8-weeks-old C57BL/6 (B6, H-2b) and BALB/c (H-2d) female mice were purchased from Orient Bio (Sungnam, South Korea). The mice were maintained under specific-pathogen-free conditions in an animal facility with controlled humidity (55 ± 5%), 12/12 h light/dark cycle, and temperature (22 ± 1°C). Mice were fed mouse chow and tap water ad libitum. All animal experiments were performed in accordance with the animal care and use committee of The Catholic University of Korea approved the protocols
Generation of MDSCs
Bone marrow mononuclear cells cells (BMMCs) were flushed out the bone marrow cavity of tibias and femurs with α-minimum essential medium (Invitrogen). BMMCs were cultured in 6 well plate at 1 × 106 cells /mL in complete medium supplemented with GM‐CSF (20 ng/mL) and M‐CSF (20 ng/mL) (both from R&D Systems). After three days, they were harvested and stained with CD11c, CD11b, and Gr-1 antibodies after blocking Fc receptors (all from BD Biosciences). CD11c–, CD11b+, and Gr-1+ MDSC populations were sorted using a MoFlo cell sorter (Beckman Coulter). Human PBMC were cultured in 6 well plate at 1 × 106 cells/ml in complete medium supplemented with GM‐CSF(20 ng/ml) and IL-6 (20 ng/ml) (both from R&D Systems). After three days, Lineage−/HLA-DR−/CD33+/CD11b+ human MDSC subsets were sorted using a MoFlo cell sorter (Beckman Coulter). The purity of the sorted MDSCs was >95%.
Generation of Tregs
Mouse splenic CD4+T cell were isolated from spleen by using mouse anti-CD4 microbeads (Miltenyi Biotec, Germany). To isolate Treg cells, CD4+ T-cells were cultured with plate-bound anti-mouse CD3 (1 μg/mL; BD Biosciences), soluble anti-mous CD28 (1 μg/mL; BD Biosciences), anti-IFN-g (2 mg/ml), anti-IL-4 (2 mg/ml), human recombinant transforming growth factor-β (TGF-β; 5 ng/mL; PeproTech, London, UK), and Retinal (0.1 μM; Sigma-Aldrich, St. Louis, MO)(16).
Human CD4+T cells were isolated from PBMC by using human anti-CD4 microbeads (Miltenyi Biotec, Germany). To isolate human Treg cells, human CD4+T-cells were cultured with plate-bound anti-human CD3 (1 μg/mL), soluble anti-human CD28 (1 μg/mL), anti-IFN-g (2 mg/ml), anti-IL-4 (2 mg/ml), human recombinant TGF-β (5 ng/mL) and Retinal (0.1 μM).
After three days, the induced Treg cells were stained with CD4, CD25 and then sorted using a MoFlo cell sorter to obtain a ~95% pure CD4+CD25+ population.
Alloreactive T-cell responses in vitro
Responder CD4+ T cells (responder cells) were derived from Balb/c mice. Antigen-presenting cells (APCs: T-cell-depleted splenocytes) were isolated from Balb/c (syngenic) or B6 (allogenic) mice and irradiated with 3,000 cGy. Responder cells (1 × 105) and irradiated APCs(1 × 105) were cocultured in 96-well plates for 4 days, pulsed with 1 μCi tritiated thymidine (3[H]-TdR; NEN Life Science Products Inc., Boston, MA) at 18 h before the end of the experiment, and counted using an automated harvester (PHD Cell Harvester; Cambridge Technology, Inc., Cambridge, MA).
In vitro co-culture systems
In FACS analysis, mouse or human CD4+T cells (1 × 106) were cocultured with MDSC or Treg alone or combined MDSC(2 × 105) and Treg (2 × 105) cells for 3days in the presence of anti-mouse CD3 antibody (0.5ug/ml). In the mixed lymphocyte reaction assay, responder CD4+T cells (1 × 105) were cocultured with MDSC or Treg alone or combined MDSC(2 × 104) and Treg (2 × 104) cells for 4days in the presence of anti-mouse CD3 antibody (0.5ug/ml). For all experimental conditions, MDSCs or Treg to T cells ratio is 1:5.
