Brenner and Rector's The Kidney, 8th ed.

CHAPTER 66. Attaining Immunologic Tolerance in the Clinic

Alan D. Salama Mohamed H. Sayegh

Immunologic Basis of Tolerance: Self and Non-Self, 2171

Immunologic Education and Central T Cell Tolerance, 2172

Peripheral T Cell Tolerance, 2173

Deletion and Apoptosis, 2173

Anergy, 2173

Regulation or Suppression, 2174

Altered Cytokine Environments, 2176

Immunologic Ignorance, 2176

Clinical Tolerance: Where is the Need?, 2176

Transplantation, 2176

Autoimmunity, 2177

Developing Tolerogenic Protocols, 2177

Clinical Transplant Tolerance, 2177

Novel Approaches for Tolerance Induction, 2178

Bone Marrow Transplantation, 2178

Tolerogenic Dendritic Cells, 2179

Regulatory T Cell Manipulation, 2179

Costimulatory Blockade Strategies, 2179

T Cell Depletion, 2179

Altered Peptide Ligands, 2180

Tolerance and Immunosuppression, 2180

Important and Outstanding Issues, 2180

Conclusions, 2180

Immunologic tolerance is a natural state, found in healthy individuals, in which a harmful immunologic response toward self-antigens is absent. This condition is more than a passive state of unresponsiveness, rather it is a complex, active condition in which responses to self-proteins are carefully modulated, by a number of different and nonmutually exclusive mechanisms. As a result of these, autoimmunity is prevented, but it may develop if perturbations in one or more of these mechanisms occurs. Manipulating the immune system to achieve such a state would be desirable in the context of treating autoimmunity, and hence preventing pathologic relapsing-remitting or progressive conditions and in the context of transplantation, in which the foreign graft, treated as “self,” would be accepted without the risk of rejection. Both situations would allow for the treatment of the patients while avoiding the need for continuous immunosuppression, with all the established associated adverse effects. In addition, in the context of transplantation, the ongoing graft loss due to chronic rejection may be avoided and the half-life of the grafts may be extended beyond their current limitations. Clinical induction of tolerance is not yet reproducibly achievable, although cases of tolerance induction, some inadvertent, have been reported. Furthermore, pilot tolerance protocols are currently being tested in selected patient populations with autoimmune diseases and in transplant recipients under the auspices of the National Institute of Health Immune Tolerance Network (www.immunetolerance.org). Understanding the basis for such changes is necessary in order to develop clinically applicable regimens that would allow tolerance to be induced in a coordinated manner and in wider patient populations. In order to understand the methods used to achieve clinical immunologic tolerance, a review of the mechanisms underlying natural tolerance is required. In this chapter, we detail the basis for immune tolerance and the methods used to achieve clinical tolerance in transplantation and autoimmunity, and describe some potential future directions.

IMMUNOLOGIC BASIS OF TOLERANCE: SELF AND NON-SELF

The adaptive immune response has the capacity to react to a multitude of antigens to enable it to fight off infectious agents and malignantly transformed cells. It achieves this through an array of lymphocyte clones, each with a unique receptor. However, a degree of cross-reactivity exists, such that one clone may react with a number of antigens (albeit with different avidities). This produces a potential problem, in that self-antigens may also cross react and could thus be the target of immunologic responses. Therefore, the immune system has evolved to distinguish self from nonself. In this way, it is set up to efficiently eliminate all invading foreign molecules, while not reacting adversely to the multitude of self molecules, which make up the complex human organism. This pro-cess occurs during a period of immunologic development during fetal life, when the vast array of our own molecules are presented to the immune system, in order to educate it and establish the boundaries for self. The resulting tolerance is a state in which a nondestructive immune response occurs to particular self-antigens. Although in some cases there may be a complete lack of response, following removal of potentially self-reactive lymphocytes, in many cases, there are immune responses that are regulated by additional nondeletional mechanisms, limiting the responses to noninflammatory, nondestructive ones. Hence, the balance between activating self-reactive lymphocytes and regulating them dictates the outcome of the specific antigen-immune cell encounter. Under steady state, this tolerance prevents the development of autoimmunity, but when this mechanism goes wrong, either through a deficiency in immunological schooling or a breakdown of the regulatory mechanisms (and for reasons that are not always apparent), a destructive immune response is mounted towards self molecules, and autoimmunity ensues. The relatively low incidence of clinically relevant autoimmune diseases, despite measurable responses to self-antigens, is a testament to the highly efficient way in which the immune system regulates and monitors itself.

The concept of immunological “self” was first formed following Paul Ehrlich's experiments on the development of haemolysins in animals. He concluded that generation of haemolysins to self erythrocytes would result in a “horror autotoxicus” (autoimmunity as we now know it) and some, active but unknown process should therefore exist to prevent this. Landmark studies by Owen and Billingham, Brent and Medewar provided evidence that exposure to antigens during the neonatal period allowed for the development of tolerance towards those antigens regardless of their origin. Thus, demonstrating that there is a plasticity to the immune system that could allow for re-education of “self”. Indeed, it is apparent that during immunological development, a huge amount of effort and energy is expended in programming the system not to react to self-antigens. Natural tolerance is achieved through two main processes; the first termed central or clonal selection, which occurs in the thymus and involves the elimination of cells directed against self-determinants and the positive selection of those that are not. The second process, termed peripheral tolerance, utilizes a number of non-mutually exclusive mechanisms, to prevent those autoreactive cells that have escaped the clonal deletion mechanism to be efficiently regulated ( Table 66-1 ). These processes have been elucidated in vitro and in vivo and the contribution that each mechanism plays depends in part on the model systems studied. Mechanisms include functional inactivity of autoreactive cells (termed anergy); regulation of autoreactive cells by other cells (such as regulatory T cells) or factors (such as cytokines); indifference to the stimulating antigen (termed ignorance) and finally, separation of antigen targets from the autoreactive cells (sequestration) (see Table 66-1 ). Tolerance is therefore a dynamic process made up of a number of non-mutually exclusive mechanisms, operating synchronously or sequentially.


TABLE 66-1 -- Mechanisms of Tolerance

Central (Thymus/Bone Marrow)

Peripheral (Secondary Lymphoid Organs/Tissues)

Deletion of self-reactive cells through negative selection (high avidity interactions between T cells and self-MHC-antigen complex lead to cell death)

Active regulation by Treg or altered cytokine environment

Anergy (inability to proliferate)

Ignorance (cells recognize antigen but do not react)

Sequestration (cells and antigen do not meet)

MHC, major histocompatibility complex; Treg, regulatory T cells.

