体外阻断CD40-CD40L共刺激途径减轻小鼠异基因骨髓移植中移植物抗宿主病的研究

目的在异基因骨髓移植移植物抗宿主病(GVHD)小鼠模型中,观察抗CD40L单克隆抗体(单抗)体外预处理供鼠T细胞输注,观察其减轻GVHD的作用并探讨其作用机制.方法供鼠(C57BL/6H-2b)脾T细胞作为反应细胞,受鼠(BALB/cH-2d)脾细胞作为刺激细胞,分别加抗CD40L单抗或不加单抗进行混合培养,培养第5天的细胞作为经体外诱导后的脾T淋巴细胞,分别混合供鼠骨髓细胞后移植给接受8.0 Gy全身照射预处理的受鼠.比较受鼠GVHD发生和造血重建.在移植后不同时间点采用流式细胞仪检测受鼠脾细胞中T细胞表型的改变,用ELISA法检测外周血血清中Th1和Th2类细胞因子的水平.结果移植后对照组受鼠均于25 d内死于GVHD,实验组GVHD的发生率为20%,与对照组小鼠相比,存活率和存活时间明显增高和延长(P<0.01);存活的实验组小鼠(8只)第40天时骨髓细胞中H-2Db阳性细胞为(93.54±2.32)%.实验组小鼠CD3+CD4+、CD3+CD8+、CD4+CD25+、CD4+CD69+、CD8+CD25+、CD4+CD40L+和CD8+CD69+T细胞比例明显低于对照组(P<0.05),CD8+CD40L+和CD4+CD45RA+T细胞比例在两组中变化差异无显著性(P>0.05).实验组受鼠血清中细胞因子水平明显低于对照组(P<0.05).结论应用抗CD40L单抗体外孵育的脾T细胞与骨髓细胞混合移植,可明显减少GVHD的发生率,且不影响供鼠造血干细胞的植入,CD4+和CD8+T细胞对异体抗原发生耐受,耐受化T 细胞的活化障碍发生于活化的早期和成熟阶段,同时抑制了Th1和Th2类细胞因子分泌,为抗CD40L单抗应用于临床移植预防GVHD提供了实验依据.

Study of reducing graft-versus-host disease by in vitro blockade of CD40-CD40 ligand co-stimulatory pathway in allogeneic bone marrow transplantation mouse model

OBJECTIVE: To investigate the effect and its mechanism of reducing graft-versus-host disease (GVHD) by in vitro blockade of CD(40)-CD(40)L pathway in vitro, the donor T lymphocytes cultured in vitro with anti-CD(40)L mAb were transfused in bone marrow transplantation (BMT) GVHD mouse model. METHODS: C57BL/6(H-2b) spleen T cells were isolated as responder cells, and BALB/c(H-2d) spleen cells as stimulator cells. They were cocultured with or without Anti-CD(40)L mAb as anti-CD(40)L mAb group and control group, respectively. At day 5, the mixed lymphocyte response (MLR)-culture cells mixed with bone marrow cells and transfused respectively into the TBI conditioned recipient mice. The mice were divided into two groups: group A, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR control group; group B, bone marrow cells (2 x 10(6)) and spleen T lymphocytes (2 x 10(6)) from MLR anti-CD(40)L mAb group. The GVHD incidence and hematopoietic reconstitution were observed. Peripheral blood sera and spleen cells of the recipients mice were harvested at scheduled time points for the measurement of cytokines and T cell immunophenotyping with flow cytometry. RESULTS: The incidence of GVHD in group A was 100% (10/10), and in group B was 20% (2/10). The percentage of H-2D(b) positive cells in group B (n = 8) was (93.54 +/- 2.32)% at day 40 after transplantation. The levels of cytokines in serum from group B were significantly lower than those from group A (P < 0.05). The expressions of CD(4)(+), CD(8)(+), CD(4)(+)CD(25)(+), CD(8)(+)CD(25)(+), CD(4)(+)CD(69)(+), CD(8)(+)CD(69)(+) and CD(4)(+)CD(40)L(+) were lower in group B than in group A (P < 0.05). The expressions of CD(8)(+)CD(40)L(+) and CD(4)(+)CD(45)RA(+) were similar in the two groups (P > 0.05). CONCLUSION: Blockade of CD(40)-CD(40)L interaction in vitro could induce immune tolerance in vivo, reduce aGVHD in aGVHD mice model and form chimerism, which was mediated by inhibiting the Th1 and Th2 cytokines production, inducing tolerance of CD(4)(+) and CD(8)(+) cells to alloantigens. The obstruction of T cells activation after tolerance happened mainly at the early and mature phase of T cells activation. These provided the experimental basis for the use of anti-CD(40)L mAb in the clinical transplantation to prevent aGVHD.