To Study The Mechanism Of IL-33 Promoting Antitumor Immune Responses Mediated By Cd8~+T Lymphocytes

As the tumor incidence is increased gradually, cancer patients tend to be younger and low cure rate, higher mortality. And cancer has become a major "killer", seriously harm to human health. On morphology,tumors can be divided into solid tumors and non-solid tumors, the former is mainly composed by the tumor cells, tumor stroma cells, tumor- infiltrating immune cells, and vasculars which supply the tumor tissue nutritions. The numbers, functions and the types of tumor infiltrating lymphocytes determined the effects of anti-tumor immune response, and all of these have a very close relationship with the prognosis of cancer patients. In the tumor infiltrating lymphocytes, CD8+T lymphocytes(CTLs) and C D4+T helper(Th cells) cells play the main role in anti-tumor immune response, especially C D8+T lymphocytes is the basis of anti- tumor immun.The complexity of tumor microenvironment can produce local tumor immune suppression, and cause the CD8+C TLs and CD4+Th1 cells of tumor infiltrate lymphocytes in an exhausted or incompetent state. Therefore, to find a new molecular to alter the tumor microenvironment immunosuppression, reactive or reverse incompetent state of tumor infiltrating lymphocytes(TILs),has become a hot topic of anti-tumor immune therapy.Interleukin 33(IL-33) was first discovered in 1999 by O nda H and his colleagues and was named as DVS27, mainly highly expressed in canine cerebral vasospasm cells, a 30 k Da protein molecular. Four years later, the corresponding gene was found that highly expressed in high endothelial venules nucleus, the gene is referred as "high endothelial venules of nuclear factor(NF-HEV). In 2005, Schmitz and his colleagues discovered and confirmed IL-33(NF-HEV) is a new IL-1 family members by computer analysis and comparison with IL-1 family sequence.IL-33 is very similar to IL-1b and IL-18, members of IL-1 family. Human IL-33 gene is located on chromosome 9(9q24.1). And subsequently found that, IL-33 is the functional ligand of orphan receptor ST2(IL-1R- like-1). ST2 is selectively expressed in macrophages, mast cells and Th2 cells. Recent studies have found that, IL-33 can promote Th1-type immune response and affect the antiviral CD8 + T lymphocytes development. It has been reported that IL-33 is a novel cytokine that can promote Th1 type and C D8+T lymphocytes response, and plays an important protective role in cellmediated anti-tumor and anti- lentivirus infection. However, the mechanism of IL-33 anti-tumor immune response in the body is still unclear.Our previous studies have found that in a variety of human tumors,compared with tumor adjacent tissues, IL-33 and its receptor ST2 m RNA levels were significantly decreased. In addition, IL-33 can synergize T cell receptor(T cell receptor, TC R) and interleukin-12(IL-12)signals to stimulate mouse T lymphocytes in vitro, and promote CD8+T cell function in mice. But it’s immune function and mechanism of anti-tumor is still not known. Cellular immune response mediated by CD8 + T lymphocytes is the key role of anti-tumor immune response. Therefore, this paper first investgate whether IL-33 can directly enhance human CD8+T lymphocytes effector function in vitro, and then construct the tumor-bearing mice model to explore the impact of IL-33 with tumor vaccine on tumor growth and metastasis. These studies will provide a new potential treatments or biological basis for the anti-tumor immunotherapy.Part I IL-33 can promote and enhance the effector functions of human CD8+T cells in vitroObjective: To investigate whether interleukin IL-33(IL-33) can directly enhance CD8+T lymphocytes effector functions in vitro, and promote the secretion of cytokines.Methods: First of all, mononuclear cells of peripheral blood(PBMCs) were isolated by density gradient centrifugation from healthy donors’ peripheral