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*
Division of Immunology, St. Luke’s Medical Center, Milwaukee, WI 53215;
Department of Immunology, Corixia Corp. (formerly Anergen Inc.), Redwood City, CA 94010; and
Department of Immunology, Mayo Medical School, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit. We find two promiscuous
determinants on this region 141–160 and 171–190 as defined by their
immunogenicity in HLA DR3-, HLA DQ8-, and HLA DQ6-transgenic mice in
the absence of endogenous mouse class II molecules. We also studied the
minimal determinants of these two regions by truncation analysis, and
the MHC binding affinity of a set of overlapping peptides spanning the
complete sequence region of human acetylcholine receptor subunit.
One of the peptide sequences strongly immunogenic in HLA DR3-transgenic
mice also had the highest binding affinity to HLA DR3. Identification
of T cell determinants restricted to an MHC molecule known to
predispose to MG may be an important step toward the development of
peptide-based immunomodulation strategies for this autoimmune
disease. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nicotinic AChR is the predominant autoantigen in MG. Autoantibodies and
T cells reactive to AChR are found in patients with MG, and symptoms of
MG could be induced in experimental animals by immunizing them with
AChR in complete adjuvant. It is reported that the major target for
autoantibodies is a domain on the extracellular region of the
subunit of AChR, termed the main immunogenic region (1).
Several T cell determinants on the extracellular region of the
subunit of AChR have also been identified. These epitopes were
generally identified in patients that were not HLA-typed; therefore,
the MHC-restricting alleles were not identified in most cases. Within
the subunit, the regions with clustered T cell epitopes have been
described to be the nontransmembrane domains, the predominant being
region 1–210 (10).
We used mice transgenic (tg) to HLA DR3 molecule to study the MHC restriction in human AChR (hAChR) presentation. Such mice, carrying human class II Ag-presenting molecules in the absence of the mouse class II Ag-presenting molecules, are a good resource to study the role of any HLA molecule in autoantigen presentation and disease pathogenesis. Attempts to identify the HLA restriction of human CD4+ T cells are complicated by the expression of three different loci (HLA-DR, -DQ, and -DP) by APCs and the potential for heterozygosity at each locus. The reductionist approach of studying AChR T cell responses in HLA DR-tg mice let us understand the Ag-presenting function of the given human MHC class II molecule in isolation rather than in the context of three to six different MHC class II molecules on the cell surface. We have previously reported the disease susceptibility of HLA DQ8- and DQ6-tg mice to Torpedo AChR (TAChR)-induced experimental autoimmune MG (EAMG) (11).
In this study we injected the tg mice with peptides corresponding to
hAChR sequences to avoid possible artifacts due to differences between
the TAChR and hAChR sequences. Here, we report the HLA DR3-restricted T
cell determinants of hAChR subunit (1–210) and compare the results
with the in vitro binding affinity of these sequences to HLA DR3. These
studies identify two epitopes that dominate the T cell response, one of
which binds DR3 with high affinity and binds promiscuously to the other
HLA class II molecules.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HLA DR3 mice were bred and maintained in the Immunogenetics
mouse colony of Mayo Clinic. HLA DR3-tg founder mice were obtained from
Gunter Hammerling (German Cancer Research Center, Heidelberg,
Germany; Ref. 12). Briefly, a 6-kb
NdeI fragment of a HLA DRA genomic clone in pUC and a
24-kb ClaIxSalI fragment of cos 4.1
containing the B gene were coinjected into fertilized
eggs from (C57BL/6xDBA/2)F1 donors mated with C57BL/6
males. The tg mice were bred on to I-A
knockout mice
(11). HLA DR3-tg mice were bred on to C57BL/10 background
for 10 generations. The line of mice used in the experiments presented
here did not express the endogenous mouse class II molecules, I-A or
I-E. HLA DQ8- and DQ6-tg mice have been described elsewhere
(11).
