Rheumatology 2002; 41: 857-868
© 2002 British Society for Rheumatology
HLA B27 in health and disease: a double-edged sword?
The 2000 Michael Mason Prize Essay
P. Bowness
Nuffield Orthopaedic Hospital, Windmill Road, Headington, Oxford OX3 7LD, UK
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Abstract
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The strong association of the HLA class 1 allele HLA B27 with
ankylosing spondylitis (AS) has been recognized for over 25
yr, however the pathogenic mechanism linking HLA B27 with AS
and other spondyloarthropathies remains a mystery. We now know
that the principal natural function of HLA B27 is an immunologic
one, namely to bind antigenic peptides and then present them
to T lymphocytes. I have shown that HLA B27 functions as an
excellent antigen-presenting molecule in both spondyloarthropathy
patients and healthy individuals. A working molecular model
of how T cells recognize HLA B27 has been generated and tested.
Evidence that T cells have a role in spondyloarthritis has also
been found. First, expanded populations of T lymphocytes were
found in both the blood and synovial fluid of patients with
reactive arthritis (ReA). Secondly, a strong cytotoxic T-cell
response to an HLA B27-restricted peptide epitope from
Chlamydia trachomatis was found in a patient with ReA. This peptide, derived
from a bacterium known to trigger ReA, is thus a candidate ‘arthritogenic’
peptide. We have also found evidence that HLA B27 has an unusual
cell biology compared with other HLA molecules. HLA B27 demonstrates
an unusual ability to form heavy chain homodimers
in vitro.
Dimerization is dependent upon disulphide bonding through an
unpaired cysteine at position 67. Remarkably these dimers lack
ß2 microglobulin, previously thought to be an essential
component of all mature MHC class 1 molecules. HLA B27 homodimer
formation has also been demonstrated in certain cell lines
in vivo, and preliminary data suggest that significant numbers
of T cells from patients with spondyloarthropathy express a
ligand for HLA B27 homodimers. These findings have extended
our understanding of the beneficial immunologic function of
HLA B27, and have also led us to propose the testable new hypothesis
that HLA B27 heavy chain dimerization may be involved in the
pathogenesis of spondyloarthritis.
KEY WORDS: HLA B27, Reactive arthritis, Ankylosing spondylitis, Immunology.
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Introduction
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Although the strong association of possession of the human leucocyte
antigen HLA B27 with the spondyloarthropathies (Table 1
) has
been recognized for over 25 yr [
1], the pathogenic link remains
elusive. Numerous hypotheses have been proposed to explain the
association of HLA B27 with spondyloarthritis, and are summarized
in Table 2
. The theories proposed derive varying support from
epidemiological studies and from animal models of disease [reviewed
in 2]. However the development of a disease resembling human
spondyloarthritis in HLA B27-transgenic rats and mice provides
direct evidence that HLA B27 itself is involved in disease pathogenesis
[
3,
4]. Since the principle natural function of HLA class 1
molecules is to bind and present short antigenic peptides to
cytotoxic T lymphocytes (CTL) [
5], Benjamin and Parham [
6] have
suggested that HLA B27 might predispose to spondyloarthritis
by binding one or more specific ‘arthritogenic’
peptides and stimulating arthritogenic T cells. The identification
of different molecular subtypes of HLA B27, which differ in
the amino acid composition of their peptide-binding groove [
7,
8], has led to studies of the disease association and peptide-binding
specificity of different subtypes. There is some evidence that
subtypes such as HLA B*2706 and *2709, which preliminary epidemiological
studies suggest are not associated with ankylosing spondylitis
(AS) [
9], may bind a subtly different set of peptides. If confirmed,
these data would support a role for peptide binding in disease
pathogenesis. Alternatively, the cell biology or immunogenicity
of HLA B27 may be affected by the presence of an unpaired cysteine
at position 67 of the
1 helix. We have recently provided new
data suggesting that HLA B27 does indeed have an unusual cell
biology (see below), and this would be consistent with data
from HLA B27 transgenic mice implicating an abnormal form of
HLA B27 molecules in disease. There is no clear evidence supporting
antibody cross-reactivity or receptor-mediated mechanisms. Superantigens
are microbial proteins capable of stimulating large numbers
of T cells, and are capable of stimulating autoimmune reactions
[
10]. The bacteria implicated in reactive arthritis (ReA) have
not been found to produce superantigens [
11], although interestingly
another gut microorganism,
Clostridium perfringens, has [
12].
