+ T-cell counts ranging from 42 to 1853 cells/μl (median 687 cells/μl), and viral loads ranging from less than 40 to 311 695 viral RNA copies/ml (median <40 viral RNA copies/ml; 12 patients had a viral load >50 copies/ml); and 20 HIV+ patients off therapy, with CD4+ T-cell counts ranging from 15 to 1156 cells/μl (median 520 cells/μl) and viral load ranging from less than 40 to 2 607 565 viral RNA copies/ml (median 55 984 viral RNA copies/ml), were recruited for the purpose of this study. All patients were selected by excluding any co-morbidity, such as hepatitis C virus, hepatitis B virus, Epstein Barr virus, Mycobacterium tuberculosis co-infections and malignancies. Sixteen HIV-seronegative donors were used as a control and were processed under the same conditions. Features of the study population are reported in Table S1, http://links.lww.com/QAD/A771 of supplementary data. The study was approved by the Institutional Review Board of INMI (n. 78 dated 21 Nov. 2013). Residual peripheral blood from HIV+ patients obtained for CD4+ T-cell counts was used.
Peripheral blood mononuclear cells separation and cell culture
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation (Lympholyte-H; Cederlane, Canada). After separation, PBMC were cultured in RPMI 1640 (EuroClone, Italy) supplemented with 10% heat-inactivated fetal bovine serum (EuroClone), 2 mmol/l L-glutamine, 10 mmol/l HEPES buffer (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 2 mmol/l penicillin and 50 μg/ml streptomycin (EuroClone). Cells were maintained at 37°C in humidified air with 5% CO2. When indicated, PBMC were treated with 500 μg/ml N-hydroxy-nor-L-arginine (NOHA, Calbiochem, Germany), 500 μg/ml N-monomethyl-L-arginine (L-NMMA, Calbiochem, Germany), 300 UI/ml Catalase (Sigma-Aldrich, St Louis, Missouri, USA) and 500 μg/ml 1-methyl tryptophan (1-MT, Sigma-Aldrich), 10 μg/ml neutralizing anti-PD-L1 (BioLegend, USA). The HIV-specific CD8+ T-cell response was induced by stimulating with a pool of HIV peptides (Group-specific antigen, Gag; Trans-activator of transcription, Tat; Negative regulator factor, Nef ) specifically designed for CD8+ T-cell response (from NIH/AIDS Reagent Program, USA) for 18 h. γδ T-cell response was evaluated stimulating PBMC with isopentenyl pyrophosphate (IPP, 100 μg/ml, Sigma-Aldrich) for 18 h. Brefeldin A (10 μg/ml, Sigma-Aldrich) was added after 1 h of stimulation in order to prevent cytokine secretion. In selected experiments, PBMC obtained from HIV+ patients and healthy donors were incubated with 10 μg/ml of actinomycin-D (Sigma-Aldrich) for 30 min, 1, 2, and 6 h.
CD3ζ expression was accomplished using FITC-conjugated anti-Vδ2, APC-conjugated anti-CD3, PerCP-conjugated anti-CD8 or anti-CD4 monoclonal antibodies (mAbs) (BD Biosciences, USA), and PE-labelled anti-CD3ζ (Beckman Coulter, France).
In brief, 0.5×106 PBMC were incubated with antibodies for membrane staining at 4°C for 15 min, and then washed and fixed with 1% paraformaldehyde (PFA) (Sigma-Aldrich). For intracellular staining, cells were permeabilized, and stained with PE-labelled anti-CD3ζ. Acquisition of 100 000 events in the lymphocyte-gated population was performed using FACS Calibur and analysed with CellQuest software (Becton Dickinson, USA). The number of CD3ζ molecules per cell was evaluated using the Phycoerythrin Fluorescence Quantization Kit (BD Biosciences) according to the manufacturer's instructions.
Evaluation of the MDSC percentage was accomplished with 0.5×106 cells stained with FITC-conjugated anti-CD15, PE-conjugated anti-CD33, PerCP-conjugated anti-HLA-DR, a cocktail of APC-conjugated antibodies anti-CD3, -CD56, -CD19 (Lin), APC-H7-conjugated anti-CD14 and PE-Cy7-conjugated anti-CD11b, PE-conjugated anti-CD124 mAbs, PE-conjugated anti-PD-L1 mAbs (BD Biosciences). Acquisition of 100 000 events was performed in the leukocyte-gated population on FACS CANTO II and analysed with FACS DIVA software (BD Biosciences).
