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Immunoregulation of Autoimmune Diseases
Immunoregulation of Autoimmune Diseases Willem van Eden ABSTRACT Since the discovery of human leukocyte antigen (HLA) as a genetic system, the search for the biological function of HLA molecules has been a very intense and attractive area of immunological research. It has been a major factor in the development of our understanding of the role of T cells in the initiation and regulation of autoimmune diseases. The currently increasing incidences of chronic inflammatory diseases, such as allergies but also several autoimmune diseases, possibly due to our Westernized modern life-style, are asking for novel intervention or prevention strategies. Heat shock
proteins (or stress proteins) can be an example of relevant microbial antigens with an intrinsic capacity to trigger anti-inflammatory T cell regulation. Heat shock proteins may be part of an ancient system, which includes several stress-induced self-antigens, seen by a self-protective immune system. Peptide-based intervention, including clinical trials with HSP60 and HSP70 peptides, is now an area of intensive clinical research. Human Immunology 67, 446 – 453 (2006). © American Society for Histocompatibility and Immunogenetics, 2006. Published by Elsevier Inc.
ABBREVIATIONS HLA human leukocyte antigens MHC major histocompatibility complex MS multiple sclerosis IBD inflammatory bowel disease RA rheumatoid arthritis
LYP HSP AA APC APL
IR GENES AND IMMUNOREGULATION Jon van Rood accepted me as a PhD student in 1978, when I just had left medical school. At that time the existence of remarkable HLA– disease associations had been uncovered already by Jon, his co-workers, and several others. Expectedly, the underlying mechanisms were to be clarified within a couple of years. It was my “easy” task to uncover the nature of HLA– disease associations for infectious diseases and the first thing for me to carry out was an HLA-linkage analysis in families with multiple cases of leprosy, under the guidance of Rene de Vries. In retrospect, it frightens one to note that the nature of most of these disease associations are still in the dark, especially when one realizes that the basic feeling of what these associations meant then was very similar to what we think nowadays. Looking back, I realize how privileged I was to work with Jon so early in my scientific career. I remember how Jon asked me to join him in a leprosy meeting in Addis Ababa and, From the Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584CL Utrecht, The Netherlands. Division of Immunology, Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584CL Utrecht, The Netherlands; Tel: ⫹302 534 358; Fax: ⫹302 533 555; E-mail: [email protected] Human Immunology 67, 446 – 453 (2006) © American Society for Histocompatibility and Immunogenetics, 2006 Published by Elsevier Inc.
lymphoid protein heat shock proteins adjuvant arthritis antigen presenting cells altered peptide ligands
despite my ignorant status as a true beginner, how he pushed me to produce a poster (and paper) advertising his idea that polymorphic immune response genes (Ir genes) were coding for determinants which manifested interaction with microorganisms leading to activation of immune responses [1]. Nothing wrong with it, one would say now. In fact and in all modesty: exactly right. Thinking about the, at that time for me hard to follow, discussions in the lab, I remember Jon advocating the idea that HLA molecules themselves were capable of catching antigens, as if they were the “vacuum cleaners” of the immune system. Despite the contempt of his more biochemistry-trained colleagues, Jon’s early gut feelings turned out to be absolutely right—as so often—when in 1985 Babbitt et al. [2] made the first claims that MHC molecules were directly binding antigens, providing a molecular basis for understanding the Zinkemagel–Doherty phenomenon of MHC restrictions and, as we expected those days, the essence of HLA– disease associations. The leprosy studies did generate some evidence that HLA genes were contributing to disease susceptibility and that they could have impact on the quality of the antimycobacterial immune response in the patients. An 0198-8859/06/$–see front matter doi:10.1016/j.humimm.2006.03.010
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aspect of the further complexity of antimycobacterial immune responses became apparent to me when I moved in 1983 for the postdoctoral period to the laboratory of Jon’s close friend and colleague Irun Cohen of the Weizmann Institute of Science in Israel. Here I did get immersed into a model system in rats where mycobacteria were producing arthritis and Holoshitz had pioneered the adoptive transfer of mycobacteria-induced arthritis using a single T cell clone raised against whole mycobacteria [3]. The latter model was part of an array of activities in various experimental models of autoimmunity in the Cohen laboratory, which all indicated the presence of T cells with autoreactive potential in the healthy immune repertoire, and the main research issue was the nature of regulation that was active to keep such potentially dangerous T cell specificities under control. Since then, stimulated by the similar ongoing research activities in the Cohen lab, my own activities have continued to be in the area of T cell regulation of autoimmunity. SUPPRESSOR T CELLS ARE MEDIATORS OF T CELL REGULATION IN AUTOIMMUNITY For decades immunologists have been fascinated by the virtually infinite number of receptor specificities covering the vast array of antigens to be recognized by the adaptive immune system. Its molecular fundament being uncovered as the gene rearrangement phenomenon, one started to realize that the immune system was an evolutionary, unique product designed with the built-in very attractive capacity to respond to an almost endless array of unanticipated antigens and pathogens. Gene rearrangements were seen to lead to a developmentally created random repertoire of clonally distinct receptors. Implicit here is the perception that such a random repertoire comprises receptors with the capacity to recognize self. And here we are at the heart of what immunologists try to address in their studies of adaptive immunity in relation to mechanisms of autoimmunity. As it seems now, nature has provided the developing immune system with several solutions. At first Burnet’s clonal selection principle was coined as the main contributor to self-tolerance. Clonal selection was and still is thought to depend on mechanisms that are mostly cell intrinsic. The mechanisms are deletion or inactivation of the majority of self-recognizing cells in central lymphoid organs leading to what we call central tolerance. Despite ample experimental findings of the existence of selfreactive T and B cells in the healthy immune repertoires of experimental animals or humans, such as produced in the Cohen lab as mentioned above, clonal selection has
been regarded as the main contributor to self-tolerance for many years. However, stimulated by the recent rediscovery of suppressor T cells as a reality, cell “extrinsic” mechanisms now have been accepted as another major contributor to self-tolerance. And herewith regulation of immune responses in the periphery of the system, mediated by specialized regulatory T cells, is now regarded a main contributor to peripheral tolerance. The concept of specialized actively regulating cellular elements in the immune system had been accepted in the early 1980s when Gershon coined his suppressor T cells. I can remember how Jon was excited about the possibilities of exploiting such a system of regulators for the immunological control of transplant tolerance. Unfortunately, due to the limited set of experimental tools of that time, the high level of complexity of the assay systems used created difficulties and the area was seen to lack solid grounds. Therefore the area was abandoned for many years by most immunologists active in this particular field. Nowadays, however, endorsed by the presentday technical tools, it has turned out to be possible to recognize subsets of T cells with regulatory potential [4]. In essence two different populations of regulators are discriminated: natural T regs which are selected in the thymus and express CD25 and FoxP3 and adaptive T regs which originate from the periphery. The latter adaptive T regs may consist of distinct subsets based on the cytokines that they produce and are known as, for example, Tr1 (T regulator 1) and Th3 cells. A recent paper of the group of Abbas [5] has given again an interesting twist to the issue of regulatory T cells. When naïve TcR transgenic T cells were transferred into a lymphopenic mouse transgenic for the antigen of the Tg T cells (secreted OVA) the cells went through a pro-inflammatory phase resulting in a GvHlike disease. In the surviving animals it was observed that the same infused T cells, after having displayed such pro-inflammatory phenotypes, went on to display a regulatory phenotype. In other words, these findings would suggest that regulation is in fact the default state of T cells, which they adopt after having featured other characteristics such as pro-inflammatory effector cell phenotypes. EPIDEMIOLOGY AND PATHOPHYSIOLOGY OF MAJOR AUTOIMMUNE DISEASES Very similar to allergies, for some chronic autoimmune diseases an alarming increase in incidence has been noted over recent decades in Western countries. The occurrence of type I diabetes (insulin-dependent diabetes mellitus) has increased to a dramatic extend. The occurrence of type I diabetes has increased from a very low prevalence
