Distant interactions between dimorphisms in HLA-DR4 radically affect recognition of defined peptides by a specific T cell clone
Hidenori Matsuo3,
Louise Corlett,
Simon Hawke4,
Michael Nicolle5,
Paul Driscoll1,
Shrikant Deshpande2,
Edward Spack2,6 and
Nicholas Willcox
Neurosciences Group, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DS, UK
1 Department Biochemistry and Molecular Biology, University College London, WC1 6BT, UK
2 Anergen Inc., Redwood City, CA 94063, USA
Correspondence to:
N. Willcox
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Abstract
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Several isolated dimorphisms recur in many HLA class II alleles, but it is not clear whether they merely influence the binding of peptides locally or have more general effects on their recognition by T cells. For example, interchanges in HLA-DRß include 86Gly
Val and 57Asp
Ser at either end of its
helix, and 71Arg
Lys in the middle. In DR4, the existence of six subtypes differing by single substitutions at these sites enabled us to assess their functional effectsboth in isolation and in their natural contexton peptide presentation to a specific T cell clone with unusually broad cross-restrictions. Unexpectedly, the restriction imposed by 86Val was much more severe in the context of 71Arg than 71Lys, but was also more readily overcome by reducing the bulk of the `p1' peptide `anchor' residue (149Trp
Phe). Moreover, when there was also a distant 57Asp
Ser substitution, compensating similarly for 86Val proved much more difficult. Thus 86Val and 57Ser in combination had far more drastic effects on peptide presentation than they did separately, when peptide binding was also largely unchanged. These and other interactions with position 71 together provide strong evidence that the configuration of the peptideDR4 complex is critical for T cell recognition, which could be affected by subtle conformational influences on the p19 core of the peptide or on the
helix of DR4ß (between positions 86 and 57). Ideally, therefore, the effects of individual class II substitutions should be considered in their natural context rather than in isolation.
Keywords: autoimmunity, HLA-DR4, MHC polymorphism, peptide presentation, rheumatoid arthritis, T cell recognition
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Introduction
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Although MHC molecules are highly polymorphic, the differences between alleles of HLA-DR are very localized (1)often to positions in the ß chain that contact the peptides in their antigen-binding grooves (2,3). There are several hypervariable regions where alternative `cassettes' of sequence occur, such as the 67L70QR motif in HLA-DR1 and many subtypes of DR4 versus 67I70DE in DR4;Dw10 and other alleles (Table 1
). However, at other isolated positions, the variety of amino acids is very limited; at one end of the
helix, for example, 57Asp is common, but some DR or DQ alleles have 57Ser, Val or Ala instead (1). Likewise, in the middle at position 71, there is an isolated 71Arg
Lys interchange in some subtypes of DR4 (Table 1
). Located at the far end of the helix is the extreme case of DRß position 86, where the only known alternatives are Val and Glywhich are often the only differences between the subtypes of DR4 (Table 1
), DR15, DR13 or DR52 (1). The crystal structures (2,3) show a hydrophobic `pocket' here that accommodates the major `p1 anchor' residue of many peptides; moreover, by restricting the access of bulkier residues to it, this 86Val can have radical overall effects on peptide binding and recognition (2,3,6,8,9).
Such interchanges must surely cause subtle local adjustments to individual side-chain-binding pockets. In addition, however, at pivotal sites such as 57, 71 and 86, they might have more general effects on the binding properties of the whole groove or on the conformation of the bound peptides. Since the latter are not easily detected, they have been studied much less. In either case, the wide recurrence of these dimorphisms suggests that they have been selected in evolution to increase the repertoire of epitopes and pathogens that can be recognized. For instance, in the Gambia, DR1302 apparently confers some protection against severe malarial anemia, unlike DR1301 which differs only by having 86Val instead of Gly (10).
Particularly intriguing examples are seen in HLA-DR4, where the naturally occurring Dw4, Dw13 and Dw15 subtypes each differ from Dw14 by a single residue, and Dw10 by only three; each of them shows the 86Gly/Val dimorphism too (Table 1
). For clarity in this report, the subtypes of DR4 with 86Val are denoted `Dw4.1', `Dw10.1', etc., and those with 86Gly `Dw4.2', `Dw10.2', etc. (Table 1
). Moreover, these differences must hold important clues for immunopathologic responses. For example, severe rheumatoid arthritis associates strongly with HLA-DR4, but only with its Dw4, Dw14 and Dw15 subtypes (1114). That could reflect differences in peptide binding, which have been studied extensively (8,9,1521), or in presentation to specific T cells, which often prove to be much greater when they are tested (6,8,9,14,1922). In most reports (e.g. 8,21), substitutions were tested in isolation by site-directed mutagenesis and it was tacitly assumed that their effects could be generally extrapolated to other alleles/subtypes, regardless of the precise context. We were able to test that by measuring peptide presentation to a T cell clone (PM-A1) that shows unusual cross-restrictions to several DR4 subtypes (14). This clone, originally selected from the thymus of a DR4;Dw14.2+ myasthenia gravis (MG) patient, is highly sensitive to a natural
149158 epitope in the human acetylcholine receptor (AChR)
subunit (6,14). When we used it to study these dimorphisms in their natural setting (in the panel of DR4 subtypes listed in Table 1
), we unexpectedly observed striking interactions between them, which we summarize here.
