BASIC SCIENCE FOR THE CLINICIAN: T CELL REGULATION IN JUVENILE ARTHRITIS

Lucy R. Wedderburn, MD PhD MRCP MRCPCH

Address and details for correspondence:

Rheumatology Unit,

Institute of Child Health, UCL and Great Ormond Street Hospital London

30 Guilford Street,

London WC1N 1EH,

England, UK

Tel: 44 207 905 2391

Fax: 44 207 813 8494

email: l.wedderburn@ich.ucl.ac.uk

Running title: immunoregulation and JIA

Key words: juvenile idiopathic arthritis, immunoregulation, CD25, foxp3

Abstract

The maintenance of immune tolerance is achieved through several mechanisms in the vertebrate immune system. For T cells these mechanisms include selection of T cells in the thymus with removal of potentially ‘autoreactive’ cells, as well as peripheral mechanisms including immune privilege sites, activation induced cell death and immunoregulatory cytokines which regulate or prevent responses to self. There is now much evidence for regulatory T cells (Treg) in the periphery: of these regulatory cells, the best characterised to date are the CD4+ CD25+ Treg cells. This paper reviews recent data on the various types of Treg and their proposed mechanisms of action, and summarises findings relating to Treg in juvenile idiopathic arthritis (JIA). Of interest is the recent demonstration that in the mild form of JIA known as persistent oligoarticular JIA, Treg are present and functional in the joint, and are at higher frequency than in the more severe, extended oligoarticular JIA. An understanding of how the balance between regulation and inflammation is controlled should allow us to design more specific and targeted therapies for the severe forms of arthritis in children, as well as other autoimmune diseases.

Introduction

Two central features of a healthy immune system are the ability to respond to a vast diversity of foreign microbes or pathogens, while at the same time preventing immune responses to self molecules. The first of these goals is achieved by antigen recognition by two inter-connected parts, the innate and the adaptive systems. The innate system recognises a set of relatively non variable molecules on microbes, using a limited number receptors known as Pattern Recognition Receptors (PRRs). In contrast, the adaptive system has evolved methods to generate vast arrays of highly variable receptors, expressed by B cells (as antibody) and T cells (T cell receptors). In the context of these variable receptors the immune system has co-evolved strategies to prevent potentially harmful responses to self proteins, known collectively as tolerance. In addition to being tolerant of self molecules, most healthy individuals maintain ‘non-responsiveness’ to a large number of dietary or inhaled antigens as well as commensal gut bacteria. It is interesting that in many animal models in which the immune system has been disrupted, chronic inflammation of the bowel occurs (1).

For T lymphocytes, tolerance is critically dependent on the function of the thymus. Within this organ, high affinity self reactive T cells are removed (deleted) during development. A mechanism to facilitate this selection process has recently been elucidated. This involves the low level thymic expression of a wide range of self proteins from tissues all over the body specifically to ‘educate’ the developing T cells there, under control of the AIRE protein (2). However, central tolerance alone would be inadequate to ensure a safe level of non-reactivity. It is now clear that a set of mechanisms also exists in the peripheral immune system which is fundamental to maintaining immune tolerance and therefore to preventing autoimmune disease. One such mechanism which has recently come under intense investigation, is the contribution of T cells themselves, by so called ‘regulatory’ T cells (3, 4).

Mechanisms of control by regulatory T cells (Treg)

Regulatory T cells (Treg) were initially identified in mice by their ability to suppress proliferation of other cells in vitro, and to control autoimmune inflammation and disease in vivo (5-7). Removal of such cells leads to the spontaneous development of autoimmune pathology in mice, such as gastritis or colitis, though interestingly in some but not all models of animal arthritis (8, 9). A major type of regulatory T cells was identified by its surface expression of CD25, which is a component of the receptor for the cytokine IL-2α (5). CD25+CD4+ T cells make up 5-10 % of normal CD4+ T cells in the blood of rodents and humans when defined by expression of CD25 of either medium or high levels (10, 11). Several studies have also suggested that the most functionally suppressive Treg reside predominantly within the population expressing very high levels of CD25 (CD25high) (7). Despite their high expression of the IL-2Rα(CD25), these cells do not divide readily in standard in vitro assays which are used to measure T cell proliferation (12). In vitro they require contact with their target cells in order to inhibit their proliferation (13, 14). However data suggesting a role for cytokines such as IL-10 or TGFβ in the function of these Treg in vivo (15) as well as the demonstration that CD25+ Treg can in fact proliferate well in response to antigen in vivo (16), suggest that the behaviour of Treg in vitro does not always reflect the in vivo situation. CD25+ Treg have been shown to exert inhibitory effects not only on other T cells, but also B cells and cells of the innate immune system including dendritic cells and NK cells (17).

