Killing Effect Of EGFRvⅢ Polypeptide Sensitized DC-CTL Cells On Expressing EGFRvⅢ Glioma Cells In Vitro

Backgroud With the rapid development of oncology and immunology, tumor immunotherapywith its fewer side effects, good tolerance has been paid more and more attention, and become a method oftreating tumor after surgery, radiotherapy, chemotherapy.Dendritic cell is the most potent professionalantigen presenting cells inhuman(APC), it can mediated immune responses through multiple pathways toplay the role of anti-tumor, tumor immunotherapy based on dendritic cell become the focus of presentresearch.Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant ofepidermal growth factor receptor (EGFR), which is highly expressed in malignant tumors such as glioma,gastric cancer, breast cancer and closely related with occurrence, development and prognosis of tumor.Thecharacteristics of activating the downstream signal transduction system without ligand and expressing onlyin tumor cells make EGFRvIII become a good target for tumor targeting therapy. To prepare DC in vitro,and load EGFRvIII polypeptide, co-culture with T lymphocytes, then activate T cell into cytotoxic Tlymphocytes (CTL), to make T-cell recognize expressing EGFRvIII of glioma cells, verify its targetedkilling effect in vitro, explore the new methods of malignant tumor immunotherapy.Objective To amplify dendritic cells by culturing peripheral blood mononuclear cells in vitro andload EGFRvIII polypeptide onto dendritic cells, then induce T cells into antigen-specific cytotoxic Tlymphocytes by dendritic cells, verify special killing of CTL against expressing EGFRvIII U87gliomacells.Methods To extract peripheral blood from healthy volunteers, isolate mononuclear cells byFicoll lymphocyte separation medium and density gradient centrifugation, induce mononuclear cellsdifferentiate into immature DC by adding cytokines IL-4and GM-CSF, then add EGFRvIII polypeptideinto the culture system, immature DC develop into mature DC by uptaking antigen,measure the change ofdendritic cell phenotype by flow cytometry instrument. obtain T lymphocytes from peripheral blood, addcytokine IL-2to stimulate the proliferation of Tcell, co-culture the Tcell and mature DC, activatecontinually T lymphocytes cells into CTL, add EGFRvIII+U87, U87glioma cells which are used as target cells into96-well culture plate and culture for24hours, then add effector cells into target cells at a20:1ratio of effector to target.use CTL and the T cell cultured with IL-2as effector cell, set three control groupin the meantime, set up three holes for the cytotoxicity group and control group, add MTT to each wellafter12h incubation, and then incubate for another4h, A490nm OD value was measured with an ELISAreader，calculate killing rate.Results Afer mononuclear cells isolated from peripheral blood were cultured for3hours, most ofthe cells were round, arranged in clusters, adherent growth under an inverted microscope, to take smallcells for counting,1ml healthy blood can obtaine1.4×106monocytes. suck out suspension cells, addRPMI1640containing10%FBS,50ng/ml IL-4,500ng/ml rhGM-CSF into the culture medium, Afterculturing for24h, the adherent cells were decreased and the suspension cells were increased, part of thecells clumping together was observed under an inverted microscope; by48h the cells began to becomeirregular shape, suspension cells gradually were increased; while on the third day cells stretched out manyburr-like projections and became larger, began to cluster, showed a small colony; by5or6d protrusions ofcells became thicker and longer than previous cells, the cells became larger, suspension cells wereincreased; on the seventh day, cells became bigger and had a variety of shapes, some chunky, some slender,most cells suspended and scattered evenly, had a typical dendritic protrusions, and the results of DCphenotypic detection showed that the expression rate of CD83(DC maturation marker) was only2.36%,HLA-DR(DC’s MHC-II molecule)was54.38%, CD80was9.27%,DC is in immature state at this time. DCphenotype was detected after loading EGFRv III polypeptide once more, the results showed that the DChighly expressed CD83/HLA-DR and costimulatory molecules CD80, immature DC differentiated intomature after loading EGFRvIII polypeptide. Mixcultured mature DC and T lymphocytes, constantlystimulated proliferation of T lymphocyte, the percentage of CD3+CD8+T cell was increased from5.4%to12.7%. The CTL induced by DC pulsed with EGFRvIII peptide had significant activity killing EGFRvIII+U87glioma cell (45.214.53)%, higher than the Tcells cultured with IL-2(12.302.15)%,(P <0.01),The CTL induced by DC pulsed with EGFRvIII polypeptide had killing the U87(14.483.50)%,compared with the T cells cultured with IL-2((18.104.16)%, the difference was not significant (P>0.05). Conclusion Cytotoxic T lymphocytes (CTL) which was induced by DC loaded EGFRvIIIpolypeptide can specifically recognize and kill EGFRvIII+U87glioma cell, It established an experimentalbase for immunotherapy of glioma cells. glioma cells.