Effects Of IFN-γ On The Immunogenic Cell Death In Lymphoma Cells Induced By TMSC-CM

Background and ObjectiveMesenchymal stem cells(mesenchymal stem cells)are derived from the ectoderm and mesoderm in the early stages of development.Mesenchymal stem cells can be isolated and cultured from bone marrow,fat,umbilical cord and other tissues in the human body.Mesenchymal stem cells have the potential for multidirectional differentiation,and different induction conditions can make them differentiate into a variety of tissue cells.As the precursor cells of most stromal cells,mesenchymal stem cells are an important part of the tumor microenvironment.They can reach the tumor focus through recycling,form the tumor microenvironment,and participate in the occurrence and development of various types of tumor diseases,including lymphoma.Among them,mesenchymal stem cells(MSC)have anti-tumor effects and are preferred because of their characteristics(such as immune regulation ability and ability to accumulate in tumor sites).Thymus derived mesenchymal stem cells(tMSCs)have similar immunological characteristics to mesenchyme from other sources.One of the main advantages of tmscs is its ability to regulate the immune response,which is due to its key role in the development and function of the immune system.There are literature studies that when tMSCs are co-cultured with blood-derived T lymphocytes,MSCs significantly inhibit the proliferation of T lymphocytes.IFN-γ is considered to have a strong pro-inflammatory effect,and mesenchymal stem cells treated with IFN-γ have enhanced immunomodulatory properties.Studies have also reported that mesenchymal stem cells or their conditioned medium(MSC-CM)can cause apoptosis in a variety of cancers.In the past few decades,apoptosis has been considered to be programmed death rather than necrosis,and apoptosis is considered to cause no immune response.However,there is increasing evidence to support that some apoptotic cells can produce immune stimulation by releasing damage-related molecular patterns(DAMPs),which can be swallowed and presented by dendritic cells(DCs)to trigger T lymphocyte-mediated immune cytotoxicity.This functionally specific type of cell death is called "immunogenic cell death"(ICD).This study wanted to explore whether the conditioned medium of thymic mesenchymal stem cells and the conditioned medium of thymic mesenchymal stem cells stimulated by IFN-γ can inhibit the proliferation of lymphoma cells through the path of immunogenic death(ICD).Methods1.The thymus tissue removed from the operation of children with congenital heart disease was digested with collagenase IV to obtain adherent cells,which were passed to 3-5 generations for subsequent experiments.The Adherent cells were identified as thymic mesenchymal stem cells(tMSCs)by flow cytometry detection of positive expression markers CD73,CD90,CD105 and negative markers CD34,CD45,Epcam.2.The expression changes of tMSC cytokines: IDO1,COX-2,PD-L1,IL-6,IL-1α,IL-1β m RNA expression changes by different concentrations of IFN-γpretreatment were detected by q RT-PCR.3.Multi-factor detection was performed by flow cytometry to detect the effect of IFN-γ on the secretion of cytokines from tMSC.4.The conditioned medium from tMSCs(tMSC-CM)and IFN-γ pretreated tMSC conditioned medium(IFN-γ-tMSC-CM)were collected and concentrated 1:1for subsequent experiments.5.For the detection of apoptosis after co-cultivation of tMSC-CM and IFN-γ-tMSC-CM with Ramos and H9 lymphoma cells,1640 medium was used as a control.After Hoecst33342 staining,fluorescence microscope observation to determine the apoptosis of lymphoma cells and taking pictures,the nucleus was densely stained and bright blue was judged to be apoptosis.After staining with Annexin Ⅴ and 7-AAD,flow cytometry was used to detect and record the apoptosis of lymphoma cells.Annexin Ⅴ+/7AAD-was judged as early apoptosis,and Annexin Ⅴ+/7AAD+ was judged as late apoptosis.6.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells,1640 medium was used as a control.1640 medium was used as a control.Stained with Calreticulin(CRT)antibody,Heat shock protein HSP70 antibody and fluorescent secondary antibody respectively,flow cytometry was used to detect the expression differences of CRT and HSP70 in each group of culture media,and fluorescence microscope was used to observe their exposure location on the surface of lymphoma cells.7.