Functional Assessment of Myeloid-Derived Suppressor Cells, Mesenchymal Stromal Cells, and Regulatory T Cells for the Control of T Cell Function: Implications for Graft-versus- Host Disease

Research Article

Ann Hematol Oncol. 2017; 4(1): 1132.

Functional Assessment of Myeloid-Derived Suppressor Cells, Mesenchymal Stromal Cells, and Regulatory T Cells for the Control of T Cell Function: Implications for Graft-versus- Host Disease

Siegmund DM¹, Schäfer I¹, Koch R¹, Singh A¹, Handgretinger R¹, Rieber N1,2, Hartl D1,3 and Mezger M¹*

¹Department of General Paediatrics, Haematology and Oncology, University Children’s Hospital Tübingen, Germany

²Department of Pediatrics, Kinderklinik Muenchen Schwabing, Klinikum Schwabing, StKM GmbH und Klinikum rechts der Isar, Technical University of Munich, Germany

³Roche Pharma Research & Early Development (pRED), Immunology, Inflammation and Infectious Diseases (I3) Discovery and Translational Area, Roche Innovation Center Basel, Switzerland

*Corresponding author: Mezger M, Department of General Paediatrics, Haematology and Oncology, University Children’s Hospital Tübingen, Hoppe-Seyler- Str. 1, 72076 Tübingen, Germany

Received: December 22, 2016; Accepted: February 11, 2017; Published: February 14, 2017


Uncontrolled T cell responses cause harm in various diseases, which lead to cell-based therapeutic approaches to dampen T cell activation, including mesenchymal stromal cells (MSCs), regulatory T cells (Tregs) and myeloidderived suppressor cells (MDSCs). One major application is graft-versus-host disease (GvHD), a severe complication caused by alloreactive T cells in patients undergoing allogenic stem cell transplantation (alloSCT). Human MSCs are already used for the treatment of GvHD, however, MSCs have to be expanded and their clinical benefit still remains unclear. Therefore, we systematically compared the functional capacity of Tregs, polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and MSCs to suppress alloreactive T cell responses. Freshly isolated PMN-MDSCs showed the strongest inhibition of T cell proliferation compared to MSCs and Tregs, but the available cell number was limited. Thus, we generated cytokine-induced PMN-MDSCs from peripheral blood mononuclear cells (PBMCs) and from bone marrow mononuclear cells (BMMCs) in vitro. BMMC-derived PMN-MDSCs effectively suppressed T cell proliferation and dampened secretion of Interferon-γ, while PMN-MDSCs generated from PBMCs showed weaker inhibition. The effects of BMMC-derived PMN-MDSCs were partially dependent on cell contact similar to freshly isolated PMN-MDSCs. In conclusion, generated PMN-MDSCs from bone marrow might represent a novel cellular therapeutic to dampen excessive T cell responses for the management of GvHD.


alloSCT: allogenic Stem Cell Transplantation; APC: Allophycocyanin; BM: Bone Marrow; BMMCs: Bone Marrow Mononuclear Cells; CFSE: Carboxyfluorescein Succinimidyl (diacetate) Ester; FITC: Fluorescein Isothiocyanate; GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor; HLA-DR: Human Leucocyte Antigen D-related; Ig: Immunoglobulin; IFNγ: Interferon-γ; IL: Interleukin; MSCs: Mesenchymal Stromal Cells; MDSCs: Myeloid-Derived Suppressor Cells; PerCP: Peridinin Chlorophyll; PBMCs: Peripheral Blood Mononuclear Cells; PE: Phycoerythrin; PMN-MDSCs: Polymorphonuclear MDSCs; Stim: Stimulated; Tregs: Regulatory T cells; TGF-ß: Transforming Growth Factor-ß