Flow cytometry
HLA DR3 expression on B10.DR3-tg mice were analyzed by flow
cytometry. B10 and B10.A0 mice were used as
controls. Briefly, PBL were isolated by Ficoll separation, and
single-color flow cytometric analysis of HLA DR expression was
performed using mAb L227 (13).
AChR purification and disease induction
TAChR was purified and quantitated from Torpedo californica (Aquatic Research Consultants, San Pedro, CA) electric organ (14). Mice were given three injections (s.c.) of TAChR (20 µg/mouse) at 4-wk intervals. The first injection was in CFA, and boosters were given in IFA in a 1:1 ratio. After 12 wk, mice were sacrificed. Clinical assessment: Muscle weakness was assessed every week (11). Briefly, mice were allowed to grip their paws on cage top grids, were pulled off the grid by tail consecutively for 25 times for forced exercise, and were scored as follows: grade 0, no weakness at rest or after exercise; grade 1, normal strength at rest, but weak with chin on the floor and unable to raise the head after exercise; grade 2, the mice exhibit grade 1 weakness at rest; and grade 3, moribundity or quadriplegia. Electrophysiological Assessment: This was performed as explained (Refs. 15, 16). Briefly, The right midcostal diaphragm muscle was excised together with phrenic nerve and mounted vertically in a glass tissue chamber containing Ringer’s solution. Neuromuscular transmission failure (NMTF) was assessed at 40 Hz. Nerve stimulation was presented at 40 Hz in 330-ms duration trains repeated 1 train per second for a period of 2 min. Every 15 s, direct muscle stimulation at 40 Hz was superimposed. The following formula was used to estimate the NMTF. NMTF = (F - MF)/(1 - MF), where F is the force loss during nerve stimulation and MF is contractile failure (15, 16).
Peptides
Twenty-amino-acid-long synthetic peptides overlapping by 10 aa
and spanning the extracellular sequences 1–210 residues of hAChR
subunit were synthesized at the peptide core laboratory at Mayo Clinic.
Fig. 1
shows the sequence of the hAChR subunit extracellular
region. Two panels of truncated analogs of the sequences
141–160 and 171–190, missing increasing
numbers of residues on the carboxyl- or amino-terminal ends of these
sequences, were also synthesized in the Mayo Peptide Core laboratory.
The actual residues included in these sequences are:
141–160, 143–160,
145–160, 147–160,
149–160, 151–160,
153–160, 141–158,
141–156, 141–154,
141–152, 141–150,
141–148 and 171–190,
173–190, 175–190,
177–190, 179–190,
181–190, 183–190,
171–188, 171–186,
171–184, 171–182,
171–180, and 171–178. The
overlapping peptides used for MHC binding studies were synthesized on a
Gilson AMS422 (Gilson, Middleton, WI) instrument at Anergen. These
peptides were designed as a series of 14-mer sequences overlapping with
adjacent peptides by 7 aa. The apo-protein B 100 peptide 1273–1291
used as the signal peptide in competition binding assays with HLA DR3
was synthesized on an Applied Biosystems (Foster City, CA) 431
instrument and biotinylated by diisopropylcarbodiimide coupling.
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Mice were immunized with the respective peptide (50 µg/mouse) in CFA, as a thick emulsion, at the base of the tail and one of the foot pads.
T cell proliferation assays
Mice were sacrificed 7–10 days after immunization and draining
lymph node cells were removed. After lysis of RBC, the cells
were cultured in the presence of individual peptide (4, 10, or 50
µg/ml), Con A, or in the absence of Ag as controls in TCM (2.5 mM
HEPES, 100 mM sodium pyruvate, 5% horse serum, 0.1% 2-ME in RPMI 1640
with glutamine, penicillin, and streptomycin). After 24 h,
[3H]thymidine was added. Eighteen hours later,
the cells were harvested and thymidine uptake was measured using a
scintillation counter. The results were presented either as
stimulation indices (cpm of the test sample/cpm of the negative
control) or cpm.
