Download Exploring the reciprocal relationship between

Rheumatology 2003;42:716–731
doi:10.1093/rheumatology/keg262, available online at www.rheumatology.oupjournals.org
Advance Access publication 31 March 2003
Review
Exploring the reciprocal relationship between
immunity and inflammation in chronic
inflammatory arthritis
A. P. Cope
Experimental models seeking to explore how susceptible individuals develop
rheumatoid arthritis (RA) propose that genetic and environmental factors shape a
complex series of molecular and cellular interactions leading to a chronic inflammatory response. T lymphocytes and MHC class II genes have featured prominently
in these models. More recent studies have suggested that perpetuation of inflammation
in a disease-susceptible host might occur through failure to down-regulate the
inflammatory process. One prediction from this model is that effective mechanisms
of immunoregulation might be most easily investigated in non-susceptible individuals. However, this has been difficult to study in man. Based on the observation
that extended MHC haplotypes are strongly associated with RA in different ethnic
groups, I have explored the function of human MHC-encoded genes in transgenic
mice using two different experimental approaches. First, by comparing the
molecular interactions between disease-associated or non-associated HLA-DR4
molecules and CD4+ T lymphocytes, it has been possible to gain insight into how
immune responses in non-susceptible individuals might differ from T-cell responses
observed in a susceptible host. This has been achieved using transgenic mice
expressing RA disease-associated and non-associated human HLA class II
molecules. Secondly, the effects of prolonged exposure of T cells to the
proinflammatory cytokine tumour necrosis factor a (TNF) have been studied
in vitro and in vivo, focusing on T-cell receptor (TCR) signalling and effector
responses. In studies of HLA class II transgenic mice, the major differences
between disease-associated and non-associated alleles in terms of T-cell responses
occur at the level of presentation of antigenic peptides, and the sustained
expression of inflammatory cytokines such as TNF. Chronic exposure of T cells
to inflammatory cytokines such as TNF induces a phenotype which resembles
RA synovial T cells, including the induction of non-deletional and reversible
hyporesponsiveness to TCR ligation and uncoupling of proximal TCR signal
transduction pathways. The experimental findings are consistent with a model in
which HLA class II-driven inflammatory cytokine expression uncouples TCR
signalling pathways in the susceptible host in such a way as to profoundly suppress
proliferative and immunoregulatory cytokine responses, while at the same time
promoting cell survival and effector responses.
KEY WORDS: Autoimmunity, Rheumatoid arthritis, T lymphocytes, HLA-DR4, Tumour necrosis
factor, T-cell activation, T-cell hyporesponsiveness.
Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London, UK.
Accepted 30 December 2002.
Correspondence to: Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, Arthritis Research Campaign
Building, 1 Aspenlea Road, Hammersmith, London W6 8LH, UK. E-mail: [email protected]
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ß 2003 British Society for Rheumatology
Immunity and inflammation in chronic inflammatory arthritis
Protection against foreign pathogens is the primary
function of the immune system. While the innate
immune system provides the first line of defence,
adaptive immunity and the acquisition of memory
responses to foreign antigens is achieved through the
generation of an extensive repertoire of lymphocyte
antigen receptors. Because this repertoire is generated
during thymic maturation and maintained in the
periphery through recognition of complexes of self
peptide and major histocompatibility complex (MHC)
molecules, all peripheral T cells have the potential for
autoreactivity. Whilst thymic maturation generates T
lymphocytes expressing low-avidity T-cell receptors
(TCR) at low precursor frequencies, this recognition
mechanism cannot discriminate between self and foreign
antigens. Therefore, protective immunity is provided to
the host at the expense of a huge propensity for crossreactivity to self tissue antigens w1x. How is it that
autoimmunity is very common while autoimmune disease
is quite rare?
Over the last few years, susceptibility to autoimmunity
has been explored not so much in terms of a predisposition to generating autoaggressive effector cells, but more
in terms of failure to regulate an autoimmune response
wreviewed in 2x. If one considers current concepts of
cognate immunity, a defect in immunoregulation could
be acquired at multiple points during both early and
later phases of an autoimmune response. For CD4+ T
lymphocytes, the initial ligand–receptor interaction between
MHC–peptide and the TCR is crucial, since this determines qualitative and quantitative aspects of the signals
transduced through the TCR w3x. Seen through the eyes
of an autoreactive T cell, the evolution of autoimmune
disease could be envisaged as a series of checkpoints
(Fig. 1). These checkpoints provide a theoretical framework for understanding not only how disease might
progress in a susceptible host, but also for exploring how
717
the evolution of autoimmune responses might differ
qualitatively in the non-susceptible host.
Evolving concepts of disease mechanisms for rheumatoid
arthritis (RA) have provided a paradigm for understanding the pathogenesis of autoimmune disease. Over the
last 10 yr my research has focused on investigating the
reciprocal relationship between the immune response
and inflammation in the context of diseases such as RA.
Studies have focused on specific questions that have to
do with the initiation of the disease process as well as its
progression. Specifically, I have addressed (i) the molecular
nature of cognate immunity to cartilage antigens in the
susceptible host, and how these initial interactions might
contribute to autoimmune disease; (ii) how this process
might differ in a non-susceptible individual; and (iii) how
the chronic inflammatory process in turn influences the
evolution of T-cell autoreactivity and effector responses.
The experimental strategy is based upon the observation that the strongest disease associations are with genes
that contribute to an extended haplotype encoded within
the MHC w4x. I will describe experiments undertaken to
explore these issues, emphasizing how the results contribute to an understanding of specific disease checkpoints (Fig. 1). I will then discuss how some unexpected
results have influenced my own perceptions of how
inflammatory arthritis evolves, and conclude by proposing a model of RA that reconciles my observations with
current concepts of disease pathogenesis, and which
hints at the possibility of new prospects for therapy.
Exploring the function of disease-associated
HLA-DRB1 genes in rheumatoid arthritis
How do we begin to study in man the complex molecular
interactions that contribute to the pathogenesis of RA?
For many laboratories, the choice of experimental strategy
FIG. 1. Immunopathological checkpoints implicated in the pathogenesis of chronic inflammatory arthritis. Activation and
subsequent expansion of autoreactive CD4+ T cells lead to cell-to-cell interactions that initiate a cascade of effector responses.
According to this model, immunoregulatory mechanisms may influence activation, expansion anduor effector responses.
