Cellular & Molecular (2013) 10, 292–302 ß 2013 CSI and USTC. All rights reserved 1672-7681/13 $32.00 www.nature.com/cmi

REVIEW

The liver works as a school to educate regulatory immune cells

Fenglei Li1 and Zhigang Tian1,2

Because of its unique blood supply, the liver maintains a special local immune tolerogenic microenvironment. Moreover, the liver can impart this immune tolerogenic effect on other organs, thus inducing systemic . The network of hepatic regulatory cells is an important mechanism underlying liver tolerance. Many types of liver-resident -presenting cells (APCs) have immune regulatory function, and more importantly, they can also induce the differentiation of circulating immune cells into regulatory cells to further extend systemic tolerance. Thus, the liver can be seen as a type of ‘school,’ where liver APCs function as ‘teachers’ and circulating immune cells function as ‘students.’ Cellular & Molecular Immunology (2013) 10, 292–302; doi:10.1038/cmi.2013.7; published online 22 April 2013

Keywords: liver; immune regulation; immune tolerance

INTRODUCTION accepted when transplanted into hosts with a mismatched The is the most important weapon used by the major histocompatibility complex (MHC) background in a body to defend itself against pathogens and unwanted self-cells, pig model of transplantation.5 Tolerance is also achieved by but abnormal immune responses sometimes cause severe intraportal antigen delivery into the liver, which can then damage to the host. Therefore, the accuracy of the immune induce systemic tolerance.6,7 Similarly, oral tolerance—the sys- system is often more important than its ability to defend. To temic tolerance induced by orally administering foreign anti- accomplish this, the first requirement is for immune cells to gens—is also dependent upon the liver.6 Under pathological distinguish between self and non-self. T cells undergo positive states, such as hepatitis viral infection, immune tolerance, and negative selection in the , where the self-reactive T- rather than an immune response, is often induced in the liver.8 cell repertoire dies by . This central prevents the There are several ways to explain why the liver is a site of immune system from retaining memory to self. However, immune tolerance. The classic hypothesis is that the liver func- many low-affinity self-reactive T cells can still escape from tions as a ‘graveyard’ for T cells. Huang et al.9 found that when the thymus and spread into the periphery.1–3 Thus, the second T-cell receptor (TCR) transgenic mice were injected with a requirement is for the immune response to maintain an appro- specific peptide for a given TCR antigen, activated T cells tra- priate level even when confronted with an enemy, as a weak veled to the liver and died. Later, anti-CD3 administration was immune response cannot effectively clear pathogens, but an also found to induce activated T-cell accumulation and apop- excessively strong immune response can lead to autoimmune tosis in the liver.10 Although this hypothesis drew the attention disease and . of many immunologists, it could not explain the observation The unique systemic circulation within the liver makes it not that effector and memory cells were also generated during viral only able to obtain blood from the hepatic artery, but also infection, especially in hepatitis, and could sometimes clear the through the portal vein, the latter containing nutrients, meta- infection. bolic products, toxins and soluble .4 Faced with hand- Another viable explanation for liver tolerance is its unique ling all of these components, the liver also plays a role hepatic regulatory mechanisms. From an evolutionary and dif- in metabolic detoxification. Moreover, increased evidence ferentiation perspective, the fetal liver has the same hemato- demonstrates that the liver is also an important immunoto- poietic function as ; interestingly, the latter was lerant organ. As a striking example of this, liver allografts are also found to have immune regulatory potential.11 Many types

1Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China and 2Institute of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China Correspondence: Dr ZG Tian, School of Life Sciences, University of Science and Technology of China, 443 Huangshan Road, Hefei 230027, China. E-mail: [email protected] Received: 14 February 2013; Accepted: 21 February 2013 The liver works as a school to educate regulatory immune cells FL Li and ZG Tian 293 of liver-resident nonparenchymal cells and hepatocytes can may be able to modulate immune responses in other organs present antigens and often exhibit a unique regulatory function and induce systemic immune tolerance. compared with their counterparts in other organs. These anti- The influenza virus causes respiratory tract infections and gen-presenting cells (APCs) can also interact with other cir- induces lung . Interestingly, influenza can also culating cells and endow them with regulatory function. In cause hepatitis even though no viral titers can be detected in turn, the newly induced regulatory cells can go on to induce the liver, because virus-specific cytotoxic (CTLs) regulatory function in other cells, even in the periphery. Thus, originally generated within the lung can encounter Kupffer resident and circulating cells work cooperatively to form a cells in the liver and induce hepatocyte apoptosis.12 A similar complex network that maintains liver tolerance. Indeed, an phenomenon has been observed between the pancreas and the increasing number of studies demonstrate that the liver func- liver, where liver-derived, ex vivo-expanded dendritic cells tions in a dramatic way to control immune responses. In this (DCs) can improve islet allograft survival.13 Taken together, review, we will discuss how the liver functions as a ‘school’ to these observations suggest that immune cells traffic between educate regulatory cells. the liver and other organs to regulate local immune responses. The liver has also been shown to interact with and induce THE LIVER EXERTS BOTH LOCAL AND SYSTEMIC immune tolerance within the central nervous system (CNS). TOLEROGENIC EFFECTS Niemann–Pick disease is characterized by excessive sphingo- Liver tolerance is not locally restricted to the liver, as crosstalk myelin buildup in the brain, and recovery of acid sphingomye- often occurs between the liver and other organs. This crosstalk linase (ASM) expression in the host is key to a cure. While regulates the extra-hepatic immune responses that finally lead intracranial delivery of an ASM-expressing vector is not viable to systemic tolerance (Figure 1). The most well-known and because ASM-specific will be produced, Cheng and accepted illustration of this is the systemic immune tolerance colleagues14 found that simultaneously delivering an ASM- induced when a liver is transplanted into a host, where to- expressing vector into both the brain and the liver promoted lerance is induced not only to the liver, but also to skin allo- tolerance to this protein and prevented secretion, thus grafts cotransplanted from the same donor.5 Additionally, liver achieving an effective therapy. This suggested that the liver- allografts can survive without immunosuppressive drugs, even generated regulatory immune response toward ASM could though other organs—such as the skin, kidney and heart—are inhibit the destructive immune response occurring in the brain. rapidly rejected. Thus, liver allograft protection against donor- Experimental autoimmune encephalomyelitis is a mouse model specific skin and kidney rejection suggests that allogeneic liver of the autoimmune disease multiple sclerosis. It is induced in

Normal organs after hepatic pathogen clearance pancreas pancreas pancreas

skin skin skin

CNS CNS CNS kidneys 2 kidneys 3 kidneys liver liver liver

pancreas hearts hearts

1 hearts lung lung lung

skin

muscle muscle

muscle LN LN LN CNS

kidneys liver

pancreas pancreas pancreas hearts lung

skin skin skin muscle LN

4

CNS CNS CNS

kidneys 5 kidneys 6 kidneys

liver liver liver

hearts hearts lung lung hearts lung

muscle muscle LN LN muscle LN

Liver infection induces systemic immune tolerance to infection

Figure 1 Crosstalk between the liver and other organs can regulate in other organs. Unlike other immune-privileged organs, the liver contains many types of immune cells. (1) The immune response often occurs in the liver during virus infection, transplantation, ectopic antigen expression and so on. (2) The liver can sometimes generate a positive immune response to eliminate various ‘enemies’, (3) which is followed by the generation of immune defense throughout the body. (4) Immune tolerance is more often induced, allowing foreign antigens to persist in the liver. (5) Additionally, the liver can crosstalk with other organs and regulate their immune responses, effectively inducing systemic tolerance. (6) After transferring immune tolerance from the liver to the entire body, systemic immune responses are suppressed, allowing for virus infection, tumor migration, transplant acceptance and so on. CNS, central nervous system; LN, lymph node.

