Immunology Letters 108 (2007) 2026

Short review

Controlling autoimmunityLessons from the study of red blood cells as model antigens
Robert N. Barker , Mark A. Vickers, Frank J. Ward
Department Medicine and Therapeutics, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeeen AB25 2ZD, UK Received 19 September 2006; received in revised form 13 October 2006; accepted 20 October 2006 Available online 13 November 2006

Abstract The characterization of human and animal red blood cell (RBC) autoantigens in autoimmune hemolytic anemia (AIHA) has provided an opportunity study the control of specic autoimmune responses of unequivocal pathogenic relevance. The results reveal that censorship of the autoimmune helper T (Th) cell repertoire by deletion and anergy is very incomplete in healthy individuals, even for widely distributed, abundant self-antigens on RBC. There is strong evidence that autoaggression by surviving Th cells is normally held in check by other mechanisms, including failure to display the epitopes that they recognize, and active immunoregulation. AIHA is one of the rst human autoimmune diseases in which regulatory T (Tr) cells that are specic for the major autoantigens have been identied. These Tr cells recognize the dominant naturally processed epitopes, and recent studies suggest that disease develops when other determinants, to which such tolerance is less secure, and which are normally inefciently presented, are displayed at higher levels. Together, the results raise the possibility that therapy for diseases such as AIHA could be based on switching the balance of the response back towards regulation, in particular by the administration of the dominant peptides recognized by specic Tr cells. 2006 Elsevier B.V. All rights reserved.
Keywords: Autoimmunity; Tolerance; Red blood cell; Regulation; Immunotherapy

1. Introduction Damaging immune responses are an important cause of disease. Current treatments for these conditions are unsatisfactory because they are palliative rather than providing a cure, rely on inducing generalized immunosuppression, and can be associated with serious side-effects. There is therefore a pressing need to understand how immunological tolerance is lost and can be restored in order to develop effective, specic therapies. One factor holding back progress in many diseases has been the inability to analyze the specic responses that are of pathogenic relevance, because the identities of the dominant antigens are uncertain. However, this obstacle has been overcome in number of conditions that are antibody-mediated, where techniques such as immunoblotting and immunoprecipitation can be used to determine the antibody specicity [1]. Red blood cells (RBC) have not only been model antigens for basic immunological

research for many decades [14], but are also important targets for pathogenic antibodies in autoimmune hemolytic anemia (AIHA) [5,6] and transfusion medicine [6,7]. The identication and characterization of key RBC auto- and allo-antigens over the last two decades has provided an opportunity to learn lessons about the control of specic, pathogenic immune responses in both human and experimental animal disease [713]. This paper will review responses against RBC in AIHA, the mechanisms by which such reactions are prevented in health, and how this knowledge can be exploited in the future to treat the disease. The focus will be on CD4+ helper T (Th) cells, which are pivotal to the development of autoimmune diseases, including those that are antibody-mediated [1], and are therefore attractive targets for specic immunotherapy. 2. AIHA AIHA was one of the rst diseases that was shown to have an autoimmune pathology [14], and has been described in a number of species, including humans and domestic dogs, cats, rabbits,

Corresponding author. Tel.: +44 1224 555868; fax: +44 1224 555766. E-mail address: r.n.barker@abdn.ac.uk (R.N. Barker).

0165-2478/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2006.10.005

R.N. Barker et al. / Immunology Letters 108 (2007) 2026 Table 1 RBC autoantigens targeted in human and animal AIHA Species Human AIHA B cell autoantigen Rh proteins [9,10] Glycophorin A [9,10] Band 3 [10] Canine AIHA Murine AIHA NZB mouse Glycophorins [11,33] Band 3 [11] Band 3 [13,17] Band 4.1 (pr) [17] Phosphatidyl choline (pr) [17] Band 3 (cr) [12] Glycophorins [33] Yes [32] Not examined Th cell autoantigen Rh proteins [25] Unprimed autoreactive Th cells present in health Yes [26] Regulatory T cell response

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IL-10 response to dominant naturally processed Rh protein epitopes [45]

Band 3 [2124]

Not applicable

Weak IL-10 response to Band 3 [24]

Induced by rat RBC

Band 3 [31]

Yes [4]

Recovery due to suppression by CD25+ cells and IL-10+ cells(auo) of auto, but not anti-rat, response [52,4]

