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Fundamentals of liver and pancreas immunology
Introduction
The immune system manifests two strategies of host defense termed innate and adaptive immunity ( Fig. 10.1 ). Innate immunity refers to the nonspecific first line of defense against danger signals from pathogens or tumor cells. The repertoire of innate immune cells includes natural killer (NK) cells, macrophages, and dendritic cells (DCs). Innate immune cells sense both tissue injury and pathogens through pattern recognition receptors (PRRs) that trigger a rapid response. PRRs bind to well-conserved molecules from microbes, including lipopolysaccharide, other bacterial cell wall moieties, and pathogen nucleic acids. PRR signaling and the ensuing response may lead to destruction of the invading pathogen or tumor via phagocytosis or release of various cytotoxic or inflammatory agents. Innate immunity may also activate antigen-presenting cells (APCs), leading to the activation of T and B cells and leading to adaptive immunity, and such crosstalk bridges the nonspecific initial response to a highly specialized system capable of long-lasting immunologic memory.
Adaptive immunity involves antigen-specific responses, which occur de novo during an initial immune response or rapidly upon repeat exposure to a particular pathogen. The adaptive immune system comprises T and B lymphocytes that circulate within the blood, lymphatic tissues, and nonlymphoid organs, including the liver. T and B cells express specific cell-surface receptors capable of recognizing particular antigens. T-cell activation requires presentation of antigen by APCs such as DCs. APCs mediate antigen presentation to T cells within the context of major histocompatibility complex molecules (MHC I or MHC II). In addition, APCs provide a critical “second signal” through co-stimulatory molecules, and the response is further modulated by secreted cytokines ( Fig. 10.2 ). Classically, CD4 + helper T cells recognize antigen in the context of MHC II, whereas CD8 + cytotoxic T cells engage antigen loaded onto MHC I molecules. Several subsets of CD4 + cells (T helper, or Th cells) orchestrate and polarize the immune response to address particular challenges.
Although activation of innate and adaptive immunity is essential for combating pathogens and malignant cells, overly exuberant immune responses can result in severe tissue damage. The immune system is normally able to distinguish self from nonself and is controlled by numerous regulatory mechanisms. During T-cell development, autoreactive cells are deleted through negative selection in the thymus. Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) regulate immune responses in the periphery and prevent autoimmunity. Immunoinhibitory receptors, including programmed death-1 (PD-1) and cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), modulate T-cell function, working in concert with suppressor cells to regulate immune responses. As will be discussed later, Tregs, MDSCs, and immunoinhibitory pathways in the liver cooperate to create a highly tolerogenic milieu. Intrahepatic tolerance is a fundamental aspect of liver immunology that reflects its position at the interface between ingested exogenous antigens and the systemic circulation.
The balance between tolerance and immunity in the liver is tightly regulated by intrahepatic immune cells and associated signaling pathways. Several features of liver immunology point to the skewing of this balance toward tolerance under normal physiologic conditions. First, the fact that the liver is one of the most common sites for metastatic disease suggests that malignant cells are able to exploit intrahepatic immunosuppression. Second, compared with other solid organ transplants, liver allografts do not require immunosuppression in certain strains of mice and require less immunosuppression in humans. Third, the immune system is often unable to clear chronic hepatitis B and C viral infections. Additionally, oral ingestion or portal vein injection of foreign proteins can lead to tolerance in animal models. Conversely, the liver is the site of several autoimmune processes, including primary sclerosing cholangitis and primary biliary cirrhosis. Despite the prominent role that the intrahepatic immune system plays in disease, the study of liver immunology remains underdeveloped.
On the other hand, in a normal physiologic state, the pancreas does not appear to have a major impact on the overall immune response. However, malignancies of the pancreas such as ductal adenocarcinoma demonstrate significant immune evasion, which pose unique therapeutic challenges. Here, we review our current understanding of liver and pancreatic immunology and the complex interplay of cells and cytokines therein.