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the CNS and can also be controlled by liver tolerance. Liver, but APCs27 or secrete regulatory , such as IL-10 and trans- not skin, expression of the disease-associated antigen, myelin forming growth factor (TGF)-b.20 basic protein, induces protection from autoimmune neuroin- Hepatic Tregs play an important role in regulating hepatic flammation in this model. Intrahepatic MBP expression immunity and maintaining liver tolerance. Decreased Treg fre- induces the production of MBP-specific CD41CD251Foxp31 quency and their impaired function in the liver have been regulatory T cells (Tregs). Adoptively transferring these cells associated with immune-induced liver injury in several into recipient mice prevents disease,15 further indicating that cases.23–25 Our group previously found that the Tregs fre- immune responses generated in the liver can regulate the quency increased in a mouse model of ConA-induced injury. immune response in the CNS and brain. Liver injury increased in the mice after Tregs were depleted by Human coagulation factor IX (hF.IX) effectively treats the removing CD251 cells; moreover, adoptive transfer of Tregs X-linked bleeding disorder hemophilia B, but immune res- attenuated ConA-induced liver injury in a TGF-b-dependent ponses to hF.IX are a concern. Intrahepatic hF.IX expression manner.28 In an independent study, Tregs cells mediated liver has been shown to induce Tregs cells that inhibit both T helper tolerance in a similar ConA-induced mouse model through IL- (Th) cell subset generation and anti-hF.IX antibody forma- 10 secretion.29 In patients infected with hepatitis C virus, Tregs tion.16 Moreover, this tolerance was maintained even after constitute nearly half of the CD41 T-cell30 population in the immunizing mice against hF.IX intramuscularly, which nor- peripheral blood; they are believed to suppress effector T-cell mally induces a strong immune response in the absence of function and induce viral persistence.31,32 Hepatic Tregs also intrahepatic hF.IX administration.17 This phenomenon illus- play an important role in liver transplantation. They increase in trates that the liver can regulate immune responses in muscle or liver grafts after transplant and are initially required for graft the draining lymph node. acceptance, as rejection occurs upon anti-CD25 antibody- mediated Treg depletion.33 Interestingly, ectopic antigen CIRCULATING REGULATORY IMMUNE CELL expression in the liver induces antigen-specific Tregs, thus sup- ‘STUDENTS’ ARE EDUCATED IN THE LIVER plying a therapeutic strategy to combat autoimmune disease in Unlike other immune-privileged sites, such as the renal capsule other organs.15 and eye, the liver contains various types and numbers of lym- In contrast to the transplant rejection observed after Treg phocytes. Thus, while the liver can potentially eliminate patho- depletion is performed prior to transplantation, Treg depletion gens in situ, immune tolerance usually develops instead. These has no effect if performed well after liver transplantation (i.e. 20 lymphocytes include both innate (natural killer (NK) and na- days).34 A similar phenomenon occurs in a ConA-induced liver tural killer T (NKT) cells) and adaptive immune cells (T and B tolerance model, where Tregs rapidly increase in the liver after cells), which are scattered throughout the parenchyma and ConA induction but later return to normal levels.29 These migrate through the hepatic sinusoids. Although these cells results may explain why steady-state livers have so few Tregs are capable of immune surveillance and pathogen clearance, cells: because the liver contains many cells with regulatory most of them acquire or display regulatory function after enter- function, Tregs may not be needed at steady-state levels or in ing the liver. We therefore call these circulating immune cells a tolerance-induced state, but they may be needed to induce ‘students’ that are educated in the liver. tolerance.

Tregs NKT cells Tregs are among the most powerful regulatory cells, playing an NKT cells are a major subset of liver lymphocytes that reside in important role in restricting immune responses toward self- the sinusoid.35 The liver contains the highest ratio of NKT/ and foreign antigens. CD25 and Foxp3 expression are two conventional T cells compared to other organs.36 NKT cells major Treg markers and are essential for their development have features of both T and NK cells. Based on TCR expression, and function.18,19 While natural Tregs develop in the thymus,20 NKT cells can be divided into classical and non-classical NKT Tregs in peripheral organs can originate from either prolifer- cells.35,37 a-GalCer is widely used as the model antigen to ated natural Tregs or inducible Tregs, which are induced from investigate NKT cell function, and the non-classical MHC Foxp3-negative conventional CD41 T cells or even differen- molecule CD1d is believed to present glycolipid antigens to tiated CD41 T cells.21,22 NKT cells.37,38 Many CD1d-expressing hepatic APCs, includ- Normal liver contains relatively few Tregs cells (0.5%–1% of ing Kupffer cells, liver sinusoidal endothelial cells (LSECs), lymphocytes) compared with the spleen, but they play an hepatocytes, DCs, B cells and hepatic stellate cells (HSC), can important role in regulating hepatic immunity. Indeed, interact with NKT cells.37 decreased Treg frequency or function leads to increasingly dys- Activated NKT cells can express interferon (IFN)-c and IL- regulated immune responses in the liver, eventually causing 17, which are strong antiviral cytokines. However, they can also autoimmune disease and primary biliary cirrhosis.23–25 Most secrete large amounts of the anti-inflammatory cytokines IL-4 liver APCs can induce Treg development and recruit circulat- and IL-10.39–41 NKT cells exhibit powerful immune regulation ing Tregs; this will be discussed in detail below. Tregs can over autoimmune disease.42 The most well-known model is the functionally suppress other cells in both direct and indirect type 1 diabetes-susceptible non-obese diabetic (NOD) mouse manners:26 they can directly interact with effector T cells and model, where adoptive transfer of NKT cells can prevent

