CAQ CORNER

Basic Concepts in Transplant Immunology

Olivia M. Martinez1 and Hugo R. Rosen2

he liver is unique among transplanted organs with

respect to its complex, and sometimes paradoxical,
interaction with the host immune system. Early liver transplantation studies with outbred swine demonstrated that a
signicant percentage of recipients maintained their graft
in the absence of immunosuppression.1 Subsequently,
spontaneous liver allograft acceptance was also observed in
transplants done in several allogeneic rat strain combinations and most allogeneic mouse strain combinations.
Moreover, other experimental studies in rodents demonstrated that allogeneic liver transplantation provides
immune protection against subsequent cardiac, kidney,
pancreas, islet, and skin grafts from the same donor.1-7
Finally, in clinical transplantation there is increasing evidence, both anecdotal and documented, that some liver
transplant recipients who cease taking immunosuppressive drugs maintain allograft function, suggesting robust
tolerance is in place. Hence, the liver shows features of
immune privilege. Nevertheless, the liver can display
destructive immunologic processes since acute liver allograft rejection does occur in approximately 50% to 75% of
liver transplant recipients, although in the majority of cases
it is readily reversed with immunosuppressive approaches
tailored to treat cellular rejection (covered in a separate
CAQ corner). Whereas immune responses within the liver
can effectively eliminate hepatotropic pathogens including
hepatitis A, other liver pathogens such as hepatitis C persist
and manage to avoid elimination by the immune system.
Finally, a variety of autoimmune diseases with unknown
Abbreviations: Th, T helper cell; IL, interleukin; DC, dendritic
cell; APCs, antigen-presenting cells; MHC, major histocompatibility
complex; LSEC, liver sinusoidal endothelial cell; NK, natural killer;
TCR, T-cell receptor; IFN, interferon; CTLA4, cytotoxic T lymphocyte associated antigen 4; Ig, immunoglobulin; ICOS, inducible
costimulator; PD, programmed death; PDL, programmed death
ligand; CTLs, cytotoxic T lymphocytes; FasL, Fas ligand; HLA,
human leukocyte antigen.
From the 1Stanford University School of Medicine, Stanford, CA;
and 2Oregon Health and Science University / Portland VAMC, Portland, OR.
Address reprint requests to Olivia Martinez, PhD, Stanford University School of Medicine, 1201 Welch Road, MSLS P312, Stanford, CA
94305-5492. Telephone: 650-498-6247; FAX: 650-498-6250;
E-mail: omm@stanford.edu
Copyright 2005 by the American Association for the Study of
Liver Diseases
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI 10.1002/lt.20406

370

etiologies target the liver, including primary biliary cirrhosis, primary sclerosing cholangitis, autoimmune hepatitis,
and biliary atresia. Together, these observations underscore the enigmatic relationship between the liver and host
immunity. In this article, we discuss the hepatic immune
system, basic principles governing the host T-cell response
to alloantigen following liver transplantation, experimental studies elucidating the cellular and molecular mechanisms of liver allograft rejection and tolerance, and immunologic perspectives of recent developments in clinical
liver transplantation.

Role of the Immune System: The Major

Players
The primary role of the immune response is to protect
the individual from invasion by infectious pathogens
and to provide self-nonself discrimination. The coordination of an efcient immune response requires the
recognition of pathogens and subsequent activation of
immune cells and soluble mediators of immunity. The
innate immune system, through its ability to rapidly
limit the spread of infectious pathogens provides the
speed component. In contrast, classical T cells provide
the specicity, which requires days to weeks to develop.
These discrete subsets function as part of a coordinated
and complementary system, and their importance for
host defense is seen in secondary responses in which
speed and specicity are united in the form of immunological memory.8 Although graft rejection is mediated primarily by T cells, as indicated by the fact that
skin grafted onto nude mice, which lack T cells, is not
rejected, it is important to understand the division of
labor by diverse cells comprising the immune system.
Moreover, increasing evidence indicates that T cells also
exert regulatory functions, favoring peripheral tolerance.9 Several subsets of CD4 T cells with suppressive
properties have been described, including naturally
occurring CD4CD25 regulatory T cells, T regulatory type 1, and T helper (Th) type 3 cells.10
CD4CD25 regulatory T cells suppress the response
of conventional T cells via a cell contact-dependent
mechanism, whereas Th3 and T regulatory type 1 cells
produce immunosuppressive cytokines, e.g., transforming growth factor- and interleukin (IL)-10.
Dendritic cells (DCs) are professional antigen-pre-

