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GASTROENTEROLOGY 2008;134:17891801g-term toxicity. This review describes the uniqueects of the hepatic immune response, the pathwaysolved in T-cell activation and alloantigen recogni-n, effector cells and pathways mediating liver allo-aft rejection, the role of regulatory T cells, andgets of current and future immunosuppressiveents.

iver transplantation is the definitive treatment ofchoice for patients with end-stage liver disease. Re-

ients of human liver allografts require less immuno-ppression than do other organ recipients, and livernsplants confer protection on other organ grafts fromsame donor.1 Nonetheless, a significant subset of

tients does develop allograft rejection. For the mostrt, early rejection episodes do not negatively affect

HLA DR allelic analysis showed that all liver recipientsshowed microchimerism within the first 3 months aftertransplantation.8 However, in accord with prior studiesby Hisanaga et al10 and Devlin et al,11 microchimerismdid not correlate with freedom from rejection or abilityto tolerate staged immunosuppressive drug withdrawal.Taken together, these data are consistent with the notionput forth by Starzl and Zinkernagel12 that microchimer-ism is a necessary prerequisite for, but is not synonymouswith, tolerance. Clearly, mechanisms mediating graft tol-

Abbreviations used in this paper: APC, antigen-presenting cell; CNI,calcineurin inhibitor; CTLA-4, cytotoxic T lymphocyteassociated anti-gen 4; DC, dendritic cell; IL, interleukin; MHC, major histocompatibilitycomplex; NK, natural killer; PD-1, programmed death-1; TCR, T-cellreceptor; TNF, tumor necrosis factor; Treg, regulatory T cell.ansplantation Immunology: What thmunotherapy

Hugo R. Rosen

ision of Gastroenterology & Hepatology, Liver Transplantation, Hepatitis C Centeorado, and Integrated Program in Immunology, National Jewish Hospital, Denve

e liver is unique among transplanted organs withpect to its interaction with the host immune sys-. There is evidence, both anecdotal and docu-nted, that some liver recipients who cease takingmunosuppressive drugs maintain allograft func-n, suggesting robust tolerance is in place. More-er, recipients of human liver allografts require lessmunosuppression than do other organ recipients,d liver transplants confer protection on other or-n grafts from the same donor. Hence, the liverows features of immune privilege. Still, the livern display destructive immunologic processes suchrejection in approximately one quarter of patients.e understanding of the cellular and molecularchanisms operant in tolerance vs allograft rejec-n is important for developing new agents to im-ove long-term outcome, minimize infectious com-g-term graft survival.2,3 A number of animal mod-46 show spontaneous, lifetime tolerance to liver allo-linician Needs to Know for

artment of Medicine, University of Colorado Health Sciences Center, Aurora,rado

fts without therapeutic manipulation; however, thequency and extent to which drug weaning can beomplished in human liver recipients has yet to befined.7 It is widely accepted that after organ transplan-ion, hematopoietic donor cells are cotransferred intorecipient and may survive for prolonged periods. The

e of the liver and its high content in hematopoieticls (both resident and circulating) may in part explainy liver transplantation is associated with a significantmber of donor hematopoietic cells in the peripheralod of recipients and permissiveness with regards toA incompatibility.8 Donor leukocytes could mediateerance by functioning as immature antigen-presentingls (APCs) or could act as surrogate targets of rejection,s protecting the graft.8,9

Hematopoietic microchimerism is defined by the per- 2008 by the AGA Institute0016-5085/08/$34.00

doi:10.1053/j.gastro.2008.02.062

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1790 HUGO R. ROSEN GASTROENTEROLOGY Vol. 134, No. 6nce are more complex than simply induction of anergytransferred donor cells.This review covers the unique aspects of the hepaticmune response; the pathways involved in T-cell acti-ion and expansion; basic mechanisms of nonself-oantigen recognition, effector cells, and pathways op-nt in allograft rejection; the role of regulatory T cells;d targets of current and future immunosuppressiveents.

The Liver Is a Specialized ImmuneOrganThe liver is among the most interesting and potent

munologic organs, rivaled only by what are consideredtrue immune organs, such as thymus and lymph

des.13 The liver is exposed continuously to a diverse andge antigenic load, including pathogens, toxins, and tu-r cells, as well as dietary and commensal proteins.14 Theer must be actively immunocompetent and simulta-ously control inappropriate inflammatory responses totary and other harmless antigens encountered in thertal circulation, thus being able to selectively induce im-nity or tolerance to antigens.15 Pathogen invasion issed through various pattern-recognition receptors in-ding Toll-like receptors (TLRs), which are expressed onmune cells as well as on some parenchymal cells.16 Formple, Toll-like receptor 4, the receptor for lipopolysac-aride, is expressed on immune cells, as well as hepato-es, endothelial cells, and stellate cells.Lymphocytes are broadly divided into B cells, T cells, andtural killer (NK) cells. Although B and T cells possessnotypic antigen receptors that confer specificity for di-se antigenic structures, NK cells possess multiple recep-s that can detect conserved antigens or danger signalst mediate major histocompatibility complex (MHC)-un-tricted cytolysis of susceptible tumor and virus-infectedls.17,18 Numerous groups have documented that the livertains an unusually high percentage of unconventionalphoid cells that rarely are present in the blood, includ-NK cells, NK T cells expressing the T-cell receptorR), and the invariant V24JQ TCR.19,20 For example,(CD3CD16CD56) cells represent 5%15% of the

