【作者】 于津浦；

【导师】 郝希山； 任秀宝；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2008， 博士

【摘要】 目的:建立供者异体反应性NK细胞（Allo-NK）为预处理的非清髓性单倍相合造血干细胞移植（haplo-HSCT）的动物模型,探讨Allo-NK诱导移植早期供者免疫耐受和抑制移植后肺癌复发的作用机制。方法:以近交系C57BL/6J（H-2D^b）雌鼠为受鼠,以C57BL/6J×BALB/c杂交F1代(H-2D^b/d)雌鼠为供鼠,以氟达拉宾与环磷酰胺联合供者来源Allo-NK作为移植前的非清髓预处理方案,建立小鼠单倍相合造血干细胞移植模型。利用高强度磁珠分选系统纯化Allo-NK细胞亚群,流式细胞仪检测其中Ly49C⁺、Ly49A⁺细胞的比例,LDH法检测其对供者淋巴细胞的异体反应性和对Yac-1淋巴瘤和LLC肺癌细胞的杀伤活性。比较清髓性方案（9gyTBI）、各种非清髓性方案（6.5gyTBI,化疗组,Auto-NK+化疗组及Allo-NK+化疗组）的体内清髓效果,移植后不同时间点骨髓和脾脏的供者嵌合率,GVHD的发生率及严重程度。建立小鼠移植后肺癌复发模型,采用放射性标记法追踪DLI的体内分布。比较不同处理组小鼠的肿瘤大小,淋巴细胞浸润程度,采用RayBio^?小鼠细胞因子芯片检测血清中22种细胞因子的变化。MTT法检测化疗组和Allo-NK+化疗组移植后不同时间点供受者混合淋巴细胞增殖反应（MLR）。并以灭活的供者淋巴细胞作为刺激细胞,正常C57BL/6小鼠和F1免疫C57BL/6小鼠的淋巴细胞作为效应细胞,分别加入不同浓度化疗组和Allo-NK+化疗组移植后23d的CD4⁺T细胞、CD8⁺T细胞、CD4⁺CD25⁺ T细胞和CD4⁺CD25^-T细胞行单向混合淋巴细胞培养,MTT法检测MLR强度,EHSA法检测培养上清中供者特异性IFN-γ的分泌量。流式细胞法比较化疗组和Allo-NK+化疗组移植后23d的脾脏CD4⁺CD25⁺CD127^-调节性T细胞（Treg）比例;用荧光定量PCR法检测两组脾脏和胸腺CD4⁺CD25⁺T细胞和CD4⁺CD25^-T细胞中IL-2、IL-4、IL-10、TGF-β、IFN-γ、FoxP3的mRNA表达水平。分选化疗组和Allo-NK+化疗组移植后14d的胸腺CD11c⁺DC细胞,流式细胞法检测其成熟度,将其与正常C57BL/6小鼠的脾脏纯化CD3⁺T细胞体外共孵育5d,收集细胞,流式细胞法检测CD4⁺CD25⁺ CD127^- Treg比例,ELISA法检测其对供者特异性IFN-γ释放的影响,荧光定量PCR法和RayBio^?小鼠细胞因子芯片检测Foxp3和多种细胞因子表达。结果:经过磁珠分选后,Allo-NK细胞纯度从（19.85±4.27）%上升至（74.63±5.49）%,活率超过95%,其中特异性识别并杀伤C57BL/6细胞的Ly49A⁺细胞占（22.24±2.95）%。效靶比为20:1时,Allo-NK对Yac-1、LLC和C57B L/6小鼠细胞的杀伤率分别为（62.27±7.36）%,（59.86±6.24）%和（54.83±5.26）%。