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Conformational Dimorphism of Self-peptides and Molecular Mimicry in a Disease-associated HLA-B27 Subtype

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An interesting property of certain peptides presented by major histocompatibility complex (MHC) molecules is their acquisition of a dual binding mode within the peptide binding groove. Using x-ray crystallography at 1.4 A resolution, we show here that the glucagon receptor-derived self-peptide pGR ((412)RRRWHRWRL(420)) is presented by the disease-associated human MHC class I subtype HLA-B*2705 in a dual conformation as well, with the middle of the peptide bent toward the floor of the peptide binding groove of the molecule in both binding modes. The conformations of pGR are compared here with those of another self-peptide (pVIPR, RRKWRRWHL) that is also displayed in two binding modes by HLA-B*2705 antigens and with that of the viral peptide pLMP2 (RRRWRRLTV). Conserved structural features suggest that the N-terminal halves of the peptides are crucial in allowing cytotoxic T lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-restricted CTL clones demonstrates that TCR from distinct clones but with comparable reactivity may share CDR3alpha but not CDR3beta regions. Therefore, the cross-reactivity of these CTLs depends on TCR-CDR3alpha, is modulated by TCR-CDR3beta sequences, and is ultimately a consequence of the conformational dimorphism that characterizes binding of the self-peptides to HLA-B*2705. These results lend support to the concept that conformational dimorphisms of MHC class I-bound peptides might be connected with the occurrence of self-reactive CTL.
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Conformational Dimorphism of Self-peptides and Molecular
Mimicry in a Disease-associated HLA-B27 Subtype
*
Received for publication, August 3, 2005, and in revised form, September 26, 2005 Published, JBC Papers in Press, October 12, 2005, DOI 10.1074/jbc.M508528200
Christine Ru¨ ckert
‡1,2
, Maria Teresa Fiorillo
§1
, Bernhard Loll
¶1,3
, Roberto Moretti
§
, Jacek Biesiadka
,
Wolfram Saenger
, Andreas Ziegler
, Rosa Sorrentino
§4
, and Barbara Uchanska-Ziegler
‡5
From the
Institut fu¨r Immungenetik, Charite´–Universita¨ tsmedizin Berlin, Campus Virchow-Klinikum, Humboldt-Universita¨tzu
Berlin, Spandauer Damm 130, 14050 Berlin, Germany, the
§
Dipartimento di Biologia Cellulare e dello Sviluppo, Universita`La
Sapienza, via dei Sardi 70, 00185 Roma, Italy, and the
Institut fu¨r Chemie und Biochemie/Kristallographie, Freie Universita¨ t Berlin,
Takustrasse 6, 14195 Berlin, Germany
An interesting property of certain peptides presented by major
histocompatibility complex (MHC) molecules is their acquisition of
a dual binding mode within the peptide binding groove. Using x-ray
crystallography at 1.4 A
˚resolution, we show here that the glucagon
receptor-derived self-peptide pGR (
412
RRRWHRWRL
420
) is pre-
sented by the disease-associated human MHC class I subtype HLA-
B*2705 in a dual conformation as well, with the middle of the pep-
tide bent toward the floor of the peptide binding groove of the
molecule in both binding modes. The conformations of pGR are
compared here with those of another self-peptide (pVIPR, RRK-
WRRWHL) that is also displayed in two binding modes by HLA-
B*2705 antigens and with that of the viral peptide pLMP2
(RRRWRRLTV). Conserved structural features suggest that the
N-terminal halves of the peptides are crucial in allowing cytotoxic T
lymphocyte (CTL) cross-reactivity. In addition, an analysis of T cell
receptors (TCRs) from pGR- or pVIPR-directed, HLA-B27-re-
stricted CTL clones demonstrates that TCR from distinct clones but
with comparable reactivity may share CDR3
but not CDR3
regions. Therefore, the cross-reactivity of these CTLs depends on
TCR-CDR3
, is modulated by TCR-CDR3
sequences, and is ulti-
mately a consequence of the conformational dimorphism that char-
acterizes binding of the self-peptides to HLA-B*2705. These results
lend support to the concept that conformational dimorphisms of
MHC class I-bound peptides might be connected with the occur-
rence of self-reactive CTL.
Autoimmunity may develop either as a consequence of cross-reactiv-
ity of antibodies with a foreign (nonself), e.g. a bacterial or viral, antigen
and a host (self) protein (1) or depend on T cells that recognize peptides
with structurally similar, but not necessarily closely related, sequences
presented by major histocompatibility complex (MHC)
6
molecules (2,
3). MHC class I antigens consist of a highly polymorphic, MHC-en-
coded heavy chain (HC), that is non-covalently associated with
2
-mi-
croglobulin (
2
m). The HC forms a groove carrying a peptide that is a
proteolytic fragment of self- or nonself-proteins within the cell (4). The
complex of HC,
2
m, and peptide is often termed pMHC.
In case of the human MHC class I allele HLA-B27, which is very
strongly associated with ankylosing spondylitis (AS) (5, 6), autoimmu-
nity and in particular molecular mimicry between foreign and self-pro-
teins or their fragments have long been suspected to play a role in patho-
genetic processes (7–11). Cytotoxic T lymphocytes (CTLs) directed
against the self-antigen pVIPR (RRKWRRWHL, derived from vasoac-
tive intestinal peptide type 1 receptor (residues 400408)) have been
found in healthy individuals with the AS-associated HLA-B27 subtype
B*2705, and their number is increased in AS patients (12). In addition, a
proportion of these T cells cross-react with the viral pLMP2 peptide
(RRRWRRLTV, derived from latent membrane protein 2 (residues
236–244) of Epstein-Barr virus) (12, 13). Extensive structural similarity
between these peptides is observed when they are displayed by B*2705,
due to a salt bridge between pArg
5
of both peptides and residue Asp
116
at the floor of the peptide binding groove (14, 15). Interestingly, pVIPR
is presented in an unusual dual conformation, of which only one binding
mode permits the formation of the pArg
5
–Asp
116
salt bridge (14). How-
ever, a causal relationship between these two peptides, the CTLs recog-
nizing them in the context of B*2705, and AS has not been established.
We were interested whether further peptides with sequences exhib-
iting similarity to pVIPR or pLMP2 exist that would share the unortho-
dox conformation found for one of the two pVIPR binding modes and
pLMP2 in B*2705 (termed “p6
”, i.e. main chain
/
torsion angles in
-helical conformation at peptide position p6, contrasting with the
common “p4
conformation (14, 15)). A peptide derived from gluca-
gon receptor (pGR, RRRWHRWRL, residues 412–420) that exhibits
extensive sequence similarities was found and chosen for further struc-
tural and functional studies. In addition, we investigated whether exten-
sive CTL cross-reactivity had consequences for those regions of T cell
receptor (TCR) sequences that typically interact with peptide residues
of pMHC complexes, i.e. residues belonging to the complementarity
determining regions (CDR) 3 of TCR
and
chains (16–20).
MATERIALS AND METHODS
HLA-B27-positive Patients—Two female (MP and AB) and three
male (EP, LV and VP) individuals, fulfilling the modified New York
criteria for diagnosis of AS, were recruited for this study. All patients
*This work was supported by the Deutsche Forschungsgemeinschaft (Grant SFB449/
B6,Z3 to W. S., A.Z., and B. U.-Z.), Volkswagen-Stiftung (Grant I/79 989 to A. Z. and
R. S.), Sonnenfeld-Stiftung Berlin, and Fonds der Chemischen Industrie. The costs of
publication of this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked advertisement in accordance with 18 U.S.C.
Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 2A83) have been deposited in the Protein
Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New
Brunswick, NJ (http://www.rcsb.org/).
1
These authors contributed equally to this work.
2
Present address: Forschungsinstitut fu¨ r Molekulare Pharmakologie, 13125 Berlin, Germany.
3
Present address: Max-Planck-Institut fu¨ r Medizinische Forschung, Abteilung fu¨r
Biomolekulare Mechanismen, 69126 Heidelberg, Germany.
4
To whom correspondence may be addressed. Tel.: 39-06-4991-7706; Fax: 39-06-4991-
7594; E-mail: rosa.sorrentino@uniroma1.it.
5
To whom correspondence may be addressed. Tel.: 49-30-4505-53517: Fax: 49-30-4505-
53953; E-mail: barbara.uchanska-ziegler@charite.de.
6
The abbreviations used are: MHC, major histocompatibility complex; HC, heavy chain;
2
m,
2
-microglobulin; AS, ankylosing spondylitis; CTL, cytotoxic T lymphocyte; TCR,
T cell receptor; CDR, complementarity determining region; B-LCL, B lymphoblastoid
cell line; rIL-2, recombinant interleukin-2.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 4, pp. 2306–2316, January 27, 2006
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
2306 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281NUMBER 4JANUARY 27, 2006
at UNIV DI ROMA LA SAPIENZA on July 15, 2008 www.jbc.orgDownloaded from
were informed about the aim of the experiments and gave their consent.