MHC-peptide binding
The relative affinity of AChR peptides for HLA DR was
measured by an europium-streptavidin dissociation-enhanced lanthanide
fluoroimmunoassay (DELFIA) developed by Jensen and colleagues
(17). Briefly, homozygous EBV-transformed B cells
expressing HLA DR3 (line AVL, GMO 6823A) were obtained from the
National Institute of General Medical Sciences (Camden, NJ). Cells were
cultured in 40-liter spinner flasks, and the cell pellet was
solubilized in 0.5% Triton X-100. HLA DR3 was purified by affinity
chromatography with the mAb L243 (R&D Systems, Minneapolis, MN).
Solubilized HLA DR3 at a concentration of 50 nM was incubated overnight
at 37°C, pH 5.5, with 1000 nM biotinylated Apo B 100 1273–1291 and
10–100,000 nM unlabeled competitor AChR peptide. HLA
DR3-peptide complexes were added to 96-well plates precoated with
anti-HLA DR mAb L243 and washed to remove unbound material. The
plates were subsequently incubated with europium-avidin, washed, filled
with enhancing solution, and measured on a Wallac (Gaithersburg, MD)
1234 DELFIA research fluorometer. Triplicate samples for each unlabeled
AChR peptide were measured at each concentration, and the
IC50 was determined by four-parameter fit
analysis with the software program SOFTmax Pro (Molecular Devices,
Sunnyvale, CA). These studies were performed with purified HLA DR3
solubilized in detergent in the presence of a mixture of protease
inhibitors. There is no evidence in our studies or in those of others
using the DELFIA method that the peptide is consumed or degraded.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of HLA DR3 molecule has been reported to have a clear
predisposing effect for MG in humans. Identifying the peptides on the
hAChR that bind to the DR3 molecule and that stimulate T cells selected
in a system where HLA DR3 is the restricting element should aid our
understanding of the immunopathogenesis of MG. When availability of
AChR is limited, peptides spanning the sequence of hAChR become a
valuable tool to identify T cell epitopes. When individual short
peptides are used to immunize the mice, in vitro T cell proliferation
assays may reveal dominant sequences as well as cryptic sequences. Both
dominant and cryptic sequence regions are important in autoimmunity as
the Ag presentation aberrations, if any, in autoimmunity are unclear.
Previously, human PBL were used as APC to identify HLA class
II-restricted epitopes. Because these APCs carry multiple HLA class II
molecules, blocking Abs specific to HLA class II Ags were used to
understand MHC restriction. Experimental studies using mice tg to human
Ag-presenting molecules allow us to study the in vivo HLA-restricted T
cell selection and Ag-specific responses. Therefore, we used mice tg to
human Ag-presenting molecule (HLA DR3) that do not express mouse MHC
class II molecules to study the DR3-restricted T cell determinants on
hAChR subunit (1–210) extracellular region.
HLA DR3-tg mice are susceptible to EAMG
As shown in Fig. 2, HLA DR3-tg mice demonstrate significant cell surface expression of HLA
DR3 molecule. Before embarking on the study of T cell determinants on
AChR recognized in HLA DR3-tg mice, we investigated the susceptibility
of these mice to EAMG. We used HLA DR3-tg mice and mice that do not
express any MHC class II molecules. When these mice were immunized with
TAChR, a widely used immunogen in the study of EAMG, 90% of the HLA
DR3-tg mice developed clinical signs of EAMG, whereas none of the mice
that lacked MHC class II molecules developed the disease (Table I). Four of the ten mice developed grade II weakness. The disease in
these mice was further confirmed by assessing NMTF (Table II) as described in Materials and Methods.