Glycophorins (cr) [12] Induced by LCMV Band 3 [16] Not examined Yes [4] Not examined

Dominant antigen in bold; pr, polyreactive antibody, also binds nuclear antigens, e.g. histones; cr, cross-reacts with rat RBC antigen; auo, authors unpublished observations.

horses and cattle [5,6]. There are also important examples of AIHA in laboratory mice [2,3]. The New Zealand Black (NZB) mouse [2,13] develops AIHA spontaneously, and the disease can also be induced in healthy murine strains by repeated immunization with rat red blood cells [4,12], or by infection of C3HeB/FeJ mice with the docile strain of lymphocytic choriomenigitis virus (LCMV) [15,16]. In all species, the condition is a classic example of type II hypersensitivity, characterized by autoantibodies that bind RBC and reduce their life span through phagocytosis by splenic macrophages and/or complement-mediated lysis [5,6]. The autoantibody can be either warm or cold reactive [5,6]. Warm autoantibodies have optimal afnity for RBC at 37 C, are the more common cause of disease, and are predominantly IgG [5,6]. In contrast, cold RBC autoantibodies react more strongly at 0 C, are typically IgM, and are often present in healthy individuals, being harmless unless active at temperatures found in the peripheral circulation [5,6]. It is the form of the AIHA caused by warm IgG antibodies that provides a well-characterized model disease in which to study the loss of immunological self-tolerance [1]. 3. RBC autoantigens The RBC antigens bound by the pathogenic warm IgG antibodies have been identied in humans [9,10], dogs [11] and mice [12,13] (Table 1). The most common targets in human AIHA, recognized in over 70% of cases, are the Rh proteins [9,10], which are also of clinical importance in transfusion medicine because they express blood groups [7,8]. Autoantibodies with other specicities, reactive against the glycophorins, which are heavily glycosylated RBC membrane proteins, or against the RBC anion channel protein, Band 3, are produced in a minority of patients [9,10]. The major canine RBC autoantigens are the glycophorins, with Band 3 targeted by autoantibodies from

some cases [11]. In mice, Band 3 is the dominant autoantigen in NZB disease [13,17] and is also recognized by RBC antibodies in the forms of AIHA induced by cross-reactivity with rat red blood cells [12], or by LCMV infection [16]. 4. Th cells in AIHA The vast majority of IgG antibody responses are T-dependent, since B cells require help to switch class from IgM production, form germinal centers and undergo somatic mutation [18]. There is now a substantial body of data showing that the production of warm IgG autoantibodies in AIHA is no exception [1]. Valuable insights have been obtained from murine AIHA, particularly the spontaneous form affecting NZB mice. NZB IgG autoantibody production in vivo is retarded by treatment with anti-CD4 monoclonal antibody (mAb) [19], or CD4 gene deletion [20], and splenic Th cells from NZB mice, but not MHCmatched healthy strains, proliferate in vitro in response to the major murine RBC autoantigen, Band 3 [21,22]. Furthermore, NZB disease is accelerated by immunization with an insoluble peptide bearing the dominant Th cell epitope [23] from Band 3, and ameliorated by mucosal administration of a soluble analogue of this sequence [24]. Other murine models are also Th dependent, since anti-CD4 mAb treated mice do not develop AIHA induced by lymphocytic choriomenigitis virus [15], and T cell depletion prevents RBC autoantibody production in response to immunization with cross-reactive rat antigens [4]. Findings in human AIHA are also consistent with the need for help. Rh autoantigen-specic effector Th cells that have been activated in vivo can be demonstrated in the peripheral blood and/or spleen from all patients with anti-Rh autoantibodies [25], but from very few healthy donors [25,26]. It is a long-standing belief that self-tolerance in the T cell compartment is less secure than for B cells, and that, in health, antibody-mediated dis-