A. P. Cope
718
has been guided by genetic studies which have focused
on defining gene polymorphisms which most clearly
discriminate between patients with RA and the nonsusceptible host. Some of the strongest associations
found in RA patients from different ethnic groups to
date are with MHC class II genes w5, 6x. This association
was first described by Stastny in the 1970s w7x, but a
significant advance in our understanding of these associations was reported more than a decade later, when it was
shown that susceptibility to RA across different ethnic
populations correlated closely with the expression of
a specific consensus amino acid sequence (the ‘shared
epitope’) within the HLA-DRb chain w8x. This sequence
was subsequently shown by several groups of investigators to be encoded by HLA-DRB1 alleles, including
HLA-DR4 (*0401, *0404, *0405 and 0408), but also
HLA-DR1 (*0101), DR6 (*1401) and DR10 (*1001)
alleles w9–11x. Significantly, some of the key polymorphisms within non-associated DRB1*04 alleles mapped to
the same region of the DRb chain a helix (Table 1). For
example, DRab1*0402 carries negatively charged residues at b70 and 71, as opposed to the positively charged
residues found at b71 in disease-associated DRB1 alleles,
while DRab1*0403 (non-associated), which differs from
*0404 (associated) by one amino acid, carries a negatively
charged residue at codon 74.
These associations, together with the finding of follicular
aggregates of lymphocytes and antigen-presenting cells
(APC) in the synovial tissue of patients with active RA,
have provided perhaps the strongest evidence that the
immune response contributes to disease chronicity. By
studying these associations in patient cohorts that are
clinically as well as genetically heterogeneous, it has been
possible to identify genotypes that cosegregate with specific
clinical features. For example, in population studies,
different HLA-DRB1 alleles appear to influence the
severity of disease, DRB1*0401 being found in patients
with severe, seropositive, erosive RA (often with extraarticular features, such as vasculitis and Felty’s syndrome, in *0401-homozygous or *0401u*0404 compound
homozygous individuals), while DRB1*0101 and *1001
are observed at a higher frequency in patients with less
severe, seronegative, non-erosive disease w12x. Inheriting
two copies of alleles expressing the consensus sequence
greatly increases disease penetrance, time of onset and
severity. These and other studies have suggested that
genetic associations may contribute more to severity
than to disease susceptibility wreviewed in 4x. Closer
inspection suggests further that, rather than a hierarchy
of phenotypes of a single disease, these genetic associations, which are by no means uniform, are reflected
clinically as distinct disease entities. These distinct entities
may be manifest at the level of synovial histomorphology w13x, somewhat analogously to the histological
features that define different forms of B-cell lymphoma.
A functional basis for the associations between
HLA-DR and RA
MHC class II molecules function by selecting and presenting immunogenic peptide fragments of protein antigens
to CD4+ T cells. They also play a role in the selection of
the TCR repertoire in the thymus. Because CD4+ T cells
recognize linear stretches of about 9–20 amino acids
derived from self or foreign protein antigens bound in
the peptide-binding groove of polymorphic MHC class
II molecules, it has been suggested that differences in the
way that HLA-DR or -DQ molecules present selected
peptides to T cells could be an important mechanism
for susceptibility to autoimmune diseases such as RA.
According to this model, RA might represent the
sequelae of pathological T-cell responses initiated and
maintained by antigens presented by disease-associated
HLA class II molecules. On the basis of early observations, two principal models were proposed to account for
the association between RA and the consensus DRb
chain sequence. Both were based on the assumption that
the shared epitope is the critical genetic element linked
directly to disease. The first model proposed that the
shared epitope determines specific peptide binding, and
that ‘pathogenic’ peptides bind only to disease-associated
HLA class II molecules w14, 15x. This model predicted
that a gradient of affinities of disease-inducing peptide
for MHC class II molecules might account for the
differences in susceptibility anduor severity conferred by
different HLA-DR molecules. The second model proposed that the shared epitope influences TCR recognition by binding and selecting autoreactive T cells during
thymic maturation, and expanding these populations in
the peripheral compartment w16, 17x.
Although these models are not mutually exclusive,
critical testing of the models in the context of RA has
been the subject of intense research in many laboratories.
TABLE 1. Amino acid composition of HLA-DR peptide-binding pockets and their associations with RA
Pocket 4
HLA-DRB1 allele
DRB*0101
DRB1*0401
DRB1*0402
DRB1*0403
DRB1*0404
DRB1*0405
Pocket 6
Pocket 7
Pocket 1
86
13
70
71
74
11
13
28
G
G
q
V
V
V
G
F
H
H
H
H
H
Q
Q
q
D
Q
Q
Q
R
K
q
E
R
R
R
A
A
A
E
A
A
L
V
V
V
V
V
F
H
H
H
H
H
E
D
D
D
D
D
67
L
L
q
I
L
L
L
Pocket 9
71
9
57
RA
R
K
q
E
R
R
R
W
E
E
E
E
E
D
D
D
D
D
S
+
+++
–
–
++
+
Circles denote amino acid residues in the DR1*0402 molecule that differ in DR1*0401. +++, strong association with RA; –, weak or no
association with RA.
Immunity and inflammation in chronic inflammatory arthritis
Progress in this area has been hampered largely by the
fact that, until recently, it has not been possible to
compare the function of disease-associated and nonassociated HLA-DR4 molecules in isolation under experimental conditions where the remainder of the genome is
fixed.
Studying HLA-DR4-restricted T-cell responses in
transgenic mice
Conceptually, an experimental approach involving the
generation of transgenic mice expressing human MHC
class II molecules has proved attractive. Such an approach
has been developed by a number of laboratories with the
intention of studying the function of disease-associated
HLA class II molecules in vivo in a way that had not
been previously possible in human subjects w18–20x.
Comprehensive reviews published recently have detailed
the experimental approaches, highlighting the constraints
of such models and discussing how these limitations have
been resolved w21, 22x. This approach in mice has turned
out to be enormously useful for probing in vivo the function of disease-associated and non-associated human MHC
class II molecules, and their potential for contributing to
the development of inflammatory arthritis w23, 24x.
Generating a HLA-DR4-restricted TCR repertoire
in transgenic mice
Using DR4 transgenic mice that my colleagues and I had
generated at Stanford University, it was first necessary to
study in detail a CD4+ T-cell response specific for cognate
antigen that was restricted to the HLA-DRB1*04 allele
encoding HLA-DRab1*0401, and to characterize the
immunogenic epitopes presented by these HLA-DR4
molecules in vivo. This allele was chosen because of its
strong associations with RA in Caucasians w11x. To this
end, it was necessary to generate a repertoire of CD4+ T
cells whose TCR had been shaped by HLA-DR4 during
thymic maturation. This was only achieved after a detailed
analysis of the peripheral CD4+ T-cell compartment in
transgenic mice expressing different genotypes w25x. For
example, while I-Ab0u0 murine MHC class II-deficient mice
have <0.5% mature CD4+ T cells in the peripheral blood
w26x, introduction of a single copy of the HLA-DR4
transgene onto this mouse class II-deficient background
increased this level to around 4%. After crossing to
human CD4-transgenic mice, the peripheral CD4 compartment increased further to 13%, while homozygous
HLA-DR4uhuman CD4-transgenic I-Ab0u0 mice had
719
peripheral mouse CD4+ T-cell numbers approaching
those observed in mice expressing endogenous mouse
MHC class II I-A molecules (;25–30%) w22, 25x. These
observations, combined with the fact that HLA-DR4
had been shown to alter the repertoire of selected Vb
TCR in I-A-expressing mice w18x, demonstrated that
human HLA-DR4 can shape, both quantitatively and
qualitatively, the TCR repertoire in mice. It also provided a molecular framework for exploring in depth the
specificity of mature CD4+ T cells that had previously
undergone maturation and selection through cognate
interactions with self-peptideuHLA-DR4 complexes in
the thymus of transgenic mice.
