Association Of CD226 Molecule With Tumor And Experimental Autoimmune Encephalomyelitis

CD226 molecule, a member of the immunoglobulin superfamily (IgSF), is a typeⅠtransmembrane glycoprotein which expressed widely by various immunocytes. Although it had several names before including"T lineage- specific activation antigen 1 (TLiSA1)","platelet and T cell activation antigen 1 (PTA1)", and"DNAX accessory molecule-1 (DNAM-1)", it was designated as CD226 in the 7th Workshop and Conference on Human Leukocyte Differentiation Antigen in 2000.Human CD226 gene, located on chromatosome 18q22.3, was successfully cloned in 1996. The full length of human CD226 cDNA is 2664bp, with an open reading frame encoding 336 amino acids. Single nucleotide polymorphisms (SNP) have been reported in human CD226. Murine CD226 gene, located on chromatosome 18 E4, was cloned in our lab in 2002. Three isoforms of murine CD226 were found in addition to its prototype, the full length of which is 2487bp. The intact CD226 is a 65~67 kDa molecule, consisting of extracellular region, transmembrane region and cytoplasmic region. It contains 2 IgSF V like domains and several potential glycosylation sites in its extracellular region and some phosphorylation sites in its cytoplasmic region. CD226 molecule is highly conserved between different species, with similar nucleotide sequences and protein structures, indicating its important roles in immune responses and other biological functions. In addition, CD226 shares high homology with CD96 and other molecules belonging to Nectin or Nectin-like molecule (Necl) family.CD226 is expressed on the surface of a variety of cells, including T cells, NK cells, NK T cells, activated vascular endothelial cells, megakaryocyte/platel- et lineage, monocytes, thymocytes, a subset of B cells, some haemopoietic stem cells and mast cells. It is also localized in murine hippocampus and cerebellum during adulthood and postnatal development. The ligands for human CD226 were identified to be CD112/Nectin-2 and CD155/Necl-5/poliovirus receptor in 2003. The recognition and interaction between CD226 and CD112/ CD155 is involved in a lot of important biological functions or pathological processes, such as: activation, differentiation, and cytotoxicity of CTL and NK cells; proliferation and polarization of CD4+ T cells; endothelial transmigration of leukocytes; activation and aggregation of platelets; regulation of haematogenesis; degranulation of mast cells; antagonism of apoptosis of NK T cells as well as thymocytes; maturation of dendritic cells; formation of immune synapse and neuro-synapse; and some pathological processes including tumor, autoimmune diseases, transplantation rejection, and virus infection diseases.Human CD226 (hCD226) molecule exists in two forms, one is membrane binding form (membrane CD226, mCD226) on cell membrane, and the other is soluble form (soluble CD226, sCD226) in serum or cell culture supernatant. Previous study on a small scale of samples demonstrated that sCD226 was significantly higher in sera from patients of tumor and autoimmune diseases than from healthy donors. However, the only available studies between CD226 and clinical diseases are focused on the expression of mCD226 and/or sCD226 or individual SNP in certain disease whereas its role and possible mechanism involved in tumor and autoimmune disease remains unclear.In this study, monoclonal antibodies (mAbs) against four frequently used tags including human IgG Fc fragment, glutathione-S-transferase (GST), maltose-binding protein (MBP) and thioredoxin (Trx) were prepared and characterized. Base on that, Sandwich ELISA systems for quantitative detection of Fc, GST, MBP, Trx or corresponding tag fusion proteins were established or improved, with the detection limits being 1.2, 15.6, 1, and 0.4 ng/ml, respectively. Using the improved Fc ELISA, hCD226-Fc level in cell culture supernatant was detected and CHO-hCD226-Fc cell line highly secreting hCD226-Fc fusion protein was re-cloned. Purified hCD226-Fc fusion protein was prepared by collection of culture supernatant from this cell line, and purified with anti-Fc mAb labeld Sepharose 4B affinity column. The purity and biological activity of the obtained hCD226-Fc fusion protein was identified by SDS-PAGE, Western