Properties of HLA class II molecules divergently associated with Goodpasture's disease

Richard G. Phelps, Victoria Jones1, A. Neil Turner and Andrew J. Rees1

Department of Clinical and Surgical Sciences (Internal Medicine), University of Edinburgh, Royal Infirmary, Edinburgh EH3 9YW, UK
1 Department of Medicine and Therapeutics, University of Aberdeen, Aberdeen AB25 2ZD, UK

Correspondence to: R. G .Phelps


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Goodpasture's disease provides an opportunity to analyse molecular mechanisms that may underlie MHC class II associations with autoimmune disease because it is caused by autoimmunity to a defined antigen [the 230 amino acid NC1 domain of the {alpha}3 chain of type IV collagen ({alpha}3(IV)NC1)] and has strong HLA class II associations. We compared the {alpha}3(IV)NC1 peptide binding of class II molecules with strong positive (DR15) and dominant negative (DR7/1) associations using an inhibition binding assay and short synthetic peptides spanning the sequence of {alpha}3(IV)NC1. DR15 in general bound the peptides with low affinity (three of 23 < 100 nM) compared to DR1 and DR7 (12 and 10 < 100 nM respectively), and no peptide bound DR15 with much higher affinity (>10-fold) than both DR1 and DR7. Thus DR15 molecules are unlikely to increase susceptibility to Goodpasture's disease by presenting a particular {alpha}3(IV)NC1-derived peptide uniquely well and DR1/7 are unlikely to protect by their inability to present particular peptides. However DR1/7 could protect by capturing {alpha}3(IV)NC1 peptides and preventing their display bound to DR15; the binding data suggest that all the major (biochemically detectable) {alpha}3(IV)NC1 peptides presented bound to DR15 by DR15 homozygous antigen-presenting cells (APC) would bind preferentially to DR1/7 in DR15, 1/7 heterozygote APC.

Keywords: autoimmunity, Goodpasture's disease, HLA class II molecules, HLA complex


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Goodpasture's disease has very striking associations with alleles inherited at MHC class II loci. Almost 80% of patients carry DRB1*1501, one of the alleles contributing to the DR2 specificity, compared with 25% of control populations, giving an odds ratio for disease of 8.5. An even more striking opposite influence is exerted by DR7 alleles which appear to protect from the disease even in the presence of DRB1*1501 (dominant protection). There are weaker positive associations with DR3 and DR4 alleles, and negative associations with DR1 alleles. The associations are similar to those reported for many other autoimmune diseases and animal models of autoimmunity (reviewed in 1).

The mechanisms that account for MHC associations with autoimmunity are not known, but there is compelling evidence that it is in large part expression of the MHC molecules themselves that account for the associations (e.g. see 2). MHC molecules influence immune responses to exogenous antigens because their peptide binding preferences determine the antigen-derived peptides displayed to T cells (3). Very likely their peptide binding preferences also influence immune responses to self proteins. Comparisons of MHC molecules with similar disease associations have demonstrated similarities in both the structures of their peptide binding grooves (e.g. 4–6) and in their peptide-binding properties (7,8), suggesting that disease association is a consequence of capacity to present particular peptides. Evidence that expression of class II alleles directly influences autoimmunity has been obtained in mice transgenic for HLA class II alleles (9,10). Exactly how MHC-determined differences in autoantigen presentation influence susceptibility to autoimmunity is controversial. The major mechanisms are probably not the same in all studied examples of autoimmune diseases and models of autoimmunity. For example, it is well established that susceptibility to diabetes is strongly influenced by MHC class II alleles in NOD mice, but three studies of transgenic NOD mouse lines aimed at elucidating the mechanisms have offered support for a distinct mechanisms in each case (1113).

