We greatly appreciate Drs

We greatly appreciate Drs. modulator of the immune system. Methods Mice C57BL/6J (B6) and OT-II mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Foxp3mRFPIL-10eGFP mice Mouse monoclonal antibody to TCF11/NRF1. This gene encodes a protein that homodimerizes and functions as a transcription factor whichactivates the expression of some key metabolic genes regulating cellular growth and nucleargenes required for respiration,heme biosynthesis,and mitochondrial DNA transcription andreplication.The protein has also been associated with the regulation of neuriteoutgrowth.Alternate transcriptional splice variants,which encode the same protein, have beencharacterized.Additional variants encoding different protein isoforms have been described butthey have not been fully characterized.Confusion has occurred in bibliographic databases due tothe shared symbol of NRF1 for this gene and for “”nuclear factor(erythroid-derived 2)-like 1″”which has an official symbol of NFE2L1.[provided by RefSeq, Jul 2008]” were a gift from J. Weinstock.32 All animal study protocols conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Reagents, Ags, and Abs The galectin competitive inhibitors (D-galactopyranosyl)–D-thiogalactopyranoside (TDG) and 2,3-sialyllactose were purchased from Carbosynth (Compton, UK), and -lactose was purchased from Fisher Scientific (Waltham, MA, USA). Ovalbumin (OVA)323-339 was purchased from Invivogen (San Diego, CA, USA). The following antibodies were purchased from eBioscience (San Diego, CA, USA), as conjugated to FITC, PE, or allophycocyanin: IFNXMG1.2), IL-17A (eBio17B7), Foxp3 (FJK-16S), CTLA-4 (UC10-4B9), IL-10 (JES5-16E3), and isotype controls. The following antibodies were purchased from BD (East Rutherford, NJ, USA), as conjugated to FITC, PE, V450, or APC: CD4 (RM4-5), CD44 (IM7), CD62L (MEL-14), CD103 (M290), IL-4 (11B11), and isotype controls. Antibodies and recombinant cytokines for T cell polarization AVE5688 were purchased from BioLegend (San Diego, CA, USA). Recombinant human glutathione CD4+ T cell differentiation CD4+ T cells were isolated from spleens by negative selection using the CD4 T Lymphocyte Enrichment Set (BD). Cells were stimulated for 96 h with 1 g/ml anti-CD3 (145-2C11; BD) and 2 g/ml anti-CD28 (37.51; BD) for polyclonal activation. Cells were differentiated to TH1 by supplementation with 10 ng/ml IL-12 and 10 g/ml anti-IL-4 mAb (11B11). TH2 differentiation was induced by supplementation with 10 ng/ml IL-4 and 10 g/ml anti-IFN AVE5688 mAb (XMG1.2). For TH17 conversion, cells were supplemented with 20 ng/ml IL-6, 10 ng/ml IL-23, 1 ng/ml TGF1, and 10 ng/ml IL-1 in the presence of 10 g/ml anti-IFN mAb and 10 g/ml anti-IL-4 mAb. To induce Treg polarization, cells were incubated with 10 ng/ml TGF1, 1 ng/ml IL-2, and 1 ng/ml all-retinoic acid (Sigma, St. Louis, MO, USA). For Ag-specific activation, CD4+ T cells from OT-II mice were incubated with 20 M OVA323-339 in the presence of -irradiated CD4? splenocytes under Treg polarizing conditions as above. For assessment of Gal-8 binding, na?ve CD4+ T cells (CD4+CD62LhiCD44lo) were prepared by FACS from spleens of B6 mice using an Influx Cell Sorter (BD). Flow cytometry Flow cytometry was performed as described previously19 on a FACS Calibur (BD), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Gates were set based on appropriate isotype controls. Treg cell suppression assay CD4+ T cells were isolated AVE5688 from the spleens of Foxp3mRFPIL-10eGFP mice and polarized to Treg in the presence or absence of 1.5 M Gal-8. At the end of the polarization, Treg cells were sorted to 99% purity as RFP+ cells using an Influx Cell Sorter. CD4+ T cells were isolated from the spleen of B6 mice and labeled with CellTracker Green CMFDA (Molecular Probes, Eugene, OR, USA). CMFDA-labeled T cells (6 x 104) were incubated with irradiated splenocytes (6 x 105) and 1 g/ml anti-CD3 for 72 h in the presence or absence of control or Gal-8-polarized sorted Treg cells (at 1 activated T cell:1 Treg, 1:0.5, and 1:0.25). Proliferation of labeled activated T cells was assessed as dilution of CMFDA by flow cytometry. Assessment of Gal-8 binding to cell surface Na?ve T cells or Foxp3+ em in vitro /em -polarized Treg cells were incubated with varying concentrations of FITC-conjugated Gal-8 (0.1 to 0.5 M) for 1 h at 4C, and binding of Gal-8 to the cell surface was measured by flow cytometry. Affinity precipitation assay Affinity precipitation with Gal-8-conjugated agarose beads was performed as described previously.34 Briefly, primary CD4+ T cell lysates were incubated with agarose-conjugated Gal-8 overnight at 4C. Non-specific binding proteins were removed by washing, bound proteins were eluted by boiling the beads in Laemmli sample buffer, and assessed by western blotting. Western blot analysis Cell lysates were electrophoresed.