Tolerância Imunológica
description
Transcript of Tolerância Imunológica
doi: 10.1111/j.1365-2796.2007.01855.x
Immune tolerance: mechanisms and application in clinicaltransplantation
M. Sykes
From the Transplantation Biology Research Center, Bone Marrow Transplantation Section, Massachusetts GeneralHospital ⁄Harvard Medical School, Boston, MA, USA
Abstract. Sykes M (Massachusetts General Hospi-
tal ⁄Harvard Medical School, Boston, MA, USA).
Immune tolerance: mechanisms and application in
clinical transplantation (Review). J Intern Med 2007;
262: 288–310.
The achievement of immune tolerance, a state of
specific unresponsiveness to the donor graft, has the
potential to overcome the current major limitations to
progress in organ transplantation, namely late graft
loss, organ shortage and the toxicities of chronic
nonspecific immumnosuppressive therapy. Advances
in our understanding of immunological processes,
mechanisms of rejection and tolerance have led
to encouraging developments in animal models,
which are just beginning to be translated into
clinical pilot studies. These advances are reviewed
here and the appropriate timing for clinical trials is
discussed.
Keywords: bone marrow transplantation, immunity,
immunology, immunosuppressive treatment, transplan-
tation immunology.
The need for immune tolerance in transplantation
Immune tolerance is a state in which the immune sys-
tem is specifically unresponsive to antigens of interest.
For example, most people enjoy a state of immune
tolerance to their own antigens, resulting in freedom
from autoimmune disease. In the case of organ and cell
transplantation, tolerance denotes a state of specific
immune unresponsiveness to the donor graft, with nor-
mal responses to other antigens. The ability to respond
normally to other antigens contrasts sharply with the
effect of nonspecific immunosuppressive agents that
are used clinically to prevent rejection, which are asso-
ciated with increased risks of infection and malig-
nancy. This paper will review the current status of
tolerance in the field of organ and tissue transplan-
tation. Achievement of transplantation tolerance is the
‘holy grail’ in clinical transplantation for three major
reasons. First, whilst improvements in nonspecific
immunosuppressive therapy have markedly improved
outcomes in organ transplantation, these drugs are
associated with many specific organ toxicities as well
as the life-long increased risks of infection and
malignancy mentioned above. Secondly, chronic
rejection is a major factor contributing to constantly
downsloping long-term survival curves for organ
allografts. The half-lives of this second, late phase of
graft loss have not changed significantly with improve-
ments in immunosuppressive therapy over the last
25 years. Chronic rejection can be avoided by
tolerance induction. Thirdly, there is a critical shortage
of allogeneic organs for transplantation, which could
be overcome by the use of other species as organ and
tissue sources, i.e. xenografts. However, immune
barriers to xenografts are even stronger than those to
allografts, and the induction of tolerance at both the
humoral and the cellular level is likely to be needed
for the successful application of xenotransplantation in
humans.
Numerous approaches to tolerance induction have
been developed in rodent models. Many of these lar-
gely reflect the strong inherent tolerogenicity of
primarily vascularized heart, liver and kidney grafts in
these animals rather than the potency of the tolerance-
inducing regimens per se. A short course of many
288 ª 2007 Blackwell Publishing Ltd
Review |
types of immunosuppression can allow these tolero-
genic effects to prevail over the rejection response,
leading to long-term graft acceptance. Because such
grafts are unfortunately less tolerogenic in large ani-
mals and humans, these tolerance strategies have not
been effectively applied in humans. Thus, before clin-
ical evaluation is appropriate, tolerance strategies
should first be tested in ‘stringent’ animal models,
including strongly immunogenic grafts such as major
histocompatibility complex (MHC)-mismatched skin
in rodents and vascularized organ graft models in
large animals.
The simple definition of tolerance in the first para-
graph above includes several different immunological
states. In one, the allograft is accepted without chro-
nic immunosuppression, but the recipient can reject a
second graft from the same donor. In a different state,
the immune system accepts any other organ or tissue
from the same donor without immunosuppression,
and in vitro studies reveal specific unresponsiveness
to the donor. This state of tolerance can be described
as systemic. There are intermediate forms of tolerance
in which some types of second graft, but not others
from the same donor, are accepted. In some forms of
tolerance, in vitro studies show normal or reduced
anti-donor responses without the complete unrespon-
siveness that characterizes systemic tolerance. In all
these states, the recipient can reject organs from a
third party donor.
The above discussion is focused on T-cell tolerance
because T cells clearly play a central role in allograft
rejection. In naı̈ve allograft recipients, B-cell tolerance
is not a separate concern, because in the absence of
help from donor-reactive T cells, de novo anti-donor
alloantibody responses are not generated. However,
there are several situations in which B-cell tolerance
would be advantageous. These include transplantation
to recipients with preexisting ‘natural’ antibodies,
which are antibodies that are present without known
prior sensitization, against the donor. Examples are
anti-isohaemagglutinins against blood group antigens
and natural antibodies in sera of primates that
recognize porcine carbohydrate antigens, as is
discussed below. Additionally, a recipient may contain
anti-donor antibodies because of prior sensitization
to antigens of that donor, for example due to preg-
nancy, blood transfusions or prior transplants.
Mechanisms of T and B-cell tolerance
As discussed above, the achievement of T-cell toler-
ance would overcome the major barriers to successful
allografting discussed above. There are three major
mechanisms of T-cell tolerance, including clonal dele-
tion, anergy and suppression (commonly referred to
as ‘regulation’). These mechanisms may act alone or
together to achieve tolerance. Clonal deletion implies
death of T cells with receptors recognizing donor
antigens. Deletion is the major mechanism of self-
tolerance induction during T-cell development in the
thymus. Mature T cells in the peripheral lymphoid
tissues can also be deleted under certain conditions.
Suppression, in which a cell population actively
downregulates the reactivity of T cells, has recently
been implicated in many rodent transplantation toler-
ance models and in the maintenance of self-tolerance.
Anergy denotes the inability of T cells to proliferate
and produce interleukin-2 (IL-2) in response to anti-
gens they recognize. In addition, a graft may simply
be ‘ignored’ by recipient T cells. These mechanisms
are discussed in more detail and in the context of
transplantation below.
T cell clonal deletion in transplantation
Most intrathymic T-cell tolerance results from deletion
of developing thymocytes whose receptors recognize
self antigens presented by haematopoietic cells and
thymic epithelial cells (reviewed in Ref. [1]). The pro-
cesses involved in T-cell ‘education’ in the thymus
are depicted and explained in Fig. 1. High avidity
interactions between immature thymocytes due, at
least in part, to a relatively high affinity interaction
between a rearranged T-cell receptor (TCR) and a
peptide ⁄MHC complex on antigen-presenting cells
(APC) in the thymus, result in deletion of the thymo-
cyte by apoptotic cell death [2, 3]. TCRs with lower
affinity for such complexes are more likely to survive
this process, and other mechanisms are required to
ensure their tolerance when they enter the periphery,
M. Sykes | Review: Transplantation tolerance
ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 289
particularly under conditions of inflammation and
antigen upregulation. Indeed, deletion is not the only
mechanism of intrathymic tolerance induction: T cells
with receptors recognizing self antigens presented by
nonhaematopoietic thymic stromal cells [4] or even
haematopoietic cells [5] may be rendered anergic.
