Inhibiting Cardiac Allograft Rejection In Mice By Interleukin-35 Gene Therapy Combined With Decitabine Treatment

The effect of IL-35-expressing plasmid vector in combination with methyltransferase inhibitor (decitabine) on immune function of mice and immune tolerance, especially on the expression level and function of CD4+CD25+Treg, was studied. First, the proper dosage of IL-35 plasmid gene transfer was determined by in vivo hydromechanics transfection through mice caudal vein injection. Then mouse model of abdominal heterotopic heart transplantation was established, and IL-35 plasmid vector in combination with decitabine were given to the recipient mice through caudal vein injection. The effect on the expression of CD4+CD25+Treg in peripheral blood and spleen and allogeneic cardiac allograft rejection effect were assessed. In this way, we could explore a new method for inducing transplant immune tolerance.Part I Effects of IL-35 plasmid vector and decitabine on mouse T lymphocytes in vitroObjective:To identify IL-35 expression plasmid vector, and investigate the effect of IL-35 expression plasmid vector and decitabine on mouse T lymphocyte. Methods: IL-35-expressing plasmid vector (pSecTag2A-IL35) was identified by double digestion using enzymes, and at the same time a set of primer was designed to amplify the IL-35 gene with polymerase chain reaction (PCR). IL-35 expression plasmid was transfected into human adrenal gland epithelial cell line 293 cells in vitro using Lipofectamine 3000. The transfection efficacy was determined by flow cytometry, and IL-35 expression in culture supernatant of transfected 293 cell was detected by ELISA. Mice splenocytes were collected and one-way mixed lymphocyte reaction (MLC) system was established to observe the effect of IL-35 and decitabine within the culture system. Then splenocytes were collected after culture, and CD3/CD4/CD8 T cell subsets and CD4+CD25+Treg were assessed by flow cytometry (FCM). Results:IL-35 expression plasmid vector was successfully identified, and with in vitro transfection the transfected 293 cell expressed IL-35. IL-35 and decitabine upregulated the level of CD4+CD25+Treg in allogeneic mixed lymphocyte culture, and downregulated the level of CD4+CD8-T lymphocytes.Part II Effect of in vivo transfection of IL-35 expression plasmid on mouse immune functionObjective:To evaluate the efficiency of in vivo IL-35 expression plasmid transfection through tail vein injection. To assess the effect of IL-35 on mice immune function and determine the proper dosage of plasmid injection. Methods:IL-35 expression plasmid vector and Fc expression plasmid vector were used for in vivo hydromechanics transfection through tail vein injection. Mice were divided into 5 groups with different plasmid injection doses, which were 10μg,25μg,50μg, and 100μg. Peripheral blood and spleen samples were collected on day 3,5, and 7 after plasmid injection to assess the proportation of CD3/CD4/CD8 T cell subset, CD4+ CD25+Treg, and NK cell. Results:IL-35 expression plasmid was successfully transfected into the liver of mice, and the doses with highest transfection efficacy was 50μg. IL-35 upregulated the level of CD4+CD25+Treg in both the peripheral blood and spleen, and at the same time downregulated the level of CD4+T cell and NK cells in the peripheral blood.Part III Effect of IL-35 gene transfection combined with decitabine on cardiac allograft rejection in miceObjective:To investigate the effect and anti-rejection mechanism of IL-35 and decitabine in mouse abdominal allogeneic heart transplantation. Methods:Mouse model of heterotopic abdominal heart transplantation was established by using Balb/C mice as the donor and C57BL/6 as the recipent. Mice were divided into allogeneic control group, Fc plasmid control group, IL-35 plasmid group, decitabine group and IL-35 plasmid combined with decitabine group. Cardiac allograft survival time was observed, and cardiac samples were collected on day 7 after transplantation for pathology examination to grade the level of acute rejection. Peripheral blood and spleen were also collected on day 7 after transplantation to assess the T cell subsets and CD4+CD25+Treg by FCM. Results:Mouse model of heterotopic abdominal heart transplantation was successfully established. Cardiac allograft median survival time of allogeneic control group and Fc control group were 8d and 7d respectively. Cardiac allograft median survival time of IL-35 plasmid group was 10d, decitabine group was 11d and IL-35 plasmid combined with decitabine group was 12d significantly increased when compare to the control groups (P<0.01). Result of pathologic grading of acute rejection on day 7 after transplantation showed significant decrease in IL-35 plasmid group, decitabine group and IL-35 plasmid combined with decitabine group when compare to the control groups (P<0.01). Peripheral blood and spleen CD4+CD25+Treg both significantly increased in IL-35 plasmid group, decitabine group and IL-35 plasmid combined with decitabine group when compare to the control groups (P<0.01). Peripheral blood CD8+T cell significantly decreased in IL-35 plasmid group, decitabine group and IL-35 plasmid combined with decitabine group when compare to the control groups (P<0.01). Conclusion: Heterotopic abdominal heart transplantation in mice was a feasible model to study the rejection after tansplantation. IL-35 gene therapy and decitabine administration resulted in the anti-rejection effect during heterotopic abdominal heart transplantation in mice and prolonged the survival time of transplanted heart. The possible anti-rejection mechanism is that IL-35 and decetabine can stimulate the generation of CD4+CD25+Treg, which can further suppress the generation and effect of effector T cells to achieve extend survival time of the transplanted heart. Immunogene therapy with IL-35 combined with decitabine can be a novel effective approach to induce immune tolerance.