The Experimental Study On In Vitro Preparation Of Adoptive Immunotherapy Cells Induced By CD40Signaling In Combination With Cytokines

Cancer is a severe disease that does harm to human health. Despite surgery,chemotherapy and radiotherapy treatments have been continuously improving， manypatients still suffer from tumor recurrence. Using tumor biological/immunologicaltherapy as a breakthrough to find an optimized multimodality therapy is a new way ofmodern cancer treatment. Adjuvant treatment，such as cancer vaccines, monoclonalantibody therapy and cytokines，and adoptive cellular immunotherapy are relativelycommon tumor immunotherapy.Adoptive cellular immunotherapy is to refuse in vitro proliferated immune cells(including specific and non-specific) with anti-tumor activity into cancer patientsdirectly or indirectly, so as to stimulate the body’s immune response to kill tumor cells,especially in immunocompromised patients. One of the main principles of it is to isolatethe patients’ peripheral blood mononuclear cells. After the interaction with specificantigen in vitro, the cells can make specific or non-specific anti-tumor ability. Whileproliferating these cells to a certain number, we reinfuse them into the patients toprevent tumor recurrence and metastasis. In a certain extent, it solves the patients’serious problems, such as poor immunity after radiotherapy and chemotherapy and lowquality of life, prolonging the survival time of patients.Currently, cytokine-induced killer cells (CIK) has been used to assist in thetreatment of various malignant tumors, because of its strong anti-tumor activity of Tcells and non-MHC-restricted killing tumor characteristics of NK cells，it is consideredto be the hope of tumor adoptive immunotherapy. However, its clinical application stillexists some problems to be solved. Further exploring the potential anti-tumoradvantages of adoptive immunotherapy cells has far-reaching significance for improving the position of tumor adoptive immunotherapy in comprehensive cancertreatment. Therefore, this study was prepared to achieve a new adoptive cellularimmunotherapy using independently developed CD40agonist antibody combined withIFN-α and IL-7in vitro, while opening a new treatment program for tumor adoptiveimmunotherapy by exploring the role of adoptive immunotherapy from cellproliferation, cell biological characteristics and cell killing effect, etc.Objective: To compare the new method (CD40agonist group) with traditionalmethod (CD3agonist group) in adoptive immunotherapy cells’ proliferation rate, cellsubsets and biological characteristics (T cells, NK-T cells, monocytes, Treg/ICOS+Tregcells and T cells expressing PD-1/CXCR-4, etc.) as well as differences in chemotaxisand anti-tumor ability in vitro, so as to establish an optimized program of culturingadoptive immunotherapy cells in vitro.Methods: Isolate PBMC conventionally from normal peripheral blood usingFicoll，and then put them into incubator to culture after adding cytokines into6-wellplates and24-well plates, while filling fluid cytokines once every two days.（1）Atdifferent time points (day6,9,12),we record cell morphology and perform live cellcount to detect cell proliferation using trypan blue;(2)At different points in time, we useimmunofluorescence and flow cytometry to detect the percentage of T cells, NK cells,NK-T cells and CD14+CD56+CD11c+cells, and detect the changes of T cellsexpressing CD62L, CCR7, CXCR-4,PD-1and the percentage of Treg/ICOS+Tregcells.(3) Collecting cells at9day, we detect the chemotaxis of adoptive immunotherapycells in vitro using the method of Transwell.(4) We co-culture the adoptiveimmunotherapy cells and A549lung cancer cells with a certain target ratio for72hoursand then assay their ability to kill tumor using CCK-8kit.Results:(1) There are colony-like cell proliferation both in CD3and CD40agonistgroup, although the cell proliferation of CD40agonist group is slightly slower, there isno significant difference between the two groups.(2)There is no significant differencebetween T lymphocyte composition of CD3and CD40agonist group，the NK-T cellsexpression of CD40group is significantly upregulated, while the percentage ofCD8+TCMcells increase and there occurs a unique CD14+CD56+CD11c+cell populationat a particular time in CD40group.(3) Compared with CD3group, the expression ofTreg cells, ICOS+Treg cells and T cells expressing PD-1was significantly lower in CD40group.(4) At9day, the adoptive immunotherapy cells of CD40group havehigher chemotactic ability in vitro, and the killing ability of CD40group for lung cancercell line A549is not weaker than that of CD3group.Conclusions: The new established adoptive immune cell therapy of this study canimprove the NK-T cell percentage with anti-tumor ability, and significantly reduce thenumber of Treg cells, while there occurs a unique CD14+CD56+CD11c+cell populationat a particular time. It does a useful exploration for the developing of adoptive tumorimmunotherapy.