Additionally, presentation of antigens by the thymic
epithelium promotes the development of specific
regulatory cells that tolerize other T cells in the
periphery [6].
Intrathymic deletion is induced most potently by anti-
gen presented on haematopoietic cell types, including
dendritic cells [7]. This is a major reason why
allogeneic haematopoietic transplantation (HCT)
provides a powerful approach to tolerance induction
in lymphoablated rodents. To avoid rejection of the
marrow, specific T-cell ablation in the thymus and the
periphery can be achieved with relatively nontoxic,
nonmyeloablative conditioning that includes T-cell-
depleting monoclonal antibodies (mAb) and local irra-
diation to the thymus [8, 9]. As might be expected,
tolerance induced by intrathymic deletion is systemic,
as shown both in vivo and in vitro (reviewed in Ref.
[1]). In this setting, the only significant mechanism
involved in maintaining transplantation tolerance is
intrathymic clonal deletion [10–12]. Anti-donor anti-
body can be given to established mixed chimeras to
(a) (b)
Fig. 1 Schematic depiction of T cell ‘education’ in the thymus (a) and the role of mixed chimerism in achieving central dele-tional tolerance (b). In the normal situation (a) thymocyte and APC progenitors migrate to the thymus from the marrow (1) tobecome double negative thymocytes (2) or APCs. Thymocyte progenitors may productively rearrange a and b T cell receptorchains, which are first expressed by CD4+CD8+ (‘double positive’) thymocytes (3). These thymocytes are then subject to posit-ive and negative selection processes. Positive selection (3), which rescues the double positive thymocyte from programmed celldeath, results from a low avidity interaction between thymocytes and thymic epithelial cells in the thymic cortex. This interac-tion requires a low-affinity interaction between the rearranged thymocyte TCR and a self MHC ⁄ peptide complex presented bythe thymic epithelial cell. Depending on whether this MHC molecule is of class I or class II, the thymocyte will lose expres-sion of CD4 or CD8, respectively, resulting in the generation of a CD8 or CD4 single positive thymocyte. The double-positiveor single-positive thymocyte may also die, however, if it interacts with higher affinity with a self MHC ⁄ peptide complex on anAPC (4) or, possibly, a thymic epithelial cell, in the thymic medulla or corticomedullary junction. The surviving thymocytesundergo further maturation (5), then leave the thymus and enter the peripheral lymphoid tissues. In mixed chimeras (b), thymo-cyte and APC progenitors of both donor and host origin migrate to the thymus from the marrow (1) to become double negativethymocytes or APCs (2). Thymocyte progenitors of both types may productively rearrange a and b T cell receptor chains andbecome CD4 ⁄CD8 double positive thymocytes (3). These thymocytes are then subject to positive and negative selection pro-cesses. Positive selection (3), which rescues the double positive thymocyte from programmed cell death, is mediated exclu-sively by thymic epithelial cells and hence MHC of host origin in the thymic cortex. Depending on whether this MHCmolecule is of class I or class II, the donor or recipient thymocyte will lose expression of CD4 or CD8, respectively, resultingin the generation of donor and recipient CD8 and CD4 single positive thymocytes. The double positive or single positivethymocyte may also die, however, if it interacts with higher affinity with an MHC ⁄ peptide complex on a donor or recipient-derived APC (4) or, possibly, a thymic epithelial cell, in the thymic medulla or corticomedullary junction. Consequently, onlymature T cells that lack strong reactivity to donor or host antigens survive this negative selection process, resulting in emer-gence from the thymus only of T cells (of both donor and recipient origin) that are tolerant of both the donor and the host (5).
M. Sykes | Review: Transplantation tolerance
290 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310
eliminate donor chimerism; this results in loss of
tolerance to donor skin grafts, and in the de novoappearance in the blood of T cells with receptors that
recognize donor antigens. However, if the recipient
thymus is removed before chimerism is eliminated
with anti-donor antibody, specific tolerance to the
donor is preserved, and donor-reactive TCR do not
appear in the circulation [11]. These results show that
chimerism is needed only in the thymus and not in
the periphery to ensure persistent tolerance. Antigen
in the periphery is not required and, once the thymus
is removed, donor antigen is not required to maintain
tolerance at all. These results are consistent with a
purely central deletional tolerance mechanism, as
tolerance resulting from peripheral anergy or suppres-
sion requires persistent antigen [13–15]. Thymic APC
continually turn over, emphasizing the need for
haematopoietic stem cell engraftment at sufficient
levels in order to ensure an uninterrupted supply of
donor APC in the recipient thymus for life when
tolerance depends solely on intrathymic deletion.
Because they lack active suppressive tolerance mecha-
nisms, such animals are vulnerable to loss of toler-
ance if nontolerant T cells are allowed to emerge
from the thymus after intentional depletion of donor
antigen, or after exogenous administration of nonto-
lerant host-type T cells [11, 16].
Exposure of mature T cells to antigen in the peri-
phery can also result in T-cell clonal deletion [17].
Self antigen cross-presentation by lymph node dend-
ritic cells under noninflammatory conditions leads to
deletion of tissue antigen-specific CD8+ cytotoxic T
lymphocytes (CTL) [18]. CD8 cells may be deleted
because of ‘exhaustion’ in the presence of a large,
persistent antigen load [19]. As an alternative to
global T-cell depletion, co-stimulatory blockade with
anti-CD154 (see below) can be used in combination
with bone marrow transplantation (BMT) to achieve
mixed chimerism and long-term central, deletional
tolerance [20, 21]. In such animals, the preexisting
alloreactive T-cell repertoire is not depleted with
mAb, and other mechanisms come into play.
Peripheral deletion, specifically, of donor-reactive
CD8 [22, 23] and CD4 [16, 20, 24, 25] cells occurs
under these conditions. A similar phenomenon has
been demonstrated for peripheral CD8 cells in mice
receiving donor-specific transfusion (DST) combined
with anti-CD154 [26]. Peripheral T cell apoptosis has
been demonstrated, though without specific markers
for alloreactive T cells, in mice tolerized with
anti-CD154 mAb, rapamycin and cardiac allografts
[27].
Additional mechanisms of peripheral deletion, such as
the activity of ‘veto’ cells, which are cells that kill
CTL that recognize them [28], can delete alloreactive
CTL precursors in the periphery. Recently,
CD4)CD8) cytotoxic regulatory cells have been
reported to delete alloreactive CD8+ T cells with the
same specificity as the regulatory cells [29].
B cell clonal deletion in transplantation
As discussed above, there are several transplant situa-
tions in which tolerance induction of B cells would
be of potential value. Immunoglobulin (Ig) receptor
transgenic mice have been widely used for the analy-
sis of mechanisms of B-cell tolerance. Such studies
indicate that immature B cells are susceptible to
deletion when they encounter membrane-bound anti-
gens expressed by haematopoietic or nonhaemato-
poietic cells [30]. Developing B cells whose
rearranged Ig receptors recognize a self antigen
undergo developmental arrest followed by Ig light
chain ‘receptor editing’. If this ‘second chance’ rear-
rangement leads to the formation of a nonautoreactive
Ig receptor, the B cell survives; if not, the B cell
dies [31].