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Methods
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Antigens
Peptides were synthesized by standard Fmoc chemistry (23). The N-terminal biotinylated myelin basic protein (MBP) peptide Tyr84102 was purified by reverse-phase HPLC, and checked by HPLC and mass spectroscopy; its sequence is YDENPVVHFFKNIVTPRTPP. Other sequences are given in the figures.
Peptide-binding assays
Cell pellets from 40 l spinner flasks were solubilized in 0.5% Triton X-100 and HLA-DR molecules were purified by affinity chromatography with the mAb L243: they were incubated overnight (at 50 nM) at 37°C in pH 5.5 citrate buffer with 1000 nM biotinylated MBP peptide plus 1100,000 nM of unlabeled competitor in 0.2% NP-40, 0.01% NaN3 and a protease inhibitor mix (24). The HLA-DR peptide complexes were added to 96-well plates precoated with mAb L243 and incubated for 2 h at 4°C. Plates were subsequently washed with 0.1% Tween 20 in Tris-buffered saline (0.5 M Tris, 1.5 M NaCl, pH 7.5) to remove unbound material, incubated with 100 ng/ml europiumstreptavidin (Wallac, Turku, Finland) for 90 min and washed again in this buffer. To detect the europium fluorescence, DELFIA enhancement solution (Wallac) was added and plates were analyzed in a Wallac 1234 DELFIA research fluorometer (Wallac). Triplicate samples for each unlabeled AChR
peptide were tested at each concentration and the IC50 (the concentration of unlabeled peptide required for 50% inhibition of the binding of the MBP peptide) was determined by four-parameter fit analysis with the software program SOFTmax Pro.
T cell proliferation assay
We used the EpsteinBarr virus-transformed B cell lines listed in Table 1
as antigen-presenting cells (APC). They were maintained in 10% FCS in RPMI 1640 (Gibco, Paisley, UK); before use, they were pre-treated with mitomycin C (Sigma, Poole, UK) at 50 µg/ml for 1 h and thoroughly washed. They were then pulsed with varying peptide concentrations at 8x106 cells/ml at 37°C in 5% A+ human serum in RPMI medium; the standard time was 90120 min, but ranged up to 512 h in some experiments. The cells were then washed thoroughly before co-culture (at 105 cells/well in RPMI/5% serum) with well rested pre-washed PM-A responder T cells (2.5x104/well) in 200 µl round-bottomed microtiter plates (Nunclon, Roskilde, Denmark). In some experiments, antigens were added as a continuous stimulus rather than a pre-pulse. After 72 h, 1 µCi of [3H-methyl]thymidine (Amersham, Little Chalfont, UK) was added to each well; 18 h later, they were harvested and counted on a Betaplate flat bed liquid scintillation counter (Wallac). The PC50 was the peptide molarity required for half-maximal T cell stimulation; each peptideDR4 subtype combination was tested at least twice to determine this value.
The responder T cells were the PM-A line (6) and its PM-A1 clone (25); since their behavior and TCR sequences are identical, they were used interchangeably. They were maintained by fortnightly re-stimulation with recombinant AChR
subunit plus irradiated DR4;Dw4.2+ irradiated peripheral blood lymphocytes and expansion with highly purified IL-2 (Biotest, Solihull, UK) (6).
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Results
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Experimental approach
We tested peptide presentation to PM-A T cells by 10 different HLA-DR4 subtypes (Table 1
); in most experiments, the APC were preincubated with peptide (and then thoroughly washed), in order to pre-load their class II molecules and assay for `functional binding' as well as to prevent the strongly class II+ responder T cells from subsequently presenting these peptides to each other.
The presenting HLA-DR4 subtypes and the p1 anchor residue
As is typical with T cells from Dw14.2+ donors (14), and is illustrated in Fig. 1
(a), the PM-A T cell responds very well to its natural epitope when presented by Dw14.2 and Dw15.2, whereas much higher doses are required with Dw13.2 and there is no detectable response with Dw13.1 or Dw10 (6,14). Unusually, however, presentation is also consistently maximal by Dw4.2 (Fig. 1a
); even though its 71Arg
Lys substitution completely prevents presentation to three other Dw14.2-restricted T cells (14), this is no obstacle to PM-A.
The natural epitope is not presented detectably by Dw14.1 (6,14) (Fig. 1a
), which differs only by 86Gly
Val (1,6). As illustrated in Fig. 1
, 149Trp is clearly the p1 anchor; merely replacing it with a less bulky 149Phe completely and consistently overcomes this restriction in Dw14.1 (Fig. 1b
) (14). However, when we tried to extend this finding to Dw4.1 and Dw15.1, we noted two surprising contrasts.
- (i) In Dw4.1 (which differs from Dw14.1 by 71Arg
Lys), this 86Val restriction had much milder effects, despite a 149Trp at peptide p1. Though 86Val still prevented presentation of shorter peptides (e.g. p143156) (14), that was overcome merely with a higher dose of the more potent p144163 (Fig. 1a
). Moreover, this minor `insensitivity' was not altered by substituting a 149Phe at p1 (Fig. 1b
).
