Preparation Of LIGHT Transfected Cells And Monoclonal Antibody And Its Costimulatory Effect On T Cell In Vitro

T cell immune response is precisely regulated by the interaction between co-stimulatory and co-inhibitory molecules, which are now classified as immunoglobulin SF, TNF/TNFR SF and cytokine SF. LIGHT is an identified TNF superfamily member transiently expressed on the surface of activated T lymphocytes and immature dendritic cells. Engagement of LIGHT with HVEM, its TNF family receptor, costimulates human T cell proliferation and preferentially induces the production of certain cytokines, whereas HVEM can also act as a ligand that binds to BTLA or CD160 delivering a coinhibitory signal. The LIGHT/HVEM/BTLA(CD160) costimulatory/coinhibitory pathway has emerged as a potential target for the development of immune therapeutic interventions. Our results suggested LIGHT transfected cells were effective for T cell activation and cytokine production in vitro. Blockade of LIGHT with monoclonal antibody or HVEMIg fusion protein inhibited its role in costimulation of T cell response.To establish the human co-stimulatory molecule LIGHT gene transfected cell line and to investigate its co-stimulatory effect on T cell activation and proliferation in vitro, the full-length human LIGHT gene coding region was cloned from the activated T cells of human peripheral blood by RT-PCR and then inserted into the eukaryotic expression vector pIRES2-EGFP to construct the recombinant pIRES2-EGFP-LIGHT after double digestion with EcoR I and BamH I. The recombinant plasmid was transfected to murine L929 cells after induction with LipfectAMINETM 2000 and the cells were further selected with G418. The effect of L929/LIGHT cells on T cell proliferation and cytokine production in vitro was studied by MTT, ELISA and flow cytometry. It was demonstrated that the stable expression of human LIGHT on the transfected cell line was identified by flow cytometry analysis and the L929/LIGHT cells could obviously promote the proliferation of T cells stimulated by anti-CD3 mAb in vitro. The up-regulation on the cytokine production, such as IL-2, IFN-γand IL-10 could be measured by ELISA. It is evident a cell line L929/LIGHT stably expressing human LIGHT gene has been obtained and it triggers a signal which can prominently stimulate the proliferation and cytokine production of T cells in vitro.Since L929 has relatively low immunogenicity to BALB/c mice, L929/LIGHT could be adopted as immunogen for the preparation of mouse anti-human LIGHT monoclonal antibody. The splenocytes harvested from the immunized mouse were fused with murine myeloma SP2/0 cells by means of cell fusion hybridoma technique. After repeated screening by L929/LIGHT (as positive screening) and L929/mock cell (as negative control), the hybridomas secreting specific mAbs were screened by immunoflurescence assay. One cell strain of hybridoma secreting anti-LIGHT mAbs continuously and steadily was obtained and designated as 7A8. Identified by test paper, the isotope of 7A8 heavy chain was mouse IgG2b while the isotope of light chain isκ. The hybridoma cells grew well after long-term storage in liquid nitrogen and culture in vitro. A series of immunotechniques such as dot-blot, FCM and SDS-PAGE were applied for further study of LIGHT 7A8 mAb characteristics. Then, HVEMIg fusion gene was inserted into pIRES2-EGFP expression vector. pIRES2-EGFP/HVEMIg was transfected into CHO cells as described above. FACS,SDS-PAGE and Western-blot were used to demonstrate that the target protein(HVEMIg fusion protein) could be expressed and secreted into the supernatant. BCS-free supernatant of CHO/HVEMIg was condensed and purified by protein G affinity chromatography column. Studies showed that the HVEMIg fusion protein has high affinity with its ligand LIGHT. And antibody of LIGHT or HVEMIg fusion protein could partially inhibit T cells proliferation response and cytokine production in vitro.On the basis of above, we have successfully cloned huLIGHT gene and constructed L929/LIGHT. Studies showed LIGHT could costimulate T cell response involving cell multiplication and cytokine production. The anti-LIGHT mAb and HVEMIg partially impeded LIGHT/HVEM signals on T cells.