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ScienceDirect Hepatic immune regulation by stromal cells Frank A Schildberg1, Arlene H Sharpe1,2 and Shannon J Turley3 A metabolic organ, the liver also has a central role in tolerance induction. Stromal cells lining the hepatic sinusoids, such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), are the first liver cells to encounter gut-derived and systemic antigens, thereby shaping local and systemic tolerance. Recent studies have demonstrated that stromal cells can modulate immune responses by antigen-dependent and independent mechanisms. Stromal cells interfere with the function of other antigen-presenting cells (APCs) and induce non-responsive T cells as well as regulatory T cells and myeloid-derived suppressor cells (MDSCs). The immunosuppressive microenvironment thus created provides a means to protect the liver from tissue damage. Such tolerized surroundings, however, can be exploited by certain pathogens, promoting persistent liver infections. Addresses 1 Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA 2 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 3 Cancer Immunology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA Corresponding author: Turley, Shannon J ([email protected])
Current Opinion in Immunology 2015, 32:1–6 This review comes from a themed issue on Innate immunity Edited by Zhijian J Chen and Sebastian Amigorena
http://dx.doi.org/10.1016/j.coi.2014.10.002 0952-7915/# 2014 Published by Elsevier Ltd.
Introduction Early experiments in orthotopic liver transplantation gave rise to the concept that immune responses in the liver are biased towards tolerance. While other organs transplanted between unrelated pigs were promptly rejected, allogeneic liver transplants generally not only were tolerated [1], but also able to confer tolerance to another solid organ transplant from the same donor [2]. Over the years, the local regulatory cues that determine the functional complexity of immune responses in the liver have been increasingly well understood [3–5]. This in-depth knowledge of the immune regulatory mechanisms in the liver is of high clinical relevance: it is crucial for protecting the www.sciencedirect.com
integrity of this essential metabolic organ and discerning how pathogens exploit this unique microenvironment. Active modulation of immune responses in the liver stems from its unique microenvironment and hepatocytes, but also results from certain types of stromal cells in the liver sinusoids. LSECs are the most abundant non-parenchymal cell population in the liver and line the hepatic sinusoids (Figure 1a). As unique micro-vascular endothelial cells that resemble lymphatic endothelial cells, they lack a basal membrane and define the space of Disse´ [3,6,7]. The perisinusoidal space between LSECs and hepatocytes is populated by HSCs. Upon activation, HSCs differentiate into myofibroblasts, which produce extracellular matrix leading to hepatic fibrosis. This overview will discuss the immune inhibitory functions, of both LSECs and HSCs.
Liver sinusoidal endothelial cells LSECs skew the differentiation and effector function of CD4+ T cells
LSECs originate from liver-derived endothelial progenitor cells and represent a unique population of APCs. LSECs can be identified by their expression of CD45, CD31 (PECAM-1) and CD146 (MCAM, Muc18), as well as by their high scavenger activity using fluorochromelabeled OVA or BSA. They are perforated by numerous fenestrations, which facilitate the passage of molecules from the sinusoidal lumen into the space of Disse´. Because of their enormous scavenger activity, they compete with dendritic cells for uptake of circulating antigen in the liver [8]. Murine LSECs express only very low levels of major histocompatibility complex II (MHC II) and the co-stimulatory molecules CD80/86. MHC II, CD80 and CD86 expression is further downregulated by lipopolysaccharide present in portal venous blood [9]. This does not, however, preclude the ability of LSECs to prime naı¨ve CD4 T cells, albeit the process does require high antigen concentration. Th1 T cell differentiation, on the other hand, is not promoted by LSECs [10]. Instead, LSECs can inhibit inflammatory cytokine secretion by Th1 and Th17 effector CD4+ T cells in an IL-10 and PD-1-dependent manner [11]. These results suggest that LSECs possess the ability to mitigate hepatic inflammatory CD4+ T cell activity, a highly valuable trait for preventing or terminating hepatic inflammation. LSECs can also restrain inflammation by promoting induction of regulatory CD4+ T cells; LSEC-mediated induction of both Foxp3 CD25low and Foxp3+ regulatory CD4+ T cells has been shown [12]. A comparative analysis Current Opinion in Immunology 2015, 32:1–6
2 Innate immunity
Cross-presentation by LSECs to naı¨ve CD8+ T cells leads to tolerance induction
Figure 1
(a) LSEC T cell
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Anatomical position and immune regulatory functions of hepatic stromal cells. (a) The fenestrated LSECs line the hepatic sinusoid and form the space of Disse´, which is populated by HSCs. Both stromal cells separate the blood stream from hepatocytes and thereby function as physical gatekeepers to prevent destructive inflammation into the liver parenchyma. LSECs represent the first barrier for the liver. However, as DCs and T cells continually migrate through the space of Disse´ to reach the hepatic lymph nodes, HSCs are no less important. Both stromal cells are optimally positioned to interact with circulating antigens and extravasating leukocytes. (b) In addition to the barrier function described in (a), LSECs and HSCs also exert potent immune regulatory mechanisms to prevent overwhelming inflammation in the liver. Both cell types can induce Tregs, and inhibit T cell responses and the function of APCs. At this time, only LSECs are known to induce non-responsive, memory-like T cells and only HSCs lead to the induction of MDSCs. Together, these mechanisms contribute to the unique immune regulatory microenvironment of the liver.