Bone marrow transplantation
To develop the aGVHD model, Balb/c were lethally irradiated with 700 cGy and infused with 5 × 106 donor BM cells plus 5 × 106 splenocytes from Balb/c(syngenic) or C57BL/6(donor, allogenic) on day 0. On day1 and day7 after bone marrow transplantation (BMT), recipient mice received MDSCs (1 × 106) and Tregs (1 × 106) individually or in combination. The mice were monitored for clinical signs and body weight. The clinical GVHD was scored twice weekly using the clinical GVHD scoring system (Additional file 1:Table S1) . Each of the five clinical parameters summed up to get the final score at indicated time points.
Histological and immunohistochemical analyses
Mice were sacrificed on day 28 after BMT and organs captured, cryoembedded, and sectioned. Tissue specimens were fixed in 10% formalin buffer and embedded in paraffin. Sections (6 mm thick) were stained with H&E and the histologic score was determined using established scoring systems [31, 32]. For immunohistochemistry staining, sections were stained with primary antibodies against IL-6 (Abcam, Cambridge, England (ab7737)), IL-17 (Abcam (ab79056)), TNF-α (Abcam (ab6671)) and Foxp3 (H-190, Santa Cruz Biotechnology (sc-28705)) overnight at 4 °C, followed by addition of a biotinylated secondary antibody and a streptavidin-peroxidase mixture for 1 h. Color was developed by addition of 3,3-diaminobenzidine (Dako, Carpinteria, CA).
Flow cytometry analysis
Mouse lymphocytes were stained with following fluorochrome conjugated antibodies: CD4(L3T4)-PerCP Cy5.5, CD25(PC61)-APC, Foxp3(FJK-16s)-PE, IFN-g(XMG1.2)-APC, IL-17(eBio17B7)-FITC, B220(RA3-6B2)-APC, CD19(eBio1D3(1D3))-PerCP, CD1d(1B1)- PE, CD5(53-7.3)-FITC, IL-10(JES5-16E3)-APC, CD138(281-2)-PE, and T- and B-Cell Activation Antigen (GL7)-FITC. Human lymphocytes were stained with following fluorochrome conjugated antibodies: CD4(RPA-T4)-PECy7, CD25(BC96)-APC, Foxp3(259D/C7)-PE, IFN-g(4S.B3)-APC, IL-17(eBio64DEC1-FITC, , CD19(HIB19)-FITC, IL-10(JES3-19F1)-APC, CD138(MI15)-PB450. Before intracellular staining, cells were stimulated for 4 h with phorbol myristate acetate (25 ng/mL) and ionomycin (250 ng/mL) in the presence of Golgistop (BD Biosciences, San Jose, CA, USA). Intracellular staining was fixed using a BD Cytofix/ Cytoperm Plus Fixation/Permeabilization Kit and BD Golgistop Kit (BD Biosciences, San Jose, CA). Foxp3, transcription factor was fixed using a Foxp3/Transcription Factor Staining buffer set (eBioscience, San Diego, CA) following the manufacturer’s instructions. Flow cytometric analysis was performed using a cytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA) and collected data were analyzed using the FlowJo software (Tree Star, Ashland, OR).
Enzyme-linked immunosorbent assay
The concentrations of IL-17 and IFN-g in culture supernatants were measured in duplicate using a sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol (DuoSet; R&D Systems, Lille, France).
Data are expressed as the means ± standard error of the mean (SEM). One-way analysis of variance followed by Bonferroni’s post hoc test was used to compare the differences between three or more groups. Statistical significance was considered as P-value < 0.05. All statistical analysis was performed with Prism (standard version 5.01; GraphPad Software, San Diego, CA).