The Preliminary Study On The Expression Of IL-33/ST2 In Tumor Microenvironment And The Role Of IL-33 On Human CD8~+T Cells In Vitro

Tumor is a mini ecological system that consisted of tumor cells, infiltrating immune cells,stromal cells,and a series of molecules distributed in the surrounding microenvironment. The type, number and function of tumor-infiltrating lymphocytes determined the outcome of the body’s immune response to the tumor.Anti-tumor immunity of human body is mostly mediated by T cells, while type I adaptive immune response mediated by CD8+ cytotoxic cells(CTLs) and CD4+I helper T cells(Th1 cells) is the basis of anti-tumor immunity.As immune suppression induced by tumor microenvironment may cause impotency of CD4+ Th1 and CD8+ CTL cells, therefore,looking for new molecules and signaling pathways that can stimulate tumor-infiltrating lymphocytes(TILs) function and reverse its anergic state and chang the tumor microenvironment is becoming a new focus in immunotherapy of tumor.IL-33 is the eleventh member of the IL-1 family,and the receptor of IL-33 is a dimer consisted of ST2 and IL-1 receptor accessory protein. IL-33 is considered as an alarmin of tissue damage and infection,IL-33/ST2 pathway has a well established function in the induction and amplification of Th2-type inflammatory responses, however its role in anti-tumor immune response is still unclear.Our previous studies have found,IL-33 collaborative TCR signaling and IL-12 can promote the secretion of IFN-γ by Tc1 cells in mice.At the same time, in mice bearing of B16 melanoma model, the expression of IL-33 in tumor microenvironment also associated with increased IFN-γ production by CD8+T TILs, and can inhibit tumor growth. Based on these findings, we investigate the expression of IL-33 and its receptor ST2 in human non-small cell lung cancer(NSCLC) tumor tissues and adjacent tissues,then we use human peripheral blood mononuclear cells(PBMCs) and CD8+T cells in vitro,in order to explore the influence of IL-33 on proliferation,activation, effects of PBMCs and CD8+T cells.As an important factor,IL-33 hopefully be applied in promoting T cell anti-tumor immune response.Such studies will provide insights with potential applications in novel treatments in tumor adoptive immunotherapy.Part ⅠThe expression of IL-33/ST2 in human non-small cell lungtumor microenvironmentObjective: To investigate the expression of cytokines IL-33 and its receptor ST2 in human non-small cell lung tumor microenvironment.Methods: Collect tumor tissue and adjacent normal tissue from the same patient with definite pathological diagnosis of non-small cell lung cancer(NSCLC) in Affiliated Hospital of Soochow University,total RNA of tumor tissue and adjacent normal tissue were extracted and assayed by q RT-PCR for the levels of IL-33 m RNA and ST2 m RNA; The expression of IL-33 protein in tumor tissue and adjacent normal tissue were determined by immunohistochemical staining; tissue-derived single cell suspension were obtained by the use of digestive digestion and mechanical grinding method, the lymphocytes were obtained by layered liquid separation,the expression of ST2 in tumor-infiltrating lymphocytes(TILs) were detected by flow cytometry analysis.Results: 1. Compared with the adjacent tissues, IL-33 m RNA and ST2 m RNA levels of non-small cell lung cancer tumor tissue were significantly decreased(P<0.0001); 2. Immunohistochemical staining showed that IL-33 protein positive expression was brown particles, localized in the cytoplasm or membrane. There was no expression of IL-33 on tumor cells in patients with non-small cell lung cancer,and there are some brown particles scattered in the normal alveolar epithelial cells in adjacent tissues; 3. ST2, the receptor of IL-33,tumor-infiltrating lymphocytes from both tumor tissue and adjacent normal tissue.Conclusion: In non-small cell lung tumor microenvironment, the expression of IL-33/ST2 were detected,and the expression of ST2 which is the receptor of IL-33 was detected in tumor-infiltrating lymphocytes.Cytokine IL-33 can enhance the effector functions of humanPBMCs and CD8+T cells in vitroObjective: To investigate whether interleukin-33(IL-33) can enhance peripheral blood mononuclear cells(PBMCs) and CD8+T cells effector functions in vitro.Methods: PBMCs were harvested from healthy volunteers, CD8+T cells were collected by positive selection of CD8 magnetic beads from PBMCs, both PBMCs and CD8+T cells were stimulated with different combination of cytokines(CD3m Ab +CD28m Ab+IL-2,CD3 m Ab+CD28m Ab+IL-2+IL-12,CD3 m Ab+CD28m Ab+IL-2+IL-12+IL-33) in vitro. The cells of each group were collected after 72 h. Total RNA were extracted and assayed by q RT-PCR for the levels of interferon-gamma(IFN-γ) m RNA and Granzyme B(GZ-B) m RNA. The cytotoxic activity of cells in vitro were detected with Cell Counting Kit-8 assay. The expression of IFN-γ and PD-1were determined by flow cytometry.Results: 1. There was no significant difference of cell morphology observed by microscope among the three groups of PBMCs; 2. The levels of IFN-γ m RNA and GZ-B m RNA of PBMCs stimulated in the presence of IL-33 were significantly increased(P<0.05); 3. The killing ability of PBMCs stimulated in the presence of IL-33 was upregulated(P<0.05); 4. Compared with the other two groups, the expression of IFN-γ increased significantly and programmed cell death 1(PD-1) decreased on the PBMCs stimulated in the presence of IL-33(P<0.05); 5. There was no significant difference of cell morphology observed by microscope among the three groups of CD8+T cells; 6. The levels of IFN-γ m RNA and GZ-B m RNA of CD8+T cells stimulated in the presence of IL-33 were significantly increased(P<0.05); 7. The killing ability of CD8+T cells stimulated in the presence of IL-33 was upregulated(P<0.05); 8. Compared with the other two groups, the expression of IFN-γ increased significantly and programmed cell death 1(PD-1) decreased on the CD8+T cells stimulated in the presence of IL-33(P<0.05).Conclusion: Cytokine IL-33 might enhance the effector functions of PBMCs and CD8+T cells, thereby promoting adaptive anti-tumor immune response. This may provide us a novel therapeutic approach for tumor immunotherapy.