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eases such as AIHA are prevented due to lack of effective help [1,4]. 5. Immunological self-tolerance of Th cells Since the development of immunology as a discipline over 100 years ago, one of the major challenges has been to understand how the bodys own tissues avoid damage from an immune system that is able to respond to a vast number of foreign or noxious substances [27]. In the last two decades, with the application of modern methods of cellular, molecular and transgenic biology, a number of different mechanisms responsible for the state of self-tolerance have been demonstrated in animal models [28]. These include the censorship of potentially autoreactive cells by killing them or rendering them inherently unresponsive, lack of effective presentation of autoantigens, and various forms of active immune regulation. The need now is to determine which of these mechanisms fail in patients with autoimmune disease and how such decits can be corrected therapeutically. It is in answering these questions that the ability to study specic pathogenic responses to well-characterized RBC autoantigens has proved of value. 6. Purging of the repertoire by deletion or anergy of autoreactive Th cells A corollary of Burnetts original clonal selection hypothesis was that forbidden clones with the potential for autoreactivity are eliminated during their development [29]. With the advent of techniques, including transgenic mice, to follow the fate of antigen-specic lymphocytes in vivo, formal proof has now been obtained that Th cells reactive with at least some classes of self-components are indeed deleted during their development in the thymus [28]. However, these approaches have been applied to relatively few T cell clonotypes, leaving open the question as to whether deletion is the major mechanism of helper selftolerance, or relevant only for particular specicities, such as those where the antigen is available in the thymus. A second mechanism of self-tolerance is the functional inactivation, or anergy, of autoreactive Th cells that have survived thymic selection but then encountered the corresponding self-antigens in the periphery [34]. Again, transgenic models provide denitive experimental evidence that anergy can be a means to silence certain autoreactive clones [28], but the completeness and general relevance of such censorship remains uncertain. Studies of responses to RBC antigens have enabled the extent of the autoreactive Th cell repertoire that escapes deletion and anergy to be assessed. The results provided a clear demonstration that these processes are very incomplete, and cannot alone explain the control of autoreactive Th cells in health. Several studies illustrate that healthy mice [4,12,31], dogs [32,33] and humans [26] harbor Th cells that can be stimulated to proliferate in vitro by RBC autoantigens, and that these are primary responses, mediated by populations that are quiescent in vivo. In particular, nave peripheral blood Th cells from all healthy human donors [26] recognize self-Rh proteins, which are the major target in AIHA [9,10,25]. Furthermore, epitope mapping

revealed that this normal autoreactive repertoire is surprisingly extensive, since, in every healthy donor, multiple Rh determinants elicit primary proliferation, with up to one-third of a panel of 15-mer peptides spanning the autoantigens being stimulatory [26]. The relative ease with which the Th cells can be induced to respond in vitro indicates that they are not anergic. Comparison with parallel studies in AIHA demonstrates that the normal immune repertoire differs from that of patients, not in the absence or ne specicity of circulating RBC-specic autoreactive Th cells, but in the nding that these lymphocytes are not activated in vivo [25,26,30]. Thus, potentially autoaggressive lymphocytes survive thymic deletion and peripheral anergy in abundance as part of normal immune development, and it is the failure of mechanisms to control their activation that drives disease. This conclusion does not exclude the possibility that factors that predispose to autoimmune conditions [1], such as inheritance of particular MHC types, mediate their inuence by shaping the helper repertoire. For example, in AIHA induced by LCMV, which only occurs in C3HeB/FeJ mice [15,16], the ne specicity or avidity of Th cells selected by the class II molecules in the thymus may help to determine a response to infection that provokes autoimmune pathology via molecular mimicry. 7. Lack of effective presentation of autoantigens If not all autoreactive Th cells are purged from the repertoire by deletion or anergy, then they must be prevented from responding by other mechanisms. One long-standing explanation is that the corresponding autoantigens are sequestered, or protected in privileged sites, where they are not accessible for effective presentation to Th cells. Although this is plausible for certain autoantigens, such as those behind the bloodbrain barrier [35], it cannot be relevant for more widely distributed self-proteins, including those expressed on circulating RBC. Instead, the concept has been developed at a molecular level, by focusing, not on the accessibility of self-antigens, but on the way that they are handled after uptake by antigen presenting cells (APC). In particular, it has been proposed that many self-epitopes are effectively hidden from Th cells, or cryptic, because they are inefciently processed and presented by APC from the intact antigen [30]. Such sequences may be poorly displayed, either because they span the cut sites of APC processing enzymes and are subject to destructive processing [36], or because they bind available MHC molecules too weakly to compete effectively with other peptides for presentation [37]. Irrespective of the cause, inefcient presentation of particular epitopes will allow the respective Th cells to escape thymic deletion and remain unstimulated in the periphery [30]. There is unequivocal evidence from rodent models, particularly experimentally induced allergic encephalomyelitis, that Th cells with these specicities are potentially autoaggressive and can cause autoimmune pathology if stimulated [37]. Such observations have led to proposals that spontaneous human and animal disease also results from activation of such Th cells, triggered by exposure to cross-reactive foreign peptides, or polyclonal activators including microbial superantigens, or by a change in antigen