The antigen specific immune response is coordinated by T and B lymphocytes and hence these are the cellular effectors determining self-tolerance. T cells represent the central orchestrators of the immune response, providing essential help to antigen-specific B cells and cytotoxic T cells, as well as specificity and help in delayed type hypersensitivity responses. They therefore act as gatekeepers of immune reactivity and non-reactivity. Mechanisms of tolerance induction operate both during development in the thymus and bone marrow but also once the mature cells have been generated and are found in the periphery. Experimental data suggest that T cell tolerance is more easily achieved than B cell tolerance and may be more long lasting. Thus, achieving T cell tolerance to an antigen(s) is critical to underpin the development of clinical tolerance. Our understanding of the mechanisms promoting tolerance come from data obtained from both allogeneic and autoimmune systems, hence the discussion that follows interweaves through both.

IMMUNOLOGIC EDUCATION AND CENTRAL T CELL TOLERANCE

During immunologic development, the T lymphocytes are matured and educated in the thymus, whereas the B lymphocytes undergo a similar process in the bone marrow.

The first and most critical step in preventing the development of autoreactive lymphocytes is the elimination of such self-reactive cells, a process termed clonal deletion. The identification of autoreactive T cells for elimination is based on the strength of interaction between the antigen-presenting cells (APCs), expressing major histocompatibility complex (MHC) molecules loaded with peptide antigen and the T cells, this is termed the affinity-avidity model.[1] Those cells in the thymus that react most strongly with antigens presented by self-MHC molecules are eliminated by apoptosis,[2] or negative selection, whereas those with lesser avidity continue to develop as a result of up-regulation of antiapoptotic molecules such as Bcl-2[3] and are subsequently exported out of the thymus into the periphery, or positive selection. Finally, the cells with very low avidity interactions fail to induce prosurvival signals in the T cells and die within the thymus. T cell selection begins after the recent thymic immigrants, arriving from hemopoietic tissues, have rearranged their T cell receptor (TcR) and up-regulated both CD4 and CD8 antigens. Ultimately, as few as 3% of the total number of CD4+CD8+ double-positive cells are exported from the thymus, having developed into single positive CD4+ or CD8+ cells.[3] The different processes appear to take place in particular thymic areas, for example, the cortex for positive selection and the medulla for negative selection,[4] although this differs for regulatory T cells, positively selected in the medulla.[5] Movement between the areas is carefully controlled by chemokine gradients and expression of key selectin molecules.[4] Within the thymic medulla, the transcription factor Aire (autoimmune regulator), allows the presentation of numerous tissue specific antigens to developing lymphocytes, so that those potentially self-reactive T cells are eliminated.[6] Deficiency of, or mutations in Aire results in clinical autoimmunity, termed the Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome. Interestingly, only specific certain organ systems are involved, and within these, particular parts of the organ tend to be affected, confirming that additional mechanisms must be operational to maintain systemic tolerance. Evidence that many tissue-specific antigens are expressed in the thymus have recently emerged, [7] [8] [9] including autoantigens such as insulin, thyroglobulin, and renal autoantigens such as alpha3(IV)NC1, the target in autoimmune Goodpasture disease. [9] [10] Moreover, for some antigens tested, the level of expression within the thymus appears to inversely correlate with the degree of susceptibility to autoimmune disease in experimental systems and in patients. These data suggest that for the majority of individuals, a significant part of the tolerance to autoantigens, including renal autoantigens, is due to central deletion.

Thus, at the end of T cell development, the thymus exports T cells to populate the secondary lymphoid organs. These express CD4 or CD8 and are weakly autoreactive, but they have the capacity to react more avidly with some other non-self antigens. Circulating autoreactive T cells have been identified in healthy individuals, as well as patients with clinical autoimmunity. [11] [12] The activation and expansion of these cells, following encounter with self-antigens, is tightly controlled through a number of peripheral regulatory mechanisms. Data in human disease on the contribution of these mechanisms are beginning to emerge.

PERIPHERAL T CELL TOLERANCE

Deletion and Apoptosis

One fate of activated T cells is programmed cell death termed activation-induced cell death (AICD). This process is mediated by the interaction of Fas (CD95) with its ligand (Fas-L or CD95L) on T cells, and can occur in developing thymocytes as well as mature T cells.[13] Interleukin-2 (IL-2) acting on its receptor and activating the STAT 5 signaling pathway potentiates the up-regulation of Fas-L and down-regulates Bcl2 expression on T cells, thus sensitizing them to AICD. [14] [15] Augmented AICD can promote tolerance through elimination of populations of reactive lymphocytes, and this underpins certain tolerogenic protocols and therapies [16] [17] [18] (see later). Although IL-2 sensitizes cells through the STAT 5 pathway to AICD, IL-15 acts as a growth and survival factor for T cells. [14] [15] [19] Manipulating and harnessing the combined action of these two cytokines by antagonizing IL-15 and using IL-2 agonists at the time of immune activation (such as transplantation) has resulted in donor-specific tolerance in stringent models and should prove a useful therapeutic approach.[20] Although these agents have not yet been tested in clinical trials, primate studies are ongoing and should provide the basis for formal human studies.

Anergy

Successful T cell activation requires the presentation of peptide on the appropriate MHC molecule to the TcR, termed “signal 1” and costimulatory signals termed “signal 2,” the most significant of which are the B7-CD28 and CD40-CD154 interactions.[21] T cells stimulated by low or high antigen doses or in a costimulation-deficient manner may be rendered anergic, that is hyporesponsive to further antigenic stimulation. In some models, these cells are capable of certain effector functions, such as production of interferon-γ, but they cannot produce IL-2 or undergo autocrine proliferation. Thus, antigen presentation by professional APCs, carrying the full complement of costimulatory molecules, stimulates T cell proliferation, whereas tolerance is achieved using nonprofessional APCs, which lack such molecules.[22] Anergy appears to play a significant role in clinical transplantation, accounting for donor-specific hyporesponsiveness found in established renal allografts.[23] Experimentally, anergic T cells with defective proliferative responses, and suppressed IL-2 and tumor necrosis factor-α (TNF-α) production, persist in vivo for a number of weeks or while antigen is still administered. In vitro anergy can be reversed by the addition of exogenous IL-2,[21] whereas in vivo, certain studies suggest that the anergic state is not overcome by supplementation with T cell growth factors derived from activated bystander cells.[24] However, whether anergy would suffice for clinical tolerance is uncertain because there is a significant concern that in patients a surge of cytokines following infection or inflammation could reverse this state. Moreover, it appears that peripheral tolerance can be maintained through anergy so long as the antigen is still present and available to the T cells, which may not be the case if cryptic or neoepitopes are the immunogenic fractions stimulating autoimmunity or antigraft responses.