HLA B27 is itself capable of providing a source of peptides
which in turn can be presented by HLA class 2 molecules to T
cells [
13,
14]. This intriguing observation undoubtedly requires
further study.
Strikingly, HLA B27 remains common in many populations, in a
distribution which is probably not compatible with a genetic
founder effect. The underlying thesis of this essay is that
some particular function or functions of HLA B27 have conferred
some selective advantage to HLA B27 positive individuals, at
least until recent evolutionary time. This function is likely
to be immunologic, very probably conferring on HLA B27 the ability
to present pathogen-derived antigenic epitopes to T cells either
more rapidly or more effectively than other HLA class 1 alleles.
This same feature most likely also predisposes to spondyloarthritis.
Thus, a proportion of HLA B27 positive individuals pay a price
for carrying this ‘super’ HLA allele, making this
molecule a powerful double-edged sword.
With the premise that by understanding the natural function of HLA B27, we may ultimately come to better understand its role in disease, I have set out to answer the following questions:
- (i) What determines the peptide-binding selectivity of HLA B27? How do T cells recognize this HLA B27/peptide combination and does HLA B27 function differently in patients with spondyloarthritis, compared with ‘healthy’ HLA B27 positive individuals?
- (ii) Are T lymphocytes indeed involved in ReA, one of the spondyloarthritides?
- (iii) Is there evidence that T cells from individuals with ReA recognize potentially ‘arthritogenic’ peptides?
- (iv) Is the cell biology of HLA B27 unusual, and does the unpaired cysteine at position 67 contribute to this?
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Understanding the natural function of HLA B27
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HLA B27 selectively binds peptides and presents them to the T-cell receptor (TCR) of CTL
In order to try and understand the rules governing the molecular
function of HLA B27, I initially chose to study the role of
HLA B27 in the immune response to influenza A virus, a common
virus responsible for many millions of deaths this century.
At the start of this work the natural role of MHC class 1 molecules
had been shown to involve the binding of short intracellular
peptide antigens and their subsequent presentation to CTL at
the cell surface (reviewed in [
15]). Huet
et al. [
16] had identified
a single antigenic epitope in the ‘flu nucleoprotein that
could be recognized by HLA B27 positive individuals. This epitope,
NP383–391, comprises nine amino acids, SRYWAIRTR in single
letter code. I developed an
in vitro HLA B27-binding assay in
which synthetic NP383–391 or substituted analogues differing
at single amino acid residues were added to lysates of radiolabelled
HLA B27-transfected T2 cells. This cell line lacks a functional
peptide transport mechanism and is thus unable to load endogenous
peptides into nascent HLA B27 molecules. However, exogenously
supplied peptides can promote assembly and folding of mature
HLA B27/
ß2m/peptide complexes. Results can be quantified
following immunoprecipitation with the conformation-specific
monoclonal antibody W6/32. I was able to show that binding to
HLA B27 was abrogated by substituting the arginine residue at
position 2 for leucine (the R2L substitution, see Table 3
).
The tyrosine at position 3 could be substituted to leucine (Y3L)
and the resultant peptide could not only bind to HLA B27 (Table
3
), but was also recognized by T cells raised against the native
peptide (Table 4
) [
11,
17]. However the position 3 substitution
to glutamic acid (Y3E) does not bind to HLA B27 and of course
is not recognized by CTL. These findings were in agreement with
crystallographic data showing that the peptide P2 arginine side
chain was tightly bound in a pocket under the HLA B27
1 helix
(the ‘B’ pocket) [
18,
19], and with the findings
that pooled peptides eluted from HLA B27 [
20] (but not from
other MHC alleles [
21]) shared an arginine residue at their
second position. Based upon these data, and my results for T-cell
recognition of NP peptide analogues (see below), I proposed
a model predicting which side chains of a peptide antigen would
be important for binding to HLA B27 and which would be critical
for recognition by T cells. The latter I called ‘flag’
residues [
11,
17]. A schema for this model is shown in Fig.