Cellular & Molecular Immunology The liver works as a school to educate regulatory immune cells FL Li and ZG Tian 295 diabetes onset,43,44 presumably by IL-4-mediated skewing of be still induced in CD82/2 mice, NK cell depletion by NK1.1 CD41 T cells toward Th2, thus preventing Th1-mediated antibody completely abrogated islet allograft persistence after autoimmune responses in NOD mice.45 Moreover, NKT cells anti-CD154 treatment. Moreover, islet allografts are rejected in can also suppress immune responses by secreting IL-10 in auto- perforin-deficient recipients, and perforin-secreting NK cells immune disease.46,47 Interestingly, although IL-17 is often con- are sufficient to restore tolerance in these mice.70 Hepatic NK sidered to be a pro-inflammatory , NKT cell-derived cells also produce chemokines that promote immune to- IL-17 prevents inflammatory infiltration in an a- lerance, including inflammatory protein-1a and GalCer-induced liver injury model, indicating that it also pos- -1b, which induce hepatocytes and LSECs to secrete CXCL9 to sesses anti-inflammatory properties.48 Indeed, neutralizing this recruit T cells into the liver, ultimately resulting in T-cell NKT-derived IL-17 exacerbates hepatitis with increased hep- tolerance.71 atic and , while pre-injecting IL-17 ame- NK cells also have a unique regulatory function that depends liorates hepatitis and inhibits inflammatory monocyte on their cytotoxic ability. Although APCs in skin allografts can recruitment to the liver.48 home to draining lymph nodes in recipient mice and activate Recently, crosstalk between NKT and other cells within the the alloreactive T cells that induce allograft rejection, NK cells liver was studied in detail. NKT cells enhance the proliferation can arrest this process by killing APCs and regulating T-cell and expression of cytotoxic T- antigen (CTLA)-4 priming.72 Additionally, NK cells can also detect and lyse auto- on Tregs through IL-2 secretion.49 Oo et al.50 found that a- reactive T cells and DCs.67,73 GalCer-activated NKT cells can indirectly recruit Tregs into the NK cells cocultured with hepatocytes promote DCs to prime liver, as follows: activated NKT cells first secrete IFN-c, which CD41 T cells, which then acquire Tregs cell properties. This then increases IP-10/CXCL10 chemokine expression by process depends on engaging NKG2A on NK cells,74 as NKG2A Kupffer cells, hepatocytes and biliary epithelial cells. CXCR3- signals induce changes in the cytokine milieu of cocultured expressing Tregs are then recruited into the liver in a CXCL10- cells, including decreased TNF-a and increased TGF-b concen- dependent manner and suppress the IL-10-mediated inflammatory trations. In contrast to classical Tregs, NK cell-primed DC- response. NKT cells also crosstalk with myeloid-derived sup- induced Tregs cells exert their suppressive functions through pressor cells (MDSCs), which constitutively express CD1d.51 a programmed death-1 (PD-1)-mediated pathway,74 indi- Immature monocyte recruitment into the liver was found in cating that NK cell receptor signaling can also regulate the the acute liver injury model, although suppressive function was immune function of other cells. not examined.52 