Cell lysis and immunoblotting Either tumor cells and APC (either monocytes or DCs) alone, or the mixture of the two were incubated at 37uC. After 2 hours, whole cell extracts were prepared using lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1 mM sodium orthovanadate, 10 mg/ml leupeptin, and 10 mg/ml aprotinin) for 20–30 min on ice. Lysates were separated by electrophoresis on 10% SDS-PAGE gels and then transferred to PVDF membranes (Bio-Rad, Hercules, CA). The blots were probed with anti-phosphorylated STAT3 mAb (Tyr705; Cell Signaling, Danvers, MA) or anti-phosphorylated Jak2 mAb (Tyr1007/1008; Cell Signaling, Danvers, MA) processed with ECL plus Western Blotting detection system (Amersham- Pharmacia Biotech), and exposed to Chemiluminescence BioMax Light Film (Kodak-Industries, Cedex, France). Following stripping the membranes were re-probed with anti-STAT3 mAb (Santa- Cruz Biotechnology, Santa-Cruz, CA) or anti-JAK2 Rabbit mAb (Cell Signaling, Danvers, MA). STAT3 transcription factor activity assay Tumor cells and APC (either monocytes or DC) were incubated either alone or co-cultured together for 2 hours and then nuclear extracts were prepared using Nuclear Extraction kit (Chemicon International, Temecula, CA). The lysates were then used to determine the specific binding of STAT3 to their respective targets using a STAT3 transcription factor assay kit (Chemicon International,) and the results were confirmed with the Human Active STAT3 DuoSet IC kit (R&D systems, Minneapolis, MN). Specificity was determined using non-specific oligomer as well by competing the binding with non-biotinylated oligomer supplied in the kit. Cytokine production Cultures containing 56104 CD4+ T cells and 56104 monocytes in the presence of the superantigen SEB (1 ng/ml; Sigma, St. Louis, MO), and in the absence or presence of tumor cells (56104), were plated in individual wells of flat-bottom 96-well plates (Corning, Corning, NY). Cells were stimulated for 72 hours and conditioned media were collected. IFN-c levels in the conditioned media were assayed by ELISA (R&D Systems). IL-10 secretion was determined in the conditioned media of either monocytes, tumor alone or co-culture of the two using commercial ELISA (R&D Systems). Intracellular staining and Flow cytometry Monocytes and tumor cells were cultured alone or mixed together for 24 hours, with the addition of 2 mM monensin for the last 5 hours of stimulation. Then the cells were harvested, washed twice in PBS, fixed in 4% paraformaldehyde for 30 minutes, washed twice in PBS 1% FCS, resuspended in 0.1% saponin, 1% FCS/PBS, and stained using PE-conjugated anti-cytokine IL-10 (Serotec, Oxford, UK) or anti-phosphorylated STAT3 antibodies (Santa-Cruz Biotechnology), for 30 minutes. Cells were washed twice in PBS, and the immunostained cells (16104 cells/sample) were analyzed on a FACS Calibure flow cytometer (Becton Dickinson, San Jose, CA) using the Cell Quest software. Immunoprecipitation For immunoprecipitations, whole cell lysates (prepared as described above) were precleared for 1 h with protein A-sepharose beads (Sigma-Aldrich St. Louis, MO, USA), and then incubated with the anti-JAK2 Rabbit mAb (Cell Signaling) for at least 1 h and then protein A-Sepharose beads were added and incubated Contact Induced Regulatory DC o.n with gentle rotation. Beads were then washed three times in 500 ml of lysis buffer. Samples were resuspended in reducing SDS sample buffer, heated at 95uC for 5 min, and separated by SDS- PAGE, and proteins were transferred to PVDF membrane (Bio- Rad, Hercules, CA) for analysis by Western blotting. APC phenotype DCs were cultured for 24 hours in the absence or presence of hMSCs, tumor cells or IL-10. CD86 (PharMingen, BD Biosci- ences, Mississauga, ON. Canada), human leukocyte antigen-DR (HLA-DR) (Serotec), CD80 (Biolegend San Diego, CA), CCR7 (BD Biosciences, Mississauga, ON. Canada), ILT3 (R&D Systems) and ILT4 (R&D Systems) expression was measured by direct immunofluorescence using conjugated antibodies, and the immu- nostained cells were analyzed on a FACSCaliber flow cytometer (Becton Dickinson, San Jose, CA) using CELLQuest software (Becton Dickinson). The data were calculated as the mean Fluorescence intensity. APC function CD4+ T-cells were stimulated with anti-CD3 mAb (OKT3; eBioscience San Diego, CA, USA) immobilized on protein A- sepharose beads (Sigma-Aldrich) in combination with soluble anti- CD28 mAb (0.5 mg/ml;R&D Systems) and placed in the upper chamber of a transwell. Either monocytes, hMSC alone or in combination were placed in the lower chamber. After 72 hr. medium was collected and IFN-c levels in the conditioned media were assayed by ELISA (R&D Systems). Supporting Information Figure S1 Cancer cells inhibit TNF-a and IFN-c secretion while inducing IL-10 production. A: Monocytes were used to activate autologuos CD4+ T cells (each 56104 cells per well) with SEB (1 ng/ml) in the presence or absence of either MCF-7 (upper panels) or PC3 (lower panels; 56104 cells), in triplicate wells of a 96 well dish. After 72 hours, conditioned media were collected and the level of IFN-c and TNF-a were determined using ELISA. The data represent the mean values of triplicate samples and standard deviations. Data represents one of three experiments. B: Upper panels: monocytes and PC3 were cultured alone or were co- cultured together in triplicate wells of a 96 well dish. After 72 hours, conditioned media was collected and the level of IL-10 was determined using ELISA. The data represent the mean values of triplicate samples and standard deviations. Lower panels: Monocytes and either MCF-7 or PC3 were cultured alone or were co-cultured together. After 24 hours, the cells were harvested and IL-10 was detected by intracellular immunostaining and flow cytometric analysis. Monocytes that were gated based on their forward- and side-scatter characteristics are shown. Dashed line: isotype control; Filled grey area: monocytes alone; Black line: tumor cells:monocytes co-culture; Comparable results were obtained in three separate experiments. Found at: doi:10.1371/journal.pone.0006846.s001 (0.11 MB TIF) Figure S2 hMSC conditioned monocytes actively inhibit T-cell activation. T cells were activated with anti-CD3 coated beads and soluble anti-CD28 and were placed in the upper chamber of a transwell. Monocytes, hMSC or their co-culture were placed in the bottom chamber. After 72 hours, conditioned media were collected and the level of IFN-c was determined using ELISA. The data represent the mean values of triplicate samples and standard deviations. Data represents one of three experiments. Found at: doi:10.1371/journal.pone.0006846.s002 (0.07 MB TIF) PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6846