The phenotype of CD25+ Treg is increasingly being characterised. However many of the proteins which are expressed on the surface of Treg, such as CTLA4 (cytotoxic T lymphocyte-associated protein-4), GITR (glucocorticoid-induced TNF receptor) and CD25 itself, are also increased upon activation of T cells, making it difficult to distinguish Treg from activated T cells, other than by their functional ability to suppress (18). The recent demonstration that the forkhead transcriptional regulator foxp3 is highly expressed in CD25+ Treg, and that forced over-expression of foxp3 induces a suppressive phenotype, has provided a specific tool with which to identify these cells (19, 20). This development is an example of ‘convergence’ of studies in mice and humans: the foxp3 mutant mouse, known as the ‘scurfy’ mouse, (the product of the foxp3 gene was originally called scurfin), has a phenotype which includes multiple autoimmune conditions and lymphocyte proliferation, from which these mice die within weeks of birth (21). Human patients in whom foxp3 is deficient have also been described; they present with a syndrome of multiple autoimmune and inflammatory symptoms known as immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance syndrome (IPEX). This syndrome has been shown to be due to mutations in the human foxp3 gene, which is located on the X chromosome (22). Many (perhaps most) CD25+ Treg arise from the thymus. However it has been demonstrated that CD25- cells, when stimulated in vitro, or when influenced by CD25+ cells (23) or regulatory cytokines such as TGFβ(24), may upregulate foxp3 and acquire a regulatory phenotype. This raises the intriguing possibility that perhaps all T cells may be regulatory under certain conditions. It is now clear that Treg have an important role both in preventing autoimmune disease and also in the normal kinetics of immune responses to a wide range of pathogens (reviewed in 18). Therefore it seems likely that the generation of a set of regulatory T cells is part of the normal immune response, without which responses might continue, unchecked.

The mechanisms by which CD25+ Treg exert inhibition are still unclear, although one outcome of suppression is the blocking of transcription of IL-2, inhibiting production of this autocrine growth factor (25). Although in vitro the actions of Treg are dependent upon contact with their targets, and are not transferred by soluble factors, the surface expression of cytokines by Treg, in particular of TGFβ, appears to play a role in suppression (26). Another interaction implicated in suppression by CD25+ Treg is that involving CTLA4 and its receptors CD80 and CD86 (27). The CD40/CD40L (CD154) interaction is also implicated in control of Treg function, since removal or blockade of this pathway during antigen exposure leads to increased CD25+ Treg-mediated suppression (28, 29).

Like all T cells, regulatory T cells require antigen presentation by specialised antigen presenting cells (APC) for function. Of the APC, the most potent are those known as dendritic cells (DC) and many studies indicate that DC are involved in generation of Treg (30). A bewildering diversity of in vitro systems have been used to generate such ‘tolerogenic ‘ DC, including culture in antigen in the absence of ‘danger’ signals (31), in the presence of steroids and Vitamin D (32) or IL-10 (33). Overall these studies suggest that either ‘immature’ or steady state DC are more likely to induce Treg and thereby tolerance, than fully activated DC: whether these represent separate developmental stages or pathways of differentiation in still unclear. However the concept of the regulatory DC as a potential tool to induce tolerance or even re-instate it during autoimmune pathology, is an emerging one (34).

In addition to CD4+CD25+ Treg, other types of regulatory T cell have been identified, in particular cells which are contact-independent but use cytokines such as IL-10 and TGFβ These cells, such as Tr1 cells (typically IL-10 producing) and Th3 cells (typically TGFβ secreting) may develop in response to the effects of CD25 Treg or independently (4). Both of these cytokines (IL-10 and TGFβ) have been implicated in protection from autoimmunity in animal models and there has been some success in model systems in treating autoimmune pathology, including arthritis, by their targeted delivery to the inflamed site (35, 36).

Whether Treg cells have a specific ‘repertoire’ of antigens to which they preferentially respond is unclear, although already a wide range of specificities, both self and foreign proteins, have been shown to be recognised by Treg (37). Evidence does suggest that certain self antigens appear to be immunodominant and involved in a protective response against autoimmunity. An example of this is the family of proteins known as heat shock proteins (hsp), highly conserved chaperone proteins which are upregulated in situations of cellular stress, and which have motifs that are shared across all species, from bacteria to mammals. In the adjuvant arthritis (AA) rat model of arthritis, T cells specific to hsp have been shown to protect against disease and nasal administration of hsp peptides reduced the onset and severity of the arthritis (38, 39).

