肝脏TCRγδ~+CD3~+CD4~-CD8~-T细胞在小鼠病毒性肝炎中的作用及其作用机制
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摘要
【研究背景及目的】
病毒性肝炎以肝细胞损伤为主要特征，然而肝细胞损伤的具体机制尚未完全明确。目前大部分学者认为肝炎病毒本身并不直接造成肝细胞的损伤，而机体抗肝炎病毒的免疫反应，特别是免疫细胞介导的免疫反应，是造成肝细胞损伤的主要原因。多种免疫细胞亚群，如CD4~+T细胞、CD8+T细胞、自然杀伤细胞(NK)、NKT细胞、调节性T细胞、巨噬细胞等，都被证实在这一病理过程中发挥重要作用。近年来随着对肝炎发病机制研究的深入，越来越多的研究表明非特异性免疫反应，特别是NK细胞和巨噬细胞介导的非特异性免疫反应，也参与了病毒性肝炎感染引起肝脏损伤的发病机理。我们对HBV诱导的暴发型肝衰竭（FHF）和慢加急性肝衰竭(HBV-inducedacute-on-chronic liver failure, HBV-ACLF)研究后发现,肝脏NK细胞通过死亡受体途径Fas/FasL和NK细胞受体配体识别途径NKG2D/NKG2DL对肝细胞造成损伤。在3型鼠肝炎病毒(MHV-3)诱导的小鼠暴发性肝衰竭中活化的巨噬细胞能够分泌炎性因子TNF, IL-1进一步促进肝脏中的炎症反应并加重肝细胞损伤。此外，活化的巨噬细胞还分泌促凝血活性物质fgl2凝血酶原酶（fibrinogen like protein2）， fgl2凝血酶原酶可以激活凝血级联反应，引起肝脏微循环障碍并导致肝细胞大块死亡。因此明确各种免疫细胞及其分泌的细胞因子形成的免疫网络在病毒性肝炎引起肝细胞损伤中的作用将有助于更加全面的认识病毒性肝炎的发病机制，并为其临床诊断和治疗提供重要的靶点。
近年研究发现，机体存在一种新型T细胞亚群——CD3~+CD4~-CD8~-细胞(双阴性T细胞，DN T)。在健康人群和正常小鼠外周血T细胞中，DNT细胞所占比例为1–5%。随着对DN T细胞的深入研究，越来越多的证据表明DNT细胞在多种疾病，如移植排斥、自身免疫性疾病、肿瘤免疫、感染性疾病、过敏性疾病以及移植物抗宿主病等疾病的病理进程中发挥重要作用。在不同的疾病中DN T细胞表达不同的表面标志细胞，分泌独特的效应因子，通过这些效应因子发挥其生物学功能。在器官移植模型中，将脾细胞来源的DN T细胞克隆后输入小鼠体内，DN T细胞能够通过Fas/Fasl、穿孔素等途径杀伤抗供者CD4~+T细胞、CD8+T细胞和B细胞，从而提高移植物的存活时间和存活率。Zhang, Z等通过对MRL/lpr小鼠模型中DNT细胞研究后发现，系统性红斑狼疮模型鼠（MRL/lpr）中DNT细胞高表达IL-17和IL-23R,并且与疾病进程及严重程度呈正相关。在感染土拉热弗郎西丝菌(Francisella tularensis)的小鼠中，研究发现感染后肺组织中DN T细胞可以分化为两个亚群，一群主要分泌IL-17，而另一群以分泌IFN-γ为主，并在疾病进程中发挥不同的生物学作用。Lis R. V等研究发现利什曼原虫感染者外周血TCRαβ~+DNT (TCRαβ~+CD4~-CD8~-）细胞可以分泌炎性细胞因子IFN-γ和TNF-α，而TCRγδ~+DNT(TCRγδ+CD4-CD8-)主要分泌调节性细胞因子IL-10。
迄今，肝脏DN T细胞是否在病毒性肝炎中发挥作用尚不明确。本实验室以鼠3型肝炎病毒（MHV-3,murine hepatitis virus3）感染C3H/Hej小鼠成功建立了小鼠病毒性肝炎模型，本研究拟利用MHV-3诱导的小鼠病毒性肝炎模型，探讨DN T细胞在病毒性肝炎发生发展中的作用。具体研究目标如下：
1.研究在病毒性肝炎模型小鼠肝脏中DN T细胞TCRγδ~+DN T细胞亚群和TCRαβ+DN T细胞亚群的比例。在疾病进程中动态观察病毒性肝炎模型小鼠肝脏TCRγδ+DN T细胞比例、数量。
2.鉴定肝脏TCRγδ~+DN T细胞的表面标志和细胞因子谱。
3.探讨肝脏TCRγδ~+DN T细胞细胞对肝细胞的损伤作用及机制。
【研究方法】
1. MHV-3腹腔感染途径建立小鼠病毒性肝炎模型。流式细胞术检测正常和感染MHV-310天后模型小鼠肝脏DNT表达TCRγδ+和TCRαβ+的比例。动态观察MHV-3感染0、3、7、10、15、20、30天后模型小鼠肝脏TCRγδ~+DN T细胞在肝脏T细胞中比例变化及其绝对计数。
2.流式细胞术检测感染MHV-310天的C3H/Hej小鼠肝脏TCRγδ~+DN T细胞表达表面标志CD30、 CD25、CD28和CD44的百分率、感染MHV-3后各时间点肝脏TCRγδ~+DN T细胞表达活化标志CD69分子的百分率。采用细胞因子染色法检测肝脏TCRγδ~+DN T细胞分泌IL-2、 IL-4、 IL-10、IL-17A、IL-21、IL-22、肿瘤坏死因子-α（TNF-α,tumor necrosis factor-α）、γ-干扰素（interferon，IFN-γ）、穿孔素（perforin）和颗粒酶B(granzyme B)的水平。采用α-GalCer:mCD1d二聚体来鉴定肝脏TCRγδ~+DN T细胞与NKT细胞之间的关系。
3.体外实验：磁珠分选肝脏TCRγδ~+DN T细胞。胶原酶原位灌流消化法分离小鼠原代肝细胞。以感染MHV-310天的C3H/Hej小鼠肝脏TCRγδ~+DN T细胞为效应细胞，肝细胞为靶细胞，采用非放射性细胞毒实验检测肝脏TCRγδ~+DN T细胞在体外对正常或感染MHV-3的原代肝细胞的杀伤活性。采用Transwell法检测肝脏TCRγδ+DNT细胞对肝脏细胞的杀伤作用是否依赖细胞与细胞间的接触。在体外，通过抗体阻断TNF-α, IFN-γ和IL-17A检测肝脏TCRγδ~+DN T细胞杀伤肝细胞的作用途径。
4.体内实验：将磁珠分选纯化的肝脏TCRγδ~+DN T细胞经尾静脉被动转移过继到MHV-3感染5天后C3H/Hej小鼠体内（3×106/只），同体积PBS液尾静脉转移过继到MHV-3感染5天后C3H/Hej小鼠体内作为对照，观察两组生存曲线、转移过继3天后各组外周血AST和ALT水平、肝脏组织H-E染色。
【结果】
1.在正常小鼠和MHV-3诱导的小鼠病毒性肝炎模型中，肝脏DN T细胞以TCRγδ+DNT细胞亚群为主。在感染MHV-3后各时间点肝脏TCRγδ~+DN T在肝脏T淋巴细胞中的比例较感染前显著升高，在感染后10天达到最高值（19.7±3.22%）；肝脏TCRγδ~+DN T细胞数量在MHV-3感染后显著增加，在感染后10天达到最高峰（6.51±1.29×105）。
2.流式细胞术检测MHV-3诱导的小鼠病毒性肝炎模型中肝脏TCRγδ~+DN T细胞的表面标志为TCRγδ+CD3~+CD4~-CD8~-CD25-CD28-CD30-CD44+。肝脏TCRγδ~+DNT细胞不能特异性识别α-半乳糖酰基鞘氨醇，表明这群细胞不属于NKT细胞亚群。在感染MHV-3后，随着病程进展，肝脏TCRγδ~+DNT表面活化标志CD69分子表达水平持续升高，在感染后10天达到最高水平（83.45±5.32%）。模型中肝脏TCRγδ~+DN T表达TNF-α、INF-γ、IL-17A和IL-2，几乎不表达IL-4、IL-10、IL-21、IL-22、FasL、穿孔素（perforin）和颗粒酶B(granzyme B)。
3.体外实验：MHV-3诱导的病毒性肝炎小鼠模型中，感染后肝脏TCRγδ~+DN T细胞对染后肝细胞有明显的杀伤作用。通过Transwell实验发现，肝脏TCRγδ~+DN T细胞对感染后肝细胞的直接杀伤作用不需要细胞与细胞间的接触。通过抗体阻断试验证明，阻断TNF-a后肝脏TCRγδ~+DN T细胞对感染后肝细胞的杀伤作用显著降低，而单独阻断IFN-γ或IL-17A后肝脏TCRγδ~+DNT细胞对感染后肝细胞的杀伤作用无显著改变。
4.体内实验：将感染MHV-310天后小鼠病毒性肝炎模型中肝脏TCRγδ~+DNT细胞过继给感染MHV-35天后小鼠病毒性肝炎模型小鼠，小鼠生存率显著降低，从41.7%下降至8.33%。在过继3天后，肝脏TCRγδ~+DN T细胞转移过继组小鼠血清ALT和AST水平较PBS过继对照组显著升高；在过继3天后，肝脏TCRγδ~+DN T细胞转移过继组肝脏病理损伤较PBS过继对照组更加明显，出现肝细胞水肿、气球样变、点状坏死、灶状坏死，甚至碎片状坏死，并伴有大量淋巴细胞浸润。
【结论】
1.本研究首次利用MHV-3诱导的小鼠病毒性肝炎模型探讨了肝脏TCRγδ~+DN T细胞在病毒性肝炎中的作用。研究发现小鼠病毒性肝炎模型中肝脏TCRγδ~+DNT细胞具有独特的表面标志。这群细胞不属于NKT细胞亚群。在感染MHV-3后，肝脏TCRγδ~+DNT细胞的数量大量增加并且大量活化，表达细胞因子TNF-α、IFN-γ、IL-17A和IL-2。
