Figure 4.
sCD83 binds CD154 on the surface of sTh2 cells. (A) PBMCs were isolated from blood samples obtained from AR patients (sensitized to HDM) and cultured in HDM (5 µg/ml) overnight, and then exposed to sCD83 (labeled with FITC) in the culture for 6 h. The cells were stained with anti-CD4 and IL-4 antibodies, and analyzed by flow cytometry. Gated dot plots show the sCD83-labeled cells (indicate that the sCD83 bound to a portion of PBMCs); these cells were also CD4+ IL-4+ (B) and CD154+ (C). (D) CD83+ CD154+ cells were isolated by FCCS and analyzed by co-IP. Immunoblots show a complex of CD83/CD154. (E-F) Bars show immunoblot image analysis data of panel D. (G) Immunoblots show results of CD154-depletion in sTh2 cells. (H) bars show immunoblot image analysis data of panel G. (I) Bars show Bcl2L12 mRNA levels in sTh2 cells. Data of bars are presented as mean ± SEM. Each dot in bars presents data obtained from each experiment. CD154d: CD154-deficient sTh2 cells (prepared by CD154 CRSPR). Control: sTh2 cells were treated with control CRISPR reagents. IL-5: IL-5 in the culture (20 ng/ml). Statistics of H-I: ANOVA (p<0.0001) followed by Bonferroni test. Data represent 6 independent experiments.