From the Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, Scotland, United Kingdom
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ABSTRACT |
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Class II molecules are believed to influence
immune responses by selectively binding antigen-derived peptides for
recognition by T cells. In Goodpasture's (anti-glomerular basement
membrane) disease, autoimmunity to the NC1 domain of the 3-chain of
type IV collagen (
3(IV)NC1) is strongly associated with HLA-DR15. We
have examined the influence of the peptide binding preferences of DR15
molecules on the selection of
3(IV)NC1-derived peptides displayed
bound to DR15 molecules on the surface of
3(IV)NC1-pulsed DR15-homozygous Epstein-Barr virus-transformed human B cells. The
preferences of DR15 molecules were investigated using a panel of 24 overlapping peptides spanning the sequence of
3(IV)NC1. The
3(IV)NC1-derived peptides selected for display to T cells were
determined by biochemical analysis as reported previously (Phelps,
R. G., Turner, A. N., and Rees, A. J. (1996)
J. Biol. Chem. 271, 18549-18553).
Three nested sets of naturally presented 3(IV)NC1 peptides were
detectable bound to DR15 molecules. Peptides representative of each
nested set bound to DR15 molecules, but almost two-thirds of the
3(IV)NC1 peptides studied had as good or better DR15 affinity than
those identified as naturally processed. Thus
3(IV)NC1 presentation to T cells is determined more by "processing factors" than by the
preferences of relatively indiscriminate DR15 molecules. The results
have important implications for the use of class II peptide binding
data to aid identification of potential T cell epitopes, especially for
antigens which, like
3(IV)NC1, contain many sequences able to bind
class II molecules.
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INTRODUCTION |
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Antigen presenting cells (APC)1 potentially exert a profound influence on immune responses, including those to self antigens, because they regulate the way T cells recognize antigens. CD4 T cells recognize antigens in the form of processed peptides presented bound to MHC class II molecules on the surface of class II positive APC types (1, 2). These include cells important in the initiation of immune responses, such as dendritic cells, B cells, and macrophages, and cells important in regulating the T cell repertoire and self-tolerance, such as thymic epithelia. How APC select antigen-derived peptides for display to T cells therefore not only constrains the peptide specificity of responding T cells, but also influences the repertoire of T cells available to mount immune responses. Furthermore, the way APC present antigens may determine the immunodominant T cell response, which at least for some exogenous antigens is directed at the antigen-derived peptide displayed at the highest level (3). There is therefore great interest in understanding how APC generate antigen-derived peptides and make a selection for display to T cells, both as an approach to identifying T cell epitopes for specific immune modulation and toward understanding the basic biology of immune responses.
APC, like many cell types, internalize extracellular proteins into their endosomal/lysosomal pathway where denaturing (low pH and reducing) conditions promote the access of endosomal proteases that degrade or "process" protein antigens into peptides and, ultimately, to amino acids and di-peptides (4, 5). APC that express MHC class II genes target newly synthesized MHC class II molecules to processing compartments where they become competent to bind antigen-derived peptides and intact denatured proteins (6). Peptides able to form stable complexes with class II molecules are protected from further proteolysis (7, 8) and may be transported to the cell surface for recognition by CD4 T cells. Accordingly, the selection of antigen-derived peptides displayed by APC is thought to be determined by an interplay between antigen denaturation and proteolysis (processing) on the one hand and MHC class II/peptide binding on the other. Antigen processing is poorly understood, and the range of antigen-derived peptides generated and made available for binding class II molecules is not yet known for any antigen. Few of the probably numerous endosomal proteases have been identified and their specificities characterized. Furthermore, how an antigen is processed is likely to depend not only upon its amino acid sequence but also on its tertiary and quaternary structure, which may influence susceptibility to denaturation and the access of endosomal proteases (9-11). In contrast, the way MHC class II molecules bind to peptides is well understood, and the ligands preferred by a particular class II molecule can fairly reliably be predicted.