Liver Transplantation, Vol 11, No 4 (April), 2005: pp 370-381

CAQ Corner

senting cells (APCs) that are present in virtually all

organs and provide a critical link between innate and
adaptive immunity. The ability of DCs to process and
present various types of antigens, including antigens
derived from dead cells, is unmatched in the human
body.11 Immature DCs express low levels of major histocompatibility complex (MHC) class II, adhesion, and
costimulatory molecules, but their expression is dramatically upregulated during maturation in response to
appropriate inammatory stimuli.12 There is expanding evidence that liver-derived DCs can downregulate
immune responses, hence inducing and maintaining
peripheral T-cell tolerance.13 Furthermore, it has been
hypothesized that following liver transplantation, the
existence of large numbers of DCs within the donor
liver that circulate and repopulate the recipient contributes to microchimerism.14

The Liver as an Immune Organ

Because of its location and function, the liver is continuously exposed to diverse and large antigenic loads,
including pathogens, toxins, and tumor cells, as well as
dietary and commensal proteins.15 The liver must be
actively immunocompetent and simultaneously control
inappropriate inammatory responses to dietary and
other harmless antigens encountered in the portal circulation, thus being able to selectively induce immunity
or tolerance to antigens.16 Hepatic suppression of sensitization to antigen absorbed by the portal system was
demonstrated as early as 1967.17 The context in which
antigen is presented to T cells determines whether the
responding T cell is activated or tolerized. This includes
the nature of the APC, the presence or absence of
costimulatory molecules and the cytokine microenvironment.17 In particular, hepatic APCs have a constitutive ability to activate and induce tolerogenic
responses in T cells.18 Liver sinusoidal endothelial cells
(LSECs) have a unique immune phenotype, expressing
markers typical of cells of myeloid lineage (CD1, CD4,
CD11c), even though recent data suggest that these
cells differentiate from hepatocyte progenitors.17
LSECs resemble immature DCs more than typical
microvascular endothelial cells from other organs and
seem to be a new type of organ-specic APCs.17 CD4
T cells primed by antigen-presenting LSECs fail to differentiate toward effector Th1 cells but, instead, express
high levels of immune suppressive IL-10. Moreover,
antigen presentation to CD8 T cells by LSECs in vivo
leads to their inability to respond to specic antigen
upon restimulation.19 Taken together, these data implicate antigen presentation by LSECsthe only cells to

371

have direct contact with immune cells passing through

the liveras a primary mechanism of systemic tolerance.
Lymphocytes are broadly divided into B cells, T
cells, and natural killer (NK) cells; whereas B and T cells
possess clonotypic antigen receptors that confer specicity for diverse antigenic structures, NK cells possess
multiple receptors that can detect conserved antigens or
danger signals that mediate MHC-unrestricted cytolysis of susceptible tumor and virus-infected cells.15,20
Golden-Mason and colleagues have documented an
unusually high percentage of unconventional lymphoid
cells in the liver that are rarely present in the blood,
including NK cells, NKT cells expressing the T-cell
receptor (TCR) and the invariant V24JQ TCR, and
DCs.21,22 Although the precise nature of the antigens
recognized by these putative regulatory cells is currently
unknown, CD1d (expressed on the surface of APCs
[Kupffer cells] and hepatocytes23) has been shown to be
a natural ligand for NKT cells.24 Figure 1 shows the
relative proportions of the major lymphocyte subsets
present in the healthy adult human liver as compared with
the lymphocyte repertoire found in the peripheral blood
(used with permission, from Doherty and OFarrelly15).
For example, NK (CD3 CD16CD56) cells represent
5% to 15% of the peripheral mononuclear cell population, but in the liver can comprise 45% of the lymphocyte
population.25,26 It has been suggested that these cells
deliver a death signal to circulating recipient derived T cells
that migrate through the liver after transplantation, contributing to tolerance.27 Thus, the adult human liver contains several populations of lymphocytes that exhibit rapid
antigen-nonspecic cytotoxicity and regulatory cytokine
secretion28; these cells play important roles in containment
of tumor and viral infections, brosis development,29 and
allograft rejection/tolerance.

Basic Aspects of T-Cell Recognition of

Alloantigen and T-Cell Activation
Recipient T lymphocytes can recognize donor alloantigen through either of two pathways. In the direct pathway, host T lymphocytes recognize native MHC molecules expressed on graft-associated APCs. In the
indirect pathway, host T lymphocytes recognize donor
alloantigen-derived peptides in the context of self
MHC molecules expressed on recipient APCs (Fig. 2).
Evidence for both pathways exists in liver transplantation.30 It is likely that the direct pathway predominates
early posttransplant and is a major factor in acute rejection since graft-derived APCs expressing donor alloan-

