效应T细胞功能，而不是生存确定小鼠肝炎的程度和持续时间

StephenW

Effector T cell function rather than survival determines extent and duration of hepatitis in mice
Jnl of Hepatology June 2016
Michelle Vo1,2, Lauren E. Holz1,2,3, Yik Chun Wong1,2, Kieran English1,2, Volker Benseler1,2,4, Claire McGuffog1,2, Miyuki Azuma5, Geoffrey W. McCaughan2,6, David G. Bowen1,2,⇑,y, Patrick Bertolino1,2,⇑,y
1Liver Immunology Program, Centenary Institute, Newtown, NSW, Australia; 2AW Morrow Gastroenterology and Liver Centre,
Royal Prince Alfred Hospital Newtown, NSW, and Faculty of Medicine, University of Sydney, NSW, Australia; 3Current address: Department of
Microbiology and Immunology, The Peter Doherty Institute, The University of Melbourne, Parkville, VIC, Australia; 4Current address:
Department of Surgery, University of Regensburg, Bavaria, Germany; 5Department of Molecular Immunology Graduate School, Tokyo Medical
and Dental University, Yushima, Tokyo, Japan; 6Liver Injury and Cancer Program, Centenary Institute, Newtown, NSW, Australia
See Editorial, pages 1208–1210

Background & Aims

Acute hepatitis is often mediated by cytotoxic T lymphocytes (CTLs); however, the intrinsic parameters that limit CTL-mediated liver injury are not well understood.
Methods

To investigate whether acute liver damage is limited by molecules that decrease the lifespan or effector function of CTLs, we used a well-characterized transgenic (Tg) mouse model in which acute liver damage develops upon transfer of T cell receptor (TCR) Tg CD8 T cells. Recipient Tg mice received donor TCR Tg T cells deficient for either the pro-apoptotic molecule Bim, which regulates CTL survival, or suppressor of cytokine signaling-1 (SOCS-1), which controls expression of common gamma chain cytokines; the effects of anti-PD-L1 neutralizing antibodies were also assessed.
Results

Use of Bim-deficient donor T cells and/or PD-L1 blockade increased the number of intrahepatic T cells without affecting the degree and kinetic of acute hepatitis. In contrast, SOCS-1-deficient T cells induced a heightened, prolonged acute hepatitis caused by their enhanced cytotoxic function and increased expansion. Although they inflicted more severe acute liver damage, SOCS-1-deficient T cells never precipitated chronic hepatitis and became exhausted.
Conclusions

The degree of acute hepatitis is regulated by the function of CD8 T cells, but is not affected by changes in CTL lifespan. Although manipulation of the examined parameters affected acute hepatitis, persistent hepatitis did not ensue, indicating that, in the presence of high intrahepatic antigen load, changes in these factors in isolation were not sufficient to prevent T cell exhaustion and mediate progression to chronic hepatitis.

Introduction

Acute liver injury has a variety of causes, but is often initiated by an immune response triggered by hepatotropic viruses and other pathogens targeting this organ. A robust and sustained immune response involving both innate and adaptive immunity is critical to clear infections by viruses targeting the liver, such as the hepatitis B (HBV) [1] and C viruses (HCV) [2]. Antiviral cytotoxic CD8 T lymphocytes (CTLs) play a crucial role in mediating liver injury during infection, and the induction and maintenance of broad vigorous virus-specific CD8 T cell responses is a positive predictor of spontaneous resolution of HCV infection [[3], [4], [5]]. Antibody-dependent depletion of CD8 T cells in a chimpanzee model of HCV infection led to prolonged viral infection [6]. The detection of CTLs in the periphery coincides with raised alanine transaminase (ALT) levels and manifests clinically as acute hepatitis [7]. Clearance of HCV is more common in symptomatic patients [5], and it is likely that the degree of liver damage that ensues during the acute phase of infection is critical in influencing the outcome of infection [8].

Although it is known that CTLs kill target cells via granzyme and/or Fas/FasL interactions (classical CTL killing pathway), or via the secretion of hepatotoxic cytokines (bystander killing), the molecular processes limiting the action of CTLs and responsible for terminating acute liver damage mediated by CD8 T cells are not well understood. It is important to elucidate these processes to derive strategies to increase the chances of clearing persistent hepatotropic infections, and also to prevent too sustained or vigorous CD8 T cell responses that might lead to eventual development of significant fibrosis and cirrhosis, or in the more acute setting, to the development of acute liver failure. Several regulatory processes intrinsic to T cells could be important in limiting liver damage, including: induction of inhibitory receptors such as PD-1, Tim-3, LAG3, and CTLA-4; regulation of CTL effector function (downregulation of cytokine and effector molecules); and apoptosis of CTLs. Other parameters such as the number of CD8 T cell precursors specific for the virus [[9], [10]], the affinity of the TCR recognizing viral epitopes [11], the availability of CD4 T cell help [[12], [13]], and the number of hepatocytes expressing antigen [14] have also been shown to influence the long-term CD8 T cell outcome.

In order to identify the T cell intrinsic parameters controlling the degree and kinetics of acute liver damage, we used the well-characterized Met-Kb transgenic (Tg) mouse model of immune-mediated hepatitis, in which acute liver injury is induced by TCR Tg CD8 T cells that are first activated in lymph nodes (LN) and subsequently recognize their cognate antigen in the liver, leading to hepatocellular injury [15].

