Fig. 6

The migration of mouse Th17 cells across the BCSFB in vitro does not require CCR6. Primary mouse choroid plexus epithelial cells (pmCPECs) were used as in vitro model for the BCSFB. (A) Confluent monolayers of non-stimulated or TNFα or IFNγ (10 ng/mL, 16 h) stimulated pmCPECs were double-stained for CCL20 (green) and for E-Cadherin (red) and for nuclei (DAPI, blue). The bar graph shows the quantification of the fluorescence intensity for CCL20 (green) relative to unstimulated pmCPECs. Scale bars = 50 μm. (B) Total amount (pg) of CCL20 secretion of unstimulated and 16 h pro-inflammatory cytokine (10 ng/mL of TNFα) stimulated pmCPECs monolayers towards the basolateral (top compartment) and apical (bottom compartment) side as determined by ELISA. Bar graphs show the mean ± SD of two independent experiments measured in triplicates. (C) The migration of MOG_{35 − 55}-specific WT or CCR6 KO Th17 cells across unstimulated or cytokine stimulated (10 ng/mL of TNFα or INFγ for 24 h prior to assay) pmCPECs from the basolateral to the apical side after 8 h is shown. The number of T cells added to the assay (imput = 4 × 10⁵ T cells) was set as 100% and the numbers of migrated T cells was assessed by flow cytometry. Bars show the mean % ± SD of 4 experiments with three filters per condition. Statistical analysis: (B, C) two-way ANOVA (p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, p < 0.0001 = ****). In B the statistical analysis is based on the combined values of the mean and the upper and lower SD.