Establishment of cell line transfected with human LIGHT gene and its co-stimulatory effect on T cell

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 LipfectamineTM2000 and the cells were further selected with G418.The effect of the recombinant vector transfected into L929/LIGHT cells on T cells 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 promote obviously the proliferation of T cells in vitro and were stimulated by anti-CD3 mAb.The up-regulation on the cytokine production,such as IL-2,IFN-γ and IL-10 could be demonstrated by ELISA.It is evident that a cell line L929/LIGHT stably expressing the 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.