The Roles Of Cytokine Pre-activated Donor NK Cells And Donor γδT Cells After Allogeneic Hematopoietic Stem Cell Transplantation

Part Ⅰ The GVL effect of cytokine pre-activated donor NK cells after allogeneic hematopoietic stem cell transplantationObjective: To explore the optimal cytokine combinations to induce memory-like NK cells in vitro.To investigate the graft versus leukemia（GVL）effect of cytokine preactivated donor NK cells after allogeneic hematopoietic stem cell transplantation.Methods: In the in vitro experiments,we first cultured NK cells from the mouse bone marrow（BM）cells.Then murine NK cells were stimulated with IL-12,IL-15,IL-18,IL-12+IL-15,IL-15+IL-18,IL-12+IL-18 and IL-12+IL-15+IL-18 for 16 h in vitro using NK cells cultured with IL-2 as control.The purity,activation,maturation and the production of cytokines of cytokine pre-activated NK cells were detected by flow cytometry.The cytotoxicity of cytokine pre-activated NK cells against tumor cells was measured by LDH assay.The memory response of cytokine pre-activated NK cells was tested by restimilation with IL-12+IL-15.Then we investigated the role of cytokineinduced memory NK cells in murine GVL model.The host is BALB/c（H2Kd）mouse and the donor is C57BL/6（H2Kb）mouse.The hosts were lethally irradiated by 137 Cs before the BM cells from C57BL/6 were transferred into irradiated hosts to establish the murine model of allogeneic hematopoietic stem cell transplantation（allo-HSCT）.On the day of transplantation,A20 cells（H2Kd,B cell lymphoma cell line）or A20-luciferise cells were transferred into hosts to establish the murine leukemia model.Cytokine pre-activated NK cells or control NK cells derived from CD45.1-C57BL/6（H2Kb）mouse were transferred into the GVL hosts on the day 0,day7 and day14.The survival and weight change of hosts were observed and recorded.The development of leukemia in the hosts was monitored by Caliper IVIS Lumina II.On the day 4 or day 7 after transplantation,the lymphocytes from the spleen,liver,and lung of hosts were harvested and the phenotypes were analyzed by flow cytometry.The activation,maturation,survival and apoptosis of transferred NK cells were also detected by flow cytometry.The proliferation of transferred NK cells in vivo was measured by CFSE labeling.Data were analyzed using Graph Pad Prism 5.Results:（1）The purities of cytokine pre-activated NK cells and control NK cells were more than 95%.Cytokine stimulation did not affect the purity of NK cells.Among all the treatments,NK cells had increased CD11 b expression and maintained the highlevel expression of CD43,indicating a mature status.While NKG2 D and NKp46 expressions were not greatly influenced by the cytokine treatments,CD25 expression was significantly up-regulated in all the treatment groups except IL-15 alone,with IL-12/18 and IL-12/15/18-preactivated NK cells showing the highest CD25 expressions.The components of the cytotoxic granules,Granzyme B and perforin,were not dramatically changed by the cytokine treatments.Strikingly,IFN-γ production was greatly promoted,with almost 100% positive for IL-12/18-and IL-12/15/18-preactivated NK cells compared to less 10% positive in control group.They both exhibited similar killing capacity against A20 target cells and there was no difference compared to control NK cells.（2）The cytokine pre-activated NK cells were restimulated with low dose IL-12/15 after resting for 4 days or 8 days in the low concentration of IL-2.Upon restimulation,the purity,the expressions of CD11 b and CD43 of the IL-12/18 and IL-12/15/18 pre-treated NK cells remained high.The production of IFN-γ by IL-12/18 and IL-12/15/18 pre-activated NK cells was higher than control NK cells after restimulation.