与其他非清髓性预处理方案,如6.5GyTBI组、化疗组和Auto-NK+化疗组相比,Allo-NK+化疗组不仅对骨髓毒性小,骨髓和脾脏的有核细胞在7-10天迅速恢复至正常水平;而且可显著提高移植后的供者嵌合率,移植后+21天后逐渐稳定在骨髓（28.70±5.90）%,脾脏（46.40±5.00）%,显著高于6.5GyTBI组的（6.08±0.46）%、化疗组的（10.2±2.40）%和Auto-NK+化疗组的（8.01±2.12）%,并持续3个月（P＜0.05）。与化疗组相比,Allo-NK+化疗组出现的GVHD反应轻微,仅有50%（5/10）受鼠出现体重减轻,Allo-NK+化疗组的肝脏、小肠、肾脏及皮肤的病理切片均未见明显的组织损伤。Allo-NK+化疗组接受DLI的小鼠,回输后8h～24h供者淋巴细胞在受者肺脏、脾脏、肾脏中明显蓄积,且浓度最高,时间最长。利用重复测量法发现,Allo-NK+化疗组复发肿瘤明显小于化疗组（P＜0.01）。至实验结束时Allo-NK+DLI组肿瘤明显小于不给治疗的Allo-NK+PBS组和化疗+DLI组,肿瘤抑制率分别为（70.62±3.75）%,（50.87±6.07）%和（55.94±3.98）%（P＜0.05）,且镜下可观察到肿瘤局部有较多的淋巴细胞浸润。Allo-NK+DLI组中MCP-1、IL-17、IL-12、和MCP-5较对照组分别升高1.56倍、1.36倍、1.20倍和1.17倍;而IL-10降低1.75倍。MLR结果显示,与正常C57小鼠相比,化疗组和Allo-NK+化疗组的增殖指数（SI）降低,而Allo-NK+化疗组降低程度尤甚（P＜0.05）,其中增殖抑制作用主要来源于CD4⁺T细胞中的CD4⁺CD25⁺T细胞,并呈剂量效应依赖关系。ELISA法检测发现,Allo-NK+化疗组的CD4⁺T细胞和CD4⁺CD25⁺T细胞可以显著抑制供者特异性IFN-γ分泌。流式细胞仪检测结果示Allo-NK+化疗组在移植后23d脾脏中出现一群新生的CD4⁺CD25⁺CD127^-Treg细胞,而化疗组组未见。荧光定量PCR扩增结果显示Allo-NK+化疗组CD4⁺CD25⁺ T细胞中Foxp3表达显著升高,高于化疗组（P＜0.05）,而IL-2、IFN-γ和IL-4的表达低于化疗组（P＜0.05）。流式细胞仪检测表明Allo-NK+化疗组移植后14d的胸腺CD11c⁺DC细胞成熟度明显低于化疗组,与正常C57BL/6小鼠CD3⁺T细胞共孵育5d后可诱导出（25.58±6.21）%的CD4⁺CD25⁺CD127^-Treg细胞,加入供受者MLR反应体系可显著抑制供者特异性IFN-γ释放。荧光定量PCR扩增显示诱导后的CD3⁺T细胞高表达FoxP3,与正常C57BL/6相比IL-2、IL-4、IL-10和TGF-β表达量升高,而IFN-γ表达量下降。RayBio^?小鼠细胞因子芯片检测显示IL-4,IL-6,IL-17,MCP-1分别升高1.42,3.57,1.87,1.57倍;而GM-CSF,IL-12p40,IFN-γ,RANTES,sTNFRI,VEGF分别降低1.49,1.52,1.61,1.56,2.76倍。结论:Allo-NK预处理可减少GVHD强度,并通过诱导移植早期供者特异性免疫耐受促进供者造血干细胞植入,延长供者淋巴细胞在受者体内停留时间,抑制移植后肿瘤复发。移植早期供者免疫耐受可能是通过胸腺中不成熟的供者DC细胞诱导生成的供者特异性CD4⁺CD25⁺CDl27^-Treg细胞介导的。更多还原

【Abstract】 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.更多还原