sTNFRI基因修饰的未成熟树突细胞对异基因骨髓移植小鼠移植物抗宿主病和移植物抗白血病作用的研究

Effects of immature dendritic cells genetically modified to express sTNFR I on graft-versus-host dis- ease （GVHD） and graft-versus-leukemia （GVL） in allogeneic bone marrow transplantation mice

目的探讨可溶性肿瘤坏死因子受体I（sTNFRI）基因修饰的未成熟树突细胞（DC）对白血病小鼠异基因骨髓移植（allo-BMT）后移植物抗宿主病（GVHD）和移植物抗白血病（GVL）效应的影响及其机制。方法以BALB／c（H-2^d）小鼠为供鼠，C57BL／6（H-2^b）小鼠为受鼠，建立小鼠T细胞白血病BMT模型。受鼠全身照射（TBI）后4h内，将供鼠骨髓细胞和脾细胞按1：1混合，经尾静脉输注于受鼠体内，同时输注T细胞白血病／淋巴瘤细胞株EIA细胞。实验分组：①A组：单纯照射组；②B组：白血病发病组；③C组：allo-BMT组（移植物未做任何处理）；④D组：pXZ9-DC组（未经修饰的未成熟DC组）；⑤E组：sTNFRI-DC组（sTNFRI修饰的未成熟DC组）。观察移植小鼠GVHD典型症状、病理分级、白血病细胞浸润情况、存活时间、生存率等，采用ELISA法测定IFN-y和IL4浓度，采用流式细胞术（FCM）检测异基因嵌合率。结果①A组小鼠均于10d内死于骨髓衰竭。B组小鼠均死于淋巴瘤／白血病。C组小鼠出现明显急性GVHD表现，而D组和E组小鼠只有部分出现GVHD表现，且GVHD评分及病理表现均较C组明显减低或减轻（P值均〈0．05），其中E组小鼠GVHD评分较c和D组均降低（P值均〈0．05），病理学改变最轻。C、D和E组小鼠存活时间分别为（11．50±3．50）、（21．70±5．80）和（25．80±5．20）d，D、E组小鼠存活时间均较C组明显延长（P值均〈0．05），其中E组小鼠平均存活时间最长。B组小鼠18d内均死于白血病，C、D和E组小鼠白血病的发病率分别为10％、20％、10％，各组发病率比较差异无统计学意义（P〉0．05）。②移植后C、D和E组小鼠血清IFN-1浓度在＋12d时达高峰，后逐渐下降，＋18d时IFN一1的浓度E组较C和D组降低（P〈0．05），D组较C组低（P〈0．05）。C组小鼠血清IL-4降低，而D和E组浓度渐升高，＋12d达高峰，三组间两两比较差异均有统计学意义（P值均〈0．05）。③＋30d，C、D和E组存活受鼠骨髓异基因嵌合率为95％-100％，证实为完全供鼠型植入。结论未成熟DC可诱导allo．BMT免疫耐受，输注sTNFRI基因修饰的未成熟DC可延长移植小鼠的存活时间，在一定程度上减轻GVHD的同时又保留GVL效应。

Objective To investigate the effect of immature dendritic cells （inDC） genetically modi- fied to express sTNFR I on acute graft-versus-host disease （aGVHD） and the graft-versus-leukemia （GVL） effect ofter allogeneic bone marrow transplantation （allo-BMT） in leukemic mice and its mechanism. Meth- oils An EL4 leukemia allo-BMT model was established with the BALB/c （ H-2a ） donor mice （ DM ） and C57BL/6 （H-2b） recipient mice （RM）. The RM received DM bone marrow （BM） cells at a 1：1 ratio withspleen cells intravenously via tail vein at 4 h after TBI. Fifty DM were separated randomly into five groups： ①Group A： total body irradiation （TBI） group, ②Group B： lymphoma cell leukemia group, ③Group C： allo-BMT group, ④Group D： pXZ9-DC group, ⑤Group E： sTNFR I -DC group. Acute GVHD scores, inci- dence of leukemic cell infiltration, histopathological analysis, survival rate, and survival rate of the recipients were estimated after allo-BMT. Enzyme-linked immunosorbent assay （ELISA） method was used to detect cy- tokines （INF-y and IL-4 ） production. Flow cytometry （FCM） analysis was used to detect allogeneic chimer- ism. Results ①The mice in group A and group B all died of the BM failure and lymphoma cell leukemia, respectively. The mice in group C developed typical clinical signs of aGVHD after BMT with an average sur- vival time（AST） of （ 11.50±3.50） d. The signs of aGVHD were less evident in the group D and E, and their AST （21.70±5.80 and 25.80±5.20 days, respectively） were all longer than that in group C （ P 〈 O. 05 ）. AST of group E was the longest （P 〈 0.05 ）. The mice in group B all died of leukemia within 18 days after engraftment of EL4 cells. There was was no significant difference in groups C, D and E in the incidence of leukemia （ P 〉 0.05 ）.② Serum IFN-y level reached peak value. At ＋ 12 d, then decreased gradually in group C, D, and E, and then reached the nadir at ＋ 18 d post-BMT, with the lowest in group E （P 〈 O. 05 ） , and the level was significantly lower in group D than in group C （ P 〈 0.05 ）. After BMT, serum IL-4 level slightly decreased in group C, but gradually elevated in group D and E and reached their peak at ＋ 12 d, and even more significantly increased in group E （ P 〈 0.05 ）. There was no statistical significance in the pair wise comparison among three group（ P 〈 0.05 ）. ③The average proportion of H-2d positive cells in RM was 95% - 100% on day 30 post-BMT, with complete donor-type implantation. Conclusion Immature DC can induce immuno tolerance. Immature DC genetically modified to express sTNFR I has been shown to prevent acute GVHD in lethally irradiated mice reconstituted with allogeneie bone marrow grafts while maintai- ning the GVL response.