（3）To address the GVL effect of IL-12/18-and IL-12/15/18-preactivated donor NK cells in vivo,we established a murine model by intravenously injecting luciferase-expressing A20 cells on the day of transplantation.Control NK cells,IL-12/18-or IL-12/15/18-preactivated NK cells（5×106 cells/mouse）were adoptively transferred into hosts on day 0.We next assessed tumor growth by in vivo bioluminescence imaging（BLI）on day 15 after transplantation.Strikingly,despite no therapeutic effect of the adoptively transferred NK cells was observed in terms of prolonged survival,single injection of control NK cells,IL-12/18-or IL-12/15/18-preactivated NK cells indeed reduced tumor burdens.Compared to transplant alone,donor NK cell infusion significantly prolonged the survival of recipient mice.Moreover,both IL-12/18-and IL-12/15/18-preactivated NK cells displayed more profound therapeutic GVL effects compared with control NK cells,while IL-12/15/18-preactivated NK cells were significantly superior to the NK control cells.（4）IL-12/18-and IL-12/15/18-preactivated NK cells expressed higher levels of maturation markers CD11 b and CD43 in vivo.Both the percentage and absolute numbers of cells positive for activation markers NKG2 D and CD25 of the IL-12/18-and IL-12/15/18-preactivated NK cells were significantly higher compared with control NK cells.Both IL-12/18-and IL-12/15/18-preactivated NK cells produced significantly higher level of IFN-γ in spleen,liver and lung compared with control NK cells.IL-12/18-and IL-12/15/18-preactivated donor NK cells display increased proliferation and reduced apoptosis after adoptive transfer.（5）The adoptive transfer of IL-12/18 or IL-12/15/18 pre-activated NK cells enhanced the production of TNF-α by CD4+T and CD8+T cells in the spleen and liver.Conclusion: IL-12/18 or IL-12/15/18 pre-activated NK cells were CD43^hiCD11b^hiCD25^hiIFN-γ^hi.Their phenotypes are consistent with memory-like NK cells.IL-12/18 or IL-12/15/18 did not affect the cytotoxicity of NK cells.The transfer of IL-12/18 or IL-12/15/18 preactivated memory-like NK cells displayed enhanced GVL function in a murine model.The transferred memory-like NK cells showed enhanced proliferation and decreased apoptosis in vivo.IL-12/18 or IL-12/15/18 preactivated NK cells promoted the production of TNF-α by CD4+T and CD8+T cells in the spleen and liver.Part Ⅱ The role of cytokine pre-activated donor NK cells in acute graft versus host disease after allogeneic hematopoietic stem cell transplantationObjective: To investigate the role of cytokine pre-activated donor NK cells in acute graft versus host disease after allogeneic hematopoietic stem cell transplantation.Methods: We established two acute graft versus host disease（a GVHD）models---mild a GVHD and severe a GVHD.Mild a GVHD model: BALB/c recipient mice received lethal TBI（750 c Gy: two doses of 375 c Gy with 4 h interval）from a 137 Cs source.3 hours later,1×107 bone marrow cells and 5×106 splenocytes from C57BL/6 mice were intravenously injected into lethally irradiated BALB/c recipient.Severe a GVHD model: BALB/c recipient mice received lethal TBI（750 c Gy: one dose of 750 c Gy）from a 137 Cs source.3 hours later,1×107 bone marrow cells and 5×106 splenocytes from C57BL/6 were intravenously injected into lethally irradiated BALB/c recipient.Control NK cells,IL-12/18 or IL-12/15/18 pre-treated NK cells were transferred into a GVHD hosts on day 0,day7 or day 14.The body weights of recipients and a GVHD clinical scores were assessed every 3 days.On the day 4 after transplantation,the phenotypes of lymphocytes from the spleen,liver,lung and intestinal epithelial lymphocytes were measured by flow cytometry.On the day 14 post transplantation,the allogeneic responses of T cells were tested by mixed lymphocyte reaction assay.The results were analyzed by Graph Pad Prism 5.Results:（1）Single infusion of control NK cells and IL-12/18-preactivated NK cells significantly prolonged survival of recipient mice compared with no NK cell infusion I mild a GVHD.In striking contrast,single infusion of IL-12/15/18-preactivated NK cells had no beneficial effect in suppressing a GVHD,and the survival of the hosts was significantly shortened compared with the mice received IL-12/18-preactivated or control NK cell infusion.