Goodpasture's disease is an autoimmune cause of kidney and lung failure that affords an unparalleled opportunity to study how HLA class II molecules might influence susceptibility to an important clinical disease. The disease is unique because, as well as having well-defined very strong positive (with DR15) and dominant negative (with DR7 and DR1) HLA class II associations (1,6,14,15), the target of autoimmune attack has been narrowed down to the relatively small 230 amino acid C-terminal NC1 domain of the {alpha}3 chain of type IV collagen [{alpha}3(IV)NC1] (16,17), and the most abundant naturally processed {alpha}3(IV)NC1-derived peptides presented bound to DR15 molecules have been defined biochemically (18,19). Knowledge of the antigen is clearly essential for analysis of antigen–class II interactions. Knowledge of putative major naturally processed peptides is important as it identifies autoantigen-derived peptides generated by the processing machinery within human antigen-presenting cells (APC) that are available to bind available class II molecules. It also identifies the major peptides actually presented to CD4 T cells in susceptible individuals. CD4 T cells are likely to be important in Goodpasture's disease at least for provision of T cell help to potentially autoreactive T cells and possibly also for driving the characteristic crescentic pattern of glomerular injury, as has been shown in a murine model (20). Evidence that a patient's CD4 T cells recognize {alpha}3(IV)NC1 comes from previous studies with purified and recombinant protein (21), and from our own recent observation that a patient's T cells proliferate and produce IFN-{gamma} to intact recombinant {alpha}3(IV)NC1 and certain peptides but not {alpha}5(IV)NC1 (unpublished data).

Here we report analysis of the {alpha}3(IV)NC1 peptide-binding preferences of three HLA-DR molecules with divergent associations with susceptibility to Goodpasture's disease. DR15b (composed the gene products of DRA and DRB1*1501), DR1 and DR7 were selected for this study because analysis of published genotype data of patients with Goodpasture's disease showed: (i) DRB1*1501 and probably 1502 are strongly associated with Goodpasture's disease (odds ratio 8.5), but DR16 alleles are not, despite also encoding the DR2 specificity; (ii) DRB1*0701 confers strong dominant protection from disease (OR = 0.3) such that individuals inheriting B1*1501 with *0701 have no higher risk of disease than the general population; (iii) a weaker protective influence of DRB1*01 (OR = 0.6); and (iv) that the positive association with DRB1*1501 and negative associations with DR1/7 were unlikely to be due to linkage disequilibrium with DQA/B or DRB4/5 alleles primarily responsible for the associations (1).

Strikingly, we find that DR molecules associated with strong dominant protection have generally much higher affinity for {alpha}3(IV)NC1 peptides than DR15, the DR molecule that is most strongly associated with increased susceptibility.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The peptides used in this study and method of preparing purified class II molecules are the same as described elsewhere (18). HLA-DR molecules were affinity purified from detergent lysates of the cell lines LDR2b (yielding DR15b) (23), CF0996 (DR7) and HOM2 (DR1) respectively using the murine mAb L243 (22). The murine L cell transfectant LDR2b (23) was used because available mAb do not distinguish between the two DR molecules carried by human DR15 homozygous APC. The DR7 preparation was contaminated by DRw53 molecules at a low level: Southwood et al. have shown that DRw53 contamination does not influence DR7 peptide-binding assays under conditions similar to those used in this study (24), presumably because of their low level relative to that of DR7, the limiting conditions used in the assay and the very different peptide binding characteristics of DRw53 molecules (25).

Peptide binding assays
Peptide binding to purified class II molecules was measured using an inhibition assay based on that described by Tompkins and Jensen (26) and fully described in (18). The myelin basic protein peptide MBP 86–98(98A) and flu haemagglutinin peptide HA307–319 were selected as reference peptides because of their reported high affinity for DR15b and DR1/7 respectively (27). As before, the concentrations of peptides [Pi] causing 50% reduction in reference peptide binding (IC50i) were extracted from experimental binding data by curve fitting to functions of the form , where B is binding in the absence of unlabelled test peptide (the equation derives from the law of mass action). IC50s were determined in at least three independent experiments. Expressed as logarithms, the experimental data was normally distributed with SD = 0.165 and the 95% confidence limits for the arithmetic means (three measurements) were ±0.19. Therefore the 95% confidence limits for measured IC50s (in moles) were between (mean/1.5) and (meanx1.5).

