The Dual Role Of Diffuse Large B Cell Lymphoma Derived Exosomes In Tumor Immunity

BackgroundDiffuse large B cell lymphoma(DLBCL)is the most common type of adult non-Hodgkin lymphoma.DLBCL is presented as an aggressive lymphoma and characterized by rapidly metastasis to lymph nodes,spleen,liver,bone marrow and other organs.For the treatment of DLBCL,CHOP has already been the standard combination chemotherapy.The incorporation of the CD20 antibody has significantly improved the outcome of DLBCL patients.However,due to the heterogeneity of DLBCL,approximately a third of DLBCL patients still have a relapse or drug resistance.Cancer immunotherapy aimed at improving the antitumor immune function of patients,has been the focus of cancer treatment.Immunotherapy has the advantage of less side effects than traditional chemotherapy and radiotherapy.The therapeutic effect of DC vaccine activated by tumor antigen to induce specific immune response has been clinically verified.Still,therapeutic efficacy can't meet our expection due to the weak immunogenicity of tumor antigens.Therefore,we are eager to find specific tumor antigen,which can reflect characteristics of different subtypes of DLBCL to induce stronger immune response and targeted therapy and improve survival rates of patients with DLBCL.Exosomes(EXOs),nanoscale vesicles,were secreted by many cells including hematopoietic cells and tumor cells.EXOs carry the unique characteristics of their origin cells and tumor microenvironment,involved in various biological processes such as immune suppression,angiogenesis,metastasis and antitumor activity.EXOs with unique biological characteristics of diseases,work as DC vaccine tumor antigens are expected to induce antitumor effects more precisely.But the biological characteristics and role of diffuse large B cell lymphoma(DLBCL)-derived EXOs have been less investigated.ObjectTo study the specific biological characteristics of the EXOs secreted by DLBCL cells,and to explore a new therapeutic model for the immunotherapy of DLBCL.MethodsThe biological characteristics of DLBCL EXOs was determined by Western blotting(WB),Flow cytometry(FCM),transmission electron microscope(TEM),nanoparticle tracking analysis(NTA),protein microarray and MicroRNA microarray.The PKH67 stainer was used to study the uptake of DLBCL EXOs by DCs and tumor cells.FCM was applied for the study of transfer molecules by DLBCL EXOs.Growth of DLBCL EXOs on stromal cells was determined by MTT assay.We use scratch wound assay to detect the migration of HUVECs treated by DLBCL EXOs.Transwell invasion assay,WB and Q-PCR were used to detect the invasion of DLBCL EXOs treated stromal cells.The angiogenesis induced by DLBCL EXOs was determined by Matrigel tube formation in vivo and vitro.DLBCL EXOs mice model was established to investigate the function of DLBCL EXOs in vivo by HE staining and immunohistochemistry.DCs pulsed by DLBCL EXOs were cocultured with T cells to dectect the function of T cells by CFSE stainer and FCM.LDH cytotoxicity assay and FCM to detect the antitumor effect of DLBCL EXOs in tumor bearing mice immunized by TEX.ResultsWe demonstrated that EXOs derived from DLBCL cell lines displayed malignancy molecules such as c-myc,Bcl-2,Mcl-1,CD 19,and CD20.There was a different protein expression pattern between DLBCL EXOs and Burkitt lymphoma TEXs.DLBCL EXOs were easily captured by DCs and lymphoma cells,and mainly acted as an immunosuppressive mediator,evidenced by induction of apoptosis and upregulation of PD-1 in T cells.Furthermore,the TEXs stimulated not only cell proliferation,migration of stromal cells but also angiogenesis.As a result,the TEXs promoted tumor growth in vivo.On other hand,DLBCL EXOs did not induce apoptosis of DCs.After pulsed with the TEXs,DCs could stimulate clonal expansion of T cells,increase the secretion of IL-6 and TNFa,and decrease the production of immunosuppressive cytokine IL-4 and IL-10.The T cells from tumor bearing mice immunized by TEXs were shown to possess superior antilymphoma potency relative to immunization of tumor lysates.ConclusionDLBCL EXOs displayed malignancy molecules and transfer molecules to other receptor cells.DLBCL EXOs stimulated not only cell proliferation,migration of stromal cells but also angiogenesis.Also,DLBCL EXOs mainly acted as an immunosuppressive mediator.Pulsed with the TEXs,DCs could enhance Tcells immune function.