nonuclear cell population, but in the liver can compriseto 45% of the lymphocyte population.21 It has beengested that these cells deliver a death signal to circulat-recipient-derived T cells that migrate through the liver

er transplantation, contributing to tolerance.22 Moreover,nificantly higher proportions of hepatic T cells expressR than matched peripheral blood T cells.23 The he-tic TCRpos population contains more than twice asny CD8pos cells and 4 times as many double-negativels (T cells that express neither CD8 nor CD4 coreceptors)the corresponding population in matched peripheral

od.23 The liver also hosts the largest population of tissuecrophages (Kupffer cells) in the body and different typesdendritic cells (DCs).16

inatiocelDCs are professional APCs that are present in virtuallyorgans and provide a critical link between innate andaptive immunity. The ability of DCs to process andsent various types of antigen (Ag) is unmatched in theman body.24 In particular, DCs can display Ag pro-sed exogenously on MHC class I (cross-presentation orss-priming)25 or complete MHC-peptide complexesuired externally from dead cells (cross-dressing) to8 T cells.26,27 Immature DCs express low levels ofC class II adhesion and costimulatory molecules, butir expression is up-regulated dramatically during mat-ation in response to inflammatory stimuli.28 There isanding evidence that liver-derived DCs can down-ulate immune responses, hence inducing and main-ning peripheral T-cell tolerance.29

Intrinsic differences between liver DCs and other tis-e-resident DCs have been noted and may have impor-t biologic implications. For example, human mono-es differentiated into DCs when cocultured with rater epithelial cells or liver-conditioned media secreteerleukin (IL)-10 but not IL-12p70, thus skewing themune response towards T helper type 2 rather than1.30 Thus, the hepatic microenvironment may supportgeneration and survival of regulatory T cells (de-

ibed later).27 Furthermore, it has been suggested thatnitoring of DC subsets and their activation status orokine production may provide a useful tool for pre-ting allograft outcome, including successful with-wal of immunosuppression.31 Independent of thee or extent of immunosuppressive therapy, circulatingsmacytoid DCs are increased relative to myeloid DCsclinically tolerant pediatric liver transplant recipientsmpared with those on maintenance immunosuppres-n.27,32

The context in which antigen is presented to T cellstermines whether the responding T cell is activated orerized; critical variables include the nature of the APC,presence or absence of costimulatory molecules, andcytokine microenvironment.33,34 Recent provocative

ta indicate that Ito (stellate) cells, known primarily fordiating hepatic fibrogenesis, are APCs that can presenttigen as efficiently as DCs.35 Liver sinusoidal endothe-l cells have a unique immune phenotype, expressingrkers typical of cells of myeloid lineage (CD1, CD4,11c) even though data suggest that these cells differ-tiate from hepatocyte progenitors.36 CD4 T cellsmed by antigen-presenting liver sinusoidal endothelialls or hepatocytes (which lack costimulatory molecules)l to differentiate toward effector T helper type 1 cellst, instead, express high levels of immune-suppressive10.37 Moreover, antigen presentation to CD8 T cells byer sinusoidal endothelial cells in vivo leads to their

bility to respond to specific antigen on restimula-n.38 Thus, liver sinusoidal endothelial cellsthe onlyls to have direct contact with immune cells passing

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May 2008 TRANSPLANTATION IMMUNOLOGY 1791ough the livermay play an important role in estab-hing tolerance after liver transplantation.14

Costimulatory Pathways andTransplantationBecause T cells are essential for allograft rejection

d many immunosuppressive drugs target T-cell signal-, it is important to understand the multistep processeswhich they become activated. T lymphocytes musteive 2 distinct coordinated signals to achieve optimalivation and expansion. The first signal is delivered byTCR after recognition of peptideMHC complexes onCs and the second signal is provided by the interactioncostimulatory molecules on the T cells and their cog-te ligands on APCs.39 Figure 1A shows this 2-signaldel, including the wide array of heretofore describedeptorligand pairs.40 Although the CD28/B7 and40/CD154 costimulatory pathways have garneredst of the attention in transplantation, there is emerg-evidence for the importance of other molecules in

oimmune responses,14 including critical negative sec-d signals that down-regulate or terminate T-cell re-onses (Figure 1B). CD28 (the prototypical T-cell co-mulatory molecule) is expressed constitutively on therface of T cells; the ligands for CD28 (B7-1 [CD80] and-2 [CD86]) are found on a variety of APCs includings, B cells, and macrophages. In naive T cells, CD28

stimulation enhances cell-cycle entry, potently stimu-ing expression of IL-2 and induction of anti-apoptoticteins.41 In contrast, cytotoxic T lymphocyteassoci-d antigen 4 (CTLA-4), which has approximately 20-d higher affinity for both B7-1 and B7-2 than does28, is up-regulated after T-cell activation and inhibitsell responses, regulating peripheral T-cell tolerance.42

recent analysis of CTLA-4 single-nucleotide polymor-isms in 483 liver transplant recipients showed that theLA-4 49A/6230G haplotype, which is associatedth reduced soluble CTLA-4 production, is a co-domi-nt risk factor for acute rejection.43