Calculation of Ki
To use IC50 measurements to compare the affinity of peptides for different class II molecules measured against different reference peptides it was necessary to determine an absolute measurement of peptide class II affinity (IC50 measurements are dependent upon the experimental conditions used). When both reference peptide (Pref) and test peptides (Pi) are used in excess of available class II, the (inhibition) dissociation constant (Ki) of the complex DR–Pi is related to the measured IC50 by the formula where [Pref] is the concentration of reference peptide and Kdref is the equilibrium dissociation constant of DR–reference peptide complexes. The Kdref of *MBPP–DR15b, *HAP–DR7 and *HAP–DR1 were estimated to be 16, 15 and 5 nM respectively by Hill transformation of binding measured at a range of concentrations of reference peptides, and reference peptides were used at 160, 88 and 27 nM for DR15b, DR7 and DR1 respectively. Therefore, for comparison purposes, Ki ~ IC50/11 for DR15b inhibition assays, IC50/6.87 for DR7 assays and IC50/6.33 for DR1 assays.

Motif analysis
Motif analysis for DR1 and DR7 was conducted using motifs reported by Southwood et al. (24). For DR15b we adapted a motif we used previously, based on reported binding data (27,28), by weighting smaller hydrophobic residues (VILM) at position 1 and larger hydrophobic residues (WYF) at position 4. The weighting is suggested by the reported relative sizes of pockets 1 and 4 in the crystal structures of DR15b and DR1 (29), and supported by our own pool sequencing data (see Processing and presentation of the Goodpasture antigen, R. Phelps, PhD Thesis, University of London, 1998) and that of other groups (Andrew Godkin, pers. commun. and 30).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Binding of {alpha}3(IV)NC1 peptides to HLA-DR7
All of the peptides bound to DR7 molecules. To permit comparison with class II–peptide binding measurements reported elsewhere, the IC50 measurements were used to calculate inhibition dissociation constants (Ki) for each interaction. Horizontal bars in Fig. 1Go demarcate peptides binding with high, intermediate and low affinity. All of the peptides bound to DR7 with intermediate (13 of 24) or good affinity (11 of 24) (Fig. 1Go, bottom panel), including the three peptides known to be naturally processed and presented in DR15-homozygous human B cells (identified in Fig. 1Go with large diamond markers) (18,19). This is in striking contrast with DR15b molecules to which the same peptides bound with mostly intermediate (14 of 24) or low/unmeasurable affinity (seven of 24), only three peptides having high affinity (Fig. 1Go, top panel). The assay conditions permitted measurement of IC50 for peptides with IC50 < ~100 µM, so did not distinguish low-affinity binding (IC50 >100 µM, Ki > ~10 µM) from non-binding.



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Fig. 1. Affinity of {alpha}3(IV)NC1 peptides for DR molecules divergently associated with Goodpasture's disease. Affinity is shown for DR molecules with strong positive (DR15b, top panel), weak negative (DR1, middle panel) and strong negative (DR7, bottom panel) associations with Goodpasture's disease. The peptides, P1–P23, are mostly 20mers overlapping by 10, and span the sequence of {alpha}3(IV)NC1. Markers indicate the means of three measurements of IC50s expressed as means and error bars depict the 95% confidence limits. Large markers identify peptides overlapping naturally processed and presented peptides. Peptides for which no binding was detected are shown on the abscissa with error bars reaching to the detection limit of the assay, ~100 µM. For comparison with other studies, horizontal lines are shown dividing the peptides into groups with high (Ki < 100 nM), intermediate (100 nM > Ki < 10 µM) and low (Ki >10 µM) affinity as proposed in (3), using the relations shown in Methods.

 
The results showed that DR7 had generally higher affinity for {alpha}3(IV)NC1 peptides than DR15b. In order to compare affinity for individual peptides, the logarithms of the calculated Ki for binding to DR7 and DR15b were subtracted (Fig. 2Go, top panel). Only two peptides, P7 and P11, had higher affinity for DR15b than DR7, and for neither was the difference substantial (~4- and ~3-fold respectively). Of the three peptides known to be generated by natural processing of intact {alpha}3(IV)NC1 within human APC (from their detection bound to DR15b molecules of {alpha}3(IV)NC1-pulsed APC (18,19), one (P17) bound DR7 with 40-fold higher affinity than to DR15b and two had indistinguishable (P3b) or just worse (P7) affinity for DR7 compared with DR15b.



