Claire McGuffog

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.

Antigen expression level threshold tunes the fate of CD8 T cells during primary hepatic immune responses

Significance The liver possesses unique immunological properties, with the capability of inducing tolerance upon transplantation, yet is also the target of immune-mediated damage in chronic viral hepatitis. To investigate the basis of these dichotomous outcomes, we manipulated several determinants capable of influencing outcomes of hepatic–immune interactions. Our findings reveal that a threshold of antigen expression within the liver is the dominant factor determining the fate of CD8 T cells recognizing intrahepatic antigen, irrespective of their affinity for antigen or the site of initial antigen encounter, with high-level antigen expression leading to exhaustion of T cell function. To our knowledge, for the first time, this study provides a unified model explaining the divergent consequences of hepatic–immune interactions. CD8 T-cell responses to liver-expressed antigens range from deletional tolerance to full effector differentiation resulting in overt hepatotoxicity. The reasons for these heterogeneous outcomes are not well understood. To identify factors that govern the fate of CD8 T cells activated by hepatocyte-expressed antigen, we exploited recombinant adenoassociated viral vectors that enabled us to vary potential parameters determining these outcomes in vivo. Our findings reveal a threshold of antigen expression within the liver as the dominant factor determining T-cell fate, irrespective of T-cell receptor affinity or antigen cross-presentation. Thus, when a low percentage of hepatocytes expressed cognate antigen, high-affinity T cells developed and maintained effector function, whereas, at a high percentage, they became functionally exhausted and silenced. Exhaustion was not irreversibly determined by initial activation, but was maintained by high intrahepatic antigen load during the early phase of the response; cytolytic function was restored when T cells primed under high antigen load conditions were transferred into an environment of low-level antigen expression. Our study reveals a hierarchy of factors dictating the fate of CD8 T cells during hepatic immune responses, and provides an explanation for the different immune outcomes observed in a variety of immune-mediated liver pathologic conditions.

Two lymph nodes draining the mouse liver are the preferential site of DC migration and T cell activation.

BACKGROUND & AIMS Lymph nodes (LNs) play a critical role in host defence against pathogens. In rodents, lymphatic anatomy and drainage have been characterized for many different organs. Surprisingly, the LNs draining the mouse liver have not been clearly identified. This knowledge is of central importance to allow accurate characterization of immune responses to pathogens infecting the liver. It is also important for exploring immune responses in hepatic tumour models, and mechanisms underlying the relative tolerogenic properties of the liver. In this study, we used both anatomical and immunological approaches to identify the LN(s) draining the mouse liver. METHODS Evans Blue and purified dendritic cells were directly injected into the hepatic parenchyma. RESULTS Using Evans Blue, we identified three LNs adjacent to the liver that stained with the dye within the first 5 min, which we termed portal, coeliac, and first mesenteric LNs. We also provide evidence that dendritic cells (DCs) injected under the liver capsule preferentially migrate to the coeliac and portal nodes, leading to local activation of antigen-specific naïve CD8 and CD4 T cells, suggesting this is a route of lymphatic drainage from the liver. Consistent with this result, cell-associated antigen injected under the liver capsule was also cross-presented to CD8 T cells in these nodes. CONCLUSIONS These results suggest for the first time that the coeliac and portal nodes are the main LNs draining the liver, and that DCs exiting the liver can elicit primary T cell activation within these lymph nodes; first mesenteric nodes play a secondary role. We propose this nomenclature to be used as common designations for the observed structures.

Intrahepatic Activation of Naive CD4+ T Cells by Liver-Resident Phagocytic Cells

Naive T cell activation is normally restricted to the lymphoid organs, in part because of their limited ability to migrate into the parenchyma of peripheral tissues. The liver vasculature is unique, however, and circulating leukocytes within the hepatic sinusoids have direct access to liver-resident cells, which include an abundant population of Kupffer cells. It is well accepted that recognition of cognate Ag within the liver leads to naive CD8+ T cell activation in situ, but it is unclear whether the liver also supports naive CD4+ T cell activation. In this study, we show that naive CD4+ T cells can be activated to proliferate in the liver when cognate Ag expression is induced in hepatocytes by recombinant adeno-associated viral vectors. Ag-specific retention and activation of naive CD4+ T cells within the liver are independent of lymphoid tissues but dependent on a clodronate liposome–sensitive population of liver-resident phagocytic cells. To our knowledge, this study provides the first unequivocal evidence that naive CD4+ T cells can be activated in a nonlymphoid organ. It also gives critical insight into how CD4+ T cells specific for Ag expressed in the liver are recruited to participate in protective or pathological responses during hepatotropic infections and autoimmune liver disease.

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.

