Mesenchymal Stem Cells Tune the Development of Monocyte-Derived Dendritic Cells Toward a Myeloid-Derived Suppressive Phenotype through Growth-Regulated Oncogene Chemokines

Hsin-Wei Chen, Hsin-Yu Chen, Li-Tzu Wang, Fu-Hui Wang, Li-Wen Fang, Hsiu-Yu Lai, Hsuan-Hsu Chen, Jean Lu, Ming-Shiu Hung, Yao Cheng, Mei-Yu Chen, Shih-Jen Liu, Pele Chong, Oscar Kuang-Sheng Lee and Shu-Ching Hsu
J Immunol May 15, 2013, 190 (10) 5065-5077; DOI: https://doi.org/10.4049/jimmunol.1202775

Abstract

Mesenchymal stem/stromal cells (MSCs) are promising potential candidates for the treatment of immunological diseases because of their immunosuppressive functions. However, the molecular mechanisms that mediate MSCs’ immunosuppressive activity remain elusive. In this article, we report for the first time, to our knowledge, that secreted growth-regulated oncogene (GRO) chemokines, specifically GRO-γ, in human MSC-conditioned media have an effect on the differentiation and the function of human monocyte-derived dendritic cells. The monocyte-derived dendritic cells were driven toward a myeloid-derived suppressor cell (MDSC)–like phenotype by the GRO chemokines. GRO-γ–treated MDSCs had a tolerogenic phenotype that was characterized by an increase in the secretion of IL-10 and IL-4, and a reduction in the production of IL-12 and IFN-γ. We have also shown that the mRNA expression levels of the arginase-1 and inducible NO synthase genes, which characterize MDSCs, were upregulated by GRO-γ–primed mouse bone marrow cells. In addition, the ability of GRO-γ–treated bone marrow–derived dendritic cells to stimulate the OVA-specific CD8+ T (OT-1) cell proliferation and the cytokine production of IFN-γ and TNF-α were significantly decreased in vivo. Our findings allow a greater understanding of how MDSCs can be generated and offer new perspectives to exploit the potential of MDSCs for alternative approaches to treat chronic inflammation and autoimmunity, as well as for the prevention of transplant rejection.

Introduction

Mesenchymal stem/stromal cells (MSCs) have been isolated and characterized by their adherent properties, surface phenotype, and capacity for controlled self-renewal. They have the potential to differentiate into cartilage, bone, tendon, adipose tissue, and muscle in vitro (1). MSCs are attractive candidates for tissue engineering, as well as for cell and gene therapy, because of their immunological activity and ability to preferentially migrate to sites of inflammation or tissue injury (2, 3). Therapeutic approaches that used allogeneic MSCs have yielded promising results in osteogenesis imperfecta, graft-versus-host disease, myocardial infarction, and organ transplantation in animal models or in the clinic (4–6). Studies analyzing the immunomodulatory properties of MSCs have established their beneficial role in both regenerative and immunoregulatory cell therapy (7–9). MSCs have been shown to modulate naive and effector T lymphocytes, B lymphocytes, dendritic cells (DCs), macrophages, and NK cells (10–15) through direct cell–cell contact or MSC-derived soluble factors. However, the specific molecular mechanisms by which MSCs regulate these immune cells remain unknown.

DCs play a critical role in the induction of the adaptive immune response, alloantigen elimination, and transplant rejection. Previous studies have reported that DCs are sensitive to the immunosuppressive effects of MSCs. Several well-studied regulatory mediators, such as PGE2, IDO, IL-6, and IL-10, contribute to the immunosuppressive properties of MSCs (16, 17). However, the regulation of the differentiation and cellular properties of DCs by MSCs are still unclear. In this study, the mechanisms that underlie the MSC-mediated suppression of DC differentiation were evaluated.

The subgroup of growth-regulated oncogenes (GROs), consisting of CXCL1/GRO-α, CXCL2/GRO-β, and CXCL3/GRO-γ, contains a Glu-Leu-Arg motif and belongs to the IL-8 angiogenic cytokine family. This subfamily was originally discovered in human melanoma cell lines (18). Currently, there is even little information available on the biological activity of GRO chemokines. Binding to their cognate receptors, which are G protein–coupled receptors that are characterized as CXCR2, can activate leukocyte migration, enhance the chemotaxis of endothelial cells, regulate inflammation and angiogenesis that is associated with tumorigenesis (19, 20), mediate cell-cycle arrest in monocytes, and activate cell migration by neutrophils (21, 22). However, the effect of GRO-γ on monocyte-derived DCs (MDDCs) and downstream effects on T cell responses have not been explored.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of early myeloid progenitors/precursors of granulocytes, macrophages, and DCs. MDSCs are commonly characterized by the expression of the myeloid lineage markers Gr-1 and CD11b, and these cells are thought to play a critical role in tumor immune escape, autoimmune diseases, transplant rejection, as well as in chronic inflammation and infection because of their ability to suppress T cell effector functions by upregulating the expression of immunosuppressive factors, such as arginase 1 (ARG-1) and NO synthase 2 in mice (23). However, generating stable and safe MDSCs ex vivo and selecting optimal approaches for cell-based therapies are still major challenges. We observed that MSCs secrete increased levels of GRO-γ. Therefore, the aim of this study was to investigate the role of GRO chemokines in the function and differentiation of MDDCs. We demonstrated that GRO chemokines that were secreted from MSCs inhibited the differentiation and function of MDDCs, and drove their differentiation toward an MDSC-like immunophenotype both in vitro and in vivo. The results from this study offer novel insights into the potential therapeutic platform that is effective to generate MDSCs capable of suppressing uncontrolled immune activation.

Materials and Methods

Mice

C57BL/6 (H2b) and BALB/c (H2d) mice were purchased from the National Laboratory Animal Center, National Applied Research Laboratories, Taiwan. C57BL/6 (H2b)/OT-1 transgenic mice (transgenic for the TCR-specific peptide OVA257–264, SIINFEKL) were a gift from Dr. John Kung (Academica Sinica, Taiwan). Six- to 8-wk-old mice were used for this study. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the National Health Research Institutes (protocol no. NHRI-IACUC-100003 and NHRI-IACUC-097077-A).

Reagents

Recombinant human GRO-α, GRO-β, GRO-γ, IL-4, GM-CSF, and recombinant mouse GM-CSF were purchased from PeproTech (Rocky Hill, NJ). Recombinant mouse GRO-α, GRO-β, and GRO-γ were purchased from R&D Systems (Minneapolis, MN). N-(2-hydroxy-4-nitrophenyl)-N′-(2-bromophenyl) urea (SB225002) was obtained from Calbiochem (San Diego, CA). LPS (from Escherichia coli 055:B5) was obtained from Sigma-Aldrich (St. Louis, MO). PE-labeled mouse anti-human CD11c, HLA-DR, and CD83 Abs were obtained from eBioscience (San Diego, CA). FITC-conjugated mouse anti-human CD86, DC-SIGN, CD40, and CD80 Abs were purchased from BioLegend (San Diego, CA).

Preparation of MSC-conditioned medium

Umbilical MSCs (uMSCs) were purified from cord blood and characterized as reported previously (24). The uMSCs were growth in expanded medium that contained α-MEM with 10% ES-FBS (HyClone, Logan, UT), then maintained for one more passage in medium that was supplemented with pooled AB-type human serum (Invitrogen, Carlsbad, CA). Each passage was 5 d in duration. The cultured uMSCs (3 × 105) were reseeded in 15 ml α-MEM with 10% pooled AB-type human serum for 5 d which under certain conditions has been used in place of human DCs in in vitro differentiation. The supernatant collected from the final uMSC cultures was centrifuged at 300 × g for 10 min at 4°C to remove cellular debris. This supernatant was subsequently used as MSC-conditioned medium (MSC-CM). Human serum containing medium without uMSCs cultured was used as control cultured medium.