Class II molecules are heterodimeric membrane glycoproteins with an extracellular peptide binding groove (12). Peptides are clasped with nine amino acid residues (the core binding sequence) accommodated in the groove, lying in extended conformation. Binding is largely stabilized by sequence nonspecific interactions with main chain nitrogen and oxygen atoms in the bound peptide. However, stable binding requires peptide side chain residues that point into the floor and walls of the groove to be accommodated in complementary pockets, the size and chemical character of which are extensively influenced by polymorphisms in class II alleles. Consequently, class II molecules bind a wide range of peptides but have allele-specific preferences for peptides with core binding sequences containing appropriately spaced, efficiently accommodated side chain residues (13). Peptide binding preferences can be described using peptide binding motifs (14), and motifs have been developed for some class II molecules which fairly reliably predict the class II affinity of a peptide from its amino acid sequence (15-17). Thus while it is currently impossible to predict how an antigen will be processed by APC, it is relatively straightforward to identify the antigen-contained peptides which, if generated by processing, should be preferentially bound by class II molecules and hence selected for presentation to T cells. Antigen-contained sequences with high class II affinity can be identified by comparing the class II binding affinity of synthetic peptides with overlapping sequences spanning the sequence of an antigen or by using peptide binding motifs. Both approaches have been successful in identifying immunologically relevant T cell epitopes (16-19).
Goodpasture's disease is an autoimmune nephritis caused by
autoimmunity to the 235 amino acid C-terminal NC1 domain of type IV
collagen (3(IV)NC1) (20, 21), a component of basement membrane in
some tissues. Almost 80% of patients carry a haplotype bearing HLA
DRB1*1501 and DRB5*0101 which encode the
-chains of the HLA class II
molecules DR15b2 and DR15a,
respectively (22). We are examining how DR15-homozygous human APC
process and present
3(IV)NC1 as an approach to identifying potential
T cell epitopes and toward understanding how antigen processing and
presentation and HLA genes influence susceptibility to autoimmune
diseases. Our approach has been to biochemically characterize
DR15-associated peptides, purified from DR15-homozygous EBV-transformed
human B cells, using matrix-assisted laser-desorption time of flight
mass spectrometry and reverse phase HPLC (23). Comparison of peptides
purified from
3(IV)NC1-pulsed and control non-pulsed APC identified
17 extra putative
3(IV)NC1-derived peptides. The difficulty with
this approach is the low frequency of antigen-derived peptides; all the
extra peptides occurred at low level within complex peptide mixtures,
and their sequences could not be directly determined. However, by
exploiting the tendency of class II-associated peptides to occur as
nested sets, we were able to define the sequences of eight extra
peptides. They comprised two nested sets centered on the common core
sequences LFCNVNDVCNF and LEEFRASPF (24).
Here we have used overlapping synthetic peptides spanning the sequence
of 3(IV)NC1 and matrix-based peptide binding motifs to identify all
sequences within
3(IV)NC1 able to bind to DR15 molecules, and we
ranked them according to DR15 affinity. Surprisingly, the peptides
previously shown to be naturally processed from
3(IV)NC1 and
selected for presentation bound to DR15 molecules have only intermediate DR15 affinity. Because DR15 molecules were expected to
preferentially bind the available
3(IV)NC1 sequences for which they
have greatest affinity, the biochemically detectable DR15-associated putative
3(IV)NC1-derived peptides purified from
3(IV)NC1-pulsed APC were re-analyzed for the possible occurrence of
3(IV)NC1 sequences with high DR15 affinity. The analysis identified a third nested set of naturally presented
3(IV)NC1-derived peptides. However, the most striking result was that most
3(IV)NC1 peptides with high DR15 affinity were undetectable among DR15-associated peptides purified from
3(IV)NC1-pulsed APC. Indeed, most of the naturally presented
3(IV)NC1-derived peptides bound to DR15
molecules have only intermediate affinity for DR15 molecules, both in
the range of affinities measured for
3(IV)NC1-derived peptides and in the range of affinities reported for DR-associated peptides. Thus
the peptide binding characteristics of DR15 molecules appear to be
largely permissive in determining which
3(IV)NC1-derived peptides
are displayed by DR15 APC, presumably sub-dominant to unknown
processing factors.