1
. The data on T-cell recognition derive from a panel of CTL
clones from the blood of influenza-exposed HLA B27-positive
patients and controls [
11,
17]. These CTL clones specifically
recognized and killed influenza-infected or NP383–391-pulsed
HLA B27-positive target cells [
11,
17,
22]. An example of such
a CTL assay is shown in Fig. 2
. The CTL clone SD17 kills HLA
B27-positive target cells pulsed with the NP383–391 peptide
(shown in black, dotted line). However, an analogue peptide
in which the position 4 tryptophan (W) is substituted to tyrosine
(W4Y) is recognized only weakly, and one with a position 4 phenyl
alanine (W4F) is not recognized at all. The latter peptide only
differs from the W4Y peptide by a single hydroxyl group. Thus,
this CTL clone has exquisite sensitivity for even the most subtle
of changes at peptide P4 residue. Data for recognition of different
NP383–391 analogues by six different CTL clones are summarized
in Table 4
. Positions 1, 4 and 8 of NP383–391 do not significantly
influence binding to HLA B27, but are critical for recognition
by the TCR. This paradigm has significant implications for ‘arthritogenic’
peptide models of disease pathogenesis, since it indicates rules
both for peptide binding to HLA B27, and for potential cross-recognition
of different peptides by the same TCR. Thus, it can be used
both to identify HLA B27-binding peptides (see below) and to
predict potential cross-reactive T-cell epitopes.
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FIG. 1. Cartoon schema showing binding of antigenic peptides to HLA B27. The side chains of ‘anchor’ residues of bound peptide are located within pockets surrounding the peptide-binding groove. In contrast, the side chains of ‘flag’ amino acids are orientated towards and contacted by the TCR of T lymphocytes recognizing the HLA B27/peptide combination (after Bowness et al. [22]).
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FIG. 2. Amino acid substitutions of the peptide fourth position greatly reduce or abrogate CTL recognition of NP383–391.
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HLA B27 is an excellent antigen-presenting molecule in both spondyloarthropathy patients and healthy individuals
In order to define the molecular rules for the recognition of HLA B27 by T cells, and to look for qualitative differences in the immune responses of patients with spondyloarthritis and healthy individuals, I sequenced the TCR genes of nine HLA B27-restricted NP383–391-specific CTL clones. Four clones were derived from two patients with spondyloarthropathy and five from a healthy HLA B27-positive control [22]. TCRs, like antibodies, are generated by combinatorial recombination of germline gene segments which, together with other mechanisms including random nucleotide addition at joining regions, generates a hugely diverse T-cell repertoire. This is shown schematically in Fig. 3, together with a cartoon representation of the mature TCR protein. Table 4
summarizes the TCR - and ß-chain usage of six of these clones, as well as showing the predicted amino acid usage and length of their third hypervariable regions [22]. Two important conclusions arise from comparison of these sequences. First, NP-specific HLA B27-restricted TCR use highly conserved TCRs, with the pattern of conservation (if not the particular segment used) suggesting a common rule for MHC class 1-restricted responses [22]. Thus, most TCR recognizing HLA B27/NP used a single Vß gene segment (Vß7.1 or using the revised WHO nomenclature BV7S1), and one of two V or J segments. This bias is not found in peripheral blood T lymphocytes from patient GR [22], implying antigen-driven selection. Secondly, the TCRs of NP383–391-specific HLA B27-restricted CTL from healthy and diseased individuals have almost identical predicted amino acid sequences [22]. This shows that the HLA B27 molecule in patients with spondyloarthritis is capable both of presenting a viral peptide and of generating a qualitatively similar T-cell response to that seen in healthy HLA B27-positive individuals. These molecular data are supported by functional recognition data both ex vivo and in vitro, with both patients and controls making strong CTL responses to influenza virus. These responses are HLA B27-restricted and always directed against NP383–391 [11, 17, 22]. I subsequently cloned the TCR from an HLA B27-restricted NP383–391-specific CTL clone (GRb), derived from a patient with spondyloarthritis, into a reporter cell line RBL-2H3. Thus, it could be formally proved that the HLA B27/NP383–391 complex was both necessary and sufficient for specific immune recognition by this TCR [11, 23]. This cell line was used to generate the monoclonal antibody to the human TCRß gene segment TCRVB7S1 [24]. The importance of the TCRVB7S1 gene segment in the human HLA B27/NP immune response has subsequently been confirmed using this antibody [25]. Unfortunately, the hope that such a reagent might prove invaluable for identifying HLA B27-restricted T cells in general (including those recognizing ‘arthritogenic’ peptides) was dashed by the finding that HLA B27-restricted CTL specific for an HIV peptide use completely different TCRVB segments [11]. Thus, TCRs that recognize different peptides bound to HLA B27 use different TCR gene segments, even though a significant part of their interaction is directly with the HLA B27 -helices.