Cytokine/chemokine array assay

The cytokines secretion profile of uMSCs was determined using the Human Cytokine Array C Kit (RayBio, Redwood City, CA) according to the manufacturer’s instructions. The chemiluminescent signal was detected using an ECL system (Amersham Pharmacia Biotech, Aylesbury, U.K.), and the signal intensity was quantified by spot densitometry using an AlphaImager 1220 Analysis and Documentation System (Alpha Innotech, Braintree, U.K.). Each spot signal was corrected for the adjacent background intensity and normalized to the positive control sample on the membrane.

Generation of human MDDCs

CD14+ monocytes were isolated using the human CD14+ Cell Isolation Kit (MACS; Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The purified CD14+ cells were cultured in MSC-CM or α-MEM complete medium that was supplemented with pooled AB-type human serum, recombinant human GM-CSF (rhGM-CSF; 80 ng/ml), and recombinant human IL-4 (rhIL-4; 40 ng/ml; PeproTech) in the presence or absence of GRO chemokines to induce differentiation of the cells into DCs. An additional 1 ml medium that contained the same concentrations of rhGM-CSF and rhIL-4 with or without GRO chemokines was added to each group of cells on day 3. Half of the volume of the culture medium was removed and replaced with an equal volume of cultured medium that contained the same concentrations of rhGM-CSF and rhIL-4 on day 5. Control cultures were replaced by normal cultured medium in parallel. Maturation of MDDCs (mature MDDCs [mDCs]) was induced by adding 1 μg/ml LPS to the culture medium of the immature MDDCs (iDCs) on day 5, then subsequently culturing for another 48 h. The study protocols were approved by the Institutional Review Board of Human Subject Research Ethics Committee of Academia Sinica (AS-IRB01-10113) and the Institutional Review Board of Research Ethics Committee of National Health Research Institutes (EC1001101).

FACS analysis

The phenotypic profiles of monocytes, iDCs, and mDCs were analyzed by staining 1 × 105 cells with fluorochrome-labeled Abs against CD11c, HLA-DR, CD80, CD86, CD83, CD40, and DC-SIGN. The fluorescence intensity was measured by flow cytometry. The following corresponding isotype-matched controls were used: FITC-IgG1, FITC-IgG2a, PE-IgG2a, and PE-IgG2b (BD Biosciences, San Jose, CA). Surface-labeled cells were analyzed using a FACSCalibur-flow cytometer (BD Biosciences). For cell purification, sorting was performed using a FACSAria cell sorter (BD Biosciences). The purity of individual sorted cell populations was >95%.

Endocytosis test

The endocytic activity was measured by analyzing the cellular uptake of FITC-dextran (40 kDa, FD40S; Sigma-Aldrich) as quantified by flow cytometry. Differentiated cells only (iDCs, 5 × 104 cells), or differentiating DCs that either supplement with MSC-CM (iDC+CM) or coculture with MSCs in 24-well Transwell plate (iDC+MSC) were collected and then incubated in RPMI 1640 medium with 1 mg/ml FITC-dextran for 30 min at 37°C, then fixed with 1% paraformaldehyde. The endocytosed signal within the cells was analyzed by FACS. The data analysis was performed using FlowJo version 5.7.2 software. The signal obtained for the cells incubated with medium without FITC-dextran was used as a negative control.

MLR assay

Monocyte-derived cells (3 × 104) at different stages of differentiation were irradiated with 30 Gy using an x-ray biological irradiator (x-ray R-2000; Rad Source Technologies, Alpharetta, GA) and then cultured with purified allogenic CD3+ T cells (3 × 105). For the allogenic T cell response in mouse system, splenocytes (1 × 105) harvested from BALB/c (H2d) mice were reacted with or without x-ray irradiated splenocytes (1 × 105) isolated from C57BL/6 (H2b) in the presence of either bone marrow (BM)–derived dendritic cells (BMDCs) (1 × 104) or GRO-γ–treated BMDCs (1 × 104). After 72 h of incubation, [3H]thymidine (1 μCi) was added to each well and the cell cultures were harvested using a Filtermate 96-well harvester followed by a 16-h incubation. The radioactivity (cpm) was measured to quantify cell proliferation using a Packard microplate scintillation and luminescence counter (Perkin-Elmer-Packard, Waltham, MA).

Preparation of GRO-γ knockdown MSCs

Lentiviral systems for gene silencing were obtained from the Taiwan National RNAi Core Facility. Sequences of short hairpin RNA in the pLKO.1 vector are the following: pLKO.1-shLuc (target sequence, 5′-GCGGTTGCCAAGAGGTTCCAT-3′), pLKO.1–shGRO-γ-2 (target sequence, 5′-ACATCCAAAGTGTGAATGTAA-3′), and pLKO.1–shGRO-γ-4 (target sequence, 5′-CCTCAAGAACATCCAAAGTGT-3′). Lentiviruses were collected from media of 293FT cells cotransfected with pLKO.1-derived plasmids and packaging vectors pCMVdelR8.91 and pMD.G according to the protocols provided by the Taiwan National RNAi Core Facility.

MSCs (2× 104 cells) were preseeded in 24-well plates for 16 h, then infected with lentiviruses at a multiplicity of infection of 8 PFU/cell in the presence of protamine sulfate (8 μg/ml; Sigma). Mock MSCs, shLuc nontarget MSCs, and GRO-γ–targeting MSCs (shGRO-γ-2 and shGRO-γ-4) were incubated for a further 24 h and then replaced culture media with the puromycin (5 μg/ml)-containing media for 2 d. The surviving MSCs were pooled and amplified for use in subsequent experiments. Sequentially, human peripheral CD14+ monocytes were added to mock MSCs or virus-infected MSCs, and iDC processes were performed in DC complete culture media. Suspended cells were collected for further endocytosis assay, and supernatants were determined for GRO-γ cytokine by commercial ELISA (Antigenix America, Huntington Station, NY).

