Tolerância Imunológica

Review
|
doi: 10.1111/j.1365-2796.2007.01855.x
Immune tolerance: mechanisms and application in clinical

transplantation
M. Sykes
From the Transplantation Biology Research Center, Bone Marrow Transplantation Section, Massachusetts General
Hospital Harvard Medical School, Boston, MA, USA
Abstract. Sykes M (Massachusetts General Hospital Harvard Medical School, Boston, MA, USA).
Immune tolerance: mechanisms and application in
clinical transplantation (Review). J Intern Med 2007;
262: 288310.
The achievement of immune tolerance, a state of
specic 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
nonspecic immumnosuppressive therapy. Advances
The need for immune tolerance in transplantation

Immune tolerance is a state in which the immune system is specically 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 specic
immune unresponsiveness to the donor graft, with normal responses to other antigens. The ability to respond
normally to other antigens contrasts sharply with the
effect of nonspecic immunosuppressive agents that
are used clinically to prevent rejection, which are associated with increased risks of infection and malignancy. This paper will review the current status of
tolerance in the eld of organ and tissue transplantation. Achievement of transplantation tolerance is the
holy grail in clinical transplantation for three major
reasons. First, whilst improvements in nonspecic
immunosuppressive therapy have markedly improved
outcomes in organ transplantation, these drugs are
associated with many specic organ toxicities as well
as the life-long increased risks of infection and
288
2007 Blackwell Publishing Ltd
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, transplantation immunology.
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 signicantly with improvements 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 largely reect the strong inherent tolerogenicity of
primarily vascularized heart, liver and kidney grafts in
these animals rather than the potency of the toleranceinducing regimens per se. A short course of many
M. Sykes
Review: Transplantation tolerance
types of immunosuppression can allow these tolerogenic effects to prevail over the rejection response,
leading to long-term graft acceptance. Because such
grafts are unfortunately less tolerogenic in large animals and humans, these tolerance strategies have not
been effectively applied in humans. Thus, before clinical evaluation is appropriate, tolerance strategies
should rst 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 denition of tolerance in the rst paragraph above includes several different immunological
states. In one, the allograft is accepted without chronic 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 specic 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 unresponsiveness 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 nave 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 pregnancy, blood transfusions or prior transplants.
Mechanisms of T and B-cell tolerance

