Lauren E. Holz

The site of primary T cell activation is a determinant of the balance between intrahepatic tolerance and immunity

Hepatic immunobiology is paradoxical: although the liver possesses unusual tolerogenic properties, it is also the site of effective immune responses against multiple pathogens and subject to immune-mediated pathology. The mechanisms underlying this dichotomy remain unclear. Following previous work demonstrating that the liver may act as a site of primary T cell activation, we demonstrate here that the balance between immunity and tolerance in this organ is established by competition for primary activation of CD8+ T cells between the liver and secondary lymphoid tissues, with the immune outcome determined by the initial site of activation. Using a transgenic mouse model in which antigen is expressed within both liver and lymph nodes, we show that while naive CD8+ T cells activated within the lymph nodes were capable of mediating hepatitis, cells undergoing primary activation within the liver exhibited defective cytotoxic function and shortened half-life and did not mediate hepatocellular injury. The implications of these novel findings may pertain not only to the normal maintenance of peripheral tolerance, but also to hepatic allograft tolerance and the immunopathogenesis of chronic viral hepatitis.

Intrahepatic Murine CD8 T-Cell Activation Associates With a Distinct Phenotype Leading to Bim-Dependent Death

BACKGROUND & AIMS Chronic infections by hepatotropic viruses such as hepatitis B and C are generally associated with an impaired CD8 T-cell immune response that is unable to clear the virus. The liver is increasingly recognized as an alternative site in which primary activation of CD8 T cells takes place, a property that might explain its role in inducing tolerance. However, the molecular mechanism by which intrahepatically activated T cells become tolerant is unknown. Here, we investigated the phenotype and fate of naïve CD8 T cells activated by hepatocytes in vivo. METHODS Transgenic mouse models in which the antigen is expressed in lymph nodes and/or in the liver were adoptively transferred with naïve CD8 T cells specific for the hepatic antigen. RESULTS Liver-activated CD8 T cells displayed poor effector functions and a unique CD25(low) CD54(low) phenotype. This phenotype was associated with increased expression of the proapoptotic protein Bim and caspase-3, demonstrating that these cells are programmed to die following intrahepatic activation. Importantly, we show that T cells deficient for Bim survived following intrahepatic activation. CONCLUSIONS This study identifies Bim for the first time as a critical initiator of T-cell death in the liver. Thus, strategies inhibiting the up-regulation of this molecule could potentially be used to rescue CD8 T cells, clear the virus, and reverse the outcome of viral chronic infections affecting the liver.

Hepatocyte entry leads to degradation of autoreactive CD8 T cells

Although most self-reactive T cells are eliminated in the thymus, mechanisms to inactivate or control T cells specific for extrathymic antigens are required and exist in the periphery. By investigating the site in which autoreactive T cells are tolerized, we identify a unique mechanism of peripheral deletion in which naïve autoreactive CD8 T cells are rapidly eliminated in the liver after intrahepatic activation. T cells actively invade hepatocytes, enter endosomal/lysosomal compartments, and are degraded. Blockade of this process leads to accumulation of autoreactive CD8 T cells in the liver and breach of tolerance, with the development of autoimmune hepatitis. Cell into cell invasion, or emperipolesis, is a long-observed phenomenon for which a physiological role has not been previously demonstrated. We propose that this “suicidal emperipolesis” is a unique mechanism of autoreactive T-cell deletion, a process critical for the maintenance of tolerance.

Early intrahepatic antigen‐specific retention of naïve CD8+ T cells is predominantly ICAM‐1/LFA‐1 dependent in mice