Immunoregulation in juvenile idiopathic arthritis

In the context of this increased understanding of immune regulation by T cells and DC, a number of studies of juvenile idiopathic arthritis (JIA), which are discussed below, suggest that such regulation may play a role in some subtypes of childhood arthritis. JIA is a group of diseases which affects 1 in 1000 children under 16. The JIA subtypes have different clinical features, courses and genetic associations. Children whose disease is known (using the ILAR classification and criteria (40), as oligoarticular JIA (previously called pauciarticular) have 4 or less joints involved at presentation and in the first 6 months of disease. Within this group, two divergent groups then emerge: children whose arthritis remains mild, responds well to simple treatments such as NSAID and local joint injection, and frequently enters prolonged remission (known as persistent oligoarticular JIA), and those children in whom arthritis extends to many joints, may be severe and destructive, and can be difficult to control (extended oligoarticular JIA) (41). The latter may show some overlap with the subtype known as rheumatoid factor (RF)-negative polyarticular JIA, also a severe clinical subtype. There are genetic and immunological data to suggest that the mild phenotype of persistent oligoarticular JIA is in part due to immune regulation which may involve regulatory T cells and cytokines. If this is the case, this subgroup, frequently thought of as the ‘easy-to-manage’ patients by practising Paediatric Rheumatologists, may be of great importance to our ability to understand how severe arthritis progresses. Thus, the mild group may hold the clues which we need to unravel, in order to treat the severe forms of JIA more effectively.

We and others have shown that within the inflamed and highly vascular synovium of children with oligoarticular and polyarticular JIA, there is a dense infiltrate of activated, Th1 skewed T cells which contain highly expanded oligoclonal populations (42-45). Despite this, children with persistent oligoarticular JIA may make detectable levels of IL-4 from synovial T cells (45, 46). Since IL-4 is typically associated with a Th2 response, this may alter the balance of cytokines in the joints compared to those children with extended oligoarticular disease. In addition, it is interesting that the expression of CCR4 (a member of the CC chemokine receptor family)(itself since demonstrated to be expressed on CD25+ Treg) was shown to identify that synovial T cell population in JIA which has an increased IL-4: IFN gamma ratio (47).

Within the synovial fluid T cell population of many children with JIA, a population which responds well to human hsp60, (defined by proliferation of synovial T cells to hsp60 protein in vitro), is frequently present (48) . Strikingly, children with the highest response to hsp60 are those with persistent oligoarticular JIA, and in a prospective study, a strong response by peripheral blood mononuclear cells to hsp60 was found to be associated with good clinical outcome and remission of disease (49). These hsp specific T cells in patients with persistent oligoarticular JIA have since been shown to express CD30 (a marker also identified on Treg) and to make IL-10 (50). It is therefore possible that this hsp60 specific response represents part of an immunoregulatory process in oligoarticular JIA.

In collaboration with the group of Dr. B. Prakken, we have recently studied the CD4+CD25+ T cells in the joints of children with persistent and extended oligoarticular JIA. We have shown that children with persistent oligoarticular JIA have significantly higher (p<0.01) levels of CD25+CD4+ cells in the joint than those with extended oligoarticular JIA (51). In addition, we found that that these CD25+ T cells contain cells which are suppressive in vitro, are positive for CTLA4, GITR, and CCR4 (all markers whose high expression is associated with regulatory T cells), and that they express foxp3 (51). Interestingly, the CD25+ cells in the joint included increased numbers of both CD25high and CD25int cells, (the latter expressing an intermediate level of CD25), but when analysed by CD25high alone the difference between the two groups of patients was less marked. In the same study it was shown that the expression of foxp3 was also greatly increased in the synovial CD25int cells and that these cells can also suppress very effectively. Thus, at least in the inflamed joint, cells with an intermediate CD25 expression may also be suppressive.