Programmed death-1 (PD-1) also is induced after acti-ion of T cells, shares sequence homology with CTLA-4,d contains an immunoreceptor tyrosine-based inhibi-y motif in the cytoplasmic tail, characteristic of cellu-receptors that deliver a negative signal.14 As shown inure 1B, when PD-1 is engaged by its ligands or CTLA-4ds to B7 and displaces CD28, the activation andosphorylation of SHP-2 (SRC homology 2domain-ntaining protein tyrosine phosphatase 2) that deacti-es downstream signal transducers recruited to the-1 tail results.44 SHP-2mediated dephosphorylationctivates multiple signaling molecules, resulting inwn-regulation of cytokine messenger RNA synthesisd inhibition of T-cell proliferation.44 A recent prelim-

ry analysis of liver perfusates collected from donorers before allograft implantation indicated a signifi-tly higher expression of PD-1 on CD8 T cells from

plagraegrliver relative to peripheral blood and suggested thisy be an important mechanism imparting tolerogene-y.45

Several members of the tumor necrosis factor (TNF)/F receptor families also can provide alternate costimu-ory signals to T lymphocytes. These receptor/ligand pairslude CD40/CD154, 4-1BB/4-1BBL (CD137), OX-40/OX-L (CD134/CD134L), and CD30-CD30L (CD153).14 Ofse, CD40/CD154 is the best characterized in alloim-ne responses. CD40 is expressed constitutively on APCsluding B cells, monocytes, macrophages, and DCs, buto can be expressed on nonimmune cells including endo-lial cells, mast cells, platelets, and epithelial cells. Intrast, CD154 is expressed on CD4 T cells after activa-n, and to a lesser extent on NK cells, B cells, and CD8 Tls. Unlike the CD28/B7 costimulatory pathway that pri-rily has been defined in the context of effects on T-cellction, CD40/CD154 ligation enhances APC function asasured by up-regulation of class II, CD80, and CD86ression and production of cytokines including IL-12.14,46

e sum effect of these changes to the APC is to signifi-tly augment B- and T-cell responses to alloantigen. Mo-ular and immunohistochemical analyses of CD80, CD86,d CD154 expression in biopsy specimens of liver recipi-ts showed an association between increased expression of86 and CD154 (but not CD80) in the graft during severete cellular rejection.47 CD154 also was detected onpffer cells and sinusoidal macrophages in livers duringronic rejection, but not in stable liver allografts or normalers.48 Thus, the ultimate fate of cellular immune re-nses is determined by the balance between positive andgative signals delivered by costimulatory molecules to Tls; undoubtedly, an expanding list of molecules will con-ue to be studied as possible therapeutic targets to preventd treat allograft rejection.

Basic Aspects of T-Cell Recognition ofAlloantigenThe term allorecognition refers to T-cell recognition

genetically encoded polymorphisms between membersthe same species.49 The primary targets are the MHClecules present on donor cells. The recognition ofograft MHC antigen is the primary event ultimatelyding to graft rejection. Recipient T lymphocytes canognize donor alloantigen through 2 distinct but nottually exclusive pathways. In the direct pathway, hostlymphocytes recognize native MHC molecules ex-ssed on graft-associated APCs. In the indirect path-y, host T lymphocytes recognize donor alloantigen-rived peptides in the context of self MHC moleculesressed on recipient APCs (Figure 2).50 Evidence forth pathways exists in liver transplantation. It is likelyt the direct pathways predominate early posttrans-

nt and is a major factor in acute rejection becauseft-derived APCs expressing donor alloantigen rapidlyess from the graft and enter secondary lymphoid tis-

FigengreccosCDsigsigbinmoinh

1792 HUGO R. ROSEN GASTROENTEROLOGY Vol. 134, No. 6ure 1. The 2-signal model for positive and negative costimulation of T-cell activation. (A) T-cell activation requires 2 signals: signal 1, TCRagement with MHCpeptide complex, and signal 2, ligation of costimulatory molecules on T cells with their respective ligands on APC. T cells thateive both signal 1 and positive costimulation show proliferation, secretion of cytokines, and differentiation into effector cells. The prominenttimulatory molecules delivering positive costimulation signals are CD28 and CD40L. Under certain circumstances, ICOS, CD134, CD30, 4-1BB,27, and CD70 also can deliver positive T-cell costimulation. Some costimulatory molecules, such as CTLA-4 and PD-1, can lead to negative T-cellnaling, resulting in decreased cell proliferation and cytokine production and cellular anergy. Reprinted from Gao et al.40 (B) Negative regulatorynaling of T cells. PD-1 and CTLA-4 are negative regulators of TCR signaling. Engagement of PD-1 by its ligands (PD ligand-1 or PD ligand-2) ording of CTLA-4 to B7 (displacing CD28) is followed by the recruitment of the phosphatase SHP-2 to the immunoreceptor tyrosine-based inhibitorytifs in the cytoplasmic tails of PD-1 and CTLA-4. Then SHP-2 dephosphorylates the CD3 chains and other signaling pathways, which results in

ibition of TCR signaling and downstream events associated with T-cell activation. Reprinted with permission from Mak and Saunders.44