As discussed above, the achievement of T-cell tolerance would overcome the major barriers to successful
allografting discussed above. There are three major
mechanisms of T-cell tolerance, including clonal deletion, 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 selftolerance 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 tolerance 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 antigens 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 processes 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 afnity 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 thymocyte by apoptotic cell death [2, 3]. TCRs with lower
afnity for such complexes are more likely to survive
this process, and other mechanisms are required to
ensure their tolerance when they enter the periphery,
2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310 289
M. Sykes
(a)
(b)
Fig. 1 Schematic depiction of T cell education in the thymus (a) and the role of mixed chimerism in achieving central deletional tolerance (b). In the normal situation (a) thymocyte and APC progenitors migrate to the thymus from the marrow (1) to
become double negative thymocytes (2) or APCs. Thymocyte progenitors may productively rearrange a and b T cell receptor
chains, which are rst expressed by CD4+CD8+ (double positive) thymocytes (3). These thymocytes are then subject to positive and negative selection processes. Positive selection (3), which rescues the double positive thymocyte from programmed cell
death, results from a low avidity interaction between thymocytes and thymic epithelial cells in the thymic cortex. This interaction requires a low-afnity interaction between the rearranged thymocyte TCR and a self MHC peptide complex presented by
the thymic epithelial cell. Depending on whether this MHC molecule is of class I or class II, the thymocyte will lose expression of CD4 or CD8, respectively, resulting in the generation of a CD8 or CD4 single positive thymocyte. The double-positive
or single-positive thymocyte may also die, however, if it interacts with higher afnity with a self MHC peptide complex on an
APC (4) or, possibly, a thymic epithelial cell, in the thymic medulla or corticomedullary junction. The surviving thymocytes
undergo further maturation (5), then leave the thymus and enter the peripheral lymphoid tissues. In mixed chimeras (b), thymocyte and APC progenitors of both donor and host origin migrate to the thymus from the marrow (1) to become double negative
thymocytes or APCs (2). Thymocyte progenitors of both types may productively rearrange a and b T cell receptor chains and
become CD4 CD8 double positive thymocytes (3). These thymocytes are then subject to positive and negative selection processes. Positive selection (3), which rescues the double positive thymocyte from programmed cell death, is mediated exclusively by thymic epithelial cells and hence MHC of host origin in the thymic cortex. Depending on whether this MHC
molecule is of class I or class II, the donor or recipient thymocyte will lose expression of CD4 or CD8, respectively, resulting
in the generation of donor and recipient CD8 and CD4 single positive thymocytes. The double positive or single positive
thymocyte may also die, however, if it interacts with higher afnity with an MHC peptide complex on a donor or recipientderived APC (4) or, possibly, a thymic epithelial cell, in the thymic medulla or corticomedullary junction. Consequently, only
mature T cells that lack strong reactivity to donor or host antigens survive this negative selection process, resulting in emergence from the thymus only of T cells (of both donor and recipient origin) that are tolerant of both the donor and the host (5).
particularly under conditions of inammation 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 specic
regulatory cells that tolerize other T cells in the
periphery [6].
Intrathymic deletion is induced most potently by antigen presented on haematopoietic cell types, including
dendritic cells [7]. This is a major reason why
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allogeneic haematopoietic transplantation (HCT)

provides a powerful approach to tolerance induction
in lymphoablated rodents. To avoid rejection of the
marrow, specic T-cell ablation in the thymus and the
periphery can be achieved with relatively nontoxic,
nonmyeloablative conditioning that includes T-celldepleting monoclonal antibodies (mAb) and local irradiation 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 signicant mechanism
involved in maintaining transplantation tolerance is
intrathymic clonal deletion [1012]. Anti-donor antibody can be given to established mixed chimeras to
2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310
M. Sykes
eliminate donor chimerism; this results in loss of

tolerance to donor skin grafts, and in the de novo
appearance 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, specic 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 suppression requires persistent antigen [1315]. Thymic APC
continually turn over, emphasizing the need for
haematopoietic stem cell engraftment at sufcient
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 mechanisms, such animals are vulnerable to loss of tolerance if nontolerant T cells are allowed to emerge
from the thymus after intentional depletion of donor
antigen, or after exogenous administration of nontolerant host-type T cells [11, 16].
Exposure of mature T cells to antigen in the periphery can also result in T-cell clonal deletion [17].
Self antigen cross-presentation by lymph node dendritic cells under noninammatory conditions leads to
deletion of tissue antigen-specic 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, specically, 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-specic transfusion (DST) combined
with anti-CD154 [26]. Peripheral T cell apoptosis has
been demonstrated, though without specic 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 specicity as the regulatory cells [29].
B cell clonal deletion in transplantation

