The contributions of T‐cell anergy to peripheral T‐cell tolerance

Intrathymic deletion of thymocytes with high affinity for self antigen cells plays a crucial role in contracting the autoreactive T-cell repertoire. However, this is manifestly an incomplete process. Not all self proteins are effectively presented in the thymus, including those that are expressed well after the bulk of the T-cell repertoire has been formed, and it is relatively easy to detect autoreactive T cells following immunization with self antigens. For this reason mechanisms of regulating peripheral T cells with unwanted specificity are crucial to survival. There are several mechanisms of peripheral T-cell unresponsiveness including ignorance, deletion by apoptosis, and cytokine-mediated regulation. The topic of this review is a further mechanism, T-cell anergy. Data will be highlighted, which suggests that the induction of T-cell anergy is an important contributor to peripheral T-cell tolerance, and that anergic T-cells are not passive, but may play an important role as regulatory cells. The term anergy was coined by Nossal and Pick in 19801 to describe the unresponsive state that was induced in B cells following the injection of soluble proteins in vivo. They observed that the antigen-specific B cells were present, but were refractory to subsequent activation by antigen or mitogen. The first observations of antigen-induced T-cell proliferative unresponsiveness were made with human T cell clones following culture with antigen in the absence of added antigen-presenting cells (APC).2 Since that time the term anergy has been used to describe a variety of forms of unresponsiveness, induced in a variety of ways, and mediated by diverse mechanisms. This has led to some confusion, and scepticism as to whether anergy was a useful or informative term. The definition of anergy that we favour is the following: a state of long-lasting, partial or total unresponsiveness induced by partial activation. This excludes unresponsiveness due to ‘blindness’ or ‘partial sightedness’ resulting from temporary T-cell receptor (TCR) modulation, and the refractory state that can be seen when T cells are prematurely exposed to activating stimuli too soon after their last antigen encounter. The large majority of reports of anergy have described the inhibition of interleukin-2 (IL-2) secretion by T cells that are normally capable of secreting IL-2 upon activation. However, anergy has also been described in T helper 2 (Th2) cells, in which the secretion of the key signature cytokine IL-4 is unaffected, but the ability of the T cells to proliferate in response to IL-4 is inhibited.3 The common theme in these examples is that anergy refers to the inability of T cells to produce or respond to proliferative signals. It is important to note that the unresponsiveness is often partial. This is well illustrated by the observations that Th0 cells continue to secrete IL-4 after the induction of anergy4 and T cells with the capability of secreting IL-2 and mediating cytotoxicity retain their cytotoxic potential after being rendered anergic even though IL-2 secretion is shut down.5 Having attempted to provide a universal definition of anergy, it may well be that different ‘levels’ of anergy exist.6 This is illustrated by the finding that anergy can sometimes be reversed by culture in exogenous IL-2, leading to several rounds of cell division,7 while in other systems this has been found not to be the case.8 These differences are likely to be explained by differences in the state of activation of the starting T-cell populations, and by differences in the partial activation signals that were used to induce anergy. As the intracellular signalling events that accompany anergy become better defined, as discussed below, it may be possible to explain these differences. There are at least four distinct sets of circumstances that have been observed to induce T-cell anergy in vitro; these are represented schematically in Fig. 1, and will be discussed in turn. The different circumstances that have been observed to induce T-cell anergy in vitro are illustrated. This is the most extensively studied set of conditions that lead to T cell unresponsiveness. The first observations of this kind were made in the artificial, but very precisely defined, system of Quill and Schwartz9 using artificial lipid bilayers, coated onto glass coverslips, and impregnated with H2-Ek molecules together with the appropriate peptide of pigeon cytochrome c. Exposure of T-cell clones specific for this peptide–major histocompatibility complex (MHC) to the impregnated planar membranes not only failed to induce proliferation, but rendered the T cells refractory to further stimulation in response to fully competent APC. In other words they had been rendered ‘anergic’. At the time, the nature of the missing ‘costimulatory’ molecules was completely unknown, however, the identity of one of the key molecules was established four years later when B7 (CD80) was cloned10 and found to ligate the T-cell surface molecule CD28.11 Definition of this major costimulatory pathway spawned an era of experimentation in vitro and in vivo during which the consequences and exploitability of costimulation-deficient antigen presentation were explored. This era has extended up to the present day, in that some of the first clinical trials of costimulatory blockade in the context of autoimmune disease and transplantation are currently taking place.12 The generation of a fusion protein, CTLA4-immunoglobulin, composed of the second receptor for B7 molecules on T cells, namely cytotoxic T-lymphocyte antigen 4 (CTLA-4 function is discussed later), fused to