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*
Neuroscience Group, Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom;
Ludwig Institute for Cancer Research, and Department of Biochemistry and Molecular Biology, University College, London, United Kingdom; and
Anergen Inc., Redwood City, CA 94063.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit (146–160) is presented to specific T cells from myasthenia
gravis patients by HLA-DRB3*0101—"DR52a"—or by DR4. Here we first
map residues critical for DR52a in this epitope by serial Ala
substitution. For two somewhat similar T cells, this confirms the
recently deduced importance of hydrophobic "anchor" residues at
peptide p1 and p9; also of Asp at p4, which complements this allele’s
distinctive Arg74 in DRß. Surprisingly, despite the 9
sequence differences in DRß between DR52a and DR3, merely reducing
the bulk of the peptide’s p1 anchor residue (Trp149
Phe)
allowed maximal cross-presentation to both T cells by DR3 (which has
Val86 instead of Gly). The shared
K71G73R74N77 motif in
the helices of DR52a and DR3 thus outweighs the five differences in
the floor of the peptide-binding groove. A second issue is that T cells
selected in vitro with synthetic AChR peptides rarely respond to longer
Ag preparations, whereas those raised with recombinant subunits
consistently recognize epitopes processed naturally even from whole
AChR. Here we compared one T cell of each kind, which both respond to
many overlapping 140–160 region peptides (in proliferation assays).
Even though both use Vß2 to recognize peptides bound to the same
HLA-DR52a in the same register, the peptide-selected line nevertheless
proved to depend on a recurring synthetic artifact—a widely
underestimated problem. Unlike these contaminant-responsive T cells,
those that are truly specific for natural AChR epitopes appear less
heterogeneous and therefore more suitable targets for selective
immunotherapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, the DRB1*
ß-chains are highly polymorphic. Some alleles differ only at isolated
sites such as position 57 in DRß, where Asp may change to Val, Ser,
or Ala. At the opposite end of the helix that forms one wall of the
peptide-binding groove, there is a recurring Gly/Val dimorphism at
position 86 (1). The Val86 side chain severely
limits access of bulky "p1 anchor" residues (near the N terminus of
the bound peptide) to the major hydrophobic "pocket 1," so reducing
their binding and presentation (2, 3, 4, 45). By contrast,
other DR alleles differ elsewhere by short stretches of sequence
("cassettes") that recur in different alleles (1).
Thus, in the middle of the helix, DR1 and some DR4 subtypes share a
Arg71-Ala73Ala74–Thr77
motif, whereas DR3 has
Lys71-Gly73Arg74–Asn77
instead (1) (see Discussion and Fig. 6
). While
certain peptides can bind "promiscuously" to several alleles
(5, 6), others show allele-specific motifs that reflect
critical interactions with polymorphic residues in the peptide-binding
grooves (5, 6, 7, 8, 9).
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). This
locus is less polymorphic than DRB1, but some of its alleles are in
tight linkage disequilibrium with others at DRB1, e.g., DR52a with DR3.
All of these DRß chains pair with an identical DR. While DRB3
molecules are apparently expressed less strongly than the DRB1
(6, 10), they can nevertheless present peptides to T cells
very efficiently (11, 12, 13). Recently, a specific
DR52a-binding motif has been deduced by aligning a series of epitopes
(13), but the importance of its individual components
still requires confirmation, for example by "Ala scanning."
Furthermore, while DR3 has a similar motif (8, 9, 14, 15),
it is not clear whether these alleles can present the same epitopes
(16), since DR3 reportedly has a distinct specificity
(9), perhaps because it differs from DR52a at positions 57
and 86 in the helix and at five others in the floor of the groove
(1).
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-subunit (19, 20), or pooled synthetic peptides covering the entire human
(or other) subunit sequences (21, 22). The T cells we have
raised against the full length -subunit consistently recognize
epitopes naturally processed from a range of recombinant polypeptides
and even from minute amounts of whole AChR (19).