HLA-B27 subtyping was performed using the Dynal AllSet
TM
SSP
HLA-B27 “High resolution” kit (Dynal Biotech Ltd., United Kingdom).
Patients EP, MP, AB, and VP were B*2705-positive, whereas LV was
B*2702-positive.
Cell Lines—Autologous B lymphoblastoid cell lines (B-LCLs) from
patients with AS were generated by in vitro immortalization of B cells
using the standard type 1 Epstein-Barr virus isolate B95.8 (21) and cul-
tured in RPMI (Invitrogen) supplemented with 10% fetal calf serum, 2
mML-glutamine, 100 units/ml penicillin, 100
g/ml streptomycin.
T2B*2705 transfectants described elsewhere (15) were cultured in the
same medium supplemented with 200
g/ml hygromycin B (Roche
Diagnostics, Mannheim Germany) to maintain the expression of HLA-
B27 molecules.
Generation of Antigen-specific CTL Lines and Clones—Peripheral
blood mononuclear cells from HLA-B27-positive patients with AS were
isolated by density gradient centrifugation with Lymphoprep and
depleted of the CD4
fraction by Dynabeads M-450 CD4 (Dynal ASA,
Oslo, Norway). Cell cultures were seeded at 2 10
4
cells/well in 96-well
flat-bottom microplates and stimulated by autologous B-LCLs at 0.5:1
antigen-presenting cells/responder ratio. The antigen-presenting cells
had been pulsed overnight with pVIPR or pGR peptides (8.5
M) before
being
-irradiated (200 Gy). CTL lines were grown in RPMI 1640
medium as above but supplemented with 10% heat-inactivated pooled
human serum. 20 units/ml human rIL-2 (Roche Applied Science) was
added to each well after 3 days. CTL lines were then restimulated on day
10. One week later, the specificity of CTL lines was tested by a standard
51
Cr release assay using as targets peptide-pulsed autologous B-LCL and
T2B*2705 transfectants. Phenotypic analysis of peptide-specific CTL
lines was performed by immunostaining using the following mono-
clonal antibodies: OKT3, OKT4, and OKT8 (Orthodiagnostics, Stan-
ford, CA). CTL lines were maintained in culture by weekly stimulation
with
-irradiated autologous B-LCL in complete RPMI medium (see
above) and human rIL-2 (20–100 units/ml), and were used for func-
tional assays 8–10 days after the last stimulation. pVIPR- and pGR-
reactive T cell clones were obtained by limiting dilution in 96-well plates
at 0.5–1 cell/well using phytohemagglutinin (0.5
g/ml) in the presence
of
-irradiated allogeneic peripheral blood mononuclear cells and
20–50 units/ml rIL-2. 12 days later, the clones were restimulated with
autologous B-LCLs pulsed with either peptide and further expanded in
the presence of rIL-2 (20–50 units/ml). The CTL lines carry the initials
of the patients from whom they are derived, with the exception of PM1,
PM16, PM31, PM41, PM45, PM49, PM65, PM69, and PM76 that are
also derived from patient MP.
51
Cr Release Assay—Specific reactivity of CTL lines toward pGR,
pVIPR, and pLMP2, and other peptides (Fig. 1) was tested by a standard
4-h
51
Cr release assay. Target cells (T2B*2705 transfectants) were incu-
bated overnight with the various peptides at 70
Mconcentration or
cultured in medium alone. One day later, target cells were labeled with
sodium
51
chromate, washed thoroughly, and plated (3 10
3
target
cells/well) with effector T cells at a 15:1 effector/target ratio, in the
absence of soluble peptide.
FIGURE 1. Cross-recognition of pVIPR-homologous peptides by pVIPR-stimulated CTLs derived from three patients with AS. T2B*2705 transfectants pulsed with peptides
(each at 70
M) or unpulsed (as control) were used as targets in a
51
Cr-release assay. Effector/target ratio was 15:1, and spontaneous release of
51
Cr-labeled cells was 15%. One of
two separate experiments is shown. The following peptides were employed: Vasoactive intestinal peptide receptor type 1, Homo sapiens (residues 400 –408), RRKWRRWHL (pVIPR);
glucagon receptor, H. sapiens (412– 420), RRRWHRWRL (pGR); voltage-dependent calcium channel
1 subunit, H. sapiens (513–521), SRRWRRWNR (pCAC); hypothetical protein yaiP,
E. coli (246 –253), RRWRRWIV (pyaiP); HXLF4 protein precursor, human cytomegalovirus (2–9), RRWLRLLV (pHXLF4); oxygen-regulated invasion protein Org A, Salmonella typhimurium
(77– 85), RQWRRLPQV (pOrgA); probable arabinosyltransferase C, Mycobacterium smegmatis (678– 686), QRRWQRLLV (pPATC); and latent membrane protein 2 (LMP2), Epstein-Barr
virus (236 –244), RRRWRRLTV (pLMP2).
Structural Basis for T Cell Cross-reactivity
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Analysis of TCR Gene Usage—Total RNA extraction from T cell
clones, cDNA synthesis, and amplification of TCR
and
chains were
performed as described (22). TCR families V
18 –29 were amplified by
PCR with the oligonucleotides reported by Kalams and co-workers (23).
The products were purified from an agarose gel using a gel band purifi-
cation kit (Amersham Biosciences). Internal primers upstream to the
TCR C
and C
reverse primers were used for direct sequencing.
Protein Preparation and Crystallization—The pGR peptide was syn-
thesized and purified by Alta Bioscience (Birmingham, UK). B*2705 HC
and
2
m were expressed separately in Escherichia coli. Inclusion bodies
containing the proteins were dissolved in aqueous 50% urea. HLA-
B27pGR complexes were reconstituted as described previously for
other pMHC (24–26). The complexes were purified by size exclusion
chromatography, concentrated, and used for crystallization at concen-
trations of 13–15 mg/ml in 20 mMTris/HCl, pH 7.5, 150 mMNaCl,
0.01% sodium azide. Crystals were obtained from drops made of 1.5
l
of protein solution and 1.5
l of precipitant solution (12–16% polyeth-
ylene glycol 8000, 100 mMTris/HCl, pH 7.5 or 8.0) in a hanging-drop
vapor diffusion setup using streak seeding techniques. Diffraction data-
sets were collected at European Synchrotron Radiation Facility,
Grenoble (ID 14-2) from cryo-cooled crystals at 100 K with glycerol and
polyethylene glycol 8000 as cryoprotectants.
Structure Determination of the B*2705pGR Complex—X-ray data
were processed with DENZO (27) and scaled with SCALEPACK (27)
(Table 1). The structure of B*2705pGR was determined by molecular
replacement with program EPMR (28) using the water- and peptide-
depleted B*2705m9 structure as search model (PDB entry 1JGE).
Restrained maximum-likelihood refinement was performed using REF-
MAC5 (29) comprising isotropic B-factor adjustment followed by iter-
ative manual model building with O (30). Water molecules were posi-
tioned with ARP/wARP (31). Data collection and refinement statistics
are given in Table 1. Intermediate and final structures were evaluated
with PROCHECK (32) and WHATCHECK (33). The figures showing
structural details were prepared with DINO (Visualizing Structural
Biology (2002), www.dino3d.org), MSMS (34), and DELPHI (35).
Data Deposition—The atomic coordinates and structure amplitudes
have been deposited in the Protein Data Bank (accession code 2A83).
RESULTS
Selection of a Peptide Recognized by HLA-B27-restricted Cross-reac-
tive CTLs—Because previous studies had suggested that the unusual
Trp-Arg-Arg motif at positions p4–p6 of the pVIPR and pLMP2 pep-
tides might be instrumental in leading to CTL cross-reactivity and allele-
dependent molecular mimicry involving the p6
peptide conformation
(14, 15), peptide sequences related to this motif were selected from
public protein databases (www.ncbi.nlm.nih.gov/) using the blastp pro-
gram (36). In addition, the peptides had to share the HLA-B27-specific
anchor residue pArg
2
and preferably an arginine at p1 as well as an
aliphatic residue at p9. Two peptides derived from human proteins were
identified: one originates from glucagon receptor (pGR, RRRWHR-
WRL, residues 412–420), the other from the voltage-dependent cal-
cium channel
1 subunit (pCAC, SRRWRRWNR, residues 513–521).
Both peptides bound to HLA-B*2705 molecules expressed on T2 cells
(results not shown), but only pGR led to stimulation by some pVIPR-
primed CTL (Fig. 1). In addition, four nonself-peptides were identified
that, however, were not recognized by the HLA-B27pVIPR-restricted
CTL, with the possible exception of the CTL line AB5, which weakly
recognized the B*2705pHXLF4 target (Fig. 1). Therefore, the pGR pep-
tide was chosen for further structural and functional studies.
Structural Features of the B*2705pGR Complex—The structural
basis for the observed CTL cross-reactivity was then investigated by
crystallographic analysis of the B*2705pGR complex (Table 1). The
peptide could be modeled unambiguously to the electron density (Fig.
