摘要
急性移植物抗宿主病(acute graft versus host disease,aGVHD)是骨髓移植后主要的并发症和死因之一,严重制约了同种异基因骨髓移植的临床应用。近年来,研究发现过继性输注体外诱导培养的供体或受体来源的耐受型树突状细胞(tolerogenic dendritic cells,tDCs)能够提高移植模型鼠的存活率,因此,耐受型树突状细胞在移植免疫中的作用日益引起人们的重视。但无关第三方tDCs是否也能在同种异基因骨髓移植中诱导免疫耐受,至今国际上尚没有定论。因此,本研究在体外诱导培养tDCs,研究无关第三方tDCs在体外对异基因CD4+T细胞增殖的影响,以及在体内对aGVHD的防治作用。
本研究首先在体外用GM-CSF.IL-10和TGF-β1诱导D1小鼠骨髓细胞(bone marrow,BM)获得tDCs,流式细胞术检测tDCs的CDllc、CD80、CD40、CD86、 I-A/Ⅰ-E(MHCⅡ类分子)等几种表型分子的表达,RT-PCR检测tDCs促炎因子IL-12p40、抑炎因子IL-10、TGF-P以及Fas-L、精氨酸酶mRNA的表达。结果表明,tDCs低表达I-A/Ⅰ-E(小鼠MHC Ⅱ分子)、共刺激分子(CD80、CD86、 CD40)和促炎因子IL-12p40,但高表达抑炎因子IL-10、TGF-β以及FasL、IDO和精氨酸酶,而且经脂多糖(lipopolysaccharide,LPS)刺激后tDCs仍能保持较稳定的耐受状态。因此,经IL-10和TGF-p诱导培养的DCs属于一种耐受型的DCs。
混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测无关第三方tDCs在体外对异基因CD4+T细胞增殖的影响,其中新鲜分离的B6小鼠CD4+T细胞作为应答细胞,培养的D2小鼠骨髓来源的成熟树突状细胞(mature dendritic cells,mDCs)作为刺激细胞,在此体系中添加不同浓度的D1小鼠骨髓来源的tDCs。培养4天后,检测荧光染料羧基荧光素二醋酸盐琥珀酰亚胺酯(carboxyfluoresceinsuccinimidylester,CFSE)的荧光强度及及碘化丙啶(PI)阳性细胞率,以检测CD4+T细胞的增殖和死亡情况。结果表明,单纯CD4+T细胞(B6)和mDCs(D2)共培养,D2骨髓来源的1mDCs能够刺激CD4+T细胞发生增殖；而在添加了不同剂量tDCs的实验组中,tDCs均能促进异基因CD4+T细胞死亡,抑制CD4+T细胞扩增。其中,添加了102个tDCs的实验组,即CD4+T细胞：tDCs比率为1000：1时,tDCs最能有效抑制异基因CD4+T细胞的增殖,促进T细胞死亡。这些结果表明,在体外无关第三方tDCs能抑制CD4+T细胞的增殖,并促进其死亡,且这种作用与tDCs的剂量有关。
本研究建立了典型的aGVHD小鼠模型,选用MHCI类分子完全不匹配的供受体进行清髓后的骨髓移植,并加以一定剂量的脾细胞促进aGVHD的发生,此模型aGVHD症状明显。在此模型基础上过继性输注不同剂量无关第三方tDCs后,观察受鼠生活状况、生存率、临床GVHD评分等,并以单纯aGVHD模型为对照组进行比较。结果显示,aGVHD对照组小鼠在移植后18d内全部死亡而接受无关第三方tDC输注组小鼠aGVHD症状均有所改善,其中输注104tDCs的受鼠有60%能存活至60d以上,无明显的GVHD症状。而输注103tDCs的小鼠仅有20%能存活至移植后第60天,输注105tDCs的小鼠则在移植后37d内全部死亡。这些结果表明,过继性输注一定量的无关第三方tDC能显著延长aGVHD小鼠的生存期,有效地预防异基因骨髓移植小鼠aGVHD的发生。
在移植后第7天和第21天,检测各组受鼠GVHD血清中Th1/Th2细胞因子IFN-γ、IL-4、IL-10和IL-12p70水平。在移植早期,tDC组和aGVHD组的多种细胞因子含量没有显著差异。但在移植后21天,104tDCs处理组移植小鼠血清中IL-10水平明显高于其他剂量tDCs处理组小鼠,而IFN-y水平比其他剂量tDCs处理组小鼠低。这表明tDCs预防急性GVHD的作用可能与IL-10的高表达有关。
因此,本实验表明：经GM-CSF、IL-10和TGF-β1诱导的无关第三方tDCs在体外能够抑制异基因CD4+T细胞的增殖,在体内能够有效缓解aGVHD的症状,显著延长aGVHD患者的生存期,预防aGVHD的发生,且这些作用与tDCs的剂量有关,并可能与IL-10的高表达有关。
Applications of allogeneic hematopoietic stem cell (HSC) transplantation have been limited due to the morbidity and mortality associated with acute graft versus host disease (aGVHD). Many researches have shown that donor or recipient's tolerogenic dendritic cells (tDCs) could improve allograft survival in mouse models. But, it is no idea whether third-party-derived tDCs also could induce tolerance in a model of allogeneic bone marrow transplantation (allo-BMT). In this research, different dose of D1-tDCs were adoptive transferred into the aGVHD model in allogeneic BMT, which chose B6mice as donors and D2mice as recipients, and explored the role of third-party-derived tDCs in the prevention aGVHD in mice after allo-BMT.