2. MHV-3诱导的病毒性肝炎小鼠模型中，感染后肝脏TCRγδ~+DNT细胞对感染后肝细胞有显著的杀伤作用。这种杀伤作用不依赖细胞和细胞间的接触。通过抗体阻断实验证明肝脏TCRγδ~+DNT细胞主要是通过TNF-a对感染肝细胞发挥细胞毒作用。
3.将肝脏TCRγδ~+DNT细胞转移过继到病毒性肝炎小鼠体内后能够加重病毒性肝炎小鼠病情发展，导致小鼠生存率降低。
4.本研究证明在小鼠病毒性肝炎模型肝脏中存在一群TCRγδ~+DNT细胞，参与了小鼠病毒性肝炎的发生发展，与肝脏损伤密切相关。这一研究发现有助于深入理解病毒性肝炎的发病机制并且可能为临床诊断和治疗提供一些新的参考。
【BACKGROUND&OBJECTIVE】
Viral hepatitis refers to the acute and chronic liver diseases induced by hepatotropicviruses. Viral hepatitis is characteristics with continuous liver injury. It is generally thoughthepatitis viruses themselves are not cytopathogenic and do not kill hepatocytes. Previousstudies have extensively reported the centrol role of activity of T cell-mediated immuneresponses, include CD4~+T cells, CD8+T cells, natural killer T (NKT) cells, and regulatoryT cells (Treg cells), in induction of hepatocellular injury during viral infection. Recentreports have shown that non-specific immune system do play roles in induction ofhepatocellular injury during viral infection. In mouse model of fulminant hepatic failure(FHF) induced by murine hepatitis virus strain3(MHV-3), activated macrophage producedproinflammatory mediators such as TNF and IL-1as well as the unique procoagulantactivity fgl2prothrombinase, whic subsequent exacerbate the liver micro-environment andled to hepatocyte apoptosis and necrosis. Zou Y et al demonstrated that NK cells viaFas/FasL and NKG2D/NKG2D ligand pathway induced hepatocyte injury in patients withFHF and acute-on-chronic liver failure (ACLF).
A small population of CD4-CD8-(double negative, DN) T cells has been shown to beinvolved in several diseases, including autoimmune diseases, immunodeficiency diseases, infectious disease, and tumor. In both mice and humans, these cells compose about1-5%ofall peripheral T lymphocytes, and express a specific set of cell surface molecules and acharacteristic cytokine profile. In mice infected with Francisella tularensis live vaccinestrain, lung DN T cells were found to expand into two subsets that produced two essentialcytokines, IFN-γ and IL-17A. TCRγδ~+DN T cells from Leishmania-infected individualspresent a regulatory profile, producing immunoregulatory cytokine IL-10.
Interestingly, DN T cells have also been reported in the livers of healthy humans andmice and those with diseases. Norris et al. demonstrated that DN T cells ranged from2.7to29%of total hepatic T lymphocytes in healthy humans. Seki et al. reported that the liverwas probably an important organ for activation and expansion of TCRγδ~+DN T cells inhumans and mice, especially under tumor-bearing conditions. In the portal areas of livercirrhosis patients, the majority of the γδ+T cells were TCRγδ~+DN T cells. These findingssuggest a potential role for liver DN T cells in the pathological process of liver disease.However, very little information about the potential role of DN T cells in viral hepatitis hasbeen reported.
In this study we use MHV-3induced murine hepatitis model to explore potential rolesof liver DN T cells in viral hepatitis. Therefore the purposes of this study are as the follows:
1. To examine the proportions of TCRγδ~+DN T cells and TCRαβ+DN T cells in liver DNT cells in MHV-3-induced hepatitis mice model.
2. To illustrate the phenotype and cytokine profiles of liver TCRγδ~+DN T cells in MHV-3induced fulminant hepatitis mice model.
3. To investigate the effect of of liver TCRγδ~+DN T cells on hepatocytes and its potentialmechanisms in MHV-3-induced hepatitis mice model.
【METHODS】
1. Viral hepatitis animal model was established by MHV-3infection of C3H/HeJ. Theproportions of TCRγδ~+DN T cells and TCRαβ+DN T cells in liver DN T cells inMHV-3-induced hepatitis mice model was examined. The proportions and number ofliver TCRγδ~+DN T cells were observed on day0,3,7,10,15,20, and35post virus infection.