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Martinez and Rosen

Figure 1. Distribution of lymphocyte subpopulations in healthy human blood and liver. (Adapted from Doherty and
OFarrelly.15)

tigen rapidly egress from the graft and enter secondary

lymphoid tissue, where they can encounter allospecic
T cells.31 Host T cells primed through the direct pathway have the ability to engage the allograft directly and,
thus, to efciently mediate effector functions. Further,
the proportion of host T cells that can respond to native
alloantigen is substantially greater than the proportion
of host T cells that can respond through the indirect
pathway to donor-derived peptides presented in the
context of self MHC.32 The indirect pathway probably
emanates from donor alloantigen that is shed from
damaged graft tissue, or perhaps in the case of the liver,
from soluble MHC class I molecules, that are picked up
and presented by self APCs. Thus, while the direct
pathway may be important in initiating the classical
form of acute rejection, the indirect pathway may be
important in sustaining an ongoing, persistent response
that is fueled by epitope spreading as a variety of
allopeptides are successively presented by self APCs.
Indeed, T cells with specicity for allopeptides were
readily detected in liver recipients undergoing chronic
rejection.33 At the same time it is possible that the
indirect pathway is involved in immune regulation
since T cells with allopeptide specicity were shown to
have regulatory function through inhibition of interferon (IFN)- production in kidney recipients.34 The
relative importance of the direct and indirect pathways
in liver allograft rejection and tolerance induction
remains to be claried.

Costimulatory Pathways and Transplantation

T lymphocytes must receive two distinct but coordinated signals in order to achieve optimal activation.
The rst signal is delivered by the TCR following recognition of peptide/MHC complexes on APCs and the
second signal is provided by the interaction of costimulatory molecules on the T cells and their cognate
ligand/s on APCs. Signal 1 in the absence of signal 2, as
likely occurs in the liver, leads to a state of T-cell nonresponsiveness, or anergy, in which T cells can recognize cognate antigen through the TCR but fail to
mount a functional response upon reencounter with
the antigen. Thus, there has been signicant effort
towards inhibiting or blocking costimulation as a
means to prolong allograft survival.
Two costimulatory molecule-ligand pairs that are
important in the generation of a complete T-cell
response are CD28/B735 and CD40/CD154.36 CD28
represents the prototypical T-cell costimulatory molecule. In humans, CD28 is expressed on 90% of CD4 T
cells and 50% of CD8 T cells. In conjunction with
TCR stimulation, ligation of CD28 leads to increased
production of cytokines such as IL-2, cellular proliferation, and induction of antiapoptotic proteins. The
ligands for CD28, B7-1 (CD80), and B7-2 (CD86),
are found on a variety of APCs including DCs, B cells,
and macrophages. The constitutive expression of CD86
is greater than for CD80 on APCs, although CD80
expression is enhanced during APC activation. The

CAQ Corner

Figure 2. Allorecognition pathways and graft rejection.

(A) Pathways of allorecognition. In the direct pathway,
T cells recognize intact major histocompatibility molecules on donor antigen-presenting cells (left). In the indirect pathway, T cells recognize processed alloantigen in
the form of peptides presented by recipient antigen-presenting cells (right). (B) Interactions among endothelial
cells, T cells, and recipient antigen-presenting cells in
allograft rejection. Recipient monocytes are recruited by
endothelial cells to the graft tissue. They are also transformed to become highly efcient antigen-presenting
dendritic cells that may need to recirculate to peripheral
lymphoid organs for maturation. The dendritic cells and
intragraft macrophages present donor peptides via the
indirect pathway to recruited CD4 T cells. CD8 T cells,
on the other hand, are activated by donor endothelial cells
and can either directly kill endothelial cells or traverse the
endothelium and kill parenchymal graft cells. (From
Briscoe and Sayegh30; used with permission from Nature
Publishing Group, http://www.nature.com)

expression of CD80 and CD86 has been examined by

immunohistochemistry and real-time polymerase chain
reaction in human livers following transplantation.37
CD86 was found on the majority of Kupffer cells in all
transplanted livers and in normal liver. CD80 was
instead differentially expressed and was present only
sporadically on normal liver but was present on at least
25% of the Kupffer cells in 45% of the transplanted
livers. However, CD80 expression did not correlate
with the occurrence of acute rejection.
Cytotoxic T lymphocyte associated antigen 4