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Figrecderthain pendforhanRe

May 2008 TRANSPLANTATION IMMUNOLOGY 1793e where they can encounter allospecific T cells.51 Hostcells primed through the direct pathway have theility to engage the allograft directly and, thus, to effi-ntly mediate effector functions. Further, the propor-

ure 2. Allorecognition pathways and graft rejection. (A) Direct and iognize intact major histocompatibility molecules on donor APCs. In theived peptides in the context of self MHC molecules expressed on recipiet is shed from damaged graft tissue, or perhaps in the case of the liver, frarticular DCs. (B) Interactions among endothelial cells, T cells, andothelial cells to the graft tissue are transformed to become highly efficiematuration. The DCs and intragraft macrophages present donor peptd, are activated by donor endothelial cells and either can directly kill en

printed with permission from Briscoe and Sayegh.50n of host T cells that can respond to native alloantigensubstantially greater than the proportion of host Tls that can respond through the indirect pathway to

moinimnor-derived peptides presented in the context of selfC.52 The indirect pathway probably emanates from

nor alloantigen that is shed from damaged graft tissue,perhaps in the case of the liver, from soluble MHC

ct pathways of allorecognition. In the direct pathway (left), T cellsrect pathway (right), T cells recognize processed donor alloantigen-Cs. The indirect pathway probably emanates from donor alloantigenluble MHC molecules that are picked up and presented by self APC,

ient APCs in allograft rejection. Recipient monocytes recruited bytigen-presenting DCs that recirculate to peripheral lymphoid organsia the indirect pathway to CD4 T cells. CD8 T cells, on the otherlial cells or traverse the endothelium and kill parenchymal graft cells.doMHdoor

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ides vdothelecules, that are picked up and presented by self APCs,particular DCs.14 Therefore, the direct pathway may beportant in initiating the classic form of acute rejection

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1794 HUGO R. ROSEN GASTROENTEROLOGY Vol. 134, No. 6cause donor-derived passenger leukocytes have a lim-d lifespan, the indirect pathway may predominate latersustaining a response fueled by epitope spreading as aiety of allopeptides are presented successively by selfC.14 Accordingly, T cells with specificity for allopep-es have been detected readily in liver recipients under-ing chronic rejection.53 At the same time, it is impor-t to recognize that the indirect pathway may beolved in immune regulation because T cells with al-eptide specificity were shown to have regulatory func-n through inhibition of interferon production iner organ recipients.52 The contribution of the indirectthway to hepatic allograft tolerance recently was con-med.54

In addition to graft rejection and tolerance, these al-ecognition pathways likely are important in recurrenceviral or autoimmune liver diseases. In this regard, livernsplantation is performed with no regard to specifictching of donor-recipient HLA alleles; this may servea barrier to the development of protective (ie, antiviral)l-mediated immunity directed against infected cellsthin the allograft. CD8 T cells are the primary effec-lymphocytes for provision of protective immunity

ainst intracellular pathogen infection of parenchymalls. Recognition of the infected allograft could occurher via recipient-derived T cells or via those derivedm the donor. For the recipient-derived T cells, recog-ion could occur either through use of shared HLAlecules, or could occur through the expansion of re-ient-derived T cells that are uniquely restricted by theA molecules of the donor liver. A study from ourup55 has shown the generation of new hepatitis Cus (HCV)-specific T cells that are restricted by donorA alleles, yet derived from the recipients original T-l pool (Figure 3). For the purposes of this study, weected HLA A2-negative recipients of HLA A2 grafts;A-A2 was selected as the restricting allele because age number of HCV HLA-A2 binding peptides haveen described as targets of HCV-specific CTLs.As shown in Figure 3A, recipient HCV-specific CTLnes cultured with HLA A2 lymphoid cell lines thatd been pulsed with cognate peptide (NS314061415,VALGINAV) but not irrelevant HCV core peptidere3544, YLLPRRGPRL) elicited a strong immune re-onse by interferon enzyme-linked immunospot.reover, when these recipient CTLs were cultured withipient-derived (syngeneic) lymphoid cell lines and cog-te peptide, there was no appreciable immune response.ese data suggest that these HCV-specific CTLs circu-e within the recipient and only display functionaltiviral activity (ie, secrete interferon and show cyto-icity; Figure 3B), when they encounter allograft-de-ed HLA molecules and viral peptide. Furthermore,

se CTLs can recognize endogenously processed viralexpressed by the HLA molecule of the donor graftgure 3C). It is likely that the microenvironment (eg,

apcytligphoid aggregates in the portal tracts or secondaryphoid tissues) in which this immune response wastiated contained DCs that engulfed HCV-infectedpatocytes. These results underscore the plasticity of theR, as well as the need to assess both allograft- andipient-restricted CTLs (and thus direct and indirectthways) to develop a comprehensive understanding oftective immunity to HCV after liver transplantation.

