| Background:MM is a malignant hematological disease characterized by abnormal proliferation of plasma cells in bone marrow.According to GLOBOCAN,global MM accounted for 0.9%of all new cancers and 1.1%of all cancer deaths in 2018.The incidence rate of hematological malignancies ranked second only to lymphoma.Currently,the main treatment methods for myeloma include proteasome inhibitors,immunosuppressants,monoclonal antibodies,and bone marrow transplantation.Although it has extended the survival period of patients to a certain extent,the recurrence and refractory are still common.In the tumor microenvironment,in order to survive,tumor cells reorganize the metabolic mechanism of the body,resulting in overexpression of inhibitory tumor immune targets such as PD-1,CTLA-4,LAG-3,Tim-3,CD244,CD160,CD38,and CD47,leading to immune escape and drug resistance of tumor cells.Therefore,it is necessary to identify immune target of tumor cells during treatment to achieve the effectiveness of tumor therapy.As a key enzyme in the adenosine pathway,CD73 has both the hydrolase and non-hydrolase.As a hydrolase,CD73 can hydrolyze extracellular AMP into adenosine and participate in inhibiting platelet aggregation,anti-inflammatory reactions,and immunosuppression.As a non-hydrolytic enzyme,CD73 participates in adhesion molecule signaling pathways between cells through laminin and fibronectin,and can regulate the invasion and metastasis of cancer cells.CD73 was found to be highly expressed in MM bone marrow stromal cells,lymphocytes,dendritic cells.Therefore,it is reasonable to believe that CD73 is involved in the formation of immunosuppressive micro environment in myeloma myeloma,and may participate in the proliferation and drug resistance of myeloma cells.Objective:By detecting the expression of CD73 on immune cells in MM patients,we explored the impact of blocking CD73 on monocytes in MM patients on their anti-tumor immunity,and provided potential experimental evidence for clinical treatment of MM.Methods:1.Flow cytometry:(1)Detection of CD73 expression levels on T lymphocytes,B lymphocytes,NK cells,and monocytes in MM patients BM and PB and control PB;(2)Detect the proportion of residual CD138+cells in monocytes co-incubated with RPMI-8226 cells in PB of MM patients and control;(3)Detect the proportion of residual CD138+cells in BM mononuclear cells of MM patients before and after added PSB-12379;(4)Detect the proportion of residual CD138+cells after co incubation of PB monocytes and RPMI-8226 cells in MM patients before and after added with PSB-12379;2.Cytometric bead array:(1)Detection of IL-2,IL-4,IL-6,IL-10,TNF-αand IFN-γin PB monocytes of MM patients and control groups in-cubated alone or with RPMI-8226 cells;(2)Detection the level of IL-2,IL-4,IL-6,IL-10,TNF-αand IFN-γsecretion by BM mononuclear cells in MM patients before and after PSB-12379intervention;(3)Detection of secretion of IL-2,IL-4,IL-6,IL-10,TNF-αand IFN-γby PB monocytes co-incubated with RPMI-8226 cells in MM patients before and after with PSB-12379;3.Microscopic:(1)Index of phagocytosis of RPMI-8226 cells by monocytes in PB of MM patient and control;(2)Before and after PSB-12379 intervention,the proportion of BM mononuclear cells in MM patients phagocytosis of plasma cell;(3)The proportion of mononuclear cells phagocytosis of RPMI-8226 cells by MM patients PB monocytes co-incubated with RPMI-8226 cells before and after PSB-12379 intervention;Results:1.The expression of CD73 on BM and PB in MM patients:CD73 is expressed on T-,B-and NK-lymphocytes,and monocytes in BM and PB of MM.The expression of CD73 on T-,B-and NK-lymphocytes and Monocyte in BM of MM patients was slightly higher than that of corresponding cells in PB,and there was no statistical difference;There was no significant difference in the expression of CD73 between T-,B-and NK-lymphocytes in PB of MM patients and the control;Compared with monocytes in the control,the expression of CD73 on monocytes of MM patients is significantly increased(P<0.000).2.Comparison of anti-tumor immunity of monocyte between MM patients and control:Before co-culture,MM monocytes secrete TNF-α(p=0.003),IFN-γ(p=0.031)and IL-10(p=0.023),increased significantly compared to control monocytes.After monocytes from MM or control group were co-cultured with RPMI-8226 cells,in the supernatant,MM group secreted lower levels of IL-2(p=0.023),TNF-α(p=0.009)and IFN-γ(p=0.006)than control;In cell precipitation,CD138+%in the control was significantly lower than that in the MM group(p<0.000).Under microscope,the phagocytic index of monocytes in the control was significantly higher than that in the MM group(p=0.001).3.The effect of CD73 inhibitor on anti-tumor immunity of BM mononuclear cells in MM patients:In patients with MM,BM mononuclear cells with CD73 inhibitors compared with those without CD73 inhibitors secrete more IL-2(p=0.026),TNF-α(p=0.027)and IFN-γ(p=0.022)in the supernatant.Fewer CD138+cells were seen in cell precipitation(p=0.047).4.The effect of CD73 inhibitor on anti-tumor immunity of PB monocytes in MM patients:PB monocytes of MM in the group with CD73 inhibitor compared with the group without CD73 inhibitor,in the supernatant,the group with CD73 inhibitor secrete higher IL-2(p=0.013),TNF-α(p=0.014)and IFN-γ(p=0.003).Fewer CD138+cells were seen in cell precipitation(p=0.003)in the group with CD73 inhibitor.Under microscope,it was observed higher phagocytic index of monocytes(p=0.020)in the group with CD73 inhibitor.Conclusion:1.CD73 is expressed on both BM and PB immune cells in MM patients.There was no significant difference in the expression of CD73 between T-,B-and NK-lymphocytes in PB of MM patients and the control;Compared with PB monocytes in the control,the expression of CD73 on PB moncytes in MM patients is significantly increased;2.Compared with the control,PB monocytes in MM patients exhibit anti-tumor immune deficiency;3.CD73 inhibitors can improve the anti-tumor immunity of BM mononuclear cells in MM patients;4.CD73 inhibitors can partially reverse the anti-tumor immunity of PB PB monocytes in MM patients. |
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