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processing that leads to the presentation of self-peptides that were previously poorly displayed [30]. It has proved difcult to determine whether human autoimmune disease is driven by Th cells specic for autoantigenic epitopes that are normally inefciently presented or cryptic. However, direct evidence to support the hypothesis has now been obtained in human AIHA. The RhD protein provides a unique opportunity to characterize defects in self-tolerance since, in different individuals, it represents a target for either autoaggressive [9,10,25] or conventional foreign [7,8,38] immune responses. In addition to being a major autoantigen in patients with AIHA [9,10,25] the RhD protein also carries a highly immunogenic and clinically important blood group, the D antigen [7,8,38], which is not expressed in Caucasian RhD-negative individuals due to deletion of the entire RHD gene [39]. The availability of healthy RhD-negative donors who have been exposed to the RhD protein as a foreign blood group antigen has allowed a comparison of Th cell responses to the same target in healthy alloimmune individuals [38] and patients with an autoimmune disease [25]. In particular, the helper epitopes have been mapped for the two groups. Th cells from both alloimmune donors and AIHA patients typically demonstrate recall proliferation against multiple epitopes from the RhD protein, but different sets of peptides are targeted in the two groups, and this striking distinction is maintained even when results from individuals who share the same MHC class II restricting elements are compared [40]. Since conventional foreign responses target abundantly presented, naturally processed peptides [30,3538] after in vivo immunization, these results lead to the conclusion that autoreactive Th cells activated in AIHA are specic for other, less effectively displayed, cryptic or subdominant, epitopes. Another inference from this work is that associations with HLA type, which are typical of many immune-mediated diseases, cannot necessarily be attributed to the greater effectiveness of certain MHC molecules in presenting the autoantigen peptides that drive pathogenic responses [41]. Both susceptibility to AIHA, and the ability to mount alloresponses to RhD, are positively associated with inheritance of HLA-DR15 [25,38,40], yet the peptides recognized by Th cells differ between the two groups, and the epitopes targeted in AIHA are those that are normally inefciently displayed. The conclusions drawn from the study of AIHA may well be applicable to other human autoimmune conditions, since detailed analyses of Th cells specic for the glomerular basement membrane antigen in Goodpastures disease [41] revealed that they also recognize epitopes that are normally subdominant or cryptic [1]. 8. Active regulation of potentially autoaggressive T cells Over three decades ago, it was established that immune responses could be controlled by active inhibitory mechanisms mediated by lymphocytes [42]. AIHA induced by rat RBC immunization of mice provided an early example of such infectious tolerance, since the autoimmune response is transient and the mice become refractory to further induction of disease, with splenocytes transferred from recovered animals providing protection to nave recipients [3,4]. The notion that T cells may