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EXPERIMENTAL PROCEDURES |
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Synthetic Peptides--
A panel of synthetic peptides was made
with sequences spanning the sequence of 3(IV)NC1 (Table I). They
were purified by HPLC and had their composition confirmed by
matrix-assisted laser-desorption time of flight mass spectrometry. All
the peptides were prepared as ~1 mM solutions in 20%
Me2SO, 5 mM dithiothreitol and stored at
4 °C because some had limited solubility in water and some tended to
form disulfide-linked dimers. Stock peptide solutions were subjected to
amino acid analysis to measure peptide concentrations more accurately.
The peptides hemagglutinin 307-319 and myelin basic protein
86-98(98A), called HAP and MBPP respectively, were biotinylated at the
N-terminal to make the labeled reference peptides designated *HAP and
*MBPP.
Purification of Class II Molecules-- HLA-DR15a and -DR15b molecules were affinity purified from the murine L cell transfectants LDR2a and LDR2b, respectively (25), because available monoclonal antibodies do not distinguish between the two DR molecules carried by human DR15 homozygous APC. DR15a is composed of the gene products of DRA and DRB5*0101 and DR15b of the gene products of DRA and DRB1*1501. Cells were lysed in 1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 8, containing protease inhibitors and DR molecules affinity purified employing the monoclonal antibody L243 conjugated to cyanogen bromide-activated Sepharose.
Peptide Binding Assays--
Peptide binding to purified class II
molecules was measured using an inhibition assay based on that
described by Tompkins et al. (26). The myelin basic protein
peptide MBP86-98(98A) and flu hemagglutinin peptide HA307-319 were
selected as reference peptides because of their reported high affinity
for DR15b and DR15a, respectively (27). The test peptides were
incubated at a range of concentrations with affinity purified DR
molecules (0.5 µM) and labeled reference peptides in
0.5% Nonidet P-40, 150 mM NaCl, 0.1 M sodium
citrate, pH 5.5, in the presence of protease inhibitors for 16 h
at 26 °C. The reference peptides were used at concentrations (160 nM for *MBPP and 86 nM for *HAP) at which much
less than 10% bound to DR molecules. DR-reference peptide complexes
were assayed by immobilizing DR-peptide complexes onto enzyme-linked
immunosorbent assay plates coated with L243, labeling immobilized
biotin with a streptavidin-Europium conjugate (Wallac Oy, Finland), and
measuring Europium by time-resolved fluorescence at 613 nm (excitation
at 340 nm). The concentrations of peptides [Pi] causing 50%
reduction in reference peptide binding
(IC50(i)) were extracted from
experimental binding data by curve-fitting to functions of the form
B/(1 + [Pi]/IC50(i)) where
B is binding in the absence of unlabeled test peptide (the equation derives from the law of mass action). IC50 values
were determined in at least three independent experiments. Expressed as
logarithms, the experimental data were normally distributed with S.D.
0.165, and the 95% confidence limits for the arithmetic means (three
measurements) were ±0.19. Therefore the 95% confidence limits for
measured IC50 values (in moles) were between (mean/1.5) and
(mean × 1.5).
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RESULTS |
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To examine the importance of the peptide binding characteristics
of DR15 molecules in shaping the selection of 3(IV)NC1 peptides displayed to T cells by DR15 homozygous APC, we sought to identify all
peptides within the sequence of
3(IV)NC1 with good affinity for DR15
molecules and analyze the results in the light of our previous
biochemical characterization of naturally processed
3(IV)NC1-derived peptides (23). Because that study examined peptides eluted from DR15
molecules purified from
3(IV)NC1-pulsed MGAR cells (a line of
EBV-transformed human B cells homozygous for DR15), and hence peptides
that could have been bound to DR15a or DR15b molecules, it was
necessary to examine peptide binding to both DR15a and DR15b molecules
separately. Two complementary approaches were used. First, a set of
synthetic peptides was made with overlapping sequences spanning the
sequence of
3(IV)NC1. They were compared for their capacity to
inhibit the binding of labeled reference peptides to affinity purified
DR15a and DR15b molecules. Second, the sequence of
3(IV)NC1 was
examined for peptides with high predicted affinity for DR15a or DR15b
molecules using matrix based motifs to identify any peptides likely to
bind well but inadequately represented within the set of overlapping
peptides.
