| OX40(CD134), a 50-kDa cell surface type I transmembrane glycoprotein of the TNFR family, is expressed predominantly on activated CD4+ T lymphocytes and a little on CD8+ T cells, such as ConA and PHA-activated CD4+ and CD8+ T cells, CD4+ T cells at the site of inflammation, adult T-cell leukemia(ATL) cells, human T-cell leukemia virus type l(HTLV-l)-infected T cell lines HuT102 and MT-2. The ligand for OX40, OX40L, is a type II transmembrane protein antigen of 34 kDa and a member of TNF family. The expression of OX40L is limited to a subset of blood derived CD40-stimulated dendritic cells(DC), activated B cells, vascular endothelial cells(VEC), human umbilical vein endothelial cells(HUVEC), macrophages and some organs as heart, skeletal muscles, testis and lung. Engagement of OX40/OX40L promotes T cell activation, proliferation and migration, increases the life-span of effector T cells and enhances the germinal center(GC) formation in a CD28-dependent manner. CD28/B7 is important for initial T cell expansion, whereas OX40/OX40L signal affects T cell numbers in the later stage or secondary immune responses, and is essential for the survival and/or responsiveness of the memory T cell pool. In addition, OX40L was shown to be capable of conducting reverse signaling to DC and of inducing the DC maturation in the presence of CD40 signal. Also OX40L reverse signal in HUVECs can induce the expression of CC chemokine RANTES (regulated upon activation, normal T cell expressed and secreted)/CCL5 at both mRNA and protein levels. Because of the limited expression of OX40 on antigen specific T cells at inflammation sites, OX40 becomes a interestingly targeted immunotherapy molecule for autoimrnunity and graft versus host disease (GVHD) by blocking the OX40-OX40L interaction or depleting OX40+ T cells. By contrast, engagement of OX40 by agonist monoclonal antibody (mAb) in the setting of active immunization has potent adjuvant properties, leading to enhanced cytokine production and increased numbers of antigen-specific memory T cells. These adjuvant effects lead to an enhancement of anti-tumor responses.1. Cloning of human OX40 gene and constructing of OX40 transfectant. cDNA fragment encoding human OX40 was obtained from the total RNA of PHA-activated tonsil T cells by RT-PCR and inserted into eukaryotic expression vector, pcDNA3.1, with gene cloning technique. The recombinant plasmid, pcDNA3.1-ox40, was transfered into mouse fibroblast cell line L929 with lipofectamine. Then OX40 transfectant named L929-OX40 was screened with G418 and grew well after long time passaging in vitro and storage in liquid nitrogen. OX40 expression on cell surface is stable.2. Effect of L929-OX40 transfectant on DC maturation. It was reported that OX40L was capable of conducting reverse signaling to DC and inducing DC maturation, which led us to use L929-OX40 transfectant to study OX40L reverse signal on DC. Peripheral blood mononuclear cells(PBMC) were obtained and cultured in RPMI1640 plus GM-CSF and IL-4 for 6 days, then cocultured with L929-OX40 in the presence of sCD40L for additional 48 hours. Our results showed that L929-OX40 have the potency to induce the differentiation of DCs in vitro and to up-regulate the expression of CD40, CD86 and CD83 by flow cytometry (FCM) analysis.3. Establishment of a hybridoma cell line specifically secreting monoclonal antiboday(mAb) against OX40. Based on obtaining L929-OX40 transfectant, we immunized BALB/c mice with this transfectant as immunogen. The immunized spleenocytes were fused with mouse myeloma cell line, SP2/0, by using polyethylene glycol (PEG) and cultured in HAT selection medium. Hybridoma cells were screened with L929-OX40 and PHA-activated T cells as OX40+ cells and L929-Mock as OX40" cells by FCM and subcloned more than 3 times. Then two hybridoma cell lines, named 7E11 and 5H1 respectively,, were obtained, which were able to secret specific mAb against human OX40 continuously and stably. Fast-strip analysis was performed to identify Ig subclass of these two mAb...
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