B cells have been divided into several subsets, inclu-
ding follicular, marginal zone and ‘B-1’ B cells. B-1
cells in mice produce ‘natural antibodies’ recogni-
zing important xenogeneic carbohydrate antigens
without known prior immunization [32]. Data sug-
gest that a similar subset may produce such natural
antibodies in nonhuman primates and man [33].
Such antibodies are responsible for xenograft hyper-
acute rejection, and can be deleted in mice via apop-
tosis when their surface Ig receptors are cross-linked
by cell-bound antigens [34]. Deletion and ⁄or receptorediting is responsible for the long-term tolerance of
M. Sykes | Review: Transplantation tolerance
ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 291
natural antibody-producing B-1 cells in mice when
mixed chimerism is induced using a bone marrow
donor that expresses an antigen for which natural
antibody-producing cells preexist in the recipient
[35–37].
T cell anergy
In addition to a signal through their T-cell receptor, T
cells require stimulation of additional receptors,
termed co-stimulatory molecules, in order to be fully
activated. CD28 is a major co-stimulatory receptor,
whose ligands consist of B7-1 (CD80) and B7-2
(CD86) molecules expressed by APC. T cell anergy
develops when T cells encounter peptide ⁄MHC com-
plexes without receiving adequate accessory or co-
stimulatory signals [38]. T cells can also be rendered
anergic if they encounter peptide ligands for which
they have low affinity [3]. Certain APC, such as
macrophages [39] and tolerogenic dendritic cells that
may be immature or matured in a specific manner
[40] have the capacity to induce T cell anergy, in part
due to secretion of suppressive cytokines and lack of
adequate co-stimulation. Anergy is associated with
altered signalling and tyrosine phosphorylation pat-
terns [38, 41]. T cell anergy can often [42], but not
always [16, 43], be overcome by providing exogenous
IL-2. Anergy has been associated with TCR down-
modulation [44]. It should be borne in mind that
anergy is reversible under pro-inflammatory condi-
tions [45, 46], including the presence of infection, so
it is unlikely to be reliable as the sole long-term
tolerance mechanism.
Deletion has followed induction of an anergic state in
the continued presence of antigen in some, but not
all, models [47, 48]. In mice receiving BMT under
the cover of co-stimulatory blockade, peripheral
donor-reactive CD4 T cells are rendered anergic prior
to their deletion over a period of weeks [16]. Anergic
T cells may also down-regulate the activity of other T
cells, so that they function as regulatory T cells
(Treg), perhaps by conditioning APC such that they
tolerize T cells recognizing presenting the same or dif-
ferent antigens presented by these APC [49]. More-
over, Treg (see below) can promote the induction of
T cell anergy [50] and may themselves have bio-
chemical properties suggestive of an anergic state
[51].
B cell anergy
As many self-reactive B cells escape deletion during
development in the bone marrow, anergy is an
important tolerance mechanism. Many of these B
cells are anergic and die within the peripheral
lymphoid tissues when they encounter abundant but
low avidity antigens [30] (reviewed in Ref. [31]).
Similar to T cell anergy, B cell anergy requires
persistent antigen and is characterized by antigen
receptor downregulation [30], altered signalling pat-
terns and increased apoptosis upon antigen encounter
[31]. T-cell tolerance and the consequent absence of
T cell help maintain B cell anergy. Anergic B cells
can nevertheless be activated in the presence of high
avidity antigen and T cell help [31]. Anergy is the
mechanism leading to early tolerance of natural anti-
body-producing B-1 cells in mice rendered mixed chi-
meric with bone marrow cells expressing an antigen
recognized by recipient natural antibody-producing
cells [35–37].
Lymphocytes ignoring graft antigens (‘ignorance’)
In some situations, antigens may simply be ignored
by T cells [44] or B cells [30] with receptors
recognizing them. This may occur when antigens
are presented by ‘nonprofessional APC’ which are
unable to activate T cells, or when T cells fail to
migrate to the antigen-bearing tissue, as documented
in murine solid tumour models [52]. Several factors
appear to determine such T-cell behaviour, including
the level of antigen expression, how recently the
responding T cell has emerged from the thymus
[44], and the presence or absence of proinflam-
matory cytokines [53] and co-stimulatory molecules
in peripheral tissues [54]. As might be easily
imagined, ‘ignorance’ is a precarious state which
can be upset by additional immunological stimuli
provoked by inflammation induced by infections
[55] or by presentation of antigen on professional
APCs [56].
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292 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310
Active suppression of T-cell responses
It has become increasingly clear in recent years that
several mechanisms exist to down-modulate immune
responses once they are initiated, and that it is the
balance of activating and modulating functions that
determine the outcome of any response. Many mecha-
nisms, including killing of APC by CTL, inhibitory
effects of cytokines, activation-induced cell death, etc.
contribute to this down-modulation of immune
responses. In addition, studies in the 1970s introduced
the concept that T cells themselves could actively sup-
press immune responses. Whilst certain T cell and
non-T cell populations were implicated in this sup-
pression, it is only in the last decade or so that
molecular markers of suppressive T cells have been
identified and that suppressive cell populations
have been isolated, cultured in vitro and adoptively
transferred.
Regulatory CD4+ T cells
Suppressive CD25+CD4+ T cells have been strongly
implicated in the induction and maintenance of self-
tolerance [57, 58]. More recent studies have shown
that these cells are generated mainly in the thymus,
require specific positive selection (Fig. 1) [6] and
express FoxP3, a transcription factor that controls the
genetic programme associated with their suppressive
activity [59, 60]. These Treg may require an interme-
diate-affinity MHC ⁄peptide ligand (too low for negat-
ive selection) expressed on cortical epithelial cells of
the thymus for their survival and maturation [6]. Con-
stitutively CD25+ Treg of this type have been termed
‘natural’ Treg [61]. In vitro suppression by these Treg
seems to require cell-to-cell contact [61]. Transform-
ing growth factor-b (TGF-b) is a cytokine that has
been strongly implicated in the maintenance of Treg
and as a mediator of their suppressive activity [62–
65]. Both CD4 and CD8 T cells are subject to
suppression by Treg, and memory as well as naı̈ve
responses have been shown to be suppressed. Several
reports indicate that the Treg require specific antigen
for their activation, but that the final effector mechan-
ism of suppression is nonantigen specific [66–68].
Rechallenge with specific antigen induces c-interferon
(IFN-c) expression by Treg, which appears to be crit-
ical for their function [69]. Generation, expansion,
survival and possibly the function of Treg is highly
dependent on IL-2, which is not produced by the Treg
themselves [70].