2. The phenotype and cytokine profiles of liver TCRγδ~+DN T cells were detected by flowcytometric analysis.
3. In vitro experiment
Liver TCRγδ~+DN T cells were purified by Magnetic bead sorting. Primary hepatic cellswere isolated from normal or MHV-3infected mice. Lactate dehydrogenase (LDH)release assay were used to detect the cytotoxicity of liver TCRγδ~+DN T cells oninfected hepatocytes or uninfected hepatocytes. A Transwell experiment was performedto determine whether the cytotoxicity required cell-cell contact. The cytotoxic effects ofliver TCRγδ~+DN T cells on infected hepatocytes were examined in the presence ofthree neutralizing Abs (anti-TNF-α, anti-IFN-γ, anti-IL-17A mAbs) or not.
4. In vivo experiment
Liver TCRγδ~+DN T cells from MHV-3infected C3H/HeJ mice (3×106cell/mouse)were adoptively transferred to C3H/HeJ mice5days post MHV-3infection. PBSinjected into mice5days post MHV-3infection was used as control group. Thesurvival rates, hepatic pathological changes, serum ALT and AST levels of two groupswere examined.
【RESULTS】
1. Predominant population of DN T cells in the liver of healthy or MHV-3-infected miceexpressed TCRγδ+. The proportion and number of TCRγδ~+DN T cells in the liverincreased markedly and peaked on day10post infection, compared with normalcontrols (19.7±3.22%vs.6.38±1.1%,6.51±1.29×105vs.0.76±0.11×105), and remainedhigh thereafter.
2. The surface phenotype of liver TCRγδ~+DN T cells was as TCRγδ+CD3~+CD4~-CD8~-CD25-CD28-CD30-CD44+. TCRγδ~+DN T cells did not recognize α-Galcer,demonstrating that these cells differed from NKT cells. The proportion of liver TCRγδ+DN T cells expressing CD69increased significantly post MHV-3infection, peaked onday10(83.45±5.32%), and then decreased gradually. These cells revealed a classicalinflammatory cytokine profile, with high production of TNF-α, IFN-γ, IL-17A and IL-2, but no IL-4, IL-10, IL-21or IL-22was observed.
3. In Vitro, infected liver TCRγδ~+DN T cells had cytotoxic effect on infected hepatocytes.In transwell assay, the cytotoxic effect was still observed at a comparable level to thatobserved without the Transwell. TCRγδ~+DN T cell cytotoxicity decreased markedlywhen TNF-α was blocked, but neither neutralizing IFN-γ nor IL-17A alone was able toinhibit TCRγδ~+DN T cell-mediated cytotoxicity.
4. In vivo, adoptive transfer of liver TCRγδ~+DN T cells led to a dramatic decrease insurvival of infected mice, from41.7%down to8.33%, and resulted in furtherdeterioration in liver histopathology. Hydropic degeneration, inflammatory cellinfiltration, and bridge necrosis in livers were more aggravated, accompanied byrobustly elevated blood levels of aspartate aminotransferase (AST) and alanineaminotransferase (ALT).
【CONCLUSION】
1. Liver of healthy C3H/HeJ mice contains abundant TCRγδ~+DN T cells, which markedlyincreased after MHV-3infection.
2. Liver TCRγδ~+DN T cells bearing a specific phenotype and highly activated afterMHV-3infection. These cells were distinct from NKT cells and produced theinflammatory cytokines TNF-α and IL-17A, and the Th1cytokines IFN-γ and IL-2, butnot Th2cytokines.
3. Highly activated liver TCRγδ~+DN T cells were cytotoxic to MHV-3-infectedhepatocytes in vitro and this effect did not require cell-cell contact. Moreover, thecytotoxic effect of liver TCRγδ~+DN T cells against hepatocytes involves TNF-αpathway, but not IL-17A or IFN-γ.
4. Adoptive transfer of TCRγδ~+DN T cells led to dramatically decreased survival inMHV-3-infected mice, accompanied by deteriorated histopathology and elevated ALTand AST levels.
病毒性肝炎以肝细胞损伤为主要特征，然而肝细胞损伤的具体机制尚未完全明确。目前大部分学者认为肝炎病毒本身并不直接造成肝细胞的损伤，而机体抗肝炎病毒的免疫反应，特别是免疫细胞介导的免疫反应，是造成肝细胞损伤的主要原因。多种免疫细胞亚群，如CD4~+T细胞、CD8+T细胞、自然杀伤细胞(NK)、NKT细胞、调节性T细胞、巨噬细胞等，都被证实在这一病理过程中发挥重要作用。近年来随着对肝炎发病机制研究的深入，越来越多的研究表明非特异性免疫反应，特别是NK细胞和巨噬细胞介导的非特异性免疫反应，也参与了病毒性肝炎感染引起肝脏损伤的发病机理。我们对HBV诱导的暴发型肝衰竭（FHF）和慢加急性肝衰竭(HBV-inducedacute-on-chronic liver failure, HBV-ACLF)研究后发现,肝脏NK细胞通过死亡受体途径Fas/FasL和NK细胞受体配体识别途径NKG2D/NKG2DL对肝细胞造成损伤。在3型鼠肝炎病毒(MHV-3)诱导的小鼠暴发性肝衰竭中活化的巨噬细胞能够分泌炎性因子TNF, IL-1进一步促进肝脏中的炎症反应并加重肝细胞损伤。此外，活化的巨噬细胞还分泌促凝血活性物质fgl2凝血酶原酶（fibrinogen like protein2）， fgl2凝血酶原酶可以激活凝血级联反应，引起肝脏微循环障碍并导致肝细胞大块死亡。因此明确各种免疫细胞及其分泌的细胞因子形成的免疫网络在病毒性肝炎引起肝细胞损伤中的作用将有助于更加全面的认识病毒性肝炎的发病机制，并为其临床诊断和治疗提供重要的靶点。