By transferring donor Tg T cells deficient for genes that control T cell death (Bim) or regulate signaling of cytokines critical for effector T cell function (suppressor of cytokine signaling-1; SOCS-1), or by treating recipient mice with anti-PD-L1 blocking antibodies, we investigated the roles of regulation of T cell death, regulation of CTL function, and T cell inhibitory molecules in limiting the degree and duration of liver damage, independently of TCR affinity, T cell help and antigen dose.

Our results suggest that strategies that enhance T cell survival promoted accumulation of CD8 T cells recognizing hepatocyte-expressed antigens in the liver, without altering the severity or tempo of liver damage. In contrast, augmenting the function of effector T cells prolonged acute hepatitis and increased the severity of liver damage. While these strategies had different effects on the fate and/or function of effector CD8 T cells, none prevented the resolution of acute hepatitis associated with the eventual development of functional exhaustion of T cells within in the liver.

In the setting of high-level persistent antigen expression in the liver, the degree and duration of liver damage is thus predominantly regulated by genes controlling the function of effector cells rather than those affecting T cell lifespan. Persistence of liver damage is ultimately limited by functional T cell exhaustion. These data suggest that, to be effective, immunotherapies aimed at boosting the number of CTLs in patients with liver disease will need to be combined with strategies that enhance T cell effector function and, more importantly, interfere with T cell exhaustion.
Excerpts

Blocking PD-1/PD-L1 interactions increased T cell numbers without augmenting the severity of acute liver damage or leading to chronic hepatitis

PD-1/PD-L1 blockade is currently used in the clinic to boost the function of exhausted CD8 T cells in a range of solid tumors, has been explored in chronic HCV infection [25], and is under investigation for treatment of hepatocellular carcinoma (HCC) [26]. It was therefore important to test the role of this molecule in our model.

PD-1 was expressed at similar low levels on donor Des RAG−/− T cells following 24 h of activation in the liver and LN of Met-Kb mice, but was not expressed at any stage on T cells transferred into B10.BR mice (Fig. 2A). At 48 h post-activation, liver-activated CD8 T cells expressed more PD-1 compared to LN-activated T cells (p <0.05) (Fig. 2A, B). Similar levels of expression were detected on liver- and LN-activated CD8 T cells less than a day later, probably due to death of liver-activated T cells and recirculation of LN-activated T cells to the liver [[18], [27]]. Over time, all Des RAG−/− T cells activated in Met-Kb mice became PD-1high and expression tended to increase with time (Fig. 2B), a result consistent with the phenotype of intrahepatic CD8 T cells reported in different models when antigen persists [[28], [29]]. PD-L1, the main PD-1 ligand, was also expressed at very low levels in the hepatic parenchyma of Met-Kb mice (Fig. 2C), but not in B10.BR mice (data not shown) at day 15 post-T cell transfer. Interestingly, PD-L1 expression was positively correlated with the number of infiltrating cells, and was increased in association with the more marked infiltrates of Des Bim−/− T cells. In these livers, PD-L1 expression was found in the vicinity of infiltrating Des Bim−/− T cells in the lobules and also in portal regions (Fig. 2C).

To examine the possibility that PD-1/PD-L1 interactions contributed to the induction of tolerance in liver-activated T cells and/or to the attenuation of acute hepatitis, Met-Kb and B10.BR mice, into which wild-type Des T cells had been transferred, were treated with neutralizing anti-PD-L1 or control antibodies (rat IgG). Treatment of Met-Kb mice with anti-PD-L1 every three days, beginning from day one after adoptive transfer of T cells, led to increased numbers of donor Des T cells in the liver at day 15, in comparison to control rat IgG-treated mice (Fig. 2D), suggesting that PD-L1 blockade increased T cell proliferation and/or survival. Despite accumulation of the Des T cell progeny in the liver, treatment with anti-PD-L1 did not have a significant effect on serum ALT levels (Fig. 2E). Interestingly, commencing administration of the anti-PD-L1 antibody one day earlier (day 0) decreased the number of activated T cells and inhibited hepatitis (data not shown), suggesting anti-PD-L1 interfered with T cell priming. Consistent with the failure of anti-PD-L1 to change the outcome of acute hepatitis, the phenotype of hepatocyte-activated T cells was not affected by anti-PD-L1 when treatment started at day 1, as all cells proliferated at the same rate, maintained low CD25 expression and did not produce IL-2 or IFN-γ (Supplementary Fig. 3). PD-1 expression was slightly decreased on hepatocyte-activated T cells isolated from the anti-PD-L1-treated mice in comparison to control mice, but its expression was high in both groups (Supplementary Fig. 3), providing further evidence that PD-1 expression was not sufficient to control the lack of effector function in these cells. Anti-PD-L1 treatment from day 1 post-transfer did not alter the naïve phenotype of donor T cells transferred into B10.BR mice (data not shown).