Additional CD4+ T-cell populations with suppressive
function include FoxP3+ CD25+ cells that arise from
FoxP3-CD25- cells in the periphery following anti-
gen-specific stimulation (‘adaptive’ Treg) [61], especi-
ally in the presence of TGF-b [71]. Additionally,
‘Tr1’ regulatory cells are induced by chronic antigenic
stimulation in the presence of IL-10 and can suppress
autoimmune diseases in mice. These cells produce
high levels of IL-10 and low amounts of IL-2
(reviewed in Ref. [72]), and immature dendritic cells
can support their development in vitro [73]. Both nat-
ural Treg and Tr1 cells are hyporesponsive to TCR-
mediated stimulation but can be grown slowly in vitroin the presence of certain cytokines, including IL-2.
The in vitro suppressive function of Tr1 is dependent
on IL-10 and TGF-b [72].
Transforming growth factor-b is clearly an important
cytokine for several suppressive populations. Besides
maintaining peripheral Treg populations and functions
[63, 65], TGF-b promotes adaptive Treg differenti-
ation [74] and suppresses T-cell activation and Th1
differentiation through several Treg-independent
mechanisms [65, 75]. It can also modulate dendritic
cell function, rendering them tolerogenic for T cells
[40]. However, TGF-b has highly pleiotropic func-
tions and cannot be viewed purely as an immuno-
suppressive cytokine. For example, TGF-b has been
implicated in chronic fibrotic conditions (e.g. chronic
graft-versus-host disease, GVHD; [76]) and in the dif-
ferentiation of naı̈ve T cells to the pro-inflammatory
IL-17-producing ‘Th17’ phenotype [77, 78].
Suppressive T cells have been implicated in numerous
experimental models leading to allograft tolerance
(reviewed in Refs. [79, 80]). Functional evidence for
specific suppressor cells was obtained in early models
of transplantation tolerance (reviewed in Ref. [81]) and
Hall et al. first identified CD25+CD4+ T cells as a
specific suppressive population in rats receiving
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ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 293
cardiac allografts with a short course of cyclosporin
[82]. Since then, Treg have been implicated in numer-
ous models involving acceptance of vascularized
allografts in rodents receiving an initial immuno-
suppressive treatment. Regimens have included donor-
specific cell infusions (termed DST), with [66] or
without [83, 84] co-stimulatory blockade [85, 86]
or partial T-cell depletion [87] or other combinations
of these. Treg promote the acceptance of MHC-
matched, minor histocompatibility antigen-mismatched
skin grafts in mice receiving nondepleting anti-CD4
and CD8 antibodies with or without anti-CD154
[88–90] or anti-CD154 and CD8 cell depletion
[91, 92]. Treg have been implicated in islet allograft
acceptance after treatment with CTLA4Ig [93].
Adaptive Treg [90, 94] have been implicated in some
of these studies.
The thymus plays an important role in several periph-
eral tolerance models, perhaps due to its role in gener-
ating Treg. For example, the thymus is needed for
tolerance induction in a porcine model involving a
short course of a high-dose calcineurin inhibitor in
combination with a renal allograft [95]. A similar phe-
nomenon has been observed in rats receiving soluble
alloantigens in combination with a vascularized allo-
graft [96] and active regulatory cell populations have
been described [96]. However, other mechanisms
involving recirculation of activated T cells to the thy-
mus have also been implicated [97], and the circula-
tion of peripheral dendritic cells to the thymus may
also play a role by promoting intrathymic deletion of
newly developing thymocytes [98] and possibly by
inducing positive selection of Treg.
There is considerable evidence for a role for natural
Treg in maintaining self-tolerance in humans. Con-
genital defects in FoxP3 in humans are associated
with an autoimmune syndrome, immune dysregula-
tion, polyendocrinopathy, enteropathy, X-linked
(IPEX), that resembles its counterpart in mice (the
‘scurfy’ mutant) [99]. Defects in IL-2 signalling
through the STAT-5 transcription factor lead to similar
defects in Treg in mice and humans [100, 101].
These and the above experimental results have led to
considerable interest in the role of Treg in clinical
transplantation, and correlative data have begun to
emerge. Chronic renal allograft rejection has been
associated with reduced circulating Treg concentra-
tions [102]. Whilst discontinuation of immunosup-
pressive medications usually leads to rejection, a
small fraction of such patients accept their grafts nev-
ertheless, i.e. they demonstrate ‘spontaneous’ toler-
ance. These patients do not show increased circulating
Treg compared with controls [102]. Increased urinary
FoxP3 mRNA has been reported to predict improved
outcome of renal allograft rejection episodes [103].
The use of calcineurin inhibitors, but not rapamycin,
has been associated with reduced percentages of Treg
in blood of kidney allograft recipients [104].
Studies in mice have demonstrated the ability of Treg
to inhibit GVHD [105] and Treg have been implicated
in a mouse model in which GVHD has been inhibited
by pre-BMT exposure of donor T cells to recipient al-
loantigens in the presence of anti-CD40L [106]. In
humans, several studies have documented increased
[107, 108] or decreased [109] Treg concentrations in
association with chronic GVHD and decreased num-
bers in association with acute GVHD [110] in HCT
recipients, resulting in some confusion at the present
time. Treatment of severe autoimmune disease with
lymphoablative therapy followed by autologous HCT
has been associated with restoration of normal Treg
populations [111].
Other suppressive cell populations
In addition to the T cell populations discussed above,
other T-cell and non-T-cell suppressive cell popula-
tions can down-modulate immune responses. Fully
differentiated CD4+ helper T-(Th) cells may polarize
their cytokine secretion patterns to that of the Th1
subset, which secretes IL-2 and IFN-c, the Th17 sub-
set that produces IL-17 [77] or the T-helper type 2
(Th2) subset that secretes IL-4 and IL-10 [112]. Th1
cells promote the generation of cytolytic CD8+ T
cells, whilst Th2 helps antibody responses but not
CTL responses [112]. A similar polarization of the
pattern of cytokine secretion occurs in CD8+ cytolytic
T cells [113]. In the early 1990s, there was consider-
able interest in the concept that polarization to Th2
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294 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310
type of response from a pro-inflammatory Th1 (IL-2-
and IFN-c-producing) response could promote allo-
graft acceptance, and data associated Th2 responses
with such acceptance. However, only a few studies
directly demonstrated a role for Th2 cells in tolerance
induction and it is now clear that Th2 cells and their
cytokines can promote allograft rejection (reviewed in
Ref. [114]).
Natural killer (NK) T cells (T cells that express NK
cell-associated markers and may utilize an invariant
TCR-a chain) are another subset of T cells with regu-
latory activity, which may be mediated in part by
Th2-type cytokines [115]. NKT cells have recently
been shown to depend on TGF-b for their develop-
ment [65, 75]. NKT cells are enriched in bone mar-
row and can suppress GVHD [116, 117], at least in
part via an IL-4-dependent mechanism [117]. Total
lymphoid irradiation (TLI) markedly enriches NKT
cells in the lymphoid tissues [118]. The markedly
reduced incidence of acute GVHD recently described
in patients receiving haematopoietic cell transplanta-
tion with a TLI-based regimen may be related to Th2
cytokine polarization induced by this population
[119].
A CD4)CD8) T cell population lacking NK cell
markers that suppresses skin graft rejection by CD8 T
cells with the same TCR has been described in a
mouse model [29], but the importance of this cell
population in other settings remains to be determined.
