Preliminary Research About Artificial Antigen Presenting Cells Specifically Presenting PR1

T cell immunity play a major role in the tumor immunity.Activated CD8+T cells can secret cytokines, and promote tumor cells to apoptosis. However,it is limited to HLA I matching that CD8+T lymphocytes recognize tumor antigen.The activation of specific CD8+T lymphocytes needs two signals,the first is the interaction between TCR and p/MHC complex, and the second is the interaction between costimulatory molecules Receptor and costimulatory molecules Ligand.Tumor adoptive immunotherapy usually use autoallergic APC activated T cells,but the production of autoallergic APC is very time consuming,exertion,and expensive.These problems limit its clinical application. The arrival of artificial antigen presenting cells(aAPC) resolves these problems.AAPC is very easy to culture. And the condition of its activating T cells is very easy to control.There needs a few time that aAPC activate and expand T cells.Therefore,the research about aAPC is gradually active for the past few years.We have succeeded building a single chain trimer(SCT) recombinant fusion gene.And it consists of MHC I,β2m and antigen peptide,connected by proper linker.In my research,we adopt HLA-A*0201whose epitope is PR1(VLQELNVTV).PR1, which is an HLA-A*0201restricted antigenic peptide derived from myeloid leukemia associated antigen protease3, can prime specific CTLs responses against myeloid leukemia cells.We will use Lentiviral Vector to transfer steadily PR1-SCT gene to aAPC K562/CD86cell lines which have been prepared and have polyclonal stimulate ability,making it providing antigen specific CD8+T cells of not only costimulatory signal(namely the second signal),but also specific antigen peptide signal (namely the first signal) through MHC molecules.Firstly we use gene engineering technology to clone PR1-SCT gene into the gene transfer vector,and Sequencing it.Then we use three plasmids co-transfect the293T cells, after48h harvest the supernatant which contains the virus particles,which is Pseudotyped virus with the ability of one infection and no copy.And it infects target cells (K562/CD86) at appropriate MOI.Through the antibiotics screening and FACS technology, high percents K562/CD86/PR1-HLA-A*0201-SCT is acquired, and is validated by the Western Blot technology.Then separated PBMCs and MMC-treated aAPCs are mixed lymphocyte culture.After several rounds of culture,we detect the frequency of PR1-specific CTLs by commercial Pentamer, validating specific stimulate ability of aAPCs.So,we have succeeded in preparing aAPCs providing two signals,which lay the foundation of adoptive immunotherapy fro myeloid leukemia,and provide convenience to the research about other disease.