（2）Three infusions of control NK cells further prevented a GVHD,while IL-12/18-preactivated NK cells were not as effective as the control NK cells,but still prolonged survival compared with no NK cell infusion in mild a GVHD.When IL-12/15/18-preactivated NK cells were infused into host mice three times,they had the trend to exacerbate a GVHD.（3）Both IL-12/18-preactivated and control NK cell infusion significantly reduced the alloresponses of donor T cells,whereas infusion of IL-12/15/18-preactivated NK cells had no effect on the alloreactivity of donor T cells in the mild a GVHD model.（4）In the severe a GVHD model,we observed that the infusion of IL-12/18-or IL-12/15/18-preactivated NK cells significantly prolonged survival of host mice compared with no NK cell infusion or control NK cell infusion.（5）IL-12/18-or IL-12/15/18-preactivated NK cell infusion significantly reduced the number of CD4+ and CD8+ T cells,as well as Th1 and Tc1 cells in the spleen and liver of severe a GVHD recipients.Conclusion: IL-12/18-preactivated NK cells suppressed a GVHD regardless of the severity,while IL-12/15/18-preactivated NK cells only exhibited beneficial effects in a severe a GVHD model.Furthermore,infusion of IL-12/18-preactivated NK cells might be a safer and better choice for adoptive therapy after allo-HSCT.Part Ⅲ The anti-leukemia effect of donor γδT cells after allogeneic hematopoietic stem cell transplantationObjective: To investigate the GVL effect of donor γδT cells in after allogeneic hematopoietic stem cell transplantation.Method: γδT cells,Vγ1 and Vγ4 T cells were cultured by using the spleen from TCRβ-/--C57BL/6 mouse.The activation marker expression and the production of cytokine were measured by flow cytometry.The cytotoxicities of γδT cells,Vγ1 and Vγ4 T cells against A20 cells were detected by LDH assay.The murine GVL model was established by transferring A20 cells and the BM cells from wild type C57BL/6 or TCRδ-/--C57BL/6 into lethally irriadiated BALB/c hosts.The survival and body weights of hosts were observed and reordered every 3 days.On the day 10 after transplantation,the phenotypes and absolute numbers of lymphocytes from the spleen,liver and lung were measured by flow cytometry.To confirm the γδT subset that displayed antileukemia effect in vivo,we transferred Vγ1 or Vγ4 cells respectively and performed Vγ1 or Vγ4 depletion experiments.To study the anti-leukemia effect of IL-17 A produced by Vγ4 cells,we cultured and transferred Vγ4 cells from wild type and IL-17A-/-mouse into the leukemia recipients.The results were analyzed by Graph Pad Prism 5.Results:（1）The survival of leukemia recipients was significantly decreased when the donor γδT cells were depleted.（2）γδT cells,Vγ1 and Vγ4 T cells expressed high level of NKG2 D.They all produced large amounts of Granzyme B,TNF-αand IFN-γ and displayed direct cytotoxicity against A20 cells in vitro,with the highest cytotoxicity in Vγ1 cells.（3）The production of IFN-γ was significantly reduced in the CD8+T cells from the hosts that received donor γδT cell depleted BM cells.（4）The transfer of Vγ4 cells prolonged the survival of the hosts.（5）The IL-17 A produced by Vγ4 cells exerted anti-leukimia function in vivo.Conclusion: Donor γδ T cells can mediate anti-leukemia effect during allo-HSCT through promoting IFN-γ production by CD8+ T cells.The subset of donor γδT cells to enhance GVL could be Vγ4 T cells throught the production of IL-17 A.