We have previously shown that naïve CD8+ T cells recognizing their cognate antigen within the liver are retained and undergo activation in situ, independent from lymphoid tissues. Intrahepatic primary T cell activation results in apoptosis and may play a crucial role in the ability of the liver to induce tolerance. Although adhesion molecules required for intrahepatic retention of T cells that have undergone previous extra‐hepatic activation have been characterized, adhesive interactions involved in selective antigen‐dependent intrahepatic retention of naïve CD8+ T cells have not been investigated. By adoptively transferring radiolabeled T cell receptor (TCR)‐transgenic CD8+ T cells into recipient animals ubiquitously expressing the relevant antigen, we show that 40% to 60 % of donor antigen–specific naïve CD8+ T cells were retained in the liver within 1 hour after transfer, despite ubiquitous expression of the antigen. Intravital microscopy showed that most donor naïve T cells slowed down and were irreversibly retained intrahepatically within the first few minutes after adoptive transfer, strongly suggesting that they were directly activated by liver cells in situ. This process was largely dependent on LFA‐1 and ICAM‐1, but was independent of blocking with antibodies against VCAM‐1, α4 integrin, P‐selectin, VAP‐1, and β1 integrin. ICAM‐2 seemed to play only a minor role in this process. Interestingly, LFA‐1 expressed by both donor T cells and liver cells was involved in retention of the antigen‐reactive T cells. In conclusion, LFA‐1–dependent intrahepatic T cell retention and activation are linked events that may play a crucial role in the establishment of liver‐induced antigen‐specific tolerance. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:1063–1071.)

Invading macrophages play a major role in the liver progenitor cell response to chronic liver injury

BACKGROUND & AIMS Although a strong association between liver progenitor cells (LPCs) and inflammation exists in many chronic liver diseases, the exact role of the immune system in LPC-mediated hepatic regeneration remains unclear. A number of pro-inflammatory factors were identified in cytokine knockout mice in which the LPC response was attenuated but neither the mechanism nor the producing cells are known. METHODS To identify the critical immune cells and cytokines required in the LPC response, we compared two diet-induced models of liver injury with two recently established transgenic models of immune-mediated hepatitis. RESULTS Despite severe inflammation being observed in all models, the generation of LPCs was highly dependent on the cause and kinetics of liver damage. The LPC response was associated with an increase of macrophages and CD8(+) T cells but not natural killer cells. T cell-deficient mice were able to mount a LPC response, albeit delayed, suggesting that T cells are not essential. Mice mounting an LPC response showed elevated numbers of Kupffer cells and invading CX(3)CR1(high)CCR2(high) macrophages secreting persistent high levels of tumour necrosis factor alpha (TNFalpha), a major cytokine involved in the LPC response. CONCLUSIONS Liver macrophages are an important determinant of LPC expansion during liver regeneration in models of diet- and immune-mediated liver injury. Invading macrophages in particular provide pro-mitogenic cytokines such as TNFalpha that underpin the process. LPC themselves are a source of chemokines (CCL2, CX(3)CL1) that attract infiltrating macrophages.

CD8!T cell tolerance following antigen recognition on hepatocytes

Hepatocytes, the predominant cell type in the liver, are the main cell infected by the hepatitis C virus (HCV) and represent important targets for immune therapy. Although early studies suggested that this parenchymal cell expresses low levels of class I MHC molecules, hepatocytes are emerging as important players in intrahepatic immune responses. Not only do they express high levels of molecules important in antigen presentation, but their expression of these molecules in vivo is also polarized towards the lumen of the sinusoids, thus maximising the efficiency of T cell activation. Electron micrographs indicate that interactions between T cells and hepatocytes occur in vivo via fenestrations in the sinusoidal endothelial layer. In this article, we will review the data showing that hepatocytes function as antigen presenting cells in vivo, and explore the fate of T cells activated by this cell type. We propose that primary activation of naïve CD8+ T cells by hepatocytes is a critical event occurring during the very early stages of a HCV infection, that contributes to progression to viral persistence via the removal of virus-specific T cells from the T cell repertoire.

Mechanisms of T Cell Death in the Liver: To Bim or Not to Bim?

Despite being a non-lymphoid organ, the liver displays immunological properties distinct from other solid organs and is associated with the induction of T cell tolerance. This property has been demonstrated in several clinical settings including transplantation and hepatotropic viral infections, such as those induced by hepatitis B and C viruses. Many models have been proposed to explain the ‘liver tolerance effect’, but the molecular and cellular mechanism(s) mediating this phenomenon remain unknown. Using transgenic mouse models, we have previously shown that the liver is the only non-lymphoid organ able to retain and activate naïve CD8+ T cells independently of lymphoid tissues in an antigen-specific manner. These findings, confirmed by other groups, have opened new possibilities to explain the remarkable capacity of the liver to induce antigen-specific tolerance in transplantation and following infection by hepatotropic viruses, such as the hepatitis C and B viruses. In our models, T cells activated by hepatocytes that proliferate die by neglect in a Bim-dependent manner. This paper will thus review the evidence showing Bim playing a critical role following intrahepatic primary T cell activation.