What do HLA-DR4-restricted T cells really see?
HLA-DR4-restricted T-cell responses were defined using
human cartilage (HC) gp-39 as a model antigen w27x.
Although HCgp-39 is expressed in many tissues, it is
produced in abundance by chondrocytes, and protein is
present in synovial membrane as well as synovial fluid
and serum w28–30x. The finding that mRNA transcripts
are not detectable in healthy cartilage explants but can
be induced in tissue from arthritic joints, as well as by
proinflammatory cytokines in vitro, made this an attractive candidate antigen for study w28, 29x. Through the
generation of immortalized T-cell hybridomas, the
specificities of literally thousands of T-cell responses
were evaluated. From more than 250 HCgp-39-specific
responses, nine immunogenic epitopes were identified,
with frequencies of responses of T cells to specific
peptides ranging from <1% to as high as 35% of the Tcell response, indicating that this was a sensitive as well
as a specific method for studying HLA-DR4-restricted
T-cell responses in vivo w27x. Epitopes 100–115, 262–277
and 322–337 contributed around 80% of the total
peptide specificity (Table 2), but, surprisingly, not one
of these peptides carried the predicted charged residue
at position 4. More than 95% of all antigen-specific
responses were restricted to HLA-DR4, the remainder
being restricted to the DRauI-Eb cross-species heterodimer, which is also expressed in these mice, albeit at
very low levels. All responses could be blocked with antiDR monoclonal antibodies, and each epitope identified
was subsequently found to be processed and presented
efficiently by human DRab1*0401-expressing APC. In a
pilot study designed to validate the relevance of HCgp39 epitopes defined in mice, all immunogenic epitopes
of HCgp-39 were capable of eliciting peripheral blood
TABLE 2. Characteristics of immunodeficient epitopes of HCgp-39 defined in HLA-DR4-transgenic mice
Frequency of HLA-DR4 restricted hybrids
Peptide residue
100–114 (106–114)
262–277 (265–273)
322–337 (328–336)
22–37 (25–33)
298–313 (302–310)
*0401 (n=250)
20
34
27
0
0
Core motif relative position
*0402 (n=141)
1
2
3
4
5
6
7
8
9
<1
<1
0
32
54
F
F
Y
V
L
S
T
D
C
R
K
L
D
Y
G
I
A
Q
Y
A
A
S
E
T
T
S
S
S
S
V
N
E
V
W
H
T
T
K
S
R
Q
G
S
Q
T
Adapted from reference 27 and reviewed in more detail in 22.
720
A. P. Cope
T-cell proliferative responses in at least some patients
with RA carrying HLA-DRB1*04 alleles encoding the
shared epitope consensus sequence w27x.
This approach made it possible to define precisely, for
HLA-DR4-restricted T cells, the immunodominant epitopes of a cartilage antigen that elicited T-cell responses
in patients with RA, and as such defined at the molecular
level the first checkpoint, defined as cartilage antigen
peptide-specific recognition by TCR (checkpoint 1, Fig. 1).
According to these data, TCR recognition appeared to
be highly specific for a large number of clones, because
strict dependence on specific peptide for reactivity was
demonstrated and TCR triggering did not occur in the
absence of specific peptide. However, more detailed
analyses indicated that seemingly monospecific T cells
were also cross-reactive.
Immunization with HCgp-39 promotes reactivity to
self-peptide–self-MHC complexes
While screening for positive responses to HCgp-39, a
subset of hybrids (;15–20%) were found to be highly
reactive to transgenic but not non-transgenic splenic
APC in the absence of exogenous antigen w31x. Similar
responses were observed when DRab1*0401-expressing
Epstein–Barr virus-transformed B cells were used as
APC. These responses were directed to complexes of self
peptide and HLA-DR4, since they could be inhibited by
anti-DR monoclonal antibodies.
Detailed analysis of self-reactive T cells revealed great
flexibility of TCR recognition at multiple levels w32x.
Activation of a subset of clones was further enhanced by
the addition of HCgp-39 or a panel of peptides derived
from HCgp-39. These intramolecular peptide mimics, while
carrying predicted HLA-DR-binding motifs, carried
sequences which were quite distinct from each other at
the amino acid level. These clones also responded to
APC expressing closely related HLA-DRB1 alleles that
encoded the shared epitope consensus sequence b67–74,
with or without exogenous antigen. Responses to
DRab1*0401 and to *0405 APC were particularly
vigorous, while responses to DRab1*0402 and *0403
were never observed (Fig. 2).
These data provided direct evidence for diversity and
flexibility of TCR recognition early in the evolution of
the immune response, and demonstrated that crossreactivity to self-peptide–self-MHC complexes following
immunization with cognate antigen is frequent. For
many responses, MHC-biased recognition by TCR predominated. For these T cells, one might predict charge
complementarity between MHC shared epitope sequences
and TCR complementarity-determining regions (Fig. 3B),
as proposed recently w33x, rather than between MHC and
cognate peptide (Fig. 3A). This cross-reactivity would also
provide a mechanism through which critical precursor
frequencies of autoreactive T cells could be achieved,
and, because of MHC-directed recognition, could evolve
by propagation through interactions largely independent
of specific antigen. According to this model, the peptide
is permissive for TCR recognition (checkpoint 2, Fig. 1).
While the fact that the repertoire of TCR is shaped on
self-peptide–self-MHC complexes during thymic maturation provides the most plausible explanation for this, the
model provides further evidence that cross-reactivity of
peripheral T cells is universal w1x. In the light of these
observations, it should be possible to demonstrate broad
self-reactivity for all TCR, including populations of
peptide-specific T cells that are apparently monospecific,
seemingly lacking the broad flexibility of other T-cell
subsets. Because immune responses to foreign antigens
are generated from the same pool of potentially selfreactive TCR, it follows from this that immunization of
HLA-DR4-transgenic mice with proteins derived from
foreign pathogens would activate and expand populations
of autoreactive T cells.
FIG. 2. Flexibility of TCR recognition by HCgp-39-specific T cells. (A) T cells were stimulated with APC plus HCgp-39 peptides or
native antigen. Note high background counts (>100 000 IL-2 fluorescence units) in the absence of antigen. (B) T cells were
stimulated with a panel of DRB1-expressing APC in the absence of exogenous antigen. Data are shown for clones 4A10 and 1A2,
both specific for peptide 262–277. Results are expressed as arbitrary IL-2 fluorescence units.
