Experimental Study Of Donor-derived NK Cell Infusion And IL-2, IL-15 Treatment Efficacy In Allogeneic And Haploidentical Hematopoietic Cell Transplantation

PartⅠ:Isolation,culture and identification of mouse splenic NK cellsObjective:To explore NK cell separation method and NK cell culture system and to study the cytotoxicity activity of expanded NK cells.Methods:Lymphoid cell subset percentages were analysed by flow cytometry (FCM).T cells were depleted by using CD90(Thy1.2) MicroBeads prior to NK cell enrichment by using CD49b(DX5) MicroBeads,an MiniMACS separator and an MS column.The purity of NK cell separation product was assaied by FCM.NK cells were cultured in complete DMEM medium containing IL-2 and/or IL-15.NK cell cytotoxicity was detected by using CytoTox96 kit.Results:CD3~-DX5~+NK accounts for 3.0-5.5%of total splenocytes of adult B6 mice.NK cell separation product had a purity of more than 95%.NK cell count increased by 1.27±0.05,3.14±0.18,6.23±0.28,10.81±0.92,23.10±1.44 folds after 3,6,9,12,15 days culture in complete DMEM medium containing 10ng/mL rhIL-2 and 100ng/mL rhIL-15. NK cell immunotype was CD3~-DX5~+,CD3~-DX5~+NK cells accounts for 95.0%.Resting NK cell had mild cytotoxicity of 28.7%at an E:T ratio of 10:1,while activated NK cell had a cytotoxicity of 96.3%at an E:T ratio of 10:1.Conclusion:NK cell could be highly pure separated from mouse splenocytes by using CD90(Thy1.2) MicroBeads and CD49b(DX5) MicroBeads.NK cell could be efficiently expanded in complete DMEM medium containing IL-2 and IL-15.The immunotype of expanded NK was CD3~-DX5~+.NK cell cytotoxicity was increased after expanded and activated by IL-2 and IL-15.PartⅡEstablishment of a murine nonlethal aGVHD model post allogeneic hematopoietic stem cell transplantation and transplantable leukemia model Objective:To establish a murine nonlethal aGVHD model post allogeneic hematopoietic stem cell transplantation and EL9611 erythroleukemia allogeneic hematopoietic stem cell transplantation model.Methods:Thirty-two SPF grade BALB/c mice were randomly assigned to four groups,mice received 6,7,8 or 9Gy X ray total body irradiation.Adult C57BL/6(H-2~b) males were donor mice and BALB/c(H-2d,♀) were recipient mice.Thirty-two SPF grade BALB/c mice were randomly assigned to four groups post TBI conditioning and were reconstituted with 1×10~6,2.5×10~6,5×10~6 or 10×10~6 BMCs via lateral tail vein injection.GVHD model was established by coinfusion of 5×10~6,10×10~6 or 20×10~6 splenocytes via tail vein.Six- to seven-weeks old SPF grade BALB/c females were inoculated with 1×10~6 EL9611 cells via lateral vein.Leukemia-loaded mice were randomly assigned to 4 groups as no treatment group,TBI group,syngeneic group,allogeneic HCT.Results:All mice survived 6 Gy TBI conditioning.The median survival time of mice conditioned with 7Gy TBI was 28.0d and for 8Gy TBI was 12.7d.The survival time was compare by Log-rank test,with x~2=47.24,P＜0.0001.The survival rate at 100d was 62.5%when mice were injected with 2.5×106BMCs.All mice survived 100d post-HCT when reconstituted with 5×10~6BMCs.No overt aGVHD was observed when mice were reconstituted with BMCs alone.Non-lethal aGVHD was induced when mice were reconstituted with 5×10~6 splenocytes.Lethal aGVHD was induced when mice were reconstituted with 10×10~6 or 20×10~6 splenocytes.Mice received syngeneic HCT died of EL9611 leukemia within 20 days.In the allo-BMT group 1/8 mouse survived 100d free of leukemia.The survival time was compare by Log-rank test,with x~2=40.22,P＜0.0001. Complete donor chimerism was achieved on +28d after allo-HCT.Conclusion:It was myeloablative conditioning when BALB/c mice received 8Gy TBI treatment.Hematopoietic recovery could achieve when BALB/c mice were reconstituted with 5×10~6 BMCs.Nonlethal aGVHD could induced when mice were injected with 5×10~6 splenocytes.Leukemia residual disease could be established when BALB/c mice were inoculated with 1×10~6 EL9611 leukemia cells on -8d.PartⅢDonor-derived NK cell infusion and IL-2,IL-15 treatment efficacy in allogeneic hematopoietic cell transplantationObjective:To explore the effects of ex vivo-expanded donor NK cell infusion and IL-2,IL- 15 treatment on GVHD,leukemia relapse,and immune reconstitution.Methods:Real-time quantitative PCR was used to assay sjTREC in splenocytes. RT-PCR and runoff PCR were used to assay TRBV family spectrum.FCM was used to assay splenic lymphoid immune reconstitution.Forty BALB/c mice were randomly assigned to BMT control group,GVHD control group,NK cell infusion group,and NK cell infusion,cytokines treatment group in the GVHD model.Seventy BALB/c mice were inoculated with 1×10~6 