Anatomic considerations in liver immunology
Because the vascular supply of the liver derives principally from the portal venous system draining the gut, a heavy antigen load is delivered to the liver. Portal venous blood flows slowly through the vast network of hepatic sinusoids, which are discontinuously lined by fenestrated endothelium lacking a basement membrane ( Fig. 10.3 ; see Chapters 2 and 5 ). The sluggish flow of blood allows for the efficient capture of antigens by leukocytes traveling in the blood within the sinusoids and by the endothelial cells lining the sinusoids. The liver reticuloendothelial system, comprising liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs), is very efficient at extracting antigens from portal blood. LSECs capture and process antigen at levels comparable to professional APCs, such as DCs. The microscopic anatomy of the liver also favors the ability of bloodborne leukocytes to interact with hepatic parenchymal cells and resident immune cells of the liver. The microanatomic and rheologic features of the hepatic sinusoids facilitate highly efficient antigen presentation and interactions among immune cells.
Tolerance and immunosuppression
From a teleologic perspective, tolerance to oral antigens is clearly advantageous. In experimental animal models and clinical liver transplantation studies, a greater propensity for graft acceptance has been noted compared with transplantation of other solid organs. A liver transplant protects a kidney allograft transplanted simultaneously from the same donor. Unfortunately, primary and metastatic tumors in the liver exploit intrahepatic immunosuppression to evade destruction by the immune system. A deeper understanding of liver tolerance may support therapeutic interventions to stimulate immunity for cancer or control liver immune cell function for inflammatory conditions.
Liver immune cells
Among the liver’s nonparenchymal cells (NPCs), one quarter are leukocytes. The composition of the intrahepatic leukocyte population is markedly different from that seen in other organs. The liver contains most of the cellular components of innate and adaptive immunity. Importantly, liver immune cells demonstrate unique functional properties that tend to promote a tolerogenic milieu.
Antigen-presenting cells
Experimental and clinical observations that antigens passing through the liver can lead to tolerance make the understanding of intrahepatic antigen presentation particularly relevant. APCs play a crucial role in driving adaptive immune responses and bridging innate to adaptive immunity. DCs, LSECs, KCs, and B cells all play a role in antigen presentation within the liver. The context in which an APC presents an antigen can dramatically alter the response of antigen-specific T cells. Specifically, when antigen presentation occurs in conjunction with the appropriate co-stimulatory molecules, T cells proliferate and develop an immunogenic phenotypic and functional profile ( Fig. 10.4 ). In contrast, antigens presented in the absence of co-stimulation or presence of immunoinhibitory signals lead to anergy or activation-induced T-cell death, two of the mechanisms of peripheral tolerance induction and maintenance.
Dendritic cells
DCs are a heterogeneous population of leukocytes primarily responsible for the capture of antigens in the periphery and subsequent presentation to immune effector cells. DCs are the most potent APCs of the immune system. Immature DCs are specialized to capture antigens and then migrate to lymph nodes, where they can interact with T cells. After an encounter with a pathogenic stimulus, such as bacterial lipopolysaccharide, DCs undergo phenotypic and functional changes, whereby their ability to capture antigens is diminished, but they increase their expression of class II and T-cell co-stimulatory molecules. Co-stimulatory molecule expression is essential in facilitating antigen presentation and efficient T-cell activation. Inflammatory conditions often promote a process of maturation, whereby DCs increase expression of MHC and co-stimulatory molecules, enabling efficient antigen presentation and T-cell activation. However, liver DC phenotype and function is somewhat unique, as we discuss later.
When compared with DCs from the spleen, CD11c + liver DCs were immature and only weakly immunostimulatory. In contrast to spleen DCs, liver DCs were heterogeneous in their expression of MHC class II and co-stimulatory molecules. Myeloid (CD11b + ) and lymphoid (CD8α + ) liver DCs, which each comprise approximately 10% of the total population of DCs in the liver, were as able to activate T cells as their splenic counterparts were. The bulk of the remaining cells, which had low-to-no expression of CD11b and CD8α, were poor T-cell stimulators. The presence of these atypical DCs accounted for the weakly activating nature of liver DCs on the whole. More recently, using a transgenic mouse in which CD11c hi DCs can be depleted selectively, we found that activation of antigen-specific CD8 + T cells in the liver only occurred in the presence of CD11c hi DCs.