Unbalanced T cell responses drive a variety of disease pathologies, ranging from autoimmune diseases to graft-versus-host disease (GvHD) [1,2]. Beyond immunosuppressants, several cell types with T cell suppressive effects have been investigated for cellbased therapeutic applications, such as mesenchymal stromal cells (MSCs), myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). But so far, the T cell suppressive capacity of these cell types has not been systematically compared side-by-side. For many hematological diseases, such as leukemia, allogenic stem cell transplantation (alloSCT) is a potentially curative approach, however, limited by the life-threatening complication of GvHD [1]. Transplant conditioning increases danger signals and inflammatory cytokines, followed by activation of alloreactive T cells, leading to tissue damage and further boosting the disease [1,3]. In first clinical trials for the treatment of GvHD patients, cell-based therapies with MSCs [4,5] as well as freshly isolated or in vitro expanded Tregs [6,7] showed feasibility, safety and encouraging outcomes. Furthermore, either BM-derived or in vitro generated MDSCs were found to inhibit GvHD in mice [8,9].

MSCs are multipotent cells that can differentiate into mesenchymal cell lineages. MSCs are found in several human tissues, such as bone marrow (BM), umbilical cord blood or adipose tissue [10]. During in vitro expansion, MSCs are plastic adherent and have a fibroblastic appearance. Human MSCs express CD73, CD90, and CD105 as surface molecules, but not CD34, CD45 and human leucocyte antigen D- related (HLA-DR). MSCs can modulate immune function of various cells, for example T cells, B cells, and natural killer cells [4,11,12].

MDSCs represent a heterogeneous population of immature cells from myeloid cell lineage [13]. MDCSs are functionally defined by their T cell suppressive capacity and are further sub-divided into two subsets. In humans, polymorphonuclear (PMN-) MDSCs express CD11b+, CD66b+, CD14-, and monocytic MDSCs are CD14+, and CD15-[14]. Several groups presented different methods to generate MDSCs in vitro from murine BM cells as well as human peripheral blood mononuclear cells (PBMCs) and BM cells [8,9,15,16].

Tregs are a subset of T lymphocytes and play a central role for the immunological self-tolerance as well as for the control of undesired immune reactions [17]. Tregs are characterized as CD4+CD25highFoxP3+[17]. In contrast to activated effector T cells, no exclusive Treg-specific cell marker is available so far and hence, the isolation of Tregs is still problematic [17,18].

In order to investigate which human cell type, MSCs, PMNMDSCs or Tregs, displays the greatest potential for T cell suppressive therapies, we systematically compared their immunomodulatory effects towards T cells side-by-side. We analyzed the capacity of each cell type to suppress T cell proliferation and the release of Interferon-γ (IFNγ). Furthermore, we analyzed if cell contact is required for the inhibitory effect and if the number of available cells is sufficient for a potential clinical application.

Materials and Methods

Isolation and expansion of human MSCs

Human MSCs were derived from excessive material of standard bone marrow biopsies. Excess material was used after informed consent in accordance with the Declaration of Helsinki and approval by the University Children’s Hospital Tübingen’s IRB (Institutional Review Board [IRB] approval 338/2013 B02). MSCs were cultured in the GMP facility at the Department of General Paediatrics, Haematology/Oncology in Tübingen using animal serum-free medium as described previously [5,19]. In brief, 10-15 ml bone marrow (BM) aspirates of healthy donors were resuspended in DMEM medium (1 g/l glucose, Lonza, Basel, Switzerland) supplemented with 80 IU/ml heparin sulfate, 1 mM L-glutamine (both from Biochrom, Berlin, Germany) and 108/ml irradiated human platelets (University of Tübingen, blood donor center). After 2-3 days of incubation at 37°C and 10 % CO2, non-adherent cells were removed. MSCs were expanded over a period of 3-4 weeks and harvested using TrypLE Select (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Microbial analyses was performed regularly and the purity (>95%) of MSCs was defined by flow cytometry on the basis of CD73, CD105, CD45, and CD3 as well as CD14 (to exclude T cells and monocytes, respectively) (all antibodies from BD Biosciences, San Jose, CA, USA). A characterization of MSCs is shown in the supplement (Supplementary Figures S1,S2).

Isolation of PMN-MDSCs and CD4+CD25+ Tregs

PBMCs were prepared from heparinized peripheral blood of healthy volunteers or buffy coats (Blood bank Tuebingen, Germany) by density gradient centrifugation with Biocoll separating solution (Biochrom) and washed twice with RPMI-1640 medium (Biochrom). Cell viability was checked by dye exclusion of Trypan blue staining solution (Sigma-Aldrich, St. Louis, MO, USA).