近年研究发现，机体存在一种新型T细胞亚群——CD3~+CD4~-CD8~-细胞(双阴性T细胞，DN T)。在健康人群和正常小鼠外周血T细胞中，DNT细胞所占比例为1–5%。随着对DN T细胞的深入研究，越来越多的证据表明DNT细胞在多种疾病，如移植排斥、自身免疫性疾病、肿瘤免疫、感染性疾病、过敏性疾病以及移植物抗宿主病等疾病的病理进程中发挥重要作用。在不同的疾病中DN T细胞表达不同的表面标志细胞，分泌独特的效应因子，通过这些效应因子发挥其生物学功能。在器官移植模型中，将脾细胞来源的DN T细胞克隆后输入小鼠体内，DN T细胞能够通过Fas/Fasl、穿孔素等途径杀伤抗供者CD4~+T细胞、CD8+T细胞和B细胞，从而提高移植物的存活时间和存活率。Zhang, Z等通过对MRL/lpr小鼠模型中DNT细胞研究后发现，系统性红斑狼疮模型鼠（MRL/lpr）中DNT细胞高表达IL-17和IL-23R,并且与疾病进程及严重程度呈正相关。在感染土拉热弗郎西丝菌(Francisella tularensis)的小鼠中，研究发现感染后肺组织中DN T细胞可以分化为两个亚群，一群主要分泌IL-17，而另一群以分泌IFN-γ为主，并在疾病进程中发挥不同的生物学作用。Lis R. V等研究发现利什曼原虫感染者外周血TCRαβ~+DNT (TCRαβ~+CD4~-CD8~-）细胞可以分泌炎性细胞因子IFN-γ和TNF-α，而TCRγδ~+DNT(TCRγδ+CD4-CD8-)主要分泌调节性细胞因子IL-10。
迄今，肝脏DN T细胞是否在病毒性肝炎中发挥作用尚不明确。本实验室以鼠3型肝炎病毒（MHV-3,murine hepatitis virus3）感染C3H/Hej小鼠成功建立了小鼠病毒性肝炎模型，本研究拟利用MHV-3诱导的小鼠病毒性肝炎模型，探讨DN T细胞在病毒性肝炎发生发展中的作用。具体研究目标如下：
1.研究在病毒性肝炎模型小鼠肝脏中DN T细胞TCRγδ~+DN T细胞亚群和TCRαβ+DN T细胞亚群的比例。在疾病进程中动态观察病毒性肝炎模型小鼠肝脏TCRγδ+DN T细胞比例、数量。
2.鉴定肝脏TCRγδ~+DN T细胞的表面标志和细胞因子谱。
3.探讨肝脏TCRγδ~+DN T细胞细胞对肝细胞的损伤作用及机制。
【研究方法】
1. MHV-3腹腔感染途径建立小鼠病毒性肝炎模型。流式细胞术检测正常和感染MHV-310天后模型小鼠肝脏DNT表达TCRγδ+和TCRαβ+的比例。动态观察MHV-3感染0、3、7、10、15、20、30天后模型小鼠肝脏TCRγδ~+DN T细胞在肝脏T细胞中比例变化及其绝对计数。
2.流式细胞术检测感染MHV-310天的C3H/Hej小鼠肝脏TCRγδ~+DN T细胞表达表面标志CD30、 CD25、CD28和CD44的百分率、感染MHV-3后各时间点肝脏TCRγδ~+DN T细胞表达活化标志CD69分子的百分率。采用细胞因子染色法检测肝脏TCRγδ~+DN T细胞分泌IL-2、 IL-4、 IL-10、IL-17A、IL-21、IL-22、肿瘤坏死因子-α（TNF-α,tumor necrosis factor-α）、γ-干扰素（interferon，IFN-γ）、穿孔素（perforin）和颗粒酶B(granzyme B)的水平。采用α-GalCer:mCD1d二聚体来鉴定肝脏TCRγδ~+DN T细胞与NKT细胞之间的关系。
3.体外实验：磁珠分选肝脏TCRγδ~+DN T细胞。胶原酶原位灌流消化法分离小鼠原代肝细胞。以感染MHV-310天的C3H/Hej小鼠肝脏TCRγδ~+DN T细胞为效应细胞，肝细胞为靶细胞，采用非放射性细胞毒实验检测肝脏TCRγδ~+DN T细胞在体外对正常或感染MHV-3的原代肝细胞的杀伤活性。采用Transwell法检测肝脏TCRγδ+DNT细胞对肝脏细胞的杀伤作用是否依赖细胞与细胞间的接触。在体外，通过抗体阻断TNF-α, IFN-γ和IL-17A检测肝脏TCRγδ~+DN T细胞杀伤肝细胞的作用途径。
4.体内实验：将磁珠分选纯化的肝脏TCRγδ~+DN T细胞经尾静脉被动转移过继到MHV-3感染5天后C3H/Hej小鼠体内（3×106/只），同体积PBS液尾静脉转移过继到MHV-3感染5天后C3H/Hej小鼠体内作为对照，观察两组生存曲线、转移过继3天后各组外周血AST和ALT水平、肝脏组织H-E染色。
【结果】
1.在正常小鼠和MHV-3诱导的小鼠病毒性肝炎模型中，肝脏DN T细胞以TCRγδ+DNT细胞亚群为主。在感染MHV-3后各时间点肝脏TCRγδ~+DN T在肝脏T淋巴细胞中的比例较感染前显著升高，在感染后10天达到最高值（19.7±3.22%）；肝脏TCRγδ~+DN T细胞数量在MHV-3感染后显著增加，在感染后10天达到最高峰（6.51±1.29×105）。
2.流式细胞术检测MHV-3诱导的小鼠病毒性肝炎模型中肝脏TCRγδ~+DN T细胞的表面标志为TCRγδ+CD3~+CD4~-CD8~-CD25-CD28-CD30-CD44+。肝脏TCRγδ~+DNT细胞不能特异性识别α-半乳糖酰基鞘氨醇，表明这群细胞不属于NKT细胞亚群。在感染MHV-3后，随着病程进展，肝脏TCRγδ~+DNT表面活化标志CD69分子表达水平持续升高，在感染后10天达到最高水平（83.45±5.32%）。模型中肝脏TCRγδ~+DN T表达TNF-α、INF-γ、IL-17A和IL-2，几乎不表达IL-4、IL-10、IL-21、IL-22、FasL、穿孔素（perforin）和颗粒酶B(granzyme B)。
3.体外实验：MHV-3诱导的病毒性肝炎小鼠模型中，感染后肝脏TCRγδ~+DN T细胞对染后肝细胞有明显的杀伤作用。通过Transwell实验发现，肝脏TCRγδ~+DN T细胞对感染后肝细胞的直接杀伤作用不需要细胞与细胞间的接触。通过抗体阻断试验证明，阻断TNF-a后肝脏TCRγδ~+DN T细胞对感染后肝细胞的杀伤作用显著降低，而单独阻断IFN-γ或IL-17A后肝脏TCRγδ~+DNT细胞对感染后肝细胞的杀伤作用无显著改变。
4.体内实验：将感染MHV-310天后小鼠病毒性肝炎模型中肝脏TCRγδ~+DNT细胞过继给感染MHV-35天后小鼠病毒性肝炎模型小鼠，小鼠生存率显著降低，从41.7%下降至8.33%。在过继3天后，肝脏TCRγδ~+DN T细胞转移过继组小鼠血清ALT和AST水平较PBS过继对照组显著升高；在过继3天后，肝脏TCRγδ~+DN T细胞转移过继组肝脏病理损伤较PBS过继对照组更加明显，出现肝细胞水肿、气球样变、点状坏死、灶状坏死，甚至碎片状坏死，并伴有大量淋巴细胞浸润。
【结论】
1.本研究首次利用MHV-3诱导的小鼠病毒性肝炎模型探讨了肝脏TCRγδ~+DN T细胞在病毒性肝炎中的作用。研究发现小鼠病毒性肝炎模型中肝脏TCRγδ~+DNT细胞具有独特的表面标志。这群细胞不属于NKT细胞亚群。在感染MHV-3后，肝脏TCRγδ~+DNT细胞的数量大量增加并且大量活化，表达细胞因子TNF-α、IFN-γ、IL-17A和IL-2。
2. MHV-3诱导的病毒性肝炎小鼠模型中，感染后肝脏TCRγδ~+DNT细胞对感染后肝细胞有显著的杀伤作用。这种杀伤作用不依赖细胞和细胞间的接触。通过抗体阻断实验证明肝脏TCRγδ~+DNT细胞主要是通过TNF-a对感染肝细胞发挥细胞毒作用。
3.将肝脏TCRγδ~+DNT细胞转移过继到病毒性肝炎小鼠体内后能够加重病毒性肝炎小鼠病情发展，导致小鼠生存率降低。
4.本研究证明在小鼠病毒性肝炎模型肝脏中存在一群TCRγδ~+DNT细胞，参与了小鼠病毒性肝炎的发生发展，与肝脏损伤密切相关。这一研究发现有助于深入理解病毒性肝炎的发病机制并且可能为临床诊断和治疗提供一些新的参考。
【BACKGROUND&OBJECTIVE】
Viral hepatitis refers to the acute and chronic liver diseases induced by hepatotropicviruses. Viral hepatitis is characteristics with continuous liver injury. It is generally thoughthepatitis viruses themselves are not cytopathogenic and do not kill hepatocytes. Previousstudies have extensively reported the centrol role of activity of T cell-mediated immuneresponses, include CD4~+T cells, CD8+T cells, natural killer T (NKT) cells, and regulatoryT cells (Treg cells), in induction of hepatocellular injury during viral infection. Recentreports have shown that non-specific immune system do play roles in induction ofhepatocellular injury during viral infection. In mouse model of fulminant hepatic failure(FHF) induced by murine hepatitis virus strain3(MHV-3), activated macrophage producedproinflammatory mediators such as TNF and IL-1as well as the unique procoagulantactivity fgl2prothrombinase, whic subsequent exacerbate the liver micro-environment andled to hepatocyte apoptosis and necrosis. Zou Y et al demonstrated that NK cells viaFas/FasL and NKG2D/NKG2D ligand pathway induced hepatocyte injury in patients withFHF and acute-on-chronic liver failure (ACLF).
