体外阻断CD137-CD137L途径控制小鼠移植物抗宿主病的研究

Study on control of graft-versus-host disease by blocking CD137-CD137L ligand costimulatory pathway in mice

目的探讨体外阻断活化的供鼠T淋巴细胞CD137-CD137L共刺激途径控制小鼠异基因骨髓移植（allo-BMT）后急性移植物抗宿主病（aGVHD）及其机制。方法供、受鼠的淋巴细胞体外混合培养，分别加入抗CD137L单抗或不加抗CD137L单抗培养后与供鼠骨髓细胞混合移植给受鼠。采用流式细胞术检测移植后受鼠T细胞亚群的变化，RT-PCR法检测细胞因子mRNA表达水平的变化，观察移植后受鼠GVHD的临床及病理改变。结果与未用抗CD137L单抗组相比，应用抗CD137L单抗组CD3^＋CD8^＋T细胞明显降低（P＜0．01）；IFN-γ表达水平明显减低（P＜0．01），IL-10的表达水平明显升高（P＜0．01）。移植后未预防GVHD组（A组）小鼠移植后15d内均死于aGVHD。采用甲氨蝶呤＋环孢素预防GVHD组（B组）小鼠100％发生aGVHD，但临床及病理改变程度较A组轻，平均存活时间[（9．5±2．5）d]较A组[（7．5±1．5）d]略有延长。采用抗CD137L单抗预防GVHD组（C组）受鼠aGVHD的发生率为70％，程度比A、B两组轻，与A、B两组相比生存率明显提高（P＜0．01），平均存活时间[（16．0±2．5）d]明显延长（P＜0．01），30％的小鼠生存时间大于30d。结论抗CD137L单抗体外阻断CD137-CD137L共刺激途径能有效控制小鼠GVHD，可能与影响Ⅰ类和Ⅱ类T细胞因子平衡有关。

Objective To explore the in vitro effect on control of graft-versus-host disease （GVHD） and its mechanism in mice by blockade of CD137-CD137L pathway. Methods Responder spleen cells from BALB/c donor mice （H-2^d） were incubated with stimulator spleen cells from C57BL/6 （H-2^b） recipient mice, with or without anti-CD137L mAb. Lethally irradiated C57BL/6 mice were transplanted with donor bone marrow cells plus primary MLC spleen T cells. Group A（Allo-BMT control group） ：allo-BMT mice not receiving any prevention measures for GVHD. Group B（ CsA ＋ MTX control group） ： CsA and MTX given to C57BL/6 mice after transplantation. Group C （ experimental group ） ： donor spleen cells from BALB/c mice treated with anti-CD137L mAb. The percentages of CD3^＋ CD8^＋ T and CD3^＋ CD4^＋ T cells in the three groups were detected by flow cytometry, and the level of cytokines （IFN-γ、IL-2、IL-10、IL-4） by RT-PCR. Results The incidence of GVHD in group C was 70%, while in group A and group B were 100%. The survival rate was higher and the median survival time was longer of group C than that of group A and B （ P 〈 0.01 ）. All mice in group A died of aGVHD within 15 ds, while 30% of mice in group C survived more than 30 ds. Symptoms and histological signs of GVHD in group C were the mildest among the three groups. The percentage of CD3^＋ CD8^＋ T cells and the levels of IFN-T were significantly lower （P 〈 0.01 ）, and the levels of IL-10 were significantly higher in group C than those in group A and B （P 〈 0.01 ）. Conclusion Treatment of donor T cells with anti-CD137L mAb in vitro may relieve GVHD, thereby improve the survival time and survival rate, which maybe related to increasing Thl cytokine（IFN-T） and decreasing Th2 cytokine（IL-10） as well as reducing CD3^＋ CD8^＋ T cells.