- (ii) There was a stark contrast with Dw15, which has a distinctive 57Asp
Ser replacement. Whereas Dw15.2 clearly presents peptides very efficiently (Fig. 1
; see also Fig. 2
), the corresponding 86Val restriction in Dw15.1 was only overcome with great difficulty. That required prolonged pulses at high molarities of variants of (a) the shorter p146158 sequencetruncated at p10which were presented remarkably well by Dw15.1 in view of their weakness in other subtypes (Table 2
) or of (b) the very potent p145163 (149Phe) sequence (see Fig. 2
). Thus this restriction was much more severe in the context of both 86Val and 57Ser than of either alone.

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Fig. 2. Identification by Ala scanning of critical residues in p145163 (149Phe). (i) For binding to isolated DR4;Dw4.2 (a) or Dw14.1 (b) molecules assayed by competition with biotinylated MBP p84-102 (83Tyr) at 1 µM. The IC50 values are shown with closed circles. (ii) For presentation to PM-A T cells by the indicated DR4 subtype, expressed as the PC50 values (i.e. after pre-pulsing the APC for 23 h). Every peptidesubtype combination was tested at least twice. Shown above is the residue in the parent sequence that was substituted by the indicated amino acid. Note that the scales for PC50 (on the left) and for IC50 (on the right) are not always the same. Both Dw13.2 and Dw15.1 are even less potent than appears, as they were tested here after prolonged pulsing (69 h), because responses were much weaker after 23 h. None of these peptides was presented detectably by Dw10.2, which may bind more weakly. With Dw13.2, the negative result with the 152Asp Ala substituent argues against simple repulsion by the 74Glu in this DRß, as also noted elsewhere (26).
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Table 2. Overcoming the 86Val restrictions of PM-A T cells in Dw14.1 and Dw15.1 with substituents of p146158 (LGTWTYDGSVVAI)
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To investigate further these strikingly different effects of the same 86Gly
Val dimorphism in the context of the 71Arg
Lys and the 57Asp
Ser substitutions, we next identified critical residues in the peptide.
Critical residues in the epitope
We systematically replaced each residue from 146160 (in turn) with Ala, apart from 157Ala, which was changed to Lys or Tyr instead. To adapt these analogues to subtypes with 86Val, we concomitantly replaced 149Trp with Phe (throughout) at p1 in the 145163 `parent' sequence and we tested their presentation to PM-A as above (Fig. 2
). A diagram of a DR4peptide complex is shown in Fig. 3
.
For peptide binding.
Firstly, we confirmed the anchoring roles of p1 and p9, and established that nearly all of the other variants bound similarly well to isolated Dw4.2 molecules (Fig. 2a
). Thus, the IC50 was ~20-fold higher for the 149Phe
Ala variant at p1; this weak but significant binding reflects contributions from secondary anchor residues (15,16,18). The IC50 was also higher for a Lys substituent for the 157Ala at p9 that is known to be favorable for DR4 (3,15,16,18). We could assay binding to Dw14.1 molecules too, as there are B cell lines homozygous for Dw14.1, though not for the autologous Dw14.2. In general, this required 5- to 10-fold more of most analogues than with Dw4.2 (note the different scales in Fig. 2a and b
), and higher concentrations still of the p6, p7 and p9 variants (Fig. 2b
). Also, binding to Dw14.1 was undetectable with the 149Tyr substituent at p1; with 149Ala, however, it was slightly stronger than with Dw4.2.
For peptide presentation to PM-A T cells.
The data on presentation of these variants highlight several general points (Fig. 2
). Firstly, they show that the binding register was the same in each subtype, and confirm the key anchoring roles of the p1 and p9 residues discussed above. Secondly, as expected from the crystal structure of the DRpeptide complex (2,3), the primary TCR contact residue is 153Gly at p5; neither the conservative 153Ala nor 153Ser substituents were recognized detectably in any subtype, despite binding well to Dw4.2 and Dw14.1 (Fig. 2a and b
). Secondary TCR contact sites include 150Thr at p2, especially 151Tyr at p3, and also 156Val at p8 in Dw4.2 (as shown by weaker presentation despite average binding). However, 147Gly and 148Thr appear less important here than in DR52a and DR3 (27). Indeed, in DR4, the substitutions at either end had much less effect than those in the core of the epitope.
In addition, these data provide further evidence for the interactions between positions 86 and 71 or 57 noted above (i) and (ii), and also add another example (iii).