As discussed above, there are several transplant situations in which tolerance induction of B cells would
be of potential value. Immunoglobulin (Ig) receptor
transgenic mice have been widely used for the analysis of mechanisms of B-cell tolerance. Such studies
indicate that immature B cells are susceptible to
deletion when they encounter membrane-bound antigens expressed by haematopoietic or nonhaematopoietic 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 rearrangement 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, including follicular, marginal zone and B-1 B cells. B-1
cells in mice produce natural antibodies recognizing important xenogeneic carbohydrate antigens
without known prior immunization [32]. Data suggest that a similar subset may produce such natural
antibodies in nonhuman primates and man [33].
Such antibodies are responsible for xenograft hyperacute rejection, and can be deleted in mice via apoptosis when their surface Ig receptors are cross-linked
by cell-bound antigens [34]. Deletion and or receptor
editing is responsible for the long-term tolerance of
M. Sykes
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
[3537].
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 complexes without receiving adequate accessory or costimulatory signals [38]. T cells can also be rendered
anergic if they encounter peptide ligands for which
they have low afnity [3]. Certain APC, such as
macrophages [39] and tolerogenic dendritic cells that
may be immature or matured in a specic 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 patterns [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 downmodulation [44]. It should be borne in mind that
anergy is reversible under pro-inammatory conditions [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 different antigens presented by these APC [49]. Moreover, Treg (see below) can promote the induction of
292
T cell anergy [50] and may themselves have biochemical 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 patterns 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 antibody-producing B-1 cells in mice rendered mixed chimeric with bone marrow cells expressing an antigen
recognized by recipient natural antibody-producing
cells [3537].
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 proinammatory 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 inammation induced by infections
[55] or by presentation of antigen on professional
APCs [56].
M. Sykes
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 mechanisms, 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 suppress immune responses. Whilst certain T cell and
non-T cell populations were implicated in this suppression, it is only in the last decade or so that
molecular markers of suppressive T cells have been
identied 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 selftolerance [57, 58]. More recent studies have shown
that these cells are generated mainly in the thymus,
require specic 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 intermediate-afnity MHC peptide ligand (too low for negative selection) expressed on cortical epithelial cells of
the thymus for their survival and maturation [6]. Constitutively 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]. Transforming 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 nave
responses have been shown to be suppressed. Several
reports indicate that the Treg require specic antigen
for their activation, but that the nal effector mechanism of suppression is nonantigen specic [6668].
Rechallenge with specic antigen induces c-interferon
(IFN-c) expression by Treg, which appears to be critical 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 antigen-specic stimulation (adaptive Treg) [61], especially 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 natural Treg and Tr1 cells are hyporesponsive to TCRmediated stimulation but can be grown slowly in vitro
in 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 differentiation [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 functions and cannot be viewed purely as an immunosuppressive cytokine. For example, TGF-b has been
implicated in chronic brotic conditions (e.g. chronic
graft-versus-host disease, GVHD; [76]) and in the differentiation of nave T cells to the pro-inammatory
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
specic suppressor cells was obtained in early models
of transplantation tolerance (reviewed in Ref. [81]) and
Hall et al. rst identied CD25+CD4+ T cells as a
specic suppressive population in rats receiving
M. Sykes
cardiac allografts with a short course of cyclosporin

[82]. Since then, Treg have been implicated in numerous models involving acceptance of vascularized
allografts in rodents receiving an initial immunosuppressive treatment. Regimens have included donorspecic 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 MHCmatched, minor histocompatibility antigen-mismatched
skin grafts in mice receiving nondepleting anti-CD4
and CD8 antibodies with or without anti-CD154
[8890] 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 peripheral tolerance models, perhaps due to its role in generating 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 phenomenon has been observed in rats receiving soluble
alloantigens in combination with a vascularized allograft [96] and active regulatory cell populations have
been described [96]. However, other mechanisms
involving recirculation of activated T cells to the thymus have also been implicated [97], and the circulation 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. Congenital defects in FoxP3 in humans are associated
with an autoimmune syndrome, immune dysregulation, 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
294
transplantation, and correlative data have begun to

emerge. Chronic renal allograft rejection has been
associated with reduced circulating Treg concentrations [102]. Whilst discontinuation of immunosuppressive medications usually leads to rejection, a
small fraction of such patients accept their grafts nevertheless, i.e. they demonstrate spontaneous tolerance. 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 alloantigens 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 numbers 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 populations 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 subset 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 considerable interest in the concept that polarization to Th2
M. Sykes
type of response from a pro-inammatory Th1 (IL-2and IFN-c-producing) response could promote allograft 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 regulatory 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 development [65, 75]. NKT cells are enriched in bone marrow 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 transplantation 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 [124126]. Natural [124] and adaptive
[126], FoxP3-expressing [122, 124], TGF-b-producing [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-modulation mediated by CD8 T cells is simply the killing
by alloreactive CTL of critical donor APC populations [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 activity. 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 relative 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 suppress alloimmunity or autoimmunity. Whilst methods
of nonspecically expanding mouse and human Treg
ex vivo have recently been developed [130, 131], animal studies suggest that antigen specicity is important 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 difcult
to apply.
Co-stimulatory blockade in transplantation