A small population of CD4-CD8-(double negative, DN) T cells has been shown to beinvolved in several diseases, including autoimmune diseases, immunodeficiency diseases, infectious disease, and tumor. In both mice and humans, these cells compose about1-5%ofall peripheral T lymphocytes, and express a specific set of cell surface molecules and acharacteristic cytokine profile. In mice infected with Francisella tularensis live vaccinestrain, lung DN T cells were found to expand into two subsets that produced two essentialcytokines, IFN-γ and IL-17A. TCRγδ~+DN T cells from Leishmania-infected individualspresent a regulatory profile, producing immunoregulatory cytokine IL-10.
Interestingly, DN T cells have also been reported in the livers of healthy humans andmice and those with diseases. Norris et al. demonstrated that DN T cells ranged from2.7to29%of total hepatic T lymphocytes in healthy humans. Seki et al. reported that the liverwas probably an important organ for activation and expansion of TCRγδ~+DN T cells inhumans and mice, especially under tumor-bearing conditions. In the portal areas of livercirrhosis patients, the majority of the γδ+T cells were TCRγδ~+DN T cells. These findingssuggest a potential role for liver DN T cells in the pathological process of liver disease.However, very little information about the potential role of DN T cells in viral hepatitis hasbeen reported.
In this study we use MHV-3induced murine hepatitis model to explore potential rolesof liver DN T cells in viral hepatitis. Therefore the purposes of this study are as the follows:
1. To examine the proportions of TCRγδ~+DN T cells and TCRαβ+DN T cells in liver DNT cells in MHV-3-induced hepatitis mice model.
2. To illustrate the phenotype and cytokine profiles of liver TCRγδ~+DN T cells in MHV-3induced fulminant hepatitis mice model.
3. To investigate the effect of of liver TCRγδ~+DN T cells on hepatocytes and its potentialmechanisms in MHV-3-induced hepatitis mice model.
【METHODS】
1. Viral hepatitis animal model was established by MHV-3infection of C3H/HeJ. Theproportions of TCRγδ~+DN T cells and TCRαβ+DN T cells in liver DN T cells inMHV-3-induced hepatitis mice model was examined. The proportions and number ofliver TCRγδ~+DN T cells were observed on day0,3,7,10,15,20, and35post virus infection.
2. The phenotype and cytokine profiles of liver TCRγδ~+DN T cells were detected by flowcytometric analysis.
3. In vitro experiment
Liver TCRγδ~+DN T cells were purified by Magnetic bead sorting. Primary hepatic cellswere isolated from normal or MHV-3infected mice. Lactate dehydrogenase (LDH)release assay were used to detect the cytotoxicity of liver TCRγδ~+DN T cells oninfected hepatocytes or uninfected hepatocytes. A Transwell experiment was performedto determine whether the cytotoxicity required cell-cell contact. The cytotoxic effects ofliver TCRγδ~+DN T cells on infected hepatocytes were examined in the presence ofthree neutralizing Abs (anti-TNF-α, anti-IFN-γ, anti-IL-17A mAbs) or not.
4. In vivo experiment
Liver TCRγδ~+DN T cells from MHV-3infected C3H/HeJ mice (3×106cell/mouse)were adoptively transferred to C3H/HeJ mice5days post MHV-3infection. PBSinjected into mice5days post MHV-3infection was used as control group. Thesurvival rates, hepatic pathological changes, serum ALT and AST levels of two groupswere examined.
【RESULTS】
1. Predominant population of DN T cells in the liver of healthy or MHV-3-infected miceexpressed TCRγδ+. The proportion and number of TCRγδ~+DN T cells in the liverincreased markedly and peaked on day10post infection, compared with normalcontrols (19.7±3.22%vs.6.38±1.1%,6.51±1.29×105vs.0.76±0.11×105), and remainedhigh thereafter.
2. The surface phenotype of liver TCRγδ~+DN T cells was as TCRγδ+CD3~+CD4~-CD8~-CD25-CD28-CD30-CD44+. TCRγδ~+DN T cells did not recognize α-Galcer,demonstrating that these cells differed from NKT cells. The proportion of liver TCRγδ+DN T cells expressing CD69increased significantly post MHV-3infection, peaked onday10(83.45±5.32%), and then decreased gradually. These cells revealed a classicalinflammatory cytokine profile, with high production of TNF-α, IFN-γ, IL-17A and IL-2, but no IL-4, IL-10, IL-21or IL-22was observed.
3. In Vitro, infected liver TCRγδ~+DN T cells had cytotoxic effect on infected hepatocytes.In transwell assay, the cytotoxic effect was still observed at a comparable level to thatobserved without the Transwell. TCRγδ~+DN T cell cytotoxicity decreased markedlywhen TNF-α was blocked, but neither neutralizing IFN-γ nor IL-17A alone was able toinhibit TCRγδ~+DN T cell-mediated cytotoxicity.
4. In vivo, adoptive transfer of liver TCRγδ~+DN T cells led to a dramatic decrease insurvival of infected mice, from41.7%down to8.33%, and resulted in furtherdeterioration in liver histopathology. Hydropic degeneration, inflammatory cellinfiltration, and bridge necrosis in livers were more aggravated, accompanied byrobustly elevated blood levels of aspartate aminotransferase (AST) and alanineaminotransferase (ALT).
【CONCLUSION】
1. Liver of healthy C3H/HeJ mice contains abundant TCRγδ~+DN T cells, which markedlyincreased after MHV-3infection.
2. Liver TCRγδ~+DN T cells bearing a specific phenotype and highly activated afterMHV-3infection. These cells were distinct from NKT cells and produced theinflammatory cytokines TNF-α and IL-17A, and the Th1cytokines IFN-γ and IL-2, butnot Th2cytokines.
