Figure 4
Anti-CD3 antibody in conjunction with FCS or albumin amplifies TH17 polarization.
TH17 polarization with coated versus soluble cytokine preparations in the absence and presence of anti-CD3 and anti-CD28 antibodies (n = 6, 3 exp., Bonferroni after ANOVA). (b) Addition of anti-CD3 and anti-CD28 together with coated and soluble preparations (n = 6, 3 indep. exp., Bonferroni after ANOVA). (c) Addition of IgG isotype (n = 6, 3 indep. exp., Bonferroni after ANOVA). (d) αβTCR expression on the T cell surface after TH17 polarization with addition of anti-CD3 and anti-CD28 antibodies together with cytokines (IL-6, IL-23 and TGFβ) in solution or coated form (n = 6 from 3 indep. exp.). (e) Pre-adsorption of the cell culture plate with 10% FCS (n = 4, 2 indep. exp., Bonferroni after ANOVA). (f) Effect of coating cell culture grade FCS (0.5 μl 10% in PBS) or 0.1% BSA (0.5 μl 0.1%, n = 4, 2 indep. exp). (g) Coating with 0.5 μl and 1.5 μl 0.1% BSA as used as carrier protein with and without an additional filtration step (0.2 μm, n = 8, 4 indep. exp., Bonferroni after ANOVA). For TH17 polarizations, cytokines were added at 1x concentration (50 ng/ml IL-6, 1 ng/ml TGFβ, 20 ng/ml IL-23) to the media of all cells, coating with 5x of these concentrations is indicated in the legends, polarizations were conducted for 4 days and all cells were restimulated with PMA/ionomycin.
