Facilitation of CD4+ CD25+ Regulatory T-Cell function in transplantation.

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Facilitation of CD4

_CD25

_{regulatory T-cell function}

in transplantation

Part of the work presented in this thesis was supported by the Roche Organ Transplant Research Foundation (rotrf 903539494) and the Dutch Kidney Foundation (C05-2106)

Financial support for the publication of this thesis was kindly provided by: Nierstichting Nederland

Nederlandse Transplantatie Vereniging Wyeth Pharmaceuticals

Facilitation of CD4

_CD25

_{regulatory T-cell function}

in transplantation

Een wetenschappelijke proeve

op het gebied van de Medische Wetenschappen

proefschrift

ter verkrijging van de graad van doctor

aan de Radboud Universiteit Nijmegen

op gezag van de

rector magnificus prof. mr. S.C.J.J. Kortmann

volgens besluit van het College van Decanen

in het openbaar te verdedigen op

donderdag 24 mei 2007

om 13.30 uur precies

door

Jeroen Johannes Antonius Coenen

geboren op 11 oktober 1976

isbn 978-90-9021751-2 © 2007 J.J.A. Coenen

Cover design: Krijn ontwerp, Nijmegen, The Netherlands Interior design: Peter Tychon, Wychen, The Netherlands Printed by drukkerij Thieme, Nijmegen, The Netherlands

Promotor:

Prof.dr. B.E. de Pauw

Co-promotores:

Dr. I. Joosten

Dr. L.B. Hilbrands

Manuscriptcommisie: Prof. dr. P.L. van Riel

Prof. dr. G.J. Adema

Prof. dr. F.H.J. Claas

Table of contents

Chapter1

General introduction 7

Chapter2

Tolerizing effects of co-stimulation blockade rest on functional dominance

of CD4+_CD25+_{regulatory T-cells 33}

Chapter3

CTLA-4 engagement and regulatory CD4+_CD25+_{T-cells independently}

control CD8+_{- mediated responses under costimulation blockade 57}

Chapter4

Allogeneic stimulation of naturally occurring CD4+_CD25+_{T cells induces}

strong regulatory capacity with increased donor-reactivity 81

Chapter5

Rapamycin, and not Cyclosporin A, preserves the highly suppressive

CD27+_{subset of human CD4}+_CD25+_{regulatory T cells 99}

Chapter6

Rapamycin, not Cyclosporine A, permits thymic generation and peripheral

preservation of CD4+_CD25+_FoxP3+_{T-cells 119}

Chapter7

Summary and Discussion 139

Samenvatting 153

Dankwoord 157

Curriculum Vitae 158

1

1

chapter 1

1. transplantation immunobiology 9

1.1. Transplantation and the immune system 9

1.2. Allorecognition 9

1.3. Inhibition of allograft rejection 10

1.4. Transplantation tolerance 12

2. regulatory t-cells 13

2.1. The self vs. non-self problem in adaptive immunity 13

2.1.1. Naturally occurring TREGas a separate lineage in the thymus 13

2.1.2. Peripheral generation of TREG 14

2.2. Phenotype of TREG 15

2.3. Mechanisms of suppression 15

2.4 Interrelationship of naturally occurring TREGand induced TREG 16

2.5. Alloantigen specific TREGin transplantation 17 3. balancing regulatory and effector cells in

transplantation 19

3.1. Regulatory T-cells in transplantation tolerance 19

3.2. Regulatory T-cells and immunosuppression 19

3.2.1. Conventional immunosuppression 19

3.2.2. Costimulation blockade 20

3.3. Expansion and preservation of regulatory T-cells 21

1. Transplantation immunobiology

1.1. Transplantation and the immune system

The immune system allows individuals to mount defensive reactions against a vast array of antigens. This system is a critical component of our defence against infection, but immune responses are unfortunately also elicited by antigens not associated with pathogens, like grafted tissue. Nevertheless, organ transplanta-tion has become a successful therapeutic modality for end-stage renal, cardiac, hepatic and pulmonary disease. Progress in transplantation medicine has been realized in great part by the advent of potent immunosuppressive drugs during the 1980’s1_{. Still, successful allotransplantation is characterized by a} non-physio-logical situation imposed upon an immune system that has evolved to function in an entirely different context2. The patient receiving the transplanted tissue has to use immunosuppressive drugs lifelong and as a consequence, will be at risk of malignancy, infection, renal failure or other adverse effects. Therefore, the “holy grail” in transplantation medicine remains to reach a state in which an individual’s immune system accepts the grafted tissue as if it were its own3,4.

1.2. Allorecognition

Allorecognition occurs when the host immune system detects same-species, non self antigens and this is the trigger for allograft rejection. The primary basis for allograft rejection is the ability of T-cells to recognize polymorphic versions of a variety of proteins, which are referred to as alloantigens. The most impor-tant series of alloantigens are those encoded within the Major Histocompatibil-ity Complex (MHC). The physiological role of MHC molecules is to present peptide determinants to T-cells as part of normal immune responses, but they were first discovered because of their dominant role as targets for rejection. A very high proportion of T-cells (0.1%-10%) is able to directly bind and respond to allogeneic molecules1,5. Allorecognition can occur by two distinct but not mutually exclusive pathways: direct and indirect. The direct pathway results from the recognition of foreign MHC molecules, intact, on the surface of donor cells. Indirect allorecognition occurs when donor MHC molecules are internalised, processed, and presented as peptides by host antigen presenting cells- the manner in which the immune system normally “sees” antigen6,7.