In addition, this situation is somewhat complicated by the recent recognition of a myriad of other costimulatory molecules, from both the B7 (Immunoglobulin) and TNF superfamilies, [25] [26] which can induce T cell activation in the absence of B7 signaling ( Fig. 66-1 ). Although initially thought of as representing multiple redundant pathways, it has recently been appreciated that some molecules appear to act on particular cellular compartments and at particular time points[25] ( Table 66-2 and Fig. 66-1 ). Furthermore, it is apparent that memory T cells, CD8+T cells and natural killer (NK) cells are less reliant on B7-CD28 interactions in order to achieve full activation (see later). Importantly, not all of these pathways result in an activating signal, rather some serve to suppress T cell responsiveness and are thus implicated in tolerance induction [27] [28] ( Fig. 66-2 ). Interactions between B7-CTLA-4 result in inhibition of T cell activation and appear critical in regulating T cell responsiveness.[29] Antagonizing this interaction augments T cell responsiveness, exemplified by the CTLA-4–deficient mice that develop a lethal lymphoproliferative disease.[30] Similarly, the programmed death-1 pathway (PD-1) mediates an inhibitory signal to T cells and has been implicated in tolerance maintenance in autoimmunity, transplant surgery, and maternal-fetal tolerance. [31] [32] [33] [34] [35] In addition, there is a suggestion that even activating pathways may play a different role when expressed on nonprofessional APCs compared with professional APCs. For example, ICOS-ligand (ICOS-L) expressed on professional APCs, interacts with its receptor ICOS, and transduces a positive signal, activating the T cell. By contrast ICOS-L expressed on kidney renal tubular cells appears to costimulate a negative signal.[36] The exact role that these alternative pathways play in inducing or overcoming T cell anergy remains to be clarified.

FIGURE 66-1 Positive and negative costimulatory pathways. Cartoon depicting the numerous costimulatory pathways, which modulate the balance between T cell activation and inhibition.


TABLE 66-2 -- Predominant Roles and Targets of the Costimulatory Pathways in Alloimmune Responses and Their Application for Clinical Transplantation

Costimulatory Pathway

Target Cell/Phase of Immune Response

Clinical Applicability

CD28-B7

Initiation phase, CD4+, CD8+ T cells

Successful in rodents. Less so in NHP. Clinical trial in renal transplant recipients: non- inferior to CsA, better GFR at 12 months and induces less chronic allograft nephropathy

ICOS-ICOS-L

Effector phase, CD4+, CD8+ T cells

Not yet tested in humans

CD154-CD40

Initiation phase, CD4+, CD8+ T cells, B cells

Successful in NHP but some preparations withdrawn due to thromboembolic complications

CD134-CD134L

Maintenance phase, CD4+ T cells[*]

Not yet tested

CD27-CD70

CD8+ T cells, NK cells, B cells[*]

Not yet tested

4-1BB-4-1BBL

Maintenance phase, CD8+ T cells[*]

Not yet tested

CsA, Cyclosporine A; GFR, glomerular filtration rate; NHP, nonhuman primates.

*

More prominent effects in the absence of CD28-B7 signaling.

FIGURE 66-2 The central role of CD4+T cells in immune responsiveness and tolerance. On antigen engagement, the presence of costimulatory signals (signal 2) may direct the T cells to an activation or inhibitory fate. In the case of activation, the T cell proliferates, produces cytokines, and provides help to CD8 T cells, monocytes/macrophages, and B cells to produce class-switched immunoglobulins; mediating the effector mechanisms of allograft rejection. If negative costimulatory pathways are triggered, T cells undergo anergy or apoptosis and may stimulate the generation of Treg cells, thus terminating immune responses.

Anergic cells may themselves act to attenuate the responsiveness of other T cells by down-regulating costimulatory molecules on dendritic cells and thus inhibiting effective antigen presentation.[37] This process represents one form of regulatory cell activity and is dependent on cell-cell contact. Recent data suggest that other regulatory cells may also exert their suppressive effect by acting on APCs[38] (see later).

Regulation or Suppression

An important mechanism for maintaining peripheral tolerance is the action of regulatory T cells (Tregs). [39] [40] The existence of a subset of T cells that exerted a suppressive effect on effector T cells was first described in rodent models of autoimmunity. Later work demonstrated that these cells played a role in modulating tumor immunity and transplant rejection. [41] [42] Subsequent studies have confirmed the existence of these cells in alloimmune rodent models, in human transplant recipients, [43] [44] [45] and in healthy individuals.[46] Compared with those patients with normal graft function, patients with chronic allograft rejection are reported as possessing a smaller Treg population, suggesting that loss of regulation may predispose to ongoing immunologic damage and graft loss.[47] In addition, numerous clinical autoimmune diseases appear to be associated with defects (in terms of cell numbers or function) of these cells [48] [49] ( Table 66-3 ). Interestingly, in Goodpasture disease, which is a single-hit disease in which relapses are uncommon, Tregs specific to the Goodpasture antigen appear to predominate in disease convalescence,[50] whereas relapsing-remitting autoimmune diseases such as systemic lupus erythematosus (SLE) appear to lack such a population (see Table 66-3 ). The implication of these data is that restoration of the number or function of these cellular regulators may restore the tolerogenic state and thus be an effective therapy for relapsing autoimmunity or in transplantation to induce tolerance.


TABLE 66-3 -- Role of CD4CD25 Treg in Renal Autoimmunity and Transplantation

Patient Population

Effect

Reference

Stable renal transplant recipients with or without history of acute rejection—indirect alloimmune response

Depletion of CD25 cells augmented response to allopeptides in half of recipients.

45

Stable renal transplant recipients—direct alloimmune response

Depletion of CD25 cells resulted in no significant change in alloimmune responsiveness to donor compared to third party.