The discovery that TCR stimulation without co-stimulation can induce anergy [132] has led to intensive
evaluation of co-stimulatory blockade in the transplantation eld. Blockade of the CD28 co-stimulatory
pathway can be achieved with specic mAb or with a
soluble receptor for the B7-1 B7-2 ligands. CTLA4,
an alternate, inhibitory T receptor with a higher afnity 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
upregulating B7 molecules, MHC, antigen processing

pathways, cytokines and other molecules. Blockade of
the CD40CD154 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 tolerance of these grafts is not reliably achieved [133
136]. These treatments can more reliably induce permanent 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 [139141]. This drug has been reported to
achieve robust allograft tolerance when used in combination 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 insufcient to prevent rejection of the skin graft by newly emerging alloreactive T cells in euthymic mice [147]. IFN-c,
which has traditionally been considered to be a Th1
proinammatory 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 rapamycin 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 central deletion of donor-reactive T cells, preventing their
emergence from the thymus after the transplant [23,
150].
Despite the achievement of prolonged allograft survival (though not tolerance) in nonhuman primates
296
[151154], attempts to apply co-stimulatory blockade

for the induction of tolerance clinically have not succeeded. The combination of rapamycin, DST and antiCD154 was reported to achieve tolerance in three of
ve nonhuman primate recipients [155]. However,
anti-CD154 use has been complicated by thromboembolic 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 leucocyte antigen (HLA)-mismatched bone marrow transplants that were exposed to recipient alloantigens ex
vivo in the presence of CD28 blockade with CTLA4Ig
[159]. Whilst several additional co-stimulatory pathways exist and combinations of blockers are showing
promise in rodent models, co-stimulatory blockade
alone has not yet proved to be sufciently powerful
to achieve tolerance in nonhuman primates or
humans.
Mixed chimerism as an approach to transplantation

tolerance
Bone marrow engraftment reliably induces tolerance
to the most immunogenic allografts, such as fully
MHC-mismatched skin and small bowel grafts, in animal models (reviewed in Ref. [160]). The ability to
achieve transplantation tolerance with HCT has been
well documented in patients who rst 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 neonatally, 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
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 preimmune human fetuses has been used successfully to
correct immunodeciency diseases diagnosed in utero
[166168].
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 aficts approximately 50% of patients who
undergo HLA-identical sibling HCT, even with posttransplant 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 benecial graft-versustumour 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 specic
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) conditioning 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. Peripheral 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 molecules [173, 174]. Anti-viral CTL responses in mixed
chimeras showed exquisite specicity for recipientderived 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 chimeras 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-specic 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. Intrathymic alloreactivity can be eliminated using thymic
irradiation [8], high doses of T-cell-depleting antibodies [176], or co-stimulatory blockers such as antiCD154 or CTLA4Ig [20, 177]. Whilst it is unclear
whether the requirement to overcome intrathymic alloreactivity would apply to older humans with involuted
M. Sykes
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 irradiation 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 expansion 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 systemic tolerance, as specic 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 specically accepted at
any time post-BMT [8, 21, 177, 182].
Many regimens achieving mixed chimerism and tolerance have been described in rodents [183188], including the use of TLI plus BMT [189], which has been
evaluated in humans without success [190, 191]. Various combinations of anti-T-cell antibodies, irradiation
and immunosuppressive drugs have been used successfully in large animal models [192195]. 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 important because of the difculty 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 individuals (reviewed in Ref. [178]). Replacement of
some [182] or all [20, 21, 181] T-cell-depleting antibodies with co-stimulatory blockade is therefore an
important advance.
298
Graft-versus-host disease does not occur in the animal