RNA preparation and quantitative RT-PCR

Total RNA was extracted using the TRIzol reagent and was converted to cDNA using a ReverTra Ace set (Toyobo Life Science, Osaka, Japan), according to the manufacturer’s instructions. Real-time PCR analysis was performed using an ABI Prism 7900 system (Applied Biosystems, Foster City, CA). Samples were subjected to the following PCR program: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Analyses were performed in triplicate. For each sample, the cycle threshold (Ct) value was determined. The results were normalized to the levels of the GAPDH gene on the same plate. The level of mRNA expression for different cell groups was calculated using the 2ΔCt method. Primers specific for each gene were designed, and their sequences are as follows: human GAPDH, 5′-GAGTCAACGGATTTGGTCGT-3′ (forward primer [F]), 5′-TTGATTTTGGAGGGATCTCG-3′ (reverse primer [R]); human IL-10, 5′-ATGCCCCAAGCTGAGAACCAAGACCC-3′ (F), 5′-AAGTCTCAAGGGGCTGGGTCAGCTATCCCA-3′ (R); human IDO, 5′-CGCCTTGCACGTCTAGTTCTG-3′ (F), 5′-TGACCTTTGCCCCACACAT-3′ (R); human matrix metallopeptidase-9 (MMP9), 5′-GAAGATGCTGCTGTTCAGCG-3′ (F), 5′-ACTTGGTCCACCTGGTTCAA-3′ (R); human IL-4, 5′-GGCAGTTCTACAGCCACCATG-3′ (F), 5′-GCCTGTGGAACTGCTGTGC-3′ (R); human IL-12p40, 5′-CGGTCATCTGCCGCAAA-3′ (F), 5′-CAAGATGAGCTATAGTAGCGGTCCT-3′ (R); human TNF-α, 5′-GGTGCTTGTTCCTCAGCCTC-3′ (F), 5′-CAGGCAGAAGAGCGTGGTG-3′ (R); human IFN-γ, 5′-CCAACGCAAAGCAATAGCTGC-3′ (F), 5′-CGCTTCCCTGTTTTAGCTGC-3′ (R); human Cox2, 5′-CGGTGAAACTCTGGCTAGACAG-3′ (F), 5′-GCAAACCGTAGATGCTCAGGGA-3′ (R); human PD-L1, 5′-TATGGTGGTGCCGACTACAA-3′ (F), 5′-TGCTTGTCCAGATGACTTCG-3′ (R); human PD-L2, 5′-TGACTTCAAATATGCCTTGTTAGTG-3′ (F), 5′-GAAGAGTTCTTAGTGTGGTTATATG-3′ (R); human TGF-β, 5′-GCAGAAGTTGGCATGGTAGC-3′ (F), 5′-CCCTGGACACCAACTATTGC-3′ (R); human IL-6, 5′-ATTCTGCGCAGCTTTAAGGA-3′ (F), 5′-AACAACAATCTGAGGTGCCC-3′ (R); IL-1β, 5′-ACGAATCTCCGACCACCACT-3′ (F), 5′-CCATGGCCACAACAACTGAC-3′ (R); mouse GAPDH, 5′-GATGCAGGGATGATGTTC-3′ (F), 5′-TGCACCACCAACTGCTTAG-3′ (R); mouse Arg-1, 5′-CTCCAAGCCAAAGTCCTTAGAG-3′ (F), 5′-AGGAGCTGTCATTAGGGACATC-3′ (R); mouse inducible NO synthase (iNOS), 5′-AAAGTGACCTGAAAGAGGAAAAGGA-3′ (F), 5′-TTGGTGACTCTTAGGGTCATCTTGTA-3′ (R); mouse IFN-γ, 5′-CATTGAAAGCCTAGAAAGTCTGAATAAC-3′ (F), 5′-TGGCTCTGCAGGATTTTCATG-3′ (R).

ELISA

Supernatants from the MDDCs alone (5 × 104), CD3+ purified T cells alone (isolated using the human CD3+ Cell Isolation Kit, MACS, 5 × 105; Miltenyi Biotec), or MDDCs (5 × 104) that were cocultured with CD3+ purified T cells (5 × 105) were harvested. The concentrations of IL-4, IL-10, IL-12, IFN-γ, IL-6, and TNF-α in the supernatant were determined in triplicate using commercial ELISA kits according to the manufacturer’s protocol (R&D Systems).

IDO activity assay

The biological activity of IDO can be determined by measuring the level of kynurenine in culture supernatants that were modified from previously described (25). In brief, culture supernatants, or standard samples with defined kynurenine concentration (0–160 μM), were mixed with 30% trichloroacetic acid and centrifuged at 2000 × g for 10 min. Seventy-five microliters of the suspension was mixed with the equal amount of Ehrlich reagent (100 mg p-dimethylbenzaldehyde in 5 ml glacial acetic acid) in a 96-well plate; then the absorbance of OD was measured at 492 nm. Thus, the concentration of kynurenine in determined samples could be calculated according to the standard curve.

Arginase activity assay

Arginase activity was measured in cell lysate using commercial arginase assay kits according to the manufacturer’s protocol (Abnova Systems, Taipei, Taiwan). In brief, cell pellets were lysed for 10 min in 100 μl of 10 mM Tris-HCl (pH 7.4) containing 1 μM pepstatin A, 1 μM leupeptin, and 0.4% (w/v) Triton X-100. Lysates were centrifuged at 14,000 × g at 4°C for 10 min. Subsequently, arginine substrate buffer was added to the supernatant and incubated at 37°C for 2 h. The reaction was stopped; then the concentration of urea was determined by reading OD at 430 nm.

Protein extraction and Western blot analysis

Cells (2 × 106/well) were lysed, and protein concentration in the supernatant was determined by bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL). Protein samples (20 μg /lane) were resolved by 5–20% SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare) according to the manufacturer’s instructions. The expression of MMP9 (BD Biosciences), COX-2 (Santa Cruz), IDO (Santa Cruz), and β-actin (Sigma-Aldrich) were determined in human MDDCs; the expression levels of iNOS and ARG-1 (BD Biosciences) in mouse BMDCs were also detected. The quantification of expression profile was manipulated by ImageJ software.

Confocal microscopy

MDDCs (5 × 104) were plated onto a poly-l-lysine–coated glass slide and fixed by adding 1% paraformaldehyde (Sigma-Aldrich). Cells were then incubated with anti-human IDO Ab (Chemicon, Temecula, CA) and the DyLight 488–conjugated secondary Ab (Sigma-Aldrich). The slides were mounted with 10% glycerol/PBS and sealed with nail polish. Fluorescent images were captured using a Leica TCS SP5 camera (Leica Camera AG, Solms, Germany).

Generation of mouse BMDCs

Murine BM cells were harvested and differentiated into DCs as previously described (26). BM cells (2 × 105 cells/ml) were cultured in RPMI 1640 medium that contained 20 ng/ml recombinant mouse GM-CSF in the presence or absence of the recombinant mouse GRO chemokine. On day 3, half of the volume of the culture medium was removed and replaced. On day 6, BMDCs from the different treatment groups were collected from each dish, washed, and characterized.

Assay to measure the immunosuppressive activity of mouse GRO-treated cells in vitro and in vivo

The in vitro immunosuppressive activity of the GRO-γ–treated cells was evaluated by measuring the inhibition of the proliferation of purified, OVA-specific OT-1 CD8+ T cells stimulated with OVA-primed DCs. Mouse (C57BL/6) BMDCs were differentiated in the presence or absence of GRO-γ collected on day 6 and then resuspended in LCM medium (RPMI 1640 supplemented with 5% FBS, 50 μg/ml gentamicin, 20.25 mM HEPES, 50 μM 2-ME, 100 U/ml penicillin, and 100 μg/ml streptomycin). OT-1 CD8+ T cells (2 × 105 cells/well) were collected by cell sorting using a FACSAria flow cytometer. These cells were cultured alone or with BMDCs (1 × 105 cells/well) or BMDC/GRO-γ cells (1 × 105 cells/well) in the presence of either the OVA257–264 CTL peptide epitope (1 μg/ml) or a human papilloma virus 16 E749–57 peptide (RAH control peptide; 1 μg/ml). The cultures were maintained in 200 μl cultured medium in 96-well U-bottom microplates. Cultures were incubated at 37°C in a 5% CO2 incubator for 3 d. Subsequently, [3H]thymidine (1 μCi) was added to each well followed by a 16-h incubation. Cells were harvested using a semiautomated sample harvester, and the radioactivity (cpm) was measured to quantify cell proliferation using a Packard microplate scintillation and luminescence counter.

