Anti b220

Single-cell suspensions of splenocytes were obtained from naive, infected/non-treated and infected/treated mice at 8 and 14 days p.i. Spleen cell suspensions were obtained by mechanical dissociation in PBS, then filtered in 0,70 µm strainer. 20% of each spleen was used for immunophenotyping by FACS, 80% left was dedicated to neutrophils and monocytes sorting. Red blood cells were lysed (ACK, Lonza) and an enrichment with biotinylated anti-B220 (BD Biosciences), anti-CD3 (BD Biosciences), following of anti-biotin Ab magnet-bead coupled (Miltenyi) and magnetic LS-columns (Miltenyi) was performed to remove spleen lymphocytes and increase the sorting efficacy. Cells were stained with specific markers of populations of interest (Ly6G-BD BioSciences, Ly6C-BioLegend, CD11b-BD BioSciences) and neutrophils (Ly6G^hi) and inflammatory monocytes (Ly6C^hi) were sorted using the BD Biosciences FACSAria device. Sorted cells (>97-98% pure) from naive, infected/non-treated and infected/treated mice showed a viability > 90%. They were cultured in U bottom 96-well plates at a density of 4 × 10⁶ cells/ml (1 × 10⁶ cells/250 µl/well) in 10% FBS-containing RPMI medium. After 24 h of cell culture, cell-free supernatants were collected and stored at −20°C, to allow cytokines and chemokines protein release quantification.

Spleens were snap-frozen in OCT compound (Sakura Finetek, Torrance, CA) at the time of sacrifice. Cryostat spleen sections (5 μm) were fixed in acetone, washed with PBS, and blocked with PBS/5% fetal bovine serum. Spleen sections were stained with: FITC-anti-IgD or -anti-CD45.1/CD45.2 (BD Biosciences); biotin-conjugated PNA (Sigma-Aldrich), HEL, anti-CD45.1/CD45.2, anti-B220 (BD Biosciences), anti-IgM^a, or anti-CD4 (Cedarlane Laboratories); and PE-anti-IgM^a or -anti-CD4 (BD Biosciences). Biotinylated Ab staining was revealed with rhodamine (tetramethylrhodamine)-conjugated streptavidin (Molecular Probes) or 7-amino-4-methylcoumarin-3-acetic acid-conjugated streptavidin (AMCA, Jackson ImmunoResearch) as a secondary reagent. Stained sections were mounted with Fluoro-Gel (Electron Microscopy Sciences), and tissue fluorescence was visualized using a Zeiss Axioplan 2 imaging microscope (Zeiss, Oberkochen, Germany).

Spleens from B6 mice infected at hind footpad with L. major were removed and depleted of red blood cells by Tris-buffered ammonium chloride treatment, followed by nylon wool filtration to obtain enriched T-cell suspensions. Purified CD4 T-cells were obtained by negative selection with a mAb mix, containing anti-CD8, anti-B220, anti-CD11b, and anti-panNK mAbs (BD Biosciences or Ebioscience), and anti-rat IgG magnetic beads (Dynal, Oslo, Norway). Cell suspensions were 85–88% CD4⁺ cells, as stained for detection of residual CD8 T cells or B cells with anti-CD4, anti-CD5, and anti-CD19 (BD Biosciences) mAbs.