Moreover, activated NKT cells crosstalk with DCs in a CD40L–CD40-dependent manner, resulting in semima- Myeloid-derived suppressor cells ture DCs that can suppress immune responses.53,54 MDSCs, which have a high frequency in the liver (approxi- mately 5% of all hepatic cells), include immature monocytes, Natural killer cells such as , and DCs, and are identified The liver contains much higher NK cell numbers than other by the following surface markers: Gr-11Mac-11 in mice and organs, comprising 20%–30% of all lymphocytes in the CD331CD11b1CD142 in humans. MDSCs have the powerful liver.55–57 As a major subset of innate immune cells, NK cells ability to suppress T-cell proliferation, inhibit NK cell cytotoxi- play an important role in early pathogen control against viruses city,75 and induce the production of regulatory M2 cells76 and and bacteria, as well as in controlling cancer cell growth.58,59 NK Tregs.77 Two distinct MDSC subsets can be distinguished: cell function is determined by the balance of activating- and the monocytic subset (CD11b1Ly6G2LY6Chigh) suppres- inhibitory-receptor expression,60,61 which may be influenced by ses other cells—particularly by producing inducible nitric the liver microenvironment.62,63 The liver contains a prominent oxide synthase and arginase 1—while the granulocytic NKG2A1Ly492 NK cell subset in a functionally hyporesponsive subset (CD11b1Ly6G1LY6Clow) primarily mediates immuno- state, as they exhibit a dampened IFN-c response to IL-12/IL-18 suppression by producing reactive oxygen species.78,79 stimulation. Additionally, adoptively transferred splenic NK cells MDSCs promote liver immune tolerance through several that migrate into the liver adopt the phenotype and function of mechanisms and in various disease models. MDSCs were first liver-resident NK cells.64 Therefore, the local liver microenviron- found in tumors. In liver carcinoma, MDSCs suppress the anti- ment may modify NK cell receptor expression and responsive- tumor activity of T cells and NK cells, thus promoting disease.80 ness to cytokine stimulation. MDSCs from hepatocellular carcinoma patients inhibit auto- NK cells possess several different regulatory functions, one of logous NK cell cytotoxicity and cytokine secretion by cell–cell which is secreting various cytokines and chemokines.65–67 NK contact, mainly through NKp30 expressed on NK cells.81 cell-derived IL-10 and TGF-b, for example, negatively regulate Preliminary data demonstrated that NKT cells could recruit immune responses and maintain tolerance during transplant MDSCs into the liver to suppress CD41 -mediated and pregnancy.68 NK cells can also inhibit autoreactive T-cell immune responses.82 In a transplant model, cotransplanting function and proliferation through IL-10 secretion.69 In a HSCs and islet cells leads to the long-term survival of islet transplant model of anti-CD154-induced long-term islet allo- allografts. While Treg induction and effector T-cell apoptosis graft survival in mice, tolerance depends on the MHC class I were considered to be the main mechanisms underlying this molecule.70 Further study indicated that while tolerance could process, MDSCs have recently been found to also induce