To further test whether PD-1/PD-L1 interactions prevented Bim-deficient Des cells from mediating hepatitis, Met-Kb mice into which Bim-deficient Des T cells had been adoptively transferred were treated with anti-PD-L1 from day 1. The phenotype of the donor T cells was not influenced by the expression of Bim or antibody treatment at day 2 post-transfer, as all Bim-deficient Des T cells transferred into B10.BR mice maintained a naïve CFSEhigh CD44low PD-1low CD25low phenotype, whereas all hepatocyte-activated Des T cells in Met-Kb recipients proliferated and expressed low levels of CD25 on their surface (Supplementary Fig. 4). Anti-PD-L1 treatment beginning at day 1 post-transfer did not decrease PD-1 expression on hepatocyte-activated Bim-deficient Des T cells (Supplementary Fig. 4). Blocking PD-1/PD-L1 interactions also induced a significant increase in donor Bim-deficient Des T cell numbers at day 15 in comparison to Met-Kb mice treated with control rat IgG (Fig. 2D), but did not interfere with the severity, kinetic, or the transient nature of acute hepatitis (Fig. 2E). In a similar manner to wild-type Des cells, a large proportion of these Bim-deficient Des T cells were found in the liver (Fig. 2D). Histological analysis of the liver correlated with T cell survival data, with large numbers of T cells detected in the portal and lobular regions of Met-Kb mice receiving Des Bim−/− T cells (data not shown). To test whether the function of Bim-deficient T cells could be restored at a late time point (when T cells were silenced), we commenced treatment of recipient mice with anti-PD-L1 or control rat IgG at day 20 post-transfer of Bim-deficient T cells, administering antibody every three days till day 45. Late treatment with anti-PD-L1 altered the cell survival and/or proliferation of donor T cells, such that greater numbers of Des Bim−/− T cells could be found in Met-Kb mice receiving anti-PD-L1 antibodies compared to those receiving control IgG (data not shown). However, anti-PD-L1 treatment of recipient mice did not enhance the CTL activity of donor Des Bim−/− cells at day 38 (Supplementary Fig. 5), and hepatitis was not triggered as assessed by serum ALT (data not shown).

Collectively, these results demonstrate that inhibition of PD-1/PD-L1 interactions promoted the proliferation and/or survival of self-reactive Bim-deficient T cells, suggesting that these two molecules increased T cell survival via distinct pathways. Most importantly, although activated Bim-deficient CD8 T cells accumulated and reached impressive numbers in the liver of mice treated with anti-PD-L1, their function was not restored, suggesting that, in a persisting high antigen load setting, anti-PD-L1 blockade was unable to overcome the robust silencing imposed on T cells within this organ, even when the lifespan of liver-reactive T cells was prolonged.

Discussion

By examining the ability of Tg Des T cells deficient for Bim or SOCS-1 to induce hepatitis and the role of PD-1/PD-L1 interactions in a well-characterized mouse model of acute immune-mediated hepatitis, this study demonstrated that the regulation of effector function was more critical than the regulation of the lifespan of effector CD8 T cells in limiting the tempo and degree of acute liver damage. However none of these factors were able to interfere with the dominant exhaustion process occurring in the liver in the presence of an ongoing high antigen load, and hence chronic hepatitis did not ensue.

Hepatocytes can be injured by many agents, including alcohol, toxins, chemicals and immune cells. Immune-mediated hepatocellular injury can be mediated by soluble factors such as inflammatory cytokines, including IFN-γ and TNF-α, that kill hepatocytes in a bystander manner [41], or by direct CTL killing [42]. CTLs kill their targets in an antigen-specific manner by delivering lethal granzymes to the target cells [43] and/or by Fas/FasL interactions [44]. Although the mediators of effector T cell-dependent liver damage have been well-characterized, the intrinsic T cell parameters that regulate CTL-mediated liver damage have not been previously investigated. This is an important unanswered question, as prolonged and uncontrolled and/or excessive CTL-mediated hepatocyte killing might lead to chronic or fulminant hepatitis, respectively. By using a Tg model of acute hepatitis mediated by a monoclonal population of CTLs of known specificity activated in LN, we manipulated intrinsic CD8 T cell factors that might regulate acute hepatitis, namely their susceptibility to apoptosis (via Bim), responsiveness to activation (via PD-1/PD-L1 interactions) and cytokine regulation/effector T cell function (via SOCS-1).

The transfer of CD8 Tg T cells deficient for the pro-apoptotic molecule Bim was clearly associated with prolonged survival of these liver-reactive T cells in recipient mice, suggesting that Bim was a critical regulator of T cells survival. The five fold lower intrahepatic numbers of Bim-deficient Tg T cells observed in Alb-Kb compared to Met-Kb mice suggested that the vast majority of T cells that accumulated in the liver of Met-Kb mice at 30 days post-transfer were originally activated in the LN. One of the remarkable findings of this study was that the striking accumulation of Bim-deficient Des T cells in the livers of Met-Kb mice was not sufficient to intensify or even prolong the severity and kinetic of acute hepatitis nor induce chronic liver damage. This observation extends the conclusions of our previous studies showing that Bim-deficient Tg T cells activated solely by intrahepatic bone-marrow derived cells were silenced after inducing acute liver damage [23]. These results also confirm previous reports that Bim regulates survival without altering the effector function and autoimmune potential of T cells [18].

Interestingly, inhibiting the binding of the inhibitory receptor PD-1 with its ligand PD-L1 early post-activation had a similar effect to Bim deficiency in hepatocyte-reactive CD8 T cells, resulting in increased accumulation of antigen-specific T cells in the liver without affecting the kinetic and severity of acute liver injury (Fig. 2D, E). This suggests that PD-1/PD-L1 interactions in the earlier phase post-activation are important in inhibiting T cell expansion and/or survival, rather than in dampening CD8 T cell effector function. This effect of PD-1/PD-L1 on survival seems to be distinct from the pro-apoptotic effect of Bim, as anti-PD-L1 treatment resulted in a pronounced accumulation of Bim-deficient T cells in the liver (Fig. 2D). Consistent with this, recent data suggests that PD-1 appears to promote T cell survival via induction of the transcription factor FOXO1, which regulates PD-1 expression in a positive feedback loop [45].