Interestingly, two of these respond to exactly the same 146-160 region,
which is presented by DR4 to clone PM-A1 (23), and by
DR52a to clone KB-D1 (19, 24). In stark contrast, T cell lines initially selected in vitro with synthetic AChR peptides are very heterogeneous and almost never, in our experience, respond to natural epitopes from longer Ags (19, 25). We suggested several explanations (25): 1) a low sensitivity; 2) specificity for cryptic epitopes or 3) specificity for peptides bound in an unnatural register to surface class II molecules, or with extra residues or truncations at either end; 4) new primary responses to contaminants in the original peptides, as we noted in 4/20 cases (25).
The recognition of a natural 146–160 epitope by clone KB-D1 has now
enabled us firstly to map a DR52a-restricted sequence and test its
cross-presentation by DR3. Secondly, it has permitted detailed
comparisons with one of our most sensitive peptide-selected lines
(P9[p138]) that has never responded to any longer Ags, even though it
recognizes multiple independent peptides that span the same 149–157
core, and thus appeared to be specific for the correct sequence
(25).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The recombinant polypeptides and synthetic peptides (p) are described in Ref. 25 and were generated in the Oxford laboratory unless stated otherwise. Selected peptides were further analyzed and separated by high resolution HPLC on a reverse-phase C-18 column with an aqueous acetonitrile gradient containing 0.1% trifluoroacetic acid. Fractions corresponding to different peaks were pooled and lyophilized.
Mass spectrometry
Mass spectrometry was performed on a Voyager Elite XL MALDI-TOF mass spectrometer (Perseptive Biosystems, Framingham, MA), equipped with a UV (337 nm) nitrogen laser and delayed extraction. Samples were analyzed in positive ion reflectron mode, and then the major component of each was identified using postsource decay. Reflectron spectra were calibrated externally using a peptide standard calibration mix covering the mass range of interest (Perseptive Biosystems). Spots were prepared by mixing samples 1:2 with matrix, which was a saturated solution of 2,5-dihydroxybenzoic acid (Sigma Aldrich, Poole, U.K.) dissolved in water, before spotting 1 µl onto the sample plate and allowing it to dry.
Peptide binding
The assay is detailed in Ref. 26 ; in brief, HLA-DR
molecules were solubilized from the B cell lines shown in Table I
and
isolated by affinity chromatography with the anti-DR mAb L243. They
were incubated overnight at 37°C with a dose range of the test
peptide plus a constant 1 µM concentration of biotinylated APO-B 100
peptide 1273–1291 (sequence IPDNLFLKSDGRIKYTLNK) (6, 8).
The HLA-DR:peptide complexes were captured (for 2 h at 4°C) onto
triplicate microtiter wells precoated with mAb L243, washed, and
incubated with europium-Streptavidin for 90 min (Wallac,
Turku, Finland), before further washing and analysis on a Wallac 1234
DELFIA Research Fluorometer. The IC50 is the peptide
concentration required to halve the binding of the indicator peptide,
and was determined using the program SOFTmax Pro.
Antigen-presenting cells
PBL were separated on Ficoll/metrizoate, washed, and irradiated
with 30 Gy from a 137Cs source (PBLx). The EBV-transformed
B cell lines and their HLA-DR alleles are summarized in Table I; before use, they were preincubated
with mitomycin C (MMC) at 50 µg/ml for 1 h and washed. In most
experiments, the APC were then incubated at 37°C for 5 h at
5 x 106/ml with 10–50 µg/ml of peptide before
thorough washing and coculture with the T cells.