Specificity for the response of T-cells to antigen is determined by the T-cell receptor. This first TCR-MHC triggering step is generally referred to as signal 1. Recognition of allogeneic MHC class-II molecules by the T-cell receptor pre-sent on CD4+_{T-cells results in activation and subsequent production of} pro-proliferative cytokines, e.g. IL-2, that are necessary to initiate allograft immuni-ty. These cytokines act in an autocrine manner on the CD4+_{T-cells and in a} paracrine way on the other cells, such as CD8+_{T-cells, macrophages and} B-cells8,9. Activation of CD8+_{T-cells by MHC class I molecules present on the} donor cells results in cytolysis of these cells. For a proper function, CD8+_T-cells need help from CD4+_{T-cells via cytokines and cell-to-cell interactions} mediat-ed via co-stimulatory pathways on antigen presenting cells (APC) or other T-cells10,11.

Full T-cell activation requires a second ‘positive’ costimulatory signal, gener-ally referred to as signal 2 (Fig. 1). This signal is provided by interaction with the APC12. The first identified molecules responsible for costimulation were identi-fied as T cell based CD28 interacting with B7 ligands (CD80 and CD86) on APC13. Additionally, a molecule structurally related to CD28, called cytotoxic T lymphocyte antigen-4 (CTLA-4) was found to also bind to the same B7 ligands. These molecules belong to the CD28:B7-family14_{. Next, the CD154(CD40L):} CD40 interaction emerged as a second set of costimulatory molecules, belong-ing to the tumor necrosis factor:tumor necrosis factor receptor (TNF:TNF-receptor) family. CD40 is expressed on APC’s and CD154 is expressed after activation on CD4+ _{T-cells as well as on some CD8}+ _{T-cells, NK cells and} eosinophils. It is the integration of positive and negative costimulatory signals that will determine the outcome of the alloreactive T-cell response.

The CD28:B7-family has now been extended by novel family members, ICOS:B7h and PD1:PDL-1/PDL-2, which have both positive and negative roles in T-cell activation, respectively. The TNF:TNFR family has grown with the receptor:ligand pairs 4-1BB:4-1BBL and CD134:CD134L (OX40:OX40L). Their functions in immune responses including alloimmunity are under intense investigation15.

1.3. Inhibition of allograft rejection

The important role of the CD40L and CD28 families in the activation of allore-active T-cells is demonstrated by the observation that CD40 blockade using anti-CD154 monoclonal antibody (mAb) has successfully been shown to pre-vent acute rejection and promote long-term allograft acceptance in murine

transplant models16,17. Also, blockade of CD80 and CD86 can prevent acute allograft rejection and can, depending on the model or strain combination, induce donor-specific tolerance in several murine transplant models18,19. Several studies indicate that intact CTLA-4 signalling is required for the induction of tolerance to alloantigen. CTLA-4 has a limiting effect on the alloreactive clone size20. Also, a role of CTLA-4 on regulatory T-cell function has also been impli-cated by some studies21,22(discussed further below).

Calcineurin inhibitors like cyclosporin and tacrolimus (FK506) are used as conventional immunosuppression to block transcription of the gene encoding the pro-proliferative cytokine IL-2. This successfully reduces the expansion of alloreactive effector cells and thereby prevents graft rejection. Unlike FK506 and CsA, rapamycin does not inhibit T-cell receptor (TCR)-induced cal-cineurin activity. Rather, rapamycin inhibits the activation of the mammalian target of rapamycin (mTOR), which is required for protein synthesis and cell-cycle progression. Thus, CsA inhibits TCR-induced activation, whereas rapamycin blocks signaling in response to growth factors23,24.

B7 Family TNF Family CD28 Family TNF-R Family APC Signal B7-1 or B7-2 (CD80 or CD 86) B7-1 or B7-2 B7-1 or B7-2 MHC/Peptide CD40 CD40 T Cell Signal CTLA-4 (CD152) CD28 TCR/CD3 complex CD40L (CD154) CD27

Figure 1. Costimulatory molecules and their ligands.

Depicted are the costimulatory molecules and their ligands. These molecules belong to the CD28:B7 and tumor necrosis factor : tumor necrosis factor receptor (TNF:TNFR) families and have either positive or inhibitory effects on signaling in T-cells. (Adapted from Rothstein et al. Imm. Reviews 2003).

1.4. Transplantation tolerance

Immunological tolerance is defined as a state in which (a) the immune system does not mount a pathological response against a specific antigen, (b) there is no requirement for exogenous immunosuppression, and (c) responses to other antigens are maintained25,26. Two major pathways leading to tolerance are dis-cerned, peripheral and central tolerance induction.

Central tolerance induction occurs in the thymus. In transplantation, cen-tral tolerance to a solid organ allograft requires the presence of donor bone mar-row derived cells in the thymus. This can be achieved by donor bone marmar-row transplantation and myeloablative conditioning prior to organ transplantation. T-cells that develop under these circumstances lack reactivity to donor antigens due to clonal selection in the thymus27,28. However, a major obstacle of this therapy is the high morbidity risk of the conditioning regimen required for bone marrow engraftment and deletion of pre-existing alloreactive T-cells.

