A total of 1 x105 CFSE-labeled CD4+ or CD8+ T cells were co-incubated with allogeneic CD40-B cells as stimulators at different B to T cell ratios ranging from 1:1 to 1:20. After 5–7 days proliferation was assessed by flow cytometry. Statistical analysis Data are reported as means ± standard deviation unless stated otherwise. Student’s t test or, where appropriate, CH5424802 purchase two-way analysis of variance followed by Bonferroni’s post-hoc test was used to compare groups. P values of <0.05 were considered statistically significant. Results Phenotype of CD40-activated B

cells Upon activation via CD40 B cells upregulate the expression of MHC class II, costimulatory molecules, and adhesion molecules and as a consequence they acquire potent T-cell stimulatory activity. We therefore first studied the effect of IL-10, TGF-β, and VEGF on the morphology and cell surface expression of HLA-DR and costimulatory molecules of CD40-activated B cells. The upregulation of adhesion

molecules such as ICAM-1 results in the formation of round clusters through homotypic adhesion of activated B cells. As shown in Figure 1 IL-10, TGF-β, and VEGF had no impact on cluster formation of CD40-activated B cells. Figure 1 Morphology of CD40-activated B cells. Cluster formation of CD40-activated B cells through homotypic adhesion is not affected by IL-10, TGF-β, or VEGF for 4 days. For the same activation protocol used in this work we have repeatedly shown a strong upregulation of CD80, CD86 and HLA-DR both for B cells of healthy donors and of cancer patients [28, 29]. Thus, we used the expression KU55933 chemical structure levels of vehicle treated CD40-activated B-cells as baselines and these were compared to the expression levels of cells exposed to the immunosuppressive cytokines. In a series of experiments no statistically significant differences between CD40-activated B cells treated with IL-10, TGF-β, or VEGF in comparison to controls were observed (Figure 2). Figure 2 Phenotype of CD40-activated B cells. CD40-activated B cells were cultured on CD40L-expressing NIH3T3 fibroblasts in the presence of 40 ng/ml IL-10, 10 ng/ml TGF-β, 4��8C 20 ng/ml VEGF or vehicle. After 4 days in culture the surface

expression of HLA-DR and the costimulatory molecules CD80 and CD86 by CD40-activated B cells was assessed by flowcytometry. Shown is the mean fluorescence intensity relative to vehicle-treated CD40-activated B cells. The bar graph shows the means of 6 independent experiments ± SD. Proliferation of CD40-activated B cells Activation via CD40 induces proliferation of B cells. We assessed whether the proliferation was inhibited by any of the three immunosuppressive factors. Table 1 Belnacasan cost summarizes the results of the proliferation of CD40-activated B cells cultured in the presence of either IL-10, TGF-β, or VEGF. After four days the cells were removed from the wells and the proliferation was determined by counting. TGF-β and VEGF exerted no effect on the proliferation of B cells activated through CD40.