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

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^＋An—nexin V^＋P1^-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IL4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。

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

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.