Expression Of PD-1 And TIM-3 In Diffuse Large B-cell Lymphoma And Its Effects On Cell Proliferation And Apoptosis

BackgroundDiffuse large B-cell lymphoma (DLBCL), the most common type of NHL, accounted for almost one-third of all cases in the United States and more than 40%of the NHL in Asia. This type of lymphoma accounts for the majority of cases, which were previously called " aggressive " or " high grade " lymphoma. DLBCL is of very heterogeneous in morphology, immune phenotype, genetics and clinical manifestations and it grows fast, but it generally responds well to treatment,5-year survival rate can reach 75-80%. After CHOP-like regimen,3/4 of the patients have no symptoms after treatment, nearly 50% of patients can be cured, but there are still 30%-40% of DLBCL patients appear initial primary resistance, residual disease or relapse after complete remission. In recent years, cell signaling pathways associated with tumor genesis and tumor suppressor genes, cytokines and proto-oncogene receptor molecule targeted therapy has been paying off. Therefore, further study of the pathogenesis of this disease and the new treatment method is particularly important. In recent years, with the deeply research of tumor immunology, the study found that in malignant tumor occurrence, development, metastasis and recurrence of each stage, tumor and the body's immune system is the complex relation exists. Many tumor associated antigen was identified as the immune system components, so the tumor immune to some extent can be said to be immune, and immune tolerance may hinder the process of tumor immune. In the mechanism of immune tolerance, the current study found that the regulatory T cells plays an important role in the immune tolerance, their defects and dysfunctional will directly cause autoimmune disease. T cell activation requires two activation signals synergy. The first signal, which were secreted from the T cell receptor (TCR) recognize antigens, transduce the signal into cells by CD3 molecules. The second signal was from the interaction between the antigen commission cell or costimulatory molecules of the target cell surface and T cell surface costimulatory molecules. Costimulatory molecules are adjusted by costimulatory factor and co-suppression factor, regulating the amplitude of T-cell activation and immune response strength. Chronic viral infection or long time tumor antigen exposure, T cells would express abnormally co-suppression factor, inhibiting T cell effector function. Therefore, the research on collaborative cues help to explore new immunotherapy strategies. PD-1 and TIM-3 is coordinated stimulus molecules important members of the family, all belong to negative regulatory factors involved in regulating immune process, also play an important role in tumor immunity. Programmed death 1 (PD-1), as one of CD28 super family, expressed in T, B lymphocytes and bone marrow cells and mainly regulated the function of activated lymphocytes. PD-1 knockout mice can induce a variety of autoimmune diseases, suggesting that PD-1 is an important immunosuppressive molecule and plays an important role in the outer peripheral tolerance. The immune regulation, targeting PD-1, is important to anti-tumor, anti-infective, anti-autoimmune diseases and survival from organ transplantation. TIM-3, as one of T lymphocyte immune globulin mucin (TIM) gene family, is highly expressed in T helper cell 1 (Th1) and cytotoxic T cells (CTL), and produce inhibitory signals, which leaded to Thl and CTL cells apoptosis. Galectin-9 (Galectin-9, Gal-9), a natural ligand TIM-3, can specifically bind TIM-3 molecules on Thl cells, to provide a negative T cell costimulatory signals, triggering Thl programmed cell death. Therefore, TIM-3 is recognized as a negative immunomodulatory molecule to down-regulate Th1-type immune reaction. Recent studies have shown that, in addition to Thl and CTL cells, TIM-3 can also regulate other types of immune cells, play an important role in a number of autoimmune diseases and cancer occurrence and development, which is causing more closely attention. It is shown that PD-1 and TIM-3 highly expressed in T lymphocytes in solid tumor tissues, and the tumor specific anti-tumor activity in T cells could be restored by inhibiting PD-1 and TIM-3 signal pathway. Closed PD-1 and/or TIM-3 gene can inhibit tumor cell proliferation, promote apoptosis of tumour