Peripheral tolerance as opposed to central tolerance occurs upon antigen encounter in the peripheral immune compartments, including lymph nodes and spleen. Several immune interventions have been shown to successfully induce peripheral tolerance in animal models4,28-30. To establish transplantation tolerance, alloreactive effector cells need to be eliminated, inactivated, or con-verted to an immunosuppressive alloreactive T cell. Mechanisms involved are deletion, anergy, immunoregulation, clonal exhaustion and ignorance of allore-active T cells4,6. Deletion of alloreactive T-cells might be triggered under subop-timal stimulation, such as costimulation blockade, or by depletion via the use of depleting mAb like anti-CD3 or CAMPATH-1H31-35_{. T-cell anergy is defined as} the functional inactivation of alloreactive T-cells due to disturbed costimulation and is often linked to immunoregulation36,37. Clonal exhaustion may occur as a result of chronic administration of alloantigen under suboptimal conditions, leading to deletion of alloantigen specific T-cells38. Ignorance may only play a minor role in transplantation tolerance, if any, as it is unlikely that the immune system is not triggered by the alloantigen presence. Of all the mechanisms implicated in transplantation tolerance, immunoregulation crucially deter-mines the difference between tolerance or immunity to alloantigen. Immunoregulation is an active process where regulatory T-cells control the response of alloreactive effector cells. Currently, much attention focuses on the naturally occurring regulatory CD4+_CD25+_T-cells.

2. Regulatory T-cells

2.1. The self vs. non-self problem in adaptive immunity

A quantitative analysis of published experimental data indicates that a CD4+_T cell restricted by a particular MHC Class II allele will recognize about one pep-tide in every 104_{presented by that allele}39_{. Each MHC allele can potentially} pre-sent 1010_{nonamer peptides. Given that, as noted above, a T cell has a 10}-4 prob-ability of recognizing one, it follows that a single T cell is able to recognize 106 different nonamers40. Clearly, these cells are highly cross-reactive and conse-quently pose a problem in self non-self discrimination. However, a unique pro-tective self-tolerance mechanism attributed to the thymus and peripheral com-partments is the generation of antigen specific regulatory CD4+_CD25+_{T cells} (T_REG). These cells are also referred to as “naturally occurring T_REG”, designat-ing them as a separate lineage39,41.

2.1.1. Naturally occurring T

as a separate lineage in the thymus

In the thymus, the generated T cell repertoire will provide a set of receptors of varying avidities for self-antigens, creating a continuous distribution (Fig. 2). Clonal deletion will remove those cells recognizing self-antigens with receptors of the highest avidity. It is hypothesized that a part of these potentially destruc-tive effector T cells is converted into TREG. These cells are the T cells with recep-tors that are on the high end of self avidity, but on the lower end of clonal dele-tion by negative selecdele-tion42,43. When activated by auto-antigen, the TREGexert their suppressive capacity and limit autoreactivity by downregulating the responses of T cells with receptors that recognize other auto-antigens in the same tissue. Thymic TREGcells were shown to make a substantial contribution to the peripheral TREGpopulation. Thus, the distinct TREGact as a safeguard for tolerance to self-antigen.

Tregcell +2 –2 Clonal deletion Positive selection

Cells with receptors

of a

vidity less than X (log %)

Low High Avidity for sell antigens

Figure 2. Selection of the T cell repertoire as a consequence of TCR avidity for self antigens in the thymus.

Cells whose receptor have an avidity greater than that indicated by the vertical bar (middle) undergo positive selection. Of those, cells with receptors of the highest avidities (striped area, far right) undergo negative selection by clonal deletion. Natural TREGcells arise from the

group of cells whose avidities are near the lower end of the negative selection spectrum and the higher end of the remaining positively selected cells. (Adapted from RH Schwartz, Nat. Immunology 2005).

2.1.2. Peripheral generation of T

Thymic selection seems not to be the only mode by which T_REGcells can be generated. It has been demonstrated that presentation of peptides in “subim-munogenic” conditions (low dose and/or lack of costimulation) can convert naïve T cells into CD4+_CD25+_T

REGcells in the periphery. This conversion also occurs in conditions in which the possibility of proliferation of pre-existing CD4+_CD25+_{T-cells can be excluded}44_{. Importantly, to achieve the conversion} it is pivotal for the antigen to be presented in “subimmunogenic” conditions 45-47_{. It is proposed that in the periphery T-cells become an effector T-cell when} the threshold for full activation is reached. However, if activation fails to reach that threshold, the incomplete activation may drive the T-cell towards regulato-ry function. TGF-βmay contribute to this phenomenon by raising the level of the full activation threshold48. On the other hand, it is also hypothesized that full activation of CD4+_CD25-_{cells may ultimately lead to conversion to a} regu-latory phenotype, thereby providing a negative feedback mechanism of conven-tional immune responses49.

2.2. Phenotype of T

The naturally occurring TREGcells found in the CD4+CD25+T cell pool con-stitute about 5-10% of mature CD4+_CD8- _{thymocytes and about 10% of} peripheral CD4+_{T cells}50,51_{. These cells may also convert to CD4}+_CD25-_{T cells} with a regulatory phenotype49. Other cell surface markers, such as CD45RB, CTLA-4, glucocorticoid-induced TNF receptor family-related receptor (GITR), CD122, CD103, CD134, CD62L and CD27, can also be used to define and isolate CD4+_CD25+_T

REG4,52. However, as with CD25, none of these mole-cules alone represents a distinct marker for naturally occurring T_REGas they are also expressed on other T cells. The forkhead/winged helix transcription factor Foxp3 was shown to be uniquely expressed by naturally occurring TREG, and is thought to be an important component in controlling TREGdifferentiation53,54. Nevertheless, recent findings indicate that this unique expression is only restricted to mice, for human CD4+_CD25-_{T cells may also express Foxp3 upon} activation52,55. It has been proposed that as a result of activation this Foxp3 upregulation directs human activated CD4+_CD25-_{cells to a regulatory} pheno-type49,56. From this perspective, Foxp3 would still be a unique marker for T cells with a regulatory phenotype.