perform such a regulatory role is attractive, but has undergone major revisions over the years. Initially, it was widely accepted that a distinct subset of suppressor T cells bearing the CD8 cytotoxic marker was critical for preventing autoimmune responses [4], but this notion fell into disrepute when such lymphocytes proved impossible to characterize, and the MHC genes purported to restrict their recognition could not be mapped [43]. With the realization that CD4+ Th cell clones can be classied into different functional types on the basis of the cytokines that they secrete, it emerged that at least some of the phenomena previously associated with suppression may be attributed to mutual antagonism between these Th1 and Th2 subpopulations [44]. Many models of autoimmune disease are driven by helper responses that are dominated by the Th1 subset, and inducing a corresponding Th2 bias can prevent or ameliorate the pathology [1,30]. This imbalance is not seen only in inammatory diseases, but also in autoantibody-mediated conditions, including AIHA [1]. For example, Th cells from AIHA patients [45] and NZB mice [22] secrete predominantly IFN- , with little or no IL-4, when proliferating in vitro against the respective RBC autoantigens or peptides. Such a Th1 bias is a feature of the disease, since it is not shared by healthy donors who have responded to the RhD protein as a foreign antigen. The notion that Th1 cells are pathogenic in AIHA is further supported by studies of NZB mice, where deviation of the response towards Th2 by nasal administration of soluble peptide containing the dominant helper epitope resulted in amelioration of disease [24]. Such a shift away from Th1 domination may be therapeutically benecial because the associated change in the IgG isotype of the autoantibody reduces the efciency with which Fc receptors remove RBC from the circulation [24]. There is currently intense interest in autoimmune pathology associated with Th17 [46] cells that secrete the inammatory cytokine IL-17, but pilot studies have yet to demonstrate a role for this subset in AIHA. Despite evidence for the role of Th1/Th2 antagonism, the dichotomy could not explain all examples of immune regulation. This led to a renewed search for other, inhibitory T cell subsets that mediate the role originally attributed to CD8+ suppressor cells. A mass of evidence from has now revealed that active suppressive phenomena, including the control of immune-mediated pathologies, are mediated by recently dened CD4+ T regulatory (Tr) cells [4749]. The current, widely accepted paradigm is that there are two major forms of Tr cell, dened as antigeninduced, or natural. Induced Tr are held to be generated in the periphery following chronic exposure to antigen, or nave T cell activation under tolerogenic conditions, and include Th3 and Tr1 cells that, respectively, secrete the inhibitory cytokines transforming growth factor- (TGF- ) or interleukin-10 (IL-10) [4749]. In contrast to the induced Tr subsets, natural Tr are believed to differentiate into a distinct suppressor lineage during thymic development, rather than arising from nave cells in the periphery [48], and, at least in vitro, mediate inhibition via undened mechanisms of direct cellcell contact, and not by secreted cytokine [48,49]. Natural Tr cells constitutively express a number of molecules that represent activation markers for conventional T cells, including the chain of the IL-2 receptor CD25, together with glucocorticoid-induced TNF receptor

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(GITR) and CTLA-4 [4749]. The transcription factor Foxp3 has recently been shown to be characteristic of this suppressor population, and to lead to differentiation into the CD25+ Tr phenotype in mice [50]. AIHA is one of the rst human diseases in which autoantigen-specic regulatory Th cells have been described and characterized [1,45,49]. Tr cells specic for the target Rh autoantigens can be found in the peripheral blood or spleen of patients with AIHA, and are capable of inhibiting the Th1 effector responses in vitro by secretion of IL-10 [45]. This Tr activity correlates with periods of remission, consistent with the view that the autoimmune disease perpetuates due to an imbalance between pathogenic and regulatory responses. The Rh-specic Tr cells have now been cloned and shown to mediate inhibitory activity only after stimulation by cognate antigen, and not in response to polyclonal activators, illustrating the importance of specicity in the function of such cells [51]. Although able to secrete the induced inhibitory cytokine IL-10, these Tr cells also express the natural marker FoxP3, revealing a less clear distinction between the different regulatory forms in humans ex vivo than was believed from previous in vitro studies [51]. Similarly, in murine AIHA induced by rat RBC, recovery is associated with both a CD25+ Tr cell population [52] and IL10 responses to the Band 3 autoantigen (authors unpublished

observations). In epitope mapping studies, when Tr cells from AIHA patients were tested for responsiveness to a panel of peptides spanning the Rh proteins, IL-10 was produced in response to particular sequences that fail to induce proliferation or IFNproduction [45]. As described earlier, studies of healthy RhDnegative donors who have been immunized with RhD-positive RBC have identied the dominant, naturally presented epitopes from the antigen, and shown that the Th1 cells associated with AIHA target a different, cryptic or subdominant set of determinants [25,38,40]. Further comparison with the Tr epitope maps in AIHA demonstrates that, unlike the Th1 cells, the regulatory population recognizes the dominant, naturally processed peptides [45]. This indicates that selection of the helper repertoire allows the survival of a potentially autoaggressive population that recognize inefciently displayed self-peptides, together with least some Th cells specic for dominant, naturally presented autoantigenic epitopes that are not deleted or anergized, but attain a Tr phenotype (Fig. 1). The studies lead us to propose further that a change in antigen processing in AIHA leads not only to the display of previously cryptic Rh

Fig. 1. Shaping of the autoreactive CD4+ T cell repertoire by deletion and/or anergy. These processes allow the escape of (A) at least some autoreactive Th cells specic for dominant, naturally processed epitopes, which can attain a regulatory phenotype; (B) autoreactive Th cells specic for autoantigenic epitopes that are cryptic or inefciently presented.