3. Highly activated liver TCRγδ~+DN T cells were cytotoxic to MHV-3-infectedhepatocytes in vitro and this effect did not require cell-cell contact. Moreover, thecytotoxic effect of liver TCRγδ~+DN T cells against hepatocytes involves TNF-αpathway, but not IL-17A or IFN-γ.
4. Adoptive transfer of TCRγδ~+DN T cells led to dramatically decreased survival inMHV-3-infected mice, accompanied by deteriorated histopathology and elevated ALTand AST levels.
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12Bendelac A, Savage PB and Teyton L. The biology of NKT cells. Annu. Rev. Immunol.2007;25,297-336.
13Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8-double-negative T cellsare prominent producers of IL-17A and IFN-gamma during primary respiratory murineinfection with Francisella tularensis live vaccine strain. J Immunol,2010,184:5791-5801.
14Wondimu Z, Santodomingo-Garzon T, Le T, Swain MG.. Protective role ofinterleukin-17in murine NKT cell-driven acute experimental hepatitis. Am J Pathol,2010,177:2334-2346.
15Harrington LE, Hatton RD, Mangan PR, et al. Interleukin17-producing CD4+effector Tcells develop via a lineage distinct from the T helper type1and2lineages. Nat Immunol,2005,6:1123-1132.
16He D, Wu L, Kim HK, et al. CD8+IL-17-producing T cells are important in effectorfunctions for the elicitation of contact hypersensitivity responses. J Immunol,2006,177:6852-6858.
17Hamada S, Umemura M, Shiono T, et al. IL-17A produced by gammadelta T cells playsa critical role in innate immunity against listeria monocytogenes infection in the liver. JImmunol,2008,181:3456-3463.
18Zhang JY, Zhang Z, Lin F, et al.Interleukin-17-producing CD4(+) T cells increase withseverity of liver damage in patients with chronic hepatitis B. Hepatology，2010，51:81-91.
19Ge J, Wang K, Meng QH, et al. Implication of Th17and Th1cells in patients withchronic active hepatitis B. J Clin Immunol,2010,30:60-67.
20秦来英，杜文军，刘葵花，等.白细胞介素17的肝内表达与慢性乙型肝炎肝纤维化的相关性[J].中华肝脏病杂志，2009，17（3）：221-222.
21Thomson CW, Lee BPL, Zhang L. Double-negative regulatory T cells-Non-conventional regulators. Immunologic Research,2006,35:163-177.
22Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virusinfection. Nature Reviews Immunology,2005,5:215-229.
23Ichiki Y, He XS, Shimoda S, Ishibashi H, Keeffe EB, et al. T cell immunity in hepatitis Band hepatitis C virus infection: implications for autoimmunity. Autoimmun Rev,2005,4:82-95.
24Manigold T, Racanelli V. T-cell regulation by CD4regulatory T cells during hepatitis Band C virus infections: facts and controversies. Lancet Infect Dis,2007,7:804-813.
25Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand andNKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.J Immunol,2010,184:466-475.
26Simsek H， Kadyifci A. Serum iterIeukin2and soIuDIe interIeukin2receptor inchroninactive hepatitis C：effect of interferon therapy. Jint Med Res，2002，24（3）：239~245.
27Gao B, Jeong WI, Tian Z. Liver: An organ with predominant innate immunity.Hepatology,2008,47:729-736.
28Seki S, Habu Y, Kawamura T, et al. The liver as a crucial organ in the first line of hostdefense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1Ag+T cells in Thelper1immune responses. Immunol Rev,2000,174:35-46.
1Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virusinfection. Nature Reviews Immunology,2005,5:215-229.
2Ichiki Y, He XS, Shimoda S, et al. T cell immunity in hepatitis B and hepatitis C virusinfection: implications for autoimmunity. Autoimmun Rev,2005,4:82-95.
3Manigold T, Racanelli V. T-cell regulation by CD4regulatory T cells during hepatitis Band C virus infections: facts and controversies. Lancet Infect Dis,2007,7:804-813.
4Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand andNKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.J Immunol,2010,184:466-475.
5D'Acquisto F, Crompton T. CD3+CD4-CD8-(double negative) T cells: saviours orvillains of the immune response? Biochem Pharmacol,2011,82:333-340.
6Thomson CW, Lee BPL, Zhang L. Double-negative regulatory T cells-Non-conventional regulators. Immunologic Research,2006,35:163-177.
7Fischer K, Voelkl S, Przybylski GK, et al. Isolation and characterization of humanantigen-specific TCR alpha beta(+) CD4(-)CD8(-) double negative regulatory T cells.Blood,2005,106:3306.
8Zhang ZX, Yang L, Young KJ, et al.Identification of a previously unknownantigen-specific regulatory T cell and its mechanism of suppression. Nat Med,2000,6:782-789.
9Duncan B, Nazarov-Stoica C, Surls J, et al. Double negative (CD3+4-8-) TCRalphabeta splenic cells from young NOD mice provide long-lasting protection againsttype1diabetes. Plos One,2010,5: e11427.
10Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8-double-negative T cellsare prominent producers of IL-17A and IFN-gamma during primary respiratory murineinfection with Francisella tularensis live vaccine strain. J Immunol,2010,184:5791-5801.
11Dean GS, Anand A, Blofeld A, et al.Characterization of CD3+CD4-CD8-(doublenegative) T cells in patients with systemic lupus erythematosus: production of IL-4.Lupus,2002,11:501-507.
12Crispin JC, Oukka M, Bayliss G, et al. Expanded double negative T cells in patients withsystemic lupus erythematosus produce IL-17and infiltrate the kidneys. J Immunol,2008,181:8761-8766.
13Ichiki, Y., X. S. He, S. Shimoda, et al. T cell immunity in hepatitis B and hepatitis Cvirus infection: implications for autoimmunity. Autoimmun Rev,2005，4,82-95.
14Walker, L. J., A. K. Sewell&P. Klenerman.T cell sensitivity and the outcome of viralinfection. Clin Exp Immunol,2010,159,245-55.
15Antonelli, L. R., W. O. Dutra, R. R. Oliveira, K. C. Torres, et al.Disparateimmunoregulatory potentials for double-negative (CD4-CD8-) alpha beta and gammadelta T cells from human patients with cutaneous leishmaniasis. Infect Immun,2006,74,6317-23.
16Kagi D, Vignaux F, Ledermann B, et al. Fas and perforin pathways as majormechanisms of T cell-mediated cytotoxicity. Science,1994,265:528-30.
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23Zou Y, Chen T, Han M, et al. Increased k illi ng of liver NK cells by Fas/Fas ligand andNKG2D/NKG2D ligand contributes to hepatocyte necrosisin virus–induced liverfailure. J I mmunol,2010,184(1):466-475.
24Kaki M IK, Guidottil G, Koezuka Y, et al. Natural killer T cell activation inhibitshepatitis B virusrep-licationinvivo. J ExpMed,2000,192(7):921-930.
25Baron J L, Gardiner L, Nishimura S, et al. Activation of a nonclassical NK T cell subsetin a transgenic mouse model of hepatitis B virus infection. Immunity,2002,16(4):583-594.
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29Gao B, Jeong WI, Tian Z. Liver: An organ with predominant innate immunity.Hepatology,2008,47:729-736.
30Seki S, Habu Y, Kawamura T, et al. The liver as a crucial organ in the first line of hostdefense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1Ag+T cells in Thelper1immune responses. Immunol Rev,2000,174:35-46.
