无关第三方耐受型树突细胞对小鼠异基因骨髓移植后急性移植物抗宿主病的预防作用

The role of third-party tolerogenic dendritic cells in the prevention of acute graft-versus-host-disease following allogeneic bone marrow transplantation in mice

目的探讨无关第三方耐受型树突细胞（tDC）的生物学特性及其对急性移植物抗宿主病（aGVHD）的预防作用。方法用GM—CSF、IL-10和TGF—B1诱导培养D1小鼠骨髓源tDC，用流式细胞术检测细胞表型，用RT—PCR方法检测细胞因子基因表达情况。采用混合淋巴细胞反应法检测其体外对异基因小鼠CD4＋T细胞增殖的影响；将D1小鼠骨髓源tDC按不同剂量分组输注给aGVHD小鼠模型（B6--D2），观察受鼠一般状况、生存率、临床GVHD评分、血清中Thl／Th2细胞因子水平的变化，并以单纯aGVHD模型为对照组进行比较。结果流式细胞术检测结果表明tDC表达低水平的MHCⅡ分子（I—A／I—E）和共刺激分子（CD80、CD86、CD40）。RT—PCR结果表明，tDC低水平表达炎性细胞因子IL-12p40，但高水平表达免疫抑制性因子IL-10、TGF—β、FasL、IDO和精氨酸酶。无关第三方tDC在体外能够有效抑制异基因CD4＋T细胞的增殖，并且这种抑制效应与tDC的剂量有关；小鼠体内实验显示，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10。tDC的受鼠有60％能存活至60d以上，无明显的GVHD症状。而输注10^3 tDC的小鼠仅有20％能存活至移植后第60天，输注10^5 tDC的小鼠则在移植后37d内全部死亡。移植21d时，输注10^3、10^4、10^5无关第三方tDC的小鼠血清内IL-10水平分别为（114．23±7．78）、（646．18±212．02）、（121．97±10．47）ng／L，10^4 tDC组显著高于其他组（P〈0．05）。结论经IL—10和TGF-β1诱导的无关第三方tDC在体外能够抑制CD4＋T细胞的增殖，对aGVHD有显著的预防作用，这种作用与tDC剂量和IL-10的高表达有关。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，有效预防异基因骨髓移植小鼠aGVHD的发生。

Objective To explore the biological characteristic of third-party-derived tolerogenic DC （tDC） and the influence of third-party-derived tDC on acute graft-versus-host-disease （aGVHD） following allogeneic bone marrow transplantation （allo-BMT） in mice. Methods tDC from bone marrow cells of D1 mice was cultured with low doses of GM-CSF, IL-10 and TGF-β1D1. The phenotype, expression of eytokines and function associated molecules were identified with FACS and RT-PCR. Mixed lymphocyte reaction was applied to analyze the influence of third-party-derived tDC on allo-CD4 ＋ T cells proliferation in vitro. Different doses of DI-tDC were adoptive transferred in the aGVHD model in allogeneic BMT which B6 mice as donors and D2 mice as recipients. Survival time, clinical GVHD score and the levels of Thl/2 cytokines in serum were monitored after allo-BMT using the aGVHD model as control. Results tDC expressed lower levels of MHC Ⅱ and eo-stinmlatory molecules, such as CD80, CD86 and CIM0, even when stimulated by LPS. The results by RT-PCR indicated that tDC expressed low levels of IL-12p40 and high levels of immunosuppressive molecules, such as IL-10, TGF-β, Fas Ligand, indoleamine 2,3-dioxygenase （IDO） and arginase. In the al- logeneic MLR, third-party tDC suppressed allo-CD4^＋T cells proliferation, which was relative to the dose of tDC. In the B6--~D2 mouse model, all aGVHD mice died within 18 days. Remarkably, if 104 third-party tDC were transferred, 60% mice survived at least 60 days. When the doses of tDC were reduced to 103 cells, only 20% of mice survived day 60, and when increased tDC to 105 , all of the mice died within day 37 after allo- BMT. The cytokine levels in serum indicated that 104 tDC-treated mice secreted in vivo high level of IL-10 21d after BMT （P 〈 0.05 ） ,the levels of IL-10 in 10^2, 10^4 and 10^5 tDC-treated mice were （114.23 ± 7.78） , （646.18 ± 212.02）, （ 121.97 ± 10.47） ng/L, respectively. Conclusion Third-party tDC could suppress allo-CD4^＋ T cells proliferation in vitro and prevent aGVHD in allogeneic BMT mode, which may be mediated by modulating tolerogenic cytokines secretion, such as IL-10. And this effect was associated with the dose of tDC. Adoptive therapy by transfusing third-party tDC cultured with low doses of GM-CSF, IL-10 and TGF-β1 could significantly prolong the survival of recipients and prevent aGVHD in allogeneic BMT.