Immunity and inflammation in chronic inflammatory arthritis
FIG. 3. Schematic representation of the amino acid charge
complementarity that could exist between (A) residue b71 of
the DRb-chain shared epitope sequence and position 4 (P4) of
the specific peptide, or (B) residue b71 of the DRb-chain shared
epitope sequence and residues encoded by the complementaritydetermining region (CDR) of the TCRb chain. Analysis of
HCgp-39-specific T-cell responses indicates that the charge
complementarity illustrated in scheme B may predominate, as
immunodominant peptide epitopes carry neutral amino acids
at P4.
Self-reactivity following immune responses to
foreign pathogens
In collaboration with the Kamradt laboratory, this
hypothesis was tested directly by repeating the immunization strategy outlined above, but this time substituting
OspA for HCgp-39, the outer surface protein A derived
from the tick-borne spirochete Borrelia burgdorferi, the
causative organism of Lyme disease w34x. This was a
relevant model antigen to study because CD4+ T-cell
responses to OspA have been implicated in chronic
treatment-resistant Lyme arthritis, and the disease is also
associated with HLA-DR4 w35–37x. Large numbers of
HLA-DR4-restricted, OspA-specific T-cell hybridomas
were generated, and the immunodominant epitopes were
identified as before w38x.
Two strategies were employed for identifying selfpeptide mimics of immunodominant OspA epitopes
capable of stimulating OspA-specific T cell-hybridomas.
An example is shown in Fig. 4 for OspA peptide 235–
246. In the first strategy, the SwissproteinuTREMBL
database was used to define mimics of OspA peptides on
the basis of conventional sequence alignment. In the
second, a systematic amino acid analysis was undertaken, replacing each residue of the core 9-mer OspA
epitope with all naturally occurring amino acids and
testing responses of OspA peptide-specific T cells to
these synthetic derivative peptides. On the basis of the
amino acid substitutions permissive for T-cell responsiveness, a structural ‘supertope’ motif was defined and
used to scan the same database. Mimics were identified and synthesized prior to testing on a panel of
OspA-specific T-cell hybridomas.
The results were remarkable in several respects w38x.
When responses to the ‘best fit’ peptides, identified by
721
FIG. 4. Schematic for demonstrating cross-reactivity of OspAspecific T cells to self-peptide mimics. Candidate peptide
mimics were identified by sequential substitution analysis of
each amino acid residue of the core epitope, or by a database
search for sequence alignment. Peptide mimics were identified
and the best-fit peptides synthesized and tested on panels
of OspA-specific T-cell hybridomas. Mimics that carried the
‘supertope’ motif were identified, and these were capable of
stimulating OspA-specific T cells.
either alignment or substitution analyses, were compared
on the panel of T cells that responded to the original
peptide, reactivity was most frequently observed with
peptide mimics fulfilling the supermotif. Intriguingly,
many of these peptide mimics, derived from both mouse
and human proteins, bore little or no resemblance to the
wild-type sequence. Indeed, for some there existed no
homologies at any residue. By contrast, few if any
peptides identified by conventional sequence alignment
stimulated OspA-specific T cells (Fig. 4). This lack of
reactivity was analogous to that which I had observed
for human HCgp-39 specific T cells when tested on the
closely homologous but not identical mouse peptides
w22x. These findings support data from other laboratories
w39–41x, and predict that multiple cross-reactive selfligands can be identified between immunodominant
epitopes of autoantigens and any protein derived from
infectious pathogens. They provide direct evidence that
T cells apparently highly specific for a given peptide
ligand are also broadly cross-reactive to self-peptide
ligands, and illustrate that recognition of MHCupeptide
complexes by TCR is degenerate w41x. The model
also indicates that progression across checkpoints 1
and 2 (Fig. 1), namely T-cell activation by cognate
antigen and expansion of cross-reactive clones, is almost
simultaneous.
Studying the progression through disease
checkpoints in the non-susceptible host
The experiments outlined above provided an experimental framework for exploring in the laboratory how
the immune system of an RA-non-susceptible host could
function in ways that might attenuate inflammatory
722
A. P. Cope
responses in synovial joints. The generation of transgenic
mice identical to HLA-DRab1*0401-expressing mice,
with the exception that they express an allele of HLADRB1*04 that is not associated with RA (DRab1*0402),
made it possible to address this w27x. A comparative
analysis between transgenic lines would also make it
possible to examine whether just four amino acid
differences, which distinguish DRB1*0401 from *0402
at codons 86, 69, 70 and 71 (Table 1), were sufficient
to alter the molecular interactions between APC and
antigen-specific T cells. Closer scrutiny of the sequence
and molecular models indicate that these four amino
acid changes would have several effects, including a
reduction in the size of the P1 peptide-binding pocket
(which may influence TCR recognition), and changing
the charge preference for peptide residues sitting in
pocket 4 from negative to positive w42x. Molecular
modelling predicts that gp-39 epitopes presented by
DRab1*0402 should carry small aliphatic hydrophobic
residues at P1, and would exclude positively charged
residues at P4 w15x. As well as studying the molecular
nature of immunodominant peptides processed and
presented by DRab1*0402-expressing APC in vivo, it
would also be possible to evaluate whether there was
evidence of an attenuated effector response in T cells
from transgenic mice carrying the non-associated
DRab1*0402 genotype, and what immunoregulatory
mechanisms were in evidence, if any.
Do HLA-DRab1*0402 molecules present different
epitopes to CD4+ T cells in vivo?
Using HCgp-39 as the model antigen, immunization of
HLA-DR4-transgenic lines expressing DRab1*0402
revealed that this molecule, differing from DRab1*0401
by only four amino acids, presents quite distinct sets of
immunodominant peptides of HCgp-39 to CD4+ T cells
in vivo w27x (Table 2). As observed for DRab1*0401
epitopes, these peptides did not carry the expected
charged residues at P4. On the other hand, DRab1*0402,
which has a smaller P1 pocket, bound by Val rather than
Gly at position b86, favoured binding of peptides with
smaller aliphatic residues at P1 (Val or Leu), as predicted
from the algorithm of Sinigaglia and Hammer w15x.
Because experiments have revealed that there exists in
DRab1*0401 mice a repertoire of T cells capable of
recognizing 298–313 peptide w27x, one possible explanation for these findings could relate to differences in
antigen processing and presentation of this peptide between
DRab1*0401 and *0402-expressing APC. Indeed, collaborative studies with the laboratory of Dr E. Mellins
suggest that dependence on HLA-DM for processing of
HCgp-39 peptides may explain some of these differences,
at least in part w43x. For example, data published by Hall
and colleagues w44x since this essay was first submitted
have confirmed a relationship between immunogenicity
of HCgp-39 peptides and MHC–peptide stability, dictated
by the relative sensitivity to HLA-DM editing at pH5.5,
the pH of the peptide loading compartment. Regardless
of the mechanisms, these data provided evidence that
non-associated alleles of HLA-DRB1 profoundly influence the molecular nature of the interactions between
TCR and peptide–MHC complexes.