Most 3(IV)NC1 Peptides Bind Detectably to
DR15a/b--
Synthetic peptides were made with the sequences shown in
Table I. Peptides P1 to P23 are 20 to 21 amino acids in length (except P23) and span the sequence of
3(IV)NC1
with a mostly 10-residue overlap. This degree of overlap was chosen to
slightly exceed the length of bound peptide accommodated within the
class II peptide binding groove (12). Peptide P3 proved difficult to
synthesize in any quantity and poorly soluble in aqueous buffers, so P3
was substituted by peptides P3a and P3b.
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Class II Binding Motifs Identify Probable Core Binding Sequences
Used by High Affinity Peptides--
The sequence of 3(IV)NC1 was
also examined using matrix-based DR15 peptide binding motifs. This was
done first to identify the probable core binding sequences of the
3(IV)NC1 peptides with high affinity for DR15 molecules, and second
to ensure all
3(IV)NC1 sequences with high DR15 affinity had been
identified. While every nine amino acid core binding sequence in
3(IV)NC1 was represented within the set of synthetic peptides, it
was possible that different
3(IV)NC1 peptides would bind to DR15
with higher affinity, perhaps through making fewer unfavorable
interactions outside the groove.
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DR15 Affinity of Major Naturally Processed and Presented
3(IV)NC1-derived Peptides--
Next we examined the DR15 affinity
of naturally processed and presented
3(IV)NC1 peptides, to assess
the degree to which their selection was dictated by the peptide binding
preferences of DR15 molecules. Our previous biochemical analysis of
DR15-associated peptides purified from
3(IV)NC1-pulsed DR15
homozygous APC identified two nested sets of naturally processed and
presented
3(IV)NC1-derived peptides centered on the core sequences
LFCNVNDVCNF and LEEFRASPF (23). The techniques used to identify these
peptides had limited sensitivity so they are likely to be among the
most abundant naturally presented
3(IV)NC1 peptides displayed bound
to DR15 molecules.
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Occurrence of 3(IV)NC1 Peptides with High Affinity for DR15
Molecules Among Naturally Processed DR15-associated
Peptides--
Processed
3(IV)NC1-derived peptides with high DR15
affinity would be expected to be more efficiently loaded onto DR15
molecules than peptides with intermediate affinity, so it was
surprising that none of the previously identified naturally presented
3(IV)NC1 peptides contained high DR15 affinity sequences. In that
experiment nine extra putative
3(IV)NC1-derived DR15-associated
peptides were identified whose sequences could not be determined
(called extra peptides because they were detected among DR15-associated peptides purified from
3(IV)NC1-pulsed APC but not from sham-pulsed APC). We therefore re-analyzed the data from that experiment to see if
any of the nine unaccounted extra peptides could be high DR15 affinity
3(IV)NC1 peptides.
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DISCUSSION |
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The results presented here and in Ref. 23 identify three nested
sets of 3(IV)NC1 peptides naturally processed from intact
3(IV)NC1 and selected for display bound to DR15 molecules by DR15
homozygous EBV-transformed human B cells (Table
VII). The peptides of the nested set
centered on the core sequence LEEFRASPF must be presented bound to
DR15b molecules, the DR molecule most likely to confer susceptibility
to Goodpasture's disease (22), because they have undetectable affinity
for DR15a. The other two nested sets of peptides bind with intermediate
or high affinity to DR15a and DR15b, so could be presented bound to
either (or both) DR15 molecule. The three nested sets of peptides are
likely to be the most abundant
3(IV)NC1 peptides displayed by the
APC used in the experiments because the techniques used to identify them were of limited sensitivity. This enables us to investigate the
factors that determine the
3(IV)NC1 peptides selected for display to
T cells. Here we have examined the influence of the peptide binding
preferences of DR15 molecules.