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subunit
When HLA DR3-tg mice were immunized with hAChR -chain
extracellular region (1–210) peptides, we found significant
(stimulation index (SI) >3) immune response to a limited number of
sequence regions (Fig. 3). Peptides 31–50, 41–60, 141–160, 171–190, 181–200, and
191–210 elicited an SI of >4.0 (Fig. 3). Peptides 81–100 and 91–110 elicited a moderate response (SI between
3 and 4). Peptides 141–160 and 171–190 induced consistently high T
cell responses in HLA DR3 mice. Coincidentally, we previously found
that these two peptides were strong T cell epitopes in HLA DQ8- and
DQ6-tg mice (Fig. 4). The sequence of the peptide 141–160 is homologous to the mouse
AChR141–160, but the sequence of
hAChR171–190 differs from the mouse at two
residues (Table III). Such minor sequence differences may alter T cell responses.
Therefore, we tested whether the T cells generated subsequent to
immunization with hAChR cross-react with the corresponding mAChR
peptide. As shown in Fig. 5, the T cell response to mouse peptide was comparable to that obtained
using the human peptide demonstrating self-recognition.
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To understand the minimal T cell determinant on the peptide
141–160, the peptide was increasingly truncated from carboxyl- and
amino-terminal by 2 aa. HLA DR3-tg mice were immunized with the
full-length peptide, and lymph node cells were in vitro challenged with
each of the truncated peptides as described in Materials and
Methods. The T cell response to each of the peptides is shown in
Fig. 6A. The truncated peptides 143–160, 145–160, 147–160,
149–160, and 141–158 elicited significant T cell response. However,
we found a decreasing T cell response on increased truncation from the
amino-terminal. This suggests that longer peptides (with flanking
regions) may be better T cell epitopes. The truncation analysis
demonstrates that 149–158 encompasses the minimum T cell determinant.
This 10-aa peptide contains Trp (tryptophan) at position 1 and Asp
(aspartic acid), a negatively charged residue, at position 4.
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T cell responses to the truncations of peptide 171–190 are shown
in Fig. 6
B. The truncated peptides 173–190, 175–190,
171–188, and 171–186 elicited strong T cell response, to the extent
of the T cell response elicited by the full-length peptide. This shows
that the 12 aa sequence 175–186 contains the minimum T cell
determinant presented by HLA DR3 molecule. The amino acid at position
176 is a Trp. Interestingly, the amino acid at position 179 is
lysine, a positively charged residue, and a negatively charged residue,
Glu, is at position 180.
MHC binding studies
We studied the binding affinity of a panel of overlapping hAChR
subunit peptides (1–210) to HLA DR3 (DRB1*0301) (Table IV). Within the extracellular region, the five peptides that demonstrated
relatively high binding affinity were 7–22, 27–42, 36–49, 145–163,
and 195–212. Among the five, three (36–49, 145–163, and 195–212)
corresponded to sequences that elicited strong T cell response in HLA
DR3. The other two peptides did not show any significant T cell
reactivity (compare Fig. 3 and Table IV).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene. They also lack the I-E
molecule, which is absent in the H-2b locus.
Hence any CD4+ class II-restricted T cells will
be restricted to the tg human class II molecule, HLA DR3.
Earlier studies have shown that most of the T cell epitopes on hAChR
subunit in MG patients are within the nontransmembrane domain.
Therefore, we used overlapping synthetic peptides corresponding to the
extracellular region of AChR subunit to examine HLA DR affinity and
antigenicity. HLA DR3-tg mice were immunized with each of these
peptides, and T cell responses were measured. A limited number of
peptide sequences elicited significant T cell responses.
Among the peptides that induced the strongest responses were sequences
141–160 and 171–190. These two peptides were strong T cell
determinants in HLA DQ8- and DQ6-tg mice also. We had earlier reported
that HLA DQ8-tg mice were more susceptible to EAMG compared with HLA
DQ6-tg mice. Thus the peptide sequences 141–160 and 171–190 are among
the promiscuous epitopes on AChR subunit, stimulating T cell
responses in HLA DR3-, DQ8-, and DQ6-tg mice. We earlier described such
promiscuous sequences on diphtheria toxin and tetanus toxin (18, 19). Similarly, the dominant T cell epitope associated with HLA
DR2 multiple sclerosis patients, myelin basic protein peptide 84–103,
binds with high affinity to HLA DR2 and promiscuously to nine of ten
other HLA DR alleles (20). Promiscuous sequence regions in
exogenous Ags may be relevant in eliciting universal immune responses
to develop vaccines. The promiscuous sequence regions on autoantigens
may be significant reagents in the immunomodulation of autoimmune
diseases.