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of 0.04 per 1000 at the turn of the 19th century. In Britain, prevalence among children under 11 years of age rose linearly from 0.1 to 0.6 to 1.3 per 1000 children, as demonstrated in three national birth cohort studies initiated in 1946, 1958, and 1970, respectively [6]. MS has also shown an impressive increment. For example, in Lower Saxony (Germany) the incidence of MS doubled over a period of 15 years [7]. Inflammatory bowel disease (IBD), including both Crohn’s disease and ulcerative colitis, likewise is on the rise [8, 9]. The incidence of childhood IBD in the British Isles increased every decade over the past 2 decades and is almost twice that in 1983 [9]. The epidemiological distribution of type I diabetes and MS has revealed a so-called north–south gradient where incidences of disease, such as of type 1 diabetes and MS, decrease from north to south in the Northern hemisphere and reciprocally from south to north in the Southern hemisphere [10]. This is the case for Europe and Africa and similarly for the America’s and Asia. This north–south gradient has been interpreted as an indicator of the infectious origin of these diseases; however, the absence of infection, or changed life-style, as argued under the hygiene hypothesis (see below) could equally well explain these geographic distributions. Among the major autoimmune diseases, rheumatoid arthritis (RA) may be an exception, in the sense that no increased incidences have been noted. In fact, decreases have been documented, although this might have been artificially caused by a further clinical subdivision of entities such as RA [11]. Be that as it may, a major increase in RA does not seem to occur. The genetics of autoimmune diseases is largely determined by the major histocompatibility complex. Certain class II alleles of the polymorphic MHC (MHC-II) are present with raised frequencies among patients with type I diabetes, MS, and RA, leading to the well-known HLA associations. As these diseases are primarily held to be T cell-mediated, the associated HLA molecules are supposedly selecting and presenting crucial autoimmune T cell epitopes for the triggering of pathogenic proinflammatory T cells. This pathogenic pathway is thought to be mechanistically connected to an inefficient negative selection of T cell repertoire by the associated HLA molecules in the thymus (leading to failing central tolerance). In addition, the associated HLA molecules may lead to inefficient peripheral tolerance, possibly due to insufficient induction of immune regulation in the peripheral secondary lymphoid organs. Although additional genetic components, such as the AIRE transcription factors, critical to central tolerance induction in the thymus, have been discovered, deficiencies in AIRE genes have not been seen to lead to major autoimmune diseases such as type I diabetes, MS, or RA [12]. On the other hand, the disturbances of
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immune regulation in the periphery, having impact on mechanisms of peripheral tolerance, have been suggested to underlie these diseases in various ways. This may be illustrated by the recently discovered genetic polymorphisms in the lymphoid protein (LYP) tyrosine phosphatase, associated with both type I diabetes and RA [13]. As the LYP is responsible for the tuning of T cell activation, genetic variation of LYP will lead to variation in lymphocyte trigger ability. Furthermore, the revival of interest in regulatory cellular subsets of T cells (the former suppressor T cells) in experimental animal models of autoimmunity has now led to first demonstrations of defective regulatory T cells in diseases such as MS and diabetes. In MS the existence of regulatory T cells, as defined by their cell surface markers, was documented. However, when the cells were tested in functional assays their capacity to regulate other disease causing T cells was found to be impaired [14]. In addition, a careful and sensitive analysis of T cells directed to a disease-relevant islet cell auto-antigen revealed that such T cells existed in healthy individuals and had a regulatory phenotype, whereas in diabetic patients these T cells were inflammatory [15]. In contrast to this, various studies on RA have shown the presence and activity of regulatory T cells, both in peripheral blood and in the synovial compartment during active disease [16, 17]. Although in one of the studies anti-TNF interventions were demonstrated to unleash suppressed activity of regulatory T cells, it seems that impaired T cell regulation is a feature more of MS and type I diabetes than of RA. If so, it seems especially that conditions that manifest defective T cell regulation during active disease are currently demonstrating the highest rises in incidence. Therefore, following the hygiene hypothesis, one may argue that a lack of microbial exposure is especially detrimental to those conditions that are characterized by a relative lack of T cell regulation. And this makes sense, as we now know that microbial exposure and infection are prime factors in the generation of active T cell regulation. In addition to the promotion of T cell regulation, antigenic exposure, such as that caused by microbial exposure, is also likely to be responsible for clonal expansions of T cells in a healthy balanced immune system. Antigenic deprivation will lead to a relative lymphopenia, which is to be compensated by homeostatic proliferation of lymphocytes. In the NOD mouse model of spontaneous type I diabetes, the existing lymphopenia and ensuing homeostatic proliferation were demonstrated to be causally related to the generation and expansion of diabetogenic T cells, leading to disease [18]. Thus, in more general terms, antigenic stimulation, such as through microbial exposure, may be a natural
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barrier against potentially disease-triggering homeostatic proliferation. INFECTION AND THE RISING INCIDENCES OF AUTOIMMUNE DISEASES A major contribution of Jon van Rood to immunology was his early discovery of HLA polymorphisms. Since then Jon and many of his colleagues have championed the idea that during evolution infectious diseases have been the driving forces behind the origin of MHC polymorphism. Infectious diseases were held to select for genetic polymorphism of Ir genes to secure higher survival chances for the population as a whole (and not the individual) during epidemics. Now with the advent of antimicrobial therapies and vaccines we have taken away the selective pressure by infection to a great extent, at least in the developed countries. Clearly enough, it will take many generations before we will be able to notice the inevitable changes in genetic makeup of individuals due to this reduced selection on the basis of infections. Nonetheless, over recent decades we have seen an alarming increase in the occurrence of allergic conditions such as atopy and asthma in the western world. Very similar to the increase in type I diabetes, some other autoimmune diseases such as MS and inflammatory diseases such as IBD, especially Crohn’s disease, have shown a disturbing increase, as already discussed above. Globally, it seems that immune-mediated disorders are more prevalent nowadays than in the past and this is especially true in the Western developed world. Based on epidemiological studies there is reason to suspect that a reduced exposure to infectious organisms has taken away some of the mechanistic factors that are utilized by a properly functioning immune system to generate internal control. It is known that children are born with an immune system that reacts preferably with a T cell response which is Th2 biased. During further development and exposure to commensal flora and probably other infectious events (childhood diseases, vaccination, etc.) Th1 responses are seen to develop and to take over. Along similar lines the development of T regs is supposed to depend on such exposures. The idea that lack of exposure to infectious organisms may result in a suboptimal internal balance in the immune system, leading to the increased prevalence of immune disorders, has received wide attention by scientists and the lay public and the idea is now known as the hygiene hypothesis. The hygiene hypothesis idea has been investigated now to a great extend in various epidemiological studies. Despite many remaining uncertainties and doubts, there seems to be hardly any evidence in support of childhood infectious diseases or household hygiene as significant factors in this. What