2A), revealing two conformations (termed pGR-A and pGR-B) with
similar B-factors (Fig. 2B). The only residue with markedly higher B-fac-
tors is pArg
6
whose guanidinium group is considerably more flexible in
both conformations than the side chains of any of the other residues.
Both conformations are present in a 1:1 ratio as judged from the elec-
tron density maps. The pGR-A and -B conformations differ from pArg
3
to pArg
8
, with clearly distinguishable C
traces from p4–p7 (Fig. 2, C
and D). The torsion angles (
,
) of the main-chain residues of the pep-
tide are of
-strand type except at residue pArg
6
, which exhibits right-
handed
-helical conformation. Therefore, irrespective of the binding
mode, pGR is bound in the p6
conformation, which had been detected
for HLA-B27peptide complexes so far in case of one of the two peptide
binding modes in B*2705pVIPR (14) as well as in B*2705pLMP2 (15).
Consequently, the side chain of pHis
5
points toward the interior of the
binding groove. Both pGR conformations lead to fully solvent-exposed
pTrp
4
and pArg
6
side chains that exhibit relatively few, weak HC con-
tacts, nearly all 3.5 Å. A notable exception is the short salt bridge (2.80
Å) between pArg6
NH2
of pGR-B and Glu155
OE1
on the
2-helix. The
two pGR conformations are also distinguished by the interactions that
are formed by the buried pHis
5
. In pGR-A, pHis5
ND1
binds to pArg3
NH2
(with the side chain of pArg
3
in extended conformation), and a water-
mediated hydrogen bond is formed to Asp
77
on the
1-helix and Asp
116
on the floor of the peptide binding groove (Fig. 3A). In the B conforma-
tion, pHis5
ND1
forms a water-mediated hydrogen bond to pArg3
NE
(with the side chain of pArg
3
in bent conformation) and a direct
intrapeptide hydrogen bond is found between pHis5
NE2
and pTrp7
O
(Fig. 3Band Table 2). The two water molecules that mediate the con-
tacts between pHis
5
in pGR-A with Asp
77
and Asp
116
are retained in
pGR-B (Fig. 3).
Table 2 lists the most pronounced differences between the two con-
formations. With the exception of pArg
1
, all solvent-exposed peptide
side chains exhibit conformational dimorphism. The two Trp residues
at p4 and p7 show distinct rotamer conformations that lead to different
TABLE 1
Data collection and refinement statistics
Data collection HLA-B*2705pGR
Space group P2
1
Unit cell (a,b,c(Å);
(°)) 51.0, 82.1, 65.3; 108.7
Resolution (Å)
a
50.0–1.40 (1.42–1.40)
Unique reflections
a
97,993 (4,386)
Completeness (%)
a
97.9 (87.7)
I/
(I)
a
28.0 (7.9)
R
syma,b
0.039 (0.112)
Refinement
Non-hydrogen atoms 4,141
R
crysta,c
0.127 (0.150)
R
freea,d
0.150 (0.170)
Heavy chain, no. of atoms/average B
factor
2
)2,383/14.8
2
m, no. of atoms/average Bfactor
2
) 867/16.6
Peptide, no. of atoms/average Bfactor
2
) 168/13.9
Water, no. of molecules/average Bfactor
2
) 710/30.9
Glycerol, no. of atoms/average Bfactor
2
) 36/36.0
r.m.s.d.
e
from ideal geometry, bond length (Å) 0.012
bond angles (°) 1.48
a
Values in parentheses refer to the highest resolution shell.
b
R
sym
⫽⌺
h
i
I
h,i
⫺具I
h
/
h
i
I
h,i
.
c
R
cryst
⫽⌺
h
F
o
F
c
/F
o
(working set, no
cut-off applied).
d
R
free
is the same as R
cryst
, but calculated on 5% of the data excluded from refine-
ment.
e
Root mean square deviation from target geometries.
Structural Basis for T Cell Cross-reactivity
2308 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281NUMBER 4JANUARY 27, 2006
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juxtaposition of their indole moieties. The termini of the peptide are
bound in the characteristic binding modes that have been observed
previously (14, 15, 37). Most notably, the side chain of pArg
1
is sand-
wiched between the HC residues Arg
62
and Trp
167
, resulting in an ener-
getically favorable stabilization (38), whereas the side chain of pLeu
9
does not contact the floor of the F pocket (Figs. 2A,3A, and 3B) but is
firmly anchored by numerous hydrophobic interactions.
Structural Comparison of pGR, pVIPR, and pLMP2 Complexed with
B*2705—A comparison of the structures of pGR, pVIPR, and pLMP2 in
complex with B*2705 is greatly facilitated by the similar, high resolu-
tions obtained and the isomorphous crystallization modes (space group
P2
1
, Table 1) (14, 15), demonstrating that intermolecular interactions
that are associated with crystal packing apply to all structures. We have
already pointed out that structural molecular mimicry in the context of
HLA-B27 is an allele- and peptide-dependent property, because the
similarity between the viral pLMP2 peptide and the self-peptide pVIPR
is much more pronounced when both peptides are displayed by the
B*2705 than by the B*2709 subtype (14, 15). However, only one of the
two pVIPR conformations, the unusual p6
binding mode, participates
in molecular mimicry with pLMP2. The side-chain orientations (Fig. 4)
as well as the surface properties (Fig. 5) show that molecular mimicry
extends to the sequence-related pGR peptide as well. This mimicry is
most obvious when pGR is in the B conformation, owing to the two
rotamer conformations of pTrp4 (Fig. 2, Cand D): only one of these is
FIGURE 2. General structural properties of the B*2705pGR complex. For sake of clar-
ity, water molecules are omitted in all representations; in A–C, the view is from the side of
the
2-helix. A, final 2F
o
F
c
electron density map (blue mesh) contoured at 1
, with pGR
in A-conformation (blue) and in B-conformation (pink) shown in ball-and-stick represen-
tation; the polymorphic residue 116 (Asp in the case of B*2705) is shown as well; it is not
contacted directly by any peptide residue. B, color scheme depicting the anisotropic
B-factor distribution in both pGR conformations. C, superimposition of both pGR confor-
mations, viewed as in A.D, superimposition of both pGR conformations, 90° rotated
toward the viewer in comparison to C.
FIGURE 3. Differential contacts of the residue pHis
5
in the two pGR conformations A
and B. Water molecules are drawn as red spheres, hydrogen bonds are depicted as red
dashed lines, and all distances are given in Å. Only peptide residues and selected
1-hel-
ical residues are shown; the view is through the
2-helix (removed), roughly along the
length of the peptide binding groove toward the N-terminal peptide residues.
B*2705pGR is shown with the peptide in A-conformation (A) and B-conformation (B),
which results in distinct contacts with water molecules, peptide residues, and HC atoms.
Structural Basis for T Cell Cross-reactivity
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TABLE 2
Comparison of pGR conformations with those of pVIPR and pLMP2 in the B*2705 subtype
Only direct intra-peptide contacts and contacts between the peptides and HC residues (up to 3.50 Å; HB, hydrogen bond; SB, salt bridge) are included; solvent-mediated interactions are omitted, and van der Waals (vdW)
contacts are not given explicitly. In pGR, the p3–p8 residues exhibit double conformations. In case of the B*2705pLMP2 complex, pTrp
4
and Asp
116
occur in alternative conformations; only one of the equally occupied pTrp
4
conformations (with higher degree of similarity to the pVIPR-p6
conformation) and the higher occupied Asp
116
conformation (occupancy 75%) are considered.
Peptide position B*2705pGR conformations A and B B*2705pVIPR conformation p6
B*2705pLMP2
Peptide residue Contact residue Distance Interaction Peptide residue Contact residue Distance Interaction Peptide residue Contact residue Distance Interaction
ÅÅÅ
p1 and p2 Contacts formed by pArg1 and pArg2 are very similar in all complexes; the side chain of pArg1 is solvent-exposed, pArg2 is buried.
p3 pArg3
N
Tyr99
OH
3.09 HB pLys3
N
Tyr99
OH
2.96 HB pArg3
N
Tyr99
OH
3.06 HB
pArg3 Tyr99, Leu156, 3.6–4.0 vdW pLys3
O
Tyr99
OH
3.43 HB
Tyr159 pLys3
NZ
pTrp4
O
2.82 HB
pArg3
NH2
(A) pHis5
ND1
(A) 2.9 HB
p4 The side chain of this residue is solvent-exposed in all complexes.
pTrp4 Gln65, Ile66 3.6–4.0 vdW pTrp4
O
pLys3
NZ
2.82 HB pTrp4 pArg6, 3.3–3.5 vdW
pTrp4 pArg6 3.3 vdW Ile66 3.5–3.7 vdW
p5 The side chain of this residue is buried in all complexes.
pHis5
ND1
(A) pArg3
NH2
(A) 2.9 HB pArg5
NH1
Asp116
OD1
3.04 SB pArg5
NH1
Asp116
OD1
3.13 SB
pHis5
NE2
(B) pTrp7
O
(B) 3.1 HB pArg5
NH2
Asp116
OD2
3.10 SB pArg5
NH2
Asp116
OD2
2.98 SB
p6 The side chain of this residue is solvent-exposed in all complexes.
pArg6
NH2
(B) Glu155
OE1
2.8 SB pArg6 pTrp4 3.3 vdW pArg6 pTrp4 3.3–3.5 vdW
p7 pTrp7
O
(B) pHis5
NE2
(B) 3.1 HB pTrp7 Leu156 3.5 vdW pLeu7 Val152 3.5 vdW
pTrp7 Ala150, 3.6 vdW
Val152
p8 The side chain of this residue is solvent-exposed in all complexes.
pArg8
NE
(A) Glu76
OE1
2.95 HB pArg8 (pGR), pHis8 (pVIPR), or pThr8 (pLMP2) are involved in different contacts in the three complexes.
pArg8
NH2
(A) Thr73
OG1
3.50 HB
pArg8
NH2
(B) Asp77
OD2
3.43 HB
pArg8
NE
(B) Asp77
OD1
3.18 HB
pArg8
O
Trp147
NE1
2.91 HB
p9 The side chain of this residue is buried in all complexes; the contacts formed by pLeu9 (pGR, pVIPR) and pVal9 (pLMP2) are very similar.