Peptide 141–160 was previously identified by others as a dominant
sequence in human MG. T cell lines and clones generated to recombinant
AChR subunit using blood from MG patients were specific to 141–160
(9, 21, 22, 23, 24). These T cells were restricted to DR4, DR3,
and DR52a. Studies using congenic mice strains also have identified the
peptide 141–160 to be a dominant sequence, and this sequence was used
to suppress AChR-induced EAMG (25, 26, 27, 28). These
studies demonstrate the extent of promiscuity of this sequence segment
on the extracellular region of the AChR subunit across species, but
also the increased frequency of autoreactive T cells directed to this
sequence segment. Our studies demonstrate that apart from this
sequence, the sequence 171–190 is also promiscuous to all three
different HLA molecules we tested (HLA DR3, DQ8, and DQ6).
The role of such immunodominant sequence segments in immunomodulation may be controversial as such sequence regions may elicit strong immune response in all individuals. However, it may also be argued that there could be functional differences in the T cell population generated by these sequences in the context of different HLA molecules. We observed the latter. Although the T cell response to 141–160 was equally strong in DQ8- and DQ6-tg mice, the responding T cells were functionally distinct as shown by dichotomy in cytokine secretion (data not shown). Such functional dichotomy in T cells generated might play a crucial role in switching a functional physicochemical milieu (e.g., cytokines) in an individual more toward disease susceptibility or to disease resistance. Hence promiscuous sequence regions may be significant in the etiopathogenesis of autoimmunity as well as in immunomodulation of autoimmunity.
We truncated the peptides hAChR141–160 and
hAChR171–190 to identify the minimum immune
recognition motifs and found that the peptide 147–158 and 175–186
contains the minimum determinants for HLA DR3-restricted T cell
response. In the case of 141–160, with the loss of every two residues
from the amino-terminal of the sequence there was a continuous loss in
the magnitude of T cell response. It is possible that although the
minimal determinant on this peptide is within the sequence region
147–158, flanking sequences are also important in effective T cell
response. The necessity of such flanking sequences in T cell responses
have been described earlier by others (29). The results
summarized in Table V
show that there is considerable overlap in the minimal determinants as
seen in HLA DR3-, DQ8-, and DQ6-tg mice in the recognition of the two
promiscuous AChR subunit sequence segments.
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171–190, an equally
strong immune response was obtained (Fig. 5). This demonstrates the
breaking of tolerance to an array of AChR sequences when immunized with
the homologous (self) sequence (e.g., 141–160) or with a heterologous
(nonself) sequence (e.g., 171–190). It may be argued that what we obtained in this study are probably not immunodominant sequences, as we used individual peptides to immunize the mice. Native hAChR is expressed at low density on muscle cells, and is difficult to purify in significant quantities. Many investigators including ourselves have used TAChR as an Ag source in disease induction studies (26, 30). In the study of autoimmunity, cryptic sequences may be as important as dominant sequences (31). Aberrations in Ag processing and presentation are hypothesized to mask identification of several determinants or reveal additional determinants. The identification of T cell determinants using individual synthetic peptides as immunogen may reveal not only cryptic determinants, but also some or most dominant determinants.