of liver APCs concluded that LSECs are the major cell type that stimulates TGFb dependent induction of CD4+ Foxp3+ Tregs. This is possible because LSECs not only can secrete TGFb but also tether LAP/TGFb to their membrane through GARP [13]. Once induced by LSECs, antigen-specific Tregs can be potent suppressors of autoimmune inflammation, as evidenced by in vivo studies in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. LSEC-mediated induction of Tregs, therefore, may be instrumental for controlling hepatic and systemic inflammatory immune responses [13]. Current Opinion in Immunology 2015, 32:1–6
LSECs have a similar cross-presentation mechanism to DCs, efficiently offering soluble antigens to CD8+ T cells [14]. Once LSECs have ingested antigen, the corresponding naı¨ve antigen-specific CD8+ T cells are, within minutes, promptly detained in liver sinusoids. This results in rapid CD8+ T cell accumulation [15]. The interaction of naı¨ve T cells with LSECs produces a non-responsive T cell mode, in which these T cells are only barely receptive to stimulation through the TCR [14]. These results suggest that these T cells are tolerized. However, this T cell non-responsiveness is not a permanent state; a recent study has demonstrated that LSEC-primed CD8+ T cells not only can survive in vivo for a long time, but that they also can be reactivated through a combination of co-stimulatory signals [16], which reinstates their capability to protect against infections. When LSECs induce CD8+ T cell priming, this does not simply lead to the generation of T cells that are ultimately tolerized. In fact, these T cells have a great similarity to memory T cells. This type of liver priming may well rescue T cells from deletion by immature antigen-presenting DCs. Instead of undergoing apoptosis, the LSEC-primed CD8+ T cell population expresses high amounts of the antiapoptotic molecule bcl-2 [15], proliferates and transiently upregulates activation markers [17,18]. Non-functional, this T cell phenotype is dependent on the co-inhibitory PD-L1 molecules expressed by LSECs interacting with the PD-1 receptor on CD8+ T cells [18]. If cognate interactions between MHC class I molecules and TCRs are present during LSEC-induced initial programming of naı¨ve CD8+ T cells, the result is increased expression of co-inhibitory PD-L1, but not of co-stimulatory CD80/86 molecules on LSECs. This further illustrates the importance of balanced delivery of stimulatory and inhibitory signals for the CD8+ T cell fate; increased co-stimulatory signaling through CD28 results in the override of PD-L1– PD1 signaling [18]. Similarly, the increase of signal strength via the TCR is also capable of breaking the non-responsive phenotype: this occurs through the production of IL-2 by T cells. An autocrine co-stimulatory factor, IL-2 production predominates over PD-L1mediated signaling [17]. Intriguingly, the non-responsiveness induced in T cells by LSECs could be a possible target for cancer immunotherapy, because LSECs are capable of cross-presenting circulating tumor antigens, thereby inducing non-responsive antigen-specific CD8+ T cells through the PD-1/PD-L1 pathway [19]. Timing is a highly important factor in the LSECmediated T cell non-responsiveness mechanism: CD28 co-stimulation only can prevent induction of non-responsive LSEC-primed T cells during the first 24–36 h; after this time, the LSEC-induced programming very likely becomes cell-intrinsic [20]. Although it is known that www.sciencedirect.com
Hepatic immune regulation by stromal cells Schildberg, Sharpe and Turley 3
antigen-presenting LSECs require several days to induce these memory-like T cells [16], it is noteworthy that there is exceedingly rapid GzmB induction in T cells in the first 18 h following contact with LSECs [21]. TCR signaling, in combination with IL-6 trans-signaling, is sufficient for this direct and rapid GzmB expression in T cells. It is as yet not established if this phenotype should be considered an intermediate developmental stage or, rather, serves a specific function during immune responses originating in the liver. Intriguingly, this LSEC-mediated direction of the CD8+ T cell fate to dysfunctional versus memory states bears similarities to T cell exhaustion.
markers, although intracellular GFAP for quiescent and aSMA for activated HSCs have been used. However, a recent publication showed that HSCs can be differentiated from other liver cells using size, granularity, and high UV-fluorescence (due to their vitamin A) in combination with the absence of scavenger activity [26]. Once HSCs are activated under inflammatory conditions, they differentiate into myofibroblasts and assume a crucial role in liver fibrogenesis. HSCs also take on the novel role of immune sentinels in the liver. Studies have demonstrated direct contact between HSCs and lymphocytes, indicating that HSCs may directly activate naı¨ve lymphocytes [27,28] (Figure 1a).
Antigen-independent immune regulatory functions of LSECs
The direct activation of lymphocytes by HSCs has, however, been challenged. HSCs express very low amounts of MHC and co-stimulatory molecules [29]; furthermore, they have low antigen uptake and inefficient processing [30]. Under non-inflammatory conditions, HSCs produce insufficient amounts of the molecules needed for antigen presentation. In cell culture, the expression of these molecules could only be upregulated in the presence of certain cytokines. Under inflammatory conditions in the liver, however, chronic liver damage and inflammatory cytokines such as IFNg, alter the HSC phenotype to increase their antigen-presenting ability [31]. As many studies of HSCs involve in vitro cultures, which are very seldom pure, such experiments may not accurately reproduce their composition in the intact liver.
