Figure 3

CD40L remains biologically active in the context of scFv:CD40L fusion proteins. A Phenotypical and morphological characterization of monocyte-derived DC (moDC) used for the study. After induction, moDC were harvested, stained with PE-conjugated antibodies specific for CD40, CD80, CD83, CD86 and HLA-DR, and expression of the indicated molecules assessed using fluorescent microscopy. B-D Immature moDC were treated for 3 days as indicated and cells with high levels of co-expressed CD80 and CD86 quantified using flow cytometry (B). In parallel, the expression of CD83 and CCR7 was assessed in HLA-DR^high and HLA-DR^low cells (C-D). E Representative flow cytometric plots of CD80, CD86, HLA-DR, CCR7 and CD83 expression on moDC matured for 3 days with 1 μg/mL anti-EpCAM:CD40L. F iDC and anti-EpCAM:CD40L-matured DC (1 μg/mL) were co-cultured with 1x10⁵ CFSE-labeled allogeneic CD3⁺ T cells at the indicated cell numbers. T cell proliferation was determined by assessing loss of CFSE. G iDC were matured using the indicated concentrations of anti-EpCAM:CD40L and allogeneic T cell proliferation assessed as described. H Representative CFSE profiles of the experiment performed in G.