146

Renal transplant patients with chronic rejection and operational tolerance (no immunosuppression and stable graft function)

Phenotype of PBMC similar in tolerant patients and healthy controls. Chronic rejectors had deficient numbers of Tregs.

47

Renal transplant recipients with acute graft rejection, chronic rejection and normal biopsy findings

Urinary foxp3 expression by Q-PCR elevated at the time of acute rejection but predictive of reversal from rejection episode and long term graft function.

147

Bone marrow recipients receiving donor cells treated ex vivo with CTLA4Ig to induce anergic regulatory cells

Reduced incidence of GVHD in recipients of anergic cells and diminished T cell responses to donor but not third party cells.

112

Bone marrow stem cell transplant recipients with or without GVHD

Lower CD4 foxp3 expression in patients who go on to develop GVHD.

148

Liver transplants

Reduced numbers of CD4CD25 Treg post transplant which recover in patients with no evidence of rejection but remain low in those with acute rejection.

149

Anti-GBM disease

Depletion of CD25 cells demonstrated augmented T cell frequencies to GBM antigens in 74% of convalescent patients.

50

SLE

Decreased levels of CD4CD25T cells in active patients compared to controls. Though some studies find levels do not correlate with clinical disease activity.

150, 151 [152] [153]

Myasthenia gravis

Normal number but decreased suppressive function of CD4CD25 Treg in patients with MG. Reduced Treg foxp3 expression.

48

Rheumatoid arthritis

Diminished ability of Treg from RA patients to suppress proinflammatory cytokine production from effector T cells and monocytes. Restoration of suppressive function and increase in number of Treg with anti-TNF therapy and methotrexate.

152

GBM, glomerular basement membrane; GVHD, graft-versus-host disease; MG, myasthenia gravis; Q-PCR, quantitative polymerase chain reaction; PBMC, peripheral blood mononuclear cells; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; TNF, tumor necrosis factor; Treg, regulatory T cells.

In clinical transplantation, a subset of patients with stable graft function appear to possess Tregs specific for their mismatched HLA antigens, and it may be that certain immunosuppressive protocols are more permissive than others in generating these populations. [45] [51]

A number of diverse cell subsets can act as regulatory cells. The most studied of these regulatory cells is a population of thymus-derived Tregs that constitute about 10% of the CD4 population; express CD4CD25, CTLA4, and GITR[52]; and are termed natural Tregs. Further characterization of the regulatory population has demonstrated that they are characterized by the expression of the foxp3 transcription factor. [53] [54] [55] Deficiency of foxp3 or neonatal thymectomy results in a loss of these Tregs and is manifested by various autoimmune phenomena.[56] This is mirrored in a human condition, in which fox-p3 is absent, termed Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance,[57] a rare and aggressive autoimmune disease characterized by diabetes, enteropathy, eczema, thyroid and hematologic abnormalities, lymphadenopathy, and premature death. Conversely, over expression of foxp3 in naïve T cells renders them regulatory.[55] The combination of foxp3 and Aire deficiency results in a fulminant autoimmune condition, with reduced survival, but interestingly involving no further organ systems than the singly deficient animals.[58]

A second population of regulatory cells develop in the periphery, in a thymic-independent manner, following antigen encounter under particular circumstances and following exposure to certain cytokines (in particular transforming growth factor-β [TGF-β]). [59] [60] These have been termed adaptive Tregs. These cells also appear to express fox-p3, although their expression of other cell surface markers is more variable.

The mechanisms of action of Tregs are only partly understood. In certain model systems and in vivo, altered cytokine profiles may underlie the inhibitory effects, with predominant IL-10, IL-5, TGF-β or IL-4 production depending on the system studied.[43] However, in vitro, cell contact appears to be essential because suppression is prevented when regulatory and effector cells are physically separated[61] and cytokine effects appear to be less important.[62]Tregs may predominantly exert their effect by acting on and modulating APCs, in particular dendritic cells, possibly through the up-regulation of indoleamine dioxygenase and subsequent tryptophan degredation. [38] [63] An alteration in APC phenotype appears to underlie the action of natural Treg and anergic T cells, and is another potential experimental tolerogenic strategy, in which immunogenic dendritic cells are altered to tolerogenic ones.

In addition, other regulatory cell phenotypes have been described, such as CD8+CD25+, CD8+CD28- T cells, and certain NK populations. [64] [65] These appear to play a role in autoimmunity (EAE) and transplant rejection[66] in both experimental systems and in patients (see Table 66-3 ).

There remain many unanswered questions regarding regulatory cells, their longevity, their antigen specificity and the relative role each plays in human disease. Numerous investigators have demonstrated a therapeutic potential for Tregs, by showing that transfer of Tregs into susceptible or immunized animals, or animals that had undergone transplant surgery may prevent development of disease or abrogate rejection. Translating these findings to the clinic and optimizing the clinical use of cellular therapies, will undoubtedly be a major focus over the next few years.

Altered Cytokine Environments

T cell effector responses in rodents, and to a lesser extent humans, can be separated according to the cytokine profile that the T cells secrete. Th1 cells produce IFN-γ, IL-2, lymphotoxin and TNF-α, and are involved in cell-mediated responses and some antibody-mediated responses, whereas Th2 cells produce IL-4, IL-5, IL-10 and IL-13 and provide help for B cells to produce immunoglobulin M (IgM), IgA and particularly IgE antibodies. Differentiation along the Th1 pathway is under the influence of IL-12, IFN-γ, and IFN-α whereas IL-4 appears to be the essential Th2 pathway differentiation cytokine. Regulation of Th1 and Th2 responses is tightly controlled by the transcription factors, T-bet and GATA3, respectively. [67] [68] [69] Th1 cytokines antagonize many of the effects of Th2 cytokines and vice versa.

More recently a distinct population of cells, termed Th17, producing IL-17 and regulated by the transcription factor RORgt, has been described as being critical in models of autoimmunity. [70] [71] The role these cells may play in human autoimmune disease and transplantation remains to be defined. This T helper cell differentiation is not as clear-cut in human subjects as it is in animals, and the relative balance between Th1 and Th2 responses is probably more relevant than absolute division of function. The balance between these different effector functions and the bias for one or other may therefore have major influences on the regulation of immune reactivity and disease.