EL9611 cell on -8d and were assigned to six groups.Results:GVHD was observed as early as +7d after allo-HCT in the control mice.The clinical GVHD score of NK cell infusion group was lower than that of GVHD control group.The lesion of skin,liver and ileum of NK cell infusion group was less severe than that of GVHD control group.All syngeneic HCT control recipient mice died of leukemia within 20 days.The median survival time of allogeneic HCT recipient mice was 28 days and 10 percent(1/10) survived +100 d.In the NK cell infusion group,44.4 percent died of leukemia and 55.6 percent survived +100 day,had prolonged survival than allogeneic HCT control group(x~2=4.487,P=0.0342).Eighty percent(8/10) mice of NK cell infusion and IL-2,IL-15 treatment for one week group survived +100 day.Eleven of 12 mice of NK cell infusion and IL-2,IL-15 treatment for two week group survived +100 day.The survival time of this group was longer than that of NK infusion group.The total splenocytes of mice in NK cell infusion and IL-2,IL-15 treatment group were(4.78±0.51)×10~7, (3.98±0.43)×10~7 and(3.45±0.23)×10~7,respectively.The splenic T,B,NK cell counts in NK cell infusion and cytokines treatment group were higher than those of control group. The sjTREC level of control group was 136.6±13.7 per 10~5 cells,while the sjTREC level of NK cell infusion group and combination treatment group were 222.2±11.4 and 287.5±10.9 per 10~5 cells,respectively.TRBV spectrum reconstitution in NK cell infusion group was faster than that of control group.Conclusion:Ex vivo-expanded NK cell infusion and IL-2,IL-15 treatment could mitigate GVHD severity,protect thymus output,promote lymphoid immune reconstitution and enhance graft-versus-leukemia effect in murine allogeneic HCT.PartⅣDonor-derived NK cell infusion and IL-2,IL-15 treatment efficacy in haploidentical hematopoietic cell transplantationObjective:To establish a nonlethal GVHD model post B6→CB6F1 haploidentical hematopoietic cell transplantation(HID) and to explore the effects of ex vivo-expanded donor NK cell infusion and IL-2,IL-15 treatment on aGVHD,leukemia relapse,and lymphoid immune reconstitution.Methods:Clinical GVHD score and histopathological examination were used to evaluate GVHD severity.Real-time quantitative PCR was used to assay sjTREC level post haploidentical HCT.RT-PCR and runoff PCR were used to assay TRBV family spectrum. FCM was used to assay splenic lymphoid immune reconstitution.In the GVHD model, forty CB6F1 mice were randomly assigned to four groups as BMT control group,GVHD control group,NK cell infusion group and NK cell infusion and IL-2,IL-15 treatment group.We established the CB6F1 leukemia model by coinfusion of 2×10~6 EL9611 leukemia cells with stem cell graft per mice at the time of HCT.In the leukemia model, fourty CB6F1 females were randomly assigned to four groups as HCT control group,NK cell infusion group,NK cell infusion and cytokine treatment for 1 week group and NK cell infusion and cytokine treatment for 2 weeks group.Results:Lethal myeloablative conditioning was achieved after CB6F1 mice were treated with 11.5 Gy TBI.GVHD was evident in GVHD control mice on +7d post HID. NK cell infusion group mice had lower clinical GVHD scores than GVHD control group mice(P＜0.05).The clinical GVHD scores of NK cell infusion and cytokines treatment group mice were lower than those of control group mice at several time points(P＜0.01). Overt GVHD lesion could be detected in skin,liver and ileum of GVHD control mice. Twenty percent(2/10) of control group mice survived +100 d post HID and eighty percent of mice died of leukemia.Ten percent of mice died of leukemia and ninety percent of mice survived +100 d post HID in the NK cell infusion group.All mice of NK cell infusion and cytokines treatment group mice survived +100d post HID.The survival time of four group mice were significantly different(x~2=30.69,P＜0.0001).Total splenocyte counts of NK cell infusion and cytokines treatment group,NK cell infusion group,control group were (5.39±0.59)×10~7,(4.42±0.39)×10~7,(3.68±0.47)×10~7 at +28d,respectively.The sjTREC level of control group was 155.3±10.7 per 10~5 cells,while the sjTREC level of NK cell infusion group and combination treatment group were 246.5±29.4 and 298.5±16.0 per 10~5 cells,respectively.TRBV spectrum reconstitution in NK cell infusion group was faster than that of control group.Conclusion:We established a nonlethal GVHD model and a leukemia model post HID.Donor NK cell infusion and IL-2,IL-15 treatment could mitigate GVHD severity, promote lymphoid immune reconstitution and enhance graft-versus-leukemia effect in murine haploidentical HCT.