| Autophagy is an evolutionarily conserved process maintaining the homeostasis of eucaryotic cells, a large number of experimental data demonstrates that autophagy considered to be a key mechanism plays an important role in regulating biological activities during physiological and pathological conditions. Autophagy dysfunction is related to most diseases, especially in cancer. The research about this aspect has become a international hot topic, however, lots of mechanisms still remained to be further investigated.Tumor associated macrophage is the major inflammatory immune cell infiltrating in the tumor microenvironment and affects formation of tumor, tumor angiogenesis, immunosuppression, tumor metastasis and chemotherapy tolerance. Macrophages are generally divided into two subtypes:classical M1 and alternative M2. M1 is usually considered to play a positive role in immune function which can kill pathogenic microorganisms and cancer cells. M2 can inhibit the Thl adaptive immune response and alleviate inflammation, it also promotes angiogenesis, tissue repair and the development of tumor Many researches have showed that tumor associated macrophages are more inclined to M2 on phenotype and function which regulate the tumor microenvironment significantly. Tumor associated macrophages promote tumor growth through multiple mechanisms, there is still a need for further define that how tumor associated macrophages produce in tumor bearing mouse and tumor patients.Our previous study had found that tumor cells cultured in vitro can release autophagosomes extracellularly, we also found that tumor cells released autophagosomes(TRAP) can induce the mouse macrophage to M2-like macrophage in vivo and in vitro, the M2-like macrophage induced by TRAP could inhibit T cell proliferation in vitro by cell to cell contact. Meanwhile, TRAP could increase the expression of PD-L1 on the mouse macrophage significantly which suggested that PD-L1 might play an important role in the suppressive function. There are malignant effusions that contain large numbers of tumor cells and macrophages produced in the tumor progression, and the level of autophay in the tumor cells will increase highly due to hypoxia and nutrient deprivation. So we suspect that there may be autophagosomes in malignant effusions and the autophagosomes may influence the function of macrophages. Because malignant effusions samples are easy to get, the research will take malignant effusions as the research object to explore the effect of hTRAP on human monocyte/macrophage combined with our previous research.Objectives:1. To detect and analyse the cell phenotype and immunoregulation function of macrophages derived from malignant effusions of cancer patients.2. To discuss the effect of hTRAP on the activation and immunoregulation function of human monocyte/macrophage.Methods:1. Detection and analysis of the cell phenotype and immunoregulation function of macrophages derived from malignant effusions of cancer patients1.1 Collected 26 malignant effusions samples, flow cytometry detected the membrane molecular expression of CD 163 and PD-L1 on the macrophages, ELISA detected the concentrations of IL-10 and IFN-y of malignant effusions, and then using SPSS 23.0 software to statistically analyse the data.1.2 Macrophages in malignant effusions was purified by magnetic beads and then incubated with anti-CD3 antibody stimulated CFSE labeled hPBMC for four days and then using flow cytometry to detect the proliferation of CD4+and CD8+ T cells.2. The study of polarization and immunoregulation function of human monocyte/macrophage induced by hTRAP2.1 Obtained autophagosome extracts in malignant effusions via differential-speed centrifugation. Flow cytometry, Western blot and electron microscopy identified autophagosome extracts qualitatively and quantitatively, then analysed them.2.2 Collected healthy human peripheral blood monocyte and induced them into macrophage by M-CSF for seven days, the hTRAP and macrophage were incubated in vitro for one day, and then the mRNA expression of NOS2, IL-10 and PD-L1 was detected by Q-PCR. The hTRAP and healthy human peripheral blood mononuclear cell were incubated in vitro for three days, and then the membrane molecular expression of HLA-DR, CD86, CD163, CD206 and PD-L1 on the macrophage was detected by flow cytometry.2.3 Macrophage induced by hTRAP for three days was incubated with anti-CD3 antibody stimulated CFSE labeled hPBMC or in the transwell chamber for four days and then using flow cytometry to detect the proliferation of CD4+ and CD8+ T cells.Results:1. Detection and analysis of the cell phenotype and immunoregulation function of macrophages derived from malignant effusions of cancer patients1.1 Compared with healthy human peripheral blood monocyte, the expression of CD 163 on the macrophage in malignant effusions of cancer patients increased obviously.1.2 The expression of PD-L1 on the macrophage in malignant effusions of cancer patients increased compared with health peripheral blood monocyte, the concentrations of IL-10 and IFN-y in malignant effusions were different from each other.1.3 There was a positive correlation between the expression ratio of CD 163 and PD-L1 on the macrophage in malignant effusions of cancer patients. The concentration of IFN-y in malignant effusions was also positively correlated with the expression ratio of PD-L1 on the macrophage.1.4 Macrophage in malignant effusions can inhibit T cell proliferation in vitro, the rate of cell division of CD4+T and CD8+T cells was reduced after adding macrophage of malignant effusions into the T cell proliferation system.2. The study of polarization and immunoregulation function of human monocyte/macrophage induced by hTRAP2.1 Malignant effusions from cancer patients contain autophagosomes.1) A high expression of LC3B in extracts was found by flow cytometry, and the expression of LC3B in extracts was more than 90%.2) The extracts contained LC3Ⅰ and LC3Ⅱ protein and the protein level of LC3II was higher than LC3I obviously detected by western blot.3) TEM examination revealed that there were a lot of autophagosomes with 100-1000nm diameter and double-membrane structure in extracts.2.2 The concentrations of autophagosomes in malignant effusions were different from each other and positively correlated with the expression of PD-L1 on the macrophage.2.3 The hTRAP and macrophage were incubated in vitro for one day, the mRNA level of NOS2 of stimulation group decreased significantly compared with the control group by Q-PCR, the mRNA level of IL-10 and PD-L1 increased obviously. The hTRAP and hPBMC were incubated in vitro for three days, the membrane molecular expression of HLA-DR and CD86 on the macrophage of stimulation group decreased compared with the control group, the membrane molecular expression of CD 163, CD206 and PD-L1 increased obviously.2.4 Compared with the control group, macrophage induced by hTRAP can suppress CD4+ T and CD8+ T cell proliferation during coculture with T cell. However, macrophage induced by hTRAP and T cell were cocultured in the transwell chamber, the rate of cell division of CD4+ T and CD8+T cells do not change significantly compared with the control group.Conclusions:1. There were lots of M2-like macrophage that the expression of PD-L1 rised in malignant effusions of cancer patients. Macrophage in malignant effusions of cancer patients can inhibit CD4+ T and CD8+ T cell proliferation in vitro. The increased expression of PD-Ll on the macrophage may be correlated with IFN-γ concentration in malignant effusions.2. Malignant effusions from cancer patients contain autophagosomes(hTRAP) and the concentrations of autophagosomes were positively correlated with the expression of PD-L1 on the macrophage.3. hTRAP can activate human monocyte/macrophage to M2-like macrophage and increase the expression of PD-L1, M2-like macrophage induced by hTRAP can suppress CD4+ T and CD8+ T cell proliferation by cell to cell contact. |
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