2.3. Mechanisms of suppression

The exact mechanism by which CD4+_CD25+_T

REGcells exert their suppressive effects remains enigmatic, but these cells have been found to suppress effector T cell proliferation in vitro through a cell contact-dependent mechanism that is largely cytokine independent57-59. A role for GITR and CTLA-4 on the surface of TREG has been implicated, but it appears that blocking CTLA-4 on CD4+_CD25-_{effector cells rather than on the CD4}+_CD25+_{cells interferes with} suppression by making the CD4+_CD25-_{cells less susceptible (this thesis).} Nev-ertheless, a role for CTLA-4 on the CD4+_CD25+_T

REGis not excluded. Ligation of CD80 and/or CD86 on effector T cells by CTLA-4 on the TREGwas postu-lated to cause “outside-in” signalling and to result in suppression of the effector cell60. Ligation of CD80 and/or CD86 on dendritic cells by CTLA-4 on the T_REGresults in the activation of indoleamine 2,3-dioxegenase (IDO). Reduced amounts of free tryptophan as a consequence of the induction of IDO are asso-ciated with decreased activation of T cells61. However, this APC dependent mechanism is not essential, for suppression is also found in APC free culture systems62.

In contrast to in vitro findings, blockade of both IL-10 and TGF-βhas been reported to abrogate TREG mediated unresponsiveness to alloantigen in vivo

implicating a role for these cytokines in CD4+_CD25+_T

REG function63-66. The apparent discrepancy could be explained by a requirement for cell contact with a third cell, such as an APC, or the de novo generation of regulatory T cells (dis-cussed below) 67,68. Recent findings may also indicate a role for TGF-βat the site of the effector cell, for TGF-βdeficient T cells were resistant to suppression by CD4+_CD25+_{T cells. Moreover, CD4}+_CD25+_T

REGcells developed normally in the absence of TGF-β, ruling out an essential role of this cytokine in CD4+_CD25+_T

REGphysiology69.

Direct cytotoxicity seems to be unlikely as a mechanism of action since it appears that CD4+_CD25+ _T

REG and suppressed CD4+CD25-T cells can co-exist for long periods of time. Separation of CD4+_CD25+_T

REGand suppressed CD4+_CD25-_{T cells demonstrates an intact effector potential of the latter, for} isolated CD4+_CD25-_{T cells can proliferate normally and produce IL-2 after} antigenic stimulation70.

2.4 Interrelationship of naturally occurring T

and induced T

As outlined above, suppression by naturally occurring CD4+_CD25+ _T REG is clearly not a result of effector T cell elimination but rather a result of active con-trol of effector cell proliferation and effector function71. Additional de novo gen-eration of T_REGcells might be another fundamental mechanism for the induc-tion and maintenance of peripheral tolerance. A different class of CD4+_T

REG has been described whose suppressive properties are in contrast to naturally occurring CD4+_CD25+_T

REGcell contact independent and mediated through suppressive cytokines like IL-10 and TGF-β72_{. Two main types of these cells} have been described as Tr1 and Th3 cells. Tr1 cells are defined by their ability to produce IL-10 and low amounts of TGF-β, whereas Th3 cells produce preferen-tially TGF-β73,74. Different types of induced TREGdevelop under the influence of suppressive agents like IL-10, TGF-β, dexamethasone, vitamin D3, modulat-ed DC, costimulation blockade or potentially other inhibitory mechanisms in different models, but may also develop from naïve conventional CD4+₂₅- T-cells as a result of cell-contact dependent interaction with naturally occurring CD4+_CD25+ _T

REG36,75-79. It appears that CD4+CD25+ TREG expressing the

α₄β₇-integrin convert anergized CD4+₂₅-_{T-cells into IL-10 producing Tr1 like} T-cells, whereas α₄β1+TREG induce TGF-βproducing Th3 like T-cell80. The suppressive properties of TREGin vivo are thus complex and based on diverse mediators like IL-10 and TGF-βto exert systemic suppressive effects81,82.

2.5. Alloantigen specific T

in transplantation

Given the earlier mentioned crossreactivity of T cells and the diversity of TCRs that are expressed by TREGit would seem highly probable that a proportion of TREGcross-reacts with alloantigens expressed on transplanted organs and tis-sues. However, the frequency of these cells must be low, for rejection is the inevitable outcome after transplantation of an allograft4,50. Nevertheless, alloantigen specific TREGactivity can be detected in various allograft models. In

vivo, TREGcells specific for donor MHC can be detected after exposing an indi-vidual to donor alloantigens before transplantation and in mice with long term surviving allografts63,65. Regulatory T cells can be identified at the site of the tol-erated allograft and CD4+_CD25+_{T cells were found to actively restrain effector} T cells, indicating that alloantigen specific TREGcontribute to allograft toler-ance63,83-86.