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Fig. 2. Comparison of affinity of {alpha}3(IV)NC1 peptides for DR15b and protective DR molecules. For each peptide, the relative affinity for DR15b and protective class II molecules [DR7 (top panel) and DR1 (bottom panel)] is shown as the ratio of the measured Ki for DR15b and DR1/7 on a logarithmic scale. Peptides that bound with higher affinity to DR15b are indicated by columns descending the horizontal lines marking equal affinity (Ki ratio of 1). Bars on the abscissa indicate peptides overlapping naturally processed and presented peptides.

 
The significance of the measured differences in DR7 and DR15b binding affinity for presentation to {alpha}3(IV)NC1 peptides to T cells must depend on how {alpha}3(IV)NC1 is processed within APC, and in particular on which {alpha}3(IV)NC1 peptides are made available for class II binding. So far there is little evidence of substantial HLA-associated polymorphism in the processing machinery so we considered the implications of the binding data for {alpha}3(IV)NC1 presentation to T cells assuming the major {alpha}3(IV)NC1 peptides we previously identified in eluates from DR15 molecules in {alpha}3(IV)NC1-pulsed DR15-homozygous APC are processed and available for class II binding in DR7 APC. The results suggest that, barring as yet unrecognized HLA-linked processing polymorphism, at least two and possibly all three of the major (biochemically detectable) naturally processed and presented {alpha}3(IV)NC1 peptides displayed bound to DR15b by DR15b-homozygous APC in disease-susceptible individuals, would also be presented bound to DR7 in disease-protected individuals.

Thus the highly disease-protective class II molecule, DR7, has high affinity for most {alpha}3(IV)NC1 peptides, both compared to HLA peptide interactions in general and compared with the affinity of DR15b for the same peptides. To see if this feature was common to different class II molecules with similar protective disease associations we went on to examine binding to HLA-DR1.

Binding of {alpha}3(IV)NC1 peptides to HLA-DR1
Like DR7, DR1 bound most of the peptides with high (12 of 24) or intermediate (nine of 24) affinity; only three peptides either did not bind or bound with unmeasurable affinity (Fig. 1Go, middle panel). Comparison of the affinity of individual peptides for DR1 and DR15b (Fig. 1Go, bottom panel) found that two peptides, P11 and P14, had convincingly higher affinity for DR15b than DR1 and only for P11 was the difference substantial (13-fold). Of the three naturally processed {alpha}3(IV)NC1 peptides, one (P3b) bound DR1 with 10-fold higher affinity than to DR15b, and two had indistinguishable affinity for DR15b and DR1. Therefore, as for DR7, all the major naturally processed and presented {alpha}3(IV)NC1 peptides displayed bound to DR15b by DR15b-homozygous APC in disease-susceptible individuals would also be expected to be presented bound to DR1 in disease-protected individuals.

It appeared that DR1 and DR7 had very similar peptide binding characteristics relative to DR15b, so we compared them with one another (Fig. 3Go). There were striking differences (>10-fold) for most (15 of 24) of the peptides, so DR1 and DR7 do not have a similar disease association simply because they are closely related class II molecules. Therefore the results confirmed that generally high affinity for {alpha}3(IV)NC1 peptides was a consistent feature of disease-protective class II molecules in Goodpasture's disease.



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Fig. 3. Comparison of affinity of {alpha}3(IV)NC1 peptides for the two disease-protective class II molecules, DR1 and DR7. For each peptide, the relative affinity for DR1 and DR7 is represented in the same way as Fig. 2Go.

 
Identification of core binding segments engaged by DR15, DR1 and DR7
Measurement of the class II-binding affinity of the {alpha}3(IV)NC1 peptides identified 20 amino acid segments (the length of most of the peptides) with high/low affinity for the DR molecules, but the data did not identify the portions of the peptides actually bound by the class II molecules. Knowledge of core binding sequences becomes very important when using peptide-binding data to consider how autoantigen-derived peptides might bind to different class II molecules in APC expressing several different HLA molecules or to identify possible T cell epitopes. We employed two techniques to gain an indication of the likely core binding sequences in the {alpha}3(IV)NC1 peptides known to be generated by processing within APC: (i) we compared the DR-binding affinities of peptides with overlapping sequences and (ii) we applied published peptide-binding motifs (Fig. 4Go).



