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).


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



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].



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



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



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



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



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



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



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,



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



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-



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]).



Tolerância Imunológica

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