Preparation Of Monoclonal Antibody And Recombinant Protein For Human CD83 And Mechanism Study Of Its Inhibition On T Cells

As a type I immunoglobulin superfamily glycoprotein, human CD83 contains 205 amino acids, with a molecular weight of ~45kD. The extracellular compartment of CD83 is composed of 128 amino acids which are highly glycosylated and possess a V-type Ig-like domain. The 39 amino acids of its cytoplasmic tail contains no tyrosine, thus possibly no direct signal is transduced by the short compartment as the lack of immunoreceptor tyrosine-based inhibition or activation motifs (ITIM or ITAM). The murine CD83 is a 196 amino acid protein which shares 63% amino acid sequence homology with that of human. The counter receptor for CD83 has not yet been identified although experiments using recombinant CD83 protein had demonstrated its presentation on monocytes as well as immature and mature DCs.The protein-coding gene for CD83 is located in 6p23 and including five exons. Exon 1 is coding for a signaling peptide, exon 2 and exon 3 are coding for the extracellular domain of CD83 (among which exon 3 is responsible for the largest part of the V-type Ig-like domain). Exon 4 and exon 5 encode the transmembrane region and the intracellular domain of CD83, respectively. Sequence alignment showed that CD83 possess five cysteine residues in its extracellular domain, including a conserved cysteine residue near the transmembrane region that can form dimmer by intermolecular disulfide bond. The other four cysteines form intramolecular disulfide bonds which are involved in protein conformation.CD83 is predominantly expressed on mature DC as well as activated T and B lymphocytes and has been a marker for the former. In thymus, CD83 expressed on thymic stromal cells is essential for the conventional development of double-positive thymocytes to single CD4-positive T cells. However, the function of membrane CD83 (mCD83) in the peripherial immune system remains elusive. In contrast, a great deal of in vitro experiments demonstrated that soluble CD83 (sCD83), either dropped from CD83-positive cells or artificial recombination, exerted significant inhibitory effects on DCs-stimulated T cells. Furthermore, recombinant CD83-Ig or CD83ext prevented the paralysis associated with experimental autoimmune encephalomyelitis (EAE) as well as delayed acute cellular rejection of MHC-mismatched mouse skin allografts. However, the underlying mechanisms for the inhibitory roles of sCD83 remain to be an engima.Here we firstly prepared human CD83-Ig fusion protein as well as mouse monoclonal antibody against human CD83 and employed them as effective tools to observe the role of CD83 on CD3+ T cells and explored the underlying mechanism. We demonstrated that CD83-Ig inhibits proliferation and production of IL-2 and IFN-γby T cells, and the inhibitory effect of CD83 is mediated by monocytes. PGE2, but not IL-10 or TGF-β, was specifically upregulated by CD83-Ig in monocytes. Consistent with higher levels of PGE2, COX-2 expression was also increased upon CD83-Ig treatment. Finally, application of COX-2 selective inhibitor NS-398 fully prevented CD83-Ig-triggered inhibition of T cell responses. Our study establishes a new immune regulatory mechanism by CD83 via stimulation of PGE2 production in monocytes. The dissertation is composed of two parts.1. Preparation and identification of human CD83-Ig fusion protein and mouse monoclonal antibody against human CD83By using overlap extension PCR techniques, we prepared CD83-Ig fusion protein which is composed of extracellular domain of human CD83 cDNA (amino acids 1-128) and the Fcγportion of human IgG1 (hinge, CH2, and CH3). The recombinant protein is a single strand as the Fc part has two site-directed Gâ†'C mutations at sites 326 and 335, which mutate cysteine to serine and thus interfere with the interchain dilsulfide bond linkage within hinge region. Using a protein G column, soluble CD83-Ig was purified from newborn calf serum free media conditioned with transfectant