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Fig. 4. Identification of core DR-binding sequences in naturally processed {alpha}3(IV)NC1 peptides. The figure is organized as three vertical panels each showing the sequences of one of the three sets of naturally DR15-presented {alpha}3(IV)NC1 peptides at the top (18), over graphs of relevant affinity and motif data for DR15b, DR1 and DR7 molecules. To display affinity data in a way that allows appreciation of how the sequences of the peptides overlap, peptides are depicted as series of open circles (representing individual amino acids) on an abscissa that is the sequence of {alpha}3(IV)NC1, drawn on an ordinate log Ki scale. For intuitive simplicity peptides with higher affinity (lower Ki) are drawn higher on the ordinate axis. Motif data is superimposed in the form of vertical bars marking on the abscissa the proposed P1 residue (that locates in pocket 1 of the class II molecules). The height of the bars indicates how well the proposed core sequences matches the motif. Motifs used are described in Methods. Likely core-binding sequences inferred from the data are identified by underlines in the abscissa labels.

 
Five peptides were available with sequences overlapping the first set of putative naturally presented peptides (shown at top of left panel of Fig. 4Go). They were P3a, P3b P4, P5 and a truncation analogue of P3b with the sequence GTVPLYSGFSFL. The affinity of the peptides for DR15b, DR1 and DR7 is shown as log(calculated Ki) in Fig. 4Go (left-hand panel) (Ki were calculated from measured IC50s). To appreciate how the sequences of the peptides overlap, each peptide is depicted by a series of open circles (representing individual amino acids) on an abscissa that is the sequence of {alpha}3(IV)NC1. Peptides drawn higher in the three graphs have higher affinity for the respective class II molecules. The analysis suggested that the core sequence by which the peptides bound to the class II molecules was VPLYSGFSFL (underlined on abscissa) for the DR15b molecule, and FSFLFVQGNQ for DR1 and DR7 molecules. The core DR15b-binding sequence of the synthetic peptides was entirely contained within the slightly longer common core sequence of the previously identified naturally DR15b-presented peptides from this region of {alpha}3(IV)NC1.

The second set of naturally presented {alpha}3(IV)NC1 peptides was overlapped by four synthetic peptides (middle panel of Fig. 4Go). The affinities for DR15b of the available synthetic peptides were very similar and most contained several DR15b motifs, so it was not possible to localize a single core-binding sequence. The sequences of the naturally presented peptides suggests FCNVNDVCNF to be the favored DR15 core-binding sequence. Two DR1 and three DR7 core-binding sequences were identified for these peptides.

The third set of naturally presented {alpha}3(IV)NC1 peptides was also overlapped by four synthetic peptides (middle panel of Fig. 4Go). The sequence LEEFRASPF was the highest DR15b affinity core-binding sequence in this segment of {alpha}3(IV)NC1. This sequence was shared by all of the five naturally presented peptides. One of the synthetic peptides (P18 with the sequence beginning EFRASP...) did not contain this sequence but bound with only 10-fold lower affinity, so it must contain a DR15b-binding, core-binding sequence not identified by the DR15b motif. P18 also bound with very high affinity to DR1 and good affinity to DR7, but as for DR15b, the available motifs did not identify a likely core binding sequences. Candidate sequences that supply a hydrophobic amino acid in pocket 1, a major determinant of binding to DR1 and DR7, are FRASPFLECH, FLECHGRGT and LECHGRGTC. The data suggested two other DR1 and DR7 core-binding sequences in the region of {alpha}3(IV)NC1.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The striking observation is that class II molecules associated with protection from Goodpasture's disease have higher affinity for most {alpha}3(IV)NC1 peptides, including those known to be naturally processed in human B cells. The results have important implications for the mechanism by which class II alleles influence susceptibility to Goodpasture's disease and in particular the mechanism of HLA-associated dominant protection in this disease.