Is the cytokine effector response different for
HLA-DRab1*0402-restricted T cells?
The effects of HLA-DRab1*0402 on this particular
disease checkpoint were addressed by comparing cytokine expression of draining lymph-node T cells from
DRab1*0401 and *0402 mice immunized with HCgp-39,
following restimulation with native antigen or the
relevant pool of immunodominant epitopes w27x. The
results were interesting in several respects. While T-cell
proliferation was no different for DRab1*0401 and
*0402-restricted responses, HCgp-39-specific lymphnode T cells from DRab1*0402-transgenic mice produced significantly less interferon c (IFN-c) w27x. On the
other hand, DRab1*0402 T cells specific for OspA
produced abundant IFN-c, indicating that DRab1*0402
mice did not have an intrinsic defect of IFN-c production. These data provided the first real clues that one
of the consequences of DRab1*0402 presenting different peptides was to induce a very different profile of
proinflammatory cytokine production. The finding of
substantially reduced TNF-a levels in DRab1*0402
cultures is also consistent with this model w27x. The
results are also compatible with models which suggest
that, unlike disease-associated HLA-DR4 molecules,
non-associated HLA-DR4 molecules, such as DRab1*0402,
could provide disease-protective effects by altering the
peptides presented to T cells and the cytokines produced
as a consequence of these molecular changes (checkpoint
4, Fig. 1). Such a notion was proposed many years ago
to account for the sequence differences in diabetessusceptible and non-susceptible alleles w45x. The data also
substantiate the view that overproduction of proinflammatory cytokines early in the evolution of an immune
response may be central to the subsequent progression of
the inflammatory process (checkpoint 3, Fig. 1). They
are consistent with the hypothesis that the proinflammatory cascade is attenuated in the non-susceptible host
through the expression of a subset of HLA-DRB1 genes
which profoundly influence the molecular nature of
CD4+ T cell–APC interactions and immune effector
responses (checkpoint 4, Fig. 1).
The chronic phase of inflammation
Much attention has focused on the role of cytokines in
the pathogenic events that ultimately result in cartilage
destruction and bone erosion wreviewed in 46x. The
experiments described above demonstrated that the
initial stimulation of T cells by complexes of antigenic
peptide and disease-associated, but not non-associated,
MHC class II molecules could profoundly influence the
expression of proinflammatory cytokines. This cytokine
drive may well be central to sustaining the chronic phase
of disease, because in mutant mouse models it is possible
to induce chronic inflammatory disease in the absence
Immunity and inflammation in chronic inflammatory arthritis
of functional T and B lymphocytes by sustained overexpression of proinflammatory cytokines in vivo. These
mouse models have been highly informative. Nonetheless, for RA patients with an intact adaptive immune
system, studying the reciprocal relationship between
inflammation and immunity and exploring how chronic
overexpression of cytokines might promote T-cell effector
responses became a priority.
T cells are chronically exposed to TNF in synovial
joints
My own studies of how the inflammatory milieu
influences the function of chronically activated T cells
were undertaken during my PhD studentship in the
Feldmann laboratory at the Kennedy Institute of
Rheumatology. I began to explore how the chronic
inflammatory process influenced the differentiation and
effector functions of chronically activated T cells in the
belief that the inflammatory environment in established
chronic inflammation might be quite different from that
which influences the very earliest events of T-helper
cell differentiation. At the time, I set out to test the
hypothesis that the inflammatory milieu promotes the
inflammatory response by enhancing T-cell proliferative
and cytokine responses. The results were unexpected,
and were partly a consequence of the experimental
design. The approach I chose was based on three
fundamental experimental observations. First, proinflammatory cytokines, such as TNF and interleukin 1
(IL-1), were expressed and up-regulated at the protein
and mRNA levels in synovial tissue from RA patients
w47–49x. Secondly, experiments in the laboratory had
confirmed that infiltrating mononuclear cells expressed
cognate cytokine receptors w50x, which were preferentially up-regulated on synovial compared with peripheral
blood cells w51x, and that ligand colocalized with cells
expressing the cognate receptor w52x. This was most
comprehensively documented for the prototypic inflammatory cytokine TNF, at a time when the possibility that
TNF could be implicated in the chronic phase of RA
pathogenesis was only beginning to emerge w53x. Thirdly,
I demonstrated for the first time that expression of the
naturally occurring TNF inhibitors, the soluble TNF
receptor (TNF-R; p55 and p75 sTNF-R), were also
substantially increased at the site of inflammation w54x.
However, despite levels 3- to 5-fold higher in the synovial
joint fluid compared with serum, levels were insufficient
to neutralize completely the biological effects of TNF
in the joint. Thus, the possibility that activated mononuclear cells, including T cells, were exposed to cytokines such as TNF for prolonged periods at sites of
inflammation in vivo was beyond doubt.
Chronic TNF induces reversible, non-deletional
T-cell hyporesponsiveness
I then set about trying to mimic chronic TNF exposure
in an in vitro model, and subsequently in vivo. The
results, summarized in Table 3, are derived from an
extensive analysis of human and murine T-cell lines and
clones in vitro, and from experiments in vivo undertaken
723
TABLE 3. TNF induces non-deletional and reversible T-cell hyporesponsiveness in vitro and in vivo
Chronic exposure to TNF suppresses T-cell activation
Proliferation and cytokine production (Th1 and Th2 cytokines)
Dose- and time-dependent
Observed at non-toxic concentrations of TNF
IL-2 and mitogen responses are spared
Inducible over days
Shedding of soluble TNF-R is impaired
Rapid reversibility in vitro and in vivo on withdrawing TNF
Data are derived from an extensive analysis of T-cell responses in man
and transgenic and mutant mice. For more detailed review see reference 75.
in TCR-transgenic mice treated with recombinant TNF
or anti-TNF, or after intercrossing to human TNF
(hTNF)–globin-transgenic mice, as well as in p55 and
p75 TNF-R deficient mice w55–57x. The principle
finding—that chronic TNF suppressed T-cell activation—was unambiguous, and could not have been
predicted from published data at that time, which
suggested that TNF was costimulatory and a growth
factor for T cells w58x. Moreover, the p55 TNF-R
appeared to be necessary and sufficient for sustained
TNF signalling to induce T-cell hyporesponsiveness.