models of mixed chimerism discussed above, despite
the use of unmodied donor bone marrow cells. This
is due in part to the continued presence of the T-celldepleting 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 induction 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 preexisting alloreactive T-cell repertoire is not depleted
with mAb but is tolerized by the combination of
BMT and co-stimulatory blockade, specic 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 45 weeks. Deletion 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 presence of CD4 cells, but only in the rst 10 days, after
which the donor-reactive CD8 cell deletion is complete. The CD4 cells that rapidly tolerize the CD8
population do not appear to be typical CD25+ natural Treg [22]. Regulatory cells do not appear to play
a signicant role in maintaining the long-term tolerance 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 maintain a regulatory-response specic for that donor
[198]. If the donor-reactive cells are completely deleted, 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
tolerance, possibly reecting less rapid or complete

peripheral deletion of donor-reactive T cells.
Bone marrow
conditioning
transplantation
without
host
In the regimens inducing mixed chimerism discussed

above, specic host conditioning is used to overcome
the immunological and physiological barriers to donor
marrow engraftment. Microchimerism, meaning chimerism 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 distinguished from the mixed chimerism discussed
above, in which multilineage chimerism is readily
measurable by ow cytometry. The signicance of the
detection of spontaneous microchimerism is unclear
[202]. Lasting microchimerism is clearly not required
for tolerance in all models [203205], and tolerance is
by no means assured in the presence of microchimerism [206]. Microchimerism neither denotes a state of
tolerance nor is required to maintain an allograft
under all circumstances [202, 206208].
Based on the reports of microchimerism in organ allograft recipients, several groups have intentionally
boosted microchimerism in patients receiving conventional 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 [211213], ongoing studies
combining marrow infusion with T-cell-depleting antibodies 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 engraftment of allogeneic haematopoietic cells given in
sufcient numbers with adequate T-cell immunosuppression [216]. Once mixed chimerism is achieved
in the rat to mouse species combination, both T cell
and B-cell tolerance is observed [214, 217220].
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 specicity recognized by these
Nab is a ubiquitous carbohydrate epitope, Gala13Galb1-4GlcNAc-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 producing 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 chimerism 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]. AntiGal-producing cells are tolerized [35, 219] by an early
anergy mechanism and later by clonal deletion and or
receptor editing [37].
Mixed allogeneic chimeras [222] and mixed xenogeneic 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 chimeras, due to a competitive advantage of recipient
mouse marrow over xenogeneic rat marrow [224].
Species specicity or selectivity of haematopoietic
cytokines, adhesion molecules, etc. [225] probably
account for this advantage. Achievement of xenogeneic 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
that human T cells can be centrally tolerized to porcine 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 ligands on xenogeneic donor haematopoietic cells [232].
the aGal knockout pig is a major advance in overcoming the obstacles posed by anti-aGal Nab [243,
245247], antibodies of other, less dominant specicities are a signicant obstacle to success using aGal
knockout pigs as organ source animals to nonhuman
primates [248]. The ability of mixed chimerism to
tolerize Nab-producing cells of all specicities [217]
may therefore be an important advantage of this
approach.
Current clinical trials of tolerance induction

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 inuence of mouse MHC [237, 238]. Likewise,
excellent immune function is achieved in humans
receiving HLA-mismatched allogeneic thymic transplantation for the treatment of congenital thymic aplasia (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. Signicantly, 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 donorspecic 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
300
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 centres 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 immmunosuppression in cohorts of liver transplant recipients, it
is not yet clear whether this approach will successfully 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 justied 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-mismatched kidney and BMT with nonmyeloablative
conditioning [192].
At the Massachusetts General Hospital, we have
recently developed clinical regimens aimed at achieving mixed chimerism with nonmyeloablative conditioning 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
GVHD [252255]. GVH-reactive donor T cells that

