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Current Anaesthesia & Critical Care 21 (2010) 229e232

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Current Anaesthesia & Critical Care

journal homepage: www.elsevier .com/locate/cacc

FOCUS ON: TRANSPLANTATION

Update on transplantation tolerance

Anne Cunningham*

Department of Pharmacy Health and Wellbeing, Faculty of Applied Sciences, University of Sunderland, City Campus, Sunderland SR1 3SD, UK

Keywords:TransplantationToleranceTregsFoxP3

* Tel.: þ44 (0)01915152979.E-mail address: anne.cunningham@sunderland.ac.

0953-7112/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.cacc.2010.07.008

s u m m a r y

The induction of transplantation tolerance has become a major goal, because modern immunosup-pressive therapy has not improved chronic rejection rates, and is associated with significant side effects.This article aims to explain the principles of immunological tolerance. Mechanisms of central toleranceinvolve deletion of self-reactive T cells. Mechanisms of peripheral tolerance are reviewed and also theidentification of a subset of regulatory T cells which are characterised by the expression of the tran-scription factor FoxP3.

Interesting recent insights on the role of the ‘anti-inflammatory’ cytokine transforming growth factorb which can ultimately lead to the generation of inhibitory Tregs or inflammatory Th17 cells (CD4 helperT cells which secrete the pro-inflammatory cytokine IL17) are discussed.

There are many ways to induce experimental tolerance in animals, however these are difficult totranslate tolerance into the clinical context. In addition, standard immunosuppressive agents are calci-neurin inhibitors which block T cell activation and IL-2 production. These drugs not only inhibit theactivation of effector T cells, but also Tregs, therefore inhibiting Treg driven tolerance induction. Otherclasses of immunosuppressive drugs should be introduced into the clinic to allow for the possibility oftolerance induction. Strategies to modulate immune responses following transplantation and theirpotential risks are discussed.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Transplantation is an effective treatment for end stage organfailure. However, it requires that patients take immunosuppressivedrugs for life. These have side effects (eg nephrotoxicity), increasethe risk of infection and cancer, butmost importantly fail to preventchronic graft rejection. Chronic rejection is arguably the biggestproblem following transplantation, and its development is linkedto the incidence and severity of acute rejection episodes. The goal oftransplant immunologists has been to harness the immune systemto specifically ignore the graft, but respond fully to pathogens/tumour cells, without long term immunosuppression.

The idea of self tolerance was first put forward by Paul Elrich in1901 when he failed to immunise goats with autologous red cells.He reasoned there must be mechanisms to prevent immuneresponses to self tissue under normal circumstances, and coinedthe term ‘horror autotoxicus’ as a prediction of what would happenif this were not the case.1 However understanding the mechanismsby which we are self tolerant and exploiting them to preventdisease has proved extremely difficult.

uk.

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Sir Peter Medawar was awarded a Nobel Prize in 1960 for hisdiscovery of ‘acquired immunological tolerance’. He demonstratedthat transplant rejection was an immunological response and thattolerance to skin allografts could be induced experimentally in fetalmice and chick embryos.2 So where are we fifty years later e areimmunologists any nearer their goal of turning theory into realityand inducing a donor specific tolerance following transplantation?.

In order to explain where the field of transplantation toleranceis now, a brief overview of tolerance and how this is linked toclinical and experimental tolerance induction will be made.

2. Central tolerance

Immature thymocytes are produced in the bone marrow andtravel to the thymus where they undergo a random process ofreceptor rearrangement followed by thymic selection. The newlyformed T cell antigen receptors (TCR) are first positively selectedby their ability to bind with low affinity to self MHC/peptidecomplexes on thymic epithelial cells ie if the newly formed TCRare not ‘useful’, they die by neglect. However, since the process israndom, it is essential to delete those TCR with high affinity forself MHC/peptide and reduce the risk of autoimmunity. In recentyears, it has been appreciated how much effort is made toexpress tissue specific proteins within the thymus under the

