Studies On Biological Properties And Anti-leukemia Effects Of TGF-?1?silenced Leukemia Cell?derived Exosomes

Background and Aim:Minimal residual leukemia cells?MRL?is the major cause for leukemia relapse after remision.Therefore,there is pressing need to develop effective strategies to eradicate MRL for prolonging the long-term suvival of leukemia patients.Traditional treatments including chemotherapy and hematopoietic stem-cell transplantation?HSCT?may cause serious adverse effect due to their unspecific effect.However,this limitation can be overcomed by immunotherapy which is designed to elicit specific immune response to killing tumor cells.Therefore,with this advantage,immunotherapy has been attracted more attentions in recent years.Exosomes are a subtype of membrane bound nano-vesicles released by both healthy and tumor cells including leukemia cells.Leukemia cell derived exosomes?LEX?,a kind of tumor cell derived exosomes?TEX?,have been demonstrated to be enriched in tumor associated antigen?TAA?as well as a series of immun-related proteins including HSP and MHC moleculars.It has been found that LEX can be uptake by APC efficiently,and thereby initiate antigen-specific immune response.Besides,exosome can be aquired by non-traumatic method.Moreover,it has been reported that exosomal cancer immunotherapy is independent of MHC restriction,and is capable of crossing blood brain barrier and well tolerated.Therefore,LEX can be developed as a potential candidate for a novel source of anti-leukemia vaccines.However,the data from preclinical studies has demonstrated that non-modified TEX based vaccine did not manifest satisfactory effect in inducing efficient anti-tumor effect on cancer patients,or worse,mediated immune tolerance.Therefore the relatively poor immunogenicity of TEX-vaccine in patients limited its further clinical application.It has been reported that TEXs carry a series of immunosuppressive factors including TGF-?1,which plays a crucial role on impairing the efficacy of TEX based vaccine through multiple mechanism.In the present study,we silenced TGF-?1 in L1210 leukemia cells with a lentiviral shRNA vector and prepared the LEX_TGF-?1si.Moreover,we investigated the immunogenicity of the modified LEX-based vaccine in vitro and in vivo.Methods:Exosomal TGF-?1 was downregulated via lentiviral shRNA silencing of TGF-?1on leukemia cells.All exosomes were isolated and purified by ultracentrifugation.The characteristics of LEX_TGF-?1si were determined via electron microscopy,western blot analysis,and flow cytometry?FCM?.The uptake of exosomes by DCs were investigated by confocal and FCM.The impact of exosomes on DCs'phenotype and function were detected by FCM,enzyme linked immunosorbent assay?ELISA?and mixed lymphocyte reaction?MLR?,and the related mechanism was investigated by Real-time PCR and western blot analysis.DC_LEX-TGF-?1si induced CD4⁺T-cell proliferation,Th1 cytokine secretion,specific CTL activity were analyzed by cell proliferation assay,cytotoxicity assays and ELSIA assay.Moreover,to verify the superiority of DC_LEX-TGF-?1si immunization in vivo,DC_LEX-TGF-?1si was subcutaneously injected into DBA/2 mice:either followed by tumor challenge or tumor bearing,and the prolonged survival and tumor growth were compared.Results1.Using lentiviral shRNA can successfully silence TGF-?1 on L1210 cells.Exosomes from TGF-?1 silenced L1210 cells were accord with the typical exosome characteristics.Moreover,compared with non-modified LEX,LEX_TGF-?1siGF-?1si contained downregulated TGF-?1 protein.2.LEX_TGF-?1siGF-?1si can be efficiently uptake by DCs,and more potently upregulated CD80,CD86 and MHC II expression on DC surface.Moreover,the secretion of IL-12p70 and TNF-?from DC were upregulated by LEX_TGF-?1si.While,TGF-?1production was downregulated.Besides,DC_LEX-TGF-?1si could facilitated T cell proliferation more effecicently.It has been found that during the process of DC maturation,LEX inhibited the activity of TLRs/MyD88/NF-?B signaling pathway in DCs through ubiquitination and proteasomal degradation of the adapter protein,MyD88.However,LEX_TGF-?1siGF-?1si can effectively reverse the degradation of MyD88protein,and therefore more effectively promote the maturation of DCs than non-modified LEX.3.Compared to DC_LEX,DC_LEX-TGF-?1siEX-TGF-?1si induced antigen-specific CD4⁺T cell proliferation and Th1 cytokine secretion more potently.Besides,the cytotoxicity assays demonstrated that DC_LEX-TGF-?1si could induced a more potent CTL response than DC_LEX.4.In both protective and therapeutic antitumor tests,immunization with DC_LEX-TGF-?1si resulted in a more potent capability to inhibit tumor growth and to prolong survival.It has been found DC_LEX-TGF-?1siEX-TGF-?1si vaccination could effectively upregulated the percentage of CD4⁺T,CD8⁺T,Th1 and Tc1,but downregulated the percentage of Tregs in tumor-bearing mice.Conclusion:These data suggested that down-regulation of exosomal TGF-?1through gene modification on parental cells by lentiviral shRNA is a feasible strategy.LEX_TGF-?1siGF-?1si could more effeciently promoted the maturation and function of DCs.Vaccination with DC_LEX-TGF-?1siEX-TGF-?1si could induce more powerful antigen-specific CD4⁺T cells proliferation,Th1 cytokine secretion and CTL response,therefore effectively induced a more potent anti-tumor immunity in animal models.Our strategy of optimizing exosome vaccine may have promising potential for leukemia immunotherapy.