Figure 4
EpCAM-restricted paracrine maturation of immature DC by anti-EpCAM:CD40L. A Indicated mono- and/or co-cultures of HT1080, HT1080.CD40 and DLD-1 were treated with increasing concentrations of anti-EpCAM:CD40L and CD40 signaling assessed by measuring production of IL-8. B Indicated mono- and co-cultures of HEK.YFP, HEK.EpICD-YFP, HEK.EpCAM-YFP and immature moDC (iDC) were treated overnight with increasing concentrations of anti-EpCAM:CD40L. iDC maturation was assessed by determining the concentration of IL-12/23 in the culture supernatant. To determine antigen-dependent effects, cells were pre-treated with parental anti-EpCAM blocking antibody. C iDC were co-cultured overnight with the indicated tumor cell lines in the presence or absence of 100 ng/mL anti-EpCAM:CD40L ± anti-EpCAM blocking antibody. iDC maturation was assessed by determining the concentration of IL-12/23 in the culture supernatant. D HEK293.YFP, HEK293.EpICD-YFP or HEK293.EpCAM-YFP cells were co-cultured for 2 h with iDC at a 1:1 ratio in the presence or absence of anti-EpCAM:CD40L. Phagocytosis by the CD80high/CD86high population was determined using flow cytometry. E iDC were co-cultured for 3 days with the indicated tumor cell lines in the presence or absence of anti-EpCAM:CD40L. Co-cultures were harvested, inactivated and replated at the indicated ratio with PBMCs. T cell proliferation was assessed 6 days later as described.