Structural Basis for T Cell Cross-reactivity
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congruent with the corresponding residue in the pVIPR-p6
and
pLMP2 structures. A calculation of the root mean square deviations for
the C
atoms between the different peptides and their conformations in
B*2705 (results not shown) supports this conclusion and demonstrates
that pGR-B, pVIPR-p6
, and pLMP2 exhibit the highest degree of
structural similarity.
TCR-accessible, exposed side chains of the peptide that exhibit struc-
tural equivalence between the three peptides include at least pArg
1
and
pTrp
4
but possibly also pArg
6
because of its considerable flexibility in
pGR (Fig. 2B). In addition, the surfaces above the peptide binding
groove-embedded residues pArg
3
/Lys
3
,pHis
5
/Arg
5
, and pTrp
7
/Leu
7
are comparable (Fig. 5). As observed previously (15), the similarity is
most pronounced around the N-terminal half of the pVIPR and pLMP2
peptides, and this is also true for the pGR peptide. The electrostatic
surface properties of the three complexes in B*2705 are similar as well
and are clearly most pronounced for the regions surrounding the N-ter-
minal halves of the three peptides (Fig. 5).
CTL Cross-reactivity between pGR, pVIPR, and pLMP2 in the HLA-
B27 Context—The cross-reactive potential of pGR-stimulated CTL
from AS patients typing as B*2705 and B*2702 was then investigated.
The fact that the CTL lines are not clones could be relevant in the case
of CTL exhibiting cross-reactivity, as pointed out previously (14, 15).
However, it is unlikely that lack of reactivity is influenced by oligoclonal-
ity of the CTL lines, because this feature would be expected to enhance,
and not to diminish cross-reactivity. Of 23 CTL lines from four patients
(VP, MP, EP, and LV) (Fig. 6), four reacted also with B*2705pVIPR and
FIGURE 4. Comparison of the binding modes of
pGR, pVIPR, and pLMP2 in B*2705. Superimpo-
sition of the peptides pGR (in B-conformation,
pink) with pVIPR (in p6
binding mode, light
green), (Aand B) or pLMP2 (dark green)(Cand D).
The peptides are viewed from the side (Aand C)or
from the top (rotation by 90° toward the viewer) (B
and D). The representations resemble those in Fig.
2, Cand D, respectively. E,left panel, schematic rep-
resentation of side-chain orientations when
viewed from the N to the C termini of pGR (A- or
B-conformation), pVIPR (in p6
binding mode),
and pLMP2 in B*2705. The shaded areas indicate
regions of structural similarity between the pep-
tides. The orientations of the peptide side chains in
the binding pockets are indicated and the primary
sequence of the peptides is shown. *, the indole
moieties of the exposed pTrp
4
residues of pGR and
pLMP2 exhibit conformational dimorphism, and
this is also the case for certain other exposed resi-
dues of pGR (pArg
6
and pArg
8
). E,right panel, floor
of peptide binding groove indicated by
-sheet
and binding region for a TCR by TCR.”
Structural Basis for T Cell Cross-reactivity
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B*2705pLMP2 (VP7G, VP78G, VP90G, and LV3G). One CTL line rec-
ognized pGR and pLMP2 (VP52G), and another exhibited cross-reac-
tivity between pGR and pVIPR (EP31G). The reactivity with pGR was
usually much stronger than with pVIPR or pLMP2. These results dem-
onstrate that pGR-stimulated CTL lines, which can recognize also one
or even both other peptides in the context of B*2705, are readily detect-
able, although some differences between the CTL donors were appar-
ent. pGR-stimulated CTLs derived from patients MP and EP were
nearly never cross-reactive, whereas about half of the CTLs from donor
VP exhibited cross-reactivity. These results reveal also that the com-
plexes of each of the three peptides with B*2705 must exhibit structural
(14, 15) or dynamic (39) properties (or both) that lead to the prevention
of cross-reactivity in the majority of the CTL lines.
Analysis of TCR
and
Chains from pVIPR- and pGR-stimulated T
Cell Clones—TCR gene usage was assessed for 40 clones derived from
pVIPR- or pGR-stimulated CTL. 17 of these clones have been reported
earlier (15). Four clonotypes reacted only with the peptide employed for
stimulation, whereas three exhibited different degrees of cross-reactiv-
ity. The clones mimicked the reactivity of the CTL from which they
originated (Tables 3 and 4) and revealed that the CDR regions of the
TCR
and -
chains contribute differentially to the cross-reactivity
exhibited by these clones.
All 23 cross-reactive clones shared the (D/N)RDDKIIFG motif within
their CDR3
regions, although they belonged to different TCR
chain
families, whereas the non-cross-reactive clones lacked this motif
(Table 3). However, also the latter exhibited some similarities: the
majority shared the motif SSYKLIFG or a closely related sequence.
Interestingly, clones from the most highly cross-reactive CTL AB5 and
from the mono-specific PM65 shared the SGGSYIPTFG motif in one of
their two
-chains. This could be connected to the fact that the PM65
CTL gave only borderline reactivity with pLMP2, thus partly resembling
the fully cross-reactive AB5 CTL (Fig. 1). All clones derived from patient
FIGURE 5. Surface representation of B*2705
complexed with pGR, pVIPR, and pLMP. A, C,
and E, electrostatic surface potential, colored red
and blue for negative and positive potential,
respectively, gray areas are uncharged. B*2705 is
shown in complex with pVIPR (A), pGR (C), and
pLMP2 (E). B,D, and F, molecular surface represen-
tations of B*2705 in complex with pVIPR (B), pGR
(D), and pLMP2 (F), as viewed by an approaching
TCR.
Structural Basis for T Cell Cross-reactivity
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EP shared also a DSMD motif, just before the previously mentioned
SSYKLIFG sequence, which was absent from all other sequences and
may thus be connected with the exclusive specificity for B*2705pGR
that characterizes these clones.
In marked contrast to these results, no consistent sequence motif
could be discerned among the
-chains of these clones, irrespective of
whether they belonged to the group of non-cross-reactive or to the
cross-reactive clones (Table 4). Only the TXXXQXFG motif was pres-
ent in several of the CDR3
sequences but independent of the clonal
reactivity. There was also no similarity in the usage of V
and J-region
sequences. For example, although the cross-reactive clones derived
from AB4 and AB5 shared the V
22 family, their J-region sequences
were different. Despite some similarities in CDR3
regions with various
CTL or clones from patients with AS or reactive arthritis, another HLA-
B27-associated disease, no consistent pattern emerges from a compar-
ison of these TCR
chains with those of the clones analyzed in the
present study (Table 4), except perhaps that the CDR3
region of the
more stringent TCR tended to be shorter. Either the V
families, the
J-regions, or both were distinct. Several T cell clones express a dual TCR
with two
-chains (PM65 and AB5; Table 3) or
-chains (EP16G;
Table 4), sharing common motifs in their CDR3 regions with T cell
clones from the same or a different patient. Although this dual expres-
sion might explain the recognition of the different peptides in some
cases (AB5), some other cross-reactive clones contain only single
or
chains (AB4, MPVPAC7). Moreover, some mono-specific T cells
(PM65 and EP16G) express more than one TCR
or -
chain. EP16G
clones in particular display two
chains that share the same CDR3
region but have two related variable regions (3 and 3.1).