T cells from patients with RA are hyporesponsive to
TCR engagement
While histological and flow-cytometric analysis of
RA synovial T cells and knowledge of the HLA class
II associations discussed above have done much to
perpetuate the notion that a chronic antigen-driven
immune response is central to the disease process w13,
59x, functional studies of synovial T cells have continued
to perplex workers in this field. The laboratory of Panayi
was one of the first to appreciate the potential importance of the finding that proliferative T-cell responses to
recall antigens, such as purified protein derivative (PPD),
or to mitogens or following cross-linking of the TCR,
were dramatically attenuated w60x. Since then a hierarchy
has been documented from peripheral blood to synovial
tissue, in increasing order of hyporesponsiveness, suggesting not only that loss of T-cell reactivity is acquired,
but also that this phenotype may be induced by the
inflammatory process itself w61, 62x. According to the
results described above, chronic exposure to TNF could
provide one possible mechanism for this effect.
Chronic TNF exposure uncouples proximal TCR
signal transduction pathways
To explore this at the molecular level, a mouse T-cell
hybridoma model has been studied in my laboratory,
because with this model it is possible to reproduce many
of the functional T-cell defects of primary T cells chronically exposed to TNF in vivo, but in the absence of
accessory cells w63x. Several lines of experimental evidence suggested that chronic TNF exposure impairs T-cell
activation by attenuating TCR signalling pathways
w63x. First, T cells chronically exposed to TNF required
more peptide for longer periods to commit to IL-2
production, suggesting that the threshold of activation
724
A. P. Cope
was dramatically altered. Secondly, cell surface TCR
expression was reduced, indicating that not only did
chronic TNF uncouple TCR signalling pathways, but
also that TNF interrupted assembly of the TCR–CD3
complex and its transport to the cell surface. Thirdly, a
detailed biochemical analysis of proximal TCR signalling pathways in TNF-treated T cells confirmed that,
through persistent expression and signalling of inflammatory mediators such as TNF, the chronic inflammatory process was capable of uncoupling antigen receptor
signalling directly.
In the light of this, it became imperative to explore
how chronic TNF targeted the expression of the TCR–
CD3 complex, and what effect this might have on
downstream signalling pathways (for a schematic of
TCR–CD3 assembly and signalling pathways, see
references 64 and 65 and Fig. 5). TCR-z was one
possible target of TNF. Indeed, following closely the
kinetics of IL-2 down-regulation, western blotting analysis
of whole-cell lysates revealed that chronic TNF exposure
suppressed the expression of TCR-z in a dose- and timedependent fashion, while levels of CD3-e, -c and -d, as
well as the protein tyrosine kinases ZAP-70, p56Lck and
p59Fyn, were not altered w63x. By immunoprecipitation
analysis it was possible to demonstrate that cell surface
CD3-e was in fact reduced compared with levels in
immunoprecipitates derived from whole cell lysates. This
was consistent with a model in which TNF appeared
to disrupt the assembly anduor cell surface stability of
TCR–CD3 complexes through its effects on TCR-z
expression w63x.
A second unexpected experimental observation provided further evidence that persistent TNF signalling
in T cells influenced TCR–CD3 complex assembly and
stability at the T-cell surface. Immunoblotting analysis
of unstimulated and TNF-stimulated T cells revealed
that the expression of the novel transmembrane adapter
TRIM (T-cell receptor interacting molecule) was markedly
down-regulated by TNF treatment. Closer examination
demonstrated that TRIM expression was reduced by
TNF before changes in TCR-z expression could be
detected, and that reconstitution of both TRIM and
TCR-z expression was required to fully restore TCR
responsiveness in TNF-treated cells. The implications
of these findings have only recently become apparent,
through studies of TRIM expression in human peripheral blood and Jurkat T cells w66, 67x. In collaboration
with Dr Burkhart Schraven, we demonstrated that
FIG. 5. TCR signal transduction pathways. Polymorphic TCR-ab chains associate with the invariant chains (CD3-c, - d and -e and
TCR-z), consisting of non-covalently linked ce and de heterodimers and disulphide linked z–z homodimers, which transmit signals
inside the cell. TCR ligation leads to the phosphorylation of tandemly arranged tyrosine residues within immunoreceptor tyrosinebased activation motifs (ITAMs) of TCR-z chain and CD3-c, -d and -e chains by src family kinases, notably Lck and Fyn. The
phosphorylation of adapter proteins, such as p36LAT by ZAP-70 and src kinases, serves as a crucial link between membrane
proximal phosphorylation events and the activation of downstream RasuMAPK (ERK) and calcium signalling pathways. Sustained
activation of these pathways is required for transactivation of the transcription factor AP-1 complex and NFAT, leading to IL-2
production, T-cell proliferation and effector responses. LAT, linker for activation of T cells; MAPK, mitogen-activated protein
kinase; AP-1, activating protein 1; NFAT, nuclear factor of activated T cells; PLC, phospholipase C; PIP, phosphoinositol
phosphate; InsP3, inositol triphosphate; DAG, diacylglycerol; PKC, protein kinase C; SLP-76, SH2-domain containing leucocyte
protein of 76 kDa; Vav, a Rac/Rho-specific guanine nucleotide exchange factor; SOS, son of sevenless.
Immunity and inflammation in chronic inflammatory arthritis
TRIM plays a role in regulating the cycling of TCR–
CD3 complexes at the cell surface w67x. For example, the
half-life of TCR–CD3 complexes in stable Jurkat clones
overexpressing TRIM is increased. This in turn leads to
increased cell surface expression of TCR and enhanced
signalling responses, as determined by intracellular
calcium mobilization w67x. We can speculate from these
experiments that sustained TNF signals in T cells could
impair TCR–CD3 assembly not only through its effects
on TCR-z expression, but also by reducing the half-life
of assembled complexes at the cell surface by downregulating the expression of TRIM. The kinetics of these
changes, as well as the precise interactions between
TCR-z and TRIM, are currently under investigation.
Nevertheless, the findings provided a molecular basis for
the profound hyporesponsiveness of T cells following
TNF stimulation, and predicted that downstream TCR
signalling pathways might be significantly attenuated as
a result.
Selective uncoupling of downstream TCR signalling
pathways by TNF
A comprehensive analysis of signalling pathways in
control and TNF-treated T cells has been undertaken
to establish to what extent distal TCR signal transduction pathways were attenuated as a consequence of the
proximal defects outlined above. As a direct consequence of TCR-z phosphorylation being reduced in
TNF-treated T cells, downstream signalling events, such
as recruitment of ZAP-70 to phospho-TCR-z through its
SH2 domains and its subsequent phosphorylation, were
also impaired w63x. The transmembrane adapter protein
linker for activation of T cells (LAT) is an in vivo substrate for ZAP-70 kinase, and plays a key role in linking
membrane proximal events with both calcium and
RasuMAPK (mitogen-activated protein kinase) pathways w68, 69x (Fig. 5x. LAT and PLCc1 phosphorylation
were substantially reduced in TNF-treated cells, and as
predicted intracellular calcium mobilization was also
dramatically attenuated w63x. On the other hand, Rasdriven pathways, including activation of the MAPK
ERK activation, c-Fos induction and TCR-induced CD69
expression, were preserved (J. Clark, K. Aleksiyadis
and A. Cope, unpublished data). These findings identify
a novel molecular basis for T-cell hyporesponsiveness.