The more marked hepatitis observed following adoptive transfer of hepatocyte-reactive T cells deficient for SOCS-1, a molecule that regulates signaling of several cytokines binding to the common γ chain, including IL-2 [36], suggests that cytokine expression by T cells changed the effector function of T cells with an associated increase in the severity of hepatitis. The similar T cell numbers in the Met-Kb LN during the first days after transfer regardless of SOCS-1 expression (Fig. 4D) suggests that the effect of SOCS-1 on hepatitis did not occur during primary activation in LN. Instead, SOCS-1-deficient Tg T cells acquired enhanced function at day 5 after initial LN priming. This seemed to occur not only via increased expansion of CD8 T cells in the liver, probably due to their increased sensitivity to IL-2, but also by enhanced cytotoxic function. It is not clear whether this enhanced effector function was programmed during primary activation in LN or, alternatively, acquired upon secondary restimulation in the liver. However, the high expression of CD25 by SOCS-1-deficient, but not SOCS-1-sufficient, CD8 T cells in the Met-Kb recipient liver (Fig. 5B), suggests that the absence of SOCS-1 regulation extended cytokine expression in T cells and favored increased proliferation within the liver and enhanced effector function. This data is consistent with the known regulatory role of SOCS-1 [[36], [37]]. SOCS-1 gene transcription is induced in response to initial IL-2R/IL-2 binding that occurs just after T cell activation. SOCS-1 inhibits JAK/STAT signaling that lies downstream of the IL-2R [[31], [32], [33]], acting in a negative feedback loop to regulate the IL-2 signaling pathway [36]. SOCS-1 deficiency is thus thought to augment the “responsiveness” of T cells to cytokines at a later stage of the immune response when these molecules accumulate.

Although we were able to prolong effector function and alter the outcome of hepatitis by ablating SOCS-1 in donor T cells, liver damage was still controlled and did not evolve into chronic hepatitis in this model. SOCS-1 and SOCS-3 have been reported to have redundant inhibitory activities. Thus it is possible that other cytokine inhibitory mechanisms such as SOCS-3 compensate for SOCS-1 deficiency to restrict acute hepatitis. Regardless of cytokine regulation, most Tg SOCS-1−/− T cells were also deleted following early increases in survival and expansion, suggesting that like SOCS-1-sufficient Tg T cells, SOCS-1-deficient Tg T cells also died by Bim-dependent apoptosis. It would be interesting to know whether Tg T cells deficient for both SOCS-1 and Bim, and thus displaying enhanced survival as well as dysregulated effector function, would induce fulminant hepatitis or become exhausted following transfer into Met-Kb mice. Unfortunately SOCS-1−/− Bim−/− Des TCR Tg mice could not be bred despite our several attempts to generate this line.

The exact mechanisms responsible for T cell silencing after acute hepatitis in these different models remain unclear. However, our recent study showed that CTLs become exhausted in the presence of a persisting high intrahepatic antigen load [14]. Together with the results from this study showing that Bim- and SOCS-1-deficient T cells express PD-1 and Tim-3 and become functionally exhausted, it would appear that T cell exhaustion occurs in the face of ongoing high levels of intrahepatic antigen expression, regardless of enhanced T cell function or resistance to apoptosis. Based on these findings, we predict that strategies that tend to increase the survival of antigen-specific T cells would fail to interfere with robust T cell exhaustion mechanisms that occur in the liver in this setting.

This data is highly relevant for therapies that attempt to restore T cell effector function in patients chronically infected with HBV or HCV. It is thought that in these clinical settings, persisting high antigen load might play an important role in evolution towards chronic infection by promoting T cell exhaustion; similar mechanisms may also play a role in promoting resistance of hepatocellular carcinoma to tumor-specific CTL responses [46]. The relatively limited efficacy of PD-1 inhibition in HCV infection in vivo [[25], [47]], suggests that other parameters need to be taken into consideration. The compensatory roles of other inhibitory molecules, including Tim-3, TGIT, and CTLA-4, have been proposed as key to understand these observations [48]. An alternative, non-exclusive possibility suggested by our findings is that T cell responses are difficult to restore or enhance in the presence of high antigen load (e.g. large tumor burden in HCC or high levels of intrahepatic antigen expression in HBV and HCV). This study and previous results [14] would predict that inhibition of PD-1/PD-L1 interactions would be most effective in combination with other treatments that interfere with T cell exhaustion, or in the presence of lower antigen loads, such as lowered tumor burden following resection in HCC or in combination with antiviral therapy to lower viral titers in chronic HBV and HCV.

In conclusion, this study demonstrates that a highly efficient population of CTLs that secrete hepatotoxic cytokines is critical to potentiate severe acute hepatitis. Although promoting survival of CD8 T cells enhanced their clonal expansion and accumulation in the liver, it was not sufficient to amplify liver damage or mediate chronic hepatitis. This would suggest that once activated, in the presence of high levels of hepatocyte-expressed antigen, CD8 T cells have a limited window of opportunity during which effector function is mediated, regardless of their lifespan. Past this phase, if antigen is not cleared in the liver, effector CD8 T cells become exhausted. This pathway might have been selected during evolution to avoid irreversible damage to the liver and prevent death by fulminant hepatitis. The adverse effect of this protection mechanism is, however, the persistence of viruses (e.g. HBV and HCV) that chronically infect the host by promoting silencing of the antiviral T cell response.

Collectively, these results assist in our understanding of why T cells become exhausted after acute hepatitis in the presence of ongoing high-level antigen expression, and why the extent of viral antigen-specific T cell infiltrates do not always correlate with ALT levels in patients chronically infected with HBV and HCV [49]. This current study predicts that antiviral strategies that boost the priming or effector function of CTLs would be more effective than those that increase CTL survival. The combination of treatments that increase CD8 T cell survival and function and interfere with T cell exhaustion might also improve the efficacy of these therapies.