Allograft tolerance induced through “central” allograft tolerance rests on deletional mechanisms in the thymus after bone marrow transplantation rather than active regulation87,88. Theoretically, a role for the thymus in the generation of CD4+_CD25+_T

REG specific for alloantigen cannot be discarded, for expres-sion of peptide in thymic epithelial cells resulted in the generation of CD4+_CD25+_T

REGto that specific peptide. Alloantigens might thus induce the development of T_REGcells by positive selection in the thymus. Nevertheless, the thymus is not essential for the generation of alloantigen specific T_REG. Alloantigen specific TREG can also be generated in the periphery by thymec-tomized recipients of allografts in vivo, resulting in peripheral tolerance. Repeated antigen exposure in the periphery has been suggested to induce T_REG cells, so it can be hypothesized that naturally occurring TREGcrossreactive to alloantigen might clonally expand in this fashion89. In support of both the pro-posed central and peripheral mechanism it was found that the persistent pres-ence of the graft itself as a permanent source of alloantigen is pivotal for the maintenance of tolerance in vivo90. Additionally, the earlier mentioned infec-tious de novo generation of TREG cells by naturally occurring CD4+CD25+ TREGmight further expand the pool of allospecific TREGcells. Alloantigen spe-cific T_REG may thus arise from direct development in the thymus or in the periphery from crossreactive naturally occurring TREGand conversion of effec-tor T cells into a regulaeffec-tory phenotype (Fig. 3).

Figure 3. Origin of alloantigen-specific regulatory T cells.

Theoretically alloantigens might induce the development of TREGcells by positive selection in

the thymus, by clonal expansion in the periphery of naturally occurring TREGcells that

cross-react with alloantigens, or by conversion of naive or activated alloantigen-specific peripheral T cells to a regulatory phenotype. (Adapted from KJ Wood, Nat. Immunology 2003).

Alloantigen Alloantigen Thymus Developing Naive T cells Periphery Alloantigen-specific TREGcells Conversion to TREGcells phenotype

Clonal expansion Naturally occurring alloantigen-cross-reactive TREGcells

3. Balancing regulatory and effector cells in

transplantation

3.1. Regulatory T-cells in transplantation tolerance

Accumulating evidence indicates that a balance between regulation by T_REG and deletion of effector T-cells is an effective strategy to control immune responsiveness after organ transplantation91. The large number of alloreactive T-cells requires an extensive deletion of effectors and a reduction of these graft destructive cells will facilitate the emergence of immunoregulation by T_REG subsets. As a result it is increasingly proposed that clinical transplantation toler-ance protocols that target alloreactive T-lymphocytes need to specifically spare TREGto induce proper transplantation tolerance. Once induced, immunoregu-latory mechanisms like linked suppression and infectious tolerance contribute to the maintenance of an immunoregulatory network, which is also essential to harness alloreactivity of recent thymic emigrants92. Linked suppression occurs when a potentially alloreactive T-cell comes under the tolerizing influence of a T_REGas both cells recognize their respective alloantigens presented by the same APC93,94. This leads to a re-education of the destructive allo-responsive T-cell with the induction of a TREGphenotype. The re-educated aggressive cell can in turn induce other naïve CD4+_{T-cells to adopt a regulatory phenotype, hereby} propagating the tolerant state. These mechanisms have been implicated in tol-erance achieved through numerous strategies including costimulation blockade or treatment with immunosuppressive drugs95-97. For a proper clinical applica-tion of T_REGin the induction of transplantation tolerance it is thus of high sig-nificance to accurately determine the effects of immunosuppression for the reduction of the alloreactive clone size on the development and functional activity of TREG.

3.2. Regulatory T-cells and immunosuppression

3.2.1. Conventional immunosuppression

CsA inhibits TCR-induced activation, whereas rapamycin blocks signaling in response to growth factors23,24. Albeit efficient in preventing allograft rejection by reducing effector T-cell expansion, it remains to be elucidated how these

immunosuppressive drugs may antagonize the induction of tolerance by affect-ing the naturally occurraffect-ing TREGpopulation. Signalling by IL-2 is also required for the function of CD4+_CD25+_T

REGand there has been concern about the impact of these drugs on the development and function of CD4+_CD25+_T

REG in transplantation tolerance4,98,99. Considering the different modes of action mentioned above, calcineurin inhibitors like CsA and FK506 and rapamycin may thus have differential effects on CD4+_CD25+ _{regulatory T-cells. Indeed,} high doses of calcineurin inhibition administered at the time of tolerance induction can prevent long term allograft survival and the development of CD4+_CD25+ _T

REG, whereas rapamycin was found to selectively expand CD4+_CD25+_T

REGin vitro, to increase the CD4+CD25+TREG to CD4+CD25 -T-cell ratio and to preserve CD4+_CD25+_T

REG dependent immunoregulation

in vivo95,100-104. However, timing of calcineurin inhibition may be an important factor on its effect on transplantation tolerance, for a delayed CsA administra-tion was to preserve the inducadministra-tion of anti-CD40 and anti-CD86 mediated hyporesponsiveness in vitro and to induce a prolonged drug-free allograft sur-vival in a rhesus monkey kidney-allograft model in combination with anti-CD40 and anti-CD86 costimulation blockade105,106_.

Nevertheless, in the thymus, signaling through the common γ-chain of cytokine receptors is essential for naturally occurring CD4+_CD25+_T

REG devel-opment. CD4+_CD25+ _T

REG are generated from thymocytes that express FoxP354,107_{. In the periphery, IL-2 signaling is required for sustaining the TREG} population at a size necessary to maintain immune homeostasis108,109. This rais-es considerable concern about the impact of blocking IL-2 signaling by different immunosuppressive agents on CD4+_CD25+_T

REGhomeostasis and the mainte-nance of tolerance to self-antigens or the induction of tolerance to alloantigens. If signaling of the growth factor IL-2 is as crucial for the functional activity of T_REGas recently proposed, both types of agents might thus affect T_REG func-tion after activafunc-tion98,99,110,111.