They are of interest because attenuation of intracellular
calcium mobilization and preservation of RasuERK
(extracellular signal-regulated kinase) signalling is the
reverse of the aberrant TCR signalling pathways defined
in anergic T cells by other laboratories, whereby calcium responses are spared and RasuERK signals are
attenuated w70, 71x.
TCR signalling is also defective in RA synovial
T cells
Impaired TCR signalling has been well documented in
RA synovial T cells. Down-regulation of TCR-z chain
expression and abnormal recruitment and phosphorylation of p36 LAT have also been documented w72, 73x.
Significantly, calcium mobilization is also reduced in
725
peripheral blood as well as synovial T cells from patients
with RA w74, 75x, while unpublished data indicate
that Ras activation is up-regulated in synovial T cells
(C. Verweij, personal communication). On the basis of
our own studies, TNF could be one of several factors,
including other cytokines or reactive oxygen species,
responsible for the uncoupling of TCR signals in chronic
inflammatory diseases such as RA in man w55, 76x.
Studying T-cell hyporesponsiveness in the mouse:
lessons from in vivo models
The precise significance of T-cell hyporesponsiveness in
the context of chronic inflammatory disease in vivo is not
clear. Nevertheless, the distinctive cell phenotypes shared
by RA synovial T cells and T cells chronically exposed to
TNF in vitro or in vivo (summarized in Table 4) have
made it possible to draw some tentative conclusions
based upon results derived from experiments in the
mouse. I have recently reviewed the possible contributions that hyporesponsive T cells could make to the
pathogenesis of autoimmune disease w32, 76, 77x; these
are illustrated in Fig. 6. Several points are worthy of
note. Jacob and McDevitt w78x first demonstrated that
TNF therapy was beneficial in a murine model of lupus.
Similar results followed in the NOD mouse w79, 80x, a
model of spontaneous type I diabetes, and subsequently
TABLE 4. Chronic exposure of T cells to TNF induces a phenotype
resembling CD4+ T cells derived from inflamed RA synovial joints
Up-regulation of activation antigens
Induction of proliferative hyporesponsiveness
Suppression of T-cell cytokine production
Repression of CD28 gene transcription
Uncoupling of proximal TCR signal transduction
FIG. 6. Possible mechanisms through which hyporesponsive T
cells could contribute to the pathogenesis of chronic inflammatory arthritis. Hyporesponsive T cells could reflect an
adaptive response aimed at suppressing T-cell autoreactivity.
According to this model, T cells would play little or no role in
promoting disease, since hyporesponsiveness constitutes a
protective response (left panel). Alternatively, impaired T-cell
activation could promote and perpetuate the chronic inflammatory response, through loss of tolerance mechanisms that
require intact TCR signalling pathways (right panel). Host
defence would be compromised through similar mechanisms.
726
A. P. Cope
in studies of experimental autoimmune encephalomyelitis
w81x, the murine counterpart of multiple sclerosis in man.
My own studies using similar treatment protocols, but in
a TCR-transgenic mouse model, demonstrated conclusively that TNF could suppress T-cell effector responses
in vivo, either when TNF was administered intraperitoneally or when TCR-transgenic mice were crossed to
human TNF–globin transgenic mice w57x. The finding
that neutralizing TNF in healthy TCR-transgenic mice
enhanced T-cell responses provided the first evidence
that physiological expression of this cytokine could
suppress T-cell responses in vivo. More recently, the
outcome of disease progression in autoimmune-prone
mice carrying null mouse TNF or TNF-R (knockout)
genes has substantiated the view that TNF has potent
immunomodulatory effects in vivo w81–84x. These data
point to the possibility that the suppressive effects of
chronic TNF are protective, perhaps reflecting an adaptive response to inflammatory signals which attenuate
pathogenic T-cell effector responses w57, 76x (Fig. 6, left
panel).
Studying T-cell hyporesponsiveness in man: lessons
from the clinic
Back in 1992, I was provided with a unique opportunity
to test in patients whether TNF could suppress T-cell
activation in man. At this time, a cohort of RA patients
with active disease had been recruited to an open-label
trial of anti-TNF monoclonal antibody therapy winfliximab (Remicade2)x for the first time. By studying
peripheral blood T-cell proliferative responses to recall
antigens and mitogens before and after treatment, I
found that T-cell responses were dramatically restored
w55x. These findings were entirely in keeping with the
in vitro data w55x, and have since been reproduced by
several groups w85–87x, including a more recent study of
T-cell responses to influenza haemagglutinin peptide and
collagen II in RA patients treated with the p75 TNF-R–
Fc fusion protein etanercept (Enbrel2) w88x. Despite this
enhanced T-cell reactivity, multicentre trials of anti-TNF
have demonstrated conclusively a consistent and rapid
improvement in clinical parameters in ;70% of RA
patients w89–91x. Equally striking clinical responses
to anti-TNF have been documented in inflammatory
bowel disease, particularly Crohn’s disease w92, 93x.
These therapeutic responses have prompted an evaluation of TNF blockade in psoriatic arthritis, ankylosing
spondylitis and vasculitis.
While it has been postulated that the clinical response
reflects the potent anti-inflammatory properties of antiTNF, including effects on cell trafficking and angiogenesis w94, 95x, the sustained clinical response to a single
infusion of anti-TNF for periods that extend far beyond
the time point at which antibody is cleared from the
body w89x suggests that anti-TNF may induce lasting
immunomodulatory effects (Fig. 6, right panel). We are
currently exploring in the laboratory the possibility that,
through restoring thresholds of T-cell activation, the
immune system is reset, regaining the regulatory functions
of T cells which in the non-susceptible host effectively
suppress inflammatory responses. By restoring
the integrity of TCR signal transduction pathways,
autoreactive T cells would also acquire the capacity to
undergo activation-induced cell death. The finding that
gut CD4+ lymphocytes from patients with Crohn’s
disease undergo apoptosis following anti-TNF therapy
is consistent with this model w96x. This sudden increase
in apoptosis might also explain, at least in part, the
development of antinuclear antibodies in ;8% of
patients treated with anti-TNF w97x. On the other
hand, the concept that restoring T-cell responses would
enhance host responses to foreign pathogens, as well as
anti-tumour immunity, is more obvious. In diseasesusceptible individuals, therefore, sustained overexpression of TNF could contribute to disease pathogenesis
through effects which include attenuation of TCR
signalling. This might lead to the failure of immunoregulatory mechanisms critical for maintaining peripheral tolerance, such as activation-induced cell death and
immune deviation (Fig. 6, right panel). According to
the proposed model, TCR signalling and, by inference,
immunoregulation would be predicted to be intact in
non-susceptible individuals (checkpoint 4, Fig. 1).