Effector Pathways of Graft InjuryAcute cellular rejection occurred historically in 50%

75% of liver allograft recipients, although recent advancesimmunosuppression have yielded lower rates (ie, 30%ording to Scientific Registry of Transplant Recipientsta; http://www.ustransplant.org/glossary). The majorityepisodes occur within 90 days of transplant surgery,56

d the majority of cases respond to high-dose corticoste-ds. Three main types of allograft rejection are described:extremely rare hyperacute rejection characterized by

dothelial injury and mediated by deposition of antibodyd fibrin but no lymphocytic infiltration or bile ductury; acute rejection characterized by the diagnostic triadportal inflammation, bile duct damage, and venular en-thelial inflammation; chronic rejection characterized bys of small bile ducts (ie, ductopenic rejection) and anliterative vasculopathy affecting large- and medium-sizederies and the portal microvasculature.56,57

For both acute and chronic rejection, the targets ofture effector T cells are donor-derived bile duct epi-lial cells and vascular endothelium, whereas directolvement of hepatocytes is uncommon.57 Cytotoxicphocytes kill cholangiocytes via either perforin-de-

ndent pathways or activation of members of the TNFeptor superfamily (particularly Fas)57 (Figure 4). Theeraction between CTL and target cell is facilitated byegrins, notably LFA-1, which help form an immuno-ic synapse containing the secretory domain.57 In ad-ion, as shown in Figure 4B, other integrins such as verye antigen-4 may enhance adhesion and provide sur-al signals for effector cells.57

Recent data57 indicate that bile ducts constitutively ex-ss the chemokine CCL19, which attracts DCs and pro-tes their localization at the biliary epithelium where theyideally situated to respond to Ag. Although human

olangiocytes lack CD80 and CD86, minimizing their abil-to activate naive T cells,58 the majority of T cells thate into contact with activated cholangiocytes are already

med cells, and therefore, cholangiocytes may promotelocal proliferation and survival of such cells.57 Given theding that themajority of lymphocytes infiltrating humaner allografts in rejection express functional chemokineeptors CXCR3, CXCR4, and CCR559 indicates that ther-

eutic strategies in the future may aim to prevent lympho-e infiltration of liver allografts by inhibiting chemokineand/receptor-mediated pathways.

FigrecdongetculnotlelelinkwitexpdleLCtideGIN(cotivitceltidemecificperafteeffegetgatshoacecenCTHL(b),VTKcendiffor arescloreccelforeithralenclovtroassassRo

May 2008 TRANSPLANTATION IMMUNOLOGY 1795ure 3. Recipient-derived T cells thatognize HCV peptides in the context ofor HLA molecules (HLA A2). Taken to-

her, these results suggest that T cells cir-ate in liver transplant recipients and areactivated until they encounter donor al-

s containing HCV peptides. (A) Enzyme-ed immunospot assay was performedh 1000 T cells, lymphoid cell lines (LCLs)ressing A2 allele (top row), A3 allele (mid-row), or syngeneic (recipient-derived)

Ls (bottom row) co-cultured with no pep-, cognate peptide (NS314061415, KLVAL-AV), or irrelevant HCV core peptide

re3544, YLLPRRGPRL). (B) Cytotoxic ac-y of HCV-specific tetramerCD8 Tls. T-cell clones were assayed for pep--specific cytotoxicity using the fluoro-tric assessment of T lymphocyte Ag-spe-

lysis assay, which determines thecentage of labeled target cells survivingr a 5-hour incubation with effector cells;ctor:target ratio is 50:1 with 1 106 tar-cells. The target cells are selected by

ing on the PKH-26high population andw the reduction in carboxyfluorescein di-tate succinimidyl ester (CFSE) fluores-ce after incubation with NS31406-specific

L, cognate peptide (KLVALGINAV), andA-A2 LCLs (a), compared with no peptideirrelevant peptide NS525942602 (ALYDV-L) (c), and no effector cells (d). The per-tage killing of syngeneic LCLs was not

erent after incubation in the presence (e)bsence (f) of cognate peptide. FITC, fluo-

cein isothiocyanate. (C) NS31406-specificnes (HCV1HCV4, represented by bars)ognize endogenously processed Ag. T2ls were pulsed with HCV14061415 peptide2 hours. Cell lines were transduced wither empty retroviral vector or with retrovi-vector containing the HCV minigene thatodes NS314061415 peptide. Cytomega-

irus-specific T cells were used as a con-l. Interferon secretion (mean SD) was

essed by enzyme-linked immunosorbentay. Reprinted with permission fromsen et al.55