For the in vivo immunosuppression assay, mouse BMDCs that were exposed to different treatments were prepared on day −6. OT-1 splenocytes (4 × 107/mouse) were prepared and administered i.v. into 6- to 8-wk-old x-ray preirradiated (2 Gy) B6 mice on day −1. BMDCs that were differentiated in the presence or absence of GRO-γ were collected and incubated with or without the OVA257–264 peptide (1 μg/ml) at 37°C for 1 h. The cells were then washed to remove unbound peptide on the day of injection. The various BMDC preparations (5 × 104 cells /mouse) were s.c. injected into OT-1 splenocyte-reconstituted B6 mice to initiate Ag-specific activation on day 0 in vivo. Mice from each group that were treated with HBSS, BMDCs, BMDCs/GRO-γ, BMDCs/OVA257–264, or BMDCs/GRO-γ/OVA257–264 were sacrificed on day 7. Splenocytes (2 × 105 cell/well) harvested from individual mice were stimulated with the OVA257–264 (1 μg/ml) or an RAH control peptide (1 μg/ml) in 96-well U-bottom plates. Cultures were incubated at 37°C in 5% CO2 for 12, 36, and 60 h. The proliferation of splenocytes from the HBSS group or the different groups of BMDC-primed mice was determined by [3H]thymidine incorporation. Assays were performed in duplicate. Representative data from groups of three mice from three independent experiments are presented.

Statistical analysis

Statistical analyses were performed using GraphPad Prism, version 5.02 (GraphPad Software). The data are presented as the mean ± SEM from at least three independent experiments. The statistically significant differences between the groups were assessed using a one-tailed Student t test.

We considered p values <0.05 to be significant. The degree of significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001.

Results

MSC-CM exerts a suppressive effect on MDDC differentiation and function

We established a standard operating procedure in vitro to induce the differentiation of human CD14+ monocytes into MDDCs and established assays to characterize the phenotypes of myeloid cells at each stage of their differentiation pathways as shown in Fig. 1. To investigate whether the MSC-CM affected the differentiation of MDDCs, the normal culture medium was replaced with MSC-CM during the differentiation and maturation process of human CD14+ cells. We observed a significant reduction in the surface expression of CD40, CD80 HLA-DR, CD11c, DC-SIGN, and CD83 on iDCs that were treated with MSC-CM compared with untreated iDCs (Fig. 2A, upper panel). Similar results were obtained for mDCs (Fig. 2A, lower panel). To further examine whether the MSC-CM affected the endocytic activity of iDCs, iDCs that differentiated in the presence or absence of collected MSC-CM, or differentiated iDCs cultured with MSCs in the separated transwell condition have been prepared. The capability of FITC-dextran uptake for various treatments of iDCs was further analyzed by flow cytometry. The results revealed that the endocytic ability of both iDCs from the MSC-CM and the transwell cultured with MSCs treated groups were significantly reduced compared with controls (Fig. 2B). In addition, the capacity of the mDCs to stimulate allogeneic T cell proliferation in an MLR was also downregulated in the MSC-CM cultured mDCs (Fig. 2C). These results show that MSC-CM exhibits a suppressive effect on MDDC differentiation and functionality.

FIGURE 1.
FIGURE 1.

Phenotypic analysis of the differentiation stages of myeloid-derived DCs by flow cytometry. Purified human CD14+ monocytes (Mo) and MDDCs differentiated using α-MEM medium that contained 10% human AB+ serum in the presence of IL-4 (80 ng/ml) and GM-CSF (80 ng/ml) without (iDCs) or with (mDCs) LPS (1 μg/ml) were surface labeled. Cells were analyzed by flow cytometry. Gray lines indicate isotype controls. The percentages of positive cells that were obtained for the different Abs, CD14, CD64, CD80, CD86, CD40, CD83, HLA-DR, and DC-SIGN, are indicated. Representative results of five independent experiments are shown.

FIGURE 2.
FIGURE 2.

MSC-CM exerts a suppressive effect on MDDC differentiation. Purified CD14+ monocytes from human PBMCs were cultured in DC differentiation medium in the presence or absence of MSC-CM (1/2× volume). MDDCs were untreated (iDC) or treated (mDC) with LPS (1 μg/ml) on day 5 and for an additional 2 d. The phenotype and function of the MDDCs were analyzed on day 7. (A) Surface markers associated with DC maturation were stained and analyzed by flow cytometry. Mean fluorescence intensity (MFI) was determined after analyzing 10,000 cells. (B) The endocytic activities of differentiated iDCs that supplemented with MSC-CM or iDCs cultured in MSC-containing system (transwell) were executed. iDCs were pulsed with FITC-dextran (1 mg/ml) for 30 min at 37°C, and their endocytic ability was assessed by FITC-dextran uptake measured by flow cytometry. FACS profiles are shown for iDCs in the absence of FITC-dextran as a negative control (NC, gray line), iDCs (dashed line), iDCs in the presence of MSC-CM (iDC+MSC-CM, solid black line, left panel), or iDCs in MSCs containing transwell environment (iDC+MSC, solid black line, right panel) after FITC-dextran uptake. Data are representative of three independent experiments. (C) mDCs were cocultured with allogeneic T cells (DC:T = 1:10) for 4 d. Thymidine incorporation was measured after a 16-h pulse with 1 μCi/well [3H]thymidine. T cell proliferation was determined by [3H]thymidine incorporation in triplicate cultures of three different donors. Data are expressed as the fold change relative to the group without MSC-CM supplementation (mean ± SEM in MFI) (A) or cpm (mean ± SEM) of [3H]thymidine uptake (C). Representative results of three separate experiments performed in triplicate are shown. *p < 0.05, **p < 0.01, ***p < 0.0001.

GRO-γ plays a key role in mediating the inhibitory effects of MSCs on the differentiation and function of MDDCs

Next, we investigated which soluble factors in the MSC-CM were responsible for the observed suppressive activity on the differentiation of MDDCs. A significant increase in the GRO chemokine concentration was detected in the MSC-CM by the human cytokine array analysis (Fig. 3A). We further examined the biological effects of the different isoforms of GRO on the phenotype of human iDCs. GRO-α, GRO-β, and GRO-γ were analyzed, and it was determined that only GRO-α and GRO-γ had significant inhibitory effects on CD40 expression (Fig. 3B). Because there was only a very minor increase in the intensity of GRO-α in the cytokine/chemokine array (Fig. 3A), we focused our study on GRO-γ in subsequent experiments. To further confirm the effect of GRO-γ present in MSC-CM on MDDC differentiation, we showed that the suppressive effect of the conditioned medium on iDCs and mDCs was partially reversed with the anti–GRO-γ Ab, but not by the corresponding isotype control (Fig. 3C). In the presence of the neutralizing Ab, the surface-level expression of CD80, DC-SIGN, and CD83 on iDCs was significantly increased compared with cells that were treated with the isotype control or MSC-CM. The anti–GRO-γ Ab also substantially reversed the suppressive effect of the MSC-CM on the surface expression of CD80, CD11c, DC-SIGN, and CD40 on mDCs (Fig. 3C, lower panel).

FIGURE 3.
FIGURE 3.