Is a shift from ‘antigen mode’ to ‘inflammation
mode’ characteristic of effector T cells in chronic
inflammatory disease?
As TCR hyporesponsiveness in chronic inflammation
appears to correlate with disease severity anduor chronicity
regardless of immunosuppressive therapy w55, 61x, it is
difficult to reconcile the long-held belief that T-cell
effector responses in inflamed joints are exclusively antigendriven. Rather, data suggest that during the chronic
phase of the disease process it may be the cytokine milieu
that sustains and maintains pathogenic T cells w98, 99x.
According to this model there is a gradual decline of
antigen-induced proliferative responsiveness as cytokine
drive increases (Fig. 7). Nevertheless, antigen drive and
cytokine drive are inextricably linked in the sense that
the initial antigen response initiates both a cytokine
cascade and the acquisition of cytokine responsiveness
through up-regulation of cytokine receptors, while T-cell
hyporesponsiveness arises through subsequent sustained
expression of inflammatory cytokines (Fig. 7).
How could hyporesponsive T cells contribute to the
effector phase of inflammation? Or are they passive
bystanders? One possible explanation lies in the observation that depletion of hyporesponsive T cells from RA
synovial mononuclear cell cultures significantly reduces
the expression of proinflammatory cytokines w100x. Evidence suggests that this process of activation may be
mediated by cell-to-cell contact, by as yet poorly defined
receptor–ligand interactions w101–103x. Failure of synovial joint T cells from RA patients to undergo apoptosis
could further facilitate cell-to-cell signals and perpetuate
the inflammatory response w98x. Perhaps B-cell help and
autoantibody production is enhanced through similar
cell-to-cell mechanisms wreviewed in 104x. In order to
Immunity and inflammation in chronic inflammatory arthritis
FIG. 7. A model for the role of CD4+ T cells in the
pathogenesis of chronic inflammation. Antigen drive may
predominate during the early phase of inflammatory
responses. T cells could be recruited through bystander
activation, or by stimulation with self antigens released from
inflamed tissues. As the inflammatory process progresses,
chronic cytokine production induces profound non-deletional
T-cell hyporesponsiveness. Hyporesponsive T cells function as
effector cells and sustain the chronic inflammatory process
through mechanisms less dependent on antigen signals (see
text). It is proposed that, by reversing T-cell hyporesponsiveness, antigen-dependent responses that serve to regulate the
inflammatory process (e.g. through expression of immunoregulatory cytokines) are restored.
address this issue specifically, we have recently undertaken gene expression profiling of control and TNFtreated mouse T cells. Preliminary data suggest there is a
TNF-induced gene expression signature which, in vivo,
would be predicted to favour Th1 cell differentiation
[induction of interferon regulatory factor-1 (IRF-1), IL12Rb1 and IFN-cR genes], recruitment of T cells to
inflamed joints wfucosyltransferase VII, interferongamma-inducible protein 10 (IP-10) and RANTES (regulated upon activation, normal T-cell expressed and
secreted cytokine)x, effector responses initiated through
cell surface ligands such as RANK (receptor activator of
nuclear factor kB) ligand and cell survival (induction of
anti-apoptotic genes cIAP-2, A20 and Bis). These may
turn out to be important results because if the data can
be reproduced in vivo they imply that hyporesponsive
synovial T cells are not inert, but have the potential to
become potent effector cells through dominant cytokinedriven signal transduction pathways. Selective uncoupling of TCR signalling and sparing of antigen-specific
RasuERK activation would contribute significantly to this
phenotype by promoting cell survival and the persistence
of activated cells in inflamed joints.
Concluding remarks
Confirming unequivocally that hyporesponsive T cells
are capable of promoting the inflammatory process in
RA is a priority because an understanding of the precise
mechanisms involved could provide a molecular basis
for testing hitherto unexplored immunotherapeutic
727
strategies. Studying the effects of suppressing the Rasu
ERK pathway is just one example. To date, the assumption has been that T cells in RA are harmful, because
effector responses are sustained by chronic activation by
tissue-specific antigens presented by disease-associated
HLA class II molecules (Fig. 1). The assumption that
synovial T cells are harmful is probably correct, but
perhaps for the wrong reasons. For example, T-celltargeted strategies have focused almost exclusively on
depressing T-cell function further. The results have been
disappointing w105, 106x, perhaps because the calciumucalcineurin pathway and transactivation of nuclear
factor of activated T cells (NFAT) are already profoundly depressed in cytokine-activated T cells. However,
results from several laboratories, including my own,
suggest that therapeutic strategies aimed at restoring the
function of subsets of T cells should now be considered.
A major challenge will be to identify which specific T-cell
subsets in vivo are hyporesponsive. For example, if these
subsets include regulatory T cells, such as IL-10producing Tr1-like CD4+ cells or IL-16-producing
CD8+ T cells w107, 108x, restoring TCR responsiveness
could suppress the inflammatory activities of offending effector T-helper cell subsets, macrophages and
fibroblasts.
This therapeutic approach is entirely in line with the
thesis proposing that susceptibility to autoimmunity
arises through failure of the adaptive immune system to
regulate the inflammatory response. In the short term,
restoring T-cell responsiveness towards ‘normal’ (but
not beyond) could be achieved by a combination of
immunotherapeutic approaches targeting the peptide or
altered peptide ligand specificities of regulatory T cells
(which could be determined in HLA class II-transgenic
mice as described above) together with anti-TNF.
This approach would provide anti-inflammatory cover,
while at the same time enhancing responsiveness to
the therapeutic peptide by restoring TCR signalling
pathways. A detailed knowledge of the specific TCR
signalling defects could provide clues as to how best to
monitor such therapy, and how to avoid rebound T-cell
hyper-reactivity and its inevitable consequences, systemic
autoimmune disease and acute cytokine release syndromes. Anti-TNF in combination with non-depleting
anti-CD4 or anti-CD3 monoclonal antibody could
have similar beneficial therapeutic effects w109x. If, on
the other hand, this hypothesis is incorrect and T-cell
hyporesponsiveness turns out to be beneficial to the
disease process, then a more precise understanding of
how the inflammatory process uncouples signalling
pathways in cytokine-activated T cells would greatly
facilitate the development of novel immunosuppressive
agents.
Acknowledgements
I gratefully acknowledge Professors Marc Feldmann,
David Wallach and Hugh McDevitt, in whose laboratories
728
A. P. Cope
much of the work was undertaken, my mentor Professor
Sir Ravinder Maini for continued support, and members
of the Cope Laboratory who are continuing with this
work. These studies would not have been possible
without the support of the Wellcome Trust over the
last 12 years, and the Arthritis Research Campaign.
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