新生期小鼠CD4^(+) Foxp3^(-)细胞在肝脏中的免疫耐受研究

Study on immune tolerance of CD4^(+) Foxp3^(-) cells in liver of neonatal mice

目的研究小鼠CD4^(+) Foxp3^(-)T细胞在新生期肝脏获得的免疫耐受表型以及可能的机制。方法选取新生14 d及相应周龄的雄性C57BL/6或Foxp3-GFP小鼠,通过流式细胞术、体外刺激培养及酶联免疫吸附试验法检测其肝脏、脾脏及胸腺中CD4^(+) Foxp3^(-)细胞的比率、耐受表型,增殖及凋亡等情况,并初步明确影响新生期肝脏CD4^(+) Foxp3^(-)T细胞增殖及存活的关键因素。结果新生后14 d小鼠肝脏CD4^(+) Foxp3^(-)细胞比率显著低于脾脏及胸腺,在出生后第14天达到最低点,随后迅速回升,在出生后第49天其比率达到胸腺水平,显著高于脾脏,差异有统计学意义(P<0.05)。相较于脾脏,新生14 d小鼠肝脏CD4^(+) Foxp3^(-)T细胞显著活化,并CD39、CD73、Helios等耐受相关分子表达显著升高,差异有统计学意义(P<0.05)。进一步的体外实验显示,新生期肝脏CD4^(+) Foxp3^(-)T细胞的增殖水平显著低于脾脏,差异有统计学意义(P<0.05)。此外,新生14 d肝脏CD4^(+) Foxp3^(-)T细胞的凋亡水平明显高于脾脏,差异有统计学意义(P<0.05)。结论出生后14 d内可能是肝脏调控其局部免疫稳态的关键时间窗,尤其是肝内CD4^(+) Foxp3^(-)T细胞在该阶段获得了与脾脏完全不同的耐受表型,这些耐受表型可能对新生期肝脏甚至机体的发育具有重要影响。

Objective Investigate the tolerant phenotype of mouse CD4^(+)Foxp3-T cells acquired in neonatal liver and illustrate the possible mechanisms of it.Methods The percentage,phenotype,proliferation and apoptosis of CD4^(+)Foxp3-cells in liver,spleen and thymus were compared by flow cytometry,in vitro stimulation and ELISA in C57BL/6 and Foxp3-GFP male mice at 14-days-old or the corresponding age.Results The ratio of CD4^(+)Foxp3-cells in mouse liver was significantly lower than that of the spleen and thymus at 14 days after birth,reaching the lowest point on the 14th day after birth,and then rising rapidly,reaching the level of the thymus on the 49th day after birth,significantly higher than that of the spleen,the difference was statistically significant(P<0.05).Compared with spleen,neonatal liver CD4^(+)Foxp3-T cells included more cells with an activated phenotype and positive expressing CD39,CD73,Helios,the difference was statistically significant(P<0.05).Further experiments showed that the proliferation level of CD4^(+)Foxp3-T cells in the neonatal liver was significantly lower than that of the spleen,the difference was statistically significant(P<0.05).In addition,we also found that the apoptosis of CD4^(+)Foxp3-T cells in liver was significantly higher than that in spleen at 14 days after birth,the difference was statistically significant(P<0.05).Conclusion The first two weeks after birth may be a critical window for the liver to regulate its local immune homeostasis.Especially during this stage,CD4^(+)Foxp3-T cells in liver acquired a tolerance phenotype completely different from the cells of spleen.These tolerant phenotypes may have a crucial influence on the development of the liver and even the body during the neonatal period.