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1796 HUGO R. ROSEN GASTROENTEROLOGY Vol. 134, No. 6The perforin pathway is mediated by granzymes and

ure 4. Cholangiocyte apoptosis is mediated by CTLs via activation ofarget cells by the release of chemokines, secreting specialized granuleshe target cell. Inflamed cholangiocytes express increased MHC classognition and cellcell adhesion. On CTL activation, perforin- and granzymbrane fusion occurs and granzymes are delivered into the target ceptosis; granzyme B activates Bcl2-interacting domain and caspase-3as and other TNF receptors (TNFRs). Fas ligand is contained in the secr

he immunologic synapse. Cross-linking of Fas with FasL leads to caspress TNF ligands, implicating them as potential contributors to bile duexpression of FasL via nuclear factor B-dependent pathways, resultinLFA-1 are critical for the formation of the immunologic synapse, othe

vide survival signals for effector cells. Reprinted with permission fromrforin stored in specialized secretory lysosomes that areeased into the immunologic synapse after engagementth a target cell.60 Granzymes cleave specific substrates,

ingcuwaluding caspases and Bcl2-interacting domain, result-

receptors or the granzyme/perforin pathway. (A) CTLs are attractedontain proteinases and activate the caspase cascade and apoptosisd intercellular cell adhesion molecules (ICAM-1), promoting antigencontaining secretory granules are mobilized to the cell surface whereanzyme A makes nicks in single-stranded DNA, resulting in directndent apoptosis. (B) CTLs also kill cholangiocytes by the activationgranules and with other members of the TNF family is concentratedependent apoptosis. A wide range of cells (including macrophages)ry. CD40 does not directly activate caspases but instead increasesutocrine or juxtacrine apoptosis via Fas activation. Although ICAM-1rins such as very late antigen-4 (VLA-4) may enhance adhesion ands and Afford.57inc

TNFthat c

I anme-ll. Grdepe

etoryase-dct injug in ar integAdamin the induction of apoptosis.57 There is strong cir-mstantial evidence that the granzyme/perforin path-y is activated in human allograft rejection.57,61 Cross-

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May 2008 TRANSPLANTATION IMMUNOLOGY 1797king of Fas (ubiquitously expressed on lymphoid andnlymphoid tissue including the liver) with trimerizedsL or agonist antibodies leads to formation of the death-ucing signaling complex in target cells, activation ofpase 8, and propagation of a death signal that culmi-tes in apoptosis.57 The Fas/FasL pathway has been shownvincingly to play critical roles in a variety of hepatic

thologies,62,63 and there is evidence that this pathway alsoctive during liver allograft rejection.64,65 A wide range ofls (including macrophages) express TNF ligands, impli-ing them as potential contributors to bile duct injury.57

her TNF receptors likely involved in acute and chronicection (and thus potential targets for pharmacologic in-vention) are shown in Figure 4B. CD40 ligation onolangiocytes results in up-regulation of FasL; thus, al-ugh CD40 does not directly activate caspases, it en-nces the expression of FasL via nuclear factor Bdepen-nt pathways, resulting in autocrine or juxtacrineoptosis via Fas activation.57

Regulatory T Cells and TransplantationThe balance of effector (proinflammatory) re-

onses and regulatory responses may ultimately deter-ne whether rejection or tolerance occurs. Several sub-s of CD4 T cells with suppressive properties haveen described, including naturally occurring4CD25 regulatory T cells (Treg), T regulatory typeand T helper type 3 cells.66 CD4CD25 Treg sup-ss the response of conventional T cells via a cellntact-dependent manner, whereas T helper type 3 andregulatory type 1 cells produce immunosuppressiveokines (eg, IL-10 and transforming growth factor-).hough immunoregulatory activity specific for donoroantigens is enriched in the CD4 T-cell populations,8 T cells, double-negative (CD4-CD8-) T cells, asll as NK T cells also have shown regulatory activities inferent situations.67 Treg cells also may mediate theirects by modifying the functions of other T cells, eitherectly or indirectly through APCs.67

CD25 (the -subunit of the IL-2 receptor) is used as arker for Tregs, but is by no means a definitive or specificrker. Further, CD4CD25high Tregs have been character-d by the constitutive expression of CD62L, intracellularression of CTLA-4, forkhead transcription factor 3xP3, the most specific marker for Tregs), lymphocyteivation gene-3 (LAG-3), and, most recently, the down-ulation of IL-7 receptor (CD127) expression.6871 Tregquency in the peripheral blood72 and intrahepaticxP3 cells73 are reduced in liver allograft recipients withte rejection, and T regulatory type 1 cells are increased inipients developing spontaneous graft tolerance. A recentdy showed that donor CD4CD25 Tregs originatingm the liver graft are able to suppress responder T cells

m both recipient and donor.74 These findings indicatet Treg can suppress across an MHC barrier, congruenth previous observations that Tregs suppress Ag nonspe-

tiotorinccally once activated through their TCR. Thus, chimerismdonor Treg may contribute to suppression of the directthway alloresponse, which, as described earlier, is theminant Ag presentation mechanism early after livernsplantation.74

How Immunosuppressive Agents WorkThis section builds on the understanding of basic

chanisms outlined earlier to explain how current im-nosuppressive agents work (Table 1) and to describetential new targets for immune manipulation. Theder is referred to excellent reviews that additionallyscribe important drugdrug interactions and side ef-ts of these medications.7577

In general, immunosuppression can be attained by block-lymphocyte response pathways, depleting lymphocytes,diverting lymphocyte traffic.76 Calcineurin inhibitors

