Experimental Study On Alloreactive NK Cells In Inducing Donor Tolerance And Inhibiting Cancer Relapse After Transplantation

Objective:To construct a nonmyeloablative haploidentical allogeneic hemopoietic stem cell transplantation(haplo-HSCT)mouse model with a novel conditioning regimen composed of donor derived alloreactive NK cells(Allo-NKs) and low dose of fludarabine.To study the effects and underlying machenism of Allo-NKs in inducing donor immunotolerance and inhibiting cancer relapse after haplo-HSCT.Methods:The C57BL/6J(H-2D^b)female mice were used as recipients and C57BL/6J×BALB/c F1(H-2D^b/d)female mice as donors.A haploidentical allogeneic hemotopoietic stem cell transplantation(Haplo-HSCT) mouse model was constructed and donor-derived Allo-NK combined with fludarabine and cyclophosphamide was used as the nonmyeloablative conditioning regimen.The Allo-NK cells subset was isolated by magnetic beads separation columns,in which the proportions of the Ly49C⁺ and Ly49A⁺ cells were detected by flowcytometry,the alloreactivity against donor lymphocytes and the antitumor cytotoxicity against Yac-1 and LLC cell lines were measured by LDH method.The myeloablativity in vivo,donor engraftment after Haplo-HSCT and intensity of GVHD were compared among different conditioning regimens, including myeloablative(9Gy TBI)and some nonmyeloablative regimens:such as 6.5Gy TBI,low dose of chemotherapy,Auto-NK combined with chemotherapy and Allo-NK combined with chemotherapy.The relapse model of lung cancer after Haplo-HSCT was constructed.The distribution kinetic of infused donor lymphocytes in vivo was analyzed.The inhibition of relapse tumor,infiltration of lymphocytes in situ and fluctuation of 22 kinds of cytokines in serum were compared among different groups.The conventional donor-recipient mixed lymphocyte proliferation responses(MLRs)were compared between chemotherapy group and All-NK+chemotherapy group at different time points after Haplo-HSCT using MTT method.The one-way MLRs composed of inactivated F1-derived stimulators and C57BL/6-derived effectors were conducted to compare the regulatory effects of the different subsets by adding various numbers of CD4⁺ T cells,CD8⁺ T cells,CD4⁺CD25⁺ T cells and CD4⁺CD25^- T cells.Donor-specific IFN-γsecretion was measured by ELISA method.The proportions of CD4⁺CD25⁺ CD127^- regulatory T cells in different groups were analyzed using flowcytometry.The RNA expression of IL-2,IL-4,IL-10,TGF-β, IFN-γ,and FoxP3 of CD4⁺CD25⁺ and CD4⁺CD25^- T cells in different groups were detected using real time quantitative RT-PCR amplification.The CD11c⁺DC cells in thymus of different groups were purified and the phenotypes were analyzed using flowcytometry.Then the thymus CD11c⁺DC cells were cocultured with purified CD3⁺ T cells from normal C57BL/6 mice to induce Tregs.The proportions of CD4⁺CD25⁺ CD127^- Treg,the donor-specific IFN-γsecretion and expression of Foxp3 and variable cytokines were compared between different groups.Results:The purity of Allo-NK cells increased from(19.85±4.27)%to (74.63±5.49)%after magnetic separation with validity higher than 95%,in which Ly49A⁺ cells occupied(22.24±2.95)%.At E/T ratio of 20:1,the cytotoxicity of Allo-NK cells against Yac-1 cells,LLC cells and C57B L/6-derived lymphocytes were(62.27±7.36)%,(59.86±6.24)%and(54.83±5.26)%,respectively.Compared with other nonmyeloablative conditioning regimens,Allo-NK+ chemotherapy groups showed lower toxicity to bone marrow,higher rate of donor engraftment and little GVHD.The number of karyocytes in spleen and BM recovered 7-10 days later.The rates of donor chimerism reached(28.70±5.90)%in BM, (46.40±5.00)%in spleen on 21d after Haplo-HSCT and steady stayed at a higher level for about 3 months compared with 6.5GyTBI group,chemotherapy group and Auto-NK+ chemotherapy group(P＜0.05).The intensity of GVHD was slight in the Allo-NK+ chemotherapy group compared with the chemotherapy group,in which only half of C57BL/6 recipient experienced weight loss,and no distinct pathological damages observed in the liver,intestine,kidney and skin samples. The infused donor cells of Allo-NK+ chemotherapy group were mostly accumulated in lung,spleen and kidney which reached peak 8-24h after infusion with a considerable higher level and longer time than other groups according to the distribution kinetic curve.The sizes of relapse tumors were smaller in Allo-NK+PBS group compared with chemotherapy+PBS group(P＜0.01).At the end of test,the relapse tumors were the smallest in Allo-NK+ DLI group among all groups with inhibitory rate of(70.62±3.75)%,which is significantly smaller than(50.87±6.07)%in Allo-NK +PBS group and(55.94±3.98)%in chemotherapy+DLI group(P＜0.05),together with increased infiltration of lymphocytes in situ.The levels of multiple cytokines in serum of Allo-NK+DLI group ascended compared with the control group,such as MCP-1,IL-17,IL-12 and MCP-5,though the level of IL-10 descended simultaneously.The MLRs demonstrated that the stimulatory index(SI)in both Allo-NK+ chemotherapy group and chemotherapy group decreased,and especially lower in Allo-NK+ chemotherapy group(P＜0.05).The inhibitory function mainly derived from the CD4⁺CD25⁺ T cells subset which showed a distinct dose-effect relationship.The inhibition of donor specific IFN-γsecretion by either CD4⁺ T cells or CD4⁺CD25⁺ T cells subset from Allo-NK+ chemotherapy group was identified using ELISA method.The newly emerged CD4⁺CD25⁺ CD127^- Treg cells were detected only in Allo-NK+ chemotherapy group on 23d after Haplo-HSCT.Significantly increased mRNA expression of Foxp3 and decreased expression of IL-2,IFN-γand IL-4 were identified in the CD4⁺CD25⁺ T cells subset from Allo-NK+ chemotherapy group(P＜0.05).The immature phenotypes were observed in thymus CD11c⁺DCs from Allo-NK+ chemotherapy group.After coculturing with C57BL/6 derived CD3⁺T cells for 5 days,(25.58±6.21)%CD4⁺CD25⁺CD127^-Treg were induced which inhibited the donor specific IFN-γsecretion in donor-recipient MLR system significantly.Higher mRNA expression of Foxp3,IL-2,IL-4,IL-10 and TGF-β, lower expression of IFN-γwere detected using real time quantitative RT-PCR amplification.The results of RayBio^?cytokine microarray demonstrated considerable increase of IL-4,IL-6,IL-17 and MCP-1,decrease of GM-CSF, IL-12p40,IFN-γ,RANTES,sTNFRI and VEGF at protein level.Conclusion: Donor derived- alloreactive NK cells will reduce the intensity of GVHD and facilitate engraftment of the haploidentical allogeneic hemopoietic stem cell by inducing donor specific immonotolerance,which will prolong the half-life of infused donor lymphocytes in vivo and inhibit cancer relapse after transplantation. The donor specific immonotoelerance is mediated by newly generated CD4⁺CD25⁺CD127^-Tregs early after transplantation that induced by immature thymus donor-derived DCs.