remains and what seems to have received sufficient epidemiological support is the possibility that contact with so-called “old friends” is critical [19]. In the developed countries we may have lost our ancient parasitic relationships that existed between humans and their gutresiding helminths, their environmental saprophytic mycobacteria, their endogenously residing mycobacteria, and so on. Recent successes of experimentally confronting patients with immune disorders, such as allergies and Crohn’s disease, with worms or mycobacteria can be seen to support this idea [20, 21]. Despite the fact that there may be some attraction in the concept of re-infesting individuals with worms, mites, and some bacteria— think for instance of the current commercial successes of probiotics— one can foresee that clean and safer alternatives will be needed. Thus, infectious diseases seem to have had their evolutionary contribution to the origin of genes coding for MHC polymorphisms and there is evidence that in addition the presence of microbial agents has a continuous balancing impact on immune behavior. That the chronic disturbance of such a balance may predispose to immunemediated disorders is then a logical consequence. Along similar lines of reasoning it is not unexpected that under such unbalanced circumstances infection may be the actual trigger that starts the development of an unwanted autoimmune response, as for example is seen in the form of arthritis when autoimmune disease-prone Lewis rats are immunized with mycobacteria, the equivalent in humans most likely being the arthritis that is seen following cancer treatment using high doses of living BCG [22]. HSP AND T CELL REGULATION OF AUTOIMMUNITY Mycobacteria-induced adjuvant arthritis (AA) in Lewis rats is a T-cell-mediated autoimmune disease which can be transferred into naïve rats using mycobacteria-specific CD4⫹ T cells. When the exact antigen specificity of the arthritogenic T cells was analyzed, the mycobacterial antigen involved was found to be the GroEL homologue of mycobacteria, which is mycobacterial heat shock protein 60 (mtHSP60). Despite the fact that mtHSP60 was the mycobacterial antigen that stimulated the arthritisproducing T cells, when the mtHSP60 molecule was isolated from the context of the entire mycobacterium and used to immunize rats, no disease was seen to develop. Interestingly, however, induction of AA with prior mtHSP60 immunization was found to be impossible [23]. Apparently, mtHSP60 immunization led to the production of resistance to subsequent disease induction. This was also found in a similar model in Lewis rats,
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which does not involve any microbial agent, produced by administration of avridine or CP20961 [24]. Subsequent experiments carried out by various groups using various experimental autoimmune models have now substantiated the disease-inhibitory effect of mtHSP60 immunization [25–29]. A comprehensive analysis of HSP60 T cell epitopes in adjuvant arthritis indicated the induction of self-HSP (host HSP) cross-recognition as an underlying mechanistic principle of the arthritis-suppressive potential of mtHSP60. By testing an overlapping set of 15-mer peptides spanning the complete mtHSP60 sequence, nine distinct dominant T cell epitopes were detected. Subsequent adoptive transfer studies, using T cell lines generated to all epitopes, revealed that only T cells directed to a very conserved 256 –265 sequence transferred protection [28]. The same T cells were shown to recognize the tissue or mammalian HSP60 homologous peptide and heat-shocked autologous cells. Furthermore, active immunization with the conserved peptide protected against the induction of both mycobacteriainduced adjuvant arthritis and avridine arthritis. All other (nonconserved) peptides failed to produce such protection. Recently the mtHSP60 mode of action in suppressing arthritis has been reproduced very similarly for mtHSP70 by others [30] and ourselves [29]. Also, in these latter cases T cells recognizing the very conserved mtHSP70 peptides were found to produce protection. Disease-suppressive activities of HSP have been observed also in other experimental models of autoimmunity, such as in diabetes of NOD mice and in EAE. Interestingly, also in more distantly related models of inflammation, such as atherosclerosis and allergies, disease-protective effect of exposures to HSP have been noted (reviewed in [31]). Given the multitude of models involving different triggering substances or antigens, where single molecules such as mtHSP60 and 70 were found to be protective, the induction of regulatory self-HSP cross-reactive T cell responses may well explain the observations made so far. T CELL RESPONSES TO STRESSED APC The cell stress response or heat shock response is an evolutionarily ancient cellular response to stresses that cells may experience. The various kingdoms of life share the cell stress proteins and most of them have appeared essential for life. Critical to the stress response is the relationship with protein folding. Under stress, proteins are less well folded or are misfolded, and some of them are denatured. The evolutionary solution to this has been the creation of cellular folding catalysts or chaperones. Some of these catalysts or chaperones are constitutively
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expressed, but most of them are induced under stress. The latter proteins are heat shock proteins or stress proteins. Most HSP are intracellular proteins. However, evidence that molecular chaperones are also secreted has accumulated. In this manner it seems that these proteins also have cell– cell signalling qualities, for instance to myeloid or vascular endothelial cells. This would indicate a true signaling function for HSP, which herewith may have an early warning function in signalling stress. The fact that some HSP may have pro-inflammatory activities and some may have anti-inflammatory activities suggests that these proteins can act as a factor in the control of inflammation and immunity. It is assumed that the regulatory potential of self-HSP reactive T cells is exerted at the site of inflammation where, under the influence of the stressed environment caused by toxic mediators of inflammation, cells do express enhanced levels of HSP. The potential of these cells to recognize endogenously produced self-HSP peptides, captured in the MHC molecules of stressed antigen presenting cells, was demonstrated in a variety of experiments. We have demonstrated that our microbial HSP reactive T cells cross-recognize stressed APC under certain conditions. Self HSP60 cross-reactive T cells in the rat were produced by repeated restimulations with the conserved core epitope (256 –265) of MtHSP60. Subsequent testing of these T cell lines for their proliferative responses in the presence of either normal or heat-shocked (30 minutes culture at 43°C and a recovery period of 4 hours at 37°C) spleen cells revealed moderate responses in the presence of normal APC and high proliferative responses in the presence of the heat-shocked APC [32, 33]. Thus, T cells with specificity for self-HSP60 were responsive to endogenously produced self-HSP, as presented by cells in the absence of added antigens. These responses in the absence of added antigens were demonstrated to be MHC restricted, as these responses were fully inhibited by adding an antibody specific for RT1-B (OX6) to the culture. An antibody specific for RT1-D (OX17) had no inhibitory effect for the T cell lines tested sofar. In some cases, the self-HSP60 reactive T cell line became auto-proliferative and expanded without the need of restimulations with antigens. These lines were also seen to produce IL4, IL10, and INF␥. Upon transfer they were demonstrated to have arthritis suppressive potential. In coculture experiments the suppressive potential of self-HSP specific T cells was analyzed. T cell lines were generated by immunizing with the rat homologous sequence of MtHSP60 256 –265 (R256 –265). For control purposes, the same protocol was followed for the generation of T cell lines with specificity for OVA 323– 339. Following isolation of the draining lymph node T
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cells, the cells were restimulated in vitro with normal or heat-shocked (protocol as above) spleen cells. Following an expansion phase on IL2, these T cells were cocultured with responder cells A2b in the presence of its antigen MtHSP60 180 –188. It turned out that HSP60 specific T cells which had been restimulated with stressed spleen cells had a very significant suppressive effect on the level of A2b proliferation. In other words, self-HSP reactive T cells not only are responsive to stressed APC but also they seem to develop a regulatory phenotype upon recognition of stressed APC.