the Fc portion of immunoglobulin, facilitated many of these experiments. Most of the observations of anergy in response to TCR ligation in the absence of full costimulation have involved established mouse or human T-cell clones.13 In contrast, naïve T cells appear not to be susceptible to the induction of anergy under most of these conditions in vitro.14-16 However, several of the descriptions of anergy in vivo have involved the injection of bacterial superantigens, as discussed in detail below, implying that naïve T cells can be rendered unresponsive in this way. In the process of screening peptides for those with improved binding characteristics to MHC molecules, Sette and colleagues discovered that some analogues, usually differing by a single amino acid from the parent peptide, acted as antagonists.17 These antagonists induced no detectable signalling to the T cell, and had no effect on subsequent T cell reactivity. Soon after this, Sloan-Lancaster and colleagues described amino acid-substituted peptide variants that not only failed to induce T-cell proliferation, but that induced T-cell anergy.18 In contrast with the first conditions that were discovered to induce anergy, the effect of these peptide partial agonists was observed even when the APC provided full costimulation. Similar observations were made by Germain and colleagues using transfectants expressing amino acid-substituted MHC class II molecules as APC.19 As the nature of TCR–CD3-transduced signals was elucidated it became clear that these altered peptide ligands (APL), or altered MHC molecules, induced a characteristic pattern of early signalling events, suggestive of partial activation.20, 21 This will be discussed further, below. There has been considerable interest in the possibility that APL could be used therapeutically.22 However their potential is limited by the fact that a peptide that acts as an APL for one T-cell clone/TCR may well be an agonist or entirely neutral for other clones with the same antigen specificity. Therapeutic use of APL in autoimmune disease or in transplantation would require therefore that the T cell affected by the APL could regulate other T cells with the same autoantigen or alloantigen specificity. The finding that certain viral escape mutants encode APL, in place of a CTL epitope may be taken to indicate the potential of this approach.23, 24 The importance of cell division in the maintenance of T-cell reactivity was demonstrated by experiments in which mouse T-cell clones were cultured with costimulation-positive antigen-pulsed APC, but were prevented from dividing by the addition of anti-IL-2 and anti-IL-2 receptor (IL-2R) antibodies.25 This observation could be explained in two ways. First, it could be that cell division is necessary to avoid unresponsiveness. This would be consistent with the suggestion that inhibitors of activation, ‘anergy proteins’, are invariably generated in the context of T-cell activation, but that the T cell escapes from their inhibitory effects by the dilution that results from division. A second possible explanation for the anti-IL-2/R result is that IL-2R signalling is itself important in maintaining T-cell responsiveness. These possibilities have recently been addressed using a drug, rapamycin, that blocks IL-2R signalling and prevents transition from G1 to S phase of the cell cycle. T cells were cultured with immobilized anti-CD3 and anti-CD28 in the presence or absence of rapamycin. Despite TCR/CD3- and CD28-mediated signals the cells cultured in the presence of rapamycin became anergic.26 The additional finding in this study was that cell cycle arrest by addition of hydroxyurea did not have the same effect. This suggests that IL-2R-mediated signals, independent of the induction of G1 to S phase transition prevent the induction of anergy. IL-10 is clearly an important regulatory cytokine, and has been implicated in some models of transferrable T-cell tolerance. The first connection between T cell anergy and IL-10 was made by Groux et al.8 who noted that stimulation of human CD4+ T cells in the presence of IL-10 induced a state of profound unresponsiveness, that could not be reversed with IL-2. These findings may be explained by later observation that stimulation of T cells in the presence of IL-10 favoured the emergence of IL-10-secreting T cells. Such cells have regulatory properties, and have been labelled as T regulatory (Tr) cells.27 In retrospect, the description of the IL-10 effect could be re-interpreted as ‘immune deviation’ rather than as the induction of anergy. However, as will be argued below, a spectrum of non-responsive T cells appears to exist, and the distinction between anergy and immune deviation may be semantic. The earliest observations of T-cell anergy arose from the culture of human T-cell clones with antigen in the absence of APC, as described above.2, 28 Initially this was thought to require high antigen concentrations, however, it was subsequently shown to occur in the presence of the same antigen concentration that induced optimal proliferation when presented by professional APC.29 This is an anomalous finding, in that activated human T cells, as used in these experiments, not only express high levels of MHC class II molecules, but also high levels of B7 family molecules.30-32 The molecular mechanisms responsible for this form of anergy remain to be adequately explained, although some information is available which suggests that TCR–CD3 signalling is altered in some way.33 The possibility that CTLA-4 (CD152) ligation contributes to the effects of T–T interactions has not been fully explored; given that CD152 signalling can alter CD3-transduced signals,34 this