Human CD8+CD28) T cells have been reported to
suppress alloresponses and xenoresponses in vitro
[120], and recent studies have implicated CD8 cells
as regulatory cells in models of autoimmunity [121],
heart graft acceptance [122], skin grafting [123] and
GVHD [124–126]. ‘Natural’ [124] and ‘adaptive’
[126], FoxP3-expressing [122, 124], TGF-b-produ-cing [127], and IL-10-producing [126] regulatory
CD8 cells have been described, and extensive data
are emerging on the role of these cells in various
models. One mechanism of immune down-modula-
tion mediated by CD8 T cells is simply the killing
by alloreactive CTL of critical donor APC popula-
tions [128].
Some CD8+ CTL-mediated suppressive phenomena
might be attributable to ‘veto’ activity of these cells.
‘Veto’ cells inactivate CTL recognizing antigens
expressed on the veto cell surface [28], resulting in
suppression of CTL responses to antigens shared by
the veto cells. CTL, various bone marrow cell subsets
and NK cells have been reported to have such activ-
ity. Veto cells may promote GVH tolerance, promote
allogeneic marrow engraftment and promote tolerance
induction with DST (reviewed in Refs [28, 81]). Veto
activity has been suggested to involve TGF-b [129].
Thus, whilst many types of Treg have been recently
described, much remains to be learned about the relat-
ive importance of each of these, their potential in
large animal models and the circumstances under
which they can be optimally generated. Several
groups are exploring the approach of expanding Treg
in vitro and then administering them in vivo to sup-
press alloimmunity or autoimmunity. Whilst methods
of nonspecifically expanding mouse and human Treg
ex vivo have recently been developed [130, 131], ani-
mal studies suggest that antigen specificity is import-
ant for the achievement of effective suppression
following adoptive transfer [130]. As alloreactivity
includes many different donor antigens and donor
cells will not be available pretransplant for cadaveric
donor transplantation, this approach may be difficult
to apply.
Co-stimulatory blockade in transplantation
The discovery that TCR stimulation without co-stimu-
lation can induce anergy [132] has led to intensive
evaluation of co-stimulatory blockade in the transplan-
tation field. Blockade of the CD28 co-stimulatory
pathway can be achieved with specific mAb or with a
soluble receptor for the B7-1 ⁄B7-2 ligands. CTLA4,
an alternate, inhibitory T receptor with a higher affin-
ity than CD28 for these ligands, has been studied in
experimental models as a soluble CTLA4-Ig fusion
protein. Another pathway that has been targeted
recently involves the interaction between CD154 on
activated T cells with the CD40 receptor on APC.
This interaction plays an important role in
allowing APC to achieve full activating capacity by
M. Sykes | Review: Transplantation tolerance
ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 295
upregulating B7 molecules, MHC, antigen processing
pathways, cytokines and other molecules. Blockade of
the CD40–CD154 pathway alone or in combination
with CTLA4-Ig can achieve marked prolongation of
fully MHC-mismatched skin graft survivals in some
mouse strain combinations. However, permanent toler-
ance of these grafts is not reliably achieved [133–
136]. These treatments can more reliably induce per-
manent acceptance of tolerogenic rodent allografts
such as hearts [137]. Anergy of donor-reactive cells
and an important role for Treg have been implicated
in such models [138]. Rapamycin, a pharmacological
inhibitor of mammalian target of rapamycin, appears
to selectively allow expansion, activation and survival
of Treg whilst blocking proliferation of effector T
cells [139–141]. This drug has been reported to
achieve robust allograft tolerance when used in com-
bination with anti-CD154 [142] or with anti-IL-15
and a long-acting form of IL-2 [143].
The combination of DST and anti-CD154 leads to
long-term acceptance of several types of allografts
[144] and to prolongation of fully MHC-mismatched
skin graft survival, which can be permanent in
thymectomized mice [145]. Again, both anergy and a
role for Treg have been implicated [144, 145], as well
as peripheral CD8 cell deletion [26, 146]. However,
these mechanisms are apparently insufficient to pre-
vent rejection of the skin graft by newly emerging al-
loreactive T cells in euthymic mice [147]. IFN-c,which has traditionally been considered to be a Th1
proinflammatory cytokine, has been shown to play an
important role in the tolerance achieved in this and
other models [145, 146, 148], possibly because of its
role in supporting Treg function [69]. DST with rapa-
mycin and anti-CD154 has been reported to markedly
prolong islet allograft survival in nonhuman primates
[149]. The combination of anti-CD154, BMT and
DST allows the achievement of mixed chimerism and
robust tolerance; the durable chimerism ensures cen-
tral deletion of donor-reactive T cells, preventing their
emergence from the thymus after the transplant [23,
150].
Despite the achievement of prolonged allograft survi-
val (though not tolerance) in nonhuman primates
[151–154], attempts to apply co-stimulatory blockade
for the induction of tolerance clinically have not suc-
ceeded. The combination of rapamycin, DST and anti-
CD154 was reported to achieve tolerance in three of
five nonhuman primate recipients [155]. However,
anti-CD154 use has been complicated by thromboem-
bolic phenomena [156], resulting in termination of the
trials evaluating it. Anti-CD40 agents may have less
pro-thrombotic activity [157] and may be evaluated in
future trials. Whilst CTLA4Ig alone did not lead to
optimal renal allograft survival in nonhuman primates
[151], it is currently being evaluated as a calcineurin
inhibitor-sparing immunosuppressant in clinical trials
[158]. In another clinical trial, acute GVHD was
reduced in leukaemic patients receiving human leuco-
cyte antigen (HLA)-mismatched bone marrow trans-
plants that were exposed to recipient alloantigens exvivo in the presence of CD28 blockade with CTLA4Ig
[159]. Whilst several additional co-stimulatory path-
ways exist and combinations of blockers are showing
promise in rodent models, co-stimulatory blockade
alone has not yet proved to be sufficiently powerful
to achieve tolerance in nonhuman primates or
humans.
Mixed chimerism as an approach to transplantationtolerance
Bone marrow engraftment reliably induces tolerance
to the most immunogenic allografts, such as fully
MHC-mismatched skin and small bowel grafts, in ani-
mal models (reviewed in Ref. [160]). The ability to
achieve transplantation tolerance with HCT has been
well documented in patients who first received HCT
with conventional myeloablative conditioning to treat
a haematological malignancy, and later accepted an
organ transplant from the same donor without chronic
immunosuppressive therapy (reviewed in Ref. [161]).
Haematopoietic cell administration in utero or neo-
natally, in immunologically immature hosts, has long
been known to be associated with transplantation
tolerance in animals (reviewed in Ref. [162]) and
durable chimerism and renal allograft tolerance have
recently been achieved in a porcine model involving
in utero transplantation of T-cell-depleted adult bone
marrow [163]. Both intrathymic and extrathymic
M. Sykes | Review: Transplantation tolerance
296 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310
mechanisms have been implicated in neonatally
induced tolerance [164, 165]. As prenatal diagnosis of
congenital diseases has become possible, injection of
allogeneic pluripotent haemopoietic stem cells to pre-
immune human fetuses has been used successfully to
correct immunodeficiency diseases diagnosed in utero[166–168].