HSP REGULATION AND MICROBIAL EXPOSURE Heat shock proteins are immunodominant proteins. HSP60 was known as the common antigen of gram negative bacteria, already before it’s molecular definition. In other words, immune exposure to microbes leads to a vigorous B and T cell response directed against HSP, such as HSP60 and HSP70. In addition, in patients with inflammatory diseases, responses (B and T cells) against self-HSP60 and 70 can be easily detected. Apparently, the immune system has an interest in recognizing these molecules and exploits in such a manner the ubiquitous presence of these evolutionarily ancient molecules. Based on the above-mentioned stress signalling function of these molecules and given their observed anti-inflammatory potential, it seems that immune responses to HSP are part of the intrinsic immune mechanisms to regulate peripheral tolerance. And, in line herewith, HSP have already shown their capacity to induce regulatory T cells [31]. The fact that bacterial HSP have been effective in inducing protection against experimental autoimmunity further suggests that HSP-mediated immune regulation may also be one of the underlying mechanisms through which environmental flora can contribute to disease resistance. There are many experimental findings that have indicated that bacterial recontamination of germ-free animals induced resistance against immune disorders. In a more wider perspective, one may conclude that HSP may constitute a molecular factor in the mechanisms underlying the hygiene hypothesis. As it is now, the interest in the concept of regulatory T cells as an important factor in the control of autoimmunity has become very broad and intense. The detection of relevant antigens in this area is now of high priority. Especially here, the proof of the pudding will be in the eating. Clinical trials exploiting these novel concepts are therefore now eagerly awaited.
MHC-TARGETED PEPTIDE INTERVENTIONS IN AUTOIMMUNITY So far, peptide therapies in autoimmunity have focused on so-called altered peptide ligands (APL) and on T cell receptor-derived peptides as a result of interventions previously known as T cell vaccination. Altered peptide ligands were originally created by introducing minor amino acid substitutions in autoimmune T cell epitopes and were defined by their potential to induce a tolerogenic response in the autoreactive T cells. Since then, various studies have indicated the potential of APL to block the development of autoimmune diseases. In the experimental model of adjuvant arthritis Marca Wauben et al. developed an APL on the basis of the arthritis-producing T cell epitope. Interestingly, it turned out that the APL was effectively inducing regulation by triggering a disease-suppressive APL-specific T cell which produced suppression of disease following transfer into naïve recipients [34]. Possibly very similar to the principle of microbial HSP, it is now proposed to use microbial epitopes as natural APL for the control of autoimmune diseases such as multiple sclerosis [35]. The principle of immunization with inactivated autoreactive T cells (T cell vaccination) selected from an individual’s own T cell repertoire also has led to the development of TcR peptide therapies for autoimmunity [36]. In the model of rat adjuvant arthritis, Chris Broeren has shown that activated T cells do internalize their T cell receptors and that immunogenic fragments can become loaded into upregulated MHC-II molecules [37]. This cell surface presentation of TcR peptide was seen to be recognized by TcR-specific T cells with disease regulatory potential. The current status of these TcR peptide approaches has been reviewed recently [36]. First clinical trials using HSP peptides have been initiated. Peptide 277 of HSP60 has been tested by parenteral administration in type I diabetes and the results have indicated that peptide-specific T cells changed their production of pro-inflammatory cytokines into the production of anti-inflammatory cytokines, such as IL4 and IL10 [38]. Interestingly, in the treated patients a remarkable beneficial effect on the insulinproducing capacity of the pancreas and insulin dependency was noted. Similarly, oral administration of peptide dnaJP1, derived from a bacterial HSP40 (cochaperone for HSP70), in RA patients induced a tolerogenic T cell response in T cells which manifested a pro-inflammatory T cell phenotype prior to oral treatment [39]. Follow-up phase 2 trials are currently under analysis. In a recent study, a computer algorithm was used to identify multiple HLA-DR binding peptides of HSP60. Interestingly, one of the epitopes was very similar to the peptide that was
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protective against experimental arthritis in Lewis rats [40]. Evidently, this peptide can be a candidate for HSP60-based immunotherapy in patients with arthritis. In conclusion, peptide interventions are on their way to become a novel addition to the therapeutic armamentarium against autoimmune diseases. Clearly, this attractive development would not have been possible without detailed knowledge of HLA polymorphisms, as generated by giants such as Jon van Rood.
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Lee AT, Ortmann W, Behrens TW, Gregersen PK: Analysis of families in the multiple autoimmune disease genetics consortium (MADGC) collection: the PTPN22 620W allele associates with multiple autoimmune phenotypes. Am J Hum Genet 76(4):561, 2005. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA: Loss of functional suppression by CD4⫹CD25⫹ regulatory T cells in patients with multiple sclerosis. J Exp Med 199(7):971, 2004. Arif S, Tree TI, Astill TP, Tremble JM, Bishop AJ, Dayan CM, Roep BO, Peakman M: Autoreactive T cell responses show proinflammatory polarization in diabetes but a regulatory phenotype in health. J Clin Invest 113(3):451, 2004. Cao D, Malmstrom V, Baecher-Allan C, Hafler D, Klareskog L, Trollmo C: Isolation and functional characterization of regulatory CD25brightCD4⫹ T cells from the target organ of patients with rheumatoid arthritis. Eur J Immunol 33(1):215, 2003. van Amelsfort JM, Jacobs KM, Bijlsma JW, Lafeber FP, Taams LS: CD4(⫹)CD25(⫹) regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, and function between peripheral blood and synovial fluid. Arthritis Rheum 50(9):2775, 2004. King C, Ilic A, Koelsch K, Sarvetrick N: Homeostatic expansion of T cells during immune insufficiency generates autoimmunity. Cell 117(2):265, 2004. Rook GA, Stanford JL: Give us this day our daily germs. Immunol Today 19(3):113, 1998. Weinstock JV, Summers RW, Elliott DE: Role of helminths in regulating mucosal inflammation. Springer Semin Immunopathol 27(2):249, 2005. Rook GA, Adams V, Hunt J, Palmer R, Martinelli R, Brunet LR: Mycobacteria and other environmental organisms as immunomodulators for immunoregulatory disorders. Springer Semin Immunopathol 25(3– 4):237, 2004. Ochsenkuhn T, Weber MM, Caselmann WH: Arthritis after Mycobacterium bovis immunotherapy for bladder cancer [letter, comment]. Ann Intern Med 112(11):882, 1990. van Eden W, Thole JE, van der Zee R, Noordzij A, van Embden JD, Hensen EJ, Cohen IR: Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature 331(6152):171, 1988. Kingston AE, Hicks CA, Colston MJ, Billingham ME: A 71-kD heat shock protein (HSP) from Mycobacterium tuberculosis has modulatory effects on experimental rat arthritis. Clin Exp Immunol 103(1):77, 1996. van Eden W, Hogervorst EJ, Wauben MH, van der Zee R, Boog CJ: Heat-shock proteins as antigens in autoimmunity. Biochem Soc Trans 19(1):171, 1991. van den Broek MF, Hogervorst EJ, Van Bruggen MC, Van Eden W, van der Zee R, van den Berg WB: Protection against streptococcal cell wall-induced arthritis by pretreatment with the 65-kD mycobacterial heat shock protein. J Exp Med 170(2):449, 1989.