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells co-cultured for 48 hours were collected,and the level of ATP released into the supernatants was determined by chemiluminescence.8.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells co-cultured for 48 hours were collected,and the level of HMGB1 released into the supernatants was measured by enzyme-linked immunosorbent assay(ELISA).9.Mononuclear cells are separated from human venous whole blood,and human CM-CSF and IL-4 factors are added to induce dendritic cells.After dendritic cells were co-cultured with tMSC-CM and IFN-γ-tMSC-CM,flow cytometry was used to detect the expression of dendritic cell maturation markers CD80,CD83 and CD86.10.Flow cytometry was used to detect the expression of PD-L1 on the surface of lymphoma cells after co-cultured with Ramos and H9 lymphoma cells in each group for 48 hours.Experimental result1.Acquisition and identification of thymic mesenchymal stem cells(tMSCs).The adherent cells were successfully isolated from fresh human thymus specimens.The morphology of the adherent cells was similar to that of fibroblasts,with a long spindle shape.The positive expression rates of CD73,CD90 and CD105 on the surface of adherent cells were 97.8%,87.8%,and 96.0%,while the expression of CD34,CD45 and Epcam are all negative.Therefore,adherent cells isolated from thymus tissue were identified as thymic mesenchymal stem cells(tMSCs).2.Set the final concentration of IFN-γ to 25 μg/ml,50 μg/ml and 100 μg/ml to incubate tMSC for 48 hours.Compared with tMSCs cultured without IFN-γ,the expressions of IDO1(P <0.005),COX-2(P <0.005),PD-L1(P <0.005),IL-6(P<0.001),IL-1α(P <0.001),and IL-1β(P <0.001)in tMSCs cultured with IFN-γ were all up-regulated in a concentration-dependent manner.3.After IFN-γ cultured tMSC for 48 hours,compared with normal tMSC without IFN-γ,the secretion of MCP-1(P <0.01),IFN-γ(P <0.001),IL-6(P <0.01),and IL-33(P <0.01)increased,while the secretion of IL-8 decreased.4.Different conditioned media were co-cultured with Ramos and H9 lymphoma cells for 48 hours.Compared with the 1640 group,the early apoptosis rate of lymphoma cells increased in both the tMSC-CM group(P <0.01)and the IFN-γ-tMSC-CM group(P <0.005),and the IFN-γ-tMSC-CM group increased significantly.The late apoptosis rate increased in both the tMSC-CM group(P<0.05)and the IFN-γ-tMSC-CM group(P <0.005),and the IFN-γ-tMSC-CM group increased significantly.5.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells for 48 hours.Compared with the 1640 group,the expression of calreticulin(CRT)and heat shock protein(HSP70)on the surface of lymphoma cells were up-regulated in tMSC-CM and IFN-γ-tMSC-CM,and IFN-γ-tMSC-CM was up-regulated significantly.6.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells for 48 hours.Compared with the 1640 group,tMSC-CM released extracellular ATP(P <0.005)and IFN-γ-tMSC-CM released extracellular ATP(P<0.01),and the tMSC-CM group increased significantly.7.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells for 48 hours.Compared with the 1640 group,the level of HMGB1 released into the extracellular space increased significantly in the tMSC-CM group(P <0.05)and the IFN-γ-tMSC-CM group(P <0.01),and the IFN-γ-tMSC-CM group increased significantly.8.tMSC-CM and IFN-γ-tMSC-CM and lymphoma cells were co-cultured with i DC for 48 hours and compared with the 1640 group,the dendritic cell marker CD38 in the tMSC-CM group(P <0.01).And the expression of mature dendritic cell markers CD80(P <0.01),CD83(P <0.05)and CD86(P <0.005)increased;The expression of CD38(P <0.01),CD80(P <0.005),CD83(P <0.005),and CD86(P<0.005)in the IFN-γ-tMSC-CM group increased significantly.9.After tMSC-CM and IFN-γ-tMSC-CM were co-cultured with Ramos and H9 lymphoma cells for 48 hours,and then flow cytometry was used to detect the expression of PD-L1 on the lymphoma cells.ConclusionThymus-derived Mesenchymal Stem Cell Conditioned Medium(tMSC-CM)has the effect of inducing apoptosis of lymphoma cells.And IFN-γ pretreated thymic mesenchymal stem cell conditioned medium(IFN-γ-tMSC-CM)up-regulated this effect.tMSC-CM induces lymphoma cells that undergo apoptosis to express DAMPs molecules,down-regulates the expression of PD-L1,and promotes the maturation of dendritic cells.Therefore,it is believed that tMSC-CM can participate in the induction of immunogenic death(ICD)of lymphoma cells.In addition,tMSC-CM pretreated with IFN-γ can make the effect of ICD on lymphoma cells more significant.