3.2.2. Costimulation blockade

In the expansion of the alloreactive effector T-cell pool, costimulation is required for optimal survival, cytokine production and proliferation of effector T-cells112-115. Conversely, blockade or absence of costimulatory pathways has been shown to result in the prevention of allograft rejection and the induction of tolerance16-18,110,116-119. Jenkins and Schwartz showed that absence of costimu-lation led to T-cell non-responsiveness both in vitro and in vivo120. This non-responsive state of the T-cells could be rescued by antigen specific TCR

stimula-tion in the presence of exogenous IL-2, a phenomenon referered to as T-cell anergy121. Correspondingly, blockade in vivo of the costimulatory pathways CD80/CD86-CD28 and or CD40-CD40L by monoclonal antibodies proved an attractive target for immunosuppressive therapy110,122,123.

It was found that the application of anti-CD40L induces infectious toler-ance and enables the emergence of donorspecific CD4+_CD25+ _regulatory T-cells30,93. It can be hypothesized that a regimen that blocks pro-mitotic signal-ing but ensures antigen driven T-cell activation, like co-stimulation blockade, may facilitate a functional dominance of regulatory T-cells by the synergizing effects of selective reduction of alloreactive effector pool and activation of alloantigen specific regulatory T-cells. In line with this, it was found that succes-full application of anti-CD40L and anti-CD86 blockade depends on the pres-ence of CD4+_CD25+_{regulatory T-cells}124_.

3.3. Expansion and preservation of regulatory T-cells

In vivo, the optimal exploitation of the suppressive capacity of TREG may be accomplished either by the generation of new regulatory T cells or by a selective reduction of harmful T-cells, both resulting in a shift of balance towards domi-nance of regulatory mechanisms. As an appealing therapeutic, the balance of T_REGvs. effector cells may also be shifted by infusion of T_REG after large scale expansion ex-vivo. Already, expanded T_REG have been used as a therapeutic in models of autoimmune disease and expanded TREG have been used to treat ani-mal models of Graft Versus Host Disease (GVHD)125-128. In these models it appeared that antigen-specific T_REG were more effective than polyclonal expanded TREG. Importantly, the antigen specific TREG were able to reverse dia-betes with new onset disease. With respect to human allograft tolerance a subset that emerges after in vitro expansion with cytokines may be of particular inter-est; antigen-specific CD27+_T

REG were shown to have an increased suppressive capacity and were able to suppress ongoing and memory allograft responses, in contrast to their CD27- _counterpart52_{. The physiological relevance of potent} CD27+_{suppressive T-cells in vivo has been established by identification of these} cells in synovial fluid of juvenile arthritis patients129.

Thus, characterizing the conditions that promote the perserverence and the suppressive capacity of potent TREGin vivo may thus have important clinical implications for both in vivo and expansion-based ex-vivo therapeutic ap-proaches for the induction of transplantation tolerance.

4. Scope of this thesis

In the present thesis we explored the the role of CD4+_CD25+_{regulatory T-cells} in transplantation and the effects of different immunosuppressive regimens on these cells. In experimental immunosuppression, costimulation blockade is a promising approach. In Chapter 2 we found that the tolerizing effects of anti-CD40L and anti-CD86 costimulation blockade rest on the induction of func-tional dominance of naturally occurring CD4+_CD25+_{T-cells over alloreactive} effector cells. Subsequently, in Chapter 3 we elaborate on the importance of bal-ancing alloreactive effectors and regulatory T-cells as observed in chapter 2 by demonstrating that negative co-stimulatory signalling via CTLA-4 is pivotal for the containment of alloreactive effectors rather than the activation of CD4+_CD25+_{regulatory T-cells. CD4}+_CD25+_{regulatory T-cells are thus} identi-fied as key players in the regulation of the alloreactive effector response.

The suppressive characteristics of CD4+_CD25+_{regulatory T-cells have made} these cells themselves attractive candidates for immunotherapy in transplanta-tion. Therefore, we analysed in Chapter 4 to what extent donor-specific CD4+_CD25+_{regulatory T-cells arise from the naturally occurring CD4}+_CD25+ regulatory T-cell pool during different ex-vivo expansion protocols and how this population of cells may facilitate an enhanced contribution to containment of alloreactive effector cells.

Subsequently, in Chapter 5 and Chapter 6 we analyzed to what extent cur-rently applied immunosuppressive drugs may facilitate or hamper clinical implementation of regulatory T-cell therapy and may differentially influence the induction of transplantation tolerance by affecting naturally occurring CD4+_CD25+ _{regulatory T-cell homeostasis. In Chapter 5, we found that in} human cells - in contrast to CsA - rapamycin preserves a highly suppressive CD27+_CD25+_CD4+_{regulatory T-cell subset. In Chapter 6 we demonstrate in a} murine in vivo model that the use of rapamycin preserves CD4+_CD25+ regula-tory T-cell generation in the thymus and persistence in the periphery, whereas the use of CsA results in a specific reduction of these cells in all immune com-partments. Finally, Chapter 7 summarizes this thesis and provides general con-clusions.