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27. Thompson SJ, Francis JN, Siew LK, Webb GR, Jenner PJ, Colston MJ, Elson CJ: An immunodominant epitope from mycobacterial 65-kDa heat shock protein protects against pristane-induced arthritis. J Immunol 160 (9):4628, 1998. 28. Anderton SM, van der Zee R, Prakken B, Noordzij A, van Eden W: Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis. J Exp Med 181(3):943, 1995. 29. Wendling U, Paul L, van Der Zee R, Prakken B, Singh M, van Eden W: A conserved mycobacterial heat shock protein (HSP) 70 sequence prevents adjuvant arthritis upon nasal administration and induces IL-10-producing T cells that cross-react with the mammalian self-HSP70 homologue. [In Process Citation]. J Immunol 164(5):p2711, 2000. 30. Tanaka S: Activation of T Cells Recognizing an Epitope of Heat-Shock Protein 70 Can Protect Against Rat Adjuvant Arthritis. [Record Supplied By Publisher]. J Immunol 163(10):p5560, 1999. 31. van Eden W, Hauet-Broere F, Berlo S, Paul L, van der Zee R, de Kleer I, Prakken B, Taams L: Stress proteins as inducers and targets of regulatory T cells in arthritis. Int Rev Immunol 24(3– 4):181, 2005. 32. Paul AG, van Der Zee R, Taams LS, van Eden W: A self-HSP60 peptide acts as a partial agonist inducing expression of B7-2 on mycobacterial HSP60-specific T cells: a possible mechanism for inhibitory T cell regulation of adjuvant arthritis? Int Immunol 12(7):1041, 2000. 33. van Eden W, Wendling U, Paul L, Prakken B, van Kooten P, van der Zee R: Arthritis protective regulatory potential of self-heat shock protein cross-reactive T cells. Cell Stress Chaperones 5(5):452, 2000.
34. Wauben MH, Boog CJ, van der Zee R, Joosten I, Schlief A, van Eden W: Disease inhibition by major histocompatibility complex binding peptide analogues of diseaseassociated epitopes: more than blocking alone. J Exp Med 176(3):667, 1992. 35. Steinman L, Utz PJ, Robinson WH: Suppression of autoimmunity via microbial mimics of altered peptide ligands. Curr Top Microbiol Immunol 296:55, 2005. 36. Zhang JW: T cell vaccination as an immunotherapy for autoimmune diseases. Cell Mol Immunol 1(5):321, 2004. 37. Broeren CP, Lucassen MA, van Stipdonk MJ, van der Zee R, Boog CJ, Kusters JG, van Eden W: CDR1 T-cell receptor beta-chain peptide induces major histocompatibility complex class II-restricted T-T cell interactions. Proc Natl Acad Sci USA 91(13):5997, 1994. 38. Raz I, Elias D, Avron A, Tamir M, Metzger M, Cohen IR: Beta-cell function in new-onset type 1 diabetes and immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double-blind, phase II trial. Lancet 358(9295):1749, 2001. 39. Prakken BJ, Samodal R, Le TD, Giannoni F, Yung GP, Scavulli J, Amox D, Roord S, de Kleer I, Bonnin D, Lanza P, Berry C, Massa M, Billetta R, Albani S: Epitopespecific immunotherapy induces immune deviation of proinflammatory T cells in rheumatoid arthritis. Proc Natl Acad Sci USA 101(12):4228, 2004. 40. Kamphuis S, Kuis W, de Jager W, Teklenburg G, Massa M, Gordon G, Boerhof M, Rijkers GT, Uiterwaal CS, Otten HG, Sette A, Albani S, Prakken BJ: Tolerogenic immune responses to novel T-cell epitopes from heatshock protein 60 in juvenile idiopathic arthritis. Lancet 366(9479):50, 2005.
proteins (or stress proteins) can be an example of relevant microbial antigens with an intrinsic capacity to trigger anti-inflammatory T cell regulation. Heat shock proteins may be part of an ancient system, which includes several stress-induced self-antigens, seen by a self-protective immune system. Peptide-based intervention, including clinical trials with HSP60 and HSP70 peptides, is now an area of intensive clinical research. Human Immunology 67, 446 – 453 (2006). © American Society for Histocompatibility and Immunogenetics, 2006. Published by Elsevier Inc.
ABBREVIATIONS HLA human leukocyte antigens MHC major histocompatibility complex MS multiple sclerosis IBD inflammatory bowel disease RA rheumatoid arthritis
LYP HSP AA APC APL
IR GENES AND IMMUNOREGULATION Jon van Rood accepted me as a PhD student in 1978, when I just had left medical school. At that time the existence of remarkable HLA– disease associations had been uncovered already by Jon, his co-workers, and several others. Expectedly, the underlying mechanisms were to be clarified within a couple of years. It was my “easy” task to uncover the nature of HLA– disease associations for infectious diseases and the first thing for me to carry out was an HLA-linkage analysis in families with multiple cases of leprosy, under the guidance of Rene de Vries. In retrospect, it frightens one to note that the nature of most of these disease associations are still in the dark, especially when one realizes that the basic feeling of what these associations meant then was very similar to what we think nowadays. Looking back, I realize how privileged I was to work with Jon so early in my scientific career. I remember how Jon asked me to join him in a leprosy meeting in Addis Ababa and, From the Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584CL Utrecht, The Netherlands. Division of Immunology, Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584CL Utrecht, The Netherlands; Tel: ⫹302 534 358; Fax: ⫹302 533 555; E-mail: [email protected] Human Immunology 67, 446 – 453 (2006) © American Society for Histocompatibility and Immunogenetics, 2006 Published by Elsevier Inc.