Independent of their APC properties, LSECs also have other immune regulatory functions; so-called ‘bystander functions’. One particularly important property is the surface expression of LSECtin (also known as CLEC4G), a ligand for CD44. LSECs are therefore capable of interacting with activated, but not resting, T cells since CD44 is not expressed on resting T cells. LSECtin/CD44 interaction can inhibit T cell activation and proliferation [22] or induce apoptosis [23]. The functional significance of this interaction is illustrated by the aggravated autoimmune liver disease that develops in the absence of LSECtin [24]. All of these characteristics unite to paint a picture of LSEC-based restraint of T cell effector function, contributing to the prevention of immune-mediated liver damage. In their function as immune regulatory bystander cells, LSECs also contribute to the tolerogenic environment in the liver by interacting and influencing other APCs. LSECs can negatively regulate the APC function of neighboring DCs. This ‘veto effect’ occurs independently of the LSEC antigen presentation, but depends on physical contact. This contact causes DCs to lose their ability to prime naı¨ve T cells by downregulating DC costimulatory signals [25]. This may be the mechanism that leads to inhibition of immunogenic T cell priming in the liver. Since LSEC-mediated regulation of T cell and APC function is not antigen-specific, this likely controls the local threshold to ‘fine tune’ immune effector functions.
Hepatic stellate cells Weak antigen-presenting function of HSCs and their cross-talk in the sinusoid
The developmental origin of HSCs, also called Ito cells, is still under discussion because they express marker genes of all three germ layers. However, based on current knowledge they seem to stem from mesenchymal precursors in the liver. Under non-inflammatory conditions, HSCs have central roles in vitamin A storage and regulating blood flow through the sinusoids. In contrast to LSECs, HSCs are hard to identify based on surface www.sciencedirect.com
In a recent study highly pure HSCs were examined in vitro and found to, completely lack cross-presentation competence [29]. Interestingly, this same study identified bidirectional, intercellular transfer of MHC I between the cell-surfaces of HSCs and LSECs; this is a mechanism similar to trogocytosis [32], where membrane fragments with MHC molecules are exchanged between immune cells. This exchange of MHC I molecules also was shown to occur with other sinusoidal liver cell populations. As an exchange of membrane fragments provides the opportunity for exchange of co-stimulatory or inhibitory molecules, this mechanism could explain how HSCs and other sinusoidal liver cells may acquire both activating and inhibitory properties to facilitate T cell activation in liver infection or T cell tolerance in the healthy liver. The identification of this mechanism necessitates reassessment of the antigen-presenting capacity of all sinusoidal liver cells. Antigen-independent immune regulatory functions of HSCs
Much less controversial than their APC function, HSCs manifest immunosuppressive activities as bystander cells in the context of T cell immunity. HSCs are a rich source of anti-inflammatory mediators [33], interfere with local T cell activation [34] and cause induction as well as the proliferation of Tregs [29]. HSCs seem to regulate adaptive Current Opinion in Immunology 2015, 32:1–6
4 Innate immunity
immunity by several mechanisms, but further work is needed in light of potential contamination with other APCs or in vitro differentiation of HSCs. Best characterized is HSC expression of PD-L1, especially upon exposure to IFNg or activated T cells, and secretion of the inhibitory cytokines IL-10 and TGFb. Activation of HSCs leads to their fibrogenic phenotype, as well as their acquisition of potent T cell suppressive functions. This is partially mediated by PD-L1 ligation [35,36] but an essential role for B7-H4 [37], TRAIL [38] and ICAM-1 [34,39] has also been described. HSCs also can induce and expand Foxp3+ Tregs; however, the underlying mechanism is still controversial due to their questioned APC function. Most likely HSCs act as a crucial bystander to promote liver DC-induced activation of Tregs [40]. Since HSCs form a second layer of cells between the bloodstream and hepatocytes, the immune regulatory properties of HSCs can limit the effector function of those T cells that have extravasated from the sinusoidal lumen, preventing tissue damage and loss of organ function. This immune regulatory function is so strong that HSCs are even able to promote pancreatic islet allograft survival if they are co-transplanted [35,41]. HSCs not only function as immune regulatory bystander cells during T cell activation and Treg induction, but also during contact with myeloid cells: HSCs also diminish the APC function of DCs [42,43,44]. In addition, during chronic inflammation in the liver, HSCs facilitate the differentiation of inflammatory monocytes into MDSCs that impair T cell proliferation and effector function [45,46]. The induction of multiple types of immune inhibitory cells by HSCs leads to a complex suppressive microenvironment.