373

(CTLA4) is a CD28-related protein that is upregulated

upon T-cell activation and, like CD28, binds to B7-1
and B7-2. Whereas CD28 delivers a positive costimulatory signal to T cells, CTLA4 delivers a negative signal
that attenuates T-cell function. Because CTLA4 expression is enhanced following T-cell activation, and
because it has a higher afnity for B7 than CD28 does,
it has been proposed that the physiologic function of
CTLA4 is to dampen or downregulate T-cell responses.
Thus, specic activation of CTLA4 could potentially
yield immunoinhibitory function and tolerance induction, but this approach has been hampered by the lack
of suitable reagents to deliver the negative signal.
The CD40/CD154 costimulation pathway is a second receptor/ligand pair that is critical in alloimmune
responses. CD40 is constitutively expressed on APCs,
including B cells, monocytes, macrophages, and DCs,
but can also be expressed on nonimmune cells including endothelial cells, mast cells, platelets, and epithelial
cells. In contrast, CD154 is expressed on CD4 T cells
following activation, and to a lesser extent on NK cells,
B cells, and CD8 T cells. Unlike the CD28/B7
costimulatory pathway that has primarily been dened
in the context of effects on T-cell function, CD40/
CD154 ligation augments APC function as measured
by upregulation of class II, CD80, and CD86 expression and production of cytokines, including IL-12. The
sum effect of these changes to the APC is to signicantly
enhance B- and T-cell responses to alloantigen. Molecular and immunohistochemical analysis of CD80,
CD86, and CD154 expression in biopsies of liver recipients demonstrated an association between increased
expression of CD86 and CD154, but not CD80, in the
graft during severe acute cellular rejection.38 CD154
was also detected on Kupffer cells and sinusoidal macrophages in livers during chronic rejection but not in
stable liver allografts or normal liver.39
Approaches to inhibit the CD28/B7 and CD40/
CD154 pathways have relied upon specic blocking
antibodies or soluble fusion proteins such as CTLA4immunoglobulin (Ig). In the orthotopic rat liver transplantation model, repeated administration of
CTLA4-Ig beginning at the time of transplantation, or
delayed administration of CTLA4-Ig in combination
with donor splenocytes, lead to extended graft survival
of 100 days, whereas delayed administration of
CTLA4-Ig alone or donor splenocytes alone did not.40
Gene therapy approaches to deliver CTLA4-Ig to liver
allografts have been used with some success. Delivery of
CTLA4-Ig by adeno-associated virus vectors or low
dose FK506 alone failed to induce long-term graft survival. In contrast, the combination of adeno-associated

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Martinez and Rosen

virus CTLA4-Ig transduction and low dose FK506

resulted in long-term graft survival, with most animals
maintaining their graft over 180 days.41 Adenoviralmediated gene delivery of CTLA4-Ig through ex vivo
perfusion of cold preserved livers lead to indenite survival of rat liver allografts and the generation of donorspecic unresponsiveness.42 Adenoviral-mediated
delivery of CD40-Ig fusion proteins also resulted in
indenite rat liver allograft survival and donor specic
tolerance.43
The efcacy of anti-CD154 and CTLA4-Ig were
compared in a rearterialized rat liver allograft model.44
Both treatments lead to prolonged allograft survival but
treatment with anti-CD154 was associated with less
biliary and endothelial cell injury compared with
CTLA4-Ig. The CD40/CD154 pathway was also
shown to play a role in ischemia/reperfusion injury.45
Mice decient in CD154 or wild-type animals treated
with anti-CD154, had diminished hepatic injury and
neutrophil inltration in a warm ischemia reperfusion
model compared with control mice.
While the CD28/B7 and CD40/CD154 costimulatory pathways have garnered the bulk of attention in
transplantation, there is emerging evidence that other
receptor/ligand pairs also play important roles in alloimmune responses.46,47 Recently, there has been considerable interest in two new members of the CD28/B7
family. Inducible costimulator (ICOS) is expressed following T-cell activation and augments cellular proliferation and cytokine production. The ligand for ICOS,
B7h, is constitutively expressed on B cells, macrophages
and dendritic cells as well as many nonlymphoid tissues,
including the liver.48 The ICOS/B7h pathway was originally thought to provide a costimulatory signal for Th2
cytokine production, although it is now apparent that,
under certain circumstances, ICOS can also trigger the
production of the Th1 cytokine IFN-. Further, ICOS
costimulation plays a central role in T-dependent B-cell
activation and isotype switching. Intriguingly, ICOS
may also participate in development of T regulatory
cells.49
Programmed death-1 (PD-1) is induced upon activation of T cells, shares sequence homology with
CTLA4, and contains an immunoreceptor tyrosinebased inhibitory motif in the cytoplasmic tail, characteristic of cellular receptors that deliver a negative signal. Indeed, mice decient in PD-1 develop
autoimmune-like syndromes. PD-1 binds to two
ligands, programmed death ligand (PDL)-1 and
PDL-2, each differentially regulated by Th1 and Th2
cytokines, respectively. PDL-1 is constitutively
expressed in the liver on Kupffer cells and sinusoidal

