The Expression and Characterization of Functionally Active Soluble CD83 by Pichia pastoris Using High-Density Fermentation

Abstract

CD83 is a highly glycosylated type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD83 is upregulated during dendritic cell (DC) maturation, which is critical for the initiation of adaptive immune responses. The soluble isoform of CD83 (sCD83) is encoded by alternative splicing from full-length CD83 mRNA and inhibits DC maturation, which suggests that sCD83 acts as a potential immune suppressor. In this study, we developed a sound strategy to express functional sCD83 from Pichia pastoris in extremely high-density fermentation. Purified sCD83 was expressed as a monomer at a yield of more than 200 mg/L and contained N-linked glycosylation sites that were characterized by PNGase F digestion. In vitro tests indicated that recombinant sCD83 bound to its putative counterpart on monocytes and specifically blocked the binding of anti-CD83 antibodies to cell surface CD83 on DCs. Moreover, sCD83 from yeast significantly suppressed ConA-stimulated PBMC proliferation. Therefore, sCD83 that was expressed from the P. pastoris was functionally active and may be used for in vivo and in vitro studies as well as future clinical applications.

Nsp1α of Porcine Reproductive and Respiratory Syndrome Virus Strain BB0907 Impairs the Function of Monocyte-Derived Dendritic Cells via the Release of Soluble CD83

“…HCMV also infects human DCs and suppresses allogeneic T cell proliferation by inducing the release of sCD83 (51). E. coli-expressed sCD83 displays high levels of suppression in mixed lymphocyte reactions (MLRs) in vitro and can reverse established experimental autoimmune encephalomyelitis (EAE) in vitro and in vivo (58). Although CD83 plays essential roles in both the central and peripheral immune systems, the underlying mechanisms by which CD83 regulates immune responses remain unknown, and how CD83 and sCD83 exert opposite regulatory effects on the immune system is not understood.…”

The CD83 Molecule – An Important Immune Checkpoint

“…Human CD83 contains five cysteine residues in the extracellular domain that form two pairs of disulfide bonds. It is hypothesized that the fifth cysteine is hindered from forming an intermolecular disulfide bond by the presence of several protein surface-attached oligosaccharides (11,12).…”

Continuous expression of CD83 on activated human CD4+ T cells is correlated with their differentiation into induced regulatory T cells

“…Being absent from the majority of resting cells, CD83 is predominantly induced on the surface of mature dendritic cells (mDCs) and activated T and B lymphocytes ( 1 – 3 ). Increasing evidence has demonstrated the significant regulatory roles of CD83 in the central and peripheral immune system ( 4 – 14 ). CD83 was demonstrated to be essential for the lineage commitment of CD4 + T cells in the thymus ( 4 ).…”

“…The co-stimulatory effects of mCD83 on mDCs per se for CD4 + T cells have remained controversial ( 4 , 6 – 8 ). However, soluble CD83 (sCD83), which is produced predominantly by ectodomain shedding, has clear suppressive effects in vitro and in vivo ( 9 – 14 ). A previous study by our group demonstrated that sCD83 suppresses T-cell proliferation and the secretion of interleukin (IL)-2 and interferon (IFN)-γ through prostaglandin E2 (PGE 2 ) produced by monocytes ( 15 ).…”