Conclusions LSECs and HSCs are highly involved in the maintenance of local and systemic immune tolerance. In the hepatic microenvironment they establish a functional barrier to protect hepatocytes from immune-mediated liver injury, which potentially may lead to loss of organ function. Both cell types act as immunological bystander cells by impairing the APC function of dendritic cells, interfering with T cell activation and inducing suppressive Tregs and MDSCs (Figure 1b). In contrast, HSCs lack significant APC functions, whereas LSECs use their APC function for tolerance induction. The combination of these mechanisms indicates that the hepatic sinusoid surface area is a potent immunoregulatory entity capable of modulating immune responses. Recent studies challenging the APC function of HSCs show the crucial need for developing new isolation methods to obtain highly pure liver cell populations in the future [26]. Although cellular contamination issues may be easier to resolve, artifacts based on in vitro differentiation will require novel model systems and technologies that reflect the complex regulation of Current Opinion in Immunology 2015, 32:1–6
intrahepatic immunity. Above all, more human studies and advanced cell-type-specific mouse models are needed. Although there is clear evidence that HSCs become more suppressive during fibrosis, sufficient data whether this is also true for LSECs are still missing. We stand but at the very beginning of decoding the triggers regulating the bystander and APC function of these key liver stromal cells and determining how they could be manipulated. Another important remaining question is which of these immune regulatory mechanisms are exploited by chronic hepatic infections. Although HSCs and LSECs set a high threshold for adaptive T cell responses, they can drive liver inflammation through innate immune mechanisms [47–49]. A recent publication describes a mechanism to overcome the hepatic regulatory cues by applying Tolllike receptor signaling or viral infection which leads to intrahepatic myeloid cell aggregates associated with T cell expansion (iMATEs) [50]. It would be of tremendous relevance to understand if and how the immune regulatory capacity of LSECs and HSCs are regulated in this context because this knowledge could be used for novel therapeutic vaccination strategies. In summary, the liver ‘stromal cell’ populations LSECs and HSCs control hepatic immune regulation by multiple mechanisms and we are only beginning to elucidate their immunoregulatory properties. Further understanding of hepatic immune tolerance will provide the basis for developing new immunotherapies targeting chronic viral infection and cancer.
Acknowledgements This work was supported by grants from the National Institutes of Health (R01 AI40614, to A.H.S.) and the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354) which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, NIDDK, NIGMS, FIC, and OAR (to F.A.S.). F.A.S. acknowledges the International Society for Advancement of Cytometry for support as an ISAC Scholar. Because of space restrictions, we were able to cite only a fraction of the relevant literature and apologize to colleagues whose contributions may not be appropriately acknowledged. The authors thank Cristina A. Hagmann for editing the manuscript.
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42. Sumpter TL, Dangi A, Matta BM, Huang C, Stolz DB, Vodovotz Y, Thomson AW, Gandhi CR: Hepatic stellate cells undermine the allostimulatory function of liver myeloid dendritic cells via STAT3-dependent induction of IDO. J Immunol 2012, 189:3848-3858. 43. Bhatt S, Qin J, Bennett C, Qian S, Fung JJ, Hamilton TA, Lu L: All trans retinoic acid induces arginase-1 and inducible nitric oxide synthase-producing dendritic cells with T cell inhibitory function. J Immunol 2014, 192:5098-5108. This report describes the mechanism by which HSC-secreted ATRA leads to the development of tolerogenic DCs. Mechanistically, ATRA enhances the expression of both Arg-1 and iNOS in IFNg-treated DCs, which leads to a tolerogenic DC phenotype.
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48. McHedlidze T, Waldner M, Zopf S, Walker J, Rankin AL, Schuchmann M, Voehringer D, McKenzie AN, Neurath MF, Pflanz S et al.: Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis. Immunity 2013, 39:357-371.
50. Huang LR, Wohlleber D, Reisinger F, Jenne CN, Cheng RL, Abdullah Z, Schildberg FA, Odenthal M, Dienes HP, van Rooijen N et al.: Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection. Nat Immunol 2013, 14:574-583. This study demonstrates how the immunosuppressive microenvironment in the liver, created by liver stromal cells and other sinusoidal cells, can be overcome. Applying Toll-like receptor ligands, myeloid cells form folliclelike structures, which support T cell expansion. Mechanistically, these structures form upon TNF signaling and facilitate an OX40-dependent T cell expansion.
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ScienceDirect Hepatic immune regulation by stromal cells Frank A Schildberg1, Arlene H Sharpe1,2 and Shannon J Turley3 A metabolic organ, the liver also has a central role in tolerance induction. Stromal cells lining the hepatic sinusoids, such as liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), are the first liver cells to encounter gut-derived and systemic antigens, thereby shaping local and systemic tolerance. Recent studies have demonstrated that stromal cells can modulate immune responses by antigen-dependent and independent mechanisms. Stromal cells interfere with the function of other antigen-presenting cells (APCs) and induce non-responsive T cells as well as regulatory T cells and myeloid-derived suppressor cells (MDSCs). The immunosuppressive microenvironment thus created provides a means to protect the liver from tissue damage. Such tolerized surroundings, however, can be exploited by certain pathogens, promoting persistent liver infections. Addresses 1 Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA 2 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 3 Cancer Immunology, Genentech, 1 DNA Way, South San Francisco, CA 94080, USA Corresponding author: Turley, Shannon J ([email protected])
Current Opinion in Immunology 2015, 32:1–6 This review comes from a themed issue on Innate immunity Edited by Zhijian J Chen and Sebastian Amigorena
http://dx.doi.org/10.1016/j.coi.2014.10.002 0952-7915/# 2014 Published by Elsevier Ltd.
