A Cell-Based Artificial Antigen-Presenting Cell K32/4-1BBL/CD86 For Stimulation Of Human T Cells

Tumor Adoptive immunotherapy always is the most active field of tumor biology therapy, injection antigen specific CTL of oneself not only can kill tumor cells but also can conquer the difficulty that the low effective of tumor bacterin therapy. So the sufficient activation of antigen specific CTL in vitro are very important. After antigen sppecigic CTL is injected into human body, it could be expanded without apoptosis, and get together into the tumor to kill tumor cells but have no effect on normal cells in vivo. All the demands are making reference to index sign that doctor choose on tumor adoptive immunotherapy. With the development of artificial antigen-presenting cells (aAPC), that active CTL cells in vitro are going into simple, cheape and have good repetition. Artificial APC will play a more and more important role in tumor adoptive immunotherapy.In the base of that we have got two recombination expressing vectors pV4-1BBL-CD86 and pcDNA3.1(+)-CD32a. Through the gene transfection mediated by liposome, the recombinant plasmid pV4-1BBL-CD86 and pcDNA3.1(+)-CD32a were transfected into K562 cells separately. The cell line K32/4-1BBL/CD86 which expressing 4-1BBL, CD86 and CD32a stablely have been got by filtration of G418, hygromycin and FACS. The human peripheral blood lymphocytes were stimulated with this cell-based aAPC (K32/4-1BBL/CD86) which is co-culture with CD3 monoclonal antibody in advance. The active state of CD4+ and CD8+ T cells, the proliferation ability of CD3+T cells, the anti-apoptosis ability of PBMC, the ability to secrete perforin and IFN-γof CTL were observed respectively one week later. And the artificial APC's fuction on tumor adoptive immunotherapy in vivo were estimated with an animal tumor model.1. Construction of a cell-based artificial APC line K32/4-1BBL/CD86Two signals provided by APC are essential in lymphocytes'activation, the first is antigen peptide presented by MHC I molecule, the second is co-stimulation molecule such as CD86, 4-1BBL and ICOSL. It's very important for the lymphocyte to co-stimulate with this artificial APC in activation of CTL cells. In order to active the peripheral blood lymphocytes adequately in vitro, we choose 4-1BBL and CD86 as the co-stimulater. 4-1BBL and CD86 gene were cloned into the vector pVITRO2-mcs. The 4-1BBL and CD86 gene were promoted by different promoter respectively. CD32a gene is cloned into the vector pCDNA3.1(+) and got the recombinant vector pCDNA3.1(+)CD32a. The recombinant plasmid pV4-1BBL-CD86 and pcDNA3.1(+)-CD32a were transfected into K562 cells separately to got the modified K562 cell (K32/4-1BBL/CD86) which express 4-1BBL CD86 and CD32a stabley. K32/4-1BBL/CD86 cells through molecule CD32a to recombinat with CD3 monoclonal antibody to construct a cell-based artifical antigen-presenting cell. This aAPC will provide two basic signals to active human peripheral blood lymphocytes.2. Analysis the ability of K32/4-1BBL/CD86 cells to active human peripheral blood lymphocytes in vitroOn the condition that we have succeeded in having artificial APC K32/4-1BBL/CD86 line, we evaluate CTL cells are actived by this artificial APC from the following aspect: the activation state, proliferation, cell division, anti-apoptosis and the ability to secrete perforin and IFN-γ. From the experimental results we have found that CD69 are up-regulated observably in CD4+ and CD8+ T cells comparied with the control groups (P﹤0.001); The proliferation ability of actived CD3+T cells increase 2 times than of the control group. The anti-apoptosis of actived CD3+ T cell increase 3.5 times than of the control group. And the ability of secreting perforin and IFN-γin actived CD8+T cells is high than in control groups.3. Analysis the ability of actived PBMC controlling tumor growth in vivo in tumor animal modelNOD-SCID mice were transplanted with human colonic tumor cell line LS-174-T in amount of 3×106 per mouse to form a transplanted tumor mice model. When the transplanted tumor come into being mass in armpit subcutaneous of NOD-SCID mice,the actived human PBMC by K32/4-1BBL/CD86 cell and anti-CD3 monoclone antibody were injected into tumor mass once a week in the following 3 weekes. The total amount of PBMC cells injected into mouse is 3×107 per mouse. From the observed results we founded that transplanted tumor in the treated group is smaller than the control group. It clue on that actived human CTL by aAPC K32/4-1BBL/CD86 line maybe have the ability to inhibite the tumor growth in vivo.