- (i) The Arg
Lys dimorphism at position 71. We noted four major effects of this dimorphism. Firstly, when there was a 71Lys (in Dw4.1), the 86Val had no effect on presentation of the 149Tyr substituent (Fig. 2a
), whereas, in the context of 71Arg, it caused all-or-nothing differences, i.e. between both Dw14.2/14.1 (Fig. 2b
) and Dw15.2/15.1 (Fig. 2c
). Secondly, with all of the other analogues, Dw4.2 and Dw4.1 gave almost identical PC50 values (Fig. 2a
), whereas there were significant discrepancies between Dw14.2 and Dw14.1, especially at p69 (cf. the open symbols in Fig. 2b
). Thirdly, with Dw4.2, the molarities required for the stimulatory analogues were close to their IC50 values (within a factor of 3, Fig. 2a
). In sharp contrast, with Dw14.1, presentation was, in general, 5- to 10-fold more efficient, even though the binding was 5- to 10-fold weaker (note the different scales in Fig. 2a and b
). Thus, many PC50s were 301000 times lower than the corresponding IC50s, so that functional binding was disproportionately potent in the context of the autologous 71Arg. Fourthly, these differences from Dw4 were most extreme for the substituents for the core residues, especially p24, p6 and p8, several of which are secondary TCR contact sites (2,3); these variants were not recognized detectably in the context of 71Lys, even though they bound well (Fig. 2a
), whereas, with Dw14.1, presentation generally correlated much better with peptide binding (despite requiring lower molarities). Moreover, the overall profile for presentation was broadly similar for the three main subtypes with 71Arg, and differed starkly from those with 71Lys (cf. Fig. 2b and c
with a; open symbols).
- (ii) Position 57. Despite this very similar functional binding by Dw14.1, Dw14.2 and Dw15.2, presentation by Dw15.1 was >100-fold less efficient for almost all the peptides tested. The responses we found again depended not only on high molarities but also on a prolonged peptide pulse (9 h; Fig. 2
legend). Relatively, they were stronger to the Ala substituent at p4 and also to those at p1012 that reduce the C terminal bulk; in general, they correlated with those with Dw15.2 (Fig. 2d
).
Thus the 86Val and 57Ser substitutions evidently had far more drastic effects in combination (in Dw15.1) than they did separately (cf. Dw14.1 and Dw15.2 with Dw14.2).
- (iii) Notably, too, the analogues for p1012 were presented by Dw14.1 at 10 times lower molarities than by Dw14.2. Since these subtypes differ only at position 86 near the far end of the peptide, this is another unexpected and remote interaction.
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Discussion
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This study on peptide presentation to a specific T cell clone has yielded three novel groups of findings.
- (i) The major effects of the 86Gly
Val substitution in HLA-DRß depend greatly on the context, i.e. both on the particular peptide and on the exact DR4 subtype. Only in Dw14.1 were they easily overcome with a smaller peptide anchor residue at p1.
- (ii) Even for an unusual T cell that tolerates both 71Lys and 71Arg, this interchange still radically influences not only the effects of 86Val but also the recognition of peptide residues as far apart as p2 and p11.
- (iii) A concomitant 57Asp
Ser substitution near the peptide's C-terminus also greatly reduced our ability to overcome the 86Val restriction (in Dw15.1) with a smaller p1 anchor near the N-terminus.
We were able to obtain such striking evidence for these long-range interactions because of the existence of isolated dimorphisms at positions 86, 71 and 57 in several DR4 subtypes, and because our PM-A T cell shows unusually broad cross-restrictions to them. As we propose below, they are most simply explained by subtle effects on the conformation of the peptideDR complex, though definitive proof awaits detailed structural comparisons.
The role of 86Val
Gly
Our findings on both peptide binding and presentation support the importance of this widespread dimorphism noted previously (6,8,9,1419,21). As the crystal structures for HLA-DR1 and DR4 show (2,3), the side-chain of 86Val protrudes into the `pocket 1' that accommodates the major hydrophobic `anchor' residue near the N-terminus of the peptide (Fig. 3
) and it often hinders the binding of bulkier side-chains there (8,9,1419,21,2628). In the present case, the 149Trp clearly plays this p1 anchor role; merely changing it to a less bulky Phe (or Leu) consistently and completely overcame this restriction in Dw14.1 (leading to maximal stimulation). Interestingly, that also applied not only to two other T cell clones from Dw14.2+ donors (14), but also to three further T cells restricted to DR52a/DR3 (27)which supports the generality of this phenomenon. Notably, in both Dw14.1 and DR3, it was not overcome by a 149Tyr; presumably its effective bulk is increased by hydration. Unexpectedly, for the sensitive PM-A, the small 149Ala at p1 allowed significant functional binding of this epitope, especially to Dw4.1, and Dw14.1 (Fig. 2
), presumably because it has a high overall binding score (16,29), reflecting multiple interactions along its core (1618,26,29,30).
More surprisingly, the effects of this 86Gly
Val substitution depended greatly both on the length and potency of the peptide and especially on other substitutions elsewhere in DRß. As confirmed by site-directed mutagenesis (31), the 71Arg
Lys replacement in Dw4 is crucial. Thus the 86Val restriction proved to be less completebut also less readily overcomein the context of 71Lys (in Dw4.1) than of 71Arg (in Dw14.1). These side-chains clearly point `sideways' at the peptide (2,3) and the lesser bulk or hydrogen bonding with 71Lys (see below) evidently enhances binding even of the large 149Trp or Tyr in pocket 1 in Dw4.1, e.g. by permitting more flexibility in the middle of the peptide. Presumably, similarly permissive changes in DRß must allow accommodation of the promiscuous HA 307319 peptide in other alleles with 86Val (despite its Tyr at p1), even though it generally binds better to those with 86Gly (18,19). Conversely, Krieger et al. (8) were also able to compensate for this 86Val by substituting smaller residues at p46 in the middle of the peptide; they concluded that some changes in its orientation enhanced binding, whereas others had much greater effects on its recognition by T cells (8), as also noted in mouse class II molecules (32). Analogous changes in peptide orientation can also result from alterations in the p1 anchor residue itself (22).