The MHC-peptide binding studies mostly correlate with the T cell
response studies. It is significant to note that the two lines of
studies were planned and executed in independent laboratories and
compared at the end. This is one reason for the differing design of the
overlapping synthetic peptides used in the two studies. We found that
within the extracellular region sequence of hAChR, among the five
peptides (7–22, 27–42, 36–49, 145–163, and 195–212) that bound HLA
DR3 with the highest affinity, three (36–49, 145–163, and 195–212)
were T cell stimulatory. Also, as seen in Table 6, of the five peptides on the complete sequence of hAChR subunit
that bound HLA DR3 with highest affinity, four were within the
extracellular region sequence (1–210). Three of the four (36–49,
144–163, and 195–212) contained sequences that elicited very strong T
cell response in the HLA DR3-tg mice. These sequences are aligned
according to previously defined HLA DR3 binding motifs (Table IV)
(32, 33, 34, 35). Amino acids that correspond to consensus anchor
residues are indicated in bold. Peptide 195–212 does
not obviously match the predicted DR3 motif; two potential alignments
are indicated.
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-chain. These predicted key residues occur within the
sequences of minimal determinants observed with truncated analogs of
full-length T cell determinants 141–160 and 171–190. Published
binding studies with single alanine substitutions in
AChR145–163 demonstrated that the Phe at 149
and Asp at 152 were critical for binding to HLA DR3 (9).
The minimum T cell epitope of the DR3-tg mice was within aa 149–157,
conforming to the predicted position of the DR3 binding sequence. The
MHC binding studies did not demonstrate any significant binding of
sequences corresponding to 171–190. However, these binding studies
were performed with a different set of peptides than those used for the
tg mouse studies. Based on the minimal T cell epitope and analogy to
other DR3 binding peptides, we predict that Glu at 180 corresponds to
position 4 of the core binding epitope, anchored by the positively
charged pocket in the middle of the DR3 binding groove, and that Val at
177 is anchor position 1. Alternatively, if some kinking of the peptide
occurs, the anchor residue in the hydrophobic pocket could be Trp at
176. The poor binding of AChR176–189 may
indicate that additional residues are required beyond the core sequence
to stabilize the binding of this epitope. Alternatively, this may
represent a case in which a low-affinity peptide stimulates a potent T
cell response. Additional peptide binding studies may help to resolve
this question.
This is the first scan of hAChR peptide binding to HLA DR3. This is
also the first report on the T cell determinants on hAChR subunit
using HLA DR3-tg mice. This reductionist approach to studying the role
of HLA Ags in Ag presentation and disease pathogenesis can aid in the
design of peptide-based immunotherapeutic measures for the treatment
and/or prevention of MG.
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Acknowledgments |
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0 mice, Michelle Smart for tissue typing the mice, and
Julie Hanson and her crew for breeding and maintaining the mice used in
this study. We thank Dr. Bianca Conti-Fine for TAChR, and Dr. Gary
Sieck and Wen-Zhi Zhan for measuring the NMTF. We thank Shrikant
Deshpande for the synthesis and biotinylation of the peptide used in
the binding analysis. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Raghavanpillai Raju, Division of Immunology, St. Luke’s Medical Center, 2900 West Oklahoma Avenue, Milwaukee, WI 53215. E-mail address: raju_55902{at}yahoo.com
3 Current address: InterMune Pharmaceuticals, Burlingame, CA 94010.
4 Abbreviations used in this paper: MG, myasthenia gravis; EAMG, experimental autoimmune MG; AChR, acetylcholine receptor; hAChR, human AChR; TAChR, Torpedo AChR; tg, transgenic; DELFIA, dissociation-enhanced lanthanide fluoroimmunoassay; SI, stimulation index; NMTF, neuromuscular transmission failure.
Received for publication December 26, 2000. Accepted for publication May 9, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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values in Caucasians, Orientals and Negroids. E. D. Albert, and M. P. Baur, and W. R. Mayr, eds. Histocompatibility Testing 756.-760. Springer, New York.
-subunit. Ann. Neurol. 31:311.[Medline]
subunit. Eur. J. Immunol. 20:2563.[Medline]
chain epitope within 146–162 in the development of experimental autoimmune myasthenia gravis in C57BL6 mice. Clin. Immunol. Immunopathol. 73:338.[Medline]
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