Dendritic Cells Transfected With Immunoglobulin Heavy Chain Variable Gene Framework Region Vaccine Can Induce Anti B-cell Malignance CTL Response

ObjectiveTo transfect dendritic cells(DC)with the fusional family-DNA vaccine combined with immunoglobulin heavy chain variable(IgHV)gene framework region(FR)and cytokine sequence,and stimulate peripheral blood lymphocyte in vitro,to determine whether the framework region DNA vaccine can induce the antigen-specific cytotoxic T lymphocytes(CTL)response and provide the experimental basis for further clinical trials of active immunization or adoptive T-cell therapy.MethodsThe EndoFree Plasmid Purification kit was used to prepare the fusional family-DNA vaccine combined with the human immunoglobulin heavy chain variable gene framework region and granulocyte macrophage-colony stimulating factor(GM-CSF)sequence that have been constructed by our laboratory.The human Peripheral Blood Mononuclear Cells(PBMC)were collected to culture DC.Adherent cells were induced by GM-CSF,IL-4 to generate DC with a cytokine cocktail for promoting maturation.The flow cytometry assay was used to detect the phonetype of DC.The DC were transfected with DNA vaccine by genegun before maturation and the expression of the transfected plasmid was detected with reverse-transcription polymerase chain reaction(RT-PCR)and Enzyme-Linked Immunoabsorbent(ELISA)method.At the same time,the suspended PBMC were cultured under the induction of IL-2 and other cytokines, pulsed with the transfected DC to induce the antigen-specific CTL response, which can be determined with the synthesized MHC pentamer by flow cytometry assay.The function of cultured T lymphocyte lysising the B cell lymphoma with the same sub-family IgHV was further detected by the fluorometric assessment of T lymphocyte antigen specific lysis(FATAL).Gene scan by genetic analyzer was used to analyze the T cell receptorβchain variable region(TCRBV)to observe the changes oft cell clones before and after culture.ResultsThe endotoxin-free plasmid IgHV1-GM-CSF/pcDNA3.0 and the control plasmid pcDNA3.0 were prepared for transfection.The sequencing showed that the recombinant vector included the leader peptide,FR region and connected GM-CSF cDNA.The FR region contained the coding sequence QLVQSGAEV of the HLA-A~*0201 restricted antigen nonamer peptide.With the induction of cytokines,PBMC could generate the typical morphology DC,which were about 85.0%～96.5%of the adhered cells while about 6.9%～11.3%of PBMC.After adding the cytokine cocktail,the surface molecules of CD80,CD83,CD86 were significantly increased on DC.The result showed that we got the maturated DC.DC were transfected with the comparable 4 kinds of methods,that is,nucleofector electrotransfer,genegun,Lipofectamine 2000 and FuGENE 6.The efficiency of electrotransfer was highest,but a large part of DC were died or lost the polysynapse structure after transfection.So the genegun ranked the second was choosed for DC transfection.The expression of fusional DNA vaccine in DC was measured by RT-PCR and ELISA Then,the PBMC from 2 normal donors and 2 patients with B-cell lymphoma were cultured,following the transfection and the pulse.FCM assay displayed that the lymphocytes cultured in vitro were mainly CD8+ T cell.The results of Pentamer showed that the DC could induce the antigen specific CTL response after transfection with the IgHV1-GM-CSF fusional vaccine,the highest being 4.4%,while the corresponding antigen nonamer peptide could also induce the CTL response with a little earlier time phase.The lysis assay showed that the T cells transfected with the DNA vaccine enhanced the lysis ability to B-cell lymphoma cells with the same IgHV family.After twice stimulation,the rate of lysising tumor cell was 32.4±4.9%by cultured CTL of the patient,while 18.1±3.5%by cultured CTL pulsed with antigen nonamer peptide.TCRBV genescan showed that some lymphocyte of TCRβfamily dissapeared and the T cell clones were oligoclone distribution or complete absence,only few remained polyclonal distribution after chemo-therapy in patients with lymphoma and leukemia.However,after stimulation with cytokine and co-culture with DC,the TCRβpresented the normal polyclone,but the individual advantage clone characteristics of part family appeared after stimulation by DNA vaccine,in which the CTL clones induced by antigen maybe exist.Conclusion1.Primary DC cultured from human PBMC were successfully transfected for preparing the DC vaccine immunized by the family IgHV frame region DNA using the genegun method.2.The DNA vaccine of family IgHV frame region could induce the antigen-specific CTL response in vitro,which could kill the B-cell lymphoma cell of the same IgHV family.