Anti mr

Immature DCs (10⁶/mL) were challenged with Pb18 or Pb265 (2 x 10⁵ yeasts/mL) using a DCs/yeasts ratio of 5:1, or treated with LPS (5 μg/mL) for 1, 2, 4, 8, 12, 18, 24 or 48 h. Supernatants were harvested and assayed for PGE₂ levels using a competitive enzyme-linked immunosorbent assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). In some experiments, DCs were incubated for 2h with monoclonal antibodies: anti-TLR2 (2 μg/10⁶ cells), and/or anti-MR (2 μg/10⁶ cells) (monoclonal antibodies purchased from Biolegend, San Diego, CA, USA), anti-dectin-1 (3 μg/10⁶ cells) and anti-DC-SIGN (4 μg/10⁶ cells) before fungus challenge (monoclonal antibodies purchased from R&D Systems, Minneapolis, MN, USA). These concentrations were chosen because they induced the highest percentages of blockage in previous experiments (data not shown). The protocols were designed in order to block three receptors keeping only one available.

Spleen cells and PECs were analyzed by FACS at 2 days and 8 weeks post-infection and from non-infected mice. Cells were analyzed for the expression of different markers and were previously gated from a high forward light scatter (FSC)/high side light scatter (SSC) gate. For the assay, 1 × 10⁶ cells were incubated with anti-CD16/CD32 antibody (Biolegend, San Diego, CA, USA) to block non-specific binding. Next, cells were stained with specific anti-F4/80, anti-PD-L1, anti-PD-L2, anti-MR, anti-CD11b, anti-Ly6C, anti-Ly6G (all from Biolegend), and anti-CCR2 (R&D Systems) and incubated for 30 min at 4 °C. Cells were washed twice with 1 mL of FACS buffer (1% FBS and 0.5% of sodium azide in PBS). Cell analysis was performed using the FACsAria system and FloJo software.