FIGURE 3.

(A) (Left panel) IgM does not inhibit binding of FITC labelled LPS to BMDC. Here, 20µg of IgM was initially added to 0.2 × 10⁶ BMDC at 4°C for 1 hour and without a wash step, FITC-LPS was added. (Right panel) Polyclonal IgM does not inhibit LPS induced TLR4 downregulation. IgM was added to WT-BMDC for 1 hour at 37°C and without a wash step, LPS was added to activate WT-BMDC for 48 hours. (B) An anti-Fcα/µR blocking antibody (clone TX61) does not prevent IgM mediated downregulation of CD40. Blocking antibody was initially added to WT-BMDC at 4°C for 1hr before adding IgM and LPS. BMDC were then cultured at 37°C for 48hrs. (C) Recombinant soluble PD1-Ig and CD40-Ig (but not PDL1-Ig, CD40L and CD 80-Ig) bind to polyclonal IgM immobilized on sepharose beads. No binding was observed when soluble receptor proteins were added to isotype monoclonal murine IgM or to control polyclonal IgM (i.e., depleted of IgM-ALA with leucocyte adsorption) immobilized on sepharose beads (D) Polyclonal murine IgM does not inhibit binding of soluble CD40L-Ig and soluble PDL1-Ig to their ligands. LPS activated BMDC were pretreated with murine polyclonal IgM and human IgG for 1 hour at 4°C, washed in the cold and then exposed to the ligands at 4°C for 1 hour. Cells were exposed to human IgG to prevent binding of soluble protein via the fused human Fc. PDL1 ko BMDC were used to evaluate binding of soluble PDL-1 as the high expression of PDL-1 receptor on WT-BMDC tended to partially mask the increase in PDL1 expression after soluble PDL1-Ig bound to PD1. Data in Fig 2 are representative examples of at least 3 separate experiments. For flowcytometry panels of BMDC, dead or GR1+ cells were excluded.