As in the mouse, freshly isolated DCs from human liver exhibit tolerogenic properties when compared with autologous blood DCs. Human liver DCs are weaker stimulators of T cells and produce the antiinflammatory cytokine interleukin-10 (IL-10), which induces the differentiation of naïve CD4 + T cells into regulatory T cells with suppressive function.
Kupffer cells
Liver macrophages, referred to as Kupffer cells , are the primary phagocytic cells of the liver (see Chapter 7 ). KCs represent the largest pool of macrophages in the body, derived in part from monocytic precursors in the blood and partly from fetal precursors that seed the liver early and maintain themselves by cell division in situ. They are typically found in the hepatic sinusoids; however, they also can migrate through the space of Disse to interact with hepatocytes (see Fig. 10.3 ). KCs play a major role in antigen presentation and have been implicated in portal venous tolerance, possibly by regulating T-cell responses to antigens in the context of immune tolerance to liver allografts. On the other hand, more recent murine model work has demonstrated the ability of KCs within the sinusoids to effect CD8 T-cell activation to antigens in an intercellular adhesion molecule-1 dependent manner.
Multiple lines of evidence from flow cytometry, lineage-tracing, and single-cell RNA sequencing suggest that KCs consist of two subsets. In the mouse, there is clear evidence that one subset derives from precursors in the yolk sac and/or the fetal liver and maintains itself locally for the life of the animal. These cells express more molecules linked to endocytosis and to immune tolerance, which the alternative subset of KCs derives from blood monocytes, and are increased in abundance during emergency repopulation of the liver. Recent evidence has shown that embryo-derived KCs remain resistant to irradiation via upregulation of a kinase inhibitor Cdkn1a, which may have implications in understanding radiation-induced liver diseases. In humans, lineage-tracing experiments are not possible, but genes expressed by two clusters of macrophage-like cells identified on the basis of differential gene expression argue for the same dichotomy. The distinction between KC subsets and DCs in human liver is complicated by the concern that cell surface markers that clearly distinguish macrophages from DCs in other tissues may not be absolute among liver myeloid cells, and this remains an active area of investigation.
Liver sinusoidal endothelial cells
LSECs are highly specialized cells that line the hepatic sinusoids. They are distinguished by the presence of fenestrations in their cellular membranes (see Fig 10.3 ; see also Chapter 7 ). The fenestrations are believed to facilitate the selective passage of antigens between the sinusoid and the hepatic parenchyma and may also increase the surface area available for antigen presentation. This strategic placement puts LSECs in the ideal position to interact with antigens and immune cells passing between the liver and the portal venous system.
Several studies have shown that, in addition to serving as a structural component of the hepatic sinusoids, LSECs are immune cells with the ability to capture and present antigen to T cells. As with KCs, considerable controversy surrounds the immunologic function of LSECs. In contrast to earlier work, we have shown that although LSECs are highly capable of capturing various antigens in vivo and in vitro, they lack the ability to activate T cells in the absence of exogenous co-stimulation. The differences in results may derive from the use of more specific methods of cell isolation in the latter study. The finding that LSECs are not independently capable of triggering a T-cell–mediated immune response does not, however, exclude the possibility that LSECs, in concert with DCs or KCs, play an important role in antigen presentation in the liver.
Effector cells
T cells
Like other liver immune cell populations, intrahepatic T cells have unique properties enabling them to contribute to maintenance of a tolerogenic milieu. T cells are a heterogeneous population of adaptive immune cells with both effector and suppressor subtypes. CD4 + helper T cells orchestrate immune responses, CD8 + cytotoxic T cells destroy infected host or malignant cells, and Tregs play an immunomodulatory role. The liver also contains multiple nonclassical T-cell subsets, including NKT cells and γδ T cells. The nature of the interactions between APCs and T cells polarizes T-cell differentiation and thus determines the outcome of a particular immune response.
