Improvement Of Experimental Autoimmune Encephalomyelitis By Administration Of CD226 Specific Antibodies

Human CD226 platelet and T cell activation antigen 1 (PTA1), also named as T lineage-specific activation antigen 1 (TLiSA1,1985) and DNAX accessory molecule-1(DNAM-1,1996) was designated as CD226 in the 7th Workshop and Conference on Human Leukocyte Differentiation Antigen (HLDA7) in 2000. In 1996, Human CD226 gene was cloned. The full length of human CD226 cDNA has 2603bp, and its ORF encoding 336 amino acids is composed of 7 exons and 6 introns. There are some phosphorylation sites in its cytoplasmic region. At protein level, CD226 shows 22% homology with CD96 (Tactile) which is expressed on activated T cells and NK cells, and also shares certain homology to BGP-1, CD36, PRR, and drosophila nerve colloid protein-Neuroglian. As a member of the immunoglobulin superfamily (IgSF), CD226 is a 65kDaⅠtype transmembrane glycoprotein, and composed of 318 amino acids. Human CD226 is expressed on cell surface of T cells, NK cells, NKT cells, monocytes/macrophages, megakaryocytes and platelets, and activated vascular endothelial cells, which is involved in a variety of immunological functions, including the differentiation of T cells and megakaryocytes, the cytotoxicity of NK cells against tumor cells and virus infected cells, the activation and aggregation of platelets, and the transmigration of monocytes/macrophages through vascular endothelial cells.In 2003, human poliovirus receptor (PVR/Necl-5/CD155) and its nectin/Necl (nectin-like) family member poliovirus receptor related 2 (PRR-2/ nectin-2/CD112) were identified as the ligands for CD226. CD112 also serves as the receptor for herpes simplex virus (HSV-1 and HSV-2).The cDNA encoding murine PTA1/CD226 (mPTA1/mCD226) was cloned in 2002, which is composed of 7 exons. It was shown that the ORF of murine CD226 has 1002bp, encoding 333 amino acids. The mPTA1 isⅠtype transmembrane glycoprotein, and also belongs to the immunoglobulin superfamily. Murine PTA1 has 67% homology with human PTA1 at cDNA level, and 53% homology at amino acid level. It has been demonstrated that murine CD112 and murine CD155 (Tage 4) are the ligands for murine PTA1/CD226. Murine CD226 can mediate the costimulatory signal in antigen-specific CD8+ T cell, and also participate in cell-mediated tumor specific immunity. Nevertheless, there are not sufficient researches whether and how mPTA1 play the function of immunological regulation in the auto-immunity. Both Th17 cells and Th1 cells could induce the auto-immunity response. However the relationship of the two kinds of cells is not clear. Th1 cells were once thought to be the key induction factor in EAE. Along with more and more researchs, it recently is considered that Th17 cells are indispensable to induce whole immunopathogenesis in organ-specific autoimmunity in animal model. And the theory also has been presented that EAE is primed firstly by Th17 cells. The laboratory mouse holds a special place in biology as the most accessible mammalian genetic system and therefore the organism of first resort in modeling many human trails and disorders. Therefore, in order to explore the biological function and pathological significance, we started to investigate the effect of mCD226 on pathogeny of experimental autoimmune encephalomyelitis.At first, we cloned and stabilized the CHO-mPTA1/Ig cell strain secreting mPTA1-Fcfusion protein and prepared the mPTA1-Fc fusion protein by purifing the culture supernatant. Then we gained the immune serum after immunizing New-Zealand rabbit using mCD226-Fc fusion protein. With the affinity chromatograph technology, we purified the specific poly antibodies against mPTA1. These antibodies have the satisfactory biologic activity and higher purity detected by ELISA, SDS-PAGE electrophoresis and immunofluorescence. Meanwhile, we immunized SD rat with the fusion protein and set up the system of preparing rat monoclone antibody against murine PTA1. Five positive hybrid strains from 5400 clones were selected by ELISA screening. Specific monoclone antibodies against mPTA1 were obtained from the ascitic fluid derivated from nude mice and cell culture supernatant, and could be used for detecting both the fusion protein and the natural murine PTA1 molecule which provide a favourable tool of for research on the expression,distribution and function of murine PTA1.Using these specific antibodies against murine PTA1, the expression of mPTA1 on various murine cell lineage was investigated by immunofluorescence staining technique. The results showed that mPTA1 expressed on the surface of murine mast cell, monocyte/macrophage cell lines, murine dendritic cell and a portion of B cells, but not cells origined from epithelium, fibroblast and poor-differentiated cells.We also found that normal mice splenic cells expressed mPTA1 including T cells, NK cells and macrophages. Although CD4+ T cells and CD8+ T cells all expressed the mPTA1 molecule, and CD8+ T cells expressed higher level of mPTA1 than CD4+ T cells. The expression of mPTA1 on murine splenic lymphocytes (both CD4+ T cells and CD8+ T cells) was increased after alloatigen simulation, MLC.At the same time, we found that the level of IFN-γfrom cell culture supernants in MLC was elevated in the presence of anti-mPTA1 andibodies. The similarly results also could be gained by detecting fluorescent labeling intracellular IFN-γ.To investigate the regulatory effect of mPTA1 antibodies on autoimmune diseases in vivo, we set up the murine experimental autoimmune encephalomyelitis (EAE) model. Immunizing C57BL/6 mice with MOG35-55 peptide plus Bacillus tuberculosis and pertussis toxin, the clinical disorder in nervous system appeared on day 10 after the peptide immunization. Histological examination revealed inflammatory foci characteristic of EAE throughout the CNS of affected animals with typical perivascular infiltration of mononuclear cells. Secondary demyelination was also observed. The level of IFN-γin the EAE mice splenic lymphocyte also was decreased by intracellular cytokine staining, whereas the level of IL-17 was raised up sixty percentages.The administration of anti-mPTA1 antibody could significantly decrease the disease incidence, delay the disease onset and reduce the severity of the initial phase of EAE compared with the control group. The EAE disease incidence of the group with antibody treatment downed from 60% to 30%. The disease onset was postponed on day 14 after immunizing. The clinical mean score of treatment group was cut downed from 2.2 to 0.5. The intracellular level of IFN-γin antibody treatment mice splenic lymphocyte compared with the control group was raised up 85% whereas the level of IL-17 was decreased 87%. So we assumed that PTA1 plays an important role in autoimmunity inflammation by regulating the producing of IFN-γwhich inhibited the Th17 cells in mice.