T cells and proliferation assays
The T cells and their origins are summarized in Table II. Responses of all three T cells were
clearly blocked by the anti-HLA-DR mAb L243 and not by anti-DP
or anti-DQ (data not shown). Clone PM-A1 was maintained by
restimulation with r130-265 (
1 µg/ml) plus DR4+
(subtype 0401) PBLx every 13–15 days, and expansion with IL-2 every
3–4 days thereafter (23). Clone KB-D1 and line P9[p138]
were restimulated with PHA (1:2000) plus
DR3+-DR52a+ PBLx plus IL-2 on day 0, and
subsequent IL-2 as above; for KB-D1, 25 pg/ml of IL-4 was
also added on day 0 only (24).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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146–160 epitope recognized
by two somewhat similar T cells (Table II). We also evaluate a motif
that was recently deduced by aligning 14 DR52a-restricted epitopes; its
main features are hydrophobic anchors at p1 and p9, and especially an
Asp (or occasionally an Asn or Glu) at p4 (13). The epitope core for clone KB-D1 and line P9[p138]
The epitope core proved to be very similar for KB-D1 and
P9[p138] (Table III). It differs
slightly for the DR4-restricted T cell PM-A1 (23), which
responds maximally to p149–163, whereas Thr148 was almost
essential for KB-D1, and so apparently was Gly147 for
P9[p138]. All of these T cells clearly required Trp149.
Their near-maximal responses to p147–163 and p143–156 imply no need
for long extensions at either end of this 148–156 core. They also show
that, in potent peptides with N termini extending as far as position
143, the Ala157 is not essential (45).
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Like clone KB-D1 (24), line P9[p138] proved to be
HLA-DR52a- restricted (Figs. 1 and 2). Thus,
peptides with the natural sequence (W149) were presented
maximally by APC sharing only DR52a (Fig. 1, a and
b, and Fig. 2, open symbols), whereas APC with only DR3
(±DR52b, filled symbols) presented undetectably to KB-D1 and weakly to
P9[p138].
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), a substitution that can restrict the
access of bulky hydrophobic "p1 anchor" residues to the major
hydrophobic pocket (2–4, 27, and 28). For
both the present T cells, merely changing Trp149 to Phe
allowed strong presentation by DR3 (Figs. 1 and 2), even though these
alleles differ at eight other positions in DRß (1).
Together with their unresponsiveness to p150-163 (Table III), this
clearly shows that the p1 anchor is the Trp or Phe at position 149, as
independently confirmed in Fig. 3. None of these peptides was presented
to either T cell by DR52b-transfected mouse L cells (Fig. 2), or by the
DR52b+ B cell line JVM (data not shown). Their responses to
the native sequence + DR52a, or to the Phe149
substituents + DR3, were both blocked (>90%) (Ref.
24 and unpublished data) by the mAb 16.23 that is specific
for DR52a/DR3 (29).
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To scan the epitope and compare presentation by DR52a and DR3, we
synthesized (in a second laboratory) a series of substituents, changing
each residue from 146-160 in turn to Ala, except for Ala157
which was replaced with Lys or Tyr instead. To permit presentation by
both DR52a and DR3, we introduced a Phe149 into the
"parent" sequence of these "Alanogues." We first assayed for
binding to solubilized DR molecules (Fig. 3c), focusing on DR3 because
it is expressed more strongly than DR52a (10). Only two
positions proved to be critical. As expected, changing
Phe149 to Ala abolished binding; so also did a
Phe149Tyr substitution (Fig. 3c). Notably,
Asp152 at p4 was clearly essential too, as reported
previously (8, 9, 14, 15), whereas none of the other
substitutions had significant effects. In general (although a different
labeled indicator peptide was used), the IC50 values for
most other Alanogues were comparable with those for DR4 0404, which
also has Val86 in DRß, and were about 5-fold lower for
DR4 0401, with Gly86 (46).
Residues required for presentation by DR52a
To measure their "functional binding," we next tested the presentation of each Alanogue after prepulsing the DR52a+ or DR3+ B cell lines (and then washing) before coculture with the T cells. That also prevents the T cells’ own class II molecules from presenting these peptides to each other, as occurs when they are used as a continuous stimulus (23, 24, 25) (which we also tested).
Presentation by the natural DR52a gave very similar profiles for the
two T cells (Fig. 3a). Substituting residues at either end
(146 and 158–160) with Ala had little effect (Fig. 3a);
however, Gly147 at p -2 and Thr148 appeared
more important, especially for P9[p138], as previously noted in Table III (cf. 8, 15). As expected, the Ala149 substituent at p1
showed no detectable functional binding. By contrast, the
Tyr149 variant was presented maximally to both T cells
(Fig. 3a); it evidently binds well to DR52a, although not to
DR3 (Fig. 3, b and c). Notably, all the other
residues in the 149-157 core proved to be critical too, with the single
exception of Ser154 at p6, which is evidently less
important here than in DR4 (45).