However, haematopoietic cell transplantation has not
yet been routinely applied for the intentional induction
of allograft tolerance in humans. The early protocols
that achieved tolerance in adult rodents involved lethal
total-body irradiation (TBI) as conditioning for marrow
engraftment. Removal of mature donor T cells before
transplantation was shown to reliably prevent GVHD
[169]. However, MHC-mismatched allogeneic HCT in
larger animals, including humans, has proved to be less
successful and more dangerous than in rodents because
of several factors, including the toxicity associated with
myeloablative conditioning and the inordinately high
risks of GVHD and engraftment failure (reviewed in
[170]). The incidence of marrow rejection is increased
when donor marrow is T-cell-depleted to prevent
GVHD. Even when MHC mismatching is avoided,
GVHD still afflicts approximately 50% of patients who
undergo HLA-identical sibling HCT, even with post-
transplant immunosuppressive pharmacotherapy and
reduced-intensity conditioning [171]. Although it is a
major cause of morbidity and mortality, this GVHD
risk is acceptable in individuals with malignant disease
because it is associated with beneficial graft-versus-
tumour responses [172]. However, the severe, opposing
risks of GVHD and graft failure have precluded the
routine performance of extensively HLA-mismatched
transplantation, so that many patients with no other
curative options are not transplanted because they lack
an appropriately matched donor. The risks of GVHD
and marrow aplasia caused by graft rejection would be
completely unacceptable in a patients receiving HCT
solely for the purpose of organ allograft tolerance
induction. Therefore, the development of more specific
and effective methods of overcoming the barriers to
marrow engraftment with minimal GVHD risk will be
essential before this approach can be routinely applied
to tolerance induction in patients needing organ
transplantation.
For the purpose of allograft tolerance induction,
achievement of a state of mixed, rather than full,
donor haematopoietic chimerism would be desirable.
Mixed chimerism means that donor and host elements
both contribute to haematopoietic repopulation at
readily detectable levels. Mixed chimerism can be
achieved with less toxic (nonmyeloablative) condi-
tioning regimens than those that lead to full donor
chimerism. In addition to their reduced toxic side
effects, nonmyeloablative regimens allow recovery of
host haematopoiesis, so that life-threatening marrow
failure does not occur if donor marrow is rejected.
Furthermore, improved immunocompetence has been
observed in murine mixed compared to full allogeneic
chimeras when full MHC barriers are crossed. Periph-
eral reconstitution of mixed, but not full chimeras,
includes host-type APC, allowing optimal antigen
presentation to T cells that have developed in the host
thymus, and which therefore preferentially recognize
peptide antigens presented by host-type MHC mole-
cules [173, 174]. Anti-viral CTL responses in mixed
chimeras showed exquisite specificity for recipient-
derived MHC restricting elements [175]. Additionally,
whilst nonhaematopoietic thymic stromal cells have
some capacity to induce deletional tolerance, host
haematopoietic cells present in mixed but not full chi-
meras most reliably assure the intrathymic deletion of
host-reactive cells [10, 12].
As discussed in the section on intrathymic clonal
deletion as a mechanism of transplantation tolerance,
a nonmyeloablative conditioning approach consisting
of low dose (3 Gy) TBI, T-cell-depleting mAbs and
thymic irradiation [8] reliably achieves a state of
mixed chimerism in which intrathymic deletion is the
major mechanism maintaining donor-specific tolerance
[10, 11]. T-cell alloreactivity preexisting in both the
thymus and periphery must be eliminated in order to
permit allogeneic stem cell engraftment and early
seeding of the thymus with allogeneic APC. Intra-
thymic alloreactivity can be eliminated using thymic
irradiation [8], high doses of T-cell-depleting antibod-
ies [176], or co-stimulatory blockers such as anti-
CD154 or CTLA4Ig [20, 177]. Whilst it is unclear
whether the requirement to overcome intrathymic allo-
reactivity would apply to older humans with involuted
M. Sykes | Review: Transplantation tolerance
ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 297
thymi, the adult thymus remains functional at a low
level even with advanced age [178] and large animal
studies have shown the necessity for thymic irradi-
ation in a mixed chimerism model [179]. Elimination
of peripheral anti-donor T-cell alloreactivity can be
achieved with global T-cell depletion with mAbs [8,
9] or with co-stimulatory blockers combined with
BMT [20]. Low-dose TBI or busulfan creates an
environment that facilitates engraftment and expan-
sion of donor haematopoietic stem cells [176, 180].
Whilst the need for even mildly myelosuppressive
treatments can be avoided by administering very high
marrow doses [9, 21, 181], administration of such
high stem cell numbers is not currently clinically
feasible.
Durable, multilineage mixed chimerism leads to sys-
temic tolerance, as specific unresponsiveness to the
donor is observed in in vitro assays of alloreactivity.
Moreover, donor skin grafts, which provide the most
stringent test of tolerance, are specifically accepted at
any time post-BMT [8, 21, 177, 182].
Many regimens achieving mixed chimerism and toler-
ance have been described in rodents [183–188], inclu-
ding the use of TLI plus BMT [189], which has been
evaluated in humans without success [190, 191]. Var-
ious combinations of anti-T-cell antibodies, irradiation
and immunosuppressive drugs have been used suc-
cessfully in large animal models [192–195]. However,
the mechanisms of tolerance in these models are more
complex than simple central deletion, as complete
depletion of recipient T cells has not been achieved
prior to BMT.
The ability to replace recipient T-cell depletion with
co-stimulatory blockade in murine models is import-
ant because of the difficulty in using antibodies to
achieve T-cell depletion in large animals and humans.
Secondly, if truly exhaustive T-cell depletion could be
achieved in humans, T-cell recovery from the thymus
might be dangerously slow, especially in older indi-
viduals (reviewed in Ref. [178]). Replacement of
some [182] or all [20, 21, 181] T-cell-depleting anti-
bodies with co-stimulatory blockade is therefore an
important advance.
Graft-versus-host disease does not occur in the animal
models of mixed chimerism discussed above, despite
the use of unmodified donor bone marrow cells. This
is due in part to the continued presence of the T-cell-
depleting or co-stimulatory blocking antibodies in the
circulation of the recipients at the time of BMT [196].
These antibody levels readily prevent alloreactivity by
the relatively small number of mature T cells in the
donor marrow.
