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,
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