In allograft rejection, Th1 responses appear to be dominant, whereas Th2 cytokine deviation may confer protection from rejection in both rodents and humans. [72] [73] [74] However, it should be remembered that the situation is not so clear cut, because STAT4- (Th2 predominant responses) and STAT6-deficient animals (Th1 predominant responses) reject allografts with a similar tempo to their wild type counterparts,[73] as do IFN-γ–deficient recipients (although, interestingly, they do not develop chronic rejection).[74] The deviation of a T cell cytokine response to a single antigen may result in infectious tolerance toward other related antigens or coadministered antigens.[75] Cytokine deviation may also contribute to the tolerogenic effects of altered peptide ligands, with resultant protection from disease.[76] The importance of cytokine profiles in part relates to the strength of the immune response. For example, in an allotransplantation model, the requirement of Th1 cytokines for tolerance induction (stimulating AICD) was found only when the T cell clone size was large,[77] in a MHC-mismatched graft, whereas this was absent with lesser T cell clone size, found in minor histocompatibility-mismatched grafts. Finally, costimulation through particular molecules and ligand-receptor pairs, also alters the cytokine profile. B7 molecules on APCs interacting with CD28 on T cells, are intimately involved with T cell IL-2 production and the regulation of responsiveness to IL-12, through the up-regulation of the IL-12β2 receptor subunit. Therefore, they play a role in deviation toward Th1 responses. This explains the observation that in alloimmunity, antagonizing B7-CD28 interactions, can result in the generation of predominant Th2 responses.[70] In contrast, ICOS costimulation is critical in Th2 immune responses and appears to be critical for the development of mucosal tolerance to autoantigen.[78]

The differential effect of cytokine deviation depending on the reactive clone size emphasizes the need for a depletional strategy before induction of other peripheral tolerance mechanisms. Some evidence for this process regulating responses in human transplantation and the autoimmune responses in anti-glomerular basement membrane (GBM) disease have recently been demonstrated. [50] [74] [81] Whether therapeutic deviation of immune responses from Th1 to Th2 responses is desirable is uncertain, because it has been shown in certain circumstances that attempting such skewing may not be benign and may account for increased morbidity.[80]

Immunologic Ignorance

In some experimental models, antigen-specific tolerance is maintained without deletion or anergy of the antigen-reactive T cells. In these instances, under stable conditions, the T cells do not interact successfully with the antigen, because either they do not encounter the antigen-bearing cells or because those antigen-bearing cells do not stimulate the T cells sufficiently to attain the threshold required for full T cell activation. This may be due to a lack of the appropriate machinery, in the form of costimulatory molecules or insufficient antigen concentration.[81] However, under circumstances in which the APCs or T cells change their phenotype, allowing the T cell activation threshold to be reduced, or express greater quantities of antigen, then T cell antigen recognition can occur, the cells can be activated and thus mediate tissue damage. It remains unclear whether levels of alloantigen expression would be low enough to promote ignorance as a physiologic mechanism of nonresponsiveness.

CLINICAL TOLERANCE: WHERE IS THE NEED?

Transplantation

Transplantation has evolved into the treatment of choice for end-stage renal failure, with significant improvements in quality of life and physiologic parameters compared with dialysis therapy.[82] This success in conjunction with the increasing number of patients on end-stage renal failure programs has meant that demand for transplant surgery is outstripping organ supply. This mismatch is further compounded by the limited longevity of transplanted kidneys, meaning that the organs often do not function for the duration of the recipients' life. In turn, this necessitates the recipient to undergo subsequent retransplant surgeries, and thus, one recipient may use a number of organs in his or her lifetime. This is especially pertinent for younger adults and pediatric patients. Although few organs are now lost to acute rejection, as a result of improvements in surgical techniques and immunosuppressive therapies, the long-term graft survival has altered little over the last decades, and the current mean half-life of cadaveric kidney transplants is only 10 years. Organs are lost, still functioning, with the death of the patient in half the cases, whereas in the rest, a chronic rejection process, mediated by ongoing immune responsiveness toward the graft and by other nonimmunologic factors, precipitates the graft failure.

In order to maximize the benefit of transplantation, for the patient and the potential transplant community, a single organ should function for the patient's lifetime and this would require that at least the persistent immunologic reaction be eliminated. Achieving this while avoiding the complications of increased immunosuppression are the goals of transplantation tolerance. With respect to transplantation, tolerance can be considered a state in which no harmful immunologic responses are directed toward the graft, which would function normally, while there is a fully competent immune system in the recipient, allowing the recipient to respond appropriately to infectious agents or malignant cells.[83] However, it should be remembered that even without the ongoing immunologic injury, transplants may not last indefinitely , because few rodent models in which long-term graft survival is induced are without features of chronic rejection.

Autoimmunity

The mainstay of treatment for immunologic renal disease has changed very little over the last 30 years. Although newer drugs, such as mycophenolate mofetil and tacrolimus, and biologic agents such as anti-TNF therapies are now emerging, the cornerstone of therapy for most, severe renal immune diseases remains cyclophosphamide and steroids, and these represent the “gold standard” to which newer regimens are compared. Both agents have established adverse effects and contribute to significant comorbidities. In certain auto-immune conditions involving the kidney, the immune damage occurs as a single phase (e.g., anti-GBM disease) and relapses are rare, whereas in others, a more relapsing-remitting disease pattern emerges (for example antineutrophil cytoplasmic antibody [ANCA]–associated vasculitis with glomerulonephritis or SLE, in which up to 50% of patients will undergo some clinical relapse). In the latter cases, patients have to endure multiple courses of immunosuppression and consequently are more likely to suffer from cumulative adverse effects. Moreover, in some cases, disease appears to progress despite maximal therapy with immunosuppressive agents. Re-establishing the tolerance that had broken down to initiate the autoimmune process in the first place, should allow the patient to avoid excessive immunosuppression and arguably could be a more robust form of therapy.

Little progress has been achieved clinically or in experimental renal models. One major setback is that although reports of beneficial effects using certain protocols on experimental models are published, few authors rechallenge the subjects to confirm true and robust tolerance.

DEVELOPING TOLEROGENIC PROTOCOLS

Transplantation lends itself more easily to the development of tolerogenic protocols because the timing of the transplant is precisely known, allowing preemptive or induction therapy (unlike an ongoing autoimmune process in which patients may present later and at more variable times during their disease course). Moreover, in transplantation, more of the responding T cells may be naïve to the antigen, making certain strategies more likely to succeed (such as costimulatory blockade), whereas at the time of treatment of autoimmunity, many of the autoreactive T cells have already been activated and have a memory phenotype, which appears harder to regulate.