Residues required for cross-presentation by DR3
While presentation to KB-D1 by DR3 again depended greatly on p1
and p4 (rather than p6), there were four major differences from that by
DR52a (solid symbols in Fig. 3, a and b). At p1,
the Tyr149 variant was not now presented detectably.
However, the Ala149 substituent stimulated surprisingly
well when present continuously (70% of maximum for both T cells; data
not shown); it could evidently adopt a more recognizable conformation
in DR3 than in DR52a, although not binding firmly enough to resist
washing. Notably too, the Ala157Lys substituent at p9
was presented much better by DR3 than by DR52a, whereas the
Ile158Ala variant at p10 was now much less acceptable to
KB-D1. The failure to recognize the variants at p2, p3, p5, p7, and p8,
even though they bound well to DR3, shows that these are TCR contact
residues (27); it is particularly striking with the
conservative Gly153Ala (or Ser) substitutions at the
primary contact p5, which were not recognized by either
DR52a-restricted T cell.
For the P9[p138] line plus DR3 (dotted lines in Fig. 3), we noted
several striking contrasts with KB-D1; however, the overall binding
register was clearly the same, as shown by the closely similar patterns
not only at position 149 (p1), and from 152 (p4) through 157 (p9), but
also at both termini (Fig. 3b). There were four main
differences from KB-D1: 1) the Thr148Ala and 2) the
Tyr151Ala variants were now presented much better by DR3
than by DR52a (Fig. 3b). 3) With the
Ala157Lys substituent, this difference was less extreme
than with KB-D1 (Fig. 3b), whereas 4) the
Ile158Ala variant was now presented equally well by DR3
and DR52a to line P9[p138]. These contrasts with KB-D1 were
particularly striking because samples of the same DR3+ APC
prepulsed with the Ala151 variant were completely
unrecognizable by the KB-D1 T cells but stimulated P9[p138] very
well. That apparently reflects aberrant recognition by this
peptide-selected T cell (see below).
Further differences in recognition of variants at p3
We saw similarly contrasting perceptions of variants at p3 in the
shorter p143-156 (Fig. 4); these all had
the natural Trp149 at p1, and were presented significantly
only by DR52a, and not by DR3 (not shown). Line P9[p138] responded
well regardless of whether Tyr151 was changed to benzyl
serine (with a similar bulk) or to naphthyl alanine (similar bulk to
Trp) and even better with Ala151 (Fig. 4), whereas none of
these variants was recognized detectably by clone KB-D1 (on the same
prepulsed APC), even as a continuous stimulus (not shown). This group
of peptides was synthesized in a third laboratory (Fig. 4, legend).
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Although it recognizes many overlapping peptides synthesized in
three laboratories, line P9[p138] eventually proved to be specific
for synthetic contaminants. The final proof was its consistent failure
to respond detectably (Fig. 5b) to a supposedly identical
previous synthesis of the same p145–163 (Phe149) that
stimulated so well in the parent sequence and its many Alanogue
variants (Fig. 3). In stark contrast, both versions were recognized
very well by KB-D1 and PM-A and even by a DR4-restricted "sister
line" selected against the same p138-167 from the same donor (P9;
Fig. 5b); for KB-D1, the presentation of this "previous"
version by DR52a and DR3 was almost identical to that in Fig. 1c (not shown).
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a). When its two component peaks were
separated, they both stimulated the other three T cells well, whereas
line P9[p138] only responded to its side peak 2 (Fig. 5b).
On mass spectrometry, the main peak 1 had one major component with a
molecular mass of 2011 Da, which corresponds to the expected sequence;
there was also a minor component with a molecular mass of 1910 Da,
which lacked the Thr148. By contrast, the major component
of side peak 2 had a molecular mass of 1981 Da, simultaneously lacking
a Thr and duplicating the Ala at p9 (Fig. 5c). A minor
component (2082Da) included both the correct Thr148 and
this extra Ala (not shown). Neither of these species was detected in
the main peak 1 that line P9[p138] failed to recognize.