DISCUSSION
The present study addresses the question whether the conforma-
tional dimorphism observed for a self-peptide (pVIPR) in the context of
the B*2705 subtype (14) and the functional and structural mimicry,
which it exhibits with the viral pLMP2 peptide, were serendipitous find-
ings or would extend to further peptides. Among several sequence-
related peptides of human, bacterial, or viral origin, the pGR peptide
was, apart from the previously identified pLMP2, the only one that gave
strong responses with some B*2705pVIPR-restricted CTLs (Fig. 1). The
glucagon receptor, from which pGR is derived, is, like the vasoactive
intestinal peptide type 1 receptor, a G-protein-coupled receptor. It
interacts with the peptide hormone glucagon and performs a crucial
physiological role in glucose and insulin metabolism. Expression has
been found in many different tissues, but particularly strongly in cells
within the liver and the kidney (40). CTLs that were stimulated by pGR
in the context of B*2705 exhibited occasional cross-reactivity with
pVIPR and pLMP2 as well (Fig. 6), indicating a functional similarity
between CTLs that recognize these three peptides.
It must be borne in mind, however, that the three sequence-related
peptides are possibly not the peptides that exhibit molecular mimicry
which, if at all, may be relevant in the context of autoimmunity (15).
Such relevance might be exhibited by further, currently unknown, pep-
tides that show less pronounced sequence similarity (3) or could be the
result of post-translational splicing (41), both of which are likely to have
gone unnoticed by the type of data bank searches carried out by us.
Despite this caveat, pGR, pVIPR, and pLMP2 allow to study the struc-
tural basis of TCR cross-reactivity in the HLA-B27 context. This anal-
ysis reveals that the AS-associated subtype B*2705 shows a much more
pronounced degree of molecular mimicry between the three peptides
than the non-AS-associated B*2709 subtype (42), particularly, when the
pGR-B conformation is considered (15).
7
The surfaces above the pep-
tide residues p1 to p6 of pGR, pVIPR, and pLMP2 are nearly identical
(Fig. 5), despite the different rotamer conformations of pTrp
4
and
pArg
6
, which are very likely interconvertible under physiological con-
ditions (Fig. 2). This may provide an explanation for the occasional
cross-reactivity observed for B*2705pVIPR-primed CTL (Fig. 1), and
the same applies to B*2705pGR-primed CTL (Fig. 6). However, those
parts of the complexes that are in the vicinity of the diverging C-termi-
nal peptide sequences are distinct (Figs. 4 and 5) and might be targeted
by those CTL that lack cross-reactivity.
The high resolution of the B*2705pGR structure unequivocally dem-
onstrates the existence of two peptide conformations in the binding
groove (Fig. 2). As in the case of the B*2705pVIPR complex, in which
the peptide occurs in a dual binding mode (14), it is not possible, on the
basis of the crystal structure described here, to distinguish between a
static and a dynamic peptide binding mode. Because the difference
between the two pGR conformations is not as drastic as for pVIPR-p4
and -p6
, it may be more likely that the pGR peptide exhibits a dynamic
mode of binding. Spectroscopic methods or NMR studies could pre-
7
B. Loll, M. T. Fiorillo, C. Ru¨ ckert, J. Biesiadka, W. Saenger, R. Sorrentino, A. Ziegler, and
B. Uchanska-Ziegler, manuscript in preparation.
FIGURE 6. Cross-reactivity of CTL lines. CTL lines were obtained by stimulation with
pGR from four patients with AS (VP, MP, and EP are B*2705-positive; LV is B*2702-posi-
tive). The T2B*2705 target cells were incubated overnight with pVIPR, pGR, or pLMP2 (70
M) or in medium without a peptide (control) before being used in a standard
51
Cr-
release assay. Effector/target ratio was 15:1. Spontaneous release of
51
Cr-labeled cells
was 15%. Results are representative of two experiments.
Structural Basis for T Cell Cross-reactivity
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sumably resolve this issue. It seems also possible that local changes in
pH or ion concentrations might affect the protonation state of amino
acids that are part of the peptide binding groove, or of the peptide itself.
In case of pGR, pHis
5
could be affected by such changes, possibly leading
to altered interactions between the peptide and binding groove residues
(Fig. 3). It would be necessary to analyze the B*2705pGR complex at
lower pH to uncover such differences.
We have previously pointed out that the dual pVIPR conformation in
the B*2705 subtype might influence T cell selection within the thymus,
particularly by impairing negative selection, thereby providing an expla-
nation for the frequent presence of pVIPR-directed CTL in patients
with AS (14). The same reasoning could apply for pGR, although the
differences between the A and B conformations are not as striking as
those in case of pVIPR. It seems thus possible that the emergence of
autoreactive CTL may be a direct consequence of conformational
dimorphisms and dynamic properties of a given peptide within a dis-
tinct binding groove (39). The importance of MHC allele-dependent
dynamics and different conformational states of a peptide for recogni-
tion of a pMHC by T cells is only beginning to be considered (14, 19, 39,
43–45). These attributes of peptides bound to MHC molecules may
play a role in the context of molecular mimicry and in the differential
association of HLA-B27 subtypes such as B*2704 and B*2706 or B*2705
and B*2709 with AS (5, 6, 12). However, it is likely that the general
features of this model do not only apply to HLA-B27 antigens.
The relatively conserved binding modes of human and mouse TCR
on pMHC (16–20) permit the prediction that the conserved part of the
three B*2705 complexes is likely to interact with the CDR3
loop of a
cross-reactive TCR (e.g. those expressed by CTL MPVPAC7 or AB5),
whereas the region around the peptide C termini might be responsible
primarily for interaction with the CDR3
loop of a peptide-selective
CTL such as PM45 or EP16G (Figs. 1 and 6 and Tables 3 and 4). It is
notable that CTL clones recognizing B*2705 in complex with pVIPR,
pGR, or pLMP2 (e.g. MPVPAC7 clones or clones from donor AB; Table
3) tend to exhibit a considerable degree of similarity with regard to their
CDR3
sequences, mainly through the presence of the (D/N)RDDKI-
IFG motif and sharing of the J
9.4 region. The lack of such similarity in
the TCR
chain sequences indicates that the TCR
chains are primarily
responsible for determining whether cross-reactivity between the three
peptides can occur at all, whereas the TCR
chains seem to modulate its
degree (Table 4). Those clones that exhibited no cross-reactivity, e.g.
PM45, EP16G, or EP17G, lack the CDR3
(D/N)RDDKIIFG motif, and
they were also found to possess different J
regions and TCR
sequences (Tables 3 and 4). In case of the latter two clones, the CDR3
loops are extremely short (Table 4), so that it appears doubtful whether
this part of these TCR can participate in recognition of the pMHC
surface at all.
However, in the absence of a structure of an HLA-B27peptideTCR
complex (46), these considerations must currently be regarded as spec-
ulative, and it remains also unclear whether they extend to other MHC
class I structures. Furthermore, the recently described structure of an
autoimmune TCR complexed with an HLA class II antigen and a self-
peptide revealed a novel topography of a pMHC-TCR interaction (47).
In this complex, the reactivity of the TCR is nearly exclusively restricted
to the N-terminal half of the peptide and its surrounding, and the CDR3
loops of both chains engage in atypical contacts, resulting in a large
tilting angle of the TCR on top of the pMHC. It is therefore impossible
to predict whether the TCR that interacts with HLA-B27peptide com-
plexes will recognize their epitopes in the conventional or the novel
fashion (the latter is unlikely in case of the CTL with the short CDR3
loops), or whether they might exhibit an as yet undetected additional
binding mode. Consequently, the limited sequence similarities that we
found between the CDR3
loops of PM65 and CTL from a patient with
reactive arthritis, of AB4, or CTL from two further patients (Table 4)
might indicate similar HLA-B27TCR binding modes, but this issue
TABLE THREE
TCR
-chain sequences of pVIPR- and pGR-stimulated T cell clones from patients with AS
CTL AV N JA Peptide recognition Number of clones
PM45 23 C A V S P L R G Y Q K V T F G 8.1 pVIPR 3
PM65 11 C V S G G S Y I P T F G 15.3 pVIPR 4
15CAE I H S TDK L I F G 17.1
EP16G 7 C A DSMDS S Y K L I F G 16.5 pGR 7
EP17G 7 C A DSMDS S Y K L I F G 16.5 pGR 3
AB4 7 C A V N R DDK I I F G 9.4 pVIPR/pGR 2
AB5 7 C A V N R DDK I I F G 9.4 pVIPR/pGR/pLMP2 4
15 C A A S P S S G G S Y I P T F G 15.3
MPVPAC7
a
14CAYS DRDDK I I F G 9.4 pVIPR/pLMP2 17
a
Reported in Ref. 15.
TABLE FOUR
TCR
-chain sequences of pVIPR- and pGR-stimulated T cell clones from patients with AS
The specificities and numbers of clones are given in Table 3.