Towards a molecular model of HLA B27 recognition by T cells
I was able to predict the exact orientation in which the TCR recognizes HLA B27/NP383–391, by correlation of the fine specificity of CTL clones for P1 peptide analogues with their TCR -chain usage [17]. This predicted orientation (i.e. with the TCR -chain docking with the N-terminal part of bound peptide and ß chain with C terminal) has subsequently been found in the two crystal structures of TCRs complexed with MHC class 1 [26, 27]. Dr E. Y. Jones has created a detailed molecular model showing how the GRb TCR docks with the HLA B27/NP383–391 complex, using these data together with the atomic coordinates HLA B27 [18, 19], and of the 2C TCR [26]. This model is shown in Fig. 4, and suggests that the TCR -chain plays an important role in the recognition both of the NP position 4 side chain and also of the HLA B27 1 helix. This model must of course be regarded as hypothetical, particularly since the 2C TCR is not HLA B27 restricted. However, it is supported by experiments in which site-directed mutation of certain -chain residues of the GRb TCR, predicted to make important interactions with either the HLA B27 helices or bound peptide, did indeed abrogate recognition of HLA B27/NP383–391 by the transfected GRb TCR [23].
These experiments have given us a clear idea of the molecular
anatomy of recognition of a particular HLA B27/peptide combination
by T cells. This knowledge could be used to design potentially
therapeutic peptides capable of binding tightly to HLA B27,
and possibly also of antagonizing subsequent T-cell recognition.
In summary, these studies show that HLA B27 binds peptides according to well defined molecular rules, and presents them to the TCRs of CTL. HLA B27 is an excellent antigen-presenting molecule in both spondyloarthropathy patients and healthy individuals. A working molecular model of HLA B27/peptide recognition by T cells has been generated.
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Are T lymphocytes involved in ReA?
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If the pathogenic role of HLA B27 in the spondyloarthropathies
is to present ‘arthritogenic’ peptides to CTL, one
would expect to find evidence of expanded populations of CD8+
T cells within the joints of patients with spondyloarthritis.
Whilst a few such HLA B27-restricted CTL clones have been identified
[
28], most studies of synovial fluid in patients with ReA have
found predominant HLA class 2-restricted CD4+ responses to the
triggering organisms [
29,
30]. Other groups have questioned
whether T cells are involved at all in the pathogenesis of ReA.
Therefore, we set out to study the T-cell repertoire of patients
with ReA, using monoclonal antibodies to the TCR. This technique
has the advantage of giving quantitative and reproducible
ex vivo data, although it gives no information about T-cell specificity.
Paired samples of peripheral blood and synovial fluid lymphocytes
from 10 patients with ReA arthritis were stained with a panel
of 17 monoclonal antibodies to TCR V
ß families, together
with antibodies to CD4 or CD8. Significant T-cell expansions,
defined as being >3
S.
D. above the mean value for control
peripheral blood leucocytes (PBL), were observed in all patients.
