Tumor is a chronic progressive disease, which seriously endangers health. It is characterized by rapid growth, high metastasis, low cure rate and easy recurrence. In recent decades, despite the continuous improvement and development of surgical resection, chemotherapy, radiotherapy and other treatment methods, the effective treatment of tumors is still a difficult problem. In recent years, tumor immunotherapy has opened up a new way for tumor treatment. However, the biggest obstacle to immunotherapy is tumor immunosuppression. Especially the tumor microenvironment with strong immunosuppression. The infiltrating tumor cells and immune cells, neovascularization and its endothelial cells (bec), tumor-associated fibroblasts (CAFs) and extracellular matrix are all isomorphic to the tumor microenvironment. One of the main functions of tumor microenvironment is to form an immunosuppressive environment in tumor and weaken the killing effect of immune cells on tumor cells. For example, the aggregation of a large number of regulatory T cells (Tregs) and myeloid inhibitory cells (MDSC), the production of various inhibitory cytokines, and the formation of tumor local failure T cells. All these make tumor cells escape the killing effect of immune system, which eventually leads to the immune escape of tumor cells. Therefore, to fully understand the molecular mechanism of immunosuppression in tumor microenvironment, TIM-3 is a member of T cell immunoglobulin-and mucin-domain-containing molecule (TIM) families. Human TIM-3 is a type I membrane protein composed of 30 1 amino acid. It includes the N- terminal of an Ig-like V region (rich in cysteine), a mucin region rich in serine and threonine, a transmembrane region and an intracellular region. TIM-3 was expressed in helper Th 1, Th 17 cells and CD8T cells. It is a negative regulatory molecule that acts on its ligand galectin-9. Recently, TIM-3 molecule is considered as one of the surface markers of failed T cells and plays an important role in tumor microenvironment. Therefore, the first part of this thesis takes TIM-3 and non-small cell lung cancer (NSCLC) as the starting point. To study the depletion of T cells in tumor microenvironment and its influence on tumor. Tumor immunotherapy can restore the killing effect of immune cells on tumor cells by changing tumor microenvironment. In the second part of this paper, interleukin-33 (IL-33) is taken as the research object. To reveal its anti-tumor effect in tumor microenvironment. IL-33 belongs to the cytokine family of interleukin 1 (IL- 1). It is constitutively expressed in some epithelial and endothelial cells that constitute our body's first line of defense. In addition, it is also expressed in some necrotic cells and activated immune cells. For example, when macrophages are infected by exogenous factors, although IL-33 has been proved to induce Th2 immune response, its role in TH 1 immune response is still unclear. Our recent study found that the receptor ST2 of IL-33 is expressed in effector CD8+T cells and regulated by T-bet. IL-33 can improve the function of effector CD8+T cells. Therefore, IL-33 has potential anti-tumor effect. In the second part of this paper, the anti-tumor effect of IL-33 in vivo was preliminarily discussed. This paper is divided into two parts: (1) By discussing the expression and function of TIM-3 in non-small cell lung cancer, we can understand the depletion of T lymphocytes in tumor microenvironment and the immunosuppressive mechanism of tumor microenvironment. (2) Using IL-33 as a tool, we can study how to change the tumor microenvironment. It provides a potential means for immunotherapy of tumors. The first part is the role of TIM-3 molecule in the tumor microenvironment of human non-small cell lung cancer [Objective] Taking TIM-3 molecule as the breakthrough point, this paper studies the role of Treg, exhausted T lymphocytes and their inhibitory microenvironment in the tumor microenvironment of human non-small cell lung cancer. [Methods] The tumor and adjacent tissues of patients with non-small cell lung cancer were surgically removed, digested by collagenase IV and mechanically digested. The single cell suspension was obtained, and the infiltrated lymphocytes in the tissue were separated by density gradient centrifugation, and the lymphocytes in the peripheral blood of the patient were also separated. The expression of Tim-3 in T lymphocytes was detected by flow cytometry, and the correlation between TIM-3 and clinicopathological factors was analyzed. Intracellular staining is used to analyze the functional differences between failed T cells characterized by TIM-3 and normal T cells. [Results] Compared with normal tissue infiltrating lymphocytes (TILs) and peripheral blood T cells adjacent to cancer, the expression of TIM-3 on TILs surface in patients with non-small cell lung cancer was significantly up-regulated. TIM-3 and PD- 1 were expressed in CD4+til and CD8+til. In addition, the expression of TIM-3 in tumor infiltrating CD4+T cells of patients with non-small cell lung cancer is related to tumor lymph node metastasis and patient staging. Compared with TIM-3-CD4+ and TIM-3-CD8+TILs, the secretion of interferon-γ (IFN-γ) in TIM-3+CD4+ and Tim-3+CD8+Tils decreased significantly. We also found that in tumor tissues, about 70% of TIM-3+CD4+til expressed FOXP3, and about 60% of foxp 3+CD4+til expressed TIM-3. [Conclusion] This study found that TIM-3 was highly expressed in TILs of non-small cell lung cancer, and its expression on CD4+T cells was related to lymph node metastasis and patient staging. TIM-3 and PD 65438+.

3+Treg), which makes us further understand the tumor microenvironment and provides a new target for immunotherapy. Part II: IL-33 enhances TH 1 type immune response in tumor microenvironment and its anti-tumor effect [Objective] To study the influence of cytokine IL-33 on tumor microenvironment and its anti-tumor effect. [Methods] The secreted IL-33/pCDNA3. 1 recombinant expression vector was cloned and constructed in vitro. Transfected into melanoma cell line B 16(B 16-IL-33), and transferred into empty vectors pcdna3.1(b16-mock) and il-12/pcdna3./kl respectively. B 16-IL- 12 and B 16-IL33 tumor cells were subcutaneously inoculated into C57B/L6 mice, and the tumor growth curve was observed and tumor infiltrating lymphocytes were isolated. The related immune indexes were detected by flow cytometry. 2× 105B 16-mock and B 16-IL33 tumor cells were subcutaneously inoculated into normal C57B/L6(WT) or transcription factor T-bet and eomes gene knockout (DKO) mice, and the tumor growth curve was observed to isolate tumor infiltrating lymphocytes. [Results] The growth rate of B16-IL-33 tumor in C57B/L6 mice was obviously slowed down. Compared with b 16- pseudotumor, B 16-IL-33 tumor infiltrated CD45+ and CD8+T cells, and the proportion of NK cells increased significantly.

Cells and NK cells have higher ability to secrete cytokine IFN-γ. In mice lacking transcription factors T-bet and Eomes, the growth rate of B 16-IL-33 tumor increased significantly. Compared with wild-type (WT) mice inoculated with B 16-IL-33 tumor cells, CD45+ and CD8+ infiltrated in tumor tissues. And the ability to secrete cytokine IFN-γ is reduced. [Conclusion ]IL-33 can obviously improve the immune response of Th 1 in tumor microenvironment, which has potential anti-tumor effect and provides a new method for tumor immunotherapy.