The role of 71Arg
Lys
There is a shallow pocket `4' in the middle of the groove (Fig. 3
); the polymorphic DRß residues 70 (above), 71 (below), 74 and 78 contribute to its side wall, and those at 13, 26 and 28 to its floor (2,3). The natural substitutions found here usually have far less effect on peptide binding per se than on the recognition of the bound peptide (8,20,21,33), as we have observed both here and in a parallel study (14). Thus, many T cells are totally dependent on the autologous 71Lys (14,21) or 71Arg (14), even for recognizing an identical peptide that clearly binds well in both cases. Indeed, only four of 16 published T cells tolerate both 71Lys and 71Arg (14,20,21,33); when they do, as here with PM-A, this interchange still has major effects, such as the disproportionately greater efficiency of presentation relative to binding by Dw14.1 than by Dw4.2 (Fig. 2a and b
). Similarly also, presentation of substituents for five of the core residues was drastically reduced in the context of 71Lys. As these all-or-nothing differences were clearly not caused by better binding by Dw14.1 (which was considerably weaker, Fig. 2b
), they must result from differences in the conformation of the peptideDR complex (8,20,21,33). Since they were also greatest at secondary TCR contact sites as far apart as p2, p3 and p8, differences in the peptide seem likely, especially as 71Arg lies deep to 70Gln, and therefore contacts the peptide and not the TCR (2,3). Although this 71Lys
Arg substitution appears conservative, Arg side-chains normally form several more hydrogen bonds than Lys (34); here at position 71, these create links not only with DRß 28Asp in the floor of the groove and with the 70Gln above (Fig. 3
), but also with the backbone of the bound peptide (2,3). A consequent increase in peptide rigidity in the autologous Dw14.2 might bias heavily in favor of the optimal conformation for PM-A T cells; as a result, not only is presentation disproportionately efficient, but many substitutions along the length of the core are less critical here than in Dw4.2, where the 71Lys may allow excessive flexibility (Fig. 2
). This may be further enhanced by the 152Asp
Ala replacement at p4, which is an accessory class II contact site in DR4 (16,17,33).
Interactions between positions 86 and 57
In this most extreme/remote example, neither the 86Gly
Val (in Dw14.1) nor the 57Asp
Ser (in Dw15) interchanges alone affected presentation substantially, whereas together (in Dw15.1) they reduced the potency of our best peptides at least 100-fold (Fig. 2c
). That is probably an effect on recognition, which is generally less promiscuous/permissive, but even a loss of binding would still be compelling evidence of an indirect interaction between positions 86 and 57 in Dw15.1, again apparently mediated by the peptide conformation or orientation. The parallel effects with all the analogues suggest some global decrease in the probability of a recognizable conformation in Dw15.1 and so does the requirement for prolonged pulsing at high concentrations (as with Dw13.2) (14). Moreover, the crystal structure suggests an explanation; the 57Asp
Ser substitution here prevents formation of the salt bridge that otherwise runs from the carboxyl group of the 57Asp across to 76Arg in DR
and lies deep to the peptide (2,3) (Fig. 3
). We propose that this 57Ser in Dw15.1 alters the conformation of longer peptides at this (C-terminal) end of the groove so that they can no longer be recognized at the other end. In independent support of that, the variant peptides truncated at p10 overcame these effects surprisingly well despite their weak presentation by other subtypes (Table 2
). A theoretical alternative explanation is that there are interactions between adjacent peptideDR complexes on the APC (35) that are sensitive to alterations in these anchoring pockets, e.g. because of mutual interference by the protruding N- and C-termini of the bound peptide(s).
Sometimes from less clear-out findings, others have also invoked conformational alterations in peptideclass II complexes, whether resulting from differences in the loading process (36), or in the sequences of the class II molecules (8,21,33,37) or of the peptides presented (8,32). As observed in class I (38) and DR4 (3), there may be subtle changes in the
helices as well as in the bound peptide which may have more freedom/flexibility, e.g. because of fortuitous intervening water molecules (3,38). Moreover, both the peptide and the helices in class I can apparently adjust on binding of the TCR (39). While the X-ray crystal structures have so far shown very limited plasticity in the peptide conformation in DR molecules (2,3), even minor changes can have drastic effects on T cell recognition (38). Furthermore, all the DR structures reported so far are from molecules with 57Asp, whereas we saw the most extreme and remote effects with 57Ser (in Dw15.1); its interactions with 86Val ranged over eight to 10 residues in the (extended) peptide and >30 residues in the
helix of DRß. Analogous interactions may well occur with other polymorphic residues in the floor of the groove (33,37) and/or in the
chain in DQ or DP, though the rarity of isolated dimorphisms there would make them harder to discern than in DR4.