CTL BV N-D-N BJ
PM45 13 C A S T L Q G L T ENT G E L F F G 2.2
PM65 14 C A S S L NN E Q F F G 2.1
6CAS S PG L N NE Q FFG2.1
a
EP16G 3 C A S S TNQP QHFG1.5
3.1 C A S S
b
TNQP QHFG1.5
EP17G 3.1 C A S S TNQP QHFG1.5
AB4 22 C A S S V G L A G G M DT G E L F F G 2.2
1 C A S S V G L A G R Y E Q Y F G 2.7
c
9CAS S L E L AGG EETQYFG2.5
d
AB5 22 C A S S E W DRGDD G Y T F G 1.2
MPVPAC7 1 C A S S P V EG G S R G Y N E Q F F G 2.1
a
From synovial CD8T-cells from an HLA-B27-positive patient with reactive arthritis. Unpublished data from P. Bowness, EMBL accession AJ296358.1.
b
Clones from EP16G CTL lines express two
-chains sharing the same N-D-N and joining segments and closely related variable regions belonging to the V
3 family (see
Ref. 59).
c
From an HLA-B27-restricted autoreactive synovial CTL (P1.4.52
2c
of a patient with reactive arthritis (see Ref. 60).
d
From a clonally expanded CD8T cell clone of an HLA-B27-positive patient with AS (see Ref. 61).
Structural Basis for T Cell Cross-reactivity
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must presently remain unresolved. The same applies to the possibility
that the relatively high content of positively charged residues that shape
TCR epitopes of the B*2705pGR/pVIPR/pLMP2 complexes (Fig. 5)
may provide docking points for Asp or Glu residues in the CDR3
and
CDR3
loops of TCR on cross-reactive CTL, e.g. in AB4, AB5, and
MPVPAC7 clones (Figs. 1 and 6 and Tables 3 and 4).
Our finding that clones from different CTL often express two
-or
two
-chains could be functionally relevant. In the case of EP16G, these
clones share the same CDR3
region but possess two germ-line TCR
regions. An analogous observation regarding the TCR
chain of auto-
reactive T cell clones in autoimmune diabetes has recently been
described (48). The authors show that small amino acid variations distal
to the antigen binding site of the TCR may have a profound effect on the
avidity of individual clonotypes and are indicative of a pathogenic mat-
uration of the T cell response. Although we do not know whether the
two TCR
chains expressed in the T cell clones described here have a
different affinity for the B*2705pGR complex, the possibility of affinity
maturation of TCR via reactivation of the germ line recombinatorial
process (49) should be considered. It may occur more often than sup-
posed and might play a role in tuning autoimmune reactions.
Several hypotheses have been put forward to explain the association
of HLA-B27 and spondyloarthropathies (2, 6, 50–54), but molecular
mimicry between a foreign, i.e. microbial or viral, peptide and a self-
peptide is a central postulate of the arthritogenic peptide hypothesis (2,
6, 54). Together with the previous descriptions of the B*2705pVIPR and
B*2705pLMP2 structures (14, 15), the present study provides a molec-
ular framework that accounts for the observed CTL cross-reactivity
between B*2705 molecules in complex with self-peptides (pGR and
pVIPR) and a foreign peptide (pLMP2). Therefore, the arthritogenic
peptide hypothesis (2, 6) might well be relevant for HLA-B27 and its
association with spondyloarthropathies, although other explanations
can still not be excluded. Despite the fact that many more autoimmune
diseases are associated with HLA class II than with HLA class I alleles
(55, 56), the class I allele HLA-B27 presents a particularly interesting
case. The extremely strong association between certain HLA-B27 sub-
types and AS, e.g. B*2704 and B*2705, and its absence in individuals
harboring the B*2706 and B*2709 subtypes that differ only minimally
from the former (5, 6), warrants further functional, biochemical, struc-
tural, and other biophysical investigations. In our opinion, comparative
studies involving AS-associated and non-associated subtypes (12, 14,
15, 37, 39, 57, 58) hold the key to achieving an in-depth understanding of
the pathogenesis of HLA-B27-associated autoimmune diseases.
Acknowledgments—We thank all patients for participation in this study and
are grateful to the beamline staff at the European Synchrotron Radiation
Facility, Grenoble.
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... Functional, X-ray crystallographic, spectroscopic, and calorimetric experiments as well as molecular dynamics (MD) simulations have already shown that the Asp116His micropolymorphism of the HC, combined with differential peptide binding, leads to an overall structural polymorphism of the subtypes that is responsible for several distinct features of B2705 and B2709 [reviewed by (10)(11)(12)]. For example, cytotoxic T lymphocytes (CTL) directed against the self-peptides pVIPR [RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400-408)] and pGR [RRRWHRWRL, derived from glucagon receptor (residues 412-420)] have been found in individuals with B * 27:05 (13,14). About one sixth of these T cells cross-reacts with the viral pLMP2 peptide [RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244)] of Epstein-Barr virus (EBV) (15), suggesting the existence of B2705-restricted molecular mimicry between the three peptides (14). ...
... For example, cytotoxic T lymphocytes (CTL) directed against the self-peptides pVIPR [RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400-408)] and pGR [RRRWHRWRL, derived from glucagon receptor (residues 412-420)] have been found in individuals with B * 27:05 (13,14). About one sixth of these T cells cross-reacts with the viral pLMP2 peptide [RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244)] of Epstein-Barr virus (EBV) (15), suggesting the existence of B2705-restricted molecular mimicry between the three peptides (14). In contrast, CTL cross-reactivity between the pVIPR and pLMP2 peptides has only rarely been observed when these ligands are displayed by B2709, suggesting lack of molecular mimicry (16). ...
... (ii) Does the overall conformational plasticity of a given peptide-HLA-B27 complex allow to correlate this property with CTL reactivity? and (iii) Do the complexes of B2709 displaying pGR, pVIPR, or pLMP2 exhibit a lack of comprehensive molecular mimicry? Together with previously published studies (13)(14)(15)(16)18) the high resolution structure of pGR-B2709 that we describe here permits to shed light on these questions and indicates that the occurrence of peripheral self-reactive CTL is connected with the conformational plasticity of peptide-HLA-B27 complexes. ...
Article
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The development of autoimmune disorders is incompletely understood. Inefficient thymic T cell selection against self-peptides presented by major histocompatibility antigens (HLA in humans) may contribute to the emergence of auto-reactive effector cells, and molecular mimicry between foreign and self-peptides could promote T cell cross-reactivity. A pair of class I subtypes, HLA-B2705 and HLA-B2709, have previously been intensely studied, because they are distinguished from each other only by a single amino acid exchange at the floor of the peptide-binding groove, yet are differentially associated with the autoinflammatory disorder ankylosing spondylitis. Using X-ray crystallography in combination with ensemble refinement, we find that the non-disease-associated subtype HLA-B2709, when presenting the self-peptide pGR (RRRWHRWRL), exhibits elevated conformational dynamics, and the complex can also be recognized by T cells. Both features are not observed in case of the sequence-related self-peptide pVIPR (RRKWRRWHL) in complex with this subtype, and T cell cross-reactivity between pGR, pVIPR, and the viral peptide pLMP2 (RRRWRRLTV) is only rarely observed. The disease-associated subtype HLA-B2705, however, exhibits extensive conformational flexibility in case of the three complexes, all of which are also recognized by frequently occurring cross-reactive T cells. A comparison of the structural and dynamic properties of the six HLA-B27 complexes, together with their individual ability to interact with T cells, permits us to correlate the flexibility of HLA-B27 complexes with effector cell reactivity. The results suggest the existence of an inverse relationship between conformational plasticity of peptide-HLA-B27 complexes and the efficiency of negative selection of self-reactive cells within the thymus.
... In support of this notion, HLA-B27-restricted CD8+ T cells were detected in the synovial fluid of AS patients (57, 58). They are also found to be directed against self-peptides derived from vasoactive intestinal peptide type 1 receptor (VIP1R, aminoacids 400-408) and glucagon receptor (GR, aminoacids 412-420) (59,60). A controversial finding pointed out that HLA-B*27:09 subtype that is not associated with the disease also presents the VIP1R-derived peptide (61). ...
Article
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Spondyloarthritides (SpA) are a family of interrelated rheumatic disorders with a typical disease onset ranging from childhood to middle age. If left untreated, they lead to a severe decrease in patients' quality of life. A succesfull treatment strategy starts with an accurate diagnosis which is achieved through careful analysis of medical symptoms. Classification criterias are used to this process and are updated on a regular basis. Although there is a lack of definite knowledge on the disease etiology of SpA, several studies have paved the way for understanding plausible risk factors and developing treatment strategies. The significant increase of HLA-B27 positivity in SpA patients makes it a strong candidate as a predisposing factor and several theories have been proposed to explain HLA-B27 driven disease progression. However, the presence of HLA-B27 negative patients underlines the presence of additional risk factors. The current treatment options for SpAs are Non-Steroidal Anti-Inflammatory Drugs (NSAIDs), TNF inhibitors (TNFis), Disease-Modifying Anti-Rheumatic Drugs (DMARDs) and physiotherapy yet there are ongoing clinical trials. Anti IL17 drugs and targeted synthetic DMARDs such as JAK inhibitors are also emerging as treatment alternatives. This review discusses the current diagnosis criteria, treatment options and gives an overview of the previous findings and theories to clarify the possible contributors to SpA pathogenesis with a focus on Ankylosing Spondylitis (AS) and enthesitis-related arthritis (ERA).