cells. Thereby, PD-1 and TIM-3 may play an important role in the occurrence, development, proliferation and apoptosis of tumor. In this study, we measured the expression of PD-1 and TIM-3 in CD3+T cell in patients with DLBCL, indicate the relationship between the level of the two genes and clinical indicators and chemotherapy and compare the change of the level of PD-1 and TIM-3 after chemotherapy. Our study will provide a theoretical basis for DLBCL pathogenesis and prognosis. This study also mediated by slow virus, targeted silence people diffuse large B cell lymphoma cell lines OCI-Ly19 of PD-1 and TIM-3 genes, preliminarily silence genes PD-1 or TIM-3 genes of diffuse large B cell lymphoma cell lines OCI-Ly19 proliferation, the cloning, the influence of the biological behavior of cycle and apoptosis, and provide new targets and ideas for the treatment of DLBCL.Objectives1. To investigate the expression of PD-1 and TIM-3 in CD3+T cell in patients with diffuse large B-cell lymphoma (DLBCL).2. Indicate the relationship between the level of PD-1 and TIM-3 genes-and clinical indicators.3. To compare the changes of the level PD-1 and TIM-3 between before and after chemotherapy.4. To analyze the relationship between the level of PD-1 and TIM-3 genes and chemotherapeutic effect.5. To study proliferation and cycle distribution of OCI-Ly19 cells after sliencing PD-1 and TIM-3 gene.6. To study clone formation and apoptosis of OCI-Ly19 cells after sliencing PD-1 and TIM-3 gene.Materials and methods1. A retrospective analysis was conducted on data from 46 patients with newly diagnosed diffuse large B-cell lymphoma and 30 healthy people.2. Flow cytometry was used to detect the expression of PD-1 and TIM-3 before and after chemotherapy.3. To construct the PD-1 or TIM-3 lentivirus were transfected into 293T, then the packaged lentivirus was used to infect in OCI-Ly19 cells. The PD-1 gene silencing (Lv-PD-1), TIM-3 gene silencing group (Lv-TIM-3) as experimental group, negative control group (Lv-Con) and blank control group (Con) were also setted.4. In order to detect the expression ratio of PD-1 or TIM-3 gene silencing, the PD-1 or TIM-3 mRNA lever was determined by RT-PCR and the the protein was measured by Western-blot assay.5. The effect of PD-1 or TIM-3 gene silencing on the proliferation of OCI-Ly19 cells was detected with Cell Counting Kit-8(CCK-8). Cell cycle alternation and apoptosis were measured by Flow cytometry(FCM).6. All experiment repeated at least three times, and statistical differences were analyzed using GraphPad Prism 5 software.Results1. Compared to healthy control, the expression of PD-1 and TIM-3 significantly increased in CD3+T cell in patients with DLBCL.2. There is no significant change of PD-1 and TIM-3 in patients with phase ?/? DLBCL, however, they markedly increased in patients with phase ?/? DLBCL.3. The expression of PD-1 and TIM-3 elevated in DLBCL patients with B symptoms, IPI score>2 points and high level of LDH and Ki-67.4. After four courses of standard chemotherapy, PD-1 and TIM-3 expression level decreased.5. The treatment efficiency is higher in patients with low expression of PD-1 and TIM-3 than patients with high PD-1 and TIM-3 expression.6. Real-time PCR indicated that the expression of PD-1 gene or TIM-3 gene mRNA in interference group was significantly decreased compared with negative control group and blank control group.7. Compared to the control group and blank group, the proliferation of OCI-Lyl9 cells were decreased significantly in silencing groups. Cell cycle distribution analysis proved that G0/G1 phase cells percentage increased, S phase cells declined.8. In contrast with two of control groups, cell colony forming ability was decreased and apoptotic rate was increased in PD-1 or TIM-3 gene sliencing group.Conclusion1. DLBCL patients have high expression level of PD-1 and TIM-3, which are concerned with DLBCL staging, prognosis, and proliferation and play an important role in the development of DLBCL.2. PD-1 and TIM-3 expression levels are gradually reduced with chemotherapy, and they related with the efficiency of chemotherapy. PD-1 and TIM-3 expression levels can be used as a sensitive indicator of DLBCL patients undergoing chemotherapy and prognostic factors.3. PD-1 and TIM-3 gene sliencing can inhibit OCI-Ly19 cells proliferation and colony forming.4. PD-1 and TIM-3 gene sliencing can promote OCI-Ly19 cells apoptosis.