Hence, for this line, the more crucial difference from the natural
epitope is probably the omission of Thr148 at p -1, and
evidently recurred in several syntheses (
Figs. 1–4). The duplicated
Ala157 is clearly not essential in shorter sequences such
as p143–156 (Table III, Fig. 4), although this artifact may also be
influential in longer peptides (see Discussion). Previously,
we had also excluded the alternative explanations 1) to 3) listed in
the introduction, by carefully comparing this line with clone KB-D1. We
chose this pair because of their all-or-nothing difference in
responsiveness to natural AChR Ags (24, 25) despite their
close similarities, not only in peptide preferences and DR52a/DR3
cross-restrictions (
Figs. 1–3), but also in TCR Vß2 usage and even
in Th0 phenotype (Table II). That comparison revealed 1) no gross
differences in peptide sensitivity (Fig. 1), 2) no sign of specificity
for cryptic epitopes in the AChR subunit (e.g., requiring exposure
by trypsinization (25), not shown), and 3) no dependence
on long extensions or truncations at either end of the epitope core
(Table III), or on its binding in an unnatural register (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Scanning a polygamous DR52a/DR4-restricted natural epitope cross-presented by DR3
The 146–160 region clearly includes an important natural AChR
epitope; exactly the same sequence is recognized by two independent T
cell clones—PM-A1 and KB-D1—selected from MG patients with
recombinant AChR subunits, from which it can be processed
efficiently by various APCs including PBLx, macrophages (24, 30), and dendritic, endothelial (31), and muscle
(32) cells. It is also a major pathogenic epitope in whole
Torpedo AChR for some mouse strains (33). The
DR4-restricted clone PM-A1 is considerably more sensitive than the
DR52a-restricted KB-D1 or P9[p138], especially to low Ag doses (not
shown), possibly because of differences between the T cells (e.g., in
accessory molecules or in TCR affinity), or between DR52a and DR4,
e.g., in their affinity for the peptide. Nevertheless, the similarities
extend to the TCR contact residues as well as the binding register. For
P9[p138], as well as these two clones, Gly153 at p5 was
completely indispensable; neither the Ala153 nor the
Ser153 variants were recognized detectably, even though
they bound well (46). Likewise, Thr150 and
Tyr151 at p2 and p3 were very important too. Secondary TCR
contacts include p -2 and p -1, especially for the DR52a-restricted T
cells as noted previously (15); perhaps also
Val155 and Val156 at p7 and p8, although these
could influence the peptide binding or orientation instead (2, 16, 34, 35).
Wu et al. have recently deduced a peptide-binding motif for DR52a by
aligning a series of 14 DR52a-restricted epitopes (13). We
can now add two further very similar sequences (Fig. 6), including a second and potentially
immunodominant DR52a-restricted epitope in the AChR 
subunit
sequence (36). Furthermore, our present results strongly
support the anchor roles deduced for the hydrophobic residues at p1 and
p9, and for the Asp at p4 (13), all of which proved to be
crucial for both of our T cells.
Evidently, a bulky anchor at p1 is particularly important for DR52a; it is aromatic in 7 of the 16 epitopes, and only in one is it as small as Ala (13). Despite their similar binding motifs, until recently, DR52a and DR3 were believed to present distinct sets of peptides (9, 16). Remarkably, however, we achieved maximal cross-presentation by DR3 to both T cells merely by reducing the bulk of the peptide’s p1 residue, which further emphasizes the critical importance of this anchoring in pocket 1. Similarly, for anchoring in DR4, p1 is often the only crucial residue, although the Asp152 at p4 and the Ser154 at p6 may also be important for presentation to specific T cells (45, 46).
By contrast, for DR52a, an Asp at p4 is just as prevalent (13 of 16
cases) as it is in DR3-restricted epitopes (6, 7, 8, 9, 14, 15, 16).
