阻断诱导型共刺激分子信号通路对小鼠急性移植物抗宿主病的影响

Effect of blocking inducible costimulatory signaling pathway on mouse model of acute graft-versus-host disease

目的：观察以诱导型共刺激分子（inducible costimulator，ICOS）融合蛋白阻断ICOS-B7H途径对小鼠急性移植物抗宿主病（acute graft-versus—host disease，aGVHD）的作用。方法：建立以C57BL／6鼠为供体、致死剂量照射的BALB／c鼠为受体的aGVHD模型，分别在移植0、2、4、8d后腹腔注射100Ixg的ICOS-Ig，以注射同剂量的人Ig（h—Is）作为对照。观察小鼠体重、生存率及临床GVHD症状的改变；移植4d后取小鼠脾脏单个核细胞，流式细胞术检测H一2Kd—CD4＋及H-2Kd—CD8＋细胞的CFSE强度，确定供体T淋巴细胞的增殖率；移植10d后采用Annexin—V及PI试剂盒检测供体H-2Kd—CD4及H-2Kd—CD8＋细胞的凋亡率。结果：ICOS-Ig干预可有效减轻小鼠aGVHD的严重程度，ICOS—Ig组小鼠的生存时间比h-Ig组显著延长[（20．0±3．1）vs（10．0±2．1）d，P=0．0217，n=14]，其肠道黏膜病变及肝脏汇管区淋巴细胞浸润明显减少。移植3d后，ICOS-Ig组和h·Ig组小鼠的CD4＋CFSE-和CD8＋CFSE-细胞群比例无明显差异[（98．88±0．76）％铘（99．71±0．27）％，（98．62±0．63）％椰（99．53±0．51）％，P〉0．05]；ICOS—Ig增加了体内异基因CD8＋T淋巴细胞的凋亡率[（15．7±9．59）％掷（25．92±5．66）％，P=0．032]，不影响异基因CD4＋T细胞的凋亡率[（15．72±7．34）％vs（22．78±6．94）％，P=0．078]。结论：阻断ICOS—B7H通路可促进活化的T淋巴细胞凋亡，减轻造血干细胞移植治疗血液肿瘤中发生的aGVHD的严重程度。

Objective： To block inducible costimulator（ICOS）-B7H signal with ICOS-Ig fusion protein and study the effect of ICOS-B7H signal on mouse model of acute graft-versus-host disease （aGVHD）. Methods： Allogeneic aGVHD model was established with lethally irradiated BALB/c recipients receiving allogeneic BM and spleen T cells from C57BL/ 6 with 100μg ICOS-Ig intraperitoneally 4 times at d 0, 2, 4 and 6 of transplantation, with the same concentration of human Ig （h-Ig） as control. Survival, weight and GVHD status were monitored daily. At d 4 of transplantation, mice were sacrificed and the spleens were removed and homogenized, and the division of donor T cells was assessed by CFSE intensi- ty of H-2Kd negative, and CIM or CD8 positive ceils. H-2Kd- negative, CD4 and CD8 positive spleen T cell populations at d 10 of transplantation were stained with annexin-V and PI for evaluating the apoptosis rate. Results： Block ICOS-BTH pathway effectively controlled severe aGVHD in the control group. The median survival time was （ 10 ±2.4） days in h-Ig group over （20 ± 3.1 ） days in ICOS-Ig group （ P = 0. 0217, n = 14）. Pathologic evaluation revealed that the hepatic portal and intestinal mucosa in ICOS-Ig group had less lymphocyte infiltration and tissue damage than did those in the control group. ICOS-Ig had no obvious effect on allogeneic T cell proliferation （h-Ig： 98.88 ± 0.76, ICOS-Ig： 99.71 ± 0.27 in CD4＋ T cells, h-Ig： 98.62 ± 0.63, ICOS-Ig： 99.53 ± 0.51 in CD8 ＋ T cells by CFSE tracing assay at d 3 ）, but in- creased apoptosis of allogeneic CD3＋T cells in GVHD model at d 10： h-Ig： 15.72 ±7.34, ICOS-Ig： 22.78 ±6.94 in CD4＋ T cells （P = 0. 078, n = 5） ; h-Ig： 15.7± 9.59, ICOS-Ig： 25.92 ± 5.66 in CD8 ＋ T cells （P = 0. 032, n = 5 ）. Conclusion： Block ICOS-B7H pathway can prevent aGVHD through shortening the survival time of allo-T cells.