This substantial body of evidence may seem inferential. Nevertheless, working from precisely analogous findings, Krieger et al. (8) correctly predicted not only the extended conformation of HLA-DR-bound peptides and their p1 anchorage via pocket 1, but also the existence of a turn near p5 (2,3). Definitive proof of our deductions will require detailed crystallographic comparisons of several peptides in several alleles (not all yet available in homozygous form; Table 1
); already, however, several structurally established precedents in HLA class I (3841) lend strong support.
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Conclusions
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Whatever the final explanation, these unexpected findings must have consequences for epitope prediction/identification and for disease susceptibility as well as for MHC evolution. For example, the effects of individual substitutions in HLA-DR may depend greatly both on the exact length and potency of the peptide and on their context in the rest of the class II molecule. This might become important in vaccine design; e.g. merely by using a more potent peptide, we achieved strong presentation by Dw4.1 as well as Dw4.2, whereas, for Dw14.1, we had to change the p1 anchor too. Therefore one may also need to consider the exact length and sequence of the naturally processed epitope when designing vaccines.
Our findings must be relevant in rheumatoid arthritis, where arthritogenic peptides may be involved (1114,17), and where there are strong associations with Dw4.2, Dw15.2 and both Dw14.1 and Dw14.2, but not with Dw10.1 or Dw13.2 (1114). These striking differences may reflect the impact of positions 70, 71 and 74 on T cell recognition as well as on peptide binding (1315,17). Furthermore, our results clearly show that the 70QRRAA74 and the 70QKRAA74 sequences in DR4ß are not functionally equivalent, as was initially assumed in some theories (11). Indeed, since they largely activate distinct subsets of T cells, those that are cross-restricted to both Dw4 and Dw14 may be highly unusual (14).
Finally, the importance of the context of particular substitutions in class II may help to explain both the rarity of the 86Val57Ser combination in particular in HLA-DR alleles and their general tendency to evolve by replacements of `cassettes' of codons rather than by point substitutions. While these often point into the same pocket and contact the same residue in the peptide (13), they are sometimes more dispersed, as, for example, in DR12 (1); this allele has distinctive residues in both pockets 4 and 9, perhaps to favor long-range interactions such as those described here. In either case, they must serve to increase the diversity of peptideMHC conformations presented to specific T cells, and thus broaden the range of epitopes and pathogens that can be recognized.
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Acknowledgments
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This work was supported by the Medical Research Councils of Great Britain and Canada, the Muscular Dystrophy Group of Great Britain, and the Royal Society. We thank Messrs Leslie Jacobson and R. Cotton, and Drs Howard Grey, Angela Vincent and Corinne Savill for help and advice, and Dr E. W. Petersdorf for the Dw4.1+ and Dw15.1+ B cells.
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Abbreviations
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AChR | acetylcholine receptor |
APC | antigen-presenting cell |
MBP | myelin basic protein |
MG | myasthenia gravis |
PC50 | peptide concentration (during APC pulse) required for 50% maximal T cell stimulation |
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Notes
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Transmitting editor: E. Simpson
3 Present address: Department Neurology, Kawatana National Hospital, Nagasaki 859-36, Japan 
4 Present address: Department of Clinical Neuroscience, Charing Cross Hospital, London W6 8RF, UK 
5 Present address: Department of Clinical Neuroscience, 339 Windermere Road, London, Ontario N6A 5A5, Canada 
6 Present address: Megabios Corp., 863A Mitten Road, Burlingame, CA 94010, USA 
Received 6 November 1998,
accepted 3 February 1999.
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References
|
---|
-
Kimura, A. and Sasazuki, T. 1992. XIth histocompatibility workshop reference protocol for the HLA DNA typing technique. In Tsuji, K., Aizawa, M. and Sasazuki, T., eds, HLA 1991, p. 397. Oxford University Press, Oxford.
-
Stern, L. J., Brown, J. H., Jardetzky, T. S., Gorga, J. C., Urban, R. G., Strominger, J. L. and Wiley, D. C. 1994. Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature 368:215.[ISI][Medline]
-
Dessen, A., Lawrence, C. M., Cupo, S., Zaller, D. M. and Wiley, D. C. 1997. X-ray crystal structure of HLA-DR4 (DRA*0101, DRB*0401) complexed with a peptide from human collagen II. Immunity 7:473.[ISI][Medline]
-
Petersdorf, E. W., Smith, A. G., Martin, P. J. and Hansen, J. A. 1992. HLA-DRB1 first domain sequence for a novel DR4 allele designated DRB1*0413. Tissue Antigens 40:267.
