ICOS-Ig融合蛋白阻断ICOS信号通路可抑制异基因反应性T淋巴细胞的功能  

Blocking ICOS-B7h Signal Pathway by ICOS-Ig Fusion Protein Inhibits Function of Allogeneic T Lymphocytes

作　　者：包晓辰[1] 王健民[1] 沈茜[2] 周虹[1] 杨建民[1] 宋宁霞[1] 王斌[1] 

机构地区：[1]第二军医大学长海医院血液内科,上海200433 [2]第二军医大学长海医院实验诊断科,上海200433

出　　处：《中国实验血液学杂志》2009年第4期913-917,共5页Journal of Experimental Hematology

基　　金：国家自然科学基金(编号30871100);第二军医大学博士创新基金资助

摘　　要：本研究探讨ICOS-Ig融合蛋白阻断ICOS-B7h信号通路对异基因反应性T淋巴细胞的作用和机制。建立稳定高表达ICOS-Ig的CHO细胞株,培养制备纯化的ICOS-Ig融合蛋白;以C57BL/6小鼠脾脏来源的CD4+淋巴细胞为反应细胞,BABL/C骨髓源成熟树突状细胞为刺激细胞,应用不同浓度梯度的ICOS-Ig干预,以相同浓度的人Ig作为对照。结果显示:获得的目标蛋白的分子量为54kD,内毒素含量<10EU/ml。ICOS-Ig在≥10μg/ml时可显著减少DC刺激引起的异基因反应性T淋巴细胞的增殖效应(p<0.01)。ICOS-Ig不影响T淋巴细胞的活化,ICOS-Ig浓度与CD4+ T淋巴细胞的凋亡成正相关。单纯刺激组、ICOS-Ig10μg/ml和20μg/ml干预组的CD4+Annexin V+PI-细胞群的频率分别为15.1%、26.4%和33.6%。ICOS-Ig作用后,Th1细胞因子TNFα分泌降低,Th2细胞因子IL-4分泌增加。结论:ICOS-Ig融合蛋白阻断ICOS通路可明显减弱异基因反应性T淋巴细胞的增殖反应,并改变Th细胞的极化,对CD4+ T淋巴细胞的活化无影响,但可促进活化的T淋巴细胞凋亡。Inducible costimulatory molecule （ICOS）, a CD28 family member expressed on activated T cells, plays an important roles in T cell activation and effector function. This study was purposed to investigate the effect of blocking ICOS-B7h signal pathway by ICOS-Ig fusion protein on allogeneic reactive T cells and its mechanism. CHO cells stably and highly expressing ICOS-Ig were established, while the human ICOS-Ig fusion protein was harvested and purified from supernatant of CHO cells transfected with pSecTag2/Hygro A-ICOS-Ig. The CD4^＋ cells from spleen of C57BL/6 mice were used as reactive cells, the bone marrow derived dendritic cells （DCs） from BALB/C mice were used as stimulatory cells, these cells were treated with different concentrations of ICOS-Ig or human Ig （h-Ig） as control. The results showed that the target protein with molecular weigh 54 kD and endotoxin level 〈 10 EU/ml was gained. The ICOS-Ig（≥ 10 μg/ml） could significantly inhibited the proliferative effect of allogeneic reactive T cells resulting from stimulation of DCs （p 〈0.01 ）. ICOS-Ig did not influence the activation of CD4 ^＋ T cells. ICOS-Ig concentration positively related to the apoptosis of CD4 ^＋ T ceils. The percentages of CD4 ^＋ Annexin V ^＋ PI^- cells in simple stimulated group, ICOS-Ig 10 μg/ml group and ICOS-Ig 20 μg/ml group were 15.1%, 26.4% and 33.6% respectively. ICOS-Ig decreased secretion of TNFα and increased secretion of IL-4. It is concluded that the ICOS-Ig fusion protein has bioactivity of inhibiting T cell proliferation and altering the polarization of T helper cells to Th2 cells which promotes the apoptosis of allogeneic reactive T cells but had no effect on the activation of allo-reactive CD4 ^＋ T cells.

关 键 词：可溶性共刺激分子 ICOS—Ig融合蛋白 ICOS信号通路 异基因反应性 T淋巴细胞 

分 类 号：R392.4[医药卫生—免疫学]