1D'Acquisto F, Crompton T. CD3+CD4-CD8-(double negative) T cells: saviours orvillains of the immune response? Biochem Pharmacol,2011,82:333-340.
2Thomson CW, Lee BPL, Zhang L. Double-negative regulatory T cells-Non-conventional regulators. Immunologic Research,2006,35:163-177.
3Fischer K, Voelkl S, Przybylski GK, et al. Isolation and characterization of humanantigen-specific TCR alpha beta(+) CD4(-)CD8(-) double negative regulatory T cells.Blood,2005,106:3306.
4Zhang ZX, Yang L, Young KJ, et al. Identification of a previously unknownantigen-specific regulatory T cell and its mechanism of suppression. Nat Med,2000,6:782-789.
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15Ohteki T, Seki S, Abo T, Kumagai K.Liver is a possible site for the proliferation ofabnormal CD3+4-8-double-negative lymphocytes in autoimmune MRL-lpr/lpr mice. JExp Med,1990,172:7-12.
16Marra LE, Zhang ZX, Joe B, et al. IL-10induces regulatory T cell apoptosis byup-regulation of the membrane form of TNF-alpha. J Immunol,2004,172:1028-1035.
17Zhang ZX, Stanford WL, Zhang L. Ly-6A is critical for the function of double negativeregulatory T cells. Eur J Immunol,2002,32:1584-1592.
18Lee BP, Mansfield E, Hsieh SC, et al. Expression profiling of murine double-negativeregulatory T cells suggest mechanisms for prolonged cardiac allograft survival. JImmunol,2005,174:4535-4544.
19Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8-double-negative T cellsare prominent producers of IL-17A and IFN-gamma during primary respiratory murineinfection with Francisella tularensis live vaccine strain. J Immunol,2010,184:5791-5801.
20Antonelli LR, Dutra WO, Oliveira RR, et al. Disparate immunoregulatory potentials fordouble-negative (CD4-CD8-) alpha beta and gamma delta T cells from human patientswith cutaneous leishmaniasis. Infect Immun,2006,74:6317-6323.
21Young KJ, Yang L, Phillips MJ, Zhang L.Donor-lymphocyte infusion inducestransplantation tolerance by activating systemic and graft-infiltrating double-negativeregulatory T cells. Blood,2002,100:3408-3414.
22Chen WH, Zhou DJ, Torrealba JR, et al. Donor lymphocyte infusion induces long-termdonor-specific cardiac xenograft survival through activation of recipient double-negativeregulatory T cells. Journal of Immunology,2005,175:3409-3416.
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25Ford MS, Young KJ, Zhang Z, et al. The immune regulatory function oflymphoproliferative double negative T cells in vitro and in vivo. J Exp Med,2002,196:261-267.
26Dean GS, Anand A, Blofeld A, et al. Characterization of CD3+CD4-CD8-(doublenegative) T cells in patients with systemic lupus erythematosus: production of IL-4.Lupus,2002,11:501-507.
27Crispin JC, Oukka M, Bayliss G, et al. Expanded double negative T cells in patients withsystemic lupus erythematosus produce IL-17and infiltrate the kidneys. J Immunol,2008,181:8761-8766.
28Zhang Z, Kyttaris VC, Tsokos GC. The role of IL-23/IL-17axis in lupus nephritis. JImmunol,2009,183:3160-3169.
29Kyttaris VC, Zhang Z, Kuchroo VK et al. Cutting edge: IL-23receptor deficiencyprevents the development of lupus nephritis in C57BL/6-lpr/lpr mice. J Immunol,2010,184:4605-4609.
30Duncan B, Nazarov-Stoica C, Surls J, et al. Double negative (CD3+4-8-) TCRalphabeta splenic cells from young NOD mice provide long-lasting protection againsttype1diabetes. Plos One,2010,5: e11427.
31Bottrel RL, Dutra WO, Martins FA, et al. Flow cytometric determination of cellularsources and frequencies of key cytokine-producing lymphocytes directed againstrecombinant LACK and soluble Leishmania antigen in human cutaneous leishmaniasis.Infect Immun,2001,69:3232-3239.
32Gollob KJ, Antonelli LR, Faria DR, et al. Immunoregulatory mechanisms andCD4-CD8-(double negative) T cell subpopulations in human cutaneous leishmaniasis: abalancing act between protection and pathology. Int Immunopharmacol,2008,8:1338-1343.
33Hossain MS, Roback JD, Wang F, et al. Host and donor immune responses contribute toantiviral effects of amotosalen-treated donor lymphocytes following early posttransplantcytomegalovirus infection. J Immunol,2008,180:6892-6902.
34Seki S, Abo T, Masuda T, et al. Identification of activated T cell receptor gamma deltalymphocytes in the liver of tumor-bearing hosts. J Clin Invest,1990,86:409-415.
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38Matsuo R, Herndon DN, Kobayashi M, et al. CD4-CD8-TCR alpha/beta+suppressor Tcells demonstrated in mice1day after thermal injury. J Trauma,1997,42:635-640.
39Barwig C, Raker V, Montermann E, et al. Antigen dose-dependent suppression of murineIgE responses is mediated by CD4(-)CD8(-) double-negative T cells. Clin Exp Allergy,2010,40:891-901.
40Petitjean G, Chevalier MF, Tibaoui F, et al. Level of double negative T cells, whichproduce TGF-beta and IL-10, predicts CD8T-cell activation in primary HIV-1infection.AIDS,2012,26:139-48.
2Ichiki Y, He XS, Shimoda S, et al. T cell immunity in hepatitis B and hepatitis C virusinfection: implications for autoimmunity. Autoimmun Rev,2005,4:82-95.
3Manigold T, Racanelli V. T-cell regulation by CD4regulatory T cells during hepatitis Band C virus infections: facts and controversies. Lancet Infect Dis,2007,7:804-813.
4Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand andNKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.J Immunol,2010,184:466-475.
5Thomson CW, Lee BPL, Zhang L. Double-negative regulatory T cells Non-conventionalregulators. Immunologic Research,2006,35:163-177.
6Zhang ZX, Yang L, Young KJ, et al. Identification of a previously unknownantigen-specific regulatory T cell and its mechanism of suppression. Nat Med,2000,6:782-789.
7Fischer K, Voelkl S, Przybylski GK, et al. Isolation and characterization of humanantigen-specific TCR alpha beta(+) CD4(-)CD8(-) double negative regulatory T cells.Blood,2005,106:3306.
8Antonelli LR, Dutra WO, Oliveira RR, et al. Disparate immunoregulatory potentials fordouble-negative (CD4-CD8-) alpha beta and gamma delta T cells from human patientswith cutaneous leishmaniasis. Infect Immun,2006,74:6317-6323.
9Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8-double-negative T cellsare prominent producers of IL-17A and IFN-gamma during primary respiratory murineinfection with Francisella tularensis live vaccine strain. J Immunol,2010,184:5791-5801.
10Norris S, Collins C, Doherty DG, et al. Resident human hepatic lymphocytes arephenotypically different from circulating lymphocytes. J Hepatol,1998,28:84-90.
11Seki S, Abo T, Masuda T, et al. Identification of activated T cell receptor gamma deltalymphocytes in the liver of tumor-bearing hosts. J Clin Invest,1990,86:409-415.