StephenW

效应T细胞功能，而不是生存确定小鼠肝炎的程度和持续时间
肝病2016年6月的JNL
米歇尔Vo1,2，劳伦E. Holz1,2,3，奕淳Wong1,2，基兰English1,2，沃尔克Benseler1,2,4，克莱尔McGuffog1,2，美雪Azuma5，杰弗里W. McCaughan2,6，大卫G. Bowen1,2，⇑，Y，帕特里克Bertolino1,2，⇑，Y
1Liver免疫程序，百年学院，新城，新南威尔士，澳大利亚; 2AW莫罗胃肠病学和肝病中心，
皇家阿尔弗雷德王子医院新镇，新南威尔士，及医药，悉尼，澳大利亚新南威尔士州大学法律系; 3Current地址：系
微生物学和免疫学，彼得·多尔蒂研究所，墨尔本，帕克维尔，维多利亚，澳大利亚的大学; 4Current地址：
外科，雷根斯堡，巴伐利亚，德国的大学;分子免疫学研究生院，东京医科的5Department
齿科大学，汤岛，日本东京; 6Liver损伤和癌症项目，百周年纪念研究所，新城，新南威尔士，澳大利亚
见编辑，网页1208-1210

背景和目的

急性肝炎通常是由细胞毒性T淋巴细胞（CTL）介导的;然而，该限制的CTL介导的肝损伤的特性参数不很好地理解。
方法

调查急性肝损伤是否是由降低的CTL的寿命或效应子功能的分子的限制，我们使用了充分表征的转基因（Tg）的小鼠中的急性肝损伤的发展对T细胞受体的转移（TCR）的Tg的CD8 T模型细胞。收件人Tg小鼠接受供体的TCR Tg的T细胞缺陷的任一促凋亡分子的Bim，其中规定的CTL存活，或细胞因子信号-1（SOCS-1）中，抑制用于控制共同γ链的细胞因子的表达;抗PD-L1的中和抗体的作用也进行了评估。
结果

Bim的缺陷供体的T细胞和/或PD-L1的封锁的使用增加肝内T细胞的数目，而不会影响急性肝炎的程度和动力学。相比之下，SOCS-1缺陷的T细胞诱导引起的细胞毒性增强的功能和增加扩张加剧，长时间急性肝炎。虽然他们造成更严重的急性肝损伤，SOCS-1缺陷的T细胞从未沉淀慢性肝炎和人困马乏。
结论

急性肝炎的程度由CD8 T细胞的功能调节，但并不受CTL寿命的变化。尽管被检查的参数的操作的影响急性肝炎，迁延性肝炎没有随之而来，这表明，在高肝内抗原负载的存在，在这些因素中隔离的变化不足以阻止T细胞衰竭和调解进展为慢性肝炎。

介绍

急性肝损伤有多种原因，但往往是由嗜肝病毒等病原体瞄准了这个器官引发的免疫反应启动。同时涉及先天和适应性免疫的鲁棒和持续的免疫应答是关键的，以清除病毒靶向肝脏，感染如乙型肝炎（HBV）[1]和C病毒（HCV）的[2]。抗病毒细胞毒性CD8 + T淋巴细胞（CTL）发挥感染期间介导的肝损伤的关键作用，并诱导和广泛剧烈病毒特异性CD8 T细胞应答的维护是丙型肝炎病毒感染的自发分辨率的阳性预测值[3]，[ 4]，[5]。在HCV感染的黑猩猩模型的CD8 T细胞的抗体依赖性耗尽导致延长的病毒感染[6]。 CTL的在外围的检测与提高丙氨酸转氨酶（ALT）水平一致和临床表现为急性肝炎[7]。丙型肝炎病毒的清除率是有症状患者[5]更常见，它是可能的肝损伤的过程中感染的急性期随之而来的程度是影响感染[8]的结果是至关重要的。

虽然已知的CTL杀死通过粒酶和/或中Fas / FasL的相互作用（古典CTL杀伤途径）的靶细胞，或通过肝细胞因子（旁观者杀伤），分子过程限制性CTL的操作和负责终止急性的分泌由CD8 T细胞介导的​​肝损伤没有得到很好的理解。它阐明这些过程以导出策略，以增加清除持久嗜肝感染的机会，并且还以防止过多持续或剧烈的CD8 T细胞应答可能导致显著纤维化和肝硬化的最终发展，或在更严重的设置是很重要的，急性肝功能衰竭的发展。内在T细胞数调节过程可能是限制性的肝损伤，包括重要的：抑制性受体如PD-1，添-3，LAG3，和CTLA-4的诱导; CTL效应功能（细胞因子和效应分子的下调）的监管;而CTL的凋亡。其它参数，例如CD8 + T细胞的前体的特异于病毒的数量[[9]，[10]，所述的TCR的亲和力识别病毒表位[11]，的CD4 T细胞帮助的可用性[12]，[ 13]，并表达抗原[14肝细胞的数目]也已经显示影响长期的CD8 T细胞的结果。

为了鉴定T细胞固有参数控制程度和急性肝损伤动力学，我们使用了充分表征的Met-KB转基因（Tg）的小鼠免疫介导的肝炎的模型，其中急性肝损伤是由T细胞受体的Tg诱导即首先在淋巴结活化的CD8 T细胞（LN），并随后认识到在肝脏其关联抗原，导致肝细胞损伤[15]。