For intracellular staining, PBMC were stained with FITC-conjugated anti-Vδ2, APC-conjugated anti-IFNγ, APC-H7-conjugated anti-CD8, V500-conjugated anti-CD3 (BD Biosciences) as described above. Acquisition of 100 000 events in the lymphocyte-gated population were performed on FACS CANTO II and analysed with FACS DIVA software (BD Biosciences).
Gr-MDSC and T cells separation and cell culture
Gr-MDSC depletion was performed using magnetic selection. Briefly, PBMC from cART-treated patients were stained with anti-CD15 FITC-conjugated mAb (BD Biosciences). After washing, cells were labelled with anti-FITC microbeads (Miltenyi Biotec, Germany). The purity of sorted Gr-MDSC was >95%, as verified by flow cytometry (data not shown). T cells were purified by using a Pan T-cell isolation kit (Miltenyi Biotec) according with manufacturer's instructions.
After separation, Gr-MDSC-depleted PBMC or purified T cells were cultured with Gr-MDSC at different ratios (1 : 0, 1 : 1, 1 : 4) for 18 h. In selected experiments, Gr-MDSC-depleted PBMC were cultured with Gr-MDSC separated by a semi-permeable membrane (0.4-μm pore transwell; BD FALCON, USA), and when indicated, Gr-MDSC were fixed with 1% paraformaldehyde (1% PFA) at room temperature for 10 min and cultured with Gr-MDSC-depleted PBMC (1 : 1 ratio) for 18 h.
RNA extraction and real-time PCR
Total RNA extraction from 1.5×106 PBMC or purified T cells was performed with RNeasy mini kit (QIAGEN, USA) following the manufacturer's instructions. Reverse-transcription was conducted by TaqMan Reverse Transcription Reagent kit (Applied Biosystems, USA).
The quantification of mRNA encoding for CD3ζ was performed by a Taqman real-time PCR (RT-PCR) method. The following primers and specific probe were used: forward GGTGTCATTCTCACTGCCTTGTT, reverse TACCAGCAGGGCCAGAACC, probe CTGAGAGTGAAGTTCAGC, covalently labelled with 6-carboxyfluorescein (FAM) at the 5′-end and with 6-carboxy-tetramethylrhodamine (TAMRA) as a quencher dye at the 3′-end.
PBMC or purified T cells (2 × 106) were lysed using a protein extraction kit (Complete Lysis-M, ROCHE, Switzerland). Ten micrograms of lysate were separated on a 10% gradient SDS-PAGE and electroblotted onto Hybond ECL nitrocellulose membrane (Amersham Life Science, Germany). Membranes were blocked in PBS, 5% milk powder and 0.1% Tween 20. Membranes were stained overnight with an anti-ELF-1 (C20) (Santa Cruz Biotechnology, USA), or β-actin (C4) (Merck Millipore, Germany) Abs, followed by 1-h incubation with goat ant-rabbit IgG-HRP (Upstate Biotechnology, USA). Signals were detected by enhanced chemo-luminescence (Amersham Bioscience, Germany).
GraphPad Prism version 5.00 for Windows (GraphPad Software, USA) was used to perform the analysis. A nonparametric test (Mann–Whitney) was used to compare patient groups. The parametric Spearman test was used to evaluate correlations. A P less than 0.05 was considered statistically significant.
Granulocytic myeloid-derived suppressor cells are decreased in immunological successfully treated patients
We evaluated the frequency of Gr-MDSC in HIV+ patients by flow cytometry, and we found that Gr-MDSC, identified as Lin- HLA-DRlow/- CD11b+ CD33+ CD14– CD15+ CD124+, were expanded compared with healthy donors (Fig. 1a and b).
Moreover, we did not find any differences in Gr-MDSC levels between cART-treated and non-treated patients (Fig. 1b). Albeit no correlation was observed between MDSC frequency and CD4+ T-cell count (data not shown), grouping cART patients in those with CD4+ T-cell count over 800 cells/μl (CD4+ >800), between 800 and 600 cells/μl (600< CD4+ <800), between 600 and 400 (400< CD4+ <600) and lower than 400 cells/μl (CD4+ <400), the latest had the highest frequency of Gr-MDSC (Fig. 1c). No difference was found among the other three groups, although they were still higher than healthy donors (CD4+ >800 vs. healthy donors P = 0.0005; 600< CD4+ <800 vs. healthy donors P = 0.0005; 400< CD4+ <600 P = 0.001; CD4+ <400 vs. healthy donors P < 0.0001). No correlation with viral load was observed (data not shown).