The suppressive effect of MSC-CM on MDDCs is reversed by addition of an anti–GRO-γ neutralizing Ab to MSC-CM. (A) Comparison of the cytokine profiles from the MSC-CM and the serum-containing control medium was performed using a commercial human cytokine/chemokine Ab array (RayBio). The cytokine and chemokine levels in the culture media were determined by incubating the array membrane with biotin-labeled Abs that were reactive for specific cytokines or chemokines, followed by adding HRP-conjugated streptavidin and then exposing the array to x-ray film. The results are representative of two independent experiments. (B) The influence of GRO chemokines and IL-8 on the expression of CD40 on iDCs was assessed by flow cytometry analysis. Human CD14+ monocytes were differentiated in α-MEM medium containing 10% human AB+ serum in the presence of IL-4 (80 ng/ml) and GM-CSF (80 ng/ml). CD14+ monocytes, iDCs alone, or iDCs supplemented with GRO-α, GRO-β, GRO-γ, or IL-8 were stained for CD40 expression and analyzed by flow cytometry on day 7. Gray lines correspond to unstained controls. The percentages of CD40+ cells for the indicated chemokines are shown. The results are representative of three independent experiments. (C) GRO-γ activity in MSC-CM was neutralized with the addition of an anti–GRO-γ (10 μg/ml) or an isotype-matched control Ab at 37°C for 60 min followed by an incubation at 4°C overnight. MDDCs were differentiated in DC differentiating medium with or without MSC-CM (1/2× volume) in the presence or absence of the neutralizing anti–GRO-γ Ab. Phenotypic analyses of the MDDCs were performed by flow cytometry, and the results are expressed as the fold change of mean fluorescence intensity (MFI) relative to the values obtained for untreated iDC. The means and the SEM obtained from three independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.0001.

In addition to the neutralizing effect of anti–GRO-γ on the cellular properties of MDDCs, we have also prepared the GRO-γ knockdown MSCs to culture with differentiated iDCs to verify the roles of GRO-γ in regulating the immune function of MSCs during DC differentiation. The amount of secreted GRO-γ in the supernatants of iDCs combined with two independent GRO-γ–silenced clones, either shGRO-γ-2 or shGRO-γ-4, was significantly reduced compared with the mock control MSCs and the lentiviral control-infected MSCs (Fig. 4A). The endocytic activity of CD14+ iDCs in the absence or presence of various short hairpin RNA–treated MSCs was determined by measuring the cellular cytofluorimetry intensity after dextran-FITC uptake. The percentage of endocytotic cells in differentiated iDCs alone was 76%, and the capability of Ag uptake was down to around 39% in those either mock MSCs or shLeu-infected MSC cocultured iDCs. Remarkably, both the shGRO-γ-2– and shGRO-γ-4–targeting MSCs restored the partial Ag uptake capability in cocultured iDCs (Fig. 4B).

FIGURE 4.
FIGURE 4.

GRO-γ mediated the immune-modulated activity of MSCs in DC function. MSCs were infected with shLuc (as a nonspecific targeting control), shGRO-γ-2, shGRO-γ-4 lentiviruses or without lentiviruses (as a mock control) in the puromycin (5 μg/ml)-containing media for 2 d. The surviving MSCs (2 × 104) from each treatment were pooled and then cocultured with human peripheral purified CD14+ monocytes in the DC differentiation media for 5 d. (A) The concentration of human GRO-γ in the cultured supernatants for iDCs alone or iDCs combined with various MSCs were determined by ELISA. (B) The endocytosis activity of CD14+ cells in differentiated iDCs alone or iDC contact with various lentiviral-infected MSCs were measured as indicated. One of three experimental results was represented. Results are presented as the mean ± SD for triplicates from three separate experiments. **p < 0.01.

The ability of recombinant GRO-γ to inhibit the differentiation of MDDCs and to suppress their functions was then evaluated. In the presence of the recombinant GRO-γ during monocyte-iDC differentiation, the surface expression of CD40, CD80, CD86, CD11c, CD83, and DC-SIGN on iDCs was significantly reduced compared with the expression levels on cells that were cultured in the absence of GRO-γ (Fig. 5A, upper panel). Similar results were observed with mDCs except that the expression of DC-SIGN was not affected by the addition of GRO-γ (Fig. 5A, lower panel). The addition of SB225002, which is a CXCR2 inhibitor, to the culture medium significantly reversed the suppressive effect of GRO-γ on the expression of these select surface markers on both iDCs and mDCs (Fig. 5A). Again, the expression of DC-SIGN on iDCs and mDCs was not affected by the addition of SB225002. GRO-γ also significantly downregulated the endocytic activity of iDCs and reduced the ability of mDCs to stimulate T cell proliferation in the MLR. These effects were also reversed by the addition of SB225002 to the culture (Fig. 5B, 5C). Thus, these data demonstrate that the GRO-γ secreted by MSCs plays a key role in suppressing the differentiation and function of MDDCs.

FIGURE 5.
FIGURE 5.

GRO-γ suppresses the differentiation of MDDCs. CD14+ monocytes that were purified from human PBMCs were differentiated in the presence or absence of recombinant GRO-γ (250 ng/ml) with or without pretreatment with the CXCR2 agonist SB225002 (250 nM) for 30 min. The phenotype and function of the MDDCs were analyzed on day 7. (A) After surface marker labeling, the phenotypic analysis of the MDDCs was performed by flow cytometry. Bar graphs represent the fold change of MFI relative to the MFI that was observed for untreated iDCs (fold change of MFI ± SEM). The results are representative of three to five independent experiments. (B) iDCs were pulsed with FITC-dextran (1 mg/ml) for 30 min at 37°C, and the uptake of the FITC-dextran was measured by flow cytometry. Data are representative of three separate experiments and are expressed as the fold change of MFI ± SEM relative to the DC group. The right panel also shows the one representative cytofluorimetry profile of endocytosis activity for iDCs in the absence of FITC-dextran as a negative control (NC, gray line), iDCs alone (dashed line), iDCs in the presence of MSC-CM (iDC+ CM, solid black line), and supplement with SB225002 (iDC+ CM + SB, solid fine line) after FITC-dextran uptake. Data are representative of three independent experiments. (C) mDCs were cocultured with allogeneic T cells (DC:T = 1:10) for 4 d. Thymidine incorporation was measured after a 16-h pulse with 1 μCi/well [3H]thymidine. T cell proliferation was determined by [3H]thymidine incorporation. Data are expressed as the fold change of cpm (mean ± SEM) relative to untreated MDDCs and are representative of three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.0001.

GRO-γ drives the differentiation of MDDCs toward an MDSC-like immunophenotype

In addition to the suppressive effects of GRO-γ on the differentiation and function of MDDCs, this chemokine influences the cytokine expression profile during MDDC differentiation. Real-time PCR was performed to examine the relative mRNA levels of selected genes expressed by MDDCs in 7-d cultures. These results showed that the expression levels of inflammatory cytokine genes, such as TNF-α, IFN-γ, and IL-12, were significantly downregulated during the differentiation of MDDCs in the presence of GRO-γ. The expression levels of the IL-10, IL-4, TGF-β, IL-1β, and IL-6 cytokine genes and the genes encoding COX2, MMP9, programmed death ligands (PD-L1 and PD-L2), and IDO were significantly upregulated (Fig. 6A). Human MDSCs are characterized by their ability to secrete IL-10, IL-4, IL-1β, and IL-6, and to express COX2, PD-L1, PD-L2, MMP9, and IDO (27). This real-time PCR data revealed that treating MDDCs with GRO-γ increased the expression of these MDSC marker genes compared with untreated MDDC controls (Fig. 6A). The protein expression levels of MMP9, COX-2, and IDO were increased up to nearly 2-fold in GRO-γ–treated MDDCs compared with those untreated MDDCs (Fig 6B, left panel). Intracellular staining with the anti-IDO Ab showed an increase in the intracellular expression of IDO by iDCs that were differentiated in the presence of GRO-γ (Fig. 6B, right panel). In addition, the quantity of kynurenine, the most prominent intermediate product of tryptophan catabolism by IDO, in culture supernatants of iDC in the present or absence of GRO-γ was revealed. Not only the expression of IDO protein was improved in GRO-γ–treated DC, but the tryptophan-degraded activity was increased in DC+ GRO-γ cells (Fig. 6C). These results indicate that GRO-γ suppresses MDDC differentiation and skews cell differentiation toward an MDSC-like immunophenotype.