通过转移供体的Tg的T细胞缺陷的控制的T细胞死亡（BIM）的基因或调节细胞因子效应T细胞功能的关键的信号传导（细胞因子信号-1的抑制; SOCS-1），或通过用抗PD处理的受体小鼠-L1阻断抗体，我们研究在限制肝损伤的程度和持续时间T的细胞死亡，CTL功能的调节和T细胞抑制分子的调控中的作用，独立的TCR的亲和力，T细胞的帮助和抗原剂量。

我们的研究结果表明，增强T细胞的生存战略促进CD8 T细胞识别在肝脏肝细胞表达的抗原的积累，而不改变的严重性或肝损害的速度。与此相反，扩增延长急性肝炎的效应T细胞的功能，并增加肝损伤的严重性。虽然这些策略对命运和/或效应的CD8 T细胞的功能有不同的影响，但没有防止在肝脏内的T细胞的功能的用尽的最终发展有关的急性肝炎的分辨率。

在高水平的持久性抗原表达在肝设置，程度和肝损伤的持续时间因此主​​要由基因控制效应细胞，而不是那些影响T细胞寿命的作用调节。肝功能损害的持久性最终是由功能性T细胞耗竭的限制。这些数据表明，为了有效，旨在提高在肝病患者的CTL的数目免疫疗法将需要与增强T细胞的效应子功能，并且更重要的是，干扰T细胞耗竭策略组合。
摘录

阻断PD-1 / PD-L1的相互作用增加T细胞的数量没有充实的急性肝损伤的严重程度或导致慢性肝炎

PD-1 / PD-L1的封锁目前在临床中使用，以提高耗尽的CD8 T细胞的功能的范围内的实体瘤，已探索在慢性HCV感染[25]，并且正在研究用于治疗肝癌的（HCC）[26]。因此，重要的，以测试在我们的模型该分子的作用。

PD-1以相似的低水平表达于施主德RAG - / - 活化后的24小时中的Met-KB的小鼠的肝和LN T细胞，但在转移到B10.BR小鼠T细胞上的任何阶段不表达（图2A）。在48小时活化后，肝脏活化的CD8 T细胞表达更多的PD-1相比LN-活化的T细胞（P <0.05）（图2A，B）。上liver-和LN活化CD8 T细胞不到一天后进行检测的表达水平相似，可能是由于肝脏活化的T细胞和LN-活化T细胞的再循环的死亡至肝脏[18]，[27 ]。随着时间的推移，所有辅RAG - / - 中的Met-KB小鼠活化的T细胞变成PD-1high和表达倾向于随时间（图2B），以增加与在不同的模式时，报告肝内CD8 T细胞的表型相一致的结果抗原仍然存在[28]，[29]。 PD-L1的，主要PD-1配体，但不在B10.BR小鼠也有人在的Met-KB的小鼠的肝实质（图2C）非常低的水平（数据未示出）在第15天后T细胞转移。有趣的是，PD-L1的表达与浸润细胞的数目相关，并在相关联与Des的Bim的更显着浸润增加 - / - T细胞。在这些肝，PD-L1的表达在浸润辅Bim的附近被发现 - / - T细胞中的小叶和也在门区（图2C）。

为了检验这种可能性的PD-1 / PD-L1的相互作用有助于耐受在肝脏活化的T细胞的诱导和/或对急性肝炎，蛋氨酸KB和B10.BR小鼠的衰减，成野生型辅T细胞被转移，用中和抗PD-L1或对照抗体（大鼠IgG）处理。的Met-KB小鼠抗PD-L1的处理，每三天，从T细胞的过继转移后一天开始，导致供体辅T细胞在第15天肝脏数量增加，相比于对照大鼠IgG介处理的小鼠（图2D），表明PD-L1的封锁升高的T细胞增殖和/或存活。尽管在肝中德T细胞后代的积累，用抗PD-L1的治疗并没有对血清ALT水平（图2E）一个显著效果。有趣的是，早一天（第0天）的抗PD-L1抗体的开始施用减少活化的T细胞和抑制肝炎（数据未示出）的数量，这表明抗PD-L1干扰T细胞引发。用抗PD-L1的失败来改变急性肝炎的结果一致，肝细胞激活的T细胞的表型没有影响由抗PD-L1时开始治疗在第1天，因为所有的细胞以相同的速率增殖，维持低CD25的表达，并没有产生IL-2或IFN-γ（补充图3）。 PD-1的表达稍微从抗PD-L1处理的小鼠相比孤立对照小鼠肝细胞激活的T细胞减少，但在这两个群体中的表达较高（补充图3），提供了进一步的证据PD -1表达不足以控制缺乏在这些细胞的效应子功能的。从1日转移后抗PD-L1治疗不改变转入小鼠B10.BR供体T细胞的表型天真（数据未显示）。