Gr-MDSC regulate CD3ζ expression on T cells
We previously reported that CD3ζ expression on Vγ9Vδ2 T cells from HIV+ patients is down-modulated compared with healthy donors , thus explaining their anergy . In the present study, we found that the reduction of CD3ζ level shown on Vγ9Vδ2 T cells also occurs on CD4+ and CD8+ T cells from the same HIV+ patients (Fig. 2a), confirming previously reported data  and suggesting that all T-cell subsets are affected.
Interestingly, a strong inverse correlation was observed between Gr-MDSC frequency and the expression of CD3ζ on CD8+ and CD4+ T cells, and on Vγ9Vδ2 T cells from HIV+ patients (Fig. 2b), suggesting a direct role of Gr-MDSC in affecting CD3ζ expression.
To demonstrate that Gr-MDSC from HIV+ patients are indeed able to suppress CD3ζ expression, we depleted Gr-MDSC, and we tested the expression of CD3ζ in Gr-MDSC-depleted PBMC. Gr-MDSC depletion induced a significant restoration of CD3ζ expression on all T-cell subsets (Fig. 2c); moreover, when Gr-MDSC-depleted PBMC were cultured with purified Gr-MDSC at a different ratio (1 : 1, 1 : 4), a strong down-modulation of CD3ζ was again observed (Fig. 2c). These data indicate that Gr-MDSC are able to control CD3ζ expression during HIV infection on all T-cell subsets studied. This phenomenon was not observed when monocytes were used (Fig. S1, http://links.lww.com/QAD/A771), it was not due to the induction of cell death (Fig. S2, http://links.lww.com/QAD/A771), and it was not observed on other molecules, as CD8 (Fig. S2, http://links.lww.com/QAD/A771).
Further, we excluded that Gr-MDSC act on CD3ζ throughout a bystander effect of other cells; in fact Gr-MDSC cultured with purified T cells are able to induce CD3ζ down-modulation (Fig. S3A, http://links.lww.com/QAD/A771).
The effect on CD3ζ is a general feature of Gr-MDSC; indeed, purified Gr-MDSC from healthy donors cultured with autologous PBMC induce a decrease of CD3ζ (Fig. S3B, http://links.lww.com/QAD/A771).
Gr-MDSC-induced down-modulation of CD3ζ is associated to T-cell response impairment
We wondered whether Gr-MDSC could affect both HIV-specific T cells and Vγ9Vδ2 T-cell responses. To this aim, we stimulated PBMC from HIV+ patients with HIV peptides or phosphoantigen (IPP), and evaluated IFNγ production in CD8+ T cells and Vγ9Vδ2 T cells, respectively. We found an inverse correlation between the frequency of Gr-MDSC and the percentage of CD8+ T cells producing IFNγ (Fig. 3a). We then evaluated if Gr-MDSC depletion is able to enhance the HIV-specific T-cell response, possibly by restoring CD3ζ. To answer this question, we depleted Gr-MDSC, and stimulated the remaining PBMC with HIV peptides. Gr-MDSC depletion induced a significant enhancement of HIV-specific IFNγ response compared with whole PBMC in 11 out of 14 patients (Fig. 3b and Fig. S4, http://links.lww.com/QAD/A771), indicating that Gr-MDSC regulate the HIV-specific T-cell response, possibly by down-modulating CD3ζ. An inverse correlation was found between the Gr-MDSC proportion and the percentage of Vγ9Vδ2 T cells producing IFNγ upon IPP stimulation (Fig. 3c), even though Gr-MDSC depletion does not restore IPP-induced IFNγ production (Fig. 3d).
Gr-MDSC down-modulate CD3ζ by a cell contact-dependent mechanism
To investigate whether the effect of Gr-MDSC on CD3ζ is cell contact-dependent, we co-cultured Gr-MDSC and PBMC at a 1 : 1 ratio separated by a semi-permeable membrane. We found that when co-cultured separately, Gr-MDSC were not able to down-modulate CD3ζ expression on T cells (Fig. 4), indicating that Gr-MDSC action needs cell-to-cell contact. Furthermore, to evaluate whether the cell-contact occurs via interaction between molecules expressed on the cell membrane, or via active mechanisms requiring living cells, purified Gr-MDSC were fixed with 1% PFA and then cultured with autologous PBMC. PFA-fixed Gr-MDSC were still able to induce down-modulation of CD3ζ on T cells (Fig. 4), suggesting that this phenomenon is mediated by molecule/s constitutively expressed on the Gr-MDSC membrane, also after fixation. It was demonstrated that during HIV infection CD3ζ level correlated with PD-L1 expression on neutrophils . We found that Gr-MDSC express PD-L1 (Fig. S5A); however, when PBMCs were cultured with a neutralizing anti-PD-L1 mAb no effects were observed on the expression of CD3ζ (Fig. S5B, http://links.lww.com/QAD/A771).