Table 5
shows that expansions were found in both CD4+ and CD8+
populations, and in both synovial fluid and blood [
31]. The
greatest number of expansions was found in the synovial CD8
compartment. Although different V
ß expansions were
found in different patients, we did for example find TCRBV22
expansions in the synovial CD8 compartment of five out of six
patients with ReA following
Chlamydia infection [
31]. Although
the results of studies of synovial fluid TCR repertoire are
difficult to interpret (principally because of worries about
bias in T-cell recruitment or persistence, discussed in Bowness
and Bell [
32]), the presence of T-cell expansions in the peripheral
blood of patients with ReA is undoubtedly significant. Our results
are consistent with models in which both CD4+ and CD8+ T lymphocytes
are involved in disease, although they give little clue as to
pathogenic mechanisms. CD8+ T-cell expansions studied further
expressed both activation and memory markers. Perhaps more interestingly,
some of the predicted amino acid sequences of these CD8+ synovial
T cells [
33] show remarkable homology with those obtained by
other groups, possibly favouring an antigen-driven mechanism
(P. Bowness and E. May, unpublished results). It will be important
to compare the T-cell repertoire in ReA with that found in other
spondyloarthropathies such as psoriatic arthritis. The finding
of common patterns of TCR usage might point to common pathogenic
mechanisms.
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Does Chlamydia trachomatis, a bacterium that triggers ReA, contain potential HLA B27-binding peptides and can these stimulate immune responses?
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As a first step to testing the ‘arthritogenic’ peptide
hypothesis, HLA B27-restricted CTL specific for
C. trachomatis peptides were sought in patients with ReA. One hundred and eleven
peptides were synthesized, with sequences derived from the first
13
C. trachomatis genes characterized. Peptides were selected
according to the rules for HLA B27-binding defined previously—thus,
all were 8–10 amino acids in length and had R at P2, and
an aromatic, hydrophobic or positively charged C terminus. Sixty-two
of the peptides were found to bind to HLA B27 in
in vitro binding
assays [
34,
35] (P. Bowness, R. L. Allen and A. McMichael, manuscript
in preparation). Responses to these 62 peptides were sought
in the blood and synovial fluid of 10 patients with ReA/AS and
in blood from healthy HLA B27-positive controls. Figure 5
shows
data for synovial fluid T cells from donor WT after 17 days
in culture. Targets pulsed with different peptides are shown
on the
x-axis, and specific killing on the
y-axis. Cultured
blood and synovial fluid lymphocytes from this donor specifically
recognize and kill targets pulsed with a single peptide derived
from the
C. trachomatis heat shock protein 70 (sequence KRKSNKIIG),
but not other peptides. Recognition is HLA B27 restricted, since
peptide-induced killing only occurs for targets bearing HLA
B27 [
34].
Chlamydia trachomatis-infected HLA B27-positive targets
were also recognized (data not shown) [
35], more accurately
reflecting the route by which antigenic peptides bind to HLA
B27 within cells. Responses to this peptide were not observed
in other patients with ReA (P. Bowness and R. Allen, unpublished
results), although interestingly HLA B27-restricted CTL responses
to a nonamer peptide derived from the
Yersinia heat shock protein
60 gene product have recently been described in patients with
Yersinia-induced ReA [
36]. This finding confirms the principle
that HLA B27 can present potentially ‘arthritogenic’
peptides, derived from triggering bacteria, to CTL. However,
it is important to recognize that such responses are not found
in all patients, and their mere presence does not imply any
pathogenic role. As stated previously CD4+ responses restricted
by HLA class 2 molecules have been more frequently identified
in ReA patients [
29,
30,
37]. Furthermore, adoptive transfer
studies in transgenic rats have suggested that CD4+ T cells
are perhaps more important than CD8+ in transferring disease
([
38,
39] but note these data are now qualified by the authors
[
40]).
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FIG. 5. Synovial fluid lymphocytes from a patient with ReA were cultured for 21 days with two pools of 10 peptides each (pools nos 5 and 6). CTL activity was then assayed by specific lysis of C1R B*2705 target cells pulsed with individual peptides or with pools at an effector:target ratio of 4:1. Duplicates of each condition were averaged.
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The findings of both CD4 and CD8 T-cell expansions in the synovial
fluid of patients with ReA, and of both CD4 and CD8 T-cell responses
to triggering organisms, leads us to a model, shown schematically
in Fig. 6
, in which both CD4 and CD8 T cells are involved in
the pathogenesis of ReA [
2]. One possible scenario is that CD4
T cells initiate joint inflammation and CD8 T cells play a role
in its perpetuation. This is consistent with epidemiological
evidence suggesting that HLA B27 may be a severity marker in
ReA [
41].