PMN-MDSCs were isolated based on previously established methods [20,21]. Briefly, PMN-MDSCs were obtained from the PBMC fraction by labelling with anti-CD66b fluorescein isothiocyanate (FITC) antibodies and two sequential positive selections with anti- FITC MicroBeads (all Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s protocol. CD4+CD25+ Tregs were isolated from the PBMC fraction by using CD4+CD25+ Regulatory T Cell Isolation Kit (Miltenyi Biotec), according to the manufacturer’s protocol. The purity of all isolated cells was >95% as confirmed by flow cytometry. A characterization of PMN-MDSCs and Tregs is shown in the supplement (Supplementary Figures S3,S4).

In vitro generation of cytokine-induced MDSCs

PBMCs or bone marrow mononuclear cells (BMMCs) were isolated by density gradient centrifugation and cultured at 37°C, 5% CO2 with RPMI-1640 (Biochrom) supplemented with 10% FCS (Gibco, Thermo Fi Scientific), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom). Cell density was 5x105 PBMC or 3x105 BMMCs/ml. Cells were stimulated with 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; Genzyme, Cambridge, MA, USA) or in combination of 10 ng/ml GMCSF with 10 ng/ml Interleukin-6 (IL-6; Miltenyi Biotec). Medium and supplements were refreshed every 3-4 days. After incubation for 7 days, adherent cells were removed by Detachin (Genlantis, San Diego, CA, USA). For functional assays, cytokine-induced MDSCs were isolated with CD33 MicroBeads, human (Miltenyi Biotec), according to the manufacturer’s protocol. The purity of isolated cells was >90%, as assessed by flow cytometry. A characterization of the cytokineinduced MDSCs is shown in the supplement (Supplementary Figures S5,S6).

Characterization by flow cytometry

First, cells were isolated and if required, cultured as described above. Cells were washed with phosphate- buffered saline (Sigma- Aldrich), incubated with antibodies for 15 min at room temperature, again washed and analyzed by flow cytometry with FACSCaliburTM (BD Biosciences). MSCs were stained with anti- human CD34-FITC, CD90-Phycoerythrin (PE), CD105-FITC, CD45-FITC (Miltenyi Biotec), CD73-PE, CD271-PE and HLA-ABC-PE (BD Biosciences). PMN-MDSCs were stained with anti-human CD66b-PE, CD11b- Allophycocyanin (APC),CD33-PE, CD14-APC, CXCR4-APC, HLA-DR-Peridinin chlorophyll (PerCP) (Miltenyi Biotec) and CD16-PerCP (BioLegend, San Diego, CA, USA). CD4+CD25+Tregs were stained with anti-human CD45RA-FITC, CD3-PerCP, CD4- APC, CD25-PE, CD127-FITC and FoxP3-APC (Miltenyi Biotec). Cytokine-stimulated CD33+ MDSCs from PBMCs and from BMMCs were stained with anti-human CD14-FITC, CD66b-FITC, CD56- FITC (BD Biosciences), CD33-PE, HLA-DR-PerCP, CD11b-APC, CD3-PerCP, CXCR4-APC (Miltenyi Biotec), CD16-PerCP, CD19- PE, CCR5-PE, and CCR2-APC (BioLegend). Mouse immunoglobulin (Ig) M-FITC, Mouse IgG2a-FITC, Mouse IgG2b-FITC, Mouse IgG1- PE, REA Control (S)-PE, Rat IgG2b-APC, Mouse IgG1-APC, Mouse IgG2a-APC, Mouse IgG2a-PerCP (Miltenyi Biotec), Mouse IgG1- FITC, Mouse IgG2a-PE, Mouse IgG1-PerCP (BD Biosciences) were used as isotype controls. All experiments were at least performed three times in independent experiments.