is a reasonable candidate explanation for this unusual form of anergy. It was discovered in 1991 that the long known T-cell activation antigen, CD152, was a second ligand for the B7 family of molecules.35 It was soon apparent that CD152 had a negative regulatory role, with almost opposite effects to CD28.36, 37 The most startling results were from mice in which the CD152-encoding gene was inactivated. These mice died at approximately 3 weeks of age with overwhelming lymphocyte accumulation.38, 39 The involvement of CD152 in anergy induction was first suggested by experiments in DO11.10 TCR-transgenic mice.40 It was noted that injection of soluble ovalbumin, the antigen for which the transgenic T cells were specific, induced a cohort of unresponsive cells. If anti-CD152 antibody was coinjected with the ovalbumin, the induction of anergy was prevented. Later, Walunas and Bluestone also showed that CD152 blockade prevents SEB-induced T-cell anergy in vivo.41 CD152–B7 interactions also appear to influence the development of transplantation tolerance. Administration of anti-CD152 monoclonal antibody (mAb) to murine recipients of skin allografts completely reversed the tolerance induced by thymectomy, anti-CD154 (CD40L) antibody and donor-specific blood transfusion (DST).42 Similar findings were reported in a mouse heart graft model in which long-term graft was by with in with of anti-CD152 at the time of transplantation graft anti-CD152 in these models is the induction of anergy, or the emergence of regulatory cells has to be results have also been In a tolerance model anti-CD152 did not the antigen-induced however, it is not clear that the of unresponsiveness was T-cell In an in vitro system observed that T cells from mice were susceptible to the induction of anergy by anti-CD3 recently the majority was that anergy was to a and by it was in vitro at to cells in this the anergic cell could be studied in is clear between anergy and it is clear that they are distinct of partial activation of a T This was using human T-cell clones rendered unresponsive following T–T peptide as described clones from these but while other clones that only of T cells The distinction between these two of response to be by to in that addition of a antibody to the it from the was found to be The molecular for the between these sets of clones was not Similar findings have been observed using anti-CD3 stimulation of naïve murine T cells, in the absence of of naïve T cells with immobilized anti-CD3 antibody is with in approximately of T the T cells with a anergic being to exogenous IL-2, but to proliferation in response to full activation The between the that and the that not is Similar findings were reported by who noted that T cells from mice transgenic for under the were susceptible to the induction of anergy by anti-CD3 As for several years after the in vitro of T-cell anergy was first the majority was that anergy represented an in vitro and that anergy was a on the to This would appear to be a of anergy in B cells, the cell in which this was first mice with the of anergic B cells has been to be a the that anergy is a However, this not appear to be the for T cells. have described the of anergic T cells, in for after the injection of the antigen that was responsible for anergy. The first description of in vivo T-cell anergy arose from the study of mice transgenic for an alloantigen under the The T cells from these mice were to the when in and colleagues have made use of transgenic mice in which the alloantigen is to a variety of peripheral using to in these mice was the of the was by the and of of the In some TCR or was in anergy to be the of the in vivo have involved the injection of bacterial or shown to be murine which T cells expressing TCR to be for function further in vitro In most of these experiments, injection led to a of T-cell by the of T cells expressing the TCR but to respond to in the results with anti-CD3 antibody in of the anergy induced in vivo that from that induced in vitro was that the anergic T cells were unresponsive to exogenous IL-2, even they expressed the IL-2 The of TCR-transgenic mice has to be addressed using have anergy in TCR-transgenic T experiments were with CD4+ TCR transgenic T cells. and colleagues studied mice transgenic for a TCR with specificity for a peptide of It to be very to induce unresponsiveness in the transgenic T cells, in in the transgenic This was only after partial T-cell and of However, in transgenic generated by the with mice transgenic for the the T cells that deletion in the were TCR-transgenic T cells that were an phase of by a of and the cells that were from this have observed that the T cells from the transgenic mice large of the regulatory cytokine, upon antigen the mechanisms of unresponsiveness in this The that has been by the of limited of TCR-transgenic CD4+ T cells This the transgenic T cells to function in a and for to be using or by the cells with a this model system it is clear that T-cell anergy is induced following injection of for the presence of regulatory cells was in this of these in vivo systems have been used to the of anergic T cells. In all the the anergic T cells for a of In the of reactivity was observed with time, but the unresponsiveness was the antigen was at This is of observations made in many of the transplantation tolerance in which reactivity the was The context in which the induction of anergy be thought of as a role in the of the immune system is in the induction and maintenance of self tolerance. T-cell anergy can be induced in vivo by the injection of this is to to the induction of self tolerance. The that we have is that autoantigen presentation by cells, leading