endothelium.50 Interestingly, PDL-1 decient mice

display a spontaneous accumulation of CD8 T cells in
the liver.51 Moreover, in the absence of PDL-1, T-cell
activation lead to an accelerated form of experimental
autoimmune hepatitis suggesting that PDL-1 plays a
role in the deletion of CD8 T cells in the liver
Several members of the tumor necrosis factor/tumor
necrosis factor receptor families, of which CD40/
CD154 are members, can also provide alternate
costimulatory signals to T lymphocytes. These receptor/ligand pairs include 4-1BB/4-1BBL (CD137),
OX-40/OX-40L (CD134/CD134L), and CD30CD30L(CD153). The receptor 4-1BB is expressed on
CD4 and CD8 T cells following activation, while its
ligand, 4-1BBL, is expressed on activated B cells, macrophages, and DCs. The receptor 4-1BB appears to be
particularly important in CD8 T-cell responses. In contrast, OX-40 / OX-40L primarily acts on CD4 T cells.
CD30 is another T-cell costimulatory molecule that has
been implicated in alloimmune responses. CD30 has
been shown to mark the predominant proliferating Tcell population in response to alloantigen52 and
CD30 T cells comprise the major population of
IFN- and IL-5 producing cells following alloactivation.53 A soluble form of CD30 measured in the blood
early posttransplantation was shown to be a highly sensitive and specic marker for acute rejection in kidney
recipients.54 In general, these alternate costimulatory
molecules are expressed on T cells relatively late following activation and their relationship to CD28 signaling
is still to be determined. OX-40, 4-1BB, and CD30
may act to further modulate T-cell function in the
context of CD28 signaling or to provide distinct, independent signaling. However, their role in liver transplantation remains to be studied.

Effector Pathways of Graft Injury

Once nave T cells have received signals through the
TCR and the appropriate costimulatory molecules for
complete activation, and any additional cues from cytokines or cell surface receptors required for differentiation, they transition to effector cells. Mature effector T
cells, be they CD4 or CD8, are capable of mediating
graft injury. Recently, much work has focused on the
phenotypic distinction of effector T cells from nave
cells and memory T cells,55 and this line of investigation
will likely expand our understanding of the immune
status of graft recipients in the future. The long-standing triumvirate of effector pathways in graft rejection is
(1) T-cellmediated cytotoxicity, (2) delayed type
hypersensitivity, and (3) antibody-mediated damage.

CAQ Corner

The relative importance of each of these pathways in

graft rejection is likely to depend on a variety of factors,
including the organ transplanted, the sensitization status of the recipient, concomitant infections, and the
immunosuppressive protocols that are utilized.
Activated CD8 cytotoxic T lymphocytes (CTLs)
specic for donor class I can interact with the graft
directly and cause tissue injury through biochemical
mechanisms that induce apoptosis. Indeed, apoptosis is
a prominent component of cell death in liver allograft
rejection.56 In the perforin/granzyme pathway, effector
cell-derived perforin and the serine protease granzyme
B collaborate to induce target cell death. Perforin is
inserted into the target cell membrane in a Ca
dependent process forming pores that permit the delivery and entry of granzyme B into the target cell cytosol
from preformed cellular granules. This ultimately leads
to target cell death through apoptosis. Elevations in
granzyme B messenger ribonucleic acid are associated
with acute liver allograft rejection, suggesting that this
pathway is operative following human liver transplantation.57 The Fas/Fas ligand (FasL) pathway is another
death-inducing pathway utilized by CTL. Whereas
FasL is specically induced upon CTL activation, Fas is
ubiquitously expressed on lymphoid and nonlymphoid
tissue including the liver. Cross-linking of Fas with
trimerized FasL or agonist antibodies leads to formation
of the death inducing signaling complex in target cells,
activation of caspase 8, and propagation of a death
signal that culminates in apoptosis. The Fas/FasL pathway is thought to play an important role in a variety of
hepatic pathologies and there is evidence that this pathway is also active during liver allograft rejection.58,59 A
more recently characterized molecule, granulysin, is
expressed by CD8 T cells as well as by CD4 T cells and
NK cells and can also induce target cell apoptosis.60
However, there have been no reports on the involvement of granulysin in liver allograft rejection.
Delayed type hypersensitivity is initiated by alloantigen-primed CD4 T cells specic for donor class II.
Upon reexposure to specic alloantigen, CD4 T cells
release IFN-, a proinammatory cytokine that can
cause activation of macrophages and the subsequent
release of a variety of soluble mediators. These inammatory mediators can augment the cellular antigraft
response or can cause direct tissue damage. The presence of donor-sensitized T cells in graft recipients can
be revealed in the trans-vivo delayed type hypersensitivity response in which peripheral blood mononuclear
cells from graft recipients are injected into the ear or
footpad of immunodecient severe combined immunodeciency mice.61 The degree of swelling following