12Kanayama K, Morise K, Nagura H. Immunohistochemical study of T cell receptorgamma delta cells in chronic liver disease. Am J Gastroenterol,1992,87:1018-1022.
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16Liu MF, Yang CY, Chao SC, et al. Distribution of double-negative (CD4-CD8-, DN) Tsubsets in blood and synovial fluid from patients with rheumatoid arthritis. ClinicalRheumatology,1999,18(3):227-231.
1D'Acquisto F, Crompton T. CD3+CD4-CD8-(double negative) T cells: saviours orvillains of the immune response? Biochem Pharmaco,2011,82(4):333-340.
2Thomson CW,Lee BPL,Zhang L.Double-negative regulatory T cells-Non-conventionalregulators.Immunologic Research,2006,35(1-2):163-177..
3Fischer K, Voelkl S, Przybylski GK, et al. Isolation and characterization of humanantigen-specific TCR alpha beta(+) CD4(-)CD8(-) double negative regulatory T cells.Blood,2005,105(7):2828-2835.
4Zhang ZX, Yang L, Young KJ, et al. Identification of a previously unknownantigen-specific regulatory T cell and its mechanism of suppression. Nat Med,2000,6:782-789.
5Marra LE, Zhang ZX, Joe B, et al. IL-10induces regulatory T cell apoptosis byup-regulation of the membrane form of TNF-alpha. J Immunol,2004,172:1028-1035.
6Zhang ZX, Stanford WL, Zhang L. Ly-6A is critical for the function of double negativeregulatory T cells. Eur J Immunol,2002,32:1584-1592.
7Lee BP, Mansfield E, Hsieh SC, et al. Expression profiling of murine double-negativeregulatory T cells suggest mechanisms for prolonged cardiac allograft survival. JImmunol,2005,174:4535-4544.
8Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8-double-negative T cellsare prominent producers of IL-17A and IFN-gamma during primary respiratory murineinfection with Francisella tularensis live vaccine strain. J Immunol,2010,184:5791-5801.
9Antonelli LR, Dutra WO, Oliveira RR, et al. Disparate immunoregulatory potentials fordouble-negative (CD4-CD8-) alpha beta and gamma delta T cells from human patientswith cutaneous leishmaniasis. Infect Immun,2006,74:6317-6323.
10D'Acquisto F, Crompton T. CD3+CD4-CD8-(double negative) T cells: saviours orvillains of the immune response? Biochem Pharmacol,2011,82:333-340.
11Fischer K, Voelkl S, Przybylski GK, et al. Isolation and characterization of humanantigen-specific TCR alpha beta(+) CD4(-)CD8(-) double negative regulatory T cells.Blood,2005,106:3306.
12Bendelac A, Savage PB and Teyton L. The biology of NKT cells. Annu. Rev. Immunol.2007;25,297-336.
13Cowley SC, Meierovics AI, Frelinger JA, et al. Lung CD4-CD8-double-negative T cellsare prominent producers of IL-17A and IFN-gamma during primary respiratory murineinfection with Francisella tularensis live vaccine strain. J Immunol,2010,184:5791-5801.
14Wondimu Z, Santodomingo-Garzon T, Le T, Swain MG.. Protective role ofinterleukin-17in murine NKT cell-driven acute experimental hepatitis. Am J Pathol,2010,177:2334-2346.
15Harrington LE, Hatton RD, Mangan PR, et al. Interleukin17-producing CD4+effector Tcells develop via a lineage distinct from the T helper type1and2lineages. Nat Immunol,2005,6:1123-1132.
16He D, Wu L, Kim HK, et al. CD8+IL-17-producing T cells are important in effectorfunctions for the elicitation of contact hypersensitivity responses. J Immunol,2006,177:6852-6858.
17Hamada S, Umemura M, Shiono T, et al. IL-17A produced by gammadelta T cells playsa critical role in innate immunity against listeria monocytogenes infection in the liver. JImmunol,2008,181:3456-3463.
18Zhang JY, Zhang Z, Lin F, et al.Interleukin-17-producing CD4(+) T cells increase withseverity of liver damage in patients with chronic hepatitis B. Hepatology，2010，51:81-91.
19Ge J, Wang K, Meng QH, et al. Implication of Th17and Th1cells in patients withchronic active hepatitis B. J Clin Immunol,2010,30:60-67.
20秦来英，杜文军，刘葵花，等.白细胞介素17的肝内表达与慢性乙型肝炎肝纤维化的相关性[J].中华肝脏病杂志，2009，17（3）：221-222.
21Thomson CW, Lee BPL, Zhang L. Double-negative regulatory T cells-Non-conventional regulators. Immunologic Research,2006,35:163-177.
22Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virusinfection. Nature Reviews Immunology,2005,5:215-229.
23Ichiki Y, He XS, Shimoda S, Ishibashi H, Keeffe EB, et al. T cell immunity in hepatitis Band hepatitis C virus infection: implications for autoimmunity. Autoimmun Rev,2005,4:82-95.
24Manigold T, Racanelli V. T-cell regulation by CD4regulatory T cells during hepatitis Band C virus infections: facts and controversies. Lancet Infect Dis,2007,7:804-813.
25Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand andNKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.J Immunol,2010,184:466-475.
26Simsek H， Kadyifci A. Serum iterIeukin2and soIuDIe interIeukin2receptor inchroninactive hepatitis C：effect of interferon therapy. Jint Med Res，2002，24（3）：239~245.
27Gao B, Jeong WI, Tian Z. Liver: An organ with predominant innate immunity.Hepatology,2008,47:729-736.
28Seki S, Habu Y, Kawamura T, et al. The liver as a crucial organ in the first line of hostdefense: the roles of Kupffer cells, natural killer (NK) cells and NK1.1Ag+T cells in Thelper1immune responses. Immunol Rev,2000,174:35-46.
1Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virusinfection. Nature Reviews Immunology,2005,5:215-229.
2Ichiki Y, He XS, Shimoda S, et al. T cell immunity in hepatitis B and hepatitis C virusinfection: implications for autoimmunity. Autoimmun Rev,2005,4:82-95.
3Manigold T, Racanelli V. T-cell regulation by CD4regulatory T cells during hepatitis Band C virus infections: facts and controversies. Lancet Infect Dis,2007,7:804-813.
4Zou Y, Chen T, Han M, et al. Increased killing of liver NK cells by Fas/Fas ligand andNKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.J Immunol,2010,184:466-475.
5D'Acquisto F, Crompton T. CD3+CD4-CD8-(double negative) T cells: saviours orvillains of the immune response? Biochem Pharmacol,2011,82:333-340.
6Thomson CW, Lee BPL, Zhang L. Double-negative regulatory T cells-Non-conventional regulators. Immunologic Research,2006,35:163-177.
7Fischer K, Voelkl S, Przybylski GK, et al. Isolation and characterization of humanantigen-specific TCR alpha beta(+) CD4(-)CD8(-) double negative regulatory T cells.Blood,2005,106:3306.
8Zhang ZX, Yang L, Young KJ, et al.Identification of a previously unknownantigen-specific regulatory T cell and its mechanism of suppression. Nat Med,2000,6:782-789.
9Duncan B, Nazarov-Stoica C, Surls J, et al. Double negative (CD3+4-8-) TCRalphabeta splenic cells from young NOD mice provide long-lasting protection againsttype1diabetes. Plos One,2010,5: e11427.
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