BCR-ABL-Mediated BCR-ABL Derived Antigen Peptide Enhances Dc Cross-Presentation And Induces Anti-CML Effect

Chronic myeloid leukemia(CML)is a group of malignant proliferative tumor derived from hematopoietic stem cells.The main pathogenic mechanism is the translocation of abl gene on 9 chromosome and bcr gene on 22 chromosome,which encodes a continuously-expressing BCR-ABL protein with strong tyrosine kinase activity.At present,the application of specific tyrosine kinase inhibitors such as imatinib,dasatinib,nilotinib has made great progress in the treatment of chronic myeloid leukemia.However,drug resistance and relapse happens in 30%patients.It is urgent to explore a new treatment method.Immunotherapy has received extensive attention in the treatment of tumors.The BCR-ABL fusion protein encoded by bcr-abl gene is a genetic marker of CML with its antigen specificity limited to the fusion region.The antigenic peptides GFKQSSKAL(GF)and SSKALQRPV(SS),derived from the BCR-ABL fusion region,can be specifically presented on the surface of CML cells by MHC class I molecules.These peptides can be recognized as CML-specific antigen and is an ideal target for immunotherapy.Previous study has demonstrated that by loading dendritic cells with these antigens,CML-specific immune response can be induced.Due to the low efficiency of exogenous antigen presentation via DCs,however,the treatment effect is not significant.How to enhance the presentation ability of DC and improve the treatment effect becomes a major issue.Cytoplasmic transduction peptide(CTP)is a peptide with high cytoplasmic localization ability,which contains 11 amino acid residues.This provides the possibility for efficient transduction of exogenous antigen into the nucleus.In this study,we took advantage of CTP to locate the BCR-ABL antigen into the DC cytoplasm,endogenize the exogenous antigen.The endogenized antigen was presented by MHC class I molecules to CD8~+T lymphocytes,inducing CML-specific immune response.This experiment aims to provide a theoretical basis for CML treatment.The main research methods adopted are:1.Design and synthesis of peptides and observation of their localization effects.Through literature review,the appropriate antigen peptides derived from junctional region of BCR-ABL were selected and co-synthesized with engineered CTP.Synthetic peptides were co-cultured with mouse bone marrow-derived dendritic cells.The optimal concentration and transduction time of peptides was screened by direct fluorescence observation.The immunofluorescence and laser confocal experiments were used to observe the localization of synthetic peptides in DCs for investigating whether the CTP-mediated peptides have highly efficient cytoplasmic localization function.2.The observation of CTP fusion peptides on DC cytotoxicity and the effect of triggering cross-presentation.Cell counting kit-8 was used to detect cell viability by incubating the two CTP fusion peptides with DCs at different concentrations for different times.CTP-GF,CTP-SS,GF,SS were co-incubated with DC at optimal concentrations,then co-cultured with freshly extracted CD8~+T lymphocytes.Secreting Il-2 levels were detected by ELISA.3.The effectiveness of CTP fusion peptides in inducing CML-specific immune response.The experimental groups were set as DC/CTP-GF,DC/GF,DC/CTP-SS,DC/SS,DC/CTP,DC,Blank.Peptides of each group were co-cultured with DCs for immunizing mice and spleen lymphocytes were extracted from immunized mice.Flow cytometry was used to detect mouse lymphocyte proliferation,activation and degranulation ability after immunization.Secreting cytokines were analyzed by ELISPOT.Killing ability of effector T lymphocytes on target cells was detected by lactate dehydrogenase release assay.4.Therapeutic effect and memory effect of immune response induced by CTP-mediated peptides in mice.First,BP210 cells were injected into seven groups of mice through the tail vein.One week later,dendritic cells in DC/CTP-GF,DC/GF,DC/CTP-SS,DC/SS,DC/CTP,DC,Blank groups were injected through the groin to immunize mice once a week for a total two immunizations.Life status,body weight and white blood cell in peripheral blood were recorded weekly.When the mice showed claudication,paralysis of hind limbs and their body weight decreased rapidly,they were sacrificed.Spleens and livers were weighed and photographed.Wright’s staining was performed to check the infiltration of primitive cells.Immunofluorescence was conducted for detecting the expression of BCR-ABL in liver,spleen and bone marrow.Liver and spleen were fixed in 4%paraformaldehyde overnight,then Embedded in paraffin and sectioned.H&E staining was performed for observing infiltrating leukemic cells.Survival curve was obtained using Kaplan-Meier methods after 90 days’observation.Immune memory effect was observed after 90 days.A new Blank group was set in compared with DC/CTP-GF and DC/CTP-SS.The observation methods were the same as described above.Results and conclusions:1.Peptides in each group were successfully designed and synthesized.According to literature reports and experimental requirements,FITC was coupled with peptides on N-terminus using Acp linker.HA tag was added for further localization observation.Flow cytometry showed DCs were successfully cultured.The optimal transduction efficiency was achieved in the concentration of 10μmol/L for 30 minutes.Immunofluorescence showed that CTP fusion peptides had good cytoplasmic localization efficiency,which is further proved by laser confocal.2.CTP fusion peptides had good biological safety and successfully triggered cross-presentation procession.Cell counting kit-8 demonstrated the survival of DCs was not affected by CTP.ELISA showed that IL-2secreting level was higher in CTP-mediated groups,indicating that cross-presentation was successfully triggered in these groups and the antigen presentation efficiency was higher.3.The proliferation,activation and degranulation ability,cytokine secretion ability and target cell killing effect of T lymphocytes in CTP fusion peptide immunized group were significantly higher than those in other groups.Antigen-loaded dendritic cells via CTP fusion peptides could induce a more effective cellular immune response,thereby killing BP210 target cells.4.CTP fusion peptide immunized mice have a longer survival time,and their physical indicators are well maintained.Results of HE staining examination of leukemia cell infiltration in liver,spleen and bone marrow were significantly better than other control groups.CTP fusion peptide loaded DCs could stimulate CML-specific cellular immune response more effectively and has relatively good therapeutic effect on chronic myelogenous leukemia.Meanwhile,secondary attack of the tumor did not affect the mice in the CTP fusion peptide group,indicating that the memory T lymphocytes existed in the mice and the immune memory ability was stronger.In summary,we successfully apply CTP in mediating BCR-ABL protein-derived antigenic peptides to the cytoplasm of dendritic cells.It triggered cross-presentation of dendritic cells and presented exogenous antigens to CD8+T lymphocytes by MHC class I molecules,thus inducing the CML-specific cellular immune response to kill BP210 target cells.This study is the first time introducing CTP to the immunotherapy for CML from the perspective of cross-antigen presentation and initially achieved good experimental results,which may provide a new method for patients with CML.