Supplementary Material for: Clinical Significance and Properties of IFN-γ+IL-17+ Th17 Cells in Liver Injury Associated with Chronic Hepatitis B Virus Infection

Introduction: Our previous study demonstrated that intrahepatic Th17 cells exacerbated the progression of chronic hepatitis B virus (HBV) infection. Meanwhile, we found a small group of IFN-γ and IL-17 double-positive Th17 cells (IFN-γ+IL-17+ Th17 cells) in liver tissues. This study aimed to investigate the clinical significance and properties of IFN-γ+IL-17+ Th17 cells in liver injury associated with chronic HBV infection. Methods: The frequencies of CD4+ Th cells, Tregs, and CD4+ T cells expressing specific chemokine receptors in the blood and liver tissues were detected using flow cytometry. The chemotaxis of C C chemokine receptor 5 (CCR5) and C-X-C chemokine receptor 3 (CXCR3) toward IFN-γ+IL-17+ Th17 cells and Tregs was evaluated by transwell chemotactic assay. Analyses of different variables were performed using GraphPad Prism v 5.01 and IBM SPSS Statistics 23.0. HBV-specific IFN-γ+IL-17+ Th17 cells were investigated using a cell stimulation assay with HBV antigens in vitro. Results: The frequencies of IFN-γ+IL-17+ Th17, Th17 cells, and Tregs in the blood were increased from normal controls to chronic hepatitis B (CHB) and acute-on-chronic liver failure (ACLF). The same trend could also be observed in CHB liver tissues compared to those in CHB blood specimens. Furthermore, the frequencies of IFN-γ+IL-17+ Th17 cells were positively associated with Th17 cells, Th17 cell-related cytokines (IL-17 and IL-6), HBV DNA load, and the levels of HBsAg, HBeAg, and ALT. The ratios of IFN-γ+IL-17+ Th17 cells to Tregs extremely decreased in ACLF blood specimens compared with those in CHB blood specimens. Additionally, CCR5 and CXCR3 were conducive to the recruitment of IFN-γ+IL-17+ Th17 cells and Tregs to liver tissue. Conclusions: IFN-γ+IL-17+ Th17 cells have Th17 cell-like properties in the progression of chronic HBV infection. CCR5 and CXCR3 facilitated the recruitment of IFN-γ+IL-17+ Th17 cells and Tregs to the liver. Importantly, the ratio of IFN-γ+IL-17+ Th17 cells to Tregs might be an effective assessment indicator of the severity of liver injury.引言：本团队既往研究证实，肝内Th17细胞可加剧慢性乙型肝炎病毒（HBV）感染的病情进展。同时，我们在肝组织中检出一小群干扰素-γ（IFN-γ）与白细胞介素-17（IL-17）双阳性的Th17细胞（IFN-γ+IL-17+ Th17细胞）。本研究旨在探究IFN-γ+IL-17+ Th17细胞在慢性HBV感染相关肝损伤中的临床意义与生物学特性。 方法：采用流式细胞术（flow cytometry）检测血液与肝组织中CD4+辅助性T细胞（CD4+ Th cells）、调节性T细胞（Tregs）以及表达特异性趋化因子受体的CD4+ T细胞的占比。通过Transwell趋化实验评估CC趋化因子受体5（CCR5）与C-X-C趋化因子受体3（CXCR3）对IFN-γ+IL-17+ Th17细胞及Tregs的趋化活性。使用GraphPad Prism v5.01与IBM SPSS Statistics 23.0软件对各类变量进行统计分析。通过体外HBV抗原刺激实验，研究HBV特异性IFN-γ+IL-17+ Th17细胞的相关特性。 结果：与健康对照人群相比，慢性乙型肝炎（CHB）患者及慢加急性肝衰竭（ACLF）患者血液中的IFN-γ+IL-17+ Th17细胞、Th17细胞与Tregs的占比均显著升高。相较于CHB患者的血液标本，CHB患者肝组织中上述细胞的占比同样呈现升高趋势。进一步分析表明，IFN-γ+IL-17+ Th17细胞的占比与Th17细胞、Th17细胞相关细胞因子（IL-17与IL-6）、HBV DNA载量以及HBsAg、HBeAg和ALT水平呈正相关。相较于CHB患者的血液标本，ACLF患者血液中IFN-γ+IL-17+ Th17细胞与Tregs的比值显著降低。此外，CCR5与CXCR3可促进IFN-γ+IL-17+ Th17细胞及Tregs向肝组织募集。 结论：IFN-γ+IL-17+ Th17细胞在慢性HBV感染进程中具备Th17细胞样的生物学特性。CCR5与CXCR3可介导IFN-γ+IL-17+ Th17细胞及Tregs向肝脏募集。值得关注的是，IFN-γ+IL-17+ Th17细胞与Tregs的比值或可作为评估肝损伤严重程度的有效检测指标。