to the induction of T-cell anergy, is a key in the of autoreactive T cells. This is on all the observations of anergy induction by costimulation-deficient antigen-presenting cells. Most of these experiments have cells as APC with or costimulatory have reported the ability of of human antigen human and cells to induce anergy in T Most recently we have clinical transplantation as a model system to these events in vivo. have observed the development of donor-specific in and using dilution for CD4+ T cell as a If this was to the in vivo of the observations that we had made with cells in vitro we that the induction of would be in the T-cell the that can In a study of in were on the day, and four this was The that we addressed this donor-specific was whether it was due to deletion of T cells, or to the induction of anergy. the fact that T-cell anergy can be reversed in in vitro cultured T after the CD4+ T cells from some were used for an the were cultured for 3 in exogenous IL-2 and for day, being used in In of a in was seen following culture in IL-2. in the was these together they the following model the in vivo of T-cell anergy The peripheral T-cell repertoire autoreactive due to the incomplete nature of deletion and the of and antigens. a due to a for the cells from the to the taking with the viral and a large of that were In the and autoreactive T cells will be The activated T cells will to the due to and the in the in the the T cells will their peptide on professional APC that are the most at and exogenous The autoreactive T cells, in contrast, will their ligands on the of cells that are by this time expressing MHC class II molecules due to The cells are the cells the and are to process and present The cell autoantigen presentation will to induce anergy in the T cells, as to be in the T cells with in the This model is illustrated in Fig. The possible of antigen presentation by cells is illustrated. T cells are in the an by cells that have from the exogenous and autoreactive T cells are likely to be in response to cells presentation of sets of the the activated the T cells will their antigen in The exogenous antigen-specific T cells will be presented by antigen-presenting cells that are in antigen and This will lead to of the T cells with exogenous antigen specificity. In contrast, the major cells the will be the cells, induced to express MHC class II by and the will lead to the induction of unresponsiveness in these T cells. The model may the of anergic T cells, but it not provide a reason for the T cell in maintaining a cohort of cells in the In the of this review we would to review the that cells with an anergic have regulatory and that this may their the first of the potential for anergic T cells to as cells from a in vivo model in which mice were with cells. The cells acted as cells that were in their recently we the regulatory effects of anergic T cells using of human T cells in These cells acted as cells in a and antigen-specific In they were to effect the APC expressed the antigen for which the anergic and the T cells were Most the could not be observed in a and could not be reversed by the addition of the regulatory and These results this from all the other examples of which were by soluble At the time we these findings in of the model of first by we the same results in a murine and on to that the anergic T cells could skin in the first that anergic T cells were capable of in vivo. In most we have attempted to the mechanisms anergic T cells their Having observed that they failed to T-cell to anti-CD3 and anti-CD28 antibody in the absence of APC, we that APC were for the regulatory effects to be the effect of anergic T cells on APC, cells were generated in and with or anergic T cells specific for antigens. culture with T cells the of the was In contrast, culture with the anergic T cells rendered the almost of T-cell This of was by of MHC class II and B7 family These that inhibition of and function may be one of the in which anergic T cells their regulatory These effects are precisely the opposite of the effects of helper T cells on an effect which has been to as an the effect of anergic T cells on may be to as A of very observations have been made during the last years by two and a of regulatory cells in the These cells were first in the context of a model of autoimmune disease that arose in mice that were to at 3 the with mice of the same noted that the a of peripheral T cells that were and Most when these cells were from a to a they the from the development of autoimmune in vitro have some of these they not proliferate in the absence of exogenous IL-2, they not secrete known they require in to their they IL-2 in the T cells, and they appear to be antigen in their There are many between this cell and the anergic T cells that are described It is to that this due to with self presented by a APC, or the T cell is to a they may even be generated in the At the other of the spectrum of regulatory cells are those that regulate the of as of the in vivo models T cells is that of in which T cells from from the development of It appears that to the by these on all of the available we that a spectrum of regulatory cells At one of the spectrum are cells regulatory function is mediated by that regulate other T cells or APC, at the other of the spectrum are cells regulatory function and is mediated cell surface molecular molecules that have to be In anergic T cells are and in an altered state of Not only may the induction of anergy play a role in autoreactive T cells, but their function may this to the of other T cells. the molecules involved in anergic T-cell mediated are defined, they will be for the of and precisely