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The binding data show that almost 80% of the 3(IV)NC1 peptides
examined bound to DR15a or DR15b with intermediate or high affinity. To
assess the degree to which the peptide binding characteristics of DR15
molecules influenced the selection of
3(IV)NC1 peptides displayed by
DR15-homozygous APC, the
3(IV)NC1 peptides were ranked according to
DR15 affinity. Strikingly, the synthetic peptides representative of the
naturally processed peptides ranked only between 4th and 14th within
the set of 24
3(IV)NC1 peptides (Table VIII). For example peptide P7,
representative of the nested set centered on LFCNVNDVCNF, had the 9th
highest affinity to DR15a and 5th highest affinity to DR15b. These data
suggest that binding affinity for DR15 molecules has a relatively minor
influence on which
3(IV)NC1 peptides are displayed on the surface of
APC. However, with the exception of P3b, peptides in the overlapping set were not identical to naturally processed peptides. P7 and P17
included the entire common core sequences of naturally processed nested
sets of peptides, but it was possible that they bound less well to DR15
molecules than naturally processed peptides because of interactions
outside the peptide binding groove. This explanation was confounded by
the additional experiments in which synthetic peptides with naturally
processed sequences were shown to bind to DR15 molecules with only
intermediate affinity. Thus the major (biochemically detectable)
3(IV)NC1 peptides selected for presentation bound to DR15 molecules
are not those in
3(IV)NC1 with highest DR15 affinity, indeed
naturally presented peptides bind to DR15 molecules no better than
peptides representing almost two-thirds of the sequence of
3(IV)NC1.
This means that the peptide binding characteristics of DR15 molecules
must be largely permissive to other processing factors in determining
the selection of
3(IV)NC1-derived peptides presented on the surface
of APC in our experiment.
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Inspecting the sequences of the proposed naturally processed peptides
gives some indications of the processing factors that may have
constrained the selection of 3(IV)NC1 peptides displayed bound to
DR15. All except one of the peptides contain one or more cysteine
residues likely to be involved in disulfide bonds (based on the
reported structure of the highly homologous
1(IV) chain, see Ref.
30), and many known T cell epitopes derive from regions of proteins
involved in disulfide bonds, possibly because the presence of the
disulfide linkage affords some protection from proteolytic attack (31).
The preparation of
3(IV)NC1 used in this study was reduced during
purification but had ample opportunity to oxidize prior to and during
pulsing of the APC. It is also striking that two of the nested sets,
the sets containing LFCNVNDVCNF and LEEFRASPF, derive from
corresponding portions of the two hemidomains of
3(IV)NC1. The
content of DR15 binding motifs in these regions is not strikingly
different from that of
3(IV)NC1 as a whole, but it is likely they
have similar secondary and tertiary structure which could favor their
selection for presentation to T cells. The proximity of these regions
to the surface of intact
3(IV)NC1 is not known, but they could be
similarly inaccessible to the endosomal proteases or similarly
accessible for early binding (even as partially unfolded intact
protein) to class II molecules (32, 33) with consequent protection from
proteolysis (7, 8). Processing could also favor the naturally presented
peptides by selectively destroying some of the
3(IV)NC1 peptides
with higher DR15 affinity. The endosomal enzymes important in
3(IV)NC1 processing are unknown but presumably include those shown
to process other antigens (reviewed in Ref. 5). Recently the
specificity of the candidate endosomal enzyme cathepsin E has been
described in detail (34) permitting a search of the sequence of
3(IV)NC1 for vulnerable peptide
bonds.4 Intriguingly, the two
3(IV)NC1 sequences most likely to be cut by cathepsin E (CPHGW ISL
and KGFSF IMF) occur in peptides P14 and P15, both of which have very
high DR15 affinity and neither of which is detectably presented bound
to DR15 molecules.
A more mundane explanation for the lack of relationship between DR15
affinity and the selection of 3(IV)NC1 peptides displayed bound to
DR15 is that in vitro measurements of peptide DR interaction poorly represent peptide-DR interactions within the processing compartments of APC. Certainly the conditions are different within APC;
most antigen-derived peptides generated by processing bind to newly
synthesized class II molecules by displacing the CLIP peptide from the
peptide binding groove. The interaction is catalyzed by HLA-DM which
also appears to edit the resulting class II-peptide complexes (35, 36);
antigen-derived peptides become stably bound within peptide class II
complexes as quickly as 30 min after addition of antigen. In our
experiments high concentrations of peptide were used to drive
competitive displacement of previously bound non-CLIP peptides from
detergent-solubilized DR molecules in the absence of HLA-DM and over a
16-h time course. Binding assays were performed at endosomal pH because
pH has been shown to influence DR-peptide interactions (37). However,
there is a large body of evidence that peptide-class II interactions
measured by similar techniques to those used here have immunological
relevance. In particular, similar inhibition binding assays have been
used to show the following: (i) that major T cell epitopes in exogenous antigens bind to the restricting class II molecules with high affinity
(38, 39); (ii) that the more immunogenic peptides within exogenous
antigens are a subset of those with higher class II affinity (40, 41);
and (iii) that peptides eluted from purified class II molecules bind
well to class II molecules in vitro (23, 42, 43). Therefore,
even though the measurements of class II binding affinity did not model
loading of class II molecules within APC, they should indicate the
relative capacity of DR15 molecules to bind
3(IV)NC1
peptides.