The role of intrathymic clonal deletion in the induc-
tion and maintenance of tolerance in mixed allogeneic
chimeras was discussed in an earlier section. It was
also pointed out that, in animals in which the pre-
existing alloreactive T-cell repertoire is not depleted
with mAb but is tolerized by the combination of
BMT and co-stimulatory blockade, specific peripheral
deletion of donor-reactive CD8 [22, 23] and CD4 [16,
20, 24, 25] cells is observed. The mechanisms of
deletion of the two subsets appear to be different in
this model. Peripheral donor-reactive CD8 cells are
deleted rapidly, whereas deletion of donor-reactive
CD4 cells occurs more slowly, over 4–5 weeks. Dele-
tion is preceded by anergy towards donor antigens
[16, 197]. It is interesting that the peripheral tolerance
of CD8 cells in this model is dependent on the pres-
ence of CD4 cells, but only in the first 10 days, after
which the donor-reactive CD8 cell deletion is com-
plete. The CD4 cells that rapidly tolerize the CD8
population do not appear to be typical CD25+ ‘nat-
ural’ Treg [22]. Regulatory cells do not appear to play
a significant role in maintaining the long-term toler-
ance induced by anti-CD154 and low-dose TBI with
BMT, as tolerance and chimerism are obliterated by
the infusion of relatively small numbers of nontolerant
recipient-type spleen cells in this model and linked
suppression is not observed [16, 22], presumably
because the deletion of donor-reactive T cells is so
complete. We have speculated that the persistence of
donor-reactive T cells is required to expand and main-
tain a regulatory-response specific for that donor
[198]. If the donor-reactive cells are completely dele-
ted, no suppressive reaction is maintained. In other
models of BMT with co-stimulatory blockade, on the
other hand, typical natural Treg appear to be involved
in the induction [199] and maintenance [200] of
M. Sykes | Review: Transplantation tolerance
298 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310
tolerance, possibly reflecting less rapid or complete
peripheral deletion of donor-reactive T cells.
Bone marrow transplantation without hostconditioning
In the regimens inducing mixed chimerism discussed
above, specific host conditioning is used to overcome
the immunological and physiological barriers to donor
marrow engraftment. ‘Microchimerism’, meaning chi-
merism at low levels that requires sensitive techniques
such as polymerase chain reaction to be detected, may
be detectable for many years in patients receiving
solid organ allografts without haematopoietic cell
transplantation [201]. Microchimerism should be dis-
tinguished from the mixed chimerism discussed
above, in which multilineage chimerism is readily
measurable by flow cytometry. The significance of the
detection of spontaneous microchimerism is unclear
[202]. Lasting microchimerism is clearly not required
for tolerance in all models [203–205], and tolerance is
by no means assured in the presence of microchimer-
ism [206]. Microchimerism neither denotes a state of
tolerance nor is required to maintain an allograft
under all circumstances [202, 206–208].
Based on the reports of microchimerism in organ allo-
graft recipients, several groups have intentionally
boosted microchimerism in patients receiving conven-
tional immunosuppression by infusing donor bone
marrow cells along with solid organ transplantation
[209, 210]. Whilst no clear-cut reduction in rejection
or immunosuppressive medication doses have been
observed in these trials [211–213], ongoing studies
combining marrow infusion with T-cell-depleting anti-
bodies should provide further information on the
potential of this approach.
Approaches to xenogeneic tolerance
Mixed chimerism
Mixed xenogeneic chimerism can also be achieved
with nonmyeloablative conditioning, leading to T-cell
tolerance [214]. However, innate immune barriers
posed by NK and cd T cells must be overcome to
achieve rat marrow engraftment in mice [215],
whereas these cells do not pose major barriers to en-
graftment of allogeneic haematopoietic cells given in
sufficient numbers with adequate T-cell immuno-
suppression [216]. Once mixed chimerism is achieved
in the rat to mouse species combination, both T cell
and B-cell tolerance is observed [214, 217–220].
Pigs are considered to be the most suitable xenogeneic
donor species for transplantation to humans, but
progress in this area has been impeded due to the
presence in human sera of natural antibodies (Nab) that
cause hyperacute rejection of porcine vascularized
xenografts. The major specificity recognized by these
Nab is a ubiquitous carbohydrate epitope, Gala1-3Galb1-4GlcNAc-R (aGal). GalT knockout mice have
a targeted mutation of the a1-3Gal transferase (GalT)
enzyme and, like humans, produce anti-aGal Nab.
Both preexisting and newly developing B cells produ-
cing anti-aGal antibodies are tolerized by the induction
of mixed chimerism in GalT knockout mice receiving
aGal-expressing allogeneic or xenogeneic marrow [35,
36, 221]. The induction of mixed xenogeneic chimer-
ism thereby prevents hyperacute rejection, a delayed
antibody-mediated form of rejection termed acute
vascular rejection, as well as cell-mediated rejection of
primarily vascularized cardiac xenografts [219]. Anti-
Gal-producing cells are tolerized [35, 219] by an early
anergy mechanism and later by clonal deletion and ⁄orreceptor editing [37].
Mixed allogeneic chimeras [222] and mixed xeno-
geneic chimeras [223] also show tolerization of NK
cells towards the donor. Despite this T, B and NK cell
tolerance, the levels of xenogeneic donor chimerism
decline gradually over time in mixed xenogeneic chi-
meras, due to a competitive advantage of recipient
mouse marrow over xenogeneic rat marrow [224].
Species specificity or selectivity of haematopoietic
cytokines, adhesion molecules, etc. [225] probably
account for this advantage. Achievement of xenogene-
ic haematopoiesis is an even more formidable
challenge in highly disparate (discordant) species
combinations. Genetic engineering approaches [226–
228] can overcome these barriers, and have allowed
the demonstration, using a humanized mouse model,
M. Sykes | Review: Transplantation tolerance
ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 299
that human T cells can be centrally tolerized to por-
cine xeonantigens via induction of mixed xenogeneic
chimerism [229].
Macrophages also impose an innate immune barrier to
the engraftment of xenogeneic marrow from highly
disparate species [230, 231] that may be overcome by
a genetic engineering approach to ensure adequate
inhibition of recipient macrophage activation by lig-
ands on xenogeneic donor haematopoietic cells [232].
Xenogeneic thymic transplantation
Xenogeneic T-cell tolerance can also be achieved
across highly disparate species barriers by replacing
the recipient thymus with a xenogeneic donor thymus
after host T-cell depletion and thymectomy [233–
235]. Tolerance to both the donor and the host
develops at least in part by intrathymic deletional
mechanisms [235, 236]. Adequate immune function is
achieved [234], even though positive selection in such
grafts is mediated only by porcine thymic MHC, with
no influence of mouse MHC [237, 238]. Likewise,
excellent immune function is achieved in humans
receiving HLA-mismatched allogeneic thymic trans-
plantation for the treatment of congenital thymic apla-
sia (DiGeorge syndrome) [239, 240], suggesting that
‘restriction incompatibility’ resulting from MHC
disparity between the positive selecting epithelial cells
in the thymus and the APC in the periphery need not
be a major obstacle to the achievement of adequate
immune function. Significantly, human T cells have
been shown to be tolerant of porcine donor antigens
when they develop in xenogeneic porcine thymus
grafts [241]. This approach has been applied and
demonstrated promise in pig-to-primate xenograft
models [242, 243]. In addition to intrathymic deletion,
regulatory cells probably play a role in the donor-
specific tolerance achieved with xenogeneic thymic
transplantation [244]. Ultimately, success in clinical
xenotransplantation may require a combination
approach involving thymic transplantation to tolerize
residual host T cells and newly developing T cells,
and haematopoietic cell transplantation to tolerize the
innate immune system, including Nab-producing B
cells and NK cells. Whilst the recent development of
the aGal knockout pig is a major advance in over-
coming the obstacles posed by anti-aGal Nab [243,
245–247], antibodies of other, less dominant specifici-
ties are a significant obstacle to success using aGalknockout pigs as organ source animals to nonhuman
primates [248]. The ability of mixed chimerism to
tolerize Nab-producing cells of all specificities [217]
may therefore be an important advantage of this
approach.