Th0

Th1Th2

Th17Th12

IL4

IL5

IL10

IL12 IL23

IFNγ

IL2

IL2

IL17

IL12 IL4

APCTCR + Co-stimulation

Inhibit

Fig. 1. Simplified schematic of T cell activation and the development of polarised T cellresponses. A naïve uncommitted T cell is referred to as a Th0 cell. Depending on thecytokine signal received during T cell activation, the T cell can be polarised and willdifferentiate into a T helper 1 subset (Th1) which is typically inflammatory andassociated with graft rejection. Recently, two subpopulations of Th1 cells have beenidentified: those Th1 cells that produce interleukin 12 (are referred to as ‘Th12’ cells) orthose that produce interleukin 17 (and are referred to as ‘Th17’ cells). Alternativecytokine signals (eg interleukin 4) will drive a Th0 cell down a different pathway andthe cell will develop into a T helper 2 subset (Th2). These are typically associated withallergy/parasitic infections and the production of antibodies.

A. Cunningham / Current Anaesthesia & Critical Care 21 (2010) 229e232230

control of the ‘Auto-immune Regulator Element’ AIRE.3,4 There-fore, tissue specific proteins, like insulin, are expressed in thepancreas and the thymus. Thymic expression is driven by theAIRE promoter, so that newly rearranged TCR will be exposed toMHC/insulin peptide complexes on thymic epithelial cells. Thiswill enable the deletion of potentially auto-reactive insulin-specific T cells and therefore reduce the risk of autoimmunity.Genetic manipulation of insulin gene expression in the thymushas been shown to affect whether insulin-specific T cells surviveor not. Elimination of insulin from the thymus results in theescape of insulin-specific T cells into the periphery and thedevelopment of auto-immune diabetes in a murine model.5

The very fact that TCR are selected for their ability to bind to selfMHC/peptide complexes with a low affinity most likely explainswhy the T cells from a patient can directly recognise the MHCmolecules expressed on donor tissue.6 This high frequency of‘alloreactive’ T cells is responsible for the intensity of the rejectionresponse, and is several orders of magnitude higher than theimmune response to a pathogen.7

The original experiments by Medawar and colleagues wereessentially inducing a central tolerance to subsequent skin grafts.However manipulating the immune system of a newborn is nota feasible strategy for use in clinical transplantation.

3. Peripheral tolerance

Multiple mechanisms have been proposed to explain toleranceoutside the thymus. Interfering with antigen presentation has beenpostulated to induce tolerance. A ‘naive’ Tcell requires three signalsfor activation and differentiation into an effector T cell:

Signal 1: TCRbinding to a cognateMHC/peptidecomplex (onantigenpresenting cells).

Signal 2: Costimulation (CD28 binding to CD80/86 on antigenpresenting cells and/or CD154 binding to CD40 on antigenpresenting cells).

Signal 3: Cytokine signalling (eg Interleukin-12 will drive thegeneration of the Th1 subset of helper T cells; Interleukin-4 the polarisation of Th2 helper T cell subsets etc).

Activation of a naïve T cell requires all three signals and takesplaces in lymph nodes. Dendritic cells are the most effectiveantigen presenting cells to deliver these signals, and the originaltissue micro-environment where the dendritic cell was ‘primed’ isassociated with a particular cytokine ‘message’ which will instructthe T cell response required (Fig. 1). In the case of transplantation,Interleukin 23 (IL-23) produced by dendritic cells will drive thedifferentiation of pro-inflammatory Th17 T cells which is particu-larly associated with acute graft rejection.

In contrast, signal 1 in the absence of signal 2 leads to a specifichypo-responsiveness, or anergy.8,9 Anergic cells are functionallyinactive, and may inhibit other T cells by competition for space andgrowth factors. Significantly, this state of unresponsiveness is notovercome if an anergic T cell subsequently receives signal 1 andsignal 2 (unless high levels of the T cell growth factor, interleukin-2are also provided).10 The costimulatory molecules on a T cellinclude CD28 which binds to CD80/CD86 on antigen presentingcells and CD154 (CD40L) which binds to CD40 on antigen pre-senting cells. T cells also possess a negative regulator of cos-timulation, CTLA4 (CD152). Normally intracellular, CTLA4 isexpressed on the T cell surface at the end of an immune responsewhere it has a higher affinity for CD80/CD86 than CD28. CLTA4ligation delivers an inhibitory signal to T cells.11