FIGURE 6.
FIGURE 6.

MDDCs differentiated in the presence of GRO-γ exhibit a tolerogenic cytokine profile. Purified CD14+ monocytes from human PBMCs were differentiated in the presence or absence of recombinant GRO-γ (250 ng/ml) with or without pretreatment with SB225002 (250 nM) for 30 min. (A) The expression levels of the indicated mRNAs after 7-d cultures of MDDCs were determined by real-time PCR. Ct values were normalized to the expression of the GAPDH gene. Differences were calculated with the 2−ΔCt method, and the data are expressed as the percentage relative to the values obtained for the untreated DCs. (B) The expression of immunomodulatory factors MMP9, COX-2, and IDO were performed in GRO-γ and non–GRO-γ BMDCs. iDCs differentiated for 5 d were fixed on glass slides and were permeabilized with 0.1% (v/v) of Nonidet P-40/PBS. Intracellular IDO expression was detected by staining MDDCs with a mouse anti-human IDO Ab followed by a goat anti-mouse IgG-DyLight 488–conjugated Ab. The image was obtained using confocal microscopy. Scale bar, 10 μm. (C) The biological activity of IDO was verified by measuring the production of kynurenine in cultured supernatants of DC and DC+ GRO-γ. Results are presented as the mean ± SEM for triplicates from five separate experiments. *p < 0.05, **p < 0.01, ***p < 0.0001.

GRO-γ–primed MDDCs drive T cell differentiation toward a tolerogenic immunophenotype

To further investigate the effects of GRO-γ–treated MDDCs on T cell responses, the human CD3+ T cells were purified and exposed to autologous irradiated MDDCs that were either treated or untreated with GRO-γ. The RNA of CD3+ T cells that isolated from the T/MDDCs mixtures was then extracted, and the expression of selected cytokine genes was assessed using real-time PCR. We found that higher levels of expressed IL-4 and IL-10, but lower levels of IFN-γ and IL-12 were expressed by GRO-γ–primed MDDC cocultured T cells (Fig. 7A). The cytokine profile of the MDDC/T cell coculture medium was also analyzed by ELISA. Consistent with the real-time PCR data, we found that the levels of secreted IL-4 and IL-10 were significantly increased in the supernatants from the T cells cocultured with GRO-γ–primed MDDCs. The concentration of IL-12 and IFN-γ in the coculture medium was significantly reduced by the presence of GRO-γ–primed MDDCs (Fig. 7B). These data suggest that GRO-γ–primed MDDCs can drive T cells toward a more tolerogenic phenotype.

FIGURE 7.
FIGURE 7.

T cells primed with GRO-γ–treated MDDCs present with tolerogenic properties. Human CD3+ T cells (3 × 105) were stimulated with 3 × 104 MDDCs differentiated in the presence or absence of recombinant GRO-γ with or without SB225002. (A) The mRNA expression levels of selective genes, IL-4, IL-10, IL-12, and IFN-γ, in magnetic binding CD3+ T cells purified from MDDC and T cell cocultures were evaluated by real-time PCR. Relative gene expression was normalized to that of GAPDH. Data are expressed as the fold change of gene expression relative to values obtained for the DC-primed T cell group. Results are represented as the mean ± SEM from five separate experiments. (B) The cytokine profiles in supernatants that were collected from the GRO-γ–treated and untreated MDDCs that were cocultured with T lymphocytes were analyzed by ELISA. Data are indicated as the mean of the cytokine concentration ± SEM. Results are representative of five separate experiments. ***p < 0.0001.

GRO-γ–treated BMDCs showed MDSC-like characteristics both in vitro and in vivo

To further investigate the function of GRO-γ–primed DCs in vivo, we studied the differentiation of DCs in the presence or absence of GRO-γ in the mouse system. Differentiated BMDCs were collected and stained for mouse MDSC markers using CD11b and Gr-1 fluorescent Abs (Fig. 8A). The expression of the ARG-1 and iNOS genes, which are known to be upregulated in MDSCs and function to inhibit T cell proliferation and apoptosis in mice (28), was further analyzed in the CD11b+Gr-1+ sorted cells. As expected, the mRNA levels of ARG-1 and iNOS were increased in GRO-γ–treated BMDCs (Fig. 8B), and the protein expression levels of Arg-1 and iNOS were upregulated in BMDCs in the presence of GRO-γ (Fig. 8C). The addition of the CXCR2 inhibitor SB 225002 reversed the induction of both of these genes in the GRO-γ–treated BMDCs. Moreover, the arginase activity and the production of nitrate were significantly increased in GRO-γ–containing BMDCs (Fig. 8D). These data indicate that the functional changes in the properties of GRO-γ–treated BMDCs mediate the immunosuppressive functions.

FIGURE 8.
FIGURE 8.

GRO-γ–treated mBMDCs display the MDSC phenotype. (A) Bone marrow cells from C57BL/6 mice were collected and differentiated into BMDCs by treatment with either GM-CSF alone, GM-CSF/GRO-γ, or GM-CSF/GRO-γ/SB225002 for 6 d. Surface labeling of the BMDCs using anti-CD11b and Gr-1 Abs was analyzed by FACS. CD11b and Gr-1 double-positive cells were isolated using a FACSAria cell sorter, and the transcriptional levels of the ARG-1 and iNOS genes were determined by real-time PCR (B). The expression levels of the mRNA are presented as the fold change of gene expression relative to the mRNA levels obtained for the BMDC group and normalized to the GAPDH gene expression. Data are shown as the mean ± SEM of results from three independent experiments. (C) Cell lysates from mouse BMDCs with or without GRO-γ were immunoblotted with Abs specific for Arginase-1, iNOS, and β-actin, respectively. The experiment was repeated three times with similar results. (D) The arginase enzymatic activity was revealed in the 5-d cultured cell lysates of BMDCs and GRO-γ–treated BMDCs, and the nitrate-converting activity of iNOS was performed from the cultured supernatants. Data are shown as the mean ± SD of results from three independent experiments. (E) The biological effects of GRO-γ–treated BMDCs in the Ag-specific and allogenic T cell reactions. OT-1/CD8+ T cells were sorted using a FACSAria and cocultured with GRO-γ–treated or untreated BMDCs in the presence of the OVA257–264 peptide for 3 d. Peptide-specific T cell proliferation was determined using a [3H]thymidine incorporation assay (left panel). For the allogenic T cell response, BALB/c (H2d) splenocytes (1 × 105) were reacted with or without x-ray–irradiated C57BL/6 (H2b) splenocytes (1 × 105) in the presence of either BMDCs (1 × 104) or GRO-γ–treated BMDCs (1 × 104) for 3 d. The allogenic MLR was estimated by a [3H]thymidine incorporation assay (right panel). Data are representative of the mean ± SEM from three independent experiments. (F) Schematic experimental flow chart of assays that were used to assess the immunosuppressive activity of GRO-treated cells in vivo. Step 1: different groups of BMDCs derived from C57BL/6 mouse bone marrow cells were prepared 6 d before BMDC immunization. Steps 2 and 3: C57BL/6 mice were irradiated with 2 Gy; mice were then i.v. injected with OT-1 splenocytes (4 × 107 cells/mouse) 1 d before BMDC immunization. Step 4: the various preparations of BMDCs (5 × 104 cells/mouse) were collected and s.c. injected on day 0 into 6- to 8-wk-old OT-1 splenocyte-reconstituted B6 mice. Step 5: mice from each experimental group were sacrificed, and the splenocytes from individual mice were collected and stimulated with the OVA257–264 (1 μg/ml) or the RAH control peptide (1 μg/ml) in 96-well U-bottom plates on day 7 after BMDC stimulation. The proliferative activity of the splenocytes was determined by [3H]thymidine incorporation (Step 6). (G) BMDCs derived from C57BL/6 mouse bone marrow cells treated with or without GRO-γ were collected and pulsed with OVA257–264 peptide. After removal of the unbound peptides with wash buffer, the different preparations of the BMDCs were resuspended in 150 μl PBS and s.c. injected into irradiated C57BL/6 mice reconstituted with OT-1 splenocytes (4 × 107 cells/mouse). Splenocytes from individual mice from all of the treatment groups were isolated and analyzed for their proliferative activity after stimulation with the OVA257–264 peptide for 60 h on day 7 after BMDC administration. (H) Supernatants from the OVA257–264 peptide–stimulated cells were collected from (G), and the secretion levels of both IFN-γ and TNF-α were determined by ELISA. Data in (G) and (H) are shown as means ± SEM of two to three mice per group from three independent experiments. Statistical significance was determined using a Student t test, and p values are indicated: *p < 0.05, **p < 0.01, ***p < 0.0001. nd, None detectable; ns, no significance.

