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

Part ?: Activated allogeneic NK cells as suppressors of alloreactive responses in nonmyeloablative allogeneic hematopoietic stem cell transplantationObject: The current study attempted to elucidate the mechanism of the veto activity of donor NK cells against host cytotoxic T cell precursors, and evaluate the effect of donor NK cells administration on engraftment and immune reconstitution in a murine nonmyeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) model.Methods: We used H2b splenocytes from C57BL/6 mice as responder cells, and the irradiated H2d splenocytes from BALB/c mice as stimulator cells to set up an in vitro MLR system. Tritiated thymidine was pulsed to detect the proliferation of responder cells, cytotoxicity assay was used to detect the killing capacity of responder cells against the H2d tumor cells. Irradiated H2d NK cells from BALB/c mice were added into the MLR reactions mimicking the donor NK cells. The proliferation and killing capacity of the responder cells were detected the same way as the above. Anti-TGFβblocking antibody was added to the MLR system mentioned above to determine whether the suppressive effects of the donor NK cells were caused by the release of TGFβ. The NK cells from wild type C57BL/6 mice, gld mice (FasL mutant), and pfp mice (perforin KO, both on C57BL/6 background) were used in the MLR assay to further determine the killing mechanisms of the donor NK cells. We further demonstrate that donor NK cells can promote engraftment by suppressing host alloreactive responses in nonmyeloablative allo-HSCT model. Donor NK cells from BALB/c mice were infused into host C57BL/6 mice during nonmyeloablative allo-HSCT. To evaluate the role of donor NK cell administration in transplantation outcomes, donor-recipient pairs were divided into three groups: The recipient C57BL/6 (H-2b) mice conditioned with donor hematopoietic stem cells, the recipient mice ?conditioned with sygeneous hematopoietic stem cells, the recipient mice conditioned with donor hematopoietic stem cells and donor NK cells. After two months, proliferation 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 H2d tumor cells was detected by the cytotoxicity assay. The allo-response of the host splenocytes was also measured by the level of IL-2 expression after the three day MLR reaction mentioned above by MTT.Results: The activated H2d NK cells from BALB/c mice significantly suppressed the proliferation of H2b splenocytes from C57BL/6 mice in mixed lymphocyte responses (MLR) stimulated with irradiated H2d splenocytes from BALB/c mice (p<0.01). The ability for H2b splenocytes to kill H2d tumor targets was also significantly inhibited by activated H2d NK cells (p<0.01). The same number of H2b ALAK cells or H2d splenocytes did not show the same suppressive effect. Anti-TGFβantibody blockade did not diminish this suppressive effect of NK cells. NK cells from gld (FasL mutant) mice suppressed the allo-responses as well as the wild type NK cells. NK cells from pfp (perforin knockout) mice did not completely block the inhibitory effect. The BABL/c mice treated with donor NK cells resulted in 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 infused with donor NK cells compared with control groups (p<0.05). The proliferation of the splenocytes from donor NK treated recipient mice was lower than the splenocytes from the control groups in mixed lymphocyte responses (MLR) stimulated with irradiated H2b splenocytes from C57BL/6 (B6) mice (p<0.05). The ability for the mixed splenocyts to kill H2d 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 were lower compared with the control groups (p<0.05).Conclusion: Our study demonstrates that activated allogeneic NK cells can specifically suppress the alloreactive cells, inhibiting both their proliferation and killing capacities. Granule-mediated killing might be part of the suppressive mechanism, while Fas-FasL pathway and TGFβsignaling are not involved. Donor allogeneic NK cell could promote the engraftment as suppressors of alloreactive responses in nonmyeloablative allogeneic hematopoietic stem cell transplantation.PartП: Donor NK cells and 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 NK cells were infused into host BALB/c mice during nonmyeloablative allo-HSCT. To evaluate the role of donor NK cell 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 IL-15 administration and the recipient mice conditioned with donor hematopoietic stem cells, donor NK cells and IL-15 administration. IL-15 gene was subcloned into the transfer plasmid of the lentivirus system, which was transfected together with the packaging plasmids into 293T cells. Then the recombinant virus was obtained and used to infect donor NK cells. The murine nonmyeloablative allo-HSCT model was established and donor-recipient pairs were also 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 IL-15 administration and the recipient mice conditioned with donor hematopoietic stem cells and the viral infected donor NK cells. 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 H2b 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 treated with donor NK cells and hIL-15 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 infused with donor NK cells and hIL-15 compared with control groups (p<0.05). The proliferation of the splenocytes from donor NK and hIL-15 treated recipient mice was significantly lower than the splenocytes from the control groups in thel MLR stimulated with irradiated H2b splenocytes from C57BL/6 (B6) mice (p<0.05). The ability for the mixed splenocyts to kill H2b 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 hIL-15 were obviously lower compared with the control groups (p<0.05). In the use of donor NK cells infected by the IL-15 gene recombinant virus experimental system, the primary results show the administration of viral infected donor NK cells could also resulted in higher donor chimerism percentage, higher counts of donor derived lymphocyte subsets and more obviously suppression of the host allo-response.Conclusion: Donor NK cell and IL-15 treatment could 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.PartⅢ: The discovery and function studies on IL-15 isoformObject: To explore the gene structure, origine and function of a newly discovered IL-15 isoform.Methods: Bone marrow cell and spleen cell suspensions were prepared from C57BL/6 mice. B cells were obtaind from spleen cells by Micro Beads purification, and the macrophages were obtained from the CSF stimulation of the bone marrow cells. Collect the mouse myeloma cells SP2/0, mouse monocyte-macrophage leukemia cells RAW246.7, and the B cells macrophages from primary culture. Total RNA of these cells was extracted and the coding sequence of IL-15 was amplified by RT-PCR. The cycloheximide was added into the splenocytes after stimulated with LPS for 12 hours. Splenocytes were then cultured for another 12 hours. The splenocytes stimulated with LPS for 24 hours and the normal splenocytes were also prepared as control. Western blot was used to detect the expression of IL-15 isoform gene in these splenocytes. The PCR product of IL-15 isoform gene was cloned and sequenced correctly. IL-15 gene was subcloned into the plasmid of pET43.1 vector and expressed in E.coli BL-21(DE3). Detect the solubility of the fusion protein and purify the protein. Western blot was used to detect the puriefied protein and its function was analyzed by the proliferation of CTLL-2 cells.Results: Our analysis revealed the exon 6 of IL-15 is absent when the mouse myeloma cells SP20, mouse monocyte-macrophage leukemia cells RAW246.7, and the B cells macrophages from primary culture were stimulated with LPS. The infusion of cycloheximide can reduce the expression of this isoform. pET43.1 vector containing IL-15 gene was constructed successfully. The fusion protein was purified successfully. The in vitro data demonstrated this IL-15 isoform may mediate negative control mechanism to regulate the function of normal IL-15.Conclusion: The IL-15 mRNA isoforms lacking exon 6 may be generated by alternative splicing events of the cells stimulated with LPS. Both of B cells and Macrophages could generate this isoform when stimulated with LPS. In the states of immune activation, this isoform may mediate negative control mechanism to regulate the function of normal IL-15.