375

challenge with specic alloantigen provides an index of

prior sensitization. Further, the use of the trans-vivo
assay revealed the presence of active regulatory pathways in 3 patients, including 1 liver transplant recipient, who had suspended immunosuppression but
maintained stable graft function.62 In this study,
peripheral blood mononuclear cells from the 3 patients
showed diminished response to donor antigen, but
good recall responses to unrelated antigens such as
Epstein-Barr virus in the trans-vivo assay. Suppression of antidonor responses could be broken with antibodies to IL-10 or transforming growth factor-, indicating that these cytokines were essential to
maintaining active regulation.
Antibodies specic for graft antigens can mediate
graft injury through activation of the complement
cascade or through binding to Fc receptors on effector cells and triggering antibody-dependent cell-mediated cytotoxicity. Unlike humoral rejection caused
by ABO blood group incompatibilities, alloantibodies directed at MHC class I or class II disparities are
not considered to play a prominent role in liver allograft rejection. However, a novel mechanism was
recently proposed in which antibodies against LSECs
indirectly promote acute rejection.63 In this study, it
was determined that antibodies against LSECs were
more frequently observed in patients undergoing
rejection than in patients without rejection. Furthermore, the antibodies induced expression of CD86 on
LSECs and increased T-cell proliferation while
decreasing transforming growth factor- production
in mixed LSEC/T-cell cultures. Interestingly, a signicant fraction of the anti-LSEC reactivity was not
directed against human leukocyte antigen (HLA)
antigens, suggesting that other cellular components
are the target of these antibodies.
It has been a challenging process to try to unravel
the participation of specic effector pathways, and
their interrelationships, in liver allograft rejection. It
is possible to denitively state that CD4 T cells, CD8
T cells, macrophages, and plasma cells can be found
in the liver during rejection. However, it is much
more difcult to demonstrate that any of these cell
types are actively involved in a destructive immune
response. The multitude of studies using gene-decient mice in experimental transplantation models
has taught us that it is difcult to denitively identify
any single mediator or cell type that is absolutely
required for graft rejection. Rather, it appears that
several redundant and compensatory mechanisms are
in place that can contribute to graft rejection. Moreover, the participation of alternate pathways of graft

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Martinez and Rosen

rejection by other cell types found within the inammatory inltrate, such as eosinophils, has been proposed and should be considered as well.64

Experimental Studies on Mechanisms of Liver

Allograft Rejection
The rat orthotopic liver transplant model has been particularly useful in gaining insight into the immunologic
mechanisms that are active following liver transplantation. In the rat, the outcome of liver transplantation is
highly dependent upon the particular strain combination used, as some combinations lead to classic acute
cellular rejection, whereas others lead to spontaneous
tolerance. The strain combinations that result in acute
rejection fairly closely recapitulate the features of
human liver allograft rejection. Thus, in the dark
Agouti3 Lewis combination, a slight mixed and
mononuclear cell inltrate in the portal region and
venous endothelialitis are seen by day 2 posttransplantation.65 One day later mononuclear cell inltration,
venous endothelialitis, and parenchymal changes are
apparent. By day 7, severe acute rejection is underway,
with marked inammatory inltrates, bridging necrosis, and endothelialitis. Elevations in liver enzymes are
measurable in allografts on days 3 to 4, with progressive
increases through at least day 7. Within the graft, transcripts for IL-2, IFN-, IL-4, tumor necrosis factor-,
and IL-10 are noted,65 indicating that rejection is not
associated with a polarized Th1 or Th2 cytokine
response. Apoptosis of graft cells is a noted feature of
liver allograft rejection,59,66 and it is accompanied by
specic increases in messenger ribonucleic acid for perforin, granzyme B, and FasL in allografts, but not
isografts, suggesting that these mediators participate in
the induction of cell death in the graft.59,67 Interestingly, apoptosis and graft rejection can occur in the
absence of CD8 T cells.59 One candidate cell population that could account for the increase in FasL, perforin, and granzyme B in rejecting liver allograft are NK
cells. As described above, NK cells were originally identied on the basis of their ability to kill tumor targets
without prior activation or sensitization. Recent studies
indicate that large numbers of host NK cells inltrate
liver allografts early posttransplantation68 and express
high levels of FasL and granzyme B. Moreover, a novel
NK cell activation receptor, rNKp30, was cloned from
a rejecting liver allograft, suggesting that NK cells
within the liver have the potential to mediate cytotoxicity.69

