Expression Charactets And Clinical Significance Of Th Subsets And TEM Subsets In Tumor Immununological Microenvironment Of NSCLC

Lung cancer has become a malignant tumor with the highest morbidity and mortality rate in China,as well as has posed a serious threat on people’s health.Although the technology of clinical diagnosis and treatment of lung cancer has made significant progress,the survival rate has not radically improved.In recent years,tumor biotherapy based on immune checkpoint blocking therapy has achieved significant breakthroughs in the treatment of melanoma,bladder cancer,renal carcinoma,and lung cancer,which has brought people hope to cure the tumor.However immunotherapy has been very effective in only a select field of patients indicating that there are many scientific questions yet to be determined for it.Immunotherapy is based on familiarity with the tumor microenvironment and it is imperative to explore the characteristics and clinical significance of immune cell subsets,immune molecules,and cytokines in the tumor immune microenvironment.This study focuses on the expression,clinical significance,and biological functions of Th cell subsets such as Th1,Th2,and Th17 in the lung tumor immune microenvironment,and the myeloid-derived cell subsets,TEM,and B7-H3 positive TEM in different anatomic sites of lung cancer.PartⅠ:Investigation of the clinical significance of Th subsets（Th1,Th2 and Th17）in tumor microenvironment of patients with NSCLCObjective:CD4+Th subsets play an important role in tumor progression but their expression characteristics and clinical significance in human tumor microenvironment remains unclear.In this study,we aim to analyze the expression and clinical significance of tissue-infiltrating Th1,Th2 and Th17 in lung cancer.Methods:A total of 54 patients with lung cancer were collected in this study.Tumor tissues were taken from areas of solid tumor tissues lacking the gross aspect of massive necrosis.The tumor-free normal lung tissue samples were taken at least 5 cm away from the tumor margin;the tumor-boundary lung tissue samples were taken at most 2 cm away from the tumor margin.Lymph node is the tumor area surgery to clean the lymph nodes.All samples were all used for the isolation of tissue-infiltrating leukocytes through treated with0.1%collagenase IV to obtain single cell suspension.Single cell suspension was treated with PMA and Ionomycin and CD3⁺CD4⁺IFN-γ⁺Th1,CD3⁺CD4⁺IL-4⁺Th2 and CD3⁺CD4⁺IL-17⁺Th17 were analyzed by flow cytometry.The statistical analyses for the measurements were conducted in combination with the clinical data.Results:We found that the frequency of CD3⁺CD4⁺IFN-γ⁺Th1 in tumor nest was significantly lower than that in tumor boundary,adjacent normal lung tissue or corresponding lymph node tissue;the frequency of CD3⁺CD4⁺IL-4⁺Th2 in tumor nest was significantly higher than that in tumor boundary,adjacent normal lung tissue or corresponding lymph node tissue;the frequency of CD3⁺CD4⁺IL-17⁺Th17 in tumor nest was significantly lower than that in tumor boundary,but not adjacent normal tissue or corresponding lymph node tissue.Survival analysis of 2-years survival after surgery showed that Th1^high group was significantly lower compared with Th1^low group;Th2^high and Th17^low is a good prognosis index compared with the Th2^low and Th17^high groups respectively,but this difference failed to significance.In addition,we also found that PD-1 expression showed a high level on lung tumor tissues and adjacent non-tumor tissue infiltrating T cells,and no significant difference was found between the two groups.However PD-L1 on CD45⁺CD14⁺mononcytes/macrophages in tumor tissue show a significantly higher level compared with that in adjacent nontumor tissues.In vitro stimulation experiments showed that IFN-γcould significantly increase PD-L1 expression on monocyte.Conclusion:we for the first time found Th1^high is a poor indicator for prognosis of lung cancer.PARTⅡ:Investigation of the clinical significance and biological functions of TEM and B7-H3 positive TEM in the immune microenvironment of lung cancerObjective:Tumor immune environment is very critical for the formulation of tumor immunotherapy strategies.This study focuses on a new group of myeloid-derived cell subsets,CD14+Tie-2+TEM in the lung cancer infiltrated tissue.Based on measurement and analysis of their expression characteristics and clinical significance,we aim to further explore the clinical significance and biological function of B7-H3,an immune co-stimulatory molecule expressed by this subset.Methods:Collected 56 surgically resected samples of lung cancer tissues,adjacent regions of the cancer tissues,distal normal tissues,and some peripheral blood.The tissue samples were trypsinized,triturated,and filtered to obtain single-cell suspension,while the CD14+Tie-2+TEM subsets and expression level of B7-H3 in the above subsets were measured by flow cytometry.The statistical analyses for the measurements were conducted in combination with the clinical data.In vitro induction experiments,the analysis was done to explore the expression and regulation mechanism of B7-H3 in mononuclear phagocytes,the biological effects of B7-H3 and B7-H3 positive mononuclear phagocytes,as well as their effects on the biological function of the T lymphocytes.Results:Compared with that in the peripheral blood,the TEM subsets ratio in cancer tissues,the adjacent region of cancer tissues,and the distal normal tissues were significantly higher,while the TEM levels in the adjacent region of cancer tissues were closely related with tumor staging.It was shown in further study that,compared with peripheral TEM subsets,the major phenotypic characteristics of the TEM subsets in cancer infiltrated tissues were B7-1 low,B7-2 high,B7-H1 high,and B7-H3 high.Due to the novelty of B7-H3,this molecule was chosen for further investigation.It was found that in cancer tissues,adjacent regions of cancer tissues,and distal normal tissues,the expression level of B7-H3 in CD14+Tie-2 positive mononuclear phagocytes were significantly higher than that in the CD14+Tie-2 negative subsets.Furthermore,it was shown in both the CD14+Tie-2 positive subsets and CD14+Tie-2 negative mononuclear phagocytes that,with an increased distance from the tumor,the expression level of B7-H3 gradually dropped,with significant differences.It was revealed through clinical statistical analysis that the expression levels of B7-H3 in CD14+Tie-2 positive mononuclear phagocytes,which came from cancer tissues and adjacent regions of cancer tissues,were closely related with the tumor’s size.However,in distal normal tissues the expression level of B7-H3 in TEM subsets were closely related with lymph node metastasis,suggesting that high expressions of B7-H3 in TEM subsets were present in early stage tumors.It was demonstrated in the analysis of in vitro activation that the expression of B7-H3 on mononuclear phagocytes could be significantly upregulated by LPS,IFN-γand PGE2.It was confirmed through functional tests that B7-H3 could promote mononuclear macrophages to secrete TNF-α.The B7-H3 positive mononuclear phagocyte could significantly promote the activation of CD4+T cells and the expression of immune checkpoint molecule Tim-3.However,there was no significant effect on the CD8+T lymphocytes,which revealed that the major target cell for B7-H3 positive mononuclear phagocyte was not CD8+T,but rather CD4 positive cells.Conclusion:The expression characteristics,clinical significance,and biological functions of TEM and B7-H3+TEM subsets were studied systematically in this section.Through a biological function study it was found that B7-H3 positive mononuclear phagocytes could significantly promote the secretion of TNF-α,as well as promote the expression of Tim-3,a negative immune checkpoint molecule,and also play an important role in the activation of CD4+T cells.It might play an important role in the mechanism of tumor immune escape.