Next, we used an OVA-specific challenge system to assess whether the MDSCs that are induced by GRO-γ treatment ex vivo were tolerogenic in vitro and in vivo. OT-1/CD8+ sorted T cells were stimulated with GRO-γ–treated or untreated BMDCs in the presence of the OVA257–264 peptide. T cell proliferation was then measured by [3H]thymidine incorporation. The results revealed that the proliferation of the OT-1/CD8+ T cells stimulated by in vitro differentiated BMDCs in the presence of OVA257–264 peptide that was pulsed on day 3 were downregulated by GRO-γ–primed BMDCs (Fig. 8E, left panel). To elucidate whether GRO-γ–treated BMDCs play an actively suppressive role in controlling the proliferating activity of lymphocytes, the BALB/c (H2d) splenocytes have been stimulated with irradiated C57BL/6 (H2b) splenocytes in the presence or absence of GRO-γ BMDCs. Our results showed that the reduction of proliferating activity was only denoted by the GRO-γ–treated BMDCs and not represented by the BMDC group in the allogeneic MLR (Fig. 8E, right panel).

The suppressive effect of the GRO-γ–generated MDSCs on T cells was then tested in vivo. A schematic experimental flowchart that was designed to assess the function of GRO-γ–generated MDSCs is shown in Fig. 8F. Bone marrow cells were differentiated in the presence or absence of GRO-γ for 6 d. The various preparations of BMDCs were collected and s.c. injected into the OT-1 splenocyte-adapted B6 mice. Mice immunized with different OVA257–264 peptide–pulsed BMDC cultures were then sacrificed on day 7. Splenocytes from immunized mice were collected and stimulated with the OVA257–264 peptide in vitro for 12, 36, and 60 h. The results revealed that the proliferation of the OVA257–264 peptide–stimulated splenocytes from mice injected with the GRO-γ–primed BMDCs was significantly reduced compared with cells from mice that received injection with control BMDCs after OVA257–264 peptide restimulation at 60 h (Fig. 8G). The supernatants from the OVA257–264 peptide–stimulated splenocytes from the different experimental groups were also analyzed for cytokine secretion by ELISA. We observed reduced levels of IFN-γ and TNF-α in the supernatants from the GRO-γ–primed BMDC group compared with the untreated BMDC group (Fig. 8H). These data show that GRO-γ–primed iDCs exhibit an MDSC-like phenotype and function in vivo.

Discussion

MSCs reside in the bone marrow and can be mobilized into the bloodstream to migrate to sites of inflammation and injury in various organs and tissues. More importantly, MSCs modulate the cellular development and biological functions of different types of cells of the innate and adaptive immune system in vitro and in vivo (15, 29, 30). Therefore, the immunoregulatory properties of MSCs not only contribute to their role in tissue repair and regeneration (5), but also to their antimicrobial effector functions (31). The secretion of various cytokines/chemokines and the expression of the corresponding cytokine receptors by MSCs either alone or by interaction with other cellular effectors have been demonstrated in MSC maintenance cultures and after stimulation of MSCs (32–34). These data indicate that the fate and biological function of both MSCs and cells that they interact with are determined by the cytokine network in their local microenvironment after interacting with other cells. It has also been documented that MSCs block the differentiation of monocytes and CD34+ progenitors into CD1a+ DCs and inhibit the function of these cells, which are partially mediated by soluble factors, such as IL-6, M-CSF, PGE2, and IL-10 (35, 36). Chiesa et al. (37) showed that MSCs significantly impair the ability of DCs to migrate to lymph nodes and inhibit the T cell priming capability of the DCs. MSCs have also been shown to inhibit the formation of immune synapses by disrupting the distribution of actin within DCs (38). Therefore, the inhibitory effects of the soluble factors secreted by MSCs on DCs need to be further explored. In this study, we found that small quantities of GRO-α and large amounts of GRO-γ chemokines were present in MSC-CM (Fig. 3A), that the suppressive activity of MSC-CM on the differentiation of human MDDCs was significantly reduced when GRO-γ–specific neutralizing Abs were present (Fig. 3C), the endocytic activity of CD14+ iDCs was affected by coculturing with GRO-γ–expressed MSCs but not shGRO-γ–treated MSCs (Fig. 4), and that the addition of GRO-γ to MDDC cultures directed the differentiation of these cells toward an MDSC immunophenotype (Figs. 5, 6). This is the first report, to our knowledge, that demonstrates that GRO directly contributes to the immunomodulatory properties of MSCs by altering the biological activity of differentiated myeloid cells.

The fate of the differentiated cells of the myeloid lineage critically influences the immune response. In our study, the expression of surface markers, the ability of iDCs to take up Ag, and the reactivity of the MLR mediated by human MDDCs were all inhibited by GRO chemokines. This inhibition was reversed in the presence of the CXCR2-specific inhibitor, SB225002 (Fig. 5A–C). In contrast, the expression levels of MDSC-related genes, such as COX2, MMP9, PD-L1, PD-L2, and IDO in human MDDCs (Fig. 6A, 6B) and Arg-1 and iNOS in mouse BMDCs (Fig. 8B, 8C), were upregulated in GRO-treated cells. Furthermore, adding SB225002 to GRO-treated cells reduced the gene expression of Arg-1 and iNOS (Fig. 8B), and also prevented the initiation of the immunosuppressive activity of these cells (Fig. 5C). In this article, we have shown that GRO-γ could drive the differentiation of human MDDCs and mouse BMDCs toward an MDSC-like phenotype. GRO-γ–induced MDSC-like cells that were generated ex vivo were able to inhibit the immune response of autologous T lymphocytes and reduce the secretion of inflammatory cytokines, such as IFN-γ and TNF-α (Fig. 7B, 8H). Our data suggest that myeloid-derived cell differentiation may be regulated by local levels of GRO chemokines in the peripheral blood and bone marrow.