为了进一步测试的PD-1 / PD-L1的相互作用是否阻止Bim的缺陷辅细胞介导肝炎，蛋氨酸 - KB小鼠成Bim的缺陷辅T细胞已被过继转移用抗PD-L1的从第1天处理。供体T细胞的表型没有被Bim的或抗体的治疗在后第2天转移的表达的影响，如转移到B10.BR小鼠所有的Bim缺陷辅T细胞保持了幼稚CFSEhigh CD44low PD-;低CD25low表型，而所有肝细胞活化辅T细胞的Met-KB收件人增殖和它们的表面（补充图4）上表达的CD25的低的水平。抗PD-L1治疗在一天后1传输开始没有肝细胞活化的Bim缺陷辅T细胞降低PD-1的表达（附图4）。阻断在第15天的PD-1 / PD-L1的相互作用也诱导供体的显著增加Bim的缺陷辅T细胞数目相比，与对照大鼠IgG（图2D）处理的Met-KB的小鼠，但不与所述干扰严重性，动力学，或急性肝炎（图2E）的瞬时性质。以类似的方式，以野生型辅细胞，这些Bim的缺陷辅T细胞的相当大的比例在肝脏（图2D）被发现。 （数据未显示）T细胞 - 与T细胞的存活数据相关的肝脏组织学分析，具有大量对收到德的Bim的Met-KB小鼠的门静脉和小叶区域检测T细胞 - /。测试是否Bim的缺陷的T细胞的功能，可以在较晚的时间点恢复（当T细胞被压制）中，我们在第20天转移后开始治疗用抗PD-L1或控制大鼠IgG受体小鼠的Bim的缺陷的T细胞，施用抗体每三天直到一天45.用抗PD-L1的晚期治疗改变的供体T细胞的细胞存活和/或增殖，以使得辅Bim的更大数量的 - / - T细胞可以是在接收相比那些接受对照IgG抗PD-L1抗体的Met-KB的小鼠中发现（数据未显示）。然而，抗PD-L1处理的受体小鼠的没有加强施主德的Bim的CTL活性 - / - 细胞在38天（。补充图5），并通过血清ALT的评估没有触发肝炎（数据未示出） 。

总的来说，这些结果表明，抑制PD-1 / PD-L1的相互作用促进自反应Bim的缺陷的T细胞的增殖和/或存活，这表明这两种分子经由不同的途径升高的T细胞的存活。最重要的是，虽然活化的Bim缺陷CD8 T细胞积累，达到在用抗PD-L1的处理的小鼠的肝可观的数字，它们的功能没有恢复，这表明，在一个持续的高抗原负载设置，抗PD- L1封锁无法克服该器官内施加于T细胞的稳健的沉默，即使当肝脏反应性T细胞的寿命延长。

讨论

通过检查的Tg辅T细胞缺陷的Bim的或SOCS-1来诱导肝炎的能力和PD-1 / PD-L1的相互作用的急性免疫介导的肝炎的充分表征的小鼠模型中的作用，本研究证明了效应器功能的调节比的效应的CD8 T细胞的寿命的限制速度和急性肝损伤程度的调节更加关键。但是，没有这些因素是能够与主导用尽过程发生在肝脏中的持续的高抗原负载的存在干涉，并且因此慢性肝炎没有随之发生。

肝细胞可通过众多代理商，包括酒精，毒素，化学物质和免疫细胞受伤。免疫介导的肝细胞损伤可通过可溶性因子介导的，如炎性细胞因子，包括IFN-γ和TNF-α，即杀死旁观者方式[41]肝细胞，或通过直接CTL杀伤[42]。的CTL通过提供致命的颗粒酶为靶细胞杀死他们的抗原特异性方式的目标[43]和/或通过Fas / FasL相互作用[44]。虽然效应T细胞依赖性的肝损伤的介质已被充分表征，调节的CTL介导的肝损伤的内在T细胞参数以前没有研究。这是一个重要的悬而未决的问题，如延长和不受控制的和/或过度的CTL介导的肝细胞杀伤可能分别导致慢性或暴发性肝炎。通过使用由LN激活称为特异性CTL的单克隆群介导的急性肝炎小鼠模型中，我们操纵的可能调节急性肝炎，也就是他们的易感性凋亡（通过BIM）内在的CD8 T细胞因子，快速响应，以激活（通过PD-1 / PD-L1的相互作用）和细胞因子调节/效应T细胞功能（通过SOCS-1）。

Tg的CD8 T细胞缺陷的促凋亡分子的BIM显然与受体小鼠肝脏，这些反应性T细胞的生存期延长相关，这表明Bim的是T细胞生存的关键调节器的传输。在ALB-KB相比，气象-KB的小鼠中观察到的BIM缺陷的Tg T细胞的五倍降低肝内的数字表明，绝大多数T细胞是在30天传递后在会见-KB的小鼠肝脏积累最初在LN激活。一本研究的显着结果是，Bim的缺陷辅T细胞的Met-KB小鼠肝脏的击打积累不足以强化甚至延长急性肝炎的严重程度和动力学也不诱发慢性肝损伤。这个观察拓展了我们以前的研究显示，BIM的缺陷的Tg T细胞肝内骨髓来源的细胞激活​​单独的结论诱发急性肝损伤[23]后，被压制。这些结果也证实了以前的报告，Bim的调节存活而不改变效应子功能和T细胞的自身免疫潜力[18]。

有趣的是，抑制抑制性受体的结合PD-1与其配体PD-L1的早期活化后有在肝细胞反应性CD8 T细胞的Bim缺乏类似的效果，从而导致在肝脏抗原特异性T细胞的积累增加在不影响急性肝损伤的动能和严重程度（图2D，E）。这表明，在早期阶段活化后的PD-1 / PD-L1的相互作用，而不是在润湿的CD8 T细胞的效应子功能在抑制T细胞扩增和/或存活很重要的。 PD-1 / PD-L1的上存活的这种作用似乎是从的Bim的促凋亡效果显着，如抗PD-L1处理导致的Bim缺陷的T细胞在肝脏中有显着的积累（图2D ）。与此相一致，最近的数据显示，PD-1的出现通过转录因子FOXO1，其中规定在正反馈回路[45]的PD-1表达的诱导来促进T细胞的存活。