... This is performed by threading its sequence on the constrained backbone of a peptideepitope 3D pattern preselected by the authors. DockTope provides 5 such patterns (PDB IDs 1LK2 [87], 2V2W [42], 2A83 [88], 1WBX [89] and 1WBY [89]) covering 4 MHC allotypes, and thus allowing the docking of 8-mers into H-2-Kb, 9-mers into HLA-A*02:01, HLA-B*27:05 and H-2-Db, and 10-mers into H-2-Db. This threading is followed by an energy-minimization to mildly relax the conformation of the peptide, following a protocol identical to the one described in the third step, below. ...
Chapter
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The immune system is constantly protecting its host from the invasion of pathogens and the development of cancer cells. The specific CD8+ T-cell immune response against virus-infected cells and tumor cells is based on the T-cell receptor recognition of antigenic peptides bound to class I major histocompatibility complexes (MHC) at the surface of antigen presenting cells. Consequently, the peptide binding specificities of the highly polymorphic MHC have important implications for the design of vaccines, for the treatment of autoimmune diseases, and for personalized cancer immunotherapy. Evidence-based machine-learning approaches have been successfully used for the prediction of peptide binders and are currently being developed for the prediction of peptide immunogenicity. However, understanding and modeling the structural details of peptide/MHC binding is crucial for a better understanding of the molecular mechanisms triggering the immunological processes, estimating peptide/MHC affinity using universal physics-based approaches, and driving the design of novel peptide ligands. Unfortunately, due to the large diversity of MHC allotypes and possible peptides, the growing number of 3D structures of peptide/MHC (pMHC) complexes in the Protein Data Bank only covers a small fraction of the possibilities. Consequently, there is a growing need for rapid and efficient approaches to predict 3D structures of pMHC complexes. Here, we review the key characteristics of the 3D structure of pMHC complexes before listing databases and other sources of information on pMHC structures and MHC specificities. Finally, we discuss some of the most prominent pMHC docking software.
... De même, un autre peptide du soi, provenant du récepteur du glucagon (pGR) possède des homologies avec le peptide LMP2, mais aussi avec l'Assimilatory nitrate reductase catalytic subunit (nasA) de Klebsiella pneumoniae, lorsque celui-ci est présenté par HLA-B27. Des auteurs ont observé que ce peptide pGR pouvait se lier au produit des allèles associés à la SpA, tels que HLA-B*2705, HLA-B*2704, et HLA-B*2702, et non pas aux sous-types non associés à la SpA, tels que HLA-B*2706 et HLA-B*2709 [118]. ...
Thesis
Spondyloarthritis (SpA) is a chronic inflammatory rheumatic disorder characterized by joint manifestations affecting the spine, peripheral joints and entheses, as well as extra-articular manifestations such as psoriasis, uveitis, or intestinal inflammation. This complex disorder has a strong genetic component dominated by the HLA-B27 antigen of the major histocompatibility complex class I (MHC-I), which is present in more than 80% of SpA patients. Discovered 45 years ago, the association between HLA-B27 and SpA development remains unexplained. Several hypotheses have been proposed to explain this association at the molecular level, but all face experimental inconsistencies that seem to invalidate them. Therefore, it appeared to us essential to elaborate new and yet unexplored approaches in order to better understand the molecular role of HLA-B27 in SpA development. Drosophila melanogaster is a powerful genetic model that has led to considerable advances in understanding numerous functions of metazoan cells, as well as in describing the cellular and molecular processes of many human pathologies. To elucidate the molecular pathogenic mechanisms associated with HLA-B27, we have established several transgenic Drosophila lines for SpA-associated and non-associated of HLA-B alleles, as well as for the MHC-I invariant chain, the human 2-microglobulin (hβ2m). Expression of the HLA-B27 alleles, in the presence of hβ2m, in the Drosophila wing and eye led to two specific phenotypes. The crossveinless wing phenotype is due to a disturbance in the Bone Morphogenetic Protein (BMP) signaling pathway. Interestingly, this misregulation is associated with a co-localization of HLA-B27 and the BMP type I receptor named Sax. Our preliminary results obtained in SpA patient cells suggest that HLA-B27 also colocalizes with ALK2 receptor, which is ortholog to Sax. Altogether, our results suggest that the pathogenic role of HLA-B27 in SpA may depend on a BMP signaling misregulation at the crosstalk between ossification and inflammation.
... The accession codes for the selected structures were PDB 4MAY 51 for HLA-DQB1 and PDB 2A83 (ref. 52 ) for HLA-B. Both structures were visualized in PyMOL v. 1.8.2.1 (http://www.pymol.org/). ...
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Allergic rhinitis is the most common clinical presentation of allergy, affecting 400 million people worldwide, with increasing incidence in westernized countries1,2. To elucidate the genetic architecture and understand the underlying disease mechanisms, we carried out a meta-analysis of allergic rhinitis in 59,762 cases and 152,358 controls of European ancestry and identified a total of 41 risk loci for allergic rhinitis, including 20 loci not previously associated with allergic rhinitis, which were confirmed in a replication phase of 60,720 cases and 618,527 controls. Functional annotation implicated genes involved in various immune pathways, and fine mapping of the HLA region suggested amino acid variants important for antigen binding. We further performed genome-wide association study (GWAS) analyses of allergic sensitization against inhalant allergens and nonallergic rhinitis, which suggested shared genetic mechanisms across rhinitis-related traits. Future studies of the identified loci and genes might identify novel targets for treatment and prevention of allergic rhinitis.
... Early studies detected CD8+, and to a lesser degree CD4+, Tcell oligoclonal expansion in AS patients [20][21][22]. Mass sequencing of the TCRβ CDR3 has detected expanded populations exhibiting a pro-inflammatory phenotype and CDR3 sequence similarity to clones identified in HLA-B27-positive reactive arthritis patients, and T-cells responsive to the HLA-B27 presented self-peptide pVIPR (RRKWRRWHL) [23,24]. A true immunogenic peptide, if it exists, is predicted to be a microbial mimic of a self-peptide that leads to loss of tolerance and autoimmunity (the 'arthritogenic peptide' theory). ...
Article
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Ankylosing spondylitis (AS) is a highly heritable disease for which there is a great unmet need for improved therapies. Genetics research has identified several major pathways involved in the disease, from which treatments have either now entered clinical practice or are in development. In particular, therapies targeting the IL-23 pathway were repositioned for use in AS following the discovery of multiple genes in the pathway as determinants of AS risk. Discovery of the association of aminopeptidase genes with AS, and subsequently with psoriasis, inflammatory bowel disease and other conditions, has triggered research into therapies targeting this pathway. The AS-genetic associations point to involvement of gut mucosal immunity in driving disease, and metagenomic studies have provided strong support that AS is a disease driven by interaction between the gut microbiome and host immune system, providing a rationale for the exploration of gut-targeted therapies for the disease.
Article
The association of the human lymphocyte antigen B27 (HLA-B27) with ankylosing spondylitis (AS), also now called axial spondylarthritis (axSpA), was first described 50 years ago. This article gives an overview of the available knowledge on the topic. This is a narrative review based on the experience of the authors. The HLA-B27 is a member of the HLA class I family of genes of the major histocompatibility complex (MHC). The prevalence of HLA-B27 in the central European population is approximately 8 %, i.e., the vast majority of carriers of HLA-B27+ remain healthy. The frequency of HLA-B27 shows a decline from north to south. The HLA-B27 explains only 30 % of the genetic burden of axSpA. The prevalence of the disease correlates with the frequency of HLA-B27 in the population, i.e., there are geographic differences. Approximately 60–90 % of patients with axSpA worldwide are HLA-B27+. Some 200 subtypes of HLA-B27 can be differentiated using the polymerase chain reaction (PCR). In Thailand and Sardinia two subtypes were found that are not associated with axSpA. The physiological function of HLA class I molecules is the defence of the organism against microbes. Microbial peptides are presented to the immune system, which can be specifically attacked by CD8+ T‑cells. Genetic polymorphisms of the enzyme endoplasmic reticulum aminopeptidase 1 (ERAP1), which breaks down peptides in the endoplasmic reticulum, are associated only with HLA-B27+ diseases. The pathogenesis of axSpA is unclear but a major hypothesis is that of the arthritogenic peptides. In this it is assumed that potentially pathogenic foreign or autologous peptides can be presented by HLA-B27. If nothing else, HLA-B27 plays an important role in the diagnosis, classification and determination of the severity of axSpA.
Preprint
A structural characterization of the interaction between T cell receptors (TCR) and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the X-ray data. We found that in a large number of systems ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and discriminating between induced fit and conformational selection models of TCR binding. We show that the currently widespread practise of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from X-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data, but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.