In summary, these studies show that
C. trachomatis contains
many potential HLA B27-binding epitopes, and that CTL recognizing
one such epitope can be found in the blood and synovial fluid
of a patient with ReA.
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Does Cys67 confer an unusual cell biological behaviour on HLA B27?
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We have recently developed the methodology to study the folding
of recombinant HLA B27 together with peptides and
ß2
microglobulin
in vitro [
23,
42]. These studies were undertaken
first in order to examine the role of the unpaired cysteine
at position 67 of the HLA B27 heavy chain in peptide binding
and HLA B27 folding, and secondly in order to generate tetrameric
complexes of HLA B27, refolded with specific peptides. Such
tetrameric complexes can be fluorescently tagged, permitting
direct visualization of specific T-cell responses [
43]. We have
generated for the first time fluorescent-labelled tetrameric
complexes of HLA B27 refolded with NP383–391 and
ß2m,
and shown that these ‘tetramers’ specifically stain
HLA B27-restricted NP-specific T cells as well as transfected
TCRs [
23]. Figure 7
shows a tetrameric HLA B27 NP complex, and
also specific FACS staining of CTL specific for HLA 27/NP but
not HLA B27-restricted CTL recognizing a different peptide.
In attempting to generate these complexes, we found that the
unpaired Cys67, which lies near to the mouth of ‘B’
pocket in the HLA B27
1 helix, inhibited refolding of HLA B27
heavy chains with
ß2m and peptide [
42,
44]. However,
when Cys67 was mutated to serine, heterotrimers refolded easily
and the resultant complexes were still recognized by HLA B27-restricted
NP-specific TCRs [
23]. This shows that Cys67 is not essential
for the antigen-presenting function of HLA B27.
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FIG. 7. HLA B27-restricted T cells can be stained with HLA B27-peptide tetrameric complexes. Here the HLA B27 Cys67 residue has been mutated to serine. Specific staining of ’flu NP-specific CTL by HLA B27(Ser67)/NP tetrameric complexes shows that Cys67 is not essential for recognition by T cells (right-hand panel).
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We then re-examined the behaviour of the extracellular domains
of HLA B27, and to our surprise found that they formed homodimers
under the conditions used for refolding other HLA/
ß2m/peptide
complexes. Figure 8A
shows a Western blot of HLA B27 heavy chain
homodimers refolded
in vitro, which we have called HC-B27 [
42].
Dimerization of truncated HLA B27 molecules
in vitro is dependent
on disulphide bonding through Cys67 [
42], since site-directed
mutagenesis of this residue to a serine almost completely abolished
dimerization, and since dimers are converted to heavy chain
monomers under conditions that reduce disulphide bonds. Remarkably,
these dimers are not associated with
ß2m, previously
considered essential to the structural integrity of all mature
HLA complexes. Despite the absence of
ß2m, HC-B27
maintains the conformation of a significant part of its peptide-binding
groove, as evidenced by the binding of monoclonal antibodies
such as W6/32, and is capable of binding peptide [
42]. Expression
of HC-B27 homodimers also occurs in certain transfected cell
lines (Fig. 8B
) [
42]. However standard human cell lines and
tissues cannot yet be studied, since the antibodies currently
used to detect HC-B27 dimers also react with other HLA alleles.
We are currently trying to generate a monoclonal antibody specific
for HC-B27, in order to study its expression in healthy HLA
B27-positive individuals and patients with spondyloarthritis.
We have also used fluorescent-tagged tetrameric complexes of
HC-B27 to obtain intriguing preliminary data showing that the
T cells of patients with spondyloarthritis express a ligand
for HC-B27. Figure 9
shows an experiment in which tetrameric
HC-B27 complexes specifically bind 5–8% of peripheral
blood CD3+ T lymphocytes from two patients with spondyloarthritis.
T cells from an HLA B27-positive control were also stained.
