自身免疫性肝炎患者肝内组织驻留记忆性CD8+T细胞的活化机制研究

Activation mechanism of CD8+resident memory T cells in liver tissues of patients with autoimmune hepatitis

目的自身免疫性肝炎(AIH)患者肝内CD69^(+)CD103^(+)CD8^(+)组织驻留记忆性T细胞(T_(RM))富集,本研究旨在明确AIH患者的肝脏微环境促进CD69^(+)CD103^(+)CD8^(+)T_(RM)扩增活化的分子机制。方法体外培养健康人外周血单个核细胞(PBMC),培养体系如下:(1)空白对照组完全培养基;(2)实验组完全培养基+IL-15+转化生长因子-β(TGF-β);(3)100 nmol/L雷帕霉素处理组完全培养基+IL-15+TGF-β+10 nmol/L雷帕霉素;(4)100 nmol/L雷帕霉素处理组完全培养基+IL-15+TGF-β+100 nmol/L雷帕霉素。使用流式细胞仪检测培养细胞中CD3、CD4、CD8、CD69、CD103、磷酸化S6(pS6)、磷酸化蛋白激酶B(pAkt)的表达情况。结果IL-15+TGF-β可在体外诱导CD69^(+)CD103^(+)CD8^(+)T_(RM)细胞。IL-15处理可激活CD8+T细胞中哺乳动物雷帕霉素靶蛋白复合物1(mTORC1)/S6激酶(S6K)信号通路(实验组和空白对照组的pS6平均荧光强度分别为2233.00±195.30、839.20±79.07,P<0.0001)。IL-15处理对mTORC2/蛋白激酶B(Akt)信号通路无影响(实验组和空白对照组的pAkt平均荧光强度分别为1832.00±83.86、1790.00±272.20,P>0.05)。使用mTORC1特异性抑制剂雷帕霉素可阻断IL-15对T_(RM)细胞的诱导分化作用(实验组和100 nmol/L雷帕霉素处理组的CD69+CD103+/CD8+细胞比例分别为(10.37±1.25)%、(2.14±0.24)%,P<0.0001)。结论IL-15可通过mTORC1/S6K信号通路促进CD69^(+)CD103^(+)CD8^(+)T_(RM)细胞体外扩增,靶向这一通路对AIH具有潜在的治疗价值。

Objective CD69^(+)CD103^(+)CD8^(+)tissue resident memory T cells(T_(RM))are enriched in the liver of autoimmune hepatitis(AIH)patients.This paper aims to clarify the molecular mechanism of the liver microenvironment of AIH patients to promote the expansion and activation of CD69^(+)CD103^(+)CD8^(+)T_(RM).Methods An in vitro cellular model of CD69^(+)CD103^(+)CD8^(+)T_(RM) were established from human peripheral blood mononuclear cells(PBMC).The specific culture conditions included the blank control group with only complete medium;the experimental group with complete medium+IL-15+transforming growth factor-β(TGF-β);the 100 nmol/L rapamycin treatment group with complete medium+IL-15+TGF-β+10 nmol/L rapamycin;the 100 nmol/L rapamycin treatment group with complete medium+IL-15+TGF-β+100 nmol/L rapamycin.Flow cytometry was used to detect the expression of CD3,CD4,CD8,CD69,CD103,phosphorylated S6(pS6),and phosphorylated protein kinase B(pAkt)in cultured cells.Results IL-15+TGF-βcan induce CD69^(+)CD103^(+)CD8^(+)T RM cells in vitro.IL-15 treatment can activate the mammalian target of rapamycin complex 1(mTORC1)/S6 kinase(S6K)signaling pathway in CD8+T cells(The average fluorescence intensity of pS6 in the experimental group and the blank control group are 2233.00±195.30 and 839.20±79.07,P<0.0001).IL-15 treatment has no effect on the mTORC2/protein kinase B(Akt)signal pathway(The average fluorescence intensity of pAkt in the experimental group and the blank control group are 1832.00±83.86 and 1790.00±272.20,P>0.05).The use of mTORC1 specific inhibitor rapamycin can block the induction of differentiation of IL-15 on T RM cells(The ratio of CD69+CD103+/CD8+cells in the experimental group and the 100 nmol/L rapamycin treatment group are(10.37±1.25)%,(2.14±0.24)%,P<0.0001).Conclusions IL-15 can promote the expansion of CD69^(+)CD103^(+)CD8^(+)T RM cells in vitro through the mTORC1/S6K signaling pathway.Targeting this pathway has potential therapeutic value for AIH.