Introduction Early experiments in orthotopic liver transplantation gave rise to the concept that immune responses in the liver are biased towards tolerance. While other organs transplanted between unrelated pigs were promptly rejected, allogeneic liver transplants generally not only were tolerated [1], but also able to confer tolerance to another solid organ transplant from the same donor [2]. Over the years, the local regulatory cues that determine the functional complexity of immune responses in the liver have been increasingly well understood [3–5]. This in-depth knowledge of the immune regulatory mechanisms in the liver is of high clinical relevance: it is crucial for protecting the www.sciencedirect.com
integrity of this essential metabolic organ and discerning how pathogens exploit this unique microenvironment. Active modulation of immune responses in the liver stems from its unique microenvironment and hepatocytes, but also results from certain types of stromal cells in the liver sinusoids. LSECs are the most abundant non-parenchymal cell population in the liver and line the hepatic sinusoids (Figure 1a). As unique micro-vascular endothelial cells that resemble lymphatic endothelial cells, they lack a basal membrane and define the space of Disse´ [3,6,7]. The perisinusoidal space between LSECs and hepatocytes is populated by HSCs. Upon activation, HSCs differentiate into myofibroblasts, which produce extracellular matrix leading to hepatic fibrosis. This overview will discuss the immune inhibitory functions, of both LSECs and HSCs.
Liver sinusoidal endothelial cells LSECs skew the differentiation and effector function of CD4+ T cells
LSECs originate from liver-derived endothelial progenitor cells and represent a unique population of APCs. LSECs can be identified by their expression of CD45, CD31 (PECAM-1) and CD146 (MCAM, Muc18), as well as by their high scavenger activity using fluorochromelabeled OVA or BSA. They are perforated by numerous fenestrations, which facilitate the passage of molecules from the sinusoidal lumen into the space of Disse´. Because of their enormous scavenger activity, they compete with dendritic cells for uptake of circulating antigen in the liver [8]. Murine LSECs express only very low levels of major histocompatibility complex II (MHC II) and the co-stimulatory molecules CD80/86. MHC II, CD80 and CD86 expression is further downregulated by lipopolysaccharide present in portal venous blood [9]. This does not, however, preclude the ability of LSECs to prime naı¨ve CD4 T cells, albeit the process does require high antigen concentration. Th1 T cell differentiation, on the other hand, is not promoted by LSECs [10]. Instead, LSECs can inhibit inflammatory cytokine secretion by Th1 and Th17 effector CD4+ T cells in an IL-10 and PD-1-dependent manner [11]. These results suggest that LSECs possess the ability to mitigate hepatic inflammatory CD4+ T cell activity, a highly valuable trait for preventing or terminating hepatic inflammation. LSECs can also restrain inflammation by promoting induction of regulatory CD4+ T cells; LSEC-mediated induction of both Foxp3 CD25low and Foxp3+ regulatory CD4+ T cells has been shown [12]. A comparative analysis Current Opinion in Immunology 2015, 32:1–6
2 Innate immunity
Cross-presentation by LSECs to naı¨ve CD8+ T cells leads to tolerance induction
Figure 1
(a) LSEC T cell
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MDSC Non-responsiveness/ “Memory-like” cell Current Opinion in Immunology
Anatomical position and immune regulatory functions of hepatic stromal cells. (a) The fenestrated LSECs line the hepatic sinusoid and form the space of Disse´, which is populated by HSCs. Both stromal cells separate the blood stream from hepatocytes and thereby function as physical gatekeepers to prevent destructive inflammation into the liver parenchyma. LSECs represent the first barrier for the liver. However, as DCs and T cells continually migrate through the space of Disse´ to reach the hepatic lymph nodes, HSCs are no less important. Both stromal cells are optimally positioned to interact with circulating antigens and extravasating leukocytes. (b) In addition to the barrier function described in (a), LSECs and HSCs also exert potent immune regulatory mechanisms to prevent overwhelming inflammation in the liver. Both cell types can induce Tregs, and inhibit T cell responses and the function of APCs. At this time, only LSECs are known to induce non-responsive, memory-like T cells and only HSCs lead to the induction of MDSCs. Together, these mechanisms contribute to the unique immune regulatory microenvironment of the liver.