blood, and then CD8+T lymphocytes was separated from PBMCs by immune magnetic beads, and stimulated respectively with four cell factor combinations: CD3 monoclonal antibody(m Ab)(50ng/ml)+CD28 m Ab(200ng/ml); CD3 m Ab(50ng/ml)+CD28 m Ab(200ng/ml) +IL-12(10ng/ml); CD3 m Ab(50ng/ml)+CD28 m Ab(200ng/ml)+IL-33(30ng/ml); CD3 m Ab(50ng/ml)+CD28 m Ab(200ng/ml)+IL-12(10ng/ml)+IL-33(30ng/ml), for 24 h and 72 h, and then the cells were collected. The purity of CD8+T lymphocytes, and the expression of IFN-γ, IL-33s’ receptor ST2, CD107 a was detected by flow cytometry; Total RNA was extracted and assayed by realtime PCR(q RT-PCR) to detect the expression of interferon-γ(IFN-γ), granzyme B, IL-21, IL-10,TIM-3, TNF-α, ST2 and other molecules in m RN A levels. Use Cytometric Bead Array(CBA) kit to assay the supernatant cytokines of IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, IL-17 a.Results: 1. The purity of CD8+T lymphocytes is above 95%, comply with our test requirements. 2. ST-2,IL-33 receptor, was expressed on CD8+ T cells. 3. The stimulation and activation of IL-33 to CD8+T lymphocytes is in a dosedependent effect(P <0.05). 4. There was no significant difference of cell morphology observed by microscope among the four groups of CD8+T lymphocytes. 5. There was no significant difference of cell size after stimulated 24 or 72 hours among the four groups of CD8+T lymphocytes detected by flow cytometry. 6. IL-12 can upregulate the expression of IL-33 receptor ST2(P <0.05) in the CD8+T lymphocytes in vitro.7. IL-33 can stimulate CD8+T lymphocytes directly and promote the expression and production of IFN-γ, and combination with IL-12 group upreguleted more significant(P <0.05). 8. Compared with the medium group, the expression of CD107 a increased significantly in the CD8+T lymphocytes stimulated in the presence of IL-33(P<0.05). and costimulation with IL-12 group upreguleted more significant(P<0.05). 9. Detected by realtime-PCR, the expression of GZ-B,CCL20 etc. upreguleted(P<0.05),. 10. Negative costimulatory molecule TIM-3,LAG-3 etc. were decreased in the presence of IL-33(P<0.05), 11. IL-33 had no effect on the expression of IL-12 R, IL-2, perforin, PD-1, etc.Conclusion: Cytokine IL-33 can stimulate CD8+T cells direccty and enhance the effector functions of CD8+T cells, promoting adaptive immune response. This may provide a novel potential treatments for tumor immunotherapy.PartⅡ IL-33 enhances the effector function of tumor vaccineObjective: To explore whether IL-33 can increase the effector function of tumor antigen-specific CD8+T lymphocyte in vivo, enhance tumor vaccine effect, inhibit the tumor growth and metastasis and prolong the survival of tumor-bearing mice.Methods:The construction of tumor-bearing mice.C57BL/6 mice were first inoculated with melanoma cells B16 in the left side of abdominal subcutaneous. For tumor vaccination, B16,B16-IL-33 and B16-IL12 cells were irradiated and then injected subcutaneously(s.c.) in tumor-bearing mice starting on day 4 after tumor inoculation. The injection was repeated every 4 days and a total of four times.Tumor growth curves and survival of tumor-bearing mice curves were drawed by Graph Pad Prism 5.0. And in early stage of tumors, on day 4 after tumor inoculation, Analyze the tumor microenvironment of CD8+IFN-γ+T lymphocytes ratio by flow cytometry.Results: 1. IL-33 can enhance the effect of the tumor vaccine, and compared with the control group, IL-33 with the tumor vaccine inhibit tumor growth obviously. 2. IL-33 with the tumor vaccine can prolong the survival of tumor-bearing mice compared with the control group B16. 3. IL-33 promotes the production of IFN-γ by tumor- infiltrating CD8+T lymphocytes.Conclusion: IL-33 can enhance the effector function of tumor antigen-specific CD8+T lymphocytes in vivo to inhibit tumor growth and prolong survival of tumorbearing mice, B16IL33 + B16IL12 vaccine combination group was more significant.