The liver contains a full complement of T-cell subsets, although the relative proportions of each population are different when compared with lymphoid organs. Conventional or classical T cells express the αβ T-cell receptor in association with either CD4 or CD8. These are the most prevalent T cells in the body and account for about one third of the murine liver T-cell population. In contrast, unconventional T cells expressing NK markers or the γδ T-cell receptor comprise a greater proportion of liver T cells, approximately 50% and 10%, respectively. Immunosuppressive Tregs expressing FOXP3 are heavily represented in the liver. Among the classical T cells, the liver also contains Th17 cells , which are capable of producing the highly inflammatory cytokine IL-17. Th17 cells play an important role in the promotion of inflammatory and fibrotic disorders affecting the liver.
The diversity of conventional T cells is based on their recognition of specific peptide antigen motifs within the context of MHC class I or II molecules expressed by APCs. The αβ T-cell receptor is highly variable, and numerous T cells, each recognizing a different antigen presented by APCs, are present in the immune system. CD8 + T cells respond to peptides presented on MHC class I molecules, which are expressed by nearly every cell in the body, excluding erythrocytes. Activated CD8 + T cells become cytotoxic T lymphocytes. CD4 + helper T cells recognize antigens presented on MHC class II molecules on the surface of professional APCs. CD4 + T-cell subsets, Th1 and Th2 cells, then regulate and amplify the immune response by secreting cytokines, which affect nearby effector cells.
γδ T cells
The γδ T-cell receptor is relatively invariant and can recognize multiple nonpeptide antigens without the need for MHC presentation. γδ T cells represent 10% of liver T cells, whereas they comprise only a small proportion (<5%) of T cells in the blood or lymphoid organs. γδ T cells also are abundant at other environmental interfaces, including the skin and mucosal surfaces. Through secretion of activating and modulatory cytokines, γδ T cells help orchestrate early responses to atypical bacterial and viral pathogens. γδ T cells can also promote antitumor immunity through their early secretion of interferon-γ (IFN-γ). Conversely this cell type has immunosuppressive properties as well. The high proportion of γδ T cells in the liver suggests that they have an important immunologic role, but further investigation is required. As with most lymphocyte populations, heterogeneity among liver γδ T cells precludes simple generalizations concerning their functions.
Natural killer T cells
NKT cells share characteristics of conventional T cells and NK cells and are defined by the presence of several T cell and NK cell surface markers. Most NKT cells react against glycolipid antigens in the context of CD1d, which is an MHC class I–like glycoprotein. CD1d is expressed on APCs and hepatocytes. NKTs express invariant T-cell receptor chains that are conserved across species, suggesting an important role for NKT cells in the innate immune response to pathogens. Activated NKT cells are capable of producing IFN-γ and IL-4, which are the prototypical Th1 and Th2 cytokines, respectively.
NKT cells constitute a relatively large proportion of T cells found in the liver compared with other organs. In addition, a local expansion of NKT cells is seen in several models of liver injury, such as partial hepatectomy. NKT cells play a role in inflammatory diseases and in clearance of infection from the liver. Depletion of NKT cells abrogated the effects of experimentally induced hepatitis in a mouse model, and mice lacking NKT cells are susceptible to viral and bacterial infections. NKT cells also play a part in tumor surveillance in the liver. In murine primary and metastatic tumor models, NKT cells can mediate tumor rejection, in part because of their ability to secrete IFN-γ. Work in other murine models suggests that liver NKTs have the capacity to suppress T-cell proliferation and hence contribute to immunosuppression in the liver.
Natural killer cells
NK cells are innate responders and, unlike T cells, do not possess receptors for specific peptide antigens. By expressing a variety of activating and inhibitory receptors, NK cells can bind ligands on their target cells. The resulting activation of NK cells causes the release of lytic granules, or cytokines such as IFN-γ, which kill the infected host or tumor cell in an MHC-unrestricted fashion. NK cells are a major component of murine and human liver lymphocytes and mediate inflammatory reactions seen in viral and autoimmune hepatitis. Bulk human liver NK cells possess weaker lytic capabilities when compared with autologous blood NK cells because the liver has a greater proportion of NK cell subtypes with weaker cytolytic function when compared with blood NK cells.
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