Much of the work developing tolerogenic protocols has been carried out in rodent models, but less success has been achieved in larger animal models, using nonhuman primates (NHPs) and in clinical transplantation and autoimmunity. Buoyed by the infrequent patients that appear to have achieved a state of transplantation tolerance, the mechanisms underlying immunologic tolerance have been extensively investigated, in order to reproduce and manipulate them ( Table 66-4 ).


TABLE 66-4 -- Examples of Current Tolerance Trials in Renal Disease and Transplantation Supported by the Immune Tolerance Network

Condition and Aim

Protocol

Study Type

Renal disease

ANCA-associated vasculitis

Rituximab (anti-CD20) in inducing remission of ANCA-associated vasculitis[*]

Phase II/III

SLE

Treatment of SLE including lupus nephritis with CTLA4-IgG4

Phase II

Kidney transplantation

ESRF and myeloma

Combined bone marrow and kidney transplant for multiple myeloma with kidney failure

Phase I

Drug minimization and withdrawal

Combination immunosuppressive therapy to prevent kidney transplant rejection and aid in drug withdrawal in adults using campath, sirolimus and tacrolimus

Phase II

Achieving chimerism

Reducing the risk of transplant rejection: simultaneous kidney and bone marrow transplant using T cell depletion with MEDI-507

Phase I

ANCA, antineutrophil cytoplasmic antibody; ESRF, end-stage renal failure; ITN, www.immunetolerance.org; SLE, systemic lupus erythematosus.

*

Also in Europe trialed in Rituxivas trial under auspices of EUVAS (see www.vasculitis.org).

Two fundamental hurdles exist to the development of clinical tolerance. The first is the significant immunologic difference between rodents and humans, with the inbred animals having a less immunologically experienced immune system, meaning that many of the strategies that are effective in the former are less effective in the latter. Second, there is no robust, positive method of measuring the tolerant state, and thus it remains impossible to monitor whether such a state has been achieved, at all but the basic level.[84] It has recently been suggested that rather than pursuing true transplantation tolerance, a lesser state of near-tolerance, or so-called prope tolerance, should be strived for, which may be more easily achievable and in which a small, less toxic dose of some immunosuppressive is administered, so as to maintain graft function.[85] Although this is readily achievable with certain current drug combinations, little long-term data are available on such a strategy and its impact on graft survival.

CLINICAL TRANSPLANT TOLERANCE

Based on the mechanisms of immune regulation previously discussed, investigators have attempted to manipulate the alloimmune response so as to induce donor-specific transplant tolerance. This has mainly been attempted in rodent models and claims of transplant tolerance are generally based on grafts surviving for over 100 days with donor-specific hyporesponsiveness (indicated by acceptance of a second graft from the original donor strain and rejection of third party grafts). How this translates with regard to longevity of human transplants is impossible to know. Moreover, many reports fail to document whether chronic rejection is present in their long-term surviving grafts. Issues relating to durability are important, because clinical tolerance may be fragile and liable to break down, for example following an infectious episode.[86] Indeed, certain reports of human transplant tolerance have been revised following subsequent graft loss. [89] [90] Thus, many of these reports represent what can be achieved in model systems, and they would need to be replicated in NHPs before applicability to humans can be inferred.

Examples of tolerance in clinical organ transplantation do exist. In liver transplant recipients, planned drug withdrawal is successful with maintenance of long-term graft survival in about a quarter of patients.[89] With other transplanted organs clinical tolerance is less common and for the most part have come about inadvertently, following noncompliance or following a need for drug withdrawal to minimize toxicity or treat malignancies ( Table 66-5 ). Reviews of renal transplant patients who have stopped immunosuppression revealed that a minority maintain graft survival beyond a year from the time they stopped their drugs. Unusual cases of patients who have stopped their maintenance immunosuppression and kept their grafts have been reported[90] but suggest that these patients may be self-selected in that they may not mount similar alloresponses to those who lose their grafts, with low alloantigen production despite numerous sensitizing blood transfusions.[91] The exact basis of this immunosuppression-free unresponsiveness remains poorly understood and may not be the same for all these patients. Some have reported the development of michrochimerism, whereas others have noted that the presence of michrochimerism did not correlate with tolerance.[89] In some, greater numbers of cells with a regulatory T cell phenotype have been reported, compared with patients with chronic rejection.[91]


TABLE 66-5 -- Examples in which Clinical Renal Transplantation Tolerance Has Been Achieved

Protocol

Outcome

Clinical Application

Reference

Total lymphoid irradiation (TLI), ATG, low dose steroids

Withdrawal of all immunosuppression in 2/28.12 year graft survival in one. Not related to chimerism.

Inconsistent induction of tolerance in small proportion of patients

87, 88 [89] [90]

TLI, ATG, CD34+ stem cells from HLA-mismatched donors

Short-term follow-up. Complete withdrawal of immunosuppression in two of four patients. But subsequent rejection episodes. Transient chimerism

Demonstrates feasibility of approach, but intense induction which still requires maintenance immunosuppression

97, 142 [99] [144]

Combined bone marrow and kidney transplantation using myeloablative and nonmyeloablative regimen

Renal transplant tolerance up to 4 years of follow-up with stable graft function using nonmyeloablative regimen

High morbidity and intense induction for nonmalignant conditions. Less severe nonmyeloablative regimens more promising

92, 143 [94] [145]

Noncompliance

20% to 30% of patients admit noncompliance, but small numbers appear to develop operational tolerance. 94% of noncompliant patients experience a late rejection episode and have worse renal function

Poor predictors of who will not reject. Many appear to be low-responders based on low PRA following blood transfusion

91, 144, 145 [93] [146] [147]

Unsuitable and not to be recommended

ATG, anti-thymocyte globulin; HLA, human leukocyte antigen; PRA, panel reactive antibodies.