First, tDCs were cultured with low doses of GM-CSF, IL-10and TGF-β1from bone marrow cells of D1mice. The phenotype, expression of cytokines and function associated molecules were identified with FACS and RT-PCR. The results assessed by FACS indicated that tDCs expressed lower levels of MHC II and co-stimulatory molecules, such as CD80, CD86and CD40, even when stimulated by LPS. The results assessed by RT-PCR indicated that tDCs expressed low levels of IL-12p40and high levels of'immunosuppressive'molecules, such as IL-10, TGF-β, Fas Ligand, and arginase. Therefore, this type of cells remained to tolerogenic phenotypes.
Mixed lymphocyte reaction (MLR) analyzed the influence of third-party-derived tDC on allo-CD4+T cells proliferation in vitro. The suppressive activity was tested by CFSE staining. Freshly isolated CD4+T cells from B6mice were used as responder cells and activated by mature DCs from D2mice alone or in the presence of third-party tDCs with the different doses. After4days, CFSE progressive dilution and PI positive-expression were used as the readouts of responder cells proliferation and apoptosis. The results indicated that at all the responders versus tDCs ratios, tDCs were able to promote the apoptotic death of responder cells and suppress proliferation of allo-CD4+T cells. While102tDCs, which CD4+T cells versus tDCs ratio was1000:1, could inhibit expansion most effectively and lead to massive death of allo-CD4+T cells. In the allogeneic MLR, third-party tDCs could suppress allo-CD4+T cells proliferation, which was relative to the dose of tDCs.
In order to determine whether the third-party tDCs were able to induce tolerance in aGVHD mouse models, an aGVHD model was established which chose B6mice as donors and D2mice as recipients. The MHC molecules of these two mice are different completely. The recipients were given a lethal dose of total body irradiation. Bone marrow cells and spleen cells from donors were injected after24h. Different doses of the third-party-derived tDCs were adoptive transferred into the aGVHD models, and termed "tDCs-treated group". The aGVHD group was the control group. Survival time and clinical GVHD score were observed after allo-BMT. In the B6→D2mouse models, all aGVHD mice died within18days. Remarkably, if10third-party tDCs were transferred,60%mice survived at least60days. But when the doses of tDCs were reduced to103cells, only20%of mice survived past day60and when increased to105, all of the mice died within day37after allo-BMT. These results indicated that adoptive therapy by transfusing third-party tDCs could significantly prolong the survival time of recipients after allo-BMT, and this role was associated with the dose of tDCs.
To determine how tDCs reduce aGVHD, the cytokines IFN-y, IL-4, IL-10and IL-12p70in serum were further examined on7d and21d after allo-BMT. There was no significant difference between the tDCs-treated groups and aGVHD group in the early time post-BMT. But, on the21d, it was found that the levers of IL-10in serum were obviously higher in the mice treated with104tDCs than the mice transferred other doses of tDCs, and the IFN-y production is lower than the other two tDC-treated groups. From these results, we believed that the role of third-party tDCs in prevention aGVHD was associated with high IL-10secretion.
引文
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