Article
Objectives: HLA-B27 and the endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 genes are predisposing factors for AS. A single nucleotide polymorphism (SNP) in the ERAP2 promoter (rs75862629) coordinates the transcription of both ERAP genes. We investigated whether this SNP associates with AS and whether it affects the expression of the two major HLA-B27 alleles present in Sardinia, the AS-associated B*2705 and the non-AS-associated B*2709. Methods: Four SNPs in the ERAP region were genotyped in HLA-B*2705-positive patients with AS (n = 145), B27-positive healthy subjects (n = 126) and B27-negative controls (n = 250) and the allele and haplotype frequencies were derived. The expression of ERAP1 and ERAP2 mRNAs in 36 HLA-B27-positive B lymphoblastoid cell lines was measured by quantitative PCR. An electrophoretic mobility shift assay was performed to search for a nuclear factor binding the DNA sequence encompassing rs75862629. The expression of HLA-B27 molecules related to the SNP at rs75862629 was determined by flow cytometry. Results: The minor allele G at rs75862629 was found significantly increased in B27 healthy individuals, both B*2705 and B*2709, compared with B*2705-positive patients with AS and B27-negative controls. The electrophoretic mobility shift assay indicated the lack of binding of a transcription factor as the cause of the observed reduction in the ERAP2 concomitant with a higher ERAP1 expression. Of note, this occurs with a different cell surface expression of the HLA-B*2705 and HLA-B*2709 molecules. Conclusion: SNP rs75862629, by modulating simultaneously the expression of ERAP1 and ERAP2, provides protection from AS in HLA-B27-positive subjects in Sardinia. This has a functional impact on HLA-B27 expression and likely on disease onset.
Article
Full-text available
A structural characterization of the interaction between αβ TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.
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Publisher Summary X-ray data can be collected with zero-, one-, and two-dimensional detectors, zero-dimensional (single counter) being the simplest and two-dimensional the most efficient in terms of measuring diffracted X-rays in all directions. To analyze the single-crystal diffraction data collected with these detectors, several computer programs have been developed. Two-dimensional detectors and related software are now predominantly used to measure and integrate diffraction from single crystals of biological macromolecules. Macromolecular crystallography is an iterative process. To monitor the progress, the HKL package provides two tools: (1) statistics, both weighted (χ 2 ) and unweighted (R-merge), where the Bayesian reasoning and multicomponent error model helps obtain proper error estimates and (2) visualization of the process, which helps an operator to confirm that the process of data reduction, including the resulting statistics, is correct and allows the evaluation of the problems for which there are no good statistical criteria. Visualization also provides confidence that the point of diminishing returns in data collection and reduction has been reached. At that point, the effort should be directed to solving the structure. The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
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Human immunodeficiency virus 1 (HIV-1) infection is associated with a vigorous cellular immune response that allows detection of cytotoxic T lymphocyte (CTL) activity using freshly isolated peripheral blood mononuclear cells (PBMC). Although restricting class I antigens and epitopes recognized by HIV-1-specific CTL have been defined, the effector cells mediating this vigorous response have been characterized less well. Specifically, no studies have addressed the breadth and duration of response to a defined epitope. In the present study, a longitudinal analysis of T cell receptor (TCR) gene usage by CTL clones was performed in a seropositive person using TCR gene sequences as a means of tracking responses to a well-defined epitope in the glycoprotein 41 transmembrane protein. 10 CTL clones specific for this human histocompatibility leukocyte antigen-B14-restricted epitope were isolated at multiple time points over a 31-mo period. All clones were derived from a single asymptomatic HIV-1-infected individual with a vigorous response to this epitope that was detectable using unstimulated PBMC. Polymerase chain reaction amplification using V alpha and V beta family-specific primers was performed on each clone, followed by DNA sequencing of the V-D-J regions. All 10 clones utilized V alpha 14 and V beta 4 genes. Sequence analysis of the TCR revealed the first nine clones isolated to also be identical at the nucleotide level. The TCR-alpha junctional region sequence of the tenth clone was identical to the junctional region sequences of the other nine, but this clone utilized distinct D beta and J beta gene segments. This study provides evidence that the observed high degree of HIV-1-specific CTL activity may be due to monoclonal or oligoclonal expansion of specific effector cells, and that progeny of a particular CTL clone may persist for prolonged periods in vivo in the presence of a chronic productive viral infection. The observed limited TCR diversity against an immunodominant epitope may limit recognition of virus variants with mutations in regions interacting with the TCR, thereby facilitating immune escape.
Article
An immunological basis has been postulated for the strong association between at least five subtypes of the HLA-B27 allele (B27.01, .02, .04, .05, and .06) and ankylosing spondylitis, namely that cytotoxic T lymphocyte (CTL) responses are induced against an "arthritogenic" peptide that these different subtypes can all present. This requires a degree of overlap between the peptide binding repertoires of different B27 molecules. The present work, using CTL responses to Epstein-Barr virus (EBV) as a model system in which to identify B27-restricted epitopes, provides the first direct evidence that different disease-related alleles can present the same immunodominant peptide. We first noted that EBV-specific CTL clones, whether from B27.05-, B27.02-, or B27.04-positive donors, were largely subtype-specific in their restriction, recognizing only EBV-transformed B cell lines of the relevant B27 subtype. However, when tested against targets expressing individual EBV proteins from recombinant vaccinia virus vectors, all B27.05-restricted, all B27.02-restricted, and a proportion of B27.04-restricted clones were reactive to the same viral nuclear antigen, Epstein-Barr nuclear antigen (EBNA)3C. In subsequent peptide sensitization assays, all the EBNA3C-specific clones tested and also the EBNA3C-specific component within polyclonal CTL preparations from B27.05-, B27.02-, or B27.04-positive donors recognized the same immunodominant viral peptide RRIYDLIEL (EBNA3C residues 258-266). This sequence accords well with the proposed B27.05 peptide motif and clearly must be accommodated within the different peptide binding grooves of B27.05, B27.02, and B27.04 molecules. Clonal analysis revealed a second component of the B27.04-restricted response that was not shared with other subtypes. This was directed against an EBV latent membrane protein LMP2 epitope whose sequence RRRWRRLTV satisfies some but not all requirements of the B27.05 peptide motif. We conclude that there is indeed a degree of functional overlap between different B27 subtypes in their selection and presentation of CTL epitopes.
Article
Ankylosing spondylitis (AS) and Reiter's syndrome (RS) both show a strong correlation with the HLA B27 haplotype. We studied whether sharing of homologous amino acid sequences in the HLA B27 antigen with an invading microbe might occur, and if so, what is the biological significance of such homology. In a computer search of the Dayhoff data bank, we found a homology of six consecutive amino acids between HLA B27.1 antigen residues 72-77 and Klebsiella pneumoniae nitrogenase residues 188-193. These shared sequences are hydrophilic, suggesting locations on molecules exposed to the cell surface. Immunochemical analysis showed that 18 of 34 sera from patients with RS (53%) and 7 of 24 sera from patients with AS (29%) contained antibodies that bound to a synthesized peptide sequence representing residues 69-84 of HLA B27.1. In contrast, only 1 of 22 sera from healthy, B27+ controls (5%) contained antibodies to this peptide (p less than 0.01). Sera from three HLA B27- patients with RS did not possess antibodies to the HLA B27 peptide. Additionally, greater than 40% of HLA B27 patients with AS or RS had antibodies to Klebsiella residues 184-193, while none of the normal nonarthritic HLA B27 haplotype subjects did. Our results suggest that an autoimmune response(s) directed against HLA B27.1 may be a pathogenic mechanism in a subset of patients with AS and RS. Further, this response may initially be induced against Klebsiella pneumoniae, a microorganism that shares sequence homology with HLA B27.
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Rheumatoid arthritis is genetically linked to major histocompatibility complex (MHC) molecules (HLA-DR4 and related molecules) and characterized pathologically by high levels of HLA-DR expression and infiltration of proliferative of synovial tissue with CD4+ T lymphocytes. T-lymphocyte activation is driven by specific signaling through polymorphic alpha/beta T-cell receptors (TCRs) that are reactive with antigen-MHC complexes present at the sites of inflammation. We are interested in characterizing rheumatoid TCRs molecularly to ascertain potential binding surfaces for antigen + MHC in synovial tissue. Accordingly, we have recently investigated the TCR alpha and beta chain heterogeneity in a series of 10 rheumatoid synovia obtained at the time of joint surgery. The most frequently detected Vbeta families were Vbeta12, 14, and 17, each of which was found in 80% of specimens. We report here the molecular cloning and sequence analysis of 20 cloned Vbeta segments amplified with a Vbeta14 family-specific TCR primer, and six cloned Vbeta segments amplified with a Vbeta17 family-specific TCR primer from four rheumatoid synovia. Comparison with the data base revealed that these sequences belonged to the closely related Vbeta3, Vbeta14, and Vbeta17 families. Dominant clones were apparent in two of the individuals by the presence of identical V-D-J regions, suggesting an antigen-driven process. Amino acid sequence analysis revealed a conserved motif in the putative fourth hypervariable region or CDR4. Molecular modeling of this epitope suggests that charged side chains are available for binding to ligand structures (e.g., antigen, MHC, or superantigen). We suggest this epitope may play a role in the molecular pathogenesis of rheumatoid arthritis.
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We have examined the basis for immunodominant or “public” TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen, the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1α and CDR2α loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRα constant domain thought to form part of the docking site for CD3ϵ. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.