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2

chapter 2

Tolerizing effects of co-stimulation

blockade rest on functional

dominance of CD

4

+

CD

25

+

regulatory T-cells

Jeroen J.A. Coenen

1,2

, Hans J.P.M. Koenen

1

,

Esther van Rijssen

1

, Luuk B. Hilbrands

2

and Irma Joosten

1

1

_{Dept. of Bloodtransfusion and Transplantation Immunology,}

_{Dept. of Nephrology, University Medical Center Nijmegen,}

The Netherlands

Abstract

Clinical tolerance is the net result of regulatory and effector functions. Here we show, that tolerance induction by costimulation blockade preferentially works through CD4+_CD25+_{regulatory T-cell mediated suppression that is effectively} achieved by selective reduction of the effector T-cell load. Anti-CD 86+anti-CD40L mAb treatment during in vitro MLR typically results in the induction of a suppressive polyclonal T-cell population. This induced suppressive capacity was found to be dependent on the presence of CD4+_CD25+ _{T cells at the start of} MLR. Using a CFSE based strategy, we show that within the polyclonal T-cell population the suppressive effect was exerted by a non-dividing CD4+_CD25high T-cell subset. The cells exclusively originated from pre-existing CD4+_CD25+ regulatory T cells and proved anergic and highly suppressive upon isolation. They carried the CD45RBlow_{and CD62L}high_{phenotype and expressed GITR.} There was no indication of de novo induction of TREGby co-stimulation block-ers. Instead, we observed both in vitro and in vivo, that costimulation blockade shifted the ratio between alloreactive effectors and regulatory T-cells in favour of the latter. We therefore conclude that costimulation blockade contributes to functional dominance of regulatory T-cells by preventing expansion of alloreac-tive effector T-cells. Tolerance inducing protocols should ideally facilitate this phenomenon.

Introduction

The development of tolerance in vivo has been described as a highly dynamic process involving several mechanisms, including deletion of the alloreactive T cell pool and immunoregulation1. CD4+_CD25+ _{regulatory T-cells can exert} potent regulatory activity, both in vivo and in vitro2,3. Several in vivo studies underscore the importance of balancing CD4+_CD25+ _{regulatory T-cells and} effector T-cells in establishing T-cell homeostasis4-6. In parallel, naturally occur-ring CD4+_CD25+ _{regulatory T-cells were shown to mediate dose dependent} suppression in vitro7. Thus, it is increasingly appreciated that enhancement of in vivo regulatory T-cell function and consequently tolerance induction, may well be accomplished by inducing a balance shift between effectors and regula-tors, resulting in dominance of regulatory T-cell suppression8-11.

In the expansion of the alloreactive effector T-cell pool, costimulation is required for optimal survival, cytokine production and proliferation of T-cells12-15. Conversely, blockade or absence of costimulatory pathways has been shown to result in the prevention of allograft rejection and the induction of tolerance16-18. We reasoned that a regimen that blocks pro-mitotic signaling but ensures anti-gen driven T-cell activation, like co-stimulation blockade, may facilitate a func-tional dominance of regulatory T-cells by the synergizing effects of selective reduction of alloreactive effector pool and activation of alloantigen specific reg-ulatory T-cells. Previously, we and others showed that costimulation blockade by anti-CD40L+antiCD86 mAb treatment during in vitro MLR resulted in the induction of a suppressive polyclonal T-cell population, dependent on the pres-ence of CD4+_CD25+ _{T cells at the start of MLR}19,20_.

We here demonstrate that regulatory T-cells become functionally dominant when a shift of balance between regulatory and effector T-cells is effectuated by costimulation blockade. These findings further support the notion that toler-ance inducing protocols should allow for selective enrichment of regulatory T-cells.

Materials and methods

Cell purification

Experiments were performed with completely MHC-mismatched combina-tions of C57BL/6 (H-2b_{) or C3H (H-2}k_{) stimulators and BALB/c (H-2}d₎ responders. All mice were purchased from Charles River Lab (Charles River, Sulzfeld, Germany). Mice were used at 8 to 12 weeks of age and housed in a specified pathogen free facility. BALB/c responder cells were purified from

pooled peripheral lymph node and spleen cells. Cells were depleted of erythro-cytes by osmotic shock in lysis buffer containing 0.15mM NH4Cl (Merck, Darmstadt, Germany), 10.0 mM KHCO3 (Merck) and 0.1 mM Na2EDTA (Sigma-Aldrich, Steinheim, Germany). Cells were enriched for T-cells by deple-tion of MHC class II positive cells by coating with anti MHC class II magnetic microbeads (Miltenyi, Bergisch Gladbach, Germany), followed by passage through a LD microbead column (Miltenyi). Depletion of CD4+_CD25+_T-cells was carried out by magnetic cell separation. To this end, the T cell enriched population was inoculated with saturating amounts of biotin-labeled anti-CD25 antibody (7D4; BD Biosciences, San Diego, USA). Cells were subse-quently incubated with anti-biotin microbeads and separated using MS columns according to the manufacturer’s instructions. Depleted populations contained <1% CD4+_CD25+ _{T-cells. C57BL/6 or C3H stimulator cells were} enriched by culturing splenocytes in 9 cm petridishes (Allegiance, Zutphen, The Netherlands) for two hours and further overnight culture of the adherent cell fraction. After 18 to 24 hours the non-adherent cells were obtained by har-vesting the supernatant and gently washing the petridish.