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immune suppression in this model. Chou et al.83 found that lymphocytes, liver-resident APCs will then perform their own cotransplanting HSCs promoted MDSC generation both in immune regulatory functions and, more importantly, induce vitro and in vivo and that soluble factors mediated this IFN-c the differentiation of circulating cells toward a regulatory state. signaling-dependent process. More importantly, they also Because of these two features, hepatic APCs are worthy of earn- found that cotransplanted MDSCs could promote long-term ing the name ‘teacher’ within the context of the liver (Figure 2, islet allograft survival. MDSCs are also important during viral Table 1). infection. In a hepatitis B virus (HBV) transgenic mouse model, MDSC frequency in the liver increased from 6.05% in Kupffer cells normal mice to 13.6% in transgenic mice. In addition, HBV- Kupffer cells represent 20% of liver nonparenchymal cells and transgenic hepatic MDSCs were able to powerfully suppress the are the largest fixed macrophage population in the body. They generation of hepatitis B surface antigen-specific lympho- reside in the periportal area of the sinusoidal vascular space and cytes.84 MDSCs also can work as an important negative feed- are specifically identified by their expression of MHC-II mole- back regulator for the Th1 immune response within the liver. In cules, CD80, CD86 and ICAM-1. Due to their location and a model where TGFb2/2 mice develop acute liver inflam- slow blood flow, Kupffer cells are perfectly suited to clear endo- mation caused by CD41 T cell-derived IFN-c, MDSCs were toxins and microorganisms from the blood. These features found adjacent to Th1 cells in the liver. After CD41 T-cell additionally facilitate contact between Kupffer cells and other depletion or the inhibition of IFN-c signals, MDSCs were sig- cells, which allows Kupffer cells to ‘educate’ them, thus indu- nificantly reduced.85 cing liver tolerance to many antigen sources.93–95 In the above examples, MDSCs were located at the sites of OVA-specific OT-I T cells transferred into mice through the ongoing immune response in the liver. More interestingly, portal vein can detect Kupffer cells loaded with an OVA- however, MDSCs selectively accumulated inside the liver even derived peptide in the liver, which then induces T-cell tolerance when the immune response took place elsewhere. In a tumor to OVA by causing activation-induced apoptosis.96 The most model using DA-3 cell lines subcutaneously injected into mice, unique characteristic of Kupffer cells is their ability to secrete MDSCs homed to and increased in number within the liver. the regulatory cytokines IL-10 and TGF-b after LPS stimu- MDSCs were then able to upregulate PD-L1 expression on liver lation.97 Kupffer cells can also suppress DC-mediated T-cell 86 Kupffer cells, contributing to immunosuppression. activation through prostaglandin (PG)E2, 15d-PGJ2 and nitric oxide expression.98,99 Similarly, they can downregulate antigen LIVER-RESIDENT ‘EDUCATORS’ TEACH CIRCULATING uptake by LSECs and decrease T-cell activation though TNF-a CELLS IN THE LIVER and IL-10.100 Moreover, they can enhance IL-10 expression by The liver’s unique blood transport system strongly influences Tregs in the liver, which is very important for maintaining its ability to harbor immune regulatory function. Terminal a tolerant microenvironment.101 Studies also indicate that portal vessels function as the main blood supply, and a large Kupffer cells are important APCs that interact with NKT cells number of circulating lymphocytes contained in the blood by presenting lipid antigens.102 Thus, Kupffer cells interact in a (approximately 108 per 24 h) encounter liver-resident cells at tolerogenic way with almost all other cell subsets in the liver. liver sinusoids. One type of these cells is fenestrated LSECs, which form a layer of thin vessels that function to separate Hepatocytes the blood from hepatocytes.87 Kupffer cells and DCs, located Hepatocytes occupy two-thirds of the total cell population in the in the sinusoidal lumen, are also important liver-resident liver and are responsible for most of its metabolic functions. APCs. The small space of Disse forms another barrier separat- Facing plenty of gut antigens, as well as neoantigens synthesized ing LSECs from hepatocytes, and HSCs also reside in these during the metabolic process, hepatocytes can act like APCs and spaces. Because the sinusoids are small in diameter and exhibit function to induce tolerance. Although they are located beyond low perfusion pressure, leukocytes can easily adhere to this the sinusoid, hepatocytes can contact T cells via their microvilli location without expressing selectins. This allows sufficient that extend through the endothelium.103 Although MHC and time for resident APCs, including Kupffer cells, LSECs and CD1 expression on the surface of hepatocytes allows them to DCs, to come into contact with circulating lymphocytes.88 present antigens to both T and NKT cells, their lack of costimu- Due to the LSEC fenestrations, circulating lymphocytes can latory molecules and CD40 expression skews this process toward enter into the space of Disse and come into contact with other tolerance rather than activation by several different mechanisms. cells that reside there, such as HSCs and hepatocytes. In addi- After encountering these tolerance-inducing hepatocytes, T cells tion to functioning to present antigens, APCs first function to are clonally eliminated by apoptosis.104,105 Moreover, Th2 cells recruit lymphocytes from the blood. LSECs and hepatocytes are preferentially induced when na¨ıve CD41 T cells encounter bearing cognate antigens can recruit the corresponding anti- hepatocytes, dampening the Th1 type response and, conse- gen-specific CD81 T cells in an antigen-dependent man- quently, CTL-mediated antiviral immunity.106 This impaired ner.89,90 Immune cells can also be recruited to the liver in a Th1 cell induction may be caused by low levels of Delta-like chemokine-dependent manner during inflammation.91 For Notch ligand, a key promoter of the Th1 response, in hepato- example, expression of CXCR3 on LSECs and CCR4 on DCs cytes.107 Hepatocytes also induce antigen-specific Tregs cells, is required for Treg recruitment.92 After recruiting circulating which prevent autoimmune disease.15 Furthermore, NK

