Expression Of Tim-3 On Non-small Cell Lung Caner Infiltrating Lymphocyte And The Study Of Its Biological Activity

T cell immunoglobulin and mucin domain family numbers,Tim-3 was identified as a immune molecular expressing on Th1 but not Th2 in 2002. It involves of 301 amino acid, belongs to tapeâ… membrane protein. The protein consist of an immunoglobulin V domain, a mucin domain, a transmembrane domain and an intracellular cytoplasmic tail with one tyrosine phosphorylation .As we get more understand about this molecular, we found that Tim-3 downregulates Th1 cells. And it also express on Th17 cells, but at a lower level than Th1 cells. Tim-3 had found an very important role in autoimmune disease,inflammation,immune tolerance and tumor immunity.Now, Tim-3's biological function and mechanism in clinical disease need to further study. It's not only worth on theoretical research, but also has potential clinical application.This article consist of two parts:1. Tim-3's expression on tumor infiltrating lymphocytes of NSCLCTumor tissue and adjacent non-cancer tissue from NSCLC patients was cut into small pieces and digested in collagenase D, use Ficoll to purifying mononuclear cells, staining with mAb, then use FCM to testing Tim-3's expressing tumor infiltrating T lymphocytes. Meanwhile, testing the Tim-3's expression on peripheral T lymphocytes of both patients and healthy people. We found that Tim-3 highly express on NSCLC tissue and has no express neither on adjacent non-cancer tissue nor peripheral blood T cells of NSCLC patients and healthy donor.2. The cloning of human Tim-3 gene,the construction of its transgenic cells and study of its biological function.T cells was purified from human peripheral blood and stimulated with PHA, after 48h, collected T cells,extracted total RNA. The Tim-3 gene was then amplified by RT-PCR. The target gene was inserted into eukaryotic expression vector pIRES2-EGFP and confirmed by sequence testing. The recombinant plasmid was transfected into mammal cell line L929 using LipfectAMINE, after 72hhours, L929 cell line stably expressing human Tim-3 protein was selected in the presence of G418.T cells was purified from human peripheral blood and stimulated with CD3 and CD28 mAb, planted to 96 well plate use an appropriate density, cultured with Tim-3 transgenic cells together. After 24h,48h,72h, use MTT to measuring the proliferation of T cells. Meanwhile collected the culture supernatant, testing the IL-2 and IFN-r by ELISA. We found that Tim-3 transgenic cells promotes T cells'proliferation and secreting of cytokines.