Donor NK Cells And Hydrodynamic Injection-mediated IL-15 Treatment Promoted Engraftment During Nonmyeloablative Allo-HSCT

Part I: The effects of irradiation on hydrodynamic injection-mediated gene expression in miceObject: To explore the effects of irradiation on hydrodynamic injection-mediated luciferase reporter gene expression in mice and evaluate the gene transfer efficiency using HGT while applied to murine hematopoietic stem cell transplantation model.Methods: The mice were divided into irradiated group and non-irradiated group. The PGL3-luc plasmid expressing luciferase reporter gene was injected into mice by a rapid large-volume injection. Six hours after the injection the luciferase substrate was intraperitoneal injected into mice and the initial level of gene expression was detected through in vivo imaging system. The irradiated group mice received nonmyeloablative doses (700cGy) of total body irradiation (TBI). The level of gene expression in two groups of mice was then monitored by in vivo imaging system in real time at 12h, 24h, 48h, 72h, 96h, and 120h after the hydrodynamic injection.Results: High expression of the transgene delivered via the tail vein was observed in the mouse liver at six hours after injection and the average expression levels of 3739±924.8 RLU/(sec·mg) protein were obtained. The level of transfer gene expression was similar between the irradiated group mice and non-irradiated group mice at each time points (p>0.05) but fell to a low level three days after gene transfer.Conclusion: The irradiation after the hydrodynamic injection didn't affect the level of transfer gene expression in mice. This new type of gene transfer methods can be applied to mouse hematopoietic stem cell transplantation model effectively. PartП: Donor NK cells and hydrodynamic injection-mediated IL-15 treatment promoted engraftment during nonmyeloablative allo-HSCTObject: We carried out experiments to determine the effects of combining donor NK cell infusion and IL-15 administration in a murine nonmyeloablative allo-HSCT model.Methods: Donor NK cells were purified from C57BL/6 mice and expanded in vitro. Donor hematological stem cells and NK cells were infused into host BALB/c mice during nonmyeloablative allo-HSCT. IL-15 gene was subcloned into the eukaryotic expression vector PCDNA3.1. The PCDNA3.1 plasmid expressing IL-15 gene was injected into the recipient mice by a rapid large-volume injection the day before transplantation. To evaluate the role of donor NK cell and IL-15 administration in transplantation outcomes, donor-recipient pairs were divided into four groups: The recipient BALB/c (H-2d) mice conditioned with donor hematopoietic stem cells, the recipient mice conditioned with donor hematopoietic stem cells and donor NK cells, the recipient mice conditioned with donor hematopoietic stem cells and hydrodynamic injection-mediated IL-15 treatment and the recipient mice conditioned with donor hematopoietic stem cells, donor NK cells and hydrodynamic injection-mediated IL-15 treatment. After two months, engraftment of the donor hematopoietic stem cells was determined through flow analysis. The counts of donor-derived splenic B, T, NK, and macrophage cells were also measured by flow anlaysis. The proliferation capacity of the splenocytes from host mice was measured with 3H-thymidine assay. The killing capacity of the host splenocytes against the H2~b tumor cells was detected by the cytotoxicity assay. The allo-response of the host splenocytes was also determined by measuring IL-2 secretion after the three day MLR reaction mentioned above by MTT.Results: The BABL/c mice conditioined with donor NK cells and hydrodynamic injection-mediated IL-15 treatment resulted in obviously higher donor chimerism percentage compared with control groups (p<0.05). The counts of donor derived lymphocyte subsets were increased in the spleen of the recipient mice treated with donor NK cells and IL-15 compared with control groups (p<0.05). The proliferation of the splenocytes from donor NK and IL-15 treated recipient mice was significantly lower than the splenocytes from the control groups in thel MLR stimulated with irradiated H2~b splenocytes from C57BL/6 (B6) mice (p<0.05). The ability for the mixed splenocyts to kill H2~b tumor targets and the level of IL-2 expression during the MLR had also been analyzed, the results of the recipient mice group infused with donor NK cells and IL-15 were obviously lower compared with the control groups (p<0.05).Conclusion: Donor NK cell and IL-15 treatment could synergistically promote the engraftment and the development of donor derived cell subsets and suppress the host allo-response in the nonmyeloablative allo-HSCT murine transplant model.