Current clinical trials of tolerance induction
Most transplant clinicians have treated patients who
have chosen to withdraw their immunosuppressive
therapy. Whilst the majority of such patients reject
their allograft, occasional patients do not [248, 249],
demonstrating that tolerance can be achieved in
humans. Extensive efforts are underway in many cen-
tres to identify markers that would distinguish such
tolerant patients prospectively before withdrawal of
immunosuppression, but so far none have proved to
be reliable [102, 250]. Whilst studies are ongoing to
evaluate the ability to slowly withdraw immmuno-
suppression in cohorts of liver transplant recipients, it
is not yet clear whether this approach will success-
fully achieve tolerance. The only approach that has
been successfully used for the intentional induction
of tolerance in humans is the nonmyeloablative
induction of mixed haematopoietic chimerism. These
studies, which have so far involved only small
numbers of patients, were justified by a combination
of clinical data in patients with haematopoietic
malignancies and extensive animal data discussed
above, including the achievement of tolerance in
nonhuman primates receiving combined MHC-mis-
matched kidney and BMT with nonmyeloablative
conditioning [192].
At the Massachusetts General Hospital, we have
recently developed clinical regimens aimed at achiev-
ing mixed chimerism with nonmyeloablative condi-
tioning in patients with haematological malignancies
[251]. This approach is based on observations in mice
that lymphohaematopoietic GVH reactions induced by
donor lymphocyte infusions given to established
mixed chimeras can mediate GVL without causing
M. Sykes | Review: Transplantation tolerance
300 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310
GVHD [252–255]. GVH-reactive donor T cells that
are activated and expand in the lymphoid tissues do
not traffic to the GVHD target tissues (which include
skin, liver and gut) because of the absence of inflam-
mation in these tissues in established mixed chimeras
[256]. Attempts to replicate this approach in patients
with haematological malignancies demonstrated the
safety and anti-tumour potential of this approach
[251], providing an opportunity to evaluate the ability
of nonmyeloablative HCT to achieve transplantation
tolerance in patients with renal failure resulting from
multiple myeloma. Six patients have received a simul-
taneous nonmyeloablative bone marrow transplant and
renal allograft from HLA-identical sibling donors. All
six patients have accepted their kidney grafts, four
without any immunosuppression for periods of more
than 2–8 years. One of these four patients required
transient immunosuppression for a rejection episode
after initial immunosuppression withdrawal and
another patient later received a myeloablative trans-
plant from the same donor to treat myeloma progres-
sion, and requires immunosuppression to treat GVHD.
The two patients who were not withdrawn from im-
munosuppression required these drugs for the treat-
ment of GVHD, not for graft rejection. Similar to the
nonhuman primate model described above, in which
BMT plays an important role in inducing tolerance to
a simultaneously transplanted kidney from the same
donor [179], chimerism in these tolerant patients was
only transient [257]. Whilst not well understood, it is
possible that the kidney graft itself may participate in
tolerance induction and ⁄or maintenance after chimer-
ism has played its initial role. In vitro studies per-
formed in these patients suggest that tolerance may be
specific for donor antigens expressed by the kidney,
whilst responses to antigens expressed on haematopoi-
etic cells but not the kidney may even be sensitized
[257]. Data also suggest a possible role for regulatory
cells other than Treg in maintaining tolerance [257].
The promising results obtained in these patients have
provided an important demonstration that tolerance
can be intentionally induced in humans. A second trial
has now been initiated at the Massachusetts General
Hospital for the transplantation of HLA-mismatched
haploidentical bone marrow and kidney grafts in
patients without malignancy. These patients receive a
regimen that was previously shown in patients with
malignancies to result in transient mixed chimerism
without GVHD [258]. With this critical safety param-
eter established and the above data showing that toler-
ance could be achieved with transient chimerism in
recipients of combined kidney and bone marrow
transplants in the HLA-identical setting, it was
deemed appropriate to evaluate this approach using
these extensively HLA-mismatched donors. Whilst
still in progress, the early results of this study are
promising.
Conclusions
As short-term results of most allogeneic organ trans-
plants are excellent, it is this author’s view that
several criteria should be met before new strategies
are justifiably evaluated to replace chronic immuno-
suppressive therapy: (i) Studies in rodents should
demonstrate robust tolerance in multiple strain combi-
nations using extensively histoincompatible, highly
immunogenic grafts such as skin. Most of the studies
implicating Treg in tolerance induction in rodent mod-
els utilize highly tolerogenic, poorly immunogenic
grafts such as primarily vascularized hearts. Whilst
treatment with a short course of many different
immunosuppressive agents allows the inherent Treg-
inducing capacity to result in tolerance to such grafts,
cardiac allografts are far less tolerogenic in large
animals; (ii) Efficacy must be demonstrated in large
animal models; (iii) Acceptable toxicity must be
demonstrated in large animal models.
As discussed above, Treg and co-stimulatory blockade
have attracted considerable interest for the induction
of tolerance. However, large animal studies have not
yet been reported in which Treg have been adminis-
tered or clearly shown to achieve transplantation toler-
ance. In fact, protocols that have led to marked graft
prolongation and have been associated with the devel-
opment of Treg in rodent models have not led to
transplantation tolerance in large animals [151, 154].
The same is true of co-stimulatory blockade. Thus,
further understanding and the development of practi-
cal and effective approaches for tolerance induction
will be needed before these strategies can be attemp-
M. Sykes | Review: Transplantation tolerance
ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310 301
ted clinically. Several recent attempts at tolerance
induction in humans, for which all three criteria above
had not been met, have failed [191, 259, 260].
In the case of HCT for the induction of tolerance, the
three criteria described above have all been met, per-
mitting the evaluation of this approach in pilot clinical
trials. When complete removal of immunosuppression
can be successfully achieved, the increased short-term
toxicity associated with the conditioning for HCT
may be acceptable. Nevertheless, it will be important
to proceed cautiously to allow adequate assessment of
the risk : benefit ratio of this tolerance strategy over
time.
Conflict of interest statement
No conflict of interest was declared.
Acknowledgements
I thank Dr Nina Tolkoff-Rubin and Dr Thomas R.
Spitzer for helpful review of the manuscript and Ms
Kelly Walsh for expert assistance with its preparation.
This work was supported by NIH grants RO1
HL49915, RO1 CA79989, PO1 P01 AI39755, PO1
HL018646, PO1 CA11159, the Immune Tolerance
Network, the Juvenile Diabetes Research Foundation
and the Multiple Myeloma Foundation.
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Correspondence: Megan Sykes, MD, Transplantation Biology
Research Center, Massachusetts General Hospital ⁄Harvard Medical
School, MGH-East Building 149-5102, 13th Street, Boston, MA
02129, USA.
(fax: +617 724 9892; e-mail: [email protected]).
M. Sykes | Review: Transplantation tolerance
310 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288–310