blot, and flow cytometry (FCM), respectively. After characterization, the fusion protein was used to be standard in sCD226 ELISA kit or to mimic natural sCD226 in cytotoxicity assay in vitro. In addition, Sandwich ELISA for quantitative detection of sCD226 was also improved.The improved sCD226 ELISA kit was used to detect sera sCD226 levels in 259 cancer patients (including tumors from various origin such as digestive system, respiratory system, hematological system, gynecological system, skeletal system, and nervous system) and 129 normal subjects. It was found that the concentration of sCD226 was significantly higher in serum from cancer patients (0.2-60 ng/ml, median 11.5 ng/ml) than in serum from normal subjects (0.1-23 ng/ml, median 6.3 ng/ml, P<0.001). Meanwhile, FCM analysis demonstrated that mCD226 expression on peripheral blood mononuclear cells (PBMC) from cancer patients (14 cases, 1.1%-56.1%, median 17.45%) was significantly lower than that from healthy controls (12 cases, 43.1%-77.8%, median 65.3%, P<0.001). In vitro, hCD226-Fc fusion protein could significantly inhibit the cytotoxicity of NK cells against its target cells K562 in a dose-dependent manner and the inhibitory rate was 34% when using 1μg/ml of hCD226-Fc in this assay. Three protease inhibitors (1-10-phenanathroline: a metalloprotease inhibitor; AEBSF: a serine protease inhibitor; NEM: a cysteine protease inhibitor) could significantly decrease sCD226 level while increase mCD226 expression of the cultured PBMC or Jurkat cells treated with PMA (phorbol 12-myristate 13-acetate), indicating that the increased serum sCD226 in cancer patients might be shed from the cell surface by certain protease(s), which are often up-regulated when tumor occurs. Furthermore, molecular weight of natural sCD226 from sera samples of tumor patients or healthy controls as well as cell culture supernatants of PMA stimulated PBMC or Jurkat cells was identified by immunoprecipitation and Western blot. A band with molecular weight of ~50 kDa, coincident with the extracellular domain of CD226 molecule, was found in all the four kinds of samples. Interestingly, another weaker band, with molecular massed of ~45 kDa, was observed in sera samples, but not in supernatant samples, indicating that the variety of proteases in vivo could cause the cleavage of sCD226 from different sites of intact CD226.After investigation of the expression, function and origin of sCD226 in tumor, we speculate the following hypothesis: the up-regulated proteases in tumor could increase sCD226 level in serum and decrease mCD226 expression on PBMC by proteolysis; the increased sCD226 shed from cell surface and released into the circulation could bind membrane CD112/CD155 on tumor cells but could not transduce activating signal, which could reduce the binding between mCD226 and its membrane ligands and inhibit the cytotoxicity against tumor cells. We think this might be one of the immune escape strategies used by tumor cells.Besides, ELISA and intracellular cytokine staining was used to study the effect of anti-hCD226 mAb LeoA1 on cytokine secretion profile and cell populations/subpopulations during mixed lymphocyte culture (MLC). LeoA1 could up-regulate IL-10, GM-CSF, and G-CSF level in MLC supernatant while down-regulate IL-2, IFN-γ, TNF-α, IL-12 p40, IL-15, IL-23, and TGF-αsecretion, and the involved cell populations included Th1, Th2, CTL, NK, and monocytes/macrophages/DCs. Using inhibitors of signaling molecules, we found the regulation of LeoA1 in MLC cytokine secretion and cell population might involve p38, JNK and PI3K.Finally, experimental autoimmune encephalomyelitis (EAE) model was successfully established and we confirmed that anti-CD226 antiody treatment could delay the onset and reduce the severity of disease. The mechanism for this effect of CD226 blockade on EAE could be the reduction of inflammatory cell infiltration in central nervous system, decreased CD25+ activated cells and increased IL-10+ cells and Treg cells in spleen.In conclusion, these results provide experimental data for fully understanding the role of CD226 in tumor and autoimmune diseases, and lay foundation for further exploration of its molecule mechanism involved in multiple biological functions as well as pathological processes.