Gr-MDSC inhibit CD3ζ mRNA expression
We asked whether the modulation of CD3ζ occurs at the transcriptional level. To investigate this possibility, a real-time PCR was carried out to establish the level of mRNA coding CD3ζ. Figure 5a shows that compared with healthy donors, HIV+ patients present a lesser quantity of mRNA coding CD3ζ. This data was confirmed by using purified T cells to normalize for T-cell number (Fig. S6A, http://links.lww.com/QAD/A771). Further, we depleted Gr-MDSC, and a real-time PCR was performed on the remaining PBMC after 18 h of culture. We found that Gr-MDSC depletion restores the expression of CD3ζ mRNA (Fig. 5b), indicating that Gr-MDSC affects the expression of CD3ζ molecule by interfering with the transcription process.
Gr-MDSC inhibit the CD3ζ transcription factor ELF-1 in T cells
We wondered whether the reduction of CD3ζ mRNA was due to its lower stability. We treated PBMC from HIV+ patients and healthy donors with actinomycin D. We found that CD3ζ mRNA from PBMC treated with actinomycin D is very stable in both HIV+ patients and healthy donors and no difference was observed between the two groups (data not shown), suggesting that the inhibition of CD3ζ is not due to mRNA instability.
One of the factors regulating the transcription of the CD3ζ gene is ELF-1 ; we evaluated the expression of this factor in the PBMC of HIV+ patients and healthy donors by western blotting. Figure 5c shows the results obtained from two representative healthy donors and four HIV+ patients. As expected, two bands are present, corresponding to the cytoplasmic and nuclear forms of ELF-1 (80 and 98 kDa, respectively). We observed that in HIV+ patients, both cytoplasmic and nuclear forms are lower than in healthy donors (Fig. 5c and d). To normalize for the number of T cells, we performed the same experiment with purified T cells, confirming previous result (Fig. S6B, http://links.lww.com/QAD/A771). These data indicate that the diminished expression of this transcription factor might participate to the down-modulation of CD3ζ mRNA in HIV+ patients. To confirm this hypothesis, we depleted Gr-MDSC from the PBMC of HIV+ patients with low CD3ζ expression and after 18 h we evaluated ELF-1 expression by western blot. We found that depletion of Gr-MDSC restored the expression of ELF-1, indicating that these cells are able to modulate CD3ζ by affecting the expression of its transcription factor (Fig. 5e).
The T-cell response against pathogens occur following the initial step of recognition of specific antigen and the transmission of activation signals, which are mediated by the TCR. During the past decade, various reports have been published showing that during chronic inflammation, such as in cancer [26–28][26–28][26–28], infections [7,29–31][7,29–31][7,29–31][7,29–31] and autoimmune disorders [32,33][32,33], T cells become functionally impaired. In all of these cases, it has been shown that the immune dysfunction is associated with the loss of expression of CD3ζ molecule. Interestingly, such CD3ζ down-regulation has also been observed in T cells that do not directly participate in eliciting the antigen-specific response. However, few studies have attempted to delineate its immunological and molecular basis, and clinical implications. In the present study, we demonstrated that during HIV infection, CD3ζ down-modulation is mediated by Gr-MDSC, and their depletion is able to restore CD3ζ expression. We also showed that Gr-MDSC inhibit CD3ζ mRNA expression by silencing its transcription factor ELF-1. These data are in line with other reports showing that Gr-MDSC are expanded in other infections  and cancer , and are correlated with a decrease of CD3ζ expression on T cells. In these papers, the authors also suggested that ArgI could decrease CD3ζ expression; however, no experiments that aimed to inhibit ArgI and confirm its role in CD3ζ modulation were performed. Although it has been demonstrated that arginine deprivation and tryptophan starvation can alter CD3ζ expression in vitro[15,16][15,16], we found that the enzyme activity of ArgI, IDO, iNOS and the production of ROS (data not shown) are not involved in the modulation of CD3ζ expression during HIV infection. This observation is consistent with the requirement of cell contact. It was previously postulated that cell contact is necessary because short-lived suppressor mediators need cell proximity . However, we excluded this hypothesis since fixed Gr-MDSCs were still able to decrement CD3ζ. Recently, Bowers and colleagues showed an immune suppression activity of neutrophils from HIV+ patients mediated by PD-L1/PD1 pathway. Further, neutrophils PD-L1 expression correlated with CD3ζ expression on T cells . We found that Gr-MDSC express PD-L1; however, the PD-L1/PD1 pathway is not involved in CD3ζ down-modulation by Gr-MDSC. These data suggest that a different, unknown mechanism, mediated by molecules expressed on the Gr-MDSC membrane, drives the alteration of CD3ζ expression.