Experimental Studies on Tolerance in the

Liver and Associated Mechanisms
A series of experimental and clinical observations indicating that the liver is less immunogenic than other
vascularized organs, and that liver allografts can even
provide tolerogenic properties for other organ grants,
have led to a search for mechanisms that account for
these ndings. It is clear from rat and mouse transplantation models of spontaneous liver tolerance that an
active immune process of some type is initiated in nonrejecting mouse70 and rat strain combinations,71 since
an inammatory inltrate and early liver injury, which
ultimately resolves, can be observed. It has been considered that the large mass of the liver and its regenerative
potential could overcome the early injury, but this is
unlikely to account for the robust protection that the
liver affords to subsequent allografts. Thus, it is reasonable to consider that some active immunoregulatory
process is associated with the liver effect. Multiple
mechanisms have been proposed to explain how the
liver could inuence or modulate the host immune
system. These mechanisms can be divided into 3 categories: soluble factors, cellular components, and structural features of the liver.
Almost 20 years ago, it was noted that the liver
produced and secreted large amounts of soluble MHC
class I that could readily be measured in the circulation
of healthy individuals and liver recipients.72,73 Thus, it
has been proposed that soluble donor class I could have
immunomodulatory properties through passive blockade of alloantibodies and donor-specic effectors or
through induction of activation-induced cell death of
allospecic CTL.74 Direct tests of the effects of soluble
class I on graft survival, however, have been difcult to
reconcile. In some studies, direct in vivo administration
of rat liver derived soluble class I prolonged cardiac
allograft survival, particularly when class I proteins were
complexed with anti-class I monoclonal antibodies.75
However, in other studies soluble MHC class I failed to
modulate kidney or cardiac allograft survival or inhibit
generation of CTL in vivo.76
The nding that serum from rats that spontaneously
accept their liver allograft could transfer the protective
effect led to a search to identify the suppressive activity.
Liver suppressor factor 1 is a 40-kD protein isolated
from the serum of spontaneously tolerant recipients
(dark Agouti3 Piebald-Viral-Glaxo [PVG]). Administration of liver suppressor factor 1 can prolong rat
cardiac allograft survival,77 but elucidation of the mechanism of liver suppressor factor 1 and the broad applicability of these ndings remain to be determined.

CAQ Corner

Finally, normal human hepatocytes can produce a

soluble form of Fas that is derived from a variant transcript lacking the terminal 5 amino acids of the extracellular region and 16 of 17 amino acids of the transmembrane region.78 Hepatocyte lysates containing
soluble Fas could inhibit anti-Fas induced apoptosis
of Jurkat cells in vitro. Moreover, the levels of soluble
Fas decreased in patients undergoing graft rejection as
compared to patients with a stable course. These data
suggest that soluble Fas could modulate the Fas/FasL
pathway in liver recipients. In the case of each of these
soluble mediators, it has been shown that they can
inhibit CTL function in vitro; however, their participation in the liver effect in vivo is unclear. While it is
unlikely that any one of these factors is solely responsible for the tolerogenic properties of liver allografts, it is
possible that they act in concert with other hepatic
components to promote tolerance.
Along these lines, it has been denitively shown that
depletion of resident leukocytes from liver allografts by
irradiation markedly diminishes the tolerogenic effect,
suggesting that some cellular component of the graft is
responsible.79 Similar conclusions can be drawn from
studies by Sriwatanawaongsa et al.80 In this model,
recovery and retransplantation of a tolerant allograft
into a second recipient of the same strain as the rst
leads to graft rejection rather than tolerance. This can
be explained by the fact that temporarily parking the
allogeneic donor liver in the rst recipient results in the
replacement of the passenger leukocytes with host leukocytes, thereby creating a chimeric liver. When the
chimeric liver is retransplanted into the second recipient, spontaneous tolerance does not occur and the graft
is rejected, because donor leukocytes are required for
induction of tolerance. This concept, of course, is in
marked contrast to the traditional paradigm of the role
of passenger leukocytes in organ allografts in which
bone marrow derived leukocytes associated with the
graft are thought to be the critical cell type involved in
priming nave recipient T cells to donor antigens in
secondary lymphoid organs, and thereby precipitating
destructive alloimmune responses.81 This disparity
between the liver and other organ grafts could be due to
multiple factors, not the least of which are differences in
the number and type of passenger leukocytes in the
various organ grafts and their intrinsic potential for
modulating alloimmune responses.
As such, a prominent model proposed by Starzl et
al.14 attributes the tolerogenic properties of the liver to
the ability of graft-derived stem cells to migrate out of
the graft following transplantation, to persist, and to
interact in some fashion with host elements to establish