An unexpected feature of the synovial fluid T cells from both JIA and rheumatoid arthritis patients has been the fact that despite their activated phenotype, they proliferate very poorly in vitro and contain few cells in active cell cycle, when analysed fresh from the joint (52-54). We have shown that this hypo-responsiveness to mitogenic stimuli which signal through the T cell receptor may be explained by the presence of CD25+ T cells. Thus, after separation of synovial T cells into CD25- or CD25+, those which are CD25-, when cultured alone, respond as well as or more powerfully than their peripheral blood counterparts to stimuli through the TCR. In contrast the CD25+ synovial T cells alone are profoundly anergic (51). On the addition of cytokines such as IL-2 or IL15, or a specific antigen to which memory cells are present in the synovial T cells, a large proliferative response may be observed and the ‘hypo-responsive’ state can be overcome. By using a specific memory antigen, the influenza peptide HA307-319 to stimulate synovial T cells in vitro, and then staining with an MHC class ll tetramer reagent which labels only T cells that are specific for this peptide (55), we have shown that in such stimulation assays, a significant part of the dividing synovial T cells are antigen non specific, (as judged by failure to bind a specific HLA-peptide tetramer reagent, Figure 1), and presumably able to respond to cytokines generated within the culture.

Thus we suggest that the CD4+ CD25+ population of synovial T cells includes a population of regulatory T cells. These cells have all the phenotypic hallmarks of CD25+ Treg, they do express foxp3 and they can suppress proliferation at least in vitro. Given that these CD25+ foxp3+ cells are also at higher frequency in the joints of children with persistent oligoarticular JIA, it is possible these cells contribute to the ‘self-regulation’ of pathology seen in this form of childhood arthritis.

Interestingly, Treg have now been demonstrated in a range of human autoimmune and infectious pathologies. With specific reference to arthritis, CD25+ Treg have been found to be present at high frequency in the joints of patients with rheumatoid arthritis (RA) (56). These cells were able to suppress the proliferation of CD25- cells from both blood and joint. These cells were also stable in number both between different inflamed joints and over time. The phenotype of the CD25+ synovial cells in RA was very similar to that of the CD25+ cells in JIA. However, in this study no correlation was seen between Treg in the joint and clinical features of the patients.

In addition to the functional studies on T cells in JIA already discussed, the genetics of JIA suggest that children with particular subtypes of JIA have genetic factors which alter the ability to regulate immune pathology. Collection of DNA samples from large cohorts of children with JIA has allowed such genetic studies with sufficient power to subdivide data by JIA subtype. In one such study where allelic differences in the promoter region controlling expression of the IL-10 cytokine, progression to extended oligoarticular disease was associated with an IL-10 haplotype (‘ATA’) which was shown to correlate with low IL-10 production in LPS-stimulated whole blood cultures (57). Further genetic studies have shown that while some ‘risk’ genes for JIA are shared across all JIA subtypes, and perhaps also with multiple autoimmune disorders, other genetic associations are subtype specific (58). For example some HLA risk associations differ between the two groups of oligoarticular JIA, in particular the haplotype DRB1*13-DQA1*01-DQB1*06 which is increased specifically in persistent oligoarticular JIA (33).

Harnessing regulatory mechanisms

There has been much progress recently in our understanding of how the immune system regulates itself, although several issues, such as the relationship between different types of Treg, their generation and their control, remain unresolved. The challenge now is to unravel the ‘break points’ at which tolerance is lost during autoimmune disease. Current evidence suggests that in one group of children with persistent oligoarticular arthritis the immune system may show effective regulation of an autoimmune inflammatory process: by understanding how this is achieved we may be able to harness these mechanisms to treat more severe forms of arthritis, both in adults and children. For example, in the future it may be possible to predict which children will evolve to severe disease, based upon the balance of inflammation and regulation in the joint, and to do this early in the disease process. If the differences in Treg between disease subtypes are present early in the inflammatory process and can be shown to be predictive of disease course, then one could envisage a diagnostic test which would be based, for example, upon flow cytometry of cells from the first joint aspirate : this would be rapid, cheap, and simple to perform. In addition, it may be possible also to use regulatory pathways, such as that governed by foxp3, to design specific drugs which will tip the balance back towards regulation in arthritis and in other autoimmune diseases (59).

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Figure 1

Legend to figure 1: Flow cytometric analysis of (A) blood and (B, C) synovial fluid T cells, after stimulation assay in vitro. Cells were labelled with the green dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and then cultured in the presence of influenza haemagglutinin peptide (HA307-319) for 5 days. Cells were stained and analysed by flow cytometry. In A and B CFSE-labelled blood or synovial T cells (gated on CD3+) are shown stained for CD4, with CFSE on the x axis: those CD4 cells which have divided are in the upper left quadrant. In plot C, CFSE labelled synovial CD4+ cells, (gated on CD3+CD4+), are stained with HA/DR1 tetramer. All cells which have divided and are specific for HA peptide are in the upper left quadrant. Note the population of divided cells which are tetramer negative (i.e. presumed to be antigen non-specific), seen in the lower left quadrant of plot C.