CHO cells. Coomassie staining showed the size of fused protein was ~54KD and it can be recognized by specific anti-CD83 mAb in Dot-blot assay. Furthermore, competition analysis showed that preincubation of CD83-Ig with PE-conjugated HB15e abrogated in a dose dependent way the binding of the latter to L929/CD83 transfectants. So, it is likely that fused CD83-Ig protein is considered bioactive and thus relevant functional studies can be performed based on this protein. After pretreatment with mitomycin C, L929/CD83, a transgenic cell line highly expressing CD83 molecule, was used to immunize BALB/c mice. Spleencytes from the immunized mice were fused with mouse myeloma cells SP2/0 by using the classical hybridoma technique and L929/CD83 transfectants were employed to screen anti-CD83 mAb secreting hybridomas. Through repeated sub-cloning and screening, one hybridoma cell line (1E11) against CD83 was eventually obtained. The hybridoma grew well after long-term culturing and storage in liquid nitrogen. Fast-strip analysis showed that subclass of 1E11 is IgG1 and the light chain belongs toκ. After primed with pristine, ascite was induced in BALB/c mice by intraperitoneal injection of well-grown 1E11 hybridoma and the output is average to 3 mL each mouse. Affinity chromatography was used to purify mAb, and the concentration of protein is 1.5~2.0 mg/mL. Indirect immunofluorescence assay suggested the engagement of purified CD83 mAb to cells was 0.2-1μg/1×106 cells. Dot-blot showed that 1E11 spcifically bind to recombinant CD83-Ig the same way as commercially available anti-CD83 mAb HB15e. Competition experiment indicated that 1E11 recognized the same antigen epitope with that of commercially available HB15e. Thus 1E11 is a specific monoclonal antibody against human CD83 molecule.2. CD83-Ig stimulated monocytes suppress T cell function via production of PGE2CD83-Ig inhibited anti-CD3 mAb triggered proliferation as well as IL-2 and INF-γproduction of PBMCs in a dose dependent manner. The inhibition of proliferation at 0.5, 1, 2, 5, and 10μg/mL recombinant protein was 84.7%, 72.6%, 53.7%, 47.8%, and 43.3% respectively. Accordingly, PBMCs stimulated with 10μg/mL CD83-Ig produced 4~5 fold lower IL-2 and about 60%~70% reduction of IFN-γsecretion as compared with anti-CD3 stimulation alone. However, CD83-Ig had any significant effect on the proliferation of purified T cells stimulated with agonistic anti-CD3 and anti-CD28 mAb. Being as a binding tool, CD83-Ig was found to bind to ~65% of fresh CD14+ monocytes whereas almost no binding of fusion protein was observed in freshly isolated CD3+ cells. Upon being stimulated with anti-CD3 and anti-CD28 mAb for 3 days, the expression of counter receptor on T cells increased slightly to about 2%. Next, cell-free supernatants collected from CD83-Ig-stimulated 24h monocytes cultures resulted in 51% and 48% proliferation suppression of T cells stimulated with anti-CD3 and anti-CD28 mAbs. Likewise, secretion of IL-2 and IFN-γwas inhibited by ~78% and ~45%, respectively. Further analysis of the supernatants showed that PGE2, but not IL-10 or TGF-β, was specifically upregulated by CD83-Ig in monocytes. Consistent with higher levels of PGE2, COX-2 expression was also increased upon CD83-Ig treatment. Finally, application of COX-2 selective inhibitor NS-398 fully prevented CD83-Ig-triggered inhibition of PBMCs as well as of T cells cultured with monocyte supernatants.Taken together, the successful cloning of CD83-Ig-coding gene, construction of recombinant vector, preparation of cell lines stablly express fusion protein as well as production of anti-CD83 mAb have laid a good foundation for further study CD83 expression and functions. Furthermore, we firstly demonstrated that PGE2, produced by monocytes upon CD83 stimulation, was a key mediator of T cell suppression. Our studies reveal a novel regulatory mechanism by which sCD83 suppresses T cell functions and thus provides a new insight on the regulatory function for CD83 in modulating magnitude of immune responses.