Mechanism by which HLA class II alleles increase susceptibility to Goodpasture's disease
The data suggest that DR15b cannot be associated with increased susceptibility to Goodpasture's disease because it has high affinity for particular {alpha}3(IV)NC1 peptides. With the exception of peptide 11, all of the Goodpasture antigen peptides studied bound as well or better to DR1 or DR7, which have the opposite association with the disease. Indeed, the data predict that individuals carrying DR1 or DR7 would present almost any {alpha}3(IV)NC1 peptide that could be generated by processing just as efficiently as individuals carrying DR15, including all those known to be generated by {alpha}3(IV)NC1 processing in human B cells.

This observation is unlikely to be an artifact of the representation of {alpha}3(IV)NC1 by a set of synthetic peptides because the panel of peptides spanned the entire sequence of {alpha}3(IV)NC1 and adjacent peptides overlapped by at least 10 amino acids, the length of the class II peptide-binding groove, thereby including all the possible class II core-binding sequences in {alpha}3(IV)NC1 at least once. One peptide, P11, did have higher affinity for DR15b than for DR1 or DR7, but at least two factors are strongly against a role for this peptide in the autoimmune responses to {alpha}3(IV)NC1. (i) P11 binds better to DR1 than to DR7 and better to DR7 than to DR15, whereas the order of association from negative to positive is DR7, DR1 and DR15. (ii) P11 is not biochemically detectable bound to DR15 molecules purified from {alpha}3(IV)NC1-pulsed DR15-homozygous APC, whereas peptides with much lower DR15 affinity are. We have recently used murine T cell clones specific for P11 to interrogate peptide mixtures eluted from DR15 molecules from {alpha}3(IV)NC1-pulsed APC with much higher sensitivity (10-fold at least) and still not detected P11 peptides. Very likely P11 is not generated or rapidly destroyed during {alpha}3(IV)NC1 processing in human B cells. Thus efficient presentation of {alpha}3(IV)NC1 peptides by the DR15 molecule cannot alone account for the association of the molecule with increased susceptibility to Goodpasture's disease.

The finding is in contrast to immune responses to exogenous antigens, where efficient presentation appears to engender stronger immune responses (3,31,32), but compatible with more complex models for the relationship between presentation of autoantigen-derived peptides and propensity for autoimmunity (33,34). For example, a peptide such as P11 that has very high affinity for DR15b, but is not usually presented because of processing constraints, could be presented under conditions that induce autoimmune disease (a cryptic antigen). Alternatively, the data identified seven {alpha}3(IV)NC1 peptides (P2, P3a, P5, P13, P16, P21 and P23) that bound to DR15b with low affinity (Ki > 1x10–5 M) at best, but this could still be high enough to support autoimmune responses comparable to those reported for an encephalolitogenic peptide in the generation of experimental autoimmune encephalomyelitis (34). However, we do not favor these peptides as candidate disease-associated peptides in Goodpasture's disease because it is difficult to reconcile the strong association of Goodpasture's disease with DRB1*1501/2, but not other related DR2 alleles, if the defining feature of disease-associated class II molecules is just low-affinity binding of {alpha}3(IV)NC1 peptides. Three of the seven low DR15b-binding peptides did not bind detectably to DR1. Thus it seems likely that a wider study of the many class II molecules with neutral disease associations would identify numerous other class II molecules that like DR15b were unable to present some or all of these peptides. Moreover, central tolerance to {alpha}3(IV)NC1 is far from complete as the peripheral blood T cells of healthy individuals proliferate when incubated with purified native {alpha}3(IV)NC1 (35), recombinant {alpha}3(IV)NC1 (36) and {alpha}3(IV)NC1 peptides (our unpublished observations).

Mechanism of dominant protection
Dominant protection has been reported for many autoimmune diseases including diabetes and rheumatoid arthritis (37,38) as well as Goodpasture's disease. Three major mechanisms are supported by experimental results; two suggest protective class II molecules influence the T cell repertoire to autoantigens, either by driving central deletion or promoting peripheral tolerance of some form; the third proposes that protective class II molecules capture autoantigen-derived peptides preventing their display bound to class II molecules that restrict pathogenic T cells. High affinity for {alpha}3(IV)NC1 peptides was a feature of both DR molecules associated with dominant protection from Goodpasture's disease. So whilst not directly informing about the mechanism by which DR7 and DR1 protect from Goodpasture's disease, the results are in striking accord with epitope capture as a mechanism for dominant protection in this disease.

