IL-35对慢加急性肝衰竭患者外周血CD8^+ T淋巴细胞功能的影响

Influence of interleukin-35 on peripheral CD8^+ T cell function in patients with acute-on-chronic liver failure

目的观察慢加急性肝衰竭(ACLF)患者血清IL-35表达水平,评估IL-35对ACLF患者CD8^+T淋巴细胞功能的影响。方法纳入2018年11月-2019年4月在海南省人民医院就诊的ACLF患者28例和健康对照者14例,采用ELISA法检测血清IL-35水平。分选外周血CD8^+T淋巴细胞,使用重组人IL-35刺激培养,应用实时定量PCR法检测CD8^+T淋巴细胞中穿孔素、颗粒酶B和颗粒溶素mRNA水平,流式细胞术检测CD8^+T淋巴细胞中程序性死亡分子-1(PD-1)和细胞毒性T淋巴细胞相关抗原4(CTLA-4)的表达。应用直接接触和间接接触共培养系统将HLA-A2限制性CD8^+T淋巴细胞与HepG2细胞共培养,加入重组人IL-35后通过检测靶细胞死亡比率和细胞因子分泌评估CD8^+T淋巴细胞的细胞毒性和非细胞毒性功能的变化。符合正态分布的计量资料2组间比较采用两独立样本t检验或配对t检验;不符合正态分布的计量资料2组间比较采用Mann-Whitney U检验;相关性分析采用Spearman秩相关分析。结果 ACLF患者血清IL-35水平[72.32(54.04~111.30) pg/ml]较健康对照者[46.00(27.02~82.29) pg/ml]显著升高(Z=2.184,P=0.020)。ACLF患者存在CD8^+T淋巴细胞功能耗竭,主要表现为CD8^+T淋巴细胞中穿孔素和颗粒酶B mRNA相对表达量降低、PD-1和CTLA-4阳性细胞比例升高、细胞杀伤(诱导靶细胞死亡)和非细胞杀伤功能(分泌IFNγ)下降(P值均<0.05)。使用重组IL-35刺激后,ACLF患者和健康对照者CD8^+T淋巴细胞中穿孔素和颗粒酶B mRNA的相对表达量均显著降低,PD-1和CTLA-4的比例则显著升高,CD8^+T淋巴细胞的直接细胞杀伤功能均显著降低(P值均<0.05)。结论升高表达的IL-35抑制ACLF患者CD8^+T淋巴细胞的直接细胞杀伤功能,提示IL-35在ACLF中可能诱导细胞免疫功能耗竭。

Objective To investigate the serum level of interleukin-35( IL-35) and its influence on CD8^+T cell function in patients with acute-on-chronic liver failure( ACLF).Methods A total of 28 patients with ACLF who attended Hainan Provincial People’s Hospital from November 2018 to April 2019 and 14 healthy controls were enrolled in this study.ELISA was used to measure the serum level of IL-35.Peripheral CD8^+T cells were separated and stimulated with recombinant human IL-35,and real-time quantitative PCR was used to measure the mRNA expression of perforin,granzyme B,and granulysin in CD8^+T cells;flow cytometry was used to measure the expression of programmed death-1( PD-1) and cytotoxic T-lymphocyte-associated antigen 4( CTLA-4) in CD8^+T cells.The direct-and indirect-contact co-culture systems were used for the co-culture of HLA-A2-restricted CD8^+T cells and HepG2 cells,and after recombinant human IL-35 was added,the percentage of target cell death and the secretion of cytokines were measured to evaluate the changes in the cytolytic and noncytolytic activities of CD8^+T cells.The two-independent-samples t test or the paired t-test was used for comparison of normally distributed continuous data between two groups,and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups;the Spearman correlation analysis was performed to investigate correlation.Results Compared with the health controls,the patients with ACLF had a significant increase in the serum level of IL-35 [72.32( 54.04-111.30)pg/ml vs 46.00( 27.02-82.29) pg/ml,Z = 2.184,P = 0.020].CD8^+T cell exhaustion was observed in ACLF patients,with the manifestations of reductions in the relative mRNA expression of perforin and granzyme B,increases in the percentages of PD-1+or CTLA-4+CD8^+T cells,and reductions in the cytolytic( induction of target cell death) and noncytolytic( secretion of interferon-γ) functions of CD8^+T cells( all P<0.05).After the stimulation with recombinant IL-35,both ACLF patients and healthy controls had significant reductions in the relative mRNA expression of perforin and granzyme B in CD8^+T cells,significant increases in the percentages of PD-1+orCTLA-4+CD8^+T cells,and significant reductions in the cytolytic and noncytolytic functions of CD8^+T cells( all P<0.05).Conclusion Elevated IL-35 can suppress the cytolytic activity of CD8^+T cells in ACLF patients,suggesting that IL-35 might induce the exhaustion of cellular immune function in ACLF patients.