of liver APCs concluded that LSECs are the major cell type that stimulates TGFb dependent induction of CD4+ Foxp3+ Tregs. This is possible because LSECs not only can secrete TGFb but also tether LAP/TGFb to their membrane through GARP [13]. Once induced by LSECs, antigen-specific Tregs can be potent suppressors of autoimmune inflammation, as evidenced by in vivo studies in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. LSEC-mediated induction of Tregs, therefore, may be instrumental for controlling hepatic and systemic inflammatory immune responses [13]. Current Opinion in Immunology 2015, 32:1–6
LSECs have a similar cross-presentation mechanism to DCs, efficiently offering soluble antigens to CD8+ T cells [14]. Once LSECs have ingested antigen, the corresponding naı¨ve antigen-specific CD8+ T cells are, within minutes, promptly detained in liver sinusoids. This results in rapid CD8+ T cell accumulation [15]. The interaction of naı¨ve T cells with LSECs produces a non-responsive T cell mode, in which these T cells are only barely receptive to stimulation through the TCR [14]. These results suggest that these T cells are tolerized. However, this T cell non-responsiveness is not a permanent state; a recent study has demonstrated that LSEC-primed CD8+ T cells not only can survive in vivo for a long time, but that they also can be reactivated through a combination of co-stimulatory signals [16], which reinstates their capability to protect against infections. When LSECs induce CD8+ T cell priming, this does not simply lead to the generation of T cells that are ultimately tolerized. In fact, these T cells have a great similarity to memory T cells. This type of liver priming may well rescue T cells from deletion by immature antigen-presenting DCs. Instead of undergoing apoptosis, the LSEC-primed CD8+ T cell population expresses high amounts of the antiapoptotic molecule bcl-2 [15], proliferates and transiently upregulates activation markers [17,18]. Non-functional, this T cell phenotype is dependent on the co-inhibitory PD-L1 molecules expressed by LSECs interacting with the PD-1 receptor on CD8+ T cells [18]. If cognate interactions between MHC class I molecules and TCRs are present during LSEC-induced initial programming of naı¨ve CD8+ T cells, the result is increased expression of co-inhibitory PD-L1, but not of co-stimulatory CD80/86 molecules on LSECs. This further illustrates the importance of balanced delivery of stimulatory and inhibitory signals for the CD8+ T cell fate; increased co-stimulatory signaling through CD28 results in the override of PD-L1– PD1 signaling [18]. Similarly, the increase of signal strength via the TCR is also capable of breaking the non-responsive phenotype: this occurs through the production of IL-2 by T cells. An autocrine co-stimulatory factor, IL-2 production predominates over PD-L1mediated signaling [17]. Intriguingly, the non-responsiveness induced in T cells by LSECs could be a possible target for cancer immunotherapy, because LSECs are capable of cross-presenting circulating tumor antigens, thereby inducing non-responsive antigen-specific CD8+ T cells through the PD-1/PD-L1 pathway [19]. Timing is a highly important factor in the LSECmediated T cell non-responsiveness mechanism: CD28 co-stimulation only can prevent induction of non-responsive LSEC-primed T cells during the first 24–36 h; after this time, the LSEC-induced programming very likely becomes cell-intrinsic [20]. Although it is known that www.sciencedirect.com
Hepatic immune regulation by stromal cells Schildberg, Sharpe and Turley 3
antigen-presenting LSECs require several days to induce these memory-like T cells [16], it is noteworthy that there is exceedingly rapid GzmB induction in T cells in the first 18 h following contact with LSECs [21]. TCR signaling, in combination with IL-6 trans-signaling, is sufficient for this direct and rapid GzmB expression in T cells. It is as yet not established if this phenotype should be considered an intermediate developmental stage or, rather, serves a specific function during immune responses originating in the liver. Intriguingly, this LSEC-mediated direction of the CD8+ T cell fate to dysfunctional versus memory states bears similarities to T cell exhaustion.
markers, although intracellular GFAP for quiescent and aSMA for activated HSCs have been used. However, a recent publication showed that HSCs can be differentiated from other liver cells using size, granularity, and high UV-fluorescence (due to their vitamin A) in combination with the absence of scavenger activity [26]. Once HSCs are activated under inflammatory conditions, they differentiate into myofibroblasts and assume a crucial role in liver fibrogenesis. HSCs also take on the novel role of immune sentinels in the liver. Studies have demonstrated direct contact between HSCs and lymphocytes, indicating that HSCs may directly activate naı¨ve lymphocytes [27,28] (Figure 1a).
Antigen-independent immune regulatory functions of LSECs
The direct activation of lymphocytes by HSCs has, however, been challenged. HSCs express very low amounts of MHC and co-stimulatory molecules [29]; furthermore, they have low antigen uptake and inefficient processing [30]. Under non-inflammatory conditions, HSCs produce insufficient amounts of the molecules needed for antigen presentation. In cell culture, the expression of these molecules could only be upregulated in the presence of certain cytokines. Under inflammatory conditions in the liver, however, chronic liver damage and inflammatory cytokines such as IFNg, alter the HSC phenotype to increase their antigen-presenting ability [31]. As many studies of HSCs involve in vitro cultures, which are very seldom pure, such experiments may not accurately reproduce their composition in the intact liver.
Independent of their APC properties, LSECs also have other immune regulatory functions; so-called ‘bystander functions’. One particularly important property is the surface expression of LSECtin (also known as CLEC4G), a ligand for CD44. LSECs are therefore capable of interacting with activated, but not resting, T cells since CD44 is not expressed on resting T cells. LSECtin/CD44 interaction can inhibit T cell activation and proliferation [22] or induce apoptosis [23]. The functional significance of this interaction is illustrated by the aggravated autoimmune liver disease that develops in the absence of LSECtin [24]. All of these characteristics unite to paint a picture of LSEC-based restraint of T cell effector function, contributing to the prevention of immune-mediated liver damage. In their function as immune regulatory bystander cells, LSECs also contribute to the tolerogenic environment in the liver by interacting and influencing other APCs. LSECs can negatively regulate the APC function of neighboring DCs. This ‘veto effect’ occurs independently of the LSEC antigen presentation, but depends on physical contact. This contact causes DCs to lose their ability to prime naı¨ve T cells by downregulating DC costimulatory signals [25]. This may be the mechanism that leads to inhibition of immunogenic T cell priming in the liver. Since LSEC-mediated regulation of T cell and APC function is not antigen-specific, this likely controls the local threshold to ‘fine tune’ immune effector functions.