lymphoid protein heat shock proteins adjuvant arthritis antigen presenting cells altered peptide ligands
despite my ignorant status as a true beginner, how he pushed me to produce a poster (and paper) advertising his idea that polymorphic immune response genes (Ir genes) were coding for determinants which manifested interaction with microorganisms leading to activation of immune responses [1]. Nothing wrong with it, one would say now. In fact and in all modesty: exactly right. Thinking about the, at that time for me hard to follow, discussions in the lab, I remember Jon advocating the idea that HLA molecules themselves were capable of catching antigens, as if they were the “vacuum cleaners” of the immune system. Despite the contempt of his more biochemistry-trained colleagues, Jon’s early gut feelings turned out to be absolutely right—as so often—when in 1985 Babbitt et al. [2] made the first claims that MHC molecules were directly binding antigens, providing a molecular basis for understanding the Zinkemagel–Doherty phenomenon of MHC restrictions and, as we expected those days, the essence of HLA– disease associations. The leprosy studies did generate some evidence that HLA genes were contributing to disease susceptibility and that they could have impact on the quality of the antimycobacterial immune response in the patients. An 0198-8859/06/$–see front matter doi:10.1016/j.humimm.2006.03.010
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aspect of the further complexity of antimycobacterial immune responses became apparent to me when I moved in 1983 for the postdoctoral period to the laboratory of Jon’s close friend and colleague Irun Cohen of the Weizmann Institute of Science in Israel. Here I did get immersed into a model system in rats where mycobacteria were producing arthritis and Holoshitz had pioneered the adoptive transfer of mycobacteria-induced arthritis using a single T cell clone raised against whole mycobacteria [3]. The latter model was part of an array of activities in various experimental models of autoimmunity in the Cohen laboratory, which all indicated the presence of T cells with autoreactive potential in the healthy immune repertoire, and the main research issue was the nature of regulation that was active to keep such potentially dangerous T cell specificities under control. Since then, stimulated by the similar ongoing research activities in the Cohen lab, my own activities have continued to be in the area of T cell regulation of autoimmunity. SUPPRESSOR T CELLS ARE MEDIATORS OF T CELL REGULATION IN AUTOIMMUNITY For decades immunologists have been fascinated by the virtually infinite number of receptor specificities covering the vast array of antigens to be recognized by the adaptive immune system. Its molecular fundament being uncovered as the gene rearrangement phenomenon, one started to realize that the immune system was an evolutionary, unique product designed with the built-in very attractive capacity to respond to an almost endless array of unanticipated antigens and pathogens. Gene rearrangements were seen to lead to a developmentally created random repertoire of clonally distinct receptors. Implicit here is the perception that such a random repertoire comprises receptors with the capacity to recognize self. And here we are at the heart of what immunologists try to address in their studies of adaptive immunity in relation to mechanisms of autoimmunity. As it seems now, nature has provided the developing immune system with several solutions. At first Burnet’s clonal selection principle was coined as the main contributor to self-tolerance. Clonal selection was and still is thought to depend on mechanisms that are mostly cell intrinsic. The mechanisms are deletion or inactivation of the majority of self-recognizing cells in central lymphoid organs leading to what we call central tolerance. Despite ample experimental findings of the existence of selfreactive T and B cells in the healthy immune repertoires of experimental animals or humans, such as produced in the Cohen lab as mentioned above, clonal selection has
been regarded as the main contributor to self-tolerance for many years. However, stimulated by the recent rediscovery of suppressor T cells as a reality, cell “extrinsic” mechanisms now have been accepted as another major contributor to self-tolerance. And herewith regulation of immune responses in the periphery of the system, mediated by specialized regulatory T cells, is now regarded a main contributor to peripheral tolerance. The concept of specialized actively regulating cellular elements in the immune system had been accepted in the early 1980s when Gershon coined his suppressor T cells. I can remember how Jon was excited about the possibilities of exploiting such a system of regulators for the immunological control of transplant tolerance. Unfortunately, due to the limited set of experimental tools of that time, the high level of complexity of the assay systems used created difficulties and the area was seen to lack solid grounds. Therefore the area was abandoned for many years by most immunologists active in this particular field. Nowadays, however, endorsed by the presentday technical tools, it has turned out to be possible to recognize subsets of T cells with regulatory potential [4]. In essence two different populations of regulators are discriminated: natural T regs which are selected in the thymus and express CD25 and FoxP3 and adaptive T regs which originate from the periphery. The latter adaptive T regs may consist of distinct subsets based on the cytokines that they produce and are known as, for example, Tr1 (T regulator 1) and Th3 cells. A recent paper of the group of Abbas [5] has given again an interesting twist to the issue of regulatory T cells. When naïve TcR transgenic T cells were transferred into a lymphopenic mouse transgenic for the antigen of the Tg T cells (secreted OVA) the cells went through a pro-inflammatory phase resulting in a GvHlike disease. In the surviving animals it was observed that the same infused T cells, after having displayed such pro-inflammatory phenotypes, went on to display a regulatory phenotype. In other words, these findings would suggest that regulation is in fact the default state of T cells, which they adopt after having featured other characteristics such as pro-inflammatory effector cell phenotypes. EPIDEMIOLOGY AND PATHOPHYSIOLOGY OF MAJOR AUTOIMMUNE DISEASES Very similar to allergies, for some chronic autoimmune diseases an alarming increase in incidence has been noted over recent decades in Western countries. The occurrence of type I diabetes (insulin-dependent diabetes mellitus) has increased to a dramatic extend. The occurrence of type I diabetes has increased from a very low prevalence
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of 0.04 per 1000 at the turn of the 19th century. In Britain, prevalence among children under 11 years of age rose linearly from 0.1 to 0.6 to 1.3 per 1000 children, as demonstrated in three national birth cohort studies initiated in 1946, 1958, and 1970, respectively [6]. MS has also shown an impressive increment. For example, in Lower Saxony (Germany) the incidence of MS doubled over a period of 15 years [7]. Inflammatory bowel disease (IBD), including both Crohn’s disease and ulcerative colitis, likewise is on the rise [8, 9]. The incidence of childhood IBD in the British Isles increased every decade over the past 2 decades and is almost twice that in 1983 [9]. The epidemiological distribution of type I diabetes and MS has revealed a so-called north–south gradient where incidences of disease, such as of type 1 diabetes and MS, decrease from north to south in the Northern hemisphere and reciprocally from south to north in the Southern hemisphere [10]. This is the case for Europe and Africa and similarly for the America’s and Asia. This north–south gradient has been interpreted as an indicator of the infectious origin of these diseases; however, the absence of infection, or changed life-style, as argued under the hygiene hypothesis (see below) could equally well explain these geographic distributions. Among the major autoimmune diseases, rheumatoid arthritis (RA) may be an exception, in the sense that no increased incidences have been noted. In fact, decreases have been documented, although this might have been artificially caused by a further clinical subdivision of entities such as RA [11]. Be that as it may, a major increase in RA does not seem to occur. The genetics of autoimmune diseases is largely determined by the major histocompatibility complex. Certain class II alleles of the polymorphic MHC (MHC-II) are present with raised frequencies among patients with type I diabetes, MS, and RA, leading to the well-known HLA associations. As these diseases are primarily held to be T cell-mediated, the associated HLA molecules are supposedly selecting and presenting crucial autoimmune T cell epitopes for the triggering of pathogenic proinflammatory T cells. This pathogenic pathway is thought to be mechanistically connected to an inefficient negative selection of T cell repertoire by the associated HLA molecules in the thymus (leading to failing central tolerance). In addition, the associated HLA molecules may lead to inefficient peripheral tolerance, possibly due to insufficient induction of immune regulation in the peripheral secondary lymphoid organs. Although additional genetic components, such as the AIRE transcription factors, critical to central tolerance induction in the thymus, have been discovered, deficiencies in AIRE genes have not been seen to lead to major autoimmune diseases such as type I diabetes, MS, or RA [12]. On the other hand, the disturbances of