NIs), cyclosporine and tacrolimus, remain the backboneimmune suppression in liver transplantation. Cyclospor-engages the immunophilin cyclophilin, forming a com-x that then engages calcineurin.76 This complex in turnibits transcription of several genes, including IL-2, criti-to T-cell activation. Tacrolimus engages a different im-nophilin, FK506-binding protein 12, to create a complext also inhibits calcineurin but with greater moleculartency.76 Thus, both CNIs interfere with signal 1 T-cellnal transduction (Figures 1 and 5). Rapamycin (siroli-s) is a macrolide antibiotic structurally related to tacroli-s but with very different mechanisms of action. Rapa-cin binds to the immunophilin, FK506-binding protein, but rather than inhibiting cytokine gene transcription inells, it blocks signals transduced from a variety of growthtor receptors to the nucleus by acting on phosphatidylsitol kinases called mammalian targets of RAPA.77,78

reover, it also inactivates 70-kD-S6 kinase, which resultsselective inhibition of the synthesis of new ribosomalteins, prolonging cell-cycle progression from the G1 toS phase.77 Thus, CNIs and sirolimus differentially affect2: CNIs inhibit IL-2 production whereas sirolimus blocksIL-2 signaling pathway at a later stage after receptording.67 Interestingly, these differences are manifested byfact that CNIs (which inhibit IL-2 secretion essential for

ector cells and Treg development) prevent activation-uced cell death of effector cells, whereas sirolimuses not have the same effect.67 Moreover, CNIs areociated with depletion in the peripheral blood of4CD25FoxP3 Tregs after renal transplanta-n, whereas sirolimus is associated with preservationTregs,79 in keeping with in vitro data.80

Corticosteroids are the most frequently used non-CNIents. Their effects are primarily transcriptionalough DNA binding and proteinprotein interactionsthe steroid-receptor complex, which target transcrip-

n factors such as activator protein 1 and nuclear fac--B,76 abrogating expression of multiple cytokines,luding IL-1, IL-2, IL-3, and IL-6.75 In addition, corti-

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1798 HUGO R. ROSEN GASTROENTEROLOGY Vol. 134, No. 6steroids suppress eicosanoid production, down-regu-e adhesion molecules, and increase the expression ofnsforming growth factor-. Antimetabolites used innsplantation include azathioprine, mycophenolatefetil, and mycophenolic acid. All 3 antagonize purinethesis, which blocks differentiation and proliferationT and B lymphocytes.75 Mycophenolate mofetil andcophenolic acid inhibit synthesis of guanosine mono-osphate nucleotides, whereas azathioprine convertsercaptopurine to tissue inhibitor of metalloprotein-, which is converted to thioguanine nucleotides.76

Antibodies used in transplantation target a wide varietydifferent pathways. Antithymocyte globulin is a poly-

ble 1. Mechanisms of Action of Immunosuppressive Agents

Drug

all-molecule immunosuppressive agentsyclosporine (CyA) Binds to cyclophilin; compl

1 T-cell signal transductiInhibits IL-2 synthesis and

acrolimus (FK506) Binds to FKBP12; complexT-cell signal transduction

Inhibits IL-2 synthesis andiroliums (rapamycin) Binds to FKBP12 (but not

proliferationRelative to CyA and FK506Blocks signals transduced

phosphatidyl inositol kinInactivates 70 kilodalton S

ribosomal proteinsPreserves Tregs in vitro anInhibits B-cell stimulation,

orticosteroids Target transcription factorsexpression of multiple cy

Suppress eicosanoid produDown-regulate adhesion mIncrease the expression of

ycophenolate mofetil (MMF) andmycophenolic acid (MPA)

Inhibits synthesis of guanoproliferation of T and B c

zathioprine (AZA) Converts 6-mercaptopurinethoguanine nucleotides,purine synthesis

Inhibits proliferation and dtein immunosuppressive drugsntithymocyte globulin Polyclonal antibody prepara

CD4, CD8, CD28) and Nuromonab-CD3 (OKT3) Binds to the CD3 antigen o

1), ultimately leading toasiliximab (chimeric) and daclizumab(humanized)

IL-2receptor antibodies, breceptor -chain (CD25proliferation

lemtuzumab (campath-IH) Humanized, recombinant mmonocytes, and macrop

ituximab Chimeric monoclonal antibelatacept (LEA29Y)a CTLA-4 fusion protein imm

and signal 2 transductio

pted from Halloran.76

monstrated efficacy in renal and islet cell transplantation (no publisnal antibody preparation that targets multiple differentitopes on T cells (CD2, CD3, CD4, CD8, CD28) and NKls (CD16).75 Muromonab-CD3 (OKT) is a murine-de-

sureraroped antibody that exerts its activity by binding to the CD3tigen on the surface of T lymphocytes, inactivating thejacent TCR (signal 1). Two currently licensed IL-2recep-antibodies, basiliximab (chimeric) and daclizumab (hu-nized), bind to the IL-2 receptor -chain, achieving im-nosuppression by competitive antagonism of IL-2uced T-cell proliferation (blocking signal 3).75 Althoughrt-term treatment with CD25-specific antibodies is ef-tive at reducing the incidence of allograft rejection, the-tically, it also might compromise the subsequent devel-ment of immunoregulation to donor alloantigens.67