GRO chemokines and their cognate receptor, CXCR2, are known to mediate the recruitment of neutrophils and to stimulate metastasis by regulating angiogenesis in several tumor models and in various human cancers (39, 40). Pappa et al. (41) showed that the serum levels of IL-8, ENA-78, and GRO-α in patients with multiple myeloma were increased. Doll et al. (42) also found that there was a significant increase in the expression of GRO-β, GRO-γ, and IL-8 in colon carcinoma compared with normal tissue, and that the GRO-γ levels were related to metastasis formation. In parallel, the modulation of the expression of CXCR2 has been shown to regulate malignant melanoma growth, angiogenesis, and metastasis (43). These data suggest that GRO chemokines may contribute to cell transformation and tumor growth. Our present results strongly suggest that GRO chemokines may contribute to tumorigenesis through their capability to generate MDSCs. Therefore, it is imperative to elucidate how GRO chemokines and their receptors downregulate the antitumor immune responses. In this study, we have established that GRO chemokines mediated immunosuppression of MSCs and that the GRO family of chemokines may play a crucial role in directing the differentiation pathway of MDSCs. In addition to GRO-γ, GRO-α also exhibited a suppressive activity. These observations warrant further exploration about the role of individual GRO chemokines, the generation of MDSCs, and the physiological control of immunity by MDSCs.

MDSCs are a heterogeneous population of cells that are characterized by the coexpression of the granulocyte differentiation Ag Gr-1 and the α1βM integrin CD11b in mice (23). In healthy mice, the MDSCs represent nearly 20–30% of the bone marrow cells. Approximately 2–4% of the cells in a healthy spleen are CD11b+Gr-1+, but this percentage increases to 50% in tumor-bearing mice (23, 44). These results indicate that the number of CD11b+Gr-1+ cells may correlate with the tumorigenic status. Haile et al. (45) have previously reported that CD11b+CD49d+ monocytic MDSCs are more potent suppressors of Ag-specific T cells in vitro compared with CD11b+CD49d− granulocytic MDSCs. In most cancer patients, MDSCs are defined as cells that express the common myeloid marker CD33 but lack the expression of the markers of mature myeloid and lymphoid cells (46). The percentage of CD14+HLA-DR−/low cells is increased in hepatocellular carcinoma patients (47). However, little is known about the biological characteristics and functions of MDSCs in nontumor settings because of the lack of specific markers. In this study, we have demonstrated that GRO-γ–treated MDSC-like cells exhibit tolerogenic properties and have the ability to suppress the effector functions of T cells in both humans and mice. We have also provided evidence that GRO-γ–induced MDSCs that were generated ex vivo are able to reduce the in vivo effector activities of OT-1 T cells in B6 mice. This reduction includes their proliferative response to OVA-pulsed APCs and the secretion of the inflammatory cytokines IFN-γ and TNF-α in response to OVA peptide–specific stimulation. These data indicate that the GRO-γ–treated MDSC-like cells that were generated in vitro may be promising candidates for the treatment of diseases as a result of an exacerbated immune response. The identification of reliable markers that can distinguish MDSCs from non-MDSCs is still challenging. Therefore, the results from our study may offer a great opportunity to characterize and define subsets of human and mouse MDSCs based on the acquisition of differential suppressive properties after GRO stimulation.

Over the last decade, there has been enormous progress in MSC-based therapy in various medical fields, such as transplantation, alloreactive pathology, and autoimmunity. These therapies take advantage of the regenerative capacities, chemoattraction to tissues that are undergoing active remodeling, easy accessibility for isolation and expansion, remarkably low immunogenicity, and the ethical acceptability of MSCs (32, 48). However, numerous unresolved questions about the true identity of MSCs remain because of the absence of a specific cell-surface marker, the complexity of their interactions with other cell types, and the broad range of cytokines/growth factors that MSCs produce. Recently, we have demonstrated that MSCs regulate the functional activation of neutrophils by orchestrating the secretion of IL-17 from activated CD4+CD45RO+ T cells (15). Our results support the concept that MSCs play a significant role in linking adaptive and innate immunity because of their immunomodulatory activities. Indeed, MSCs may not be restricted to an immunosuppressive role, but they also may promote other functional activities of immune cells. This may depend on their interaction with other cell types and the cytokine/chemokine profile that they secrete into their microenvironment. Thus, further efforts are required to better understand the interaction of MSCs with other cells and to characterize the factors and sequential cellular events that dictate their response to environmental stimuli. Furthermore, the hypothesis that MDSCs mediate the immunosuppressive activity of MSCs in vivo could also be induced by MSC-derived soluble factors other than GRO chemokines. These possibilities need to be validated.

There is increasing evidence that MDSCs have the potential to suppress autoimmune responses, including type 1 diabetes (49) and CNS autoimmune diseases (50), in experimental murine models. These observations suggest that the failure of endogenous MDSCs to appropriately control auto-reactive T cell responses in vivo may contribute to the pathogenesis of autoimmune diseases. Thus, the use of well-defined preparations of MSCs and MDSCs may become crucial therapeutic requirements for clinical applications in the near future. Considering the difficulty in producing safe clinical lots of these cells and the uncertainty about the potential of MSCs to control autoimmune or inflammatory diseases, GRO-treated MDSC-like cells could be more reliable candidates for cell therapy because of their high degree of functionality and their stability in vivo. However, the use of either MSCs or MDSCs in large-scale clinical trials should be performed with caution because the underlying mechanism of action of these cells has not been fully elucidated.

In summary, we have established for the first time, to our knowledge, that GRO chemokines and their receptors play a critical role in mediating the immunosuppressive activity of MSCs on the differentiation of cells in the myeloid lineage, in particular, through the generation of MDSC-like cells. Our study may provide a novel, alternative approach to the design of cell-based therapies. GRO-induced MDSC-like cells represent a promising approach for the treatment of human pathologies that result from an exacerbated immune response.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Core Facility of the Flow Activation Cell Sorter at the National Health Research Institutes for performing all sterile cell sorting. RNAi reagents were obtained from the National RNAi Core Facility located at the Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taiwan. We are also grateful to Drs. John Kung and Michel Klein for critically reviewing the manuscript and providing suggestions.

Footnotes

  • This work was supported by the National Health Research Institutes Grants VC-099-PP-01 and VC-100-PP-01 (to H.-W.C.) and VC-099-PP-03, VC-100-PP-03, and IV-101-PP-22 (to S.-C.H.); UST-UCSD International Center of Excellence in Advanced Bio-engineering sponsored by the Taiwan National Science Council I-RiCE Program (Grant NSC100-2911-I-009-101); Taipei Veterans General Hospital Grants VGH100D-003-2, VGH101E1-012, VGH101C-015, and VN101-07; National Science Council, Taiwan, Grants NSC100-2120-M-010-001, NSC100-2314-B-010-030-MY3, NSC100-2321-B-010-019, NSC98-2314-B-010-001-MY3, NSC 100-2911-I-010-503, and NSC 99-3114-B-002-005 (to O.K.-S.L.); and the Ministry of Education, Aim for the Top University Plan.

  • Abbreviations used in this article:

    ARG-1
    arginase 1
    BM
    bone marrow
    BMDC
    bone marrow–derived dendritic cell
    Ct
    cycle threshold
    DC
    dendritic cell
    F
    forward primer
    GRO
    growth-regulated oncogene
    iDC
    immature monocyte-derived dendritic cell
    iNOS
    inducible NO synthase
    mDC
    mature monocyte-derived dendritic cell
    MDDC
    monocyte-derived dendritic cell
    MDSC
    myeloid-derived suppressor cell
    MMP9
    matrix metallopeptidase-9
    MSC
    mesenchymal stroma/stem cell
    MSC-CM
    MSC-conditioned medium
    R
    reverse primer
    rhGM-CSF
    recombinant human GM-CSF
    rhIL-4
    recombinant human IL-4
    uMSC
    umbilical MSC.

  • Received October 4, 2012.
  • Accepted March 12, 2013.

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