It was also indispensable for binding to DR3 (Fig. 3c),
probably because of critical interactions with the Arg74
(16, 27, 28, 37) and/or the Tyr26
(16) that these two alleles uniquely share
(1). Two findings further support the key role of these
shared residues. First, despite its Gly86, DR52b completely
failed to present any of these peptides/Alanogues to either T cell: it
differs crucially by Arg74Gln and
Tyr26Phe substitutions in DRß, which may either
prevent or alter these interactions (37), although it is
otherwise somewhat similar to both DR52a and DR3 (1) (Fig. 6). Conversely, in DR3, where these Tyr26 and
Arg74 residues are shared, only a single change in the p1
anchor is required for efficient cross-presentation, despite the eight
other differences from DR52a (Fig. 6). In theory, the
cross-presentation to clone KB-D1 might depend on the synthetic error
that is required by line P9[p138]. However, that seems very unlikely,
since KB-D1 is highly sensitive to the natural sequence in the native
Ag (24); also because the correct (previous) peptide that
was unrecognizable to P9[138] was cross-presented to KB-D1 exactly as
in Fig. 1c. Furthermore, we have recently obtained very
similar results with our second DR52a-restricted (AChR ) epitope
(36) (Fig. 6); changing the Trp205 to Phe,
Leu, or Met again clearly allowed efficient cross-presentation by DR3
to one clone (not shown) although, interestingly, not to two others
that may require a different peptide orientation/conformation
(16, 34, 35, 38).
At p9, near their C termini, 12 of the 16 DR52a-restricted epitopes
also have aliphatic residues (e.g., Leu, Ile), and a Pro here prevented
binding in one case (13). With our epitopes, this position
was more important for T cell recognition (Fig. 3b).
Furthermore, DR3 was more permissive than DR52a at p9, as it presented
the Ala157Lys substituent much better to both T cells
(cf. Refs. 9 and 15); indeed a basic residue
here may confer specificity for DR3 rather than DR52a (9, 14, 16). Perhaps, in DR52a, there is repulsion between the Lys at p9
and the bulky Phe37 in DRß, or alternatively with the
Arg76 in DR, which cannot form a salt bridge to the
Val57 in DR52a as it does to the Asp57 in DR3
(27, 28) (Fig. 6). By analogy, in DR4, when this
Asp57 changes to Ser (in the 0405 subtype), there is a
preference for an Asp or Glu at peptide p9 (7), but,
surprisingly, that is not evident in DR52a-restricted epitopes
(13).
In conclusion, while DR52a and DR3 similarly prefer an Asp at p4, a
range of amino acids at other core positions is evidently compatible
with both alleles (Figs. 3 and 6). Moreover, since DR52a tolerates
larger anchors at p1, whereas DR3 is more permissive at p9, they do not
merely duplicate the same specificity; hence, it is not surprising that
they are so often in such tight linkage disequilibrium. Indeed, Bontrop
et al. (12) proposed that the less polymorphic DR52
alleles have been retained in primate evolution because they present
conserved bacterial heat shock protein epitopes so well, and thus
broaden the diversity of the responding T cells.
Recognition of artificial epitopes by AChR peptide-selected T cells
In this comparison with the natural epitope of clone KB-D1 (which responds well to native AChR (24)), we expected to find that line P9[p138] required the correct epitope sequence (because of its sensitivity to so many independent overlapping peptides), but presented aberrantly (39). However, it eventually proved to be specific for a peptide contaminant, a major difficulty with peptide selection that we had substantially underestimated previously (25).