are activated and expand in the lymphoid tissues do
not trafc to the GVHD target tissues (which include
skin, liver and gut) because of the absence of inammation 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 simultaneous 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 28 years. One of these four patients required
transient immunosuppression for a rejection episode
after initial immunosuppression withdrawal and
another patient later received a myeloablative transplant from the same donor to treat myeloma progression, and requires immunosuppression to treat GVHD.
The two patients who were not withdrawn from immunosuppression required these drugs for the treatment 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 chimerism has played its initial role. In vitro studies performed in these patients suggest that tolerance may be
specic for donor antigens expressed by the kidney,
whilst responses to antigens expressed on haematopoietic 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 parameter established and the above data showing that tolerance 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 transplants are excellent, it is this authors view that
several criteria should be met before new strategies
are justiably evaluated to replace chronic immunosuppressive therapy: (i) Studies in rodents should
demonstrate robust tolerance in multiple strain combinations using extensively histoincompatible, highly
immunogenic grafts such as skin. Most of the studies
implicating Treg in tolerance induction in rodent models 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 Treginducing capacity to result in tolerance to such grafts,
cardiac allografts are far less tolerogenic in large
animals; (ii) Efcacy 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 administered or clearly shown to achieve transplantation tolerance. In fact, protocols that have led to marked graft
prolongation and have been associated with the development 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 practical and effective approaches for tolerance induction
will be needed before these strategies can be attemp-
M. Sykes
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, permitting 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 : benet ratio of this tolerance strategy over
time.
Conflict of interest statement

No conict 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: megan.sykes@tbrc.mgh.harvard.edu).

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Review

Immune tolerance: mechanisms and application in clinical

The need for immune tolerance in transplantation

2007 Blackwell Publishing Ltd

in our understanding of immunological processes,

malignancy mentioned above. Secondly, chronic

Review: Transplantation tolerance

anti-donor antibodies because of prior sensitization

Mechanisms of T and B-cell tolerance

T cell clonal deletion in transplantation

Review: Transplantation tolerance

particularly under conditions of inammation and

allogeneic haematopoietic transplantation (HCT)

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

eliminate donor chimerism; this results in loss of

been demonstrated for peripheral CD8 cells in mice

B cell clonal deletion in transplantation

natural antibody-producing B-1 cells in mice when

Review: Transplantation tolerance

Lymphocytes ignoring graft antigens (ignorance)

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

Active suppression of T-cell responses

Regulatory CD4+ T cells

cardiac allografts with a short course of cyclosporin

Review: Transplantation tolerance

transplantation, and correlative data have begun to

Other suppressive cell populations

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

Some CD8+ CTL-mediated suppressive phenomena

Co-stimulatory blockade in transplantation

upregulating B7 molecules, MHC, antigen processing

Review: Transplantation tolerance

[151154], attempts to apply co-stimulatory blockade

Mixed chimerism as an approach to transplantation

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

mechanisms have been implicated in neonatally

For the purpose of allograft tolerance induction,

thymi, the adult thymus remains functional at a low

Review: Transplantation tolerance

Graft-versus-host disease does not occur in the animal

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

tolerance, possibly reecting less rapid or complete

In the regimens inducing mixed chimerism discussed

Approaches to xenogeneic tolerance

achieve rat marrow engraftment in mice [215],

Review: Transplantation tolerance

Current clinical trials of tolerance induction

Most transplant clinicians have treated patients who

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

GVHD [252255]. GVH-reactive donor T cells that

regimen that was previously shown in patients with

ted clinically. Several recent attempts at tolerance

Conflict of interest statement

Review: Transplantation tolerance

7 Brocker T, Riedinger M, Karjalainen K. Targeted expression

2007 Blackwell Publishing Ltd Journal of Internal Medicine 262; 288310

Review: Transplantation tolerance

and tolerance without cytoreductive host treatment. Nat Med