One possible mechanism by which expression of HC-B27 could lead
to joint inflammation would be by the presentation of peptides
to either CD4+ or CD8+ T cells. Peptide presentation by MHC
class 1 molecules to CD4+ T cells has only rarely been reported,
but might be favoured for HC-B27 by partial unwinding of the
1 helix (making this complex more similar to a class 2 molecule,
see model below). Alternatively, since the complexes used in
Fig. 9
were refolded around an HIV-derived peptide, and the
patients and control had not been exposed to HIV, another mechanism
might be involved. For example HC-B27 might be a ligand for
natural killer receptors [
45] or other receptors.
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FIG. 8. HLA B27 dimer formation (A) in vitro and (B) in vivo. (a) SDS–PAGE and HC10 Western blot of HC-B27 homodimer refolded in vitro (lanes 1 and 3) and HLA B27 (Ser67 mutant)/ß2m/peptide heterotrimer complexes (lanes 2 and 4). (B) T2 HLA B27 lysates immunoprecipitated with W6/32, analysed under reducing (1) and non-reducing (2) conditions by Western blot with HC10.
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FIG. 9. Staining of peripheral blood T lymphocytes from a patient with spondyloarthritis with HC-B27 tetrameric complexes. The PBL were double-stained with PE-conjugated HC-B27 (x-axis) and FITC-stained antibodies to CD3 (A) or CD16 (B) (y-axis). Tetramer+CD3+ cells are in the upper right quadrant of (A).
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In order to investigate these observations further, a hypothetical
model of the HLA B27 dimer, HC-B27, was built using computer
graphics and molecular dynamics, based on the crystal structure
of the extracellular regions of HLA B27 [
19]. Figure 10
shows
in cartoon form a side view of HC-B27, and Fig. 11
shows a ‘top’
view of the molecular model (N. Zaccai and E. Y. Jones, unpublished
results). Figure 11
shows the
1 and
2 helices of two HLA B27
heavy chains viewed looking down towards the cell surface. One
heavy chain is shown in green with bound peptide as a ribbon,
and the other in blue with peptide in ball and stick form. Such
a model shows that dimerization through Cys67 is energetically
feasible.
Work is now underway to identify conditions that lead to HC-B27
dimer formation
in vivo. We hypothesize that dimer formation
might only occur under particular conditions. These could include
a relative lack of
ß2m (as in the
ß2m-deficient
mice) or of suitable peptides (as in cell lines with defects
in their antigen-presenting apparatus). Similar conditions could
occur during infection with intracellular bacteria, some of
which are known to trigger ReA. We also propose that cell-surface
expression of HC-B27 results in a local inflammatory response,
through either cellular or humoral mechanisms. If HC-B27 is
indeed a pro-inflammatory ligand, its production in certain
tissues at certain times might explain the pathogenic role of
HLA B27. Future experiments can readily be designed to test
this hypothesis. For example, it will be crucial to know if
HC-B27 dimers are produced in diseased transgenic rodents, and
in patients with spondyloarthropathy. Lastly, if expression
of an aberrant form of HC-B27 does have a pathogenic role in
disease, therapeutic strategies could be developed to inhibit
HC-B27 production, or to directly target the molecule, for example,
using monoclonal antibodies.
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Conclusions
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HLA B27 plays an admirable, perhaps outstanding role in the
immune response to viruses, however, it is also directly involved
in the pathogenesis of the spondyloarthropathies. Although the
exact mechanism is unknown, we have shown that HLA B27 is capable
of presenting potentially ‘arthritogenic’ peptides
to CTL. Whilst peptide binding may be important in disease pathogenesis,
we have also recently shown that the cell biology of HLA B27
is unusual. A testable new hypothesis, involving production
of pro-inflammatory HC-B27 dimers, is proposed to explain disease
pathogenesis.
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Acknowledgments
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I am particularly grateful to the supervision and encouragement
of Professor Andrew McMichael, in whose laboratory the work
described was carried out, and to the Medical Research Council
who have supported me with a Clinical Training Fellowship, and
myself and Rachel Allen with a Clinician Scientist Fellowship.
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References
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Submitted 12 April 2000;
Accepted 7 March 2002
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Abstract
Introduction