-
Pile, K. D., Willcox, N., Bell, J. I. and Wordsworth, B. P. 1992. A novel HLA-DR4 allele (DRB1*0414) in a patient with myasthenia gravis. Tissue Antigens 40:264.[ISI][Medline]
-
Ong, B., Willcox, N., Wordsworth, P., Beeson, D., Vincent, A., Altmann, D., Lanchbury, J. S. S., Harcourt, G. C., Bell, J. I. and Newsom-Davis, J. 1991. Critical role for the Val/Gly86 HLA-DRß dimorphism in autoantigen presentation to human T cells. Proc. Natl Acad. Sci. USA 88:7343.[Abstract]
-
Petersdorf, E. W., Smith, A. G., Mickelson, E. M., Martin, P. J. and Hansen, J. A. 1991. Ten HLA-DR4 alleles defined by sequence polymorphisms within the DRB1 first domain. Immunogenetics 33:267.[ISI][Medline]
-
Krieger, J. I., Karr, R. W., Grey, H. M., Yu, W. Y., O'Sullivan, D., Batovsky, L., Zheng, Z. L., Colon, S. M., Gaeta, F. C. A., Sidney, J., Albertson, M., del Guercio, M.-F., Chesnut, R. W. and Sette, A. 1991. Single amino acid changes in DR and antigen define residues critical for peptideMHC binding and T cell recognition. J. Immunol. 148:2331.[Abstract/Free Full Text]
-
Demotz, S., Barbey, C., Corradin, G., Amoroso, D. and Lanzavecchia, A. 1993. The set of naturally processed peptides displayed by DR molecules is tuned by polymorphism of residue 86. Eur. J. Immunol. 23:425.[ISI][Medline]
-
Hill, A. V. S., Allsopp, C. E. M., Kwiatkowski, D., Anstey, N. M., Twumasi, P., Rowe, P. A., Bennett, S., Brewster, D., McMichael, A. J. and Greenwood, B. M. 1991. Common West African HLA antigens are associated with protection from severe malaria. Nature 352:595.[ISI][Medline]
-
Gregersen, P. K., Silver, J. and Winchester, R. J. 1987. The shared epitope hypothesis: an approach to understanding the molecular genetics of susceptibility to rheumatoid arthritis. Arthritis Rheum. 30:1205.[ISI][Medline]
-
Wordsworth, B. P. and Bell, J. I. 1992. The immunogenetics of rheumatoid arthritis. Semin. Immunopathol. 14:59.[ISI][Medline]
-
Winchester, R. J. 1994. The molecular basis of susceptibility to rheumatoid arthritis. Adv. Immunol. 56:389.[ISI][Medline]
-
Hawke, S., Matsuo, H., Nicolle, M., Wordsworth, P., Corlett, L., Spack, E., Deshpande, S., Driscoll, P. C. and Willcox, N. 1998. Cross-restriction of a T cell clone to HLA-DR alleles associated with rheumatoid arthritis; clues to arthritogenic peptide motifs. Arthritis Rheum., in press.
-
Sette, A., Sidney, J., Oseroff, C., del Guercio, M.-F., Southwood, S., Arrhenius, T., Powell, M. F., Colon, S. M., Gaeta, F. C. A. and Grey, H. M. 1993. HLA-DR4w4-binding motifs illustrate the biochemical basis of degeneracy and specificity in peptideDR interactions. J. Immunol. 151:3163.[Abstract/Free Full Text]
-
Hammer, J., Bono, E., Gallazzi, F., Belunis, C., Nagy, Z. and Sinigaglia, F. 1994. Precise prediction of major histocompatibility complex class IIpeptide interaction based on peptide side chain scanning. J. Exp. Med. 180:2353.[Abstract]
-
Hammer, J., Gallazzi, F., Bono, E., Karr, R. W., Guenot, J., Valsasnini, P., Nagy, Z. A. and Sinigaglia, F. 1995. Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association. J. Exp. Med. 181:1847.[Abstract]
-
Marshall, K. W., Liu, A. F., Canales, J., Perahia, B., Jorgensen, B., Gantzos, R. D., Aguilar, B., Devaux, B. and Rothbard, J. B. 1994. Role of the polymorphic residues in HLA-DR molecules in allele-specific binding of peptide ligands. J. Immunol. 152:4946.[Abstract/Free Full Text]
-
Busch R., Hill, C. M., Hayball, D., Lamb, J. R. and Rothbard, J. B. 1992. Effect of natural polymorphism at residue 86 of the HLA-DRß chain on peptide binding. J. Immunol. 147:1292.[Abstract/Free Full Text]
-
Rothbard, J. B., Busch, R., Bal, V., Trowsdale, J., Lechler R. I. and Lamb, J. R. 1989. Reversal of HLA restriction by a point mutation in an antigenic peptide. Int. Immunol. 1:487.[Medline]
-
Fu, X.-T., Bono, C. P., Woulfe, S. L., Swearingen, C., Summers, N. L., Sinigaglia, F., Sette, A., Schwartz, B. D. and Karr, R. W. 1995. Pocket 4 of the HLA-DR(
,ß1*0401) molecule is a major determinant of T cell recognition of peptide. J. Exp. Med. 181:915.[Abstract]
-
Wu, S., Gorski, J., Eckels, D. D. and Newton-Nash, D. K. 1996. T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex; effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein. J. Immunol. 156:3815.[Abstract]
-
Matsuo, H., Batocchi, A.-P., Hawke, S., Nicolle, M., Jacobson, L., Vincent, A., Newsom-Davis, J. and Willcox, N. 1995. Peptide-selected T cell lines from myasthenia gravis patients and controls recognize epitopes that are not processed from whole acetylcholine receptor. J. Immunol. 155:3683.[Abstract]
-
Tompkins, S. M., Rota, P. A., Moore, J. C. and Jensen, P. 1993. A europium fluoroimmunoassay for measuring binding of antigen to class II MHC glycoproteins. J. Immunol. Methods 163:209.[ISI][Medline]
-
Nicolle, M. W., Nag, B., Sharma, S. D., Willcox, N., Vincent, A., Ferguson, D. J. P. and Newsom-Davis, J. 1994. Specific tolerance to an acetylcholine receptor epitope induced in vitro in myasthenia gravis CD4+ lymphocytes by soluble major histocompatibility complex class IIpeptide complexes. J. Clin. Invest. 93:1361.[ISI][Medline]
-
Matsushita, S., Takahashi, K., Motoki, M., Komoriya, K., Ikagawa, S. and Nishimura, Y. 1994. Allele specificity of structural requirement for peptides bound to HLA-DRB1*0405 and -DRB1*0406 complexes: implication for the HLA-associated susceptibility to methimazole-induced insulin autoimmune syndrome. J. Exp. Med. 180:873.[Abstract]
-
Nagvekar, N., Corlett, L., Jacobson, L. W., Matsuo, H., Chalkley, R., Driscoll, P. C., Desphande, S., Spack, E. G. and Willcox, N. 1998. Scanning a DRB3*0101 (DR52a)-restricted epitope cross-presented by DR3; overlapping natural and artificial determinants in the human acetylcholine receptor. J. Immunol. 162: in press.