Hepatic stellate cells Weak antigen-presenting function of HSCs and their cross-talk in the sinusoid
The developmental origin of HSCs, also called Ito cells, is still under discussion because they express marker genes of all three germ layers. However, based on current knowledge they seem to stem from mesenchymal precursors in the liver. Under non-inflammatory conditions, HSCs have central roles in vitamin A storage and regulating blood flow through the sinusoids. In contrast to LSECs, HSCs are hard to identify based on surface www.sciencedirect.com
In a recent study highly pure HSCs were examined in vitro and found to, completely lack cross-presentation competence [29]. Interestingly, this same study identified bidirectional, intercellular transfer of MHC I between the cell-surfaces of HSCs and LSECs; this is a mechanism similar to trogocytosis [32], where membrane fragments with MHC molecules are exchanged between immune cells. This exchange of MHC I molecules also was shown to occur with other sinusoidal liver cell populations. As an exchange of membrane fragments provides the opportunity for exchange of co-stimulatory or inhibitory molecules, this mechanism could explain how HSCs and other sinusoidal liver cells may acquire both activating and inhibitory properties to facilitate T cell activation in liver infection or T cell tolerance in the healthy liver. The identification of this mechanism necessitates reassessment of the antigen-presenting capacity of all sinusoidal liver cells. Antigen-independent immune regulatory functions of HSCs
Much less controversial than their APC function, HSCs manifest immunosuppressive activities as bystander cells in the context of T cell immunity. HSCs are a rich source of anti-inflammatory mediators [33], interfere with local T cell activation [34] and cause induction as well as the proliferation of Tregs [29]. HSCs seem to regulate adaptive Current Opinion in Immunology 2015, 32:1–6
4 Innate immunity
immunity by several mechanisms, but further work is needed in light of potential contamination with other APCs or in vitro differentiation of HSCs. Best characterized is HSC expression of PD-L1, especially upon exposure to IFNg or activated T cells, and secretion of the inhibitory cytokines IL-10 and TGFb. Activation of HSCs leads to their fibrogenic phenotype, as well as their acquisition of potent T cell suppressive functions. This is partially mediated by PD-L1 ligation [35,36] but an essential role for B7-H4 [37], TRAIL [38] and ICAM-1 [34,39] has also been described. HSCs also can induce and expand Foxp3+ Tregs; however, the underlying mechanism is still controversial due to their questioned APC function. Most likely HSCs act as a crucial bystander to promote liver DC-induced activation of Tregs [40]. Since HSCs form a second layer of cells between the bloodstream and hepatocytes, the immune regulatory properties of HSCs can limit the effector function of those T cells that have extravasated from the sinusoidal lumen, preventing tissue damage and loss of organ function. This immune regulatory function is so strong that HSCs are even able to promote pancreatic islet allograft survival if they are co-transplanted [35,41]. HSCs not only function as immune regulatory bystander cells during T cell activation and Treg induction, but also during contact with myeloid cells: HSCs also diminish the APC function of DCs [42,43,44]. In addition, during chronic inflammation in the liver, HSCs facilitate the differentiation of inflammatory monocytes into MDSCs that impair T cell proliferation and effector function [45,46]. The induction of multiple types of immune inhibitory cells by HSCs leads to a complex suppressive microenvironment.
Conclusions LSECs and HSCs are highly involved in the maintenance of local and systemic immune tolerance. In the hepatic microenvironment they establish a functional barrier to protect hepatocytes from immune-mediated liver injury, which potentially may lead to loss of organ function. Both cell types act as immunological bystander cells by impairing the APC function of dendritic cells, interfering with T cell activation and inducing suppressive Tregs and MDSCs (Figure 1b). In contrast, HSCs lack significant APC functions, whereas LSECs use their APC function for tolerance induction. The combination of these mechanisms indicates that the hepatic sinusoid surface area is a potent immunoregulatory entity capable of modulating immune responses. Recent studies challenging the APC function of HSCs show the crucial need for developing new isolation methods to obtain highly pure liver cell populations in the future [26]. Although cellular contamination issues may be easier to resolve, artifacts based on in vitro differentiation will require novel model systems and technologies that reflect the complex regulation of Current Opinion in Immunology 2015, 32:1–6
intrahepatic immunity. Above all, more human studies and advanced cell-type-specific mouse models are needed. Although there is clear evidence that HSCs become more suppressive during fibrosis, sufficient data whether this is also true for LSECs are still missing. We stand but at the very beginning of decoding the triggers regulating the bystander and APC function of these key liver stromal cells and determining how they could be manipulated. Another important remaining question is which of these immune regulatory mechanisms are exploited by chronic hepatic infections. Although HSCs and LSECs set a high threshold for adaptive T cell responses, they can drive liver inflammation through innate immune mechanisms [47–49]. A recent publication describes a mechanism to overcome the hepatic regulatory cues by applying Tolllike receptor signaling or viral infection which leads to intrahepatic myeloid cell aggregates associated with T cell expansion (iMATEs) [50]. It would be of tremendous relevance to understand if and how the immune regulatory capacity of LSECs and HSCs are regulated in this context because this knowledge could be used for novel therapeutic vaccination strategies. In summary, the liver ‘stromal cell’ populations LSECs and HSCs control hepatic immune regulation by multiple mechanisms and we are only beginning to elucidate their immunoregulatory properties. Further understanding of hepatic immune tolerance will provide the basis for developing new immunotherapies targeting chronic viral infection and cancer.