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Hepatocyte Hepatocyte “Teachers” HSC LSEC LSEC DC 11 3

4 7 6 5 8 15 NK

NKT 12 16 “Students” Treg CD4+T 17

13 14 1 MDSC

2 18 DC KC 9 10 LSEC “Teachers” HSC Hepatocyte

Figure 2 The regulatory network among the ‘teachers’ and ‘students’ in the liver. The resident hepatic cells (teachers), including hepatocytes, Kupffer cells, DCs, LSECs and HSCs, form a loose blood supply channel called the sinusoid. This unique structure gives the resident cells sufficient room and time to encounter and regulate circulating cells (students), including T cells, NK cells, NKT cells and MDSCs. The crosstalk between the teachers and students forms a complex regulatory network. The effect of the education process on each pair of interacting cells is summarized in Table 1. DC, ; HSC, hepatic stellate cell; LSEC, liver sinusoidal endothelial cell; MDSC, myeloid-derived suppressor cell; NK, ; NKT, . cell–hepatocyte interaction via NKG2A-Qa-1b engagement can suppress Th cell-mediated immune responses, they can also result in increased IL-10 and decreased IFN-c production by NK induce CD25lowFoxp32 regulatory T cells.115 LSECs can also cells.74 When encountering type I IFN-secreting NKT cells, directly downregulate the ability of DCs to activate T cells.116 hepatocytes can induce IL-10-expressing CD81 Tcells,which also exhibit regulatory function.108 Hepatic stellate cells HSCs are located in the subendothelial space of Disse, and their LSECs main metabolic function is storing vitamin A and fat.117 HSCs LSECs form the structural base of hepatic sinusoids. Because can express many cytokines, participate in antimicrobial hepatic sinusoid structures do not contain an organized base- immunity, and function as APCs to cross-prime CD81 T cells ment membrane layer, LSECs are the first APCs to contact and present lipid antigens to NKT cells.118 Additionally, acti- antigens. As scavenger cells, LSECs exhibit a powerful antigen vated HSCs induce B7-H1- and TNF-related apoptosis- uptake ability that is even stronger than that of Kupffer cells,109 inducing ligand-mediated T-cell apoptosis.119,120 HSCs also and they can process and present antigens to other cells. induce CD41CD251Foxp31 Tregs cell expansion from However, their lack of MHC molecule expression and IL-12 CD41CD251Foxp32 effector T cells in an IL-2-dependent secretion prevents them from functioning like professional manner; these Tregs efficiently inhibit anti-CD3-induced T- APCs, and thus, they cannot stimulate Th cells to clonally cell proliferation.121 Further study of the islet and HSC cotrans- expand or be eliminated.110 LSECs also play an important role fer mouse model (described above) showed that HSCs play in inducing oral, as well as LSEC-primed CD81 T cell tolerance, dual roles in this process: they induce donor-derived antigen- which is mostly dependent on the expression of B7-H1 on specific effector T-cell apoptosis and expand Tregs.122 LSECs and PD-1 on CD81 T cells.111,112 Moreover, HSCs from IFN-c-receptor 1 knockout mice lose Unlike other APCs in the liver, LSECs possess regulatory this ability to protect, demonstrating that HSC-derived IFN-c functions independent of . They express signaling is very important for inducing Tregs.122 various C-type lectins, such as LSECtin, which inhibit T-cell proliferation and effector-cytokine secretion and induce T-cell Dendritic cells apoptosis by interacting with CD44 on activated T cells.113,114 The liver contains more DCs than any other parenchymal Interestingly, while LSECs can induce Treg differentiation to organ.123 Liver DCs include three subsets: myeloid and

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Table 1 The effect of education on regulators Label Inducer Responder Effect of education References 1 Kupffer Treg Enhance IL-10 expression 98 2 Kupffer NKT Enhance IL-10 expression 99 3 Hepatocyte NK Increase IL-10 and decrease IFN-c expression 71 4 Hepatocyte NKT Induce IL-10-expressing CD81T cells 105 5 Hepatocyte Treg Prevent autoimmune disease 14 6 LSEC Treg Induce CD25lowfoxp32 regulatory T cells 113 7 HSC Treg Expansion of effector T cells 118,119 8 DC Treg Induce IL-10-producing ‘T cells and Tregs’ 71,130 9 Kupffer DC Suppress activation ability 95,96 10 Kupffer LSEC Downregulate antigen uptake and activation ability 97 11 LSEC DC Downregulate activation ability 113 12 NKT Treg Enhance proliferation and CTLA-4 expression Recruit Tregs into the liver 48,49 13 NKT MDSC Recruit MDSCs into the liver 50,51,79 14 NKT DC Bring about semi-maturity in DCs 52,53 15 NK DC Promote the capacity to induce Tregs 71 16 MDSC T Decrease anti-tumor activity 77 17 MDSC NK Decrease cytotoxicity 77,78 18 MDSC Kupffer Upregulate PD-L1 expression 83