It is striking that 3(IV)NC1, the autoantigen attacked in
Goodpasture's disease, contains so many sequences able to bind to DR15
molecules, the class II molecules strongly associated with this
disease. It is likely that self-tolerance is best established to
self-antigen-derived peptides constitutively displayed at highest level
and that autoimmunity is directed at other peptides usually presented
at much lower level, called cryptic epitopes (44-46). From this
standpoint, the results are intriguing in two ways. First,
3(IV)NC1
contains a higher proportion of peptides able to bind DR15a/b (17/24 or
71%) than the 15-25% observed for a range of exogenous antigens and
human DR molecules, including DR15 (39), suggesting considerable scope
for the presentation of peptides to which tolerance has been
incompletely established. Interestingly, intermediate or better DR
affinity was found for a high proportion of overlapping peptides
spanning myelin basic protein (2-11 out of 16 peptides binding to
various DR molecules, mean 8/16 or 50%, in Ref. 47), acetylcholine
receptor
-subunit (13-25/36, mean 50% in Ref. 48), glutamic acid
decarboxylase (58% in Ref. 49), acetylcholine
-subunit (14-26/32
mean 69% in Ref. 50), all known or proposed autoantigens. Second,
3(IV)NC1 contains several peptides (e.g. P15 and P14)
with very high affinity for DR15 that are not constitutively displayed
to T cells, but which under conditions of aberrant processing
(e.g. extracellular processing) could be powerful
immunogens. This would explain why HLA-DR15 is important in
susceptibility to Goodpasture's disease even though the peptide
binding characteristics of DR15 appear to have only a permissive
influence on how
3(IV)NC1 is presented, at least under the
conditions studied.
Unless 3(IV)NC1 is very atypical, our results suggest that
identifying peptides within antigens with high class II affinity may
not economically identify naturally presented peptides. By using the
conventional approach of identifying the antigen-contained peptides
with highest class II affinity and then examining their capacity to
stimulate antigen-specific T cells, we would have had to examine
peptides representing two-thirds of the sequence of
3(IV)NC1 in
order to identify the three nested sets of naturally processed
peptides. Better understanding of how antigens are processed is
required before current knowledge of class II-peptide interactions can be efficiently used to identify T cell epitopes.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Medicine and
Therapeutics, Institute of Medical Sciences, University of Aberdeen,
Foresterhill, Aberdeen, AB25 2ZD, Scotland, UK. Tel.: 1224 681818 (ext.
54533); Fax: 1224 273066; E-mail: r.phelps{at}abdn.ac.uk.
1 The abbreviations used are: APC, antigen presenting cells; MHC, major histocompatibility complex; EBV, Epstein-Barr virus; HPLC, high pressure liquid chromatography.
2
The terms DR15a and DR15b refer to class II
molecules composed of the DR -chain and DR
-chains encoded by
DRB5*0101 and DRB1*1501, respectively. This terminology echoes the more
widely used terms DR2a and DR2b and is preferred here to refer
specifically to DR molecules encoded by particular DRB5 and DRB1
alleles. This was necessary because inheritance of any of several DRB1
alleles (B1*15xx and B1*16xx) gives rise to display of DR molecules
indistinguishable by the serological techniques used to define the DR2
specificity.
3 T. Sturniolo, J. Hammer, and F. Sinigaglia (Roche Milano Ricerche, Milan), unpublished data.
4 R. G. Phelps, M. Coughlan, A. N. Turner, and A. J. Rees, manuscript in preparation.
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REFERENCES |
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