| Human OX40 ligand (OX40L/CD134L), a member of TNF superfamily, is a type II membrane protein of about 34-40kD encoding 183 aa. OX40L, cloned by Miura et al in 1991, is originally identified as human gp34 located at 1q25 with the length of 1kb, which is mainly expressed on mature dendritc cells (DCs), activated B cells, vascular endothelial cells (ECV), human umbilical vein endothelial cells (HUVEC), macrophages and some organs as heart, skeletal muscles, testis and lung. As one pair of the important costimulatory molecules, OX40/OX40L pathway provides the important costimulatory signal to activate T and B cells in the process of immune response. Engagement of OX40/OX40L promotes CD4+ T cell activation, proliferation and migration, increases the life-span of effector T cells, enhances the germinal center (GC) formation and also promotes DCs maturation and adhesion of cells. Recent research showed OX40/OX40L signal also plays a vital role in the development of anti-tumor immunity and autoimmune diseases. OX40 is an ideal target for immunotherapy because its expression is restricted to the Ag-specific T cells at the site of inflammation or tumor infiltration. Therefore, the construction of OX40L transfected cells, the obtaining of OX40L agnostic or inhibitory reagents and investigation of their effects on OX40/OX40L signal transduction may have significant theoretic and clinical values in tumor, transplant rejection and autoimmune diseases. 1. Cloning of human OX40L gene and construction of OX40L transfected cells.cDNA fragment encoding human OX40L was obtained from the total RNA of matureDCs by RT-PCR and inserted into retrovirus expression vector pEGZ-Term with gene cloning technique. Then the derived recombinant expression vector together with helper virus expression vectors were transfected into packed cell 293T by liposomes to produce recombinant retrovirus, which was used to infect L929 cells for 72h. The transfected L929 cell expressing OX40L stably was obtained through Zeocin selection and grew well after long time passaging in vitro and storage in liquid nitrogen. The expression of OX40L on cell surface stably maintains above 95%.2. The costimulation of L929/OX40L cell line to T cells.After T cells were activated by anti-CD3 with or without anti-CD28 mAbs for 2 days, the transfected cells L929/OX40L or L929/mock were added to the T cells and cocultured for 3 days. 3H-TdR incorporation assay as well as the analysis of cell cycle and cell apoptosis showed that L929/OX40L could inhibit apoptosis of T cells and effectively stimulate their proliferation and CD28 mAb strengthened the effect. And the transfected cells also could activate CD4+ T cells evidenced by phenotype analysis. In addition, the transfected cells could promote T cells to secrete IL-2, IL-10 and IFN-y by ELIS A.3. Establishment of six hybridoma cell lines specifically secreting monoclonal antiboday (mAb) against OX40L.BALB/c mice were immunized with human OX40L transfected L929 cells as an immune antigen and the splenocytes were fused with murine myeloma SP2/0 cells by the cell fusion hybridoma technique. By means of multiple cell subcloning and repeated screening with L929/OX40L as antibody screening positive cell while L929/mock as negative control, six hybridoma cell lines (named as 9H10, 4C12, 1E7, 8D10, 4H4 and 1G1) stably secreting anti-OX40L monoclonal antibodies were selected out. Then the Ig isotypes were identified with test paper. The isotopes of 9H10, 4H4, 1E7 and 1G1 were mouse IgG1 while the isotopes of 8D10 and 4C12 were IgG2a and IgG2b respectively. The hybridoma cells grew well after long-term culture in vitro (40 passages) and storage in liquid nitrogen.The ascites were produced according to the ascites-inducing procedure in mouseabdominal cavity. The ratio of ascites formation was above 95 percent and the average yield is about 5 milliliter (ml) per mouse. After the ascites were purified by protein G affinity chromatography, the protein content was between 0.8 and 10 milligram(mg)/ml with 1:10...
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