T cell suppression assay

Responder PBMCs were obtained from healthy volunteers and stained with Vybrant CFDA SE Cell Tracer Kit (CFSE: carboxyfluorescein succinimidyl (diacetate) ester) according to manufacturer’s protocol (Life Technologies). PBMCs were stimulated with 100 IU/ml Interleukin-2 and 1 mg/ml muromonab- CD3 (OKT3) (Janssen-Cilag, Neuss, Germany). In standardized way, 60,000 responder PBMCs per well in a 96-well microtiter plate were co-cultured with 10.000 (ratio 1:0.16), 15.000 (ratio 1:0.25), or 30.000 (ratio 1:0.5) immunomodulatory cells at 37°C and 5% CO2. As a positive control, stimulated responder PBMCs without immunomodulatory cells were used, and as a negative control, unstimulated PBMCs were analyzed. In case of co-culture with MSCs, seeding of MSCs was performed the day before responder PBMCs were added. Except of supplementary figure S7 and S8, all experiments were performed in an allogenic setting. The cell culture media was RPMI-1640 containing 10% donor- specific human serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Biochrom).

After co-culture for 4-5 days, supernatants were collected and frozen at -80°C for cytokine analysis. Cells were harvested and stained with anti-CD4 PE and anti-CD8a APC (BioLegend). Only propidium iodide (BD Biosciences)-negative cells were considered for analysis. To determine polyclonal T cell proliferation, the fluorescence intensity of CFSE from gated CD4+ and CD8+ T cells was analyzed by flow cytometry. Polyclonal T cell proliferation was normalized to responder PBMC without any immunomodulatory cells as 100%. Where indicated, responder PBMCs and immunosuppressive cells were separated by a semipermeable membrane with 0.4 μm pores in a transwell plate (Corning, New York, USA) in order to investigate if cell-to-cell contact is required. In transwell experiments, responder PBMCs and immunomodulatory cells were seeded in two ratios (1:0.16 and 1:0.5) without cell-to-cell contact. All experiments were at least performed three times in independent experiments.

Cytokine analysis

Supernatants from T cell suppression assays were taken on day 4 or 5 and analyzed by using IFN-γ DuoSet ELISA (R&D systems, Abingdon, United Kingdom), according to manufacturer’s protocol.

Statistical analysis

Data are reported as means ± standard derivations (SD).

Statistical analysis was performed by using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA, USA). Differences between the groups were determined by a Mann-Whitney test regarding non- Gaussian distribution. In all tests, a P value =0.05 was considered to be significant (*P = 0.05, **P =0.01, ***P =0.001).


PMN-MDSCs show stronger suppression of T cell proliferation than MSCs and CD4+CD25+ Tregs To investigate the suppressive potential of different immunomodulatory cells, we systematically performed CFSE assays to analyze polyclonal T cell proliferation and checked release of IFNγ by enzyme-linked immunosorbent assay (ELISA). Beforehand, we characterized all immunomodulatory cells by flow cytometry (Supplementary Figures S2-S6). MSCs, PMNMDSCs and CD4+CD25+ Tregs strongly decreased proliferation of T cells in a dose-dependent manner (Figure 1A). PMN-MDSCs suppressed both CD4+ and CD8+ T cell proliferation significantly stronger than MSCs (P=0.009 and P=0.002, respectively) and freshly isolated CD4+CD25+ Tregs (P=0.008 and P=0.04, respectively). At a ratio of 1:0.5, 96.3% of CD4+ and 94.3% of CD8+ T cell proliferation was inhibited by PMN-MDSCs, 85.8% of CD4+ and 83.9% of CD8+ T cell proliferation by MSCs and 91.5% of CD4+ and 88.8% of CD8+ T cell proliferation by Tregs, respectively. In an autologous experimental setting, the inhibitory effect of PMN-MDSCs was decreased compared to the allogenic setting, but in the same range as MSCs and Tregs (Supplementary Figure S7). Surprisingly, MSCs showed a significantly greater inhibition of IFNγ release than PMNMDSCs (P=0.01) and CD4+CD25+ Tregs (P=0.05) implying that different mechanisms for immunomodulation are utilized (Figure 1B). Furthermore, we analyzed the inhibitory effect of MSCs and PMN-MDSCs from the same donors with similar results compared to different donors of the cell types (Supplementary Figure S8).