The expansion of different MDSC subsets in treatment naïve HIV+ patients was previously described in two reports [20,21][20,21]. In agreement with Vollbrecht and colleagues we found a higher proportion of Gr-MDSC in HIV+ patients. The proportion of Gr-MDSC in patients on cART remains higher than the healthy donors and comparable to the Gr-MDSC level of untreated subjects. Unlike the previous studies, we did not find correlations between %Gr-MDSC and CD4+ T-cell count or viral load. This discrepancy could be due to the different features of patients included in the cited studies. In fact, our patients were mostly cART treated whereas Qin and Vollbrecht cohorts mostly comprised treatment naïve subjects.
Interestingly, we found that among treated patients, individuals whose CD4+ T-cell counts did not go beyond 400 cells/μl showed higher Gr-MDSC levels than patients who did. On the other hand, no association was observed with HIV viral load. These data indicate that in patients on cART, Gr-MDSC frequency is associated with immunological response rather than viremia, suggesting their potential use in monitoring immunological reconstitution. However, further investigations are needed to determine whether Gr-MDSC is a predictor or a result of immunological failure. In agreement with Gr-MDSC results, we did not find any correlation between CD3ζ and HIV viral load and CD4+ T-cells count.
The CD3ζ down-modulation could represent a mechanism by which T-cell anergy is induced by HIV infection. In fact, we found an inverse correlation between Gr-MDSC frequency and HIV-specific CD8+ T-cell response, and consistent with CD3ζ up-regulation, Gr-MDSC depletion induced an increase of IFNγ production by CD8+ T cells upon HIV peptide stimulation. However, it should be noted that in three patients, Gr-MDSC depletion did not increase IFNγ production, even if CD3ζ up-regulation was found. These data suggest that other suppressive mechanisms occurred in these individuals. Unexpectedly, Gr-MDSC depletion did not restore IFNγ production by Vγ9Vδ2 T cells, despite the up-regulation of CD3ζ and the inverse correlation with Gr-MDSC levels. We previously demonstrated a CD3ζ down-modulation on Vγ9Vδ2 T cells from HIV+ patients that correlated with their functionality; furthermore, treatment with phorbol myristate acetate (PMA) restored CD3ζ expression and function . PMA is a protein kinase C (PKC) activator involved in many intracellular pathways. Thus, a possible explanation of our data is that the up-regulation of CD3ζ in Vγ9Vδ2 T cells may not be sufficient to restore their functionality, perhaps because other mechanisms occur  that could be bypassed by PKC activation. However, this issue needs investigation.
In conclusion we demonstrated that Gr-MDSC are expanded in HIV+ patients undergoing cART, in particular in low CD4+ T-cell count patients, and inhibit CD3ζ expression through an undiscovered cell contact-dependent mechanism that induces the silencing of the transcription factor ELF-1. Our data provide new knowledge on mechanisms used by Gr-MDSC in immune modulation and on their role in the immune reconstitution during antiviral treatments. These findings are pivotal since they open up the possibility of testing Gr-MDSC as a predictive factor of treatment outcome.
Author contributions: N.T., F.M. and A.S. designed the study. N.T. and F.T. performed the experiments. S.M. and E.L. performed the RT-PCR. N T. analysed the data. V.B., R.C. and C.M. contributed to analyse data. N.T. and A.S. wrote the paper. C.A., V.B., R.C., E.C., C.M., V.C. and F.M. contributed to revise the paper.
Financial support: This work was supported by grants from Italian Ministry of Health (Ricerca Corrente) to INMI L. Spallanzani.
Conflicts of interest
There are no conflicts of interest.
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