A Liver Capsular Network of Monocyte-Derived Macrophages Restricts Hepatic Dissemination of Intraperitoneal Bacteria by Neutrophil Recruitment

&NA; The liver is positioned at the interface between two routes traversed by pathogens in disseminating infection. Whereas blood‐borne pathogens are efficiently cleared in hepatic sinusoids by Kupffer cells (KCs), it is unknown how the liver prevents dissemination of peritoneal pathogens accessing its outer membrane. We report here that the hepatic capsule harbors a contiguous cellular network of liver‐resident macrophages phenotypically distinct from KCs. These liver capsular macrophages (LCMs) were replenished in the steady state from blood monocytes, unlike KCs that are embryonically derived and self‐renewing. LCM numbers increased after weaning in a microbiota‐dependent process. LCMs sensed peritoneal bacteria and promoted neutrophil recruitment to the capsule, and their specific ablation resulted in decreased neutrophil recruitment and increased intrahepatic bacterial burden. Thus, the liver contains two separate and non‐overlapping niches occupied by distinct resident macrophage populations mediating immunosurveillance at these two pathogen entry points to the liver. Graphical Abstract Figure. No caption available. HighlightsA distinct subset of resident macrophages (LCMs) occupies the hepatic capsuleLCMs are replenished from blood monocytes in the steady stateLCMs recruit neutrophils in response to bacteria reaching the liver capsuleLCM depletion decreases neutrophil recruitment and increases liver pathogen load &NA; The hepatic sinusoids harbor a well‐characterized resident macrophage population, Kupffer cells. Sierro et al. report an additional liver‐resident macrophage population occupying the hepatic capsule, phenotypically and developmentally distinct from Kupffer cells, which plays a role in immunosurveillance by sensing peritoneal pathogens and recruiting neutrophils to control intrahepatic bacterial dissemination.

Naïve CD8 T cell activation by liver bone marrow-derived cells leads to a “neglected” IL-2low Bimhigh phenotype, poor CTL function and cell death

BACKGROUND & AIMS The occurrence of primary CD8 T cell activation within the liver, unique among the non-lymphoid organs, is now well accepted. However, the outcome of intrahepatic T cell activation remains controversial. We have previously reported that activation initiated by hepatocytes results in a tolerogenic phenotype characterized by low expression of CD25 and IL-2, poor cytotoxic T lymphocyte (CTL) function, and excessive expression of the pro-apoptotic protein Bim. METHODS To investigate whether this phenotype was due to activation in the absence of co-stimulation, we generated bone marrow (bm) radiation chimeras in which adoptively transferred naïve transgenic CD8 T cells were activated in the presence of co-stimulation by liver bm-derived cells. RESULTS Despite expressing pro-inflammatory cytokines, high levels of CD25 and CD54, donor T cells activated by liver bm-derived cells did not produce detectable IL-2 and displayed poor CTL function, suggesting incomplete acquisition of effector function. Simultaneously, these cells expressed high levels of Bim and died by neglect. Transfer of Bim-deficient T cells resulted in increased T cell numbers. CONCLUSIONS These results imply that expression of CD25 and CD54 is co-stimulation dependent and distinguishes T cell activated by hepatocytes and liver bm-derived cells. In contrast, low expression of IL-2, poor CTL function and excess Bim production represent a more universal phenotype defining T cells undergoing primary activation by both types of hepatic antigen presenting cells (APC). These results have important implications for transplantation, in which all liver antigen presenting cells contribute to activation of T cells specific for the allograft.

Protective immunity to liver-stage malaria

Despite decades of research and recent clinical trials, an efficacious long‐lasting preventative vaccine for malaria remains elusive. This parasite infects mammals via mosquito bites, progressing through several stages including the relatively short asymptomatic liver stage followed by the more persistent cyclic blood stage, the latter of which is responsible for all disease symptoms. As the liver acts as a bottleneck to blood‐stage infection, it represents a potential site for parasite and disease control. In this review, we discuss immunity to liver‐stage malaria. It is hoped that the knowledge gained from animal models of malaria immunity will translate into a more powerful and effective vaccine to reduce this global health problem.