Investigations of the normal phenomena of oral tolerance havedemonstrated the role of ‘anti-inflammatory’ cytokines, particu-larly transforming growth factor b (TGFb) and interleukin 10. TGFbinhibits the proliferation of Th1 and Th2 lymphocyte subsets.Weiner12 introduced a 3rd subset of T helper cells associated withmucosal surfaces, Th3, characterised by their production of TGFb.13

Most significantly, a subset of ‘regulatory’ T cells has been identi-fied that can suppress the responses of activated T cells and turn an‘aggressive’ or ‘pathogenic’ immune response off. They were firstidentified by several groups in animal models of auto-immunediseases. Adoptive transfer models demonstrated the role of ‘patho-genic’ T cells in transferring disease (such as colitis, thyroiditis), butalso indicated a population of regulatory T cells which could inhibitthem.14,15 Originally described in the CD4 þ CD25 þ memory pop-ulation, these are now characterised by the expression of a transcrip-tion factor, FoxP3. FoxP3 is the ‘master switch’ which controls Tregdevelopment, and is predominantly (but not exclusively) expressedby CD4 þ CD25 þ T cells in both the thymus and periphery.16

It has been demonstrated that these Treg normally constitutew10% of peripheral CD4 þ T cells, and they are also found in thethymus (‘natural’ Tregs), where it is proposed those T cells bearingTCR with the highest affinity for self MHC/peptide are pre-pro-grammedby FoxP3 to be inhibitory. They proliferate poorly followingTCR stimulation, don’t produce IL-2 and constitutively express highlevels of the glucocorticoid-induced TNF-related receptor (GITR) anda high proportion (w50%) express CTLA-4. Expression of the IL-2receptor (CD25) and IL7 receptor (CD127) discriminates betweenTregs and activated T cells.17 Consequently many studies have shownthat CD4 þ CD25hi CD127low/neg T cells effectively identifies Tregs(Fig. 2), and correlates with FoxP3 expression/regulatory function.

The discovery of Tregs has been a major milestone in ourunderstanding of tolerance, and consequently there has been muchspeculation about their induction to treat inflammatory diseases,including transplantation rejection. The goal of inducing a donorspecific tolerance could be closer if Tregs that control the pathogeniceffector Tcells responsible for acute graft rejection could be induced.

Interestingly TGFb has been shown to play a role in the differ-entiation of both Tregs, but also surprisingly, inflammatory Th17cells. At low concentrations, TGFb synergises with interleukin 6 andinterleukin 21 to promote the IL-23 receptor and the differentiation

Fig. 2. Peripheral blood mononuclear cells (PBMC) from a health volunteer stained with monoclonal antibodies conjugated with 3 different fluorochromes identify a subset of Tregswhich are CD4 þ CD25hi CD127low/neg.

‘Pathogenic’ T cells‘Regulatory’ T cells

A

A. Cunningham / Current Anaesthesia & Critical Care 21 (2010) 229e232 231

of Th17 inflammatory Tcells. At high concentrations TFGb repressesIL-23 receptor expression and favours FoxP3þ Treg development. Ithas been demonstrated that the transcription factor FoxP3 interactswith another transcription factor, RORgt in the nucleus whichinhibits interleukin 17 expression. Conversely, a decrease in FoxP3and an increase in RORgt expression can tip the Treg/Th17 balancetowards pro-inflammatory Th17 cells.18 Therefore the functionaloutcome depends on a balance between two cytokine regulatedtranscription factors FoxP3 and RORgt, and exposure of a naïve Tcell to TGFb can potentially lead to either the Th17 or Treg lineage.The cytokine micro-environment in a transplanted organ and thebalance between FoxP3 and RORgt in the infiltrating T cells may bekey in determining the induction of tolerance or acute rejection.

‘Pathogenic’ T cells

‘Regulatory’ T cells

‘Regulatory’ T cells

‘Pathogenic’ T cells

B

C

Fig. 3. An illustration to represent the functional outcome of a balance between Tregsand effector T cells in (A) health, (B) transplant rejection and (C) tolerance induction.

