The Role Of TIM-3and IL-33in T Cell Mediate Anti-tumor Response

Tumors, especially malignant tumors are sorts of systemic disease. They are the major deadly diseases for human being because some unique feature, such as high speed of growth, high rate of metastasis, low cure rate and easy to relapse. Recent decades, although there’s great improvement of surgery, chemo and radiation treatment, the therapy of tumor is still a difficult problem. We still need more understanding on tumor’s occurring, developing as well as its therapy.Recently, immunotherapy became a new strategy of tumor therapy, and shows a great prospect. As we mentioned tumor immunotherapy, we have to give an introduction about tumor microenvironment. The malignant features of tumor cells cannot be manifested without an important interplay between cancer cells and their local environment. When there’s occurring a tumor, meanwhile forms a local environment where tumor cells live in. Basically, tumor microenvironment consist of the tumor cells, infiltrating immune cells, blood endothelial cells (BECs), carcinoma-associated fibroblasts (CAFs), and the extracellular matrix. Tumor environment induces tumor cells tolerance, through many ways, forms a immune suppressive environment to avoid the killing of immune system, such as accumulation of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC), production of some suppressive cytokines, and also the induce the T cell exhaustion. Therefore, the tumor immunotherapy need us to fully realize the tumor microenvironment and find effective target to change the immune suppressive situation.TIM-3belongs to T cell immunoglobulin and mucin domain containing molecules (TIM) family member. Human TIM-3is a301amino acid, type I membrane protein, includes an immunoglobulin V domain, a mucin domain, a trans-membrane domain and an intracellular tail with one tyrosine phosphorylation. TIM-3is expressed Thl, Th17cells, and also CD8+T cells that negatively regulate these cells by binding to its ligand Galectin-9, and also induce peripheral tolerance. Recently TIM-3was thought to be one of the exhausted T cell marker, and plays an important role in tumor microenvironment. So, the first part of the thesis is based on TIM-3and human non-small cell lung cancer (NSCLC), studying the exhausted T cell in tumor microenvironment and its influence on cancer patient.The most important of tumor immunotherapy is finding an efficient target to change the tumor microenvironment, make the tumor infiltrating immune cells exhibiting their killing function to tumor cells. The second part of the thesis focus on interleukin33(IL-33), disclosure its antitumor effect in tumor microenvironment. IL-33is a member of the interleukin1family (IL-1) of cytokines, and constitutively expressed in the nucleus of some endothelial and epithelial cells. In addition, it also released by some necrotic cells or activated innate immune cells such as macrophages during infection. Although IL-33is realized to induce the Th2response, but its role in Thl response is still not clear. Some results identified IL-33involves in Thl response, and also there are some literatures show development of tumor induced the production of IL-33. Recently, we found ST2, the receptor of IL-33was expressed in CD8+T cells cultured with Thl condition, and T-bet was required for this expression. And IL-33could enhance the CD8+T cell function. Hence, IL-33may have potent antitumor effect. The second part of the thesis is based on IL-33and tumor microenvironment, studying the antitumor effect of IL-33in vivo.This thesis is consist of two part:(1) studying the exhausted T cells in tumor microenvironment and the mechanism of the suppressive environment in tumor through TIM-3and NSCLC.(2) Use IL-33as a tool, study how to change the tumor microenvironment, to achieve the aim of tumor immunotherapy.Part I The Function of TIM-3in Human Non-Small Cell Lung Cancer MicroenvironmentObjective:Focus on TIM-3and exhausted T cell in tumor microenvironment, studying the effect of suppressive environment to tumor progress.Methods:Tumor tissue and adjacent normal tissue from the same patient were minced and digested with collagenase IV digestion solution. The pieces were then transferred to the steel mesh and single cell suspensions were obtained by mechanical grind. TILs were further purified by the gradient protocol, washed and re-suspended in Hank’s media. Meanwhile, peripheral T cells were purified from fresh blood of patients, then use flow cytometry to test the expression of TIM-3in TILs, and analyze the correlation between TIM-3expression and clinical pathological parameters.Result:Compare to TILs from adjacent normal tissue and peripheral T cells, TIM-3were highly expressed in TILs from NSCLC tissue, and co-expressed with PD-1in CD4+/CD8+TILs. Also, the higher frequency of TIM-3+CD4+TILs showed significant association with lymph node metastasis and more advanced cancer stages. TIM-3+CD4+and TIM-3+CD8+TILs showed less IFN-γ production than TIM-3-CD4+and TIM-3-CD8+TILs. We also found that in tumor microenvironment, almost70%percent of TIM-3+CD4+TILs were FOXP3+and about60%percent FOXP3+CD4+TILs were TIM-3+.Conclusion:We found TIM-3was highly expressed in TILs of NSCLC, and the frequency of TIM-3+CD4+TILs was associating with lymph node metastasis and cancer stages. Co-expressing of TIM-3and PD-1showed the high amount of exhausted T cell in tumor microenvironment. In addition, the expression of TIM-3in Treg (Tim-3+Treg) cells characterized a new population of regulatory T cells in tumor tissue. This part of study improved our knowledge of the mechanisms of immunosuppression within tumor microenvironment, and provided a new target for tumor immunotherapy.Part Ⅱ IL-33signal enhances the Th1response and antitumor effect.Objective:Studying the antitumor effect of IL-33in tumor microenvironment.Methods:secretory IL-33was Cloned and constructed into pCDNA3.1vector (IL-33/pCDNA3.1), then transfected into melanoma cell line B16(B16-IL-33). Meanwhile, pCDNA3.1and IL-12/pCDNA3.1were also transfected into B16as control.2×105B16-mock、B16-IL-12or B16-IL33cells were intradermally (i.d.) injected into wild type C57B/L6mice, tumor size was observed and TILs were purified to test the immune parameters.2×105B16-mock、B16-IL-12or B16-IL33cells were injected i.d. into C57B/L6(WT) or transcription factor T-bet and Eomes double knockout mice(DKO), then tumor size was observed and TILs were purified to test the immune parameters. Result:Tumor growth dramatically delayed in B16-IL-33tumor. Compare to B16-mock tumor, the population of CD45+, CD8+TILs and NK cells in B16-IL33tumor were increased. Also, the IFN-Y production of CD8+TILs and NK cells were increased in B16-IL-33tumor. Without transcription factor T-bet and Eomes, B16-IL-33tumor grew faster, and compare to WT mice, tumor infiltrating CD45+, CD8+and NK cells were decreased, and also less amount of IFN-γ were produced by CD8+, and NK cells.Conclusion:IL-33can dramatically enhance the Thl response in tumor environment and has the potent antitumor effect, which may provide us a novel therapeutic approach for tumor immunotherapy.