377

a state of microchimerism that can lead to clonal

exhaustion or deletion of host alloreactive T cells. Support for this model comes from the observation that
patients with long-term surviving allografts have evidence of donor cell microchimerism in peripheral tissue. However, because it has not been feasible to establish whether donor microchimerism in clinical allograft
recipients is a cause or an effect of long-term graft survival, and because the correlation of microchimerism
and graft survival is not absolute, validation of this
model has not been achieved.
While a multitude of studies have implicated liverassociated cells with migratory potential in spontaneous
liver tolerance, the identity of the cell or cells involved
has not been solved. One such candidate cell type is the
immature DCs found in the liver, as discussed previously. Mature DCs, rich in MHC class II and costimulatory molecule expression, are exquisitely efcient antigen-presenting cells and are potent inducers of
alloreactivity. Immature DCs, by contrast, do not
express signicant levels of the costimulatory molecules
CD80 and CD86 and as such are poor inducers of Tcell activation, and may in fact inhibit T-cell responses
through incomplete activation. Indeed, administration
of immature DCs to mice did lead to prolonged cardiac
allograft survival in the absence of immunosuppression,
although the effect was relatively modest.82 Furthermore, progenitor DCs isolated from the liver and propagated in GM-CSF failed to stimulate allogeneic T cells
in MLR, and transfer of the immature DCs to islet
allograft recipients prolonged graft survival.83 Other
resident APCs in the liver have also been implicated in
liver tolerance. As already discussed, LSECs are a
unique nonmyeloid APCs found in the liver that can
efciently cross present soluble antigen to CD8 T cells
but, through this process, induces tolerance rather than
immunity.84 Finally, Kupffer cells, macrophage-like
APCs found in the hepatic sinusoids, were shown many
years ago to be required for tolerance induction by the
liver. In these studies, inactivation of Kupffer cells with
gadolinium chloride inhibited tolerance induced via the
portal vein route.85 Possible mechanisms for the effect
of Kupffer cells in liver tolerance come from recent
studies by Sun et al.,86 demonstrating that Kupffer cells
express functional FasL that can induce apoptosis of T
cells and that transfer of Kupffer cells derived from
accepted grafts can prolong graft survival in an acute
rejection model. Finally, unique aspects of the livers
architecture that allow T cells to come in close contact
with tissue cells, including the LSECs and perhaps
hepatocytes, could promote T-cell nonresponsiveness
or deletion.87 Clearly, examples of functional immune

378

Martinez and Rosen

activation and tolerance induction can be readily demonstrated in the liver. The factors that determine either
outcome remain to be determined, but unifying paradigms to explain these observations have been proposed.88

Immune Aspects of Clinical Liver

Transplantation: Rejection, Recurrence of
Autoimmune Liver Disease, and Hepatitis C
Virus Recurrence
Acute cellular rejection occurs in 50% to 75% of liver
allograft recipients and the majority of episodes occur
within 90 days of transplant surgery.89 The targets of
activated lymphocytes are donor-derived bile duct epithelial cells and vascular endothelium, whereas direct
involvement of hepatocytes is uncommon. Chronic
rejection, often termed ductopenic rejection, is characterized by ischemic injury to and paucity of bile ducts;
the frequency of chronic rejection has decreased in the
past decade and currently occurs in less than 5% of
patients. The pharmacologic agents used to prevent and
treat acute and chronic rejection are covered in great
detail in a separate CAQ Corner by Douglas and colleagues from the Mayo Clinic. The induction of transplantation tolerance, dened as the survival of a functioning allograft in the absence of continuing
immunosuppressive therapy, would obviously represent a major advance. Experimental and emerging clinical data have suggested that transplantation tolerance
could be induced by pretransplantation myeloconditioning and infusion of donor hematopoietic cells,90
but further experience is required.
A number of diseases treated by liver transplantation
are believed to be autoimmune in origin and, theoretically, any transplanted organ is as susceptible to the
autoimmune process as the organ being replaced.91 In
particular, evidence for recurrence of autoimmune hepatitis is supported by most studies, whereas data for
recurrence of primary sclerosing cholangitis and primary biliary cirrhosis (which can be histopathologically
indistinguishable from rejection) are controversial. The
immunosuppression used to prevent graft rejection is in
most cases sufcient to prevent signicant autoimmune
damage to the allograft, but a proportion of patients
may develop graft failure from disease recurrence.
Liver transplantation is performed with no regard to
specic matching of donor-recipient HLA alleles, and
this may serve as a barrier to the development of protective (i.e., antiviral) cell-mediated immunity directed
against infected cells within the allograft. CD8 T cells
are the primary effector lymphocytes for provision of

protective immunity against intracellular pathogen

infection of parenchymal cells and are effective because
of their ability to recognize infected cells as the combination of pathogen-derived peptides in the peptidebinding grooves of HLA class I molecules on the surface
of infected cells. While incompletely understood, the
immune recognition of the hepatitis C virus infected
allograft may be essential in the containment of infection. Recognition of the allograft could occur either via
recipient-derived T cells or via those derived from the
donor. For the recipient-derived T cells, recognition
could occur either through use of shared HLA molecules, or could occur through the expansion of recipient-derived T cells that are uniquely restricted by the
HLA molecules of the donor liver. Because prior
research has focused on CD8 T cells restricted by
recipient MHC molecules, little is known about the
relative contribution of those cells uniquely restricted
by the donor liver. A recent report demonstrated the
generation of hepatitis C virus specic T cells that are
restricted by donor HLA alleles,92 underscoring the
plasticity of the TCR, as well as the need to assess both
recipient and allograft restricted CTL in order to
develop a comprehensive understanding of protective
immunity to hepatitis C virus after liver transplantation.

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