4. Therapeutic tolerance induction in transplantation

Numerous strategies have been used to induce a donor specificimmunological tolerance in experimental models. However it is fairto say that this success has not been translated into the clinic.

Techniques to induce central tolerance to a graft would requiredepletion of the recipient immune system (eg by whole bodyirradiation or treatment withmyeloablative drug therapy) followedby infusion of donor bone marrow cells, ideally depleted of donor Tcells to prevent graft vs host disease. Theoretically this couldproduce a new ‘chimeric’ immune systemwhich is tolerant to boththe donor and recipient. This has been achieved in experimentalmodels, but is difficult and dangerous clinically. Stable expressionof >1% of donor cells in the recipient has been achieved. Morerecently a combination of bone marrow transplantation and cos-timulation blockade to produce stable chimerism has shownpromise.19

Early studies have demonstrated the benefits of using depletingand non-depletingmonoclonal antibodies specific to CD4 followingtransplantation. Specifically targeting CD4 helper T cells inhibitstransplant rejection in rodent models. Studies have demonstratedthat anti-CD4 depleting antibodies are immunosuppressivewhereas non-depleting anti-CD4 antibodies induce tolerance.20

This has been termed ‘infectious tolerance’ since T cells renderedtolerant by anti-CD4 non-depleting antibodies can transfer specifictolerance to naïve CD4 T cells.21

Antibodies that block costimulatorymolecules and the deliveryof ‘signal 2’ (anti-CD40, anti-CD28) have also been demonstrated toinduce a profound donor specific tolerance in animal models.CTLA4-Ig has been an extremely useful reagent to block cos-timulation and has been shown to be effective at inducing toler-ance in animal models.22 CTLA4-Ig and anti-CD40 synergise to

induce a profound tolerance in rodents. Interestingly this isinhibited by the immunosuppressive drugs ciclosporin andtacrolimus.

Sub-optimal TCR stimulation has been also reported to induceanergy. Under normal circumstances, stimulation of the TCR withMHC/peptide leads to T cell signalling via phosphorylation of CD3cytoplasmic chains and ultimately the transcription of interleukin-

A. Cunningham / Current Anaesthesia & Critical Care 21 (2010) 229e232232

2 (IL-2; T cell growth factor) and its receptor. This enables an acti-vated T cell to undergo clonal proliferation and is a key stage in thegeneration of immune responses. Sub-optimal stimuli (such as‘altered peptide ligands’)23 lead to reduced phosphorylation of CD3,and specific hypo-responsiveness rather than activation.

One very interesting observation is that T cell activationprecedes the appearance of tolerance, and persistence of antigen isnecessary to maintain tolerance. Antigen presenting cells and IL-2are required. Therefore standard immunosuppressive drugsdesigned to inhibit Tcell activation and IL-2 production (calcineurininhibitors: ciclosporin, tacrolimus) will hinder tolerance induction.Furthermore, calcineurin is known to induce TGFb production,24

which may influence the balance between transcription factors(FoxP3, RORgt) and ultimately the balance between the generationof regulatory and effector responses.

5. Clinical perspectives

The use of immunosuppressive drugs that do not inhibit calci-neurin, eg rapamycin (sirolimus), may open avenues for therapeutictolerance induction regimes in clinical transplantation.25,26

It has been proposed to treat patients with CTLA4-Ig and anti-CD40 to block costimulation and/or induce regulatory T cells.Ultimately treatments that lead to the induction of donor specifictolerance, or lead to their numerical and functional dominancehave great potential in transplantation (Fig. 3).27,28 However thereremain many unanswered questions and concerns that the balancebetween regulation and immunity could be easily tipped one wayor the other.29,30 It is significant that amonoclonal antibody specificto CD28 (TGN1412) that was alleged to induce Tregs and was underinvestigation to treat inflammatory disease (rheumatoid arthritis)caused such spectacular adverse events and ultimately led to theinsolvency of Te Genero31,32

Timewill tell whether these recent discoveries will take another50 years to be translated to the clinic.

Conflict of interestI confirm I have no conflict of interest associated with the

material in this manuscript.

Acknowledgement

Thanks to Graeme Parker for help with preparing the figures.

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