越标记肝炎后观察到缺陷的SOCS-1肝细胞反应性T细胞的过继转移，分子，调节多种细胞因子结合到共同的γ链，包括IL-2 [36]的信号，表明以T细胞的细胞因子表达与肝炎的严重程度相关联的增加改变了的T细胞的效应子功能。在的Met-KB LN期间转移后第一天，无论SOCS-1的表达（图4D）的类似的T细胞数表明，在LN初级活化期间未发生SOCS-1对肝炎的效果。相反，SOCS-1缺失的Tg的T细胞获得的增强功能在初次启动LN后第5的一天。这似乎发生不仅通过在肝脏的CD8 T细胞的扩增的增加，可能是由于其对IL-2增加的灵敏度，而且还通过增强细胞毒性功能。目前尚不清楚在LN中，或者，当在肝脏二次再刺激获得初级激活期间这种增强的效应子功能是否被编程。然而，通过高表达的CD25的SOCS-1缺失，但不SOCS-1-足够，CD8 T细胞中的Met-KB收件人肝（图5B），表明不存在SOCS-1调节的扩展因子的表达在T细胞和赞成增加的增殖肝内和增强效应子功能。这个数据是与SOCS-1的已知的调节作用[36]，[37]一致。 SOCS-1基因的转录是响应于初始的IL-2R / IL-2的结合，仅仅T细胞活化后发生诱导。 SOCS-1抑制了位于IL-2R的下游JAK / STAT信号[31]，[32]，[33]，作用在一个负反馈环路调节的IL-2信号传导途径[36]。由此SOCS-1缺乏被认为是在免疫反应的后一阶段，以增加T细胞的“响应”于细胞因子时，这些分子蓄积。

虽然我们能够延长效应功能和供体T细胞消融SOCS-1改变肝炎的结果，肝功能损害仍然受控，并没有演变成在这个模型慢性肝炎。 SOCS-1和SOCS-3已被报道具有冗余抑制活性。因此可能的是其它细胞因子抑制机制，例如SOCS-3补偿SOCS-1缺乏限制急性肝炎。不管细胞因子调节，大多数的Tg SOCS-1 - / - T细胞也会被删除，按照生存和扩张早期的增加，这表明像SOCS-1-足够的Tg T细胞，SOCS-1缺失的Tg的T细胞也Bim的死亡依赖的细胞凋亡。这将是有趣的，知道的Tg T细胞缺陷为SOCS-1和Bim，从而提高显示生存以及失调效应功能，是否会诱发暴发性肝炎或成为耗尽之后转入遇见-KB的小鼠。不幸的是SOCS-1 - / - 的BIM - / - 德TCR Tg小鼠无法尽管我们多次尝试生成此行繁殖。

负责T细胞沉默在这些不同型号急性肝炎后的确切机制尚不清楚。然而，我们最近的研究表明，CTL的成为一个持续高肝内抗原负载[14]的存在耗尽。连同从这项研究显示，Bim-和SOCS-1缺失的T细胞表达的PD-1和Tim-3和成为功能上耗尽的结果，它会出现T细胞衰竭在正在进行的高水平肝内抗原的面发生表达，无论增强T细胞功能或抵抗凋亡。基于这些发现，我们预测，往往会增加抗原特异性T细胞的生存策略会失败发生在此设置肝脏强健的T细胞耗竭机制干涉。

此数据对于试图恢复慢性乙型肝炎病毒感染或丙型肝炎患者T细胞的效应功能治疗密切相关。据认为，在这些临床设置，持续高抗原负载可能通过促进T细胞耗竭起到朝慢性感染发展的重要作用;类似的机制也可以在促进肿瘤特异性CTL应答[46]肝细胞癌的抗性中发挥作用。 PD-1抑制HCV感染体内的相对有限的功效[25]，[47]，表明其它参数需要被考虑在内。其他抑制性分子，包括添-3，TGIT，和CTLA-4的代偿作用已被建议作为关键来了解这些观察[48]。由我们的发现表明一个替代方案中，非排他的可能性是，T细胞反应是很难恢复或在高抗原负载（例如肿瘤负荷大肝癌或高水平肝内抗原表达乙型肝炎病毒和丙型肝炎病毒）的存在下增强。该研究和以前的结果[14]。将预测的PD-1 / PD-L1的相互作用的抑制是最有效的在有与T细胞衰竭干扰，或在更低的抗原负载的存在的其他治疗方法，如降低肿瘤组合负担以下切除肝癌或与抗病毒治疗的慢性乙肝和丙肝降低病毒滴度组合。

总之，这项研究表明，分泌细胞因子肝毒性CTL的高效率的人口能够增强重症急性肝炎的关键。虽然CD8 T细胞的促存活增强在肝脏的克隆扩增和积累，这是不足够的扩增肝损伤或介导慢性肝炎。这表明，一旦被激活，在高水平的肝细胞表达的抗原的存在，CD8 T细胞具有其间效应子功能是介导的，而不管他们的寿命的机会有限窗口。过去这一阶段，如果抗原未在肝脏清除，效应CD8 T细胞耗尽。此途径可能在进化过程中已被选择，以避免对肝不可逆转的损害和防止死亡由暴发性肝炎。这种保护机制的不利影响是，然而，病毒（例如HBV和HCV），该促进抗病毒的T细胞应答的沉默慢性感染宿主的持久性。

总的来说，这些结果有助于提供了为什么在正在进行的高水平抗原表达的存在急性肝炎之后T细胞耗尽，以及为什么病毒抗原特异性T细胞浸润的程度并不总是与ALT水平的病人长期关联理解感染HBV和HCV [49]。目前的研究预测，即促进CTL的启动或效应功能抗病毒策略会比那些增加CTL生存更有效。该增加CD8 T细胞的存活和功能，并与T细胞耗竭干扰的治疗方法的组合也可能会提高这些治疗的功效。