Acknowledgements This work was supported by grants from the National Institutes of Health (R01 AI40614, to A.H.S.) and the Harvard University Center for AIDS Research (CFAR), an NIH funded program (P30 AI060354) which is supported by the following NIH Co-Funding and Participating Institutes and Centers: NIAID, NCI, NICHD, NHLBI, NIDA, NIMH, NIA, NIDDK, NIGMS, FIC, and OAR (to F.A.S.). F.A.S. acknowledges the International Society for Advancement of Cytometry for support as an ISAC Scholar. Because of space restrictions, we were able to cite only a fraction of the relevant literature and apologize to colleagues whose contributions may not be appropriately acknowledged. The authors thank Cristina A. Hagmann for editing the manuscript.
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44. Lee WC, Yu MC, Chiang YJ, Wang HC, Lu L, Qian S: Liver stellate cells suppress dendritic cells through IL-10. Transplant Proc 2005, 37:10-11. 45. Hochst B, Schildberg FA, Sauerborn P, Gabel YA, Gevensleben H, Goltz D, Heukamp LC, Turler A, Ballmaier M, Gieseke F et al.: Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion. J Hepatol 2013, 59:528-535. This study reveals that activated human HSCs convert mature peripheral blood monocytes into immunosuppressive CD14+HLA-DR /low MDSCs. The HSC-mediated induction of monocyte-derived MDSCs is independent of soluble factors, but requires physical interaction. This study identifies CD44 as one crucial mediator to induce human MDSCs. 46. Hsieh CC, Chou HS, Yang HR, Lin F, Bhatt S, Qin J, Wang L, Fung JJ, Qian S, Lu L: The role of complement component 3 (C3) in differentiation of myeloid-derived suppressor cells. Blood 2013, 121:1760-1768. This work demonstrates that murine HSCs added to a bone marrow DC culture support the development of immune-inhibitory MDSCs instead of DCs. In this model system, HSC-mediated MDSC induction is highly dependent on the expression of complement component 3 by HSCs and mediated by its cleaved product iC3b.
40. Dunham RM, Thapa M, Velazquez VM, Elrod EJ, Denning TL, Pulendran B, Grakoui A: Hepatic stellate cells preferentially induce Foxp3+ regulatory T cells by production of retinoic acid. J Immunol 2013, 190:2009-2016. This article shows that HSCs contribute to the generation of Foxp3+ Tregs through an antigen-independent mechanism. The authors demonstrate that, although HSCs alone do not present antigen to naive CD4+ T cells, HSCs induce functional Tregs by all-trans retinoic acid (ATRA) in the presence of dendritic cells and TGF-b1.
47. Kern M, Popov A, Scholz K, Schumak B, Djandji D, Limmer A, Eggle D, Sacher T, Zawatzky R, Holtappels R et al.: Virally infected mouse liver endothelial cells trigger CD8+ T-cell immunity. Gastroenterology 2010, 138:336-346.
41. Chen CH, Kuo LM, Chang Y, Wu W, Goldbach C, Ross MA, Stolz DB, Chen L, Fung JJ, Lu L et al.: In vivo immune modulatory activity of hepatic stellate cells in mice. Hepatology 2006, 44:1171-1181.
49. Liu J, Jiang M, Ma Z, Dietze KK, Zelinskyy G, Yang D, Dittmer U, Schlaak JF, Roggendorf M, Lu M: TLR1/2 ligand-stimulated mouse liver endothelial cells secrete IL-12 and trigger CD8+ T cell immunity in vitro. J Immunol 2013, 191:6178-6190.
42. Sumpter TL, Dangi A, Matta BM, Huang C, Stolz DB, Vodovotz Y, Thomson AW, Gandhi CR: Hepatic stellate cells undermine the allostimulatory function of liver myeloid dendritic cells via STAT3-dependent induction of IDO. J Immunol 2012, 189:3848-3858. 43. Bhatt S, Qin J, Bennett C, Qian S, Fung JJ, Hamilton TA, Lu L: All trans retinoic acid induces arginase-1 and inducible nitric oxide synthase-producing dendritic cells with T cell inhibitory function. J Immunol 2014, 192:5098-5108. This report describes the mechanism by which HSC-secreted ATRA leads to the development of tolerogenic DCs. Mechanistically, ATRA enhances the expression of both Arg-1 and iNOS in IFNg-treated DCs, which leads to a tolerogenic DC phenotype.
Current Opinion in Immunology 2015, 32:1–6
48. McHedlidze T, Waldner M, Zopf S, Walker J, Rankin AL, Schuchmann M, Voehringer D, McKenzie AN, Neurath MF, Pflanz S et al.: Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis. Immunity 2013, 39:357-371.
50. Huang LR, Wohlleber D, Reisinger F, Jenne CN, Cheng RL, Abdullah Z, Schildberg FA, Odenthal M, Dienes HP, van Rooijen N et al.: Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection. Nat Immunol 2013, 14:574-583. This study demonstrates how the immunosuppressive microenvironment in the liver, created by liver stromal cells and other sinusoidal cells, can be overcome. Applying Toll-like receptor ligands, myeloid cells form folliclelike structures, which support T cell expansion. Mechanistically, these structures form upon TNF signaling and facilitate an OX40-dependent T cell expansion.
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