Mixed Lymphocyte Reaction

For primary MLR, responder cells were cultured with γ-irradiated (27.5 Gray) stimulator cells at concentrations of 1*105responder cells/mL to 2.5*104 stimula-tor cells/mL in flat bottom 96-well plates (Costar, Corning, USA) containing RPMI (Gibco, Paisley, UK) with 10% FCS (Invitrogen, Dieren, The Nether-lands), 50 mM 2-ME (Biorad, Hercules, USA), 1 mM sodium pyruvate, gluta-max and antibiotics (100 U/mL penicillin; 100 mg/mL streptomycin)(all from Gibco) at 37º, 95% humidity and 5% CO2. In specified cases 1 mg/mL anti-CD40L mAb (MR1, Pangenetics, Amsterdam, The Netherlands) and 1 mg/mL anti-CD86 mAb (HB253, Pangenetics, Amsterdam, The Netherlands) were added. It was established that matched isotype controls did not modify the pro-liferation of responder cells. To analyze the cells for regulatory capacities, cells were harvested at day 7 of culture and washed to remove antibodies and cytokines and subsequently rested in 5% FCS culture medium (see above). Flow-cytometric analysis (FCM) confirmed that the cells used for restimulation or suppression assays were devoid of antibodies. At day 10 the cells were γ -irradi-ated (27.5 Gray) and added to a newly setup primary MLR of freshly purified responder and stimulator cells at the indicated ratios. Regulatory capacity of the cells previously cultured in the presence of anti-CD40L and anti-CD86 was assessed by comparison to a parallel experiment with untreated cells as a

con-trol. For functional characterization of different cellular subpopulations accord-ing to CFSE dilution and CD25 expression, cells were harvested at day 6 or 7 of culture. The different subpopulations of cells were enriched by FACS (Coulter Epics Elite, Beckman Coulter, Miami, USA) and subsequently restimulated at indicated numbers by 2.5*104stimulator cells with or without exogenous rIL-2 (BD Biosciences, Erembodegem, Belgium), or assessed for regulatory capacity by addition to a newly setup primary MLR of freshly purified responder and stimulator cells at the indicated ratios. Cell proliferation was monitored by triti-ated thymidine incorporation. Wells were pulsed with trititriti-ated thymidine (0.5 mCi) for 16-18 h before harvesting at the indicated time points. Tritiated thymi-dine incorporation was analyzed by a gas scintillation counter and is expressed as mean count per 5 minutes and SD of at least triplicate measurements. Counts per 5 minutes by gas scintillation analysis resembles counts per 1 minute as mea-sured by liquid scintillation analysis. For each experiment proliferation was measured at multiple timepoints. Peak proliferation was generally at day 5-6 for primary MLR and at day 2-3 for secondary MLR. Typical results are shown.

Flow cytometry

Cells were phenotypically analyzed by direct labelling or by an indirect two-step labelling procedure. Cells were washed twice with phosphate-buffered saline supplemented with 0.2% bovine serum albumin (BSA) (Sigma). In case of indi-rect labeling, cells were labeled first with fluorochrome-conjugated or with biotin conjugated specific antibodies; conjugated anti-CD4 (L3T4); PE-conjugated anti-CD8 (Ly-2); PE-PE-conjugated anti-CD25 (3C7); biotinylated CD25 (7D4); PE-conjugated CD45RB(16A); PE-conjugated anti-CD62L (MEL-14) (all by BD Biosciences) and biotinylated anti-glucocorti-coid-induced TNF receptor (GITR goat polyclonal Ab; R&D systems, Min-neapolis, USA). In the case of biotinylated antibodies cells were labeled with streptavidin conjugated PE (BD Biosciences) or PE-Cy5 (Beckman Coulter) . All incubations were for 20 minutes room temperature and thereafter cells were washed twice. The samples were run on a Coulter Epics XL, Beckman Coulter and at least 100.000-150.000 live gate events were collected based on live lym-phocyte gating, as indicated by 5 mg/mL propidium iodide staining. Isotype matched antibodies were used to define marker settings. Data were analyzed using Coulter Epics Expo 32.

Discrimination of subpopulations through CFSE dilution and

CD25 expression

The cell division rate of allo-MHC primed T cells was studied by labelling responder T cells with Carboxyfluorescein Diacetate Succinimidyl Ester (CFDA-SE, Molecular Probe, Eugene, OR). T-cells were labelled at 0.25-0.5 mM CFSE at 1*107_{cells for 10 min. An equal volume of FCS was added, and} cells were washed with medium. Simultaneous labelling of CD25+ T cells, by fluorochrome conjugated mAb against CD25 (see above) enables the study of division kinetics of different T cell subsets by flowcytometry. Data, preferential-ly 100.000-150.000 live gate events, were anapreferential-lyzed using Coulter Epics Expo 32.

Murine cardiac allograft transplantation

Experiments were performed with completely MHC-mismatched combina-tions of C57BL/6 (H-2b_{) donors and BALB/c (H-2}d_{) recipients. Mice were} pur-chased from Charles River Lab (Charles River, Sulzfeld, Germany). Mice were used at 8 to 12 weeks of age and housed in a specified pathogen free facility. Car-diac allografts were placed in an intra-abdominal location using the technique described by Corry et al21. Graft function was assessed every two days by palpa-tion. Animals received three doses of MR1 at days 0,2 and 4 (0.25 mg each) by intra-peritoneal injection and two doses of HB253 at day 0 (0.2 mg) and day 1 (0.1 mg) by intra-venous injection. The day of rejection was defined as the day of cessation of palpable heartbeat.

Statistical analysis

Statistical Analysis was performed using the Statistical Product and Services Solutions (SPSS) package, version 10.0. For comparisons between groups we used Student’s t test, assuming unequal variances because of small sample sizes. The effect of various amounts of cells added in cocultures was analyzed by ANOVA. For post-hoc comparisons we used Dunnett’s procedure, in which the condition without adding cells was treated as control. A P value of less than 0.05 was considered statistically significant.