-
Friede, T., Gnau, V., Jung, G., Keilholz, W., Stevanovic, S. and Rammensee, H.-G. 1996. Natural ligand motifs of closely related HLA-DR4 molecules predict features of rheumatoid arthritis-associated peptides. Biochim. Biophys. Acta 1316:85.[ISI][Medline]
-
Marshall, K. W., Wilson, K. J., Liang, J., Woods, A., Zaller, D. and Rothbard, J. B. 1995. Prediction of peptide affinity to HLA DRB1*0401. J. Immunol. 154:5927.[Abstract/Free Full Text]
-
Jardetzky, T. S., Gorga, J. C., Busch, R., Rothbard, J., Strominger, J. L. and Wiley, D. C. 1990. Peptide binding to HLA-DR1: a peptide with most residues substituted to alanine retains MHC binding. EMBO J. 9:1797.[Abstract]
-
Daubenberger, C. B., Lang, B., Nickel, B., Willcox, N. and Melchers, I. 1996. Antigen processing and presentation by a mouse macrophage-like cell line expressing human class II molecules. Int. Immunol. 8:307.[Abstract]
-
Reay, P. A., Kantor, R. M. and Davis, M. M. 1994. Use of global amino acid replacements to define the requirements for MHC binding and T cell recognition of moth cytochrome c (93103). J. Immunol. 152:3946.[Abstract/Free Full Text]
-
McNicholl, J. M., Whitworth, W. C., Oftung, F., Fu, X., Shinnick, T., Jensen, P. E., Simon, M., Wohlhueter, R. M. and Karr, R. W. 1995. Structural requirements of peptide and MHC for DR(
, ß1*0401)-restricted T cell antigen recognition J. Immunol. 155:1951.[Abstract]
-
McDonald, I. K. and Thornton, J. M. 1994. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238:777.[ISI][Medline]
-
Davis, M. M., Boniface, J. J., Reich, Z., Lyons, D., Hampl, J., Arden, B. and Chien, Y. 1998. Ligand recognition by
ß T cell receptors. Annu. Rev. Immunol. 16:526.
-
Viner, N. J., Nelson, C. A., Deck, B. and Unanue, E. R. 1996. Complexes generated by the binding of free peptides to class II MHC molecules are antigenically diverse compared with those generated by interacellular processing. J. Immunol. 156:2365.[Abstract]
-
Brett, S. J., McKean, D., York-Jolley, J. and Berzofsky, J. A. 1989. Antigen presentation to specific T cells by Ia molecules selectively altered by site-directed mutagenesis. Int. Immunol. 1:130.[Medline]
-
Reid, S. W., McAdam, S., Smith, J., Klenerman, P., O'Callaghan, C. A., Harlos, K., Jakobsen, B. K., McMichael, A. J., Bell, J. I., Stuart, D. I. and Jones, E. Y. 1996. Antagonist HIV-1 gag peptides induce structural changes in HLA-B8. J. Exp. Med. 184:2279.[Abstract/Free Full Text]
-
Garboczi, D. N., Ghosh, P., Utz, U., Fan, Q. R., Biddison, W. E. and Wiley, D. C. 1996. Structure of the complex between T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[ISI][Medline]
-
Madden, D. R., Garboczi, D. N. and Wiley, D. C. 1993. The antigenic identity of peptideMHC complexes: a comparison of the conformations of five viral peptides presented by HLA-A2. Cell 75:693.[ISI][Medline]
-
O'Callaghan, C. A., Tormo, J., Willcox, B. E., Braud, V. M., Jakobsen, B. K., Stuart D. I., McMichael, A. J., Bell, J. I. and Jones, E. Y. 1998. Structural features impose tight peptide binding specificity in the non-classical MHC molecule HLA-E. Mol. Cell 1:531.[ISI][Medline]