Fig. 2. Hypothesis to explain activation of effector, rather than regulatory CD4+ T cells in autoimmune diseases such as AIHA [45]. In health (A), the balance is in favor of regulation because Tr cells have been selected or educated to recognize the dominant naturally processed, abundantly displayed, self-peptides, generated from autoantigens such as the Rh proteins from RBC. In autoimmune disease (B), a change in processing, or the recruitment of APC with different behaviors, results in the display of self-peptides that have not previously been presented efciently [30]. There has been no opportunity for regulatory cell tolerance to develop specic for such novel peptides, and any previously unstimulated CD4+ cells in the repertoire that recognize them can now be activated as autoaggressive Th1 cells. The balance between the sequences presented, leading to activation of either Tr or Th1 cells, can change during the course of chronic disease, leading to remission or relapse, and may be amenable to therapeutic manipulation by administration of peptides containing Tr epitopes.

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epitopes, and the activation of autoaggressive Th1 cells, but also promotes disease because the peptides that are necessary to stimulate protective regulatory responses are less effectively presented (Fig. 2). Thus, autoantigens such as the RhD protein carry different epitopes that stimulate pathogenic or regulatory cells, and the development of autoimmune pathology is dependent, at least in part, on the balance between the activation of these populations, determined by the relative levels of presentation of the two sets of peptides. One example of AIHA provides circumstantial evidence for such a mechanism. Many patients with chronic lymphocytic leukemia (CLL) also develop AIHA, and the neoplastic B cells, although generally regarded as inefcient APC, are the most potent cell type for processing and presenting Rh protein to the autoreactive Th1 cells [53]. One interpretation of this nding is that CLL B cells preferentially generate peptides that are not processed from antigen by other, conventional APC, enabling them to stimulate an autoreactive T cell repertoire that is skewed towards the recognition of such subdominant or cryptic peptides. By corollary, CLL cells would be poor presenters of peptides that are normally efciently processed and displayed, making them unable to recall memory responses to foreign antigens where priming was induced by other APC types. Notwithstanding the support for altered autoantigen processing as a driver of autoaggression and the loss of tolerance, diseases such as AIHA are multifactorial in origin, and so other causes of immune dysregulation are also likely to be important. For example, Rh-specic Tr cells from AIHA patients secrete only IL-10, whereas those detected in healthy donors can produce both IL10 and TGF- , suggesting that there may also be a defect in regulatory cytokine production in disease [51]. 9. Summary and potential for therapy to restore self-tolerance in autoimmune disease Immunological self-tolerance is maintained by a complex series of complementary mechanisms, presumably reecting its importance to health and survival. Diseases such as AIHA are multifactorial in origin, and a breakdown in tolerance at several levels is likely to be necessary before pathology develops. The precise shaping of the ne specicity of the Th repertoire by factors such as MHC haplotype may inuence disease susceptibility, but extensive or complete deletion or anergy of Th cells that recognize RBC autoantigens is not necessary to protect against AIHA [30]. The disease is likely to be triggered by a failure of other mechanisms that control the surviving autoreactive Th cells, including tight limits on the range of self-peptides that are normally presented at high levels, and active regulation [4749]. Recent studies indicate that immunotherapy could be designed to restore such forms of tolerance. Many animal models demonstrate that this may be achieved by inducing mucosal tolerance with autoantigenic sequences, or the delivery of short, soluble peptides, and early human trials in type I diabetes, multiple sclerosis, rheumatoid arthritis, allergy and in AIHA are being designed to exploit these approaches [54]. An alternative strategy, which can be explored in AIHA, may be to correct Tr defects or imbalances, for example by immunization with the autoantigenic peptides that preferentially activate specic regu-

latory cells to produce IL-10 [45]. The ability of such peptides effectively to block the responses of autoreactive Th1 cells from AIHA patients in vitro [45] raises the possibility that they may exert similar profound inhibitory effects in vivo [1]. References
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