In other cases, more deliberate attempts at inducing tolerance have been made (see Table 66-5 ). Patients who received total lymphoid irradiation as induction therapy for their transplants [89] [90] and patients who received combined bone marrow and kidney transplants from the same donor, mostly for hematologic diseases complicated by renal failure, have not required long-term immunosuppression. [94] [95] [96] These cases suffer from not being generally applicable as a result of the morbidity of the conditioning protocols and the risk of graft versus host disease in the combined bone marrow and solid organ transplants. Modifications of such protocols to find a suitable, less toxic regimen are actively being pursued (see later). Finally, using a combination of antilymphocyte therapy and limited post-transplant immunosuppression, Starzl has recently reported on a cohort of patients, some of whom required only weekly dosing with immunosuppressive agents one year post-transplantation.[95] Although it is not true drug-free tolerance, this prope tolerance appears to be further along the road than our current situation, and maybe a more realistic goal for the immediate future.[85]

NOVEL APPROACHES FOR TOLERANCE INDUCTION

Bone Marrow Transplantation

The use of bone marrow transplantation in order to induce tolerance has been extensively studied in animal models and to a lesser extent in patients. [98] [145] Establishing mixed chimeric immune systems, with components from the donor and recipient bone marrow, allows the re-education of the immune system with deletion and anergy of alloreactive T cells, resulting in tolerance toward the host tissues as well as the foreign graft. Interestingly, the tolerance may outlast the chimerism, suggesting that the graft itself has some tolerogenic capacity.[96] More recent approaches to using nonmyeloablative regimens have been attempted in small numbers of patients (including children), using hemopoietic stem cells, total (or targeted) lymphoid irradiation, conventional immunosuppression, [99] [145] and additional intrathymic alloantigen inoculation in some.[98] Varying degrees of success have been reported with these methods, but longer term follow-up is required. Further refinement of these clinically applicable nonmyeloablative regimens that allow bone marrow transplantation and induction of lasting chimerism in HLA-mismatched patients are required.[99]

In the autoimmune setting, hemopoietic stem cell transplantation using autologous or allogeneic cells have been attempted with variable success. Few patients appear to be tolerant following the procedure, because relapses are frequently reported. Current morbidity and mortality rates following these regimens confine them for use as salvage procedures for more severe resistant disease.[100]

Other methods of inducing thymic re-education and central deletion apart from bone marrow transplantation have been attempted, although their clinical applicability appears currently limited. Intrathymic inoculation of both auto- and alloantigens has been reported to induce tolerance experimentally, with T cell tolerance being easier to achieve than B cell tolerance, and Th1 responses easier than Th2, [103] [104] [105] although this has not been a universal finding. [106] [107] These effects appear to be dependent on thymic dendritic cells (DCs), which are potential tolerogenic targets in their own right (see later). Additionally, the critical role of the thymus in tolerance induction has been made use of by certain investigators who have cotransplanted donor thymic tissue alongside a donor organ graft, so as to encourage thymic education of recipient cells on donor thymus (encouraging clonal deletion of antidonor reactive lymphocytes), and demonstrated the development of donor-specific tolerance.[106]

Tolerogenic Dendritic Cells

Recently, the dual role of DCs in stimulating and inhibiting immune responses has been appreciated. Tolerogenic DCs have a characteristic phenotype and have been induced by genetic manipulation (ex vivo), or by pharmacologic manipulation (in- and ex-vivo). These cells have the capacity to induce peripheral and central tolerance [109] [110] through a number of nonmutually exclusive mechanisms. Generating immature DCs (from donor or recipient cells treated with donor material) and combining them with other therapeutic strategies has proven highly successful in rodent transplantation models.[109] Although, the evidence that manipulated DCs can be used successfully to induce transplantation tolerance in higher animals has yet to be obtained, the feasibility of using such a strategy has been shown in healthy volunteers in whom reduced responsiveness to a viral protein was demonstrated following immunization with autologous immature DCs pulsed with the viral peptide.[110]

Regulatory T Cell Manipulation

Extensive experimental work has demonstrated that Treg cells can induce tolerance in rodent transplantation models[111] and that this is mirrored clinically with a subset of kidney transplant patients possessing a population of donor-specific Tregs[45] (see Table 66-3 ). Moreover, some operationally tolerant patients with stable graft function, off immunosuppression, demonstrate similar levels of Treg cells to healthy controls and significantly higher levels than patients with chronic rejection.[47] Thus, it may be possible to use such a population of cells therapeutically to promote tolerance. Clinical manipulation of donor specific regulatory cell populations in transplantation has been achieved in an indirect and limited manner in bone marrow transplantation, with ex vivo induction of anergic cells using costimulatory blockade,[112] and in solid organ transplantation through the use of donor-specific transfusion, which appears to rely on the generation of a regulatory cell population.[113] However, because in vitro expansion of Treg populations has been reported, this could be a potential therapy for subgroups of pa-tients who demonstrate evidence of ongoing immune activation and graft damage. In the context of autoimmune disease, no clinical attempts at manipulating the Treg population have been attempted, although the use of immune modulators such as anti-CD3 mAb, which appear to be permissive for Treg generation appear promising.[114] In renal autoimmunity, it appears that this application would be of value in the relapsing-remitting conditions, such as SLE and vasculitis, in which a deficiency in the Treg population may be apparent, whereas in anti-GBM disease, there appears to be an effective generation of alpha3(IV)NC1-specific Treg, in the majority of patients,[50] making such an approach unnecessary.

There remain questions regarding the antigen specificity and longevity of Treg therapy that must be clarified before this can be adopted in the clinical arena.

Costimulatory Blockade Strategies

Costimulation blockade has been highly successful in inducing long-term graft survival in rodent models. However, it has not, at least in the way that it has been currently used, reproducibly achieved tolerance in primates. [117] [118] [119] One suggestion as to why there has been such a discrepancy is that this may reflect the predominance of memory cells, generated to infectious agents, in outbred primates and humans compared to laboratory rodents.[118] Based on the impressive tolerizing potential in small animal models, B7 and CD154 T cell costimulatory blockade strategies have been tested clinically. [121] [122] Such an approach was applied with some success to bone marrow transplant recipients,[112] and a recent trial using a modified form of CTLA4Ig in renal transplantation demonstrated that it was not inferior to cyclosporin A (in combination with steroids and mycophenolate mofetil) and was associated with better GFR at 1 year and a lower incidence of chronic allograft nephropathy.[121] However, there was no suggestion that these patients were clinically tolerant. CTLA4Ig has recently been introduced for the treatment of rheumatoid arthritis and will tried in a number of other autoimmune conditions; however, data on altered immune responsiveness in patients remains sparse. Certain anti-CD154 antibodies were associated with significant adverse effects when trialed in transplantation and autoimmunity, necessitating their withdrawal, but others are being developed.

