Cells were analyzed on a FACScan flow cytometer (BD Biosciences). Cytokines (IL-4,

IL-10, and IFN-γ) were determined by ELISA using commercially available kits, according to manufacturer’s instructions (BD Biosciences). The sensitivity limits of the assays were 7 pg/mL for IL-4 and 30 pg/mL for IL-10 and Cilomilast research buy IFN-γ. CD4+CD25− and CD4+CD25+ T cells were isolated from pooled draining LN cells of L. major infected mice or from spleens of normal mice (n = 4) using a mouse TREG-cell isolation kit (Miltenyi Biotec, Bergish Gladcach, Germany) according to the manufacturer’s instructions. The suppressive capacity of TREG cells was studied in co-culture suppression assays, which were set up in 96-well plates

in RPMI 1640 (Gibco, Stem Cell Compound Library cell assay CA, USA) supplemented with 10% heat-inactivated fetal bovine serum Gibco). Proliferation was assessed by (3H)-thymidine incorporation. Briefly, CD4+CD25− (TEFF) cells isolated from draining LNs of infected WT mice (or Lgals3−/− mice, when indicated) were seeded at 5 × 104 cells per well and restimulated with 20 μg/mL of L. major antigen. Then, CD4+CD25+ TREG cells or CD4+CD25− T (TEFF) cells from either WT- or Lgals3−/−-infected mice were incorporated to cultures at different ratios. At day 5, proliferation was measured by adding 0.5 μCi (3H)-thymidine (Amersham Biosciences, Piscataway, NJ, USA) to each well. After 12 h, radioactivity was measured using a β-plate counter (Packard, Canberra, Australia). Culture supernatants were collected for cytokine measurement by ELISA. Tests were set up in triplicate. For differentiation of naïve CD4+CD25− T cells into a TREG-cell phenotype, CD4+CD25− T cells were enriched from total spleen cells of WT or Lgals3−/− mice by negative selection. CD4+CD25− T cells were resuspended at 1 × 105

cells per well in RPMI 1640 medium plus 5% fetal bovine serum, seeded in a 96-well plate coated with anti-CD3 mAb (BD Biosciences) at BCKDHA the indicated concentrations, and stimulated with soluble TGF-β1 (3 ng/mL), IL-2 (20 ng/mL), and anti-CD28 mAb (at the indicated concentrations) (all from BD Biosciences). In some experiments, cells were cultured in the presence of different concentrations of DAPT(1–10 μM, Sigma-Aldrich). After 5 days of culture, cells were harvested and analyzed for CD25 and Foxp3 by flow cytometry as described above. Cytokines were measured in culture supernatants by ELISA. Footpad tissue from infected WT and Lgals3−/− mice was frozen in Tissue Tek (Qiagen, CA, USA) medium and cut into 8–10 μm sections.