Abbreviations: CTLA, cytotoxic T lymphocyte antigen; DC, dendritic cell; HSC, hepatic stellate cell; IFN, interferon; LSEC, liver sinusoidal endothelial cell; MDSC, myeloid-derived suppressor cell; NK, natural killer cell; NKT, natural killer T cell; Treg, T regulatory cell.

lymphoid DCs located around the periportal areas and plas- immune responses. When antigen is presented to T cells by macytoid DCs (pDCs) residing in the liver parenchyma.124 APCs residing in liver sinusoids, including LSECs, Kupffer cells Although DCs are often considered to be professional APCs, and DCs, the immune response can be skewed toward to- hepatic DCs exhibit an immature phenotype with tolerogenic lerance. Thus, systemic tolerance induced by this method properties.13,125,126 IL-10highIL-12low regulatory DCs can be may be very suitable for treating autoimmune diseases caused induced by MCSF and hepatocyte growth factor secreted by stro- by known antigens. One encouraging study showed that indu- mal cells,127,128 which can regulate Th2, but not Th1, responses.129 cing liver regulatory DCs by administering Toll-like receptor Liver pDCs (CD11clowb2201LY6C1CD11b2SiglecH1) are very agonists could lead to the remission of autoimmune disease.137 weak T-cell stimulators, as they lack CD40, CD80 and CD86 These liver tolerogenic mechanisms may also be useful in pre- expression.130 Resting liver DCs express PD-1 and CTLA-4 inhi- venting transplant rejection.138,139 Thus, tolerization by liver bitory molecules that induce circulating CD81 T-cell tolerance.131 APCs may be an effective approach to prevent antigen-specific Liver mDCs (CD11c1CD8a2CD11b1) exhibit ‘endotoxin tol- allograft rejection and cure autoimmune disease. erance’, because they are resistant to LPS stimulation.132 This As the liver can interact with other organs to induce systemic unique phenomenon is important for maintaining tolerance tolerance, curing extra-hepatic immune disease by manipulat- within the liver microenvironment. When transferred into allo- ing liver immunity may be possible.14,17,140 The experimental geneic recipients, liver mDCs can elicit IL-10-producing T cells, autoimmune encephalomyelitis animal model for multiple which helps to induce tolerance to pancreatic islet allografts.13,133 sclerosis described above is a good example of this, where 1 1 Furthermore, liver DCs can induce CD4 CD25 regulatory T Luth et al.15 ingeniously expressed MBP in the liver either by cells after interacting with NK cells and hepatocytes.74 transient gene transfer or in stable liver-specific MBP- pDCs have been shown to mediate oral tolerance by suppres- transgenic mice. This ectopic MBP expression induced MBP- 1 1 sing antigen-specific CD4 and CD8 T-cell function in the specific CD41CD251Foxp31 Tregs cells in the liver, which liver.134 Additionally, liver pDCs preferentially induce Th2 res- were then exported to the central nervous system to suppress 1 ponses and promote CD4 T-cell apoptosis. CpG-stimulated the MBP-specific CD41 T cells residing there, finally protecting human pDCs mediate Treg induction, and direct contact the mice from autoimmune disease. A similar therapeutic strat- 1 between pDCs and CD4 T cells is necessary to induce Tregs egy may be beneficial for other organs in which autoimmune 135 cells. Further study indicated that human liver DCs generated disease occurs, such as the heart, joints and skin. more Tregs cells in an IL-10-dependent manner than blood To optimize liver-induced systemic tolerance, efficiently and 126 DCs. Interestingly, the impaired T-cell proliferation induced accurately delivering the target gene to tolerogenic APCs (par- 136 by liver pDCs can be rescued by blocking Treg-derived IL-10. ticularly hepatocytes) is very important. One method of achieving this is to use an improved background vector and FUTURE DIRECTIONS promoter with minimal innate immune activation; this stra- As described in detail above, the liver is a unique organ with tegy is discussed in depth by LoDuca et al.140 Another solution tolerogenic properties that could be used to suppress unwanted is to combine a chemokine with nanoparticles to transfer the

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