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immune regulation in the periphery, having impact on mechanisms of peripheral tolerance, have been suggested to underlie these diseases in various ways. This may be illustrated by the recently discovered genetic polymorphisms in the lymphoid protein (LYP) tyrosine phosphatase, associated with both type I diabetes and RA [13]. As the LYP is responsible for the tuning of T cell activation, genetic variation of LYP will lead to variation in lymphocyte trigger ability. Furthermore, the revival of interest in regulatory cellular subsets of T cells (the former suppressor T cells) in experimental animal models of autoimmunity has now led to first demonstrations of defective regulatory T cells in diseases such as MS and diabetes. In MS the existence of regulatory T cells, as defined by their cell surface markers, was documented. However, when the cells were tested in functional assays their capacity to regulate other disease causing T cells was found to be impaired [14]. In addition, a careful and sensitive analysis of T cells directed to a disease-relevant islet cell auto-antigen revealed that such T cells existed in healthy individuals and had a regulatory phenotype, whereas in diabetic patients these T cells were inflammatory [15]. In contrast to this, various studies on RA have shown the presence and activity of regulatory T cells, both in peripheral blood and in the synovial compartment during active disease [16, 17]. Although in one of the studies anti-TNF interventions were demonstrated to unleash suppressed activity of regulatory T cells, it seems that impaired T cell regulation is a feature more of MS and type I diabetes than of RA. If so, it seems especially that conditions that manifest defective T cell regulation during active disease are currently demonstrating the highest rises in incidence. Therefore, following the hygiene hypothesis, one may argue that a lack of microbial exposure is especially detrimental to those conditions that are characterized by a relative lack of T cell regulation. And this makes sense, as we now know that microbial exposure and infection are prime factors in the generation of active T cell regulation. In addition to the promotion of T cell regulation, antigenic exposure, such as that caused by microbial exposure, is also likely to be responsible for clonal expansions of T cells in a healthy balanced immune system. Antigenic deprivation will lead to a relative lymphopenia, which is to be compensated by homeostatic proliferation of lymphocytes. In the NOD mouse model of spontaneous type I diabetes, the existing lymphopenia and ensuing homeostatic proliferation were demonstrated to be causally related to the generation and expansion of diabetogenic T cells, leading to disease [18]. Thus, in more general terms, antigenic stimulation, such as through microbial exposure, may be a natural
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barrier against potentially disease-triggering homeostatic proliferation. INFECTION AND THE RISING INCIDENCES OF AUTOIMMUNE DISEASES A major contribution of Jon van Rood to immunology was his early discovery of HLA polymorphisms. Since then Jon and many of his colleagues have championed the idea that during evolution infectious diseases have been the driving forces behind the origin of MHC polymorphism. Infectious diseases were held to select for genetic polymorphism of Ir genes to secure higher survival chances for the population as a whole (and not the individual) during epidemics. Now with the advent of antimicrobial therapies and vaccines we have taken away the selective pressure by infection to a great extent, at least in the developed countries. Clearly enough, it will take many generations before we will be able to notice the inevitable changes in genetic makeup of individuals due to this reduced selection on the basis of infections. Nonetheless, over recent decades we have seen an alarming increase in the occurrence of allergic conditions such as atopy and asthma in the western world. Very similar to the increase in type I diabetes, some other autoimmune diseases such as MS and inflammatory diseases such as IBD, especially Crohn’s disease, have shown a disturbing increase, as already discussed above. Globally, it seems that immune-mediated disorders are more prevalent nowadays than in the past and this is especially true in the Western developed world. Based on epidemiological studies there is reason to suspect that a reduced exposure to infectious organisms has taken away some of the mechanistic factors that are utilized by a properly functioning immune system to generate internal control. It is known that children are born with an immune system that reacts preferably with a T cell response which is Th2 biased. During further development and exposure to commensal flora and probably other infectious events (childhood diseases, vaccination, etc.) Th1 responses are seen to develop and to take over. Along similar lines the development of T regs is supposed to depend on such exposures. The idea that lack of exposure to infectious organisms may result in a suboptimal internal balance in the immune system, leading to the increased prevalence of immune disorders, has received wide attention by scientists and the lay public and the idea is now known as the hygiene hypothesis. The hygiene hypothesis idea has been investigated now to a great extend in various epidemiological studies. Despite many remaining uncertainties and doubts, there seems to be hardly any evidence in support of childhood infectious diseases or household hygiene as significant factors in this. What
remains and what seems to have received sufficient epidemiological support is the possibility that contact with so-called “old friends” is critical [19]. In the developed countries we may have lost our ancient parasitic relationships that existed between humans and their gutresiding helminths, their environmental saprophytic mycobacteria, their endogenously residing mycobacteria, and so on. Recent successes of experimentally confronting patients with immune disorders, such as allergies and Crohn’s disease, with worms or mycobacteria can be seen to support this idea [20, 21]. Despite the fact that there may be some attraction in the concept of re-infesting individuals with worms, mites, and some bacteria— think for instance of the current commercial successes of probiotics— one can foresee that clean and safer alternatives will be needed. Thus, infectious diseases seem to have had their evolutionary contribution to the origin of genes coding for MHC polymorphisms and there is evidence that in addition the presence of microbial agents has a continuous balancing impact on immune behavior. That the chronic disturbance of such a balance may predispose to immunemediated disorders is then a logical consequence. Along similar lines of reasoning it is not unexpected that under such unbalanced circumstances infection may be the actual trigger that starts the development of an unwanted autoimmune response, as for example is seen in the form of arthritis when autoimmune disease-prone Lewis rats are immunized with mycobacteria, the equivalent in humans most likely being the arthritis that is seen following cancer treatment using high doses of living BCG [22]. HSP AND T CELL REGULATION OF AUTOIMMUNITY Mycobacteria-induced adjuvant arthritis (AA) in Lewis rats is a T-cell-mediated autoimmune disease which can be transferred into naïve rats using mycobacteria-specific CD4⫹ T cells. When the exact antigen specificity of the arthritogenic T cells was analyzed, the mycobacterial antigen involved was found to be the GroEL homologue of mycobacteria, which is mycobacterial heat shock protein 60 (mtHSP60). Despite the fact that mtHSP60 was the mycobacterial antigen that stimulated the arthritisproducing T cells, when the mtHSP60 molecule was isolated from the context of the entire mycobacterium and used to immunize rats, no disease was seen to develop. Interestingly, however, induction of AA with prior mtHSP60 immunization was found to be impossible [23]. Apparently, mtHSP60 immunization led to the production of resistance to subsequent disease induction. This was also found in a similar model in Lewis rats,
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which does not involve any microbial agent, produced by administration of avridine or CP20961 [24]. Subsequent experiments carried out by various groups using various experimental autoimmune models have now substantiated the disease-inhibitory effect of mtHSP60 immunization [25–29]. A comprehensive analysis of HSP60 T cell epitopes in adjuvant arthritis indicated the induction of self-HSP (host HSP) cross-recognition as an underlying mechanistic principle of the arthritis-suppressive potential of mtHSP60. By testing an overlapping set of 15-mer peptides spanning the complete mtHSP60 sequence, nine distinct dominant T cell epitopes were detected. Subsequent adoptive transfer studies, using T cell lines generated to all epitopes, revealed that only T cells directed to a very conserved 256 –265 sequence transferred protection [28]. The same T cells were shown to recognize the tissue or mammalian HSP60 homologous peptide and heat-shocked autologous cells. Furthermore, active immunization with the conserved peptide protected against the induction of both mycobacteriainduced adjuvant arthritis and avridine arthritis. All other (nonconserved) peptides failed to produce such protection. Recently the mtHSP60 mode of action in suppressing arthritis has been reproduced very similarly for mtHSP70 by others [30] and ourselves [29]. Also, in these latter cases T cells recognizing the very conserved mtHSP70 peptides were found to produce protection. Disease-suppressive activities of HSP have been observed also in other experimental models of autoimmunity, such as in diabetes of NOD mice and in EAE. Interestingly, also in more distantly related models of inflammation, such as atherosclerosis and allergies, disease-protective effect of exposures to HSP have been noted (reviewed in [31]). Given the multitude of models involving different triggering substances or antigens, where single molecules such as mtHSP60 and 70 were found to be protective, the induction of regulatory self-HSP cross-reactive T cell responses may well explain the observations made so far. T CELL RESPONSES TO STRESSED APC The cell stress response or heat shock response is an evolutionarily ancient cellular response to stresses that cells may experience. The various kingdoms of life share the cell stress proteins and most of them have appeared essential for life. Critical to the stress response is the relationship with protein folding. Under stress, proteins are less well folded or are misfolded, and some of them are denatured. The evolutionary solution to this has been the creation of cellular folding catalysts or chaperones. Some of these catalysts or chaperones are constitutively