A second flaw, inferred by Viner et al. (39), is that
short peptides can adopt unnatural conformations/orientations after
binding directly to surface class II molecules (at neutral pH), because
they bypass the natural processing and loading steps in (acidic)
endosomes. Moreover, many T cells recognize these conformational
differences specifically (39). While a definitive
understanding would require detailed structural analyses, the
artifactual absence of Thr148 at p -1 may likewise affect
the conformation/orientation of the present epitope. Together, these
suggestions may explain the starkly contrasting perceptions by our two
T cells of certain peptides (Figs. 3b and 4), even though
they bound in the same register. For example, the Val86 of
DR3 unexpectedly permitted weak presentation to line P9[p138] of some
potent peptides, despite their Trp149 at p1, although not
to KB-D1 (Fig. 1, a and b); this bulky anchor may
be more easily accommodated in pocket 1 because of the incorrect
Gly148 at p -1 (instead of the larger Thr). Similarly, the
contrasting responses to the Ala variants at p -1 and p3 in the
contexts of DR52a and DR3 (Fig. 3, a and b) again
suggest distinct conformational influences of the
Val86
Gly interchange in DRß, which is the only
difference between these alleles at this end of the groove.
Third, line P9[p138] tolerated a remarkably greater variety of TCR
contact residues at p3 than did KB-D1 (Figs. 3b and 4). This
line presumably recognizes an alternative orientation that reveals the
peptide backbone near p3; its exposure evidently depends partly on a
C-terminal truncation after Val156 at peptide p8 (Fig. 4)
and/or on the ThrGly replacement at p -1. Finally, at the other
end, the distinctive response by P9[p138] to the
Ile158Ala variant (Fig. 3b) may reflect the
presence of the inserted Ala between p8 and p9.
The widespread recurrence of the vital impurity is much more
significant than its exact nature; it was evidently replicated several
times in three specialist laboratories (Figs. 3 and 4, legends).
Therefore, recognition of these multiple overlapping peptides at high
sensitivity (e.g., by P9[p138]) is clearly no guarantee of
specificity for the correct sequence, which we have only confirmed for
1 of the 20 sublines that we selected with synthetic peptides
(25); even that was for a cryptic epitope that had to be
unmasked by proteolytic cleavage of one recombinant polypeptide
(25).
Minority errors such as mini-deletions, insertions, persisting protecting groups, or D-amino acids can easily escape detection, even on high resolution HPLC. Therefore, it is crucial to confirm T cell specificity with longer recombinant polypeptides and preferably with the natural autoantigen, to which our recombinant AChR-selected T cells are highly sensitive (23, 24, 25, 30). However, that is seldom attempted with peptide-selected T cells, because this widespread limitation is underrecognized. A further weakness is that there may be hindering residues at either end (40), unexpected inserts (41), or posttranslational modifications in some natural Ags that are not reproduced in synthetic peptides (reviewed in Ref. 42), including loss of amide groups (42, 43). The surprising prevalence of these problems among AChR peptide-selected T cells (19) may reflect unnatural peptide conformations after direct binding to surface class II molecules (39), new primary responses in culture at high concentrations by naïve CD45RA+ T cells (44), tolerance to the natural sequence/conformation (42), or low relative frequencies of the truly AChR-specific T cells in MG.
In conclusion, our findings strongly suggest that many of the very
heterogeneous T cell responses to AChR peptides reported previously
(21, 22) may likewise be neither truly autoimmune nor
disease-specific. By contrast, the recombinant approach has revealed
recurring recognition of two epitopes naturally processed from whole
AChR, 146-160 and 201-219; also the frequent use of DR52a, an
underrecognized candidate susceptibility gene in MG. Together, these
results suggest that the truly pathogenic T cells may be much more
restricted, which would have major implications for the prospects for
specific immunotherapy.
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Acknowledgments |
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Footnotes |
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2 Current address: Dept. of Neurology, Kawatana National Hospital, Nagasaki 859-36, Japan.
3 Address correspondence and reprint requests to Dr. Nick Willcox, Neurosciences Group, Institute of Molecular Medicine, The John Radcliffe Hospital, Headington, Oxford, OX3 9DS, UK. E-mail address:
4 Abbreviations used in this paper: MG, myasthenia gravis; AChR, acetylcholine receptor; IC50, peptide concentration required for 50% inhibition of binding of indicator peptide to DR3 molecules; MMC, mitomycin C. PBLx, 30 Gy-irradiated PBL.
Received for publication August 28, 1998. Accepted for publication January 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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