Experimental Study On The Production And Functions Of IL-10~+B Cells Induced By Malignant Effusions-derived Autophagosome(MedAP) From Tumor Patient

Autophagy is an evolutionarily conserved process of degradation of intracellular substances and plays an important role in regulating the cell metabolism, energy cycle, organelles renewal. Autophagy involves in a number of physiological and pathological processes and it is related to the development and progression of many human diseases, especially cancer.However, the level of autophagy in cells is not static and it will rise in many cases such as mild oxidative stress and nutrient deprivation over a long period. Recent studies have revealed that the activity of autophagy changes in a variety of tumor cells and it makes the study of autophagy in tumors become a hot topic in the treatment of cancer. Our group have confirmed that tumor cell-derived autophagosome contained a variety of damage-associated molecular patterns (DAMPs) molecules such as high mobility group box protein 1(HMGB1) and heat shock protein 90(HSP90)and they play an important role as a natural adjuvant in the immune response.It is well known that the main functions of B cells are antibodies production, antigen presentation and cytokine secretion. However, recent studies have found that a new B cell subset-regulatory B cells (Breg) plays an important role in the immune regulation of the occurrence, development and prognosis of some infectious diseases, autoimmune diseases and cancer. The secretion of IL-10 is one of the most important ways of Breg to suppress immune responses, and therefore the secretion of IL-10 is the main feature of Breg. It has been reported that Breg is vital in the regulation of tumor immunity. On the one hand, Breg produce IL-10 to suppress the IFN-y secretion of CD8+T cells, thereby promoting the development of tumors; On the other hand, Breg can also lead to tumor cell metastasis to the lung by recruiting Tregs Although Breg is important in tumor immunity, its exact mechanism needs to further study.Our previous study have found that autophagosome released from tumor cells under certain conditions could induce mouse B cell activation and secretion of antibody and cytokines (IL-6, IL-10). In addition, we found that the tumor cell-derived autophagosome(TDAP) could induce murine B cells to become an immunosuppressive IL-10+B cell both in vivo and in vitro. Interestingly, the DAMP molecules HMGB1 in TDAP play an important role in the induction of IL-10+ B cells. Therefore, this study wanted to further investigate whether the autophagosome derived from clinical cancer patients could induce human B cells to become IL-10+ B cells on the basis of animal experiments. Many solid tumors could produce malignant effusions that contain large of tumor cells and the level of autophagosome in these cells will rise due to hypoxia and nutrient deprivation. We suspect that there may be autophagosome in malignant effusions of tumor patient. Therefore, the aim of this study wanted to explore the effect of malignant effusions-derived autophagosome (MedAP) on human peripheral blood B cells.Objective:In this study, we use autophagosome derived from malignant effusions (MedAP) to explore the follow issues:1) whether malignant effusions contain autophagosome 2) The production and immunomodulatory functions of IL-10+B cells induced by MedAP 3) The relationship between the proportion of IL-10+B cells induced by MedAP and the levels of HMGB1in MedAP.Methods:1. Collect malignant effusions and obtain extracts via high-speed centrifugation. Quantify total protein levels of the extracts and converted its content into mg/ml according to the volume of malignant effusions.2. Detection expression of LC3 by flow cytometryx, LC3I/II by Western Blot and ultrastructural by TEM to confirm whether the extracts contain autophagosome.3. The MedAP and PBMC from healthy donors were incubated in vitro for three days and then, the proportion of IL-10+B was evaluated by flow cytometry.4. B cells induced by MedAP was incubated with CFSE labeled PBMC in a 48-well plate coated with anti-CD3 antibody for four days and than using flow cytometry to detect the proliferation of CD4+and CD8+T cells.5. Examine the levels of HMGB1 in MedAP by flow cytometryx and analyze the relationship between the proportion of IL-10+ B cells induced by MedAP and the levels of HMGB1 in MedAP.Results:1. Malignant effusions contain autophagosome.1) A high expression of LC3 in extracts was found by flow cytometryx, and the expression of LC3 in extracts was as high as 90%.2) The extracts contain LC3II protein detected by western blot.3) TEM examination revealed that there were a lot of autophagosome with bilayers structure in extracts.2. The proportion of IL-10+B cells was as high as 5%-8% in PBMC after co-culture with MedAP in vitro for 3 days and it was significantly higher than that in unstimulated PBMC (1%-2%).3. Compared with the control group, the rate of cell division of CD4+T and CD8+T cells from PBMC concultured with B cells which was induced by MedAP for three days was significantly reduced from 49.9% and 61.1% to 33.1% and 45.3%.4. The level of HMGB1 in MedAP from different patient varied widely and there was a positive correlation between the proportion of IL-10+B cells induced by MedAP and the level of HMGB1in MedAP.Conclusions:1. Malignant effusions collected from tumor patient contain autophagosome.2. MedAP induce B cells to become IL-10+B cells and the IL-10+B cells can inhibit the proliferation of T cells.3. The proportion of IL-10+ B cells induced by MedAP is positive correlation with the levels of HMGB1in MedAP.