Fig. 2

a, b Tumor cells induce IgG4 class switching in B cells. A representative flow cytometry experiment showing increased IgG4 + cells in a SUM159 and b MDA-MB-231 cells co-cultured with primary peripheral B cells. Activated primary B cells were cultured with or without tumor cells for 5–7 days and analyzed by flow cytometry. Graphs show results of three separate experiments. c Graph represents a relative increase in IgG4 protein expression as detected by ELISA in SUM159 and MDA-MB-231 cells co-cultured with B cells compared to control tumor cells. B cells were cultured with tumor cells for 5 days, and then transferred to a new flask. Cells were cultured for 2 weeks and supernatant was collected an analyzed by ELISA. This experiment was performed in duplicate. Error bars represent standard deviation of technical replicants. (d, e) A representative flow cytometry dot- plot showing increased IgG4 positive cells upon co-culture with an EBV-transformed B cell line and (d) SUM159 cells or (e) MDA-MB-231 cells. Activated B cells were co-cultured with or without tumor cells and a blocking IL-10 antibody for 5–7 days and analyzed by flow cytometry. Increase in IgG4 class switching is prevented by addition of an anti-IL-10 antibody during co-culture. Graphs show the mean of three independent experiments. Error bars represent standard deviations Significance is indicated by asterisk, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.01, ns p > 0.05