Gene therapy for tolerance: high-level expression of donor major histocompatibility complex in the liver overcomes naive and memory alloresponses to skin grafts.

Background The liver has long been recognized as having tolerogenic properties. We investigated whether recombinant adenoassociated virus (rAAV)–mediated expression of donor major histocompatibility complex in recipient livers could induce tolerance to donor-strain grafts. Methods Naive B10.BR (H-2k) or B10.BR recipients primed with a H-2Kb–expressing (Kb+) skin graft were injected with rAAV-expressing H-2Kb (rAAV-Kb) to induce Kb expression on hepatocytes 7 days before challenge with a Kb+ skin graft. Kb-specific responses were measured by interferon (IFN)-&ggr; ELISpot and flow cytometric assessment of directly H-2Kb reactive cells. Fully allogeneic grafts from C57BL/6 (H-2b) donors were transplanted onto longstanding B10.BR recipients of Kb+ skin to test for linked epitope suppression. Results rAAV-Kb–treated B10.BR mice accepted Kb+ skin grafts with increased median survival time (MST) more than 169 days compared to uninoculated (MST=18.5 days) and rAAV-Kd–treated controls (MST=19 days). rAAV-Kb–treated B10.BR animals primed with Kb+ skin grafts also accepted secondary Kb+ skin grafts in the long term (MST>100 days) compared to accelerated rejection in primed, uninoculated mice (MST=12 days). Treatments did not induce liver pathology, assessed by serum alanine aminotransferase levels and histology. IFN-&ggr; ELISpot analysis of splenocytes from rAAV-Kb–treated mice indicated reduced responses to donor Kb+ antigen, but protection was not extended to fully allogeneic C57BL/6 skin or heart grafts, even in recipients that had accepted Kb+ skin grafts in the long term. Conclusions High-level expression of donor major histocompatibility complex in recipient livers promotes tolerance to skin allografts, even in animals primed to produce a memory response. This provides proof of concept for an approach using liver-targeted gene delivery for tolerance induction to donor antigen.

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.

Differential migration of passenger leukocytes and rapid deletion of naive alloreactive CD8 T cells after mouse liver transplantation.

Donor passenger leukocytes (PLs) from transplanted livers migrate to recipient lymphoid tissues, where they are thought to induce the deletion of donor‐specific T cells and tolerance. Difficulties in tracking alloreactive T cells and PLs in rats and in performing this complex surgery in mice have limited progress in identifying the contribution of PL subsets and sites and the kinetics of T cell deletion. Here we developed a mouse liver transplant model in which PLs, recipient cells, and a reporter population of transgenic CD8 T cells specific for the graft could be easily distinguished and quantified in allografts and recipient organs by flow cytometry. All PL subsets circulated rapidly via the blood as soon as 1.5 hours after transplantation. By 24 hours, PLs were distributed differently in the lymph nodes and spleen, whereas donor natural killer and natural killer T cells remained in the liver and blood. Reporter T cells were activated in both liver and lymphoid tissues, but their numbers dramatically decreased within the first 48 hours. These results provide the first unequivocal demonstration of the differential recirculation of liver PL subsets after transplantation, and show that alloreactive CD8 T cells are deleted more rapidly than initially reported. This model will be useful for dissecting early events leading to the spontaneous acceptance of liver transplants. Liver Transpl 19:1224–1235, 2013.

Effector T cell function rather than survival determines extent and duration of hepatitis in mice

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.

Direct recognition of hepatocyte-expressed MHC class I alloantigens is required for tolerance induction

Adeno-associated viral vector-mediated (AAV-mediated) expression of allogeneic major histocompatibility complex class I (MHC class I) in recipient liver induces donor-specific tolerance in mouse skin transplant models in which a class I allele (H-2Kb or H-2Kd) is mismatched between donor and recipient. Tolerance can be induced in mice primed by prior rejection of a donor-strain skin graft, as well as in naive recipients. Allogeneic MHC class I may be recognized by recipient T cells as an intact molecule (direct recognition) or may be processed and presented as an allogeneic peptide in the context of self-MHC (indirect recognition). The relative contributions of direct and indirect allorecognition to tolerance induction in this setting are unknown. Using hepatocyte-specific AAV vectors encoding WT allogeneic MHC class I molecules, or class I molecules containing a point mutation (D227K) that impedes direct recognition of intact allogeneic MHC class I by CD8+ T cells without hampering the presentation of processed peptides derived from allogeneic MHC class I, we show here that tolerance induction depends upon recognition of intact MHC class I. Indirect recognition alone yielded a modest prolongation of subsequent skin graft survival, attributable to the generation of CD4+ Tregs, but it was not sufficient to induce tolerance.