The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation
Figure 6
Biological activity determination of sCD83.
sCD83 bound to the putative CD83 receptor on monocytes but not on T cells. (A and B) PBMC were incubated with soluble CD83 or BSA, and then coated with either PE-anti-CD83 (A) or anti-His followed by FITC-Anti-mouse IgG (B). Cells were then coated with anti-CD3 or anti-CD14 to distinguish the monocyte and T cells. CD14+ monocytes and CD3+ T cells were gated and analysis for CD83 receptor expression. (C) sCD83 suppressed ConA-stimulated PBMC proliferation. PBMCs were stained with CFSE, stimulated with ConA, and cultured with or without 20 µg/mL of sCD83 for the indicated time periods. (D) The means ± the standard error of the mean (SEM) of the MFI from (C) are shown as bar graphs. (E) sCD83 activated the NF-κB pathway. Isolated human PBMCs stimulated by sCD83 (10 µg/mL) were cultured in RPMI 1640 medium that contained 10% fetal bovine sera at 37°C for 12 h. The PBMCs were collected and lysed to detect the activation of the NF-κB pathway using Western blotting.