mtuzumab or campath-1H is a humanized, recombinantnoclonal antibody that targets antigen CD52, a cell-

in Liver Transplantation

Mechanisms of action

hibits calcineurin phosphatase and T-cell activation (inhibit signal

seits calcineurin phosphatase and T-cell activation (inhibit signal 1

sehilin); complex inhibits target of rapamycin and IL-2 driven T-cell

ks IL-2 signalling pathway at a later stage after receptor bindinga variety of growth factor receptors to the nucleus by acting oncalled mammalian targets of RAPA (mTOR)ase, which results in selective inhibition of the synthesis of new

ivoasing antibody productionas activator protein 1 and nuclear factor-kB,55 abrogating

es, including IL-1, IL-2, IL-3, and IL-6

les

monophosphate nucleotides, blocks purine synthesis, preventing

ssue inhibitor of metalloproteinase, which is converted tofering with DNA synthesis; thioguanine derivatives may inhibit

ntiation of B and T cells

that targets multiple different epitopes on T cells (CD2, CD3,ls (CD16), functionally altering and depleting different cell types

surface of T lymphocytes, inactivating the adjacent TCR (signaldepletion

imab (chimeric) and daclizumab (humanized), bind to the IL-2n), depleting activated T cells and inhibiting IL-2induced T-cell

lonal antibody that targets antigen CD52 on T, B, NK cells,, causing lysis and depletion

hat binds to CD20 on B cells, mediating lysisobulin that binds to B7 on T cells, preventing CD28 signaling

ata in liver transplantation).rivanadtormamuindshofecoreopAlemo

decresuchtokinction

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iffere

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hed dface glycoprotein, expressed on the majority of periph-l blood lymphocytes, thymocytes, monocytes, and mac-hages (Figure 5).77 Campath-1H produces profound

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May 2008 TRANSPLANTATION IMMUNOLOGY 1799pletion of circulating lymphocytes for approximately 1nth, and the rationale for its initial use is to facilitateer doses of maintenance immunosuppression.77 Ritux-ab is a chimeric monoclonal antibody against CD20 on Bls; its use in liver transplantation has been limited totients who develop posttransplant lymphoproliferativeorders81 and in recipients of ABO-incompatible grafts.82

A new generation of biological agents that block theeraction between CD80/86 and CD28 costimulatorythways (signal 2) includes belatacept (LEA29Y), a CTLA-4ion protein immunoglobulin, which has shown efficacyrecipients of kidney76,83 and islet cell allografts,84 but hast yet been studied in liver recipients. Blockade of T-cell/C costimulatory pathways mediated via CD40CD154s been accomplished using the anti-CD154 monoclonaltibody, but studies have been halted because of reports ofomboembolic phenomena, perhaps related to the expres-n of CD154 on activated platelets.77

In summary, based on our understanding of cellular and

ure 5. Immunosuppressive drugs and sites of action. As described in-1) and CD86 (B7-2) on the APCs engage CD28 on the T cell to prociumcalcineurin pathway, the mitogen-activated protein kinase pathwurn, activate transcription factors nuclear factor of activated T cells (ression of CD154 (which further activates APCs), IL-2 receptor chaher inhibiting transcription of several genes (including IL-2, critical to T-ctein 1 and NF-B, abrogating expression of multiple cytokines, includhain, achieving immunosuppression by competitive antagonism of IL-2-ugh the phosphoinositide-3-kinase (PI-3K) pathway and the molecuphocytes require synthesis of purine and pyrimidine nucleotides for r

F), and mycophenolic acid (MPA) antagonize purine synthesis, whichrapies likely will interrupt signaling of multiple cytokine receptors such ashosphate (S-1-P) receptors profoundly alters lymphocyte traffic, driving

the graft. The first agent using this novel mechanism (FTY720) was dphocyte trafficking. Reprinted with permission from Halloran.76lecular mechanisms, the immune strategies currentlyd in liver transplantation target a number of mecha-ms (eg, signal 1 and 2 transduction, lymphocyte deple-

4.n). The imminent future likely will witness an evenater expansion in our armamentarium of immunosup-ssive agents than in the past decade. Our limitationsain very similar to those of the past: long-term toxicityains a concern and we have very few biomarkers toasure the induction of tolerance85 and fine-tune immu-suppression based on an individual patients characteris-s.

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eceived November 26, 2007. Accepted February 12, 2008.ddress requests for reprints to: Hugo R. Rosen, MD, Watermanfessor of Medicine and Immunology, GI & Hepatology Division,58, Academic Ofce Building 1, 12631 East 17th Avenue, Room14, PO Box 6511, Aurora, Colorado 80045. e-mail: Hugo.Rosen@HSC.edu; fax: (303) 724-1891.upported by a VA Merit Review grant and NIH U19 A1066328.

Transplantation Immunology: What the Clinician Needs to Know for ImmunotherapyThe Liver Is a Specialized Immune OrganCostimulatory Pathways and TransplantationBasic Aspects of T-Cell Recognition of AlloantigenEffector Pathways of Graft InjuryRegulatory T Cells and TransplantationHow Immunosuppressive Agents WorkReferences