The original promise for these costimulatory blocking agents in larger animals and humans has not been realized.[122] In part, this is because of the alternative mechanisms that allow full T cell activation, through other costimulatory molecules, such as ICOS, CD134, CD70, CD30, 4–1BB.[123] In particular, it has become apparent that certain immunologic compartments, such as alloreactive CD8+T cells, memory CD4+T cells, and NK cells are less reliant on B7-CD28 and CD40-CD154 signaling for their activation but may depend on these alternative pathways. Our understanding of how these newly recognized costimulatory molecules impact on each other, as well as their relation to the B7 and CD40 pathways is still unfolding [25] [126] (see Table 66-2 , and Fig. 66-1 ). Their role in tolerance induction or maintenance requires extensive investigation, because their prime action may be on immunologic compartments that are relatively insensitive to other tolerance inducing regimens. For example, CD8-mediated T cell rejection has been a hurdle in many tolerogenic strategies that has only been overcome using a potent T cell depletion strategy. However, recent data on the effects of 4-1BB,[125] ICOS,[126] and CD70 suggest that these molecules exert a preferential role on CD8+ T cells. In contrast, the CD134-CD134L pathway has been demonstrated to be of prime importance in memory T cell activation and in the absence of B7-CD28 signaling. [126] [129] Thus, understanding how these new pathways can be manipulated to attenuate alloimmune responses is critical and should lead to new (but more complex) immunotherapeutics.

T Cell Depletion

Blockade of costimulatory pathways, induction of regulatory cell populations, or use of other tolerogenic strategies may only work under circumstances in which the alloimmune response is of a manageable size for all the alloreactive T cells to be tolerized. Therefore, reduction of the alloreactive T cell repertoire, with depleting or deletional (central or peripheral T cell apoptosis) mechanisms may play a crucial role, allowing regulatory mechanisms to function in maintaining a tolerant state.[128] However, more recent data have demonstrated that tolerance based on costimulatory blockade and deletional mechanisms may be problematic. Induction of lymphopenia lead to homeostatic proliferation with the expansion of memory T cells, themselves resistant to tolerance induction by costimulatory blockade.[129] An alternative protocol that has capitalized on the roles of certain cytokines in regulating T cell death used agonistic IL-2 and antagonistic IL-15 chimeric molecules and resulted in tolerance in highly stringent models.[17] Interestingly, inducing alloreactive effector T cell death did not seem to lead to loss of alloreactive regulatory cell populations because these are relatively resistant to apoptosis[130] and the AICD-promoting effects of IL-2.[17] Therefore, such a combination augments regulatory mechanisms while attenuating effector responses through deletion. Such a strategy is currently being tested in NHPs. In autoimmunity, certain depletional strategies have been tested with some success. The use of a humanized anti-CD3 antibody in recently diagnosed diabetic patients induced long-lasting improvement in insulin production, possibly through a combination of depletion of pathogenic T cells and induction of regulatory cells.[114]

Altered Peptide Ligands

In alloimmune responses, many of the peptides presented by APCs are themselves derived from degraded allo-MHC molecules. Interestingly, certain peptides corresponding to the nonpolymorphic regions of class I or class II MHC molecules have been shown in animal models to induce transplant tolerance (in association with a course of cyclosporine), in part through alterations in cytokine profiles as well as through deletional mechanisms, with induction of apoptosis. [78] [133] [134] The usefulness of these peptides in human transplantation remains to be confirmed, because use of altered peptide ligands in human autoimmune disease disappointingly failed to show significant beneficial effects.

TOLERANCE AND IMMUNOSUPPRESSION

The newer immunosuppressive drugs may also play a role by inducing T cell apoptosis and deletion or inducing tolerogenic DCs and Tregs, and thus may be required at least for a limited period following transplant surgery. Agents such as rapamycin or its derivatives used during the induction phase,[133] along with anti-T cell antibodies such as specific polyclonal antilymphocyte antibodies[51] but not others[134] or humanized nonmitogenic anti-CD3[135]may induce Tregs and facilitate tolerance.[136] In addition, certain agents may be used ex vivo to induce changes in donor or recipient cells (such as DCs) and promote tolerogenic responses.[112]

The precise impact that conventional immunosuppressive drugs have on tolerizing strategies needs to be investigated thoroughly, because the initial suggestion that certain drugs would impair the generation of tolerance in some models [138] [139] has not proven to be founded in others.[138] For example, calcineurin inhibitors do not impair long-term graft survival if they are given with certain costimulatory blockade regimens, such as multiple rather than single doses of anti-CD154.[139] Whether some agents are more for permissive in generating regulatory cells in vivo as they do in vitro[51] remains unclear but is of considerable interest.[133] Short-term follow-up data demonstrating that withdrawal of conventional immunosuppressants following specific induction therapy can be achieved successfully, at least in a subset of patients is highly encouraging.[95]

IMPORTANT AND OUTSTANDING ISSUES

There remain many issues that need addressing including the durability of any tolerance achieved and quantifying the tolerant state. We currently rely on not detecting a positive response to demonstrate a state of tolerance toward the allo- or autoantigens. However, this is fraught with problems, and ideally what is required is a positive tolerance assay. Realistically, this may need to be made up of a number of diverse assays measuring different aspects of the immune response. What impact infectious episodes or other inflammatory conditions will have on a tolerant state remains uncertain. Defining the populations in whom the tolerance trials should be initiated is also controversial. In transplantation, current acute rejection rates are very low, and it may be ethically difficult to enter patients into trials that have significant risks for morbidity and graft loss, especially when considering living related donation. Pediatric transplant recipients may be ideal candidates for certain trials given their more immature immunologic systems. However, their immunologic state is reflected by them having the best long-term renal graft survival with less chronic rejection than other age groups, making graft failure in a tolerance trial setting less acceptable.[140] Whether pediatric and adult recipients react similarly to tolerogenic protocols is also uncertain.

CONCLUSIONS

Clinical tolerance induction in transplantation and autoimmunity is perhaps around the corner, and pilot protocols are under way in selected patient populations [143] [155] (see Table 66-4 and www.immunetolerance.org). Critical information regarding the immunobiology of tolerance in humans should allow us to get ever closer to defining the strategies that could be applicable to a wider patient population. This requires the careful evaluation of those few patients who have achieved clinical tolerance and developing reproducible methods to measure such a state. Pilot studies in selected patient groups should then allow a clinically relevant, widely applicable protocol to be developed.

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