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expressed, but most of them are induced under stress. The latter proteins are heat shock proteins or stress proteins. Most HSP are intracellular proteins. However, evidence that molecular chaperones are also secreted has accumulated. In this manner it seems that these proteins also have cell– cell signalling qualities, for instance to myeloid or vascular endothelial cells. This would indicate a true signaling function for HSP, which herewith may have an early warning function in signalling stress. The fact that some HSP may have pro-inflammatory activities and some may have anti-inflammatory activities suggests that these proteins can act as a factor in the control of inflammation and immunity. It is assumed that the regulatory potential of self-HSP reactive T cells is exerted at the site of inflammation where, under the influence of the stressed environment caused by toxic mediators of inflammation, cells do express enhanced levels of HSP. The potential of these cells to recognize endogenously produced self-HSP peptides, captured in the MHC molecules of stressed antigen presenting cells, was demonstrated in a variety of experiments. We have demonstrated that our microbial HSP reactive T cells cross-recognize stressed APC under certain conditions. Self HSP60 cross-reactive T cells in the rat were produced by repeated restimulations with the conserved core epitope (256 –265) of MtHSP60. Subsequent testing of these T cell lines for their proliferative responses in the presence of either normal or heat-shocked (30 minutes culture at 43°C and a recovery period of 4 hours at 37°C) spleen cells revealed moderate responses in the presence of normal APC and high proliferative responses in the presence of the heat-shocked APC [32, 33]. Thus, T cells with specificity for self-HSP60 were responsive to endogenously produced self-HSP, as presented by cells in the absence of added antigens. These responses in the absence of added antigens were demonstrated to be MHC restricted, as these responses were fully inhibited by adding an antibody specific for RT1-B (OX6) to the culture. An antibody specific for RT1-D (OX17) had no inhibitory effect for the T cell lines tested sofar. In some cases, the self-HSP60 reactive T cell line became auto-proliferative and expanded without the need of restimulations with antigens. These lines were also seen to produce IL4, IL10, and INF␥. Upon transfer they were demonstrated to have arthritis suppressive potential. In coculture experiments the suppressive potential of self-HSP specific T cells was analyzed. T cell lines were generated by immunizing with the rat homologous sequence of MtHSP60 256 –265 (R256 –265). For control purposes, the same protocol was followed for the generation of T cell lines with specificity for OVA 323– 339. Following isolation of the draining lymph node T
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cells, the cells were restimulated in vitro with normal or heat-shocked (protocol as above) spleen cells. Following an expansion phase on IL2, these T cells were cocultured with responder cells A2b in the presence of its antigen MtHSP60 180 –188. It turned out that HSP60 specific T cells which had been restimulated with stressed spleen cells had a very significant suppressive effect on the level of A2b proliferation. In other words, self-HSP reactive T cells not only are responsive to stressed APC but also they seem to develop a regulatory phenotype upon recognition of stressed APC.
HSP REGULATION AND MICROBIAL EXPOSURE Heat shock proteins are immunodominant proteins. HSP60 was known as the common antigen of gram negative bacteria, already before it’s molecular definition. In other words, immune exposure to microbes leads to a vigorous B and T cell response directed against HSP, such as HSP60 and HSP70. In addition, in patients with inflammatory diseases, responses (B and T cells) against self-HSP60 and 70 can be easily detected. Apparently, the immune system has an interest in recognizing these molecules and exploits in such a manner the ubiquitous presence of these evolutionarily ancient molecules. Based on the above-mentioned stress signalling function of these molecules and given their observed anti-inflammatory potential, it seems that immune responses to HSP are part of the intrinsic immune mechanisms to regulate peripheral tolerance. And, in line herewith, HSP have already shown their capacity to induce regulatory T cells [31]. The fact that bacterial HSP have been effective in inducing protection against experimental autoimmunity further suggests that HSP-mediated immune regulation may also be one of the underlying mechanisms through which environmental flora can contribute to disease resistance. There are many experimental findings that have indicated that bacterial recontamination of germ-free animals induced resistance against immune disorders. In a more wider perspective, one may conclude that HSP may constitute a molecular factor in the mechanisms underlying the hygiene hypothesis. As it is now, the interest in the concept of regulatory T cells as an important factor in the control of autoimmunity has become very broad and intense. The detection of relevant antigens in this area is now of high priority. Especially here, the proof of the pudding will be in the eating. Clinical trials exploiting these novel concepts are therefore now eagerly awaited.
MHC-TARGETED PEPTIDE INTERVENTIONS IN AUTOIMMUNITY So far, peptide therapies in autoimmunity have focused on so-called altered peptide ligands (APL) and on T cell receptor-derived peptides as a result of interventions previously known as T cell vaccination. Altered peptide ligands were originally created by introducing minor amino acid substitutions in autoimmune T cell epitopes and were defined by their potential to induce a tolerogenic response in the autoreactive T cells. Since then, various studies have indicated the potential of APL to block the development of autoimmune diseases. In the experimental model of adjuvant arthritis Marca Wauben et al. developed an APL on the basis of the arthritis-producing T cell epitope. Interestingly, it turned out that the APL was effectively inducing regulation by triggering a disease-suppressive APL-specific T cell which produced suppression of disease following transfer into naïve recipients [34]. Possibly very similar to the principle of microbial HSP, it is now proposed to use microbial epitopes as natural APL for the control of autoimmune diseases such as multiple sclerosis [35]. The principle of immunization with inactivated autoreactive T cells (T cell vaccination) selected from an individual’s own T cell repertoire also has led to the development of TcR peptide therapies for autoimmunity [36]. In the model of rat adjuvant arthritis, Chris Broeren has shown that activated T cells do internalize their T cell receptors and that immunogenic fragments can become loaded into upregulated MHC-II molecules [37]. This cell surface presentation of TcR peptide was seen to be recognized by TcR-specific T cells with disease regulatory potential. The current status of these TcR peptide approaches has been reviewed recently [36]. First clinical trials using HSP peptides have been initiated. Peptide 277 of HSP60 has been tested by parenteral administration in type I diabetes and the results have indicated that peptide-specific T cells changed their production of pro-inflammatory cytokines into the production of anti-inflammatory cytokines, such as IL4 and IL10 [38]. Interestingly, in the treated patients a remarkable beneficial effect on the insulinproducing capacity of the pancreas and insulin dependency was noted. Similarly, oral administration of peptide dnaJP1, derived from a bacterial HSP40 (cochaperone for HSP70), in RA patients induced a tolerogenic T cell response in T cells which manifested a pro-inflammatory T cell phenotype prior to oral treatment [39]. Follow-up phase 2 trials are currently under analysis. In a recent study, a computer algorithm was used to identify multiple HLA-DR binding peptides of HSP60. Interestingly, one of the epitopes was very similar to the peptide that was
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protective against experimental arthritis in Lewis rats [40]. Evidently, this peptide can be a candidate for HSP60-based immunotherapy in patients with arthritis. In conclusion, peptide interventions are on their way to become a novel addition to the therapeutic armamentarium against autoimmune diseases. Clearly, this attractive development would not have been possible without detailed knowledge of HLA polymorphisms, as generated by giants such as Jon van Rood.
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14.
15.
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