The epitope capture hypothesis proposes that the combination of class II molecules in the peptide-loading compartments of APC influences the peptides that become bound to each. The concept is illustrated in Fig. 5Go. Dominant protection is accounted for by proposing that the peptides recognized bound to disease-associated class II molecules by autoreactive T cells are displayed instead bound to protective class II molecules in APC expressing both types of class II molecule. For autoimmunity to be averted it would also be necessary that tolerance be securely established to the autoantigen-derived peptides presented on the disease-protective class II molecules, as might be predicted for efficiently presented self-peptides. The mechanism was first proposed by Werdelin (39) and suggested by Nepom et al. (40) to explain the protective effective of DR2 in diabetes mellitus. Powerful evidence that epitope capture can influence immune responses has been obtained in NOD/BALB/c F1 mice (12), and the process has been demonstrated biochemically (41). Although it has been shown that a class II molecule associated with protection from diabetes binds with high affinity and presents a GAD65 peptide that overlaps an epitope suggested to be recognized by patients' autoreactive T cells, consistent with the hypothesis (42), direct support for the mechanisms in insulin-dependent diabetes mellitus is unlikely to be forthcoming until the targets of autoimmune attack have been identified with certainty.



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Fig. 5. Competition between class II molecules for binding antigen-derived peptides. The figure depicts the compartment within APC where class II molecules bind antigen-derived peptides. Human APC express three to eight or more class II molecules (composed of products of DRA/DRB1, DRA/DRB3-5, DQA/DQB, DPA/DPB loci on maternal and paternal chromosomes plus possible trans combinations of DQ products). Antigen-derived peptides (dark strings of beads) are scarce relative to peptides from the cells own cell surface and endosomal proteins (light strings of beads). Partitioning of antigen-derived peptide between available class II molecules is hypothesized to depend on the abundance of different class II molecules and their affinity for antigen-derived peptides. Epitope capture occurs when a class II molecule with high affinity for a particular peptide sequesters sufficient peptide such that other class II molecules present negligible quantities of that peptide.

 
In Goodpasture's disease, epitope capture would be favored if DR1/7 bound core sequences in {alpha}3(IV)NC1 close to or overlapping those bound by DR15b, such that steric hindrance would force a competition in which the class II molecule with higher affinity would be favored. The location of core-binding sequences in the sequence of {alpha}3(IV)NC1 and their relative affinity for DR1, DR15b and DR7 are shown for parts of {alpha}3(IV)NC1 from which naturally presented peptides are processed in Fig. 6Go. Presentation to DR15b-restricted T cells of all the known naturally processed {alpha}3(IV)NC1 segments could be influenced by epitope capture.



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Fig. 6. Competition between DR15b and protective class II molecules for presentation of a putative disease-related {alpha}3(IV)NC1 peptide. The sequence of {alpha}3(IV)NC1 in the vicinity of the three sets of naturally DR15-presented peptides is shown under rounded squares depicting by their width the core sequence bound by the indicated class II molecules and by their height the relative binding affinity on a logarithmic scale (relative affinities are also shown numerically in brackets). Depending on the length of peptides generated by processing, all of the naturally processed peptides could be captured by the protective class II molecules DR1 and DR7 because of their higher binding affinity.

 
The other two hypotheses that explain dominant protection propose class II-driven differences in the T cell repertoire (central tolerance) or level of peripheral tolerance. Both have been demonstrated in NOD mice with skewed T cell repertoires due to transgenes encoding I-Ag7-restricted diabetogenic TCR in which `diabetes-protective' MHC class II transgenes have additionally been introduced. Schmidt et al. showed that expression of protective class II molecules resulted in disappearance from the peripheral repertoire of the diabetogenic T cell and suggested the mechanism was negative deletion on the protective class II molecule in the thymus (11). Evaluation of this hypothesis outside the refined setting of a multiple transgenic NOD mouse is required. It may be less plausible in Goodpasture's disease as there is very little structural similarity between DR7 and DR15 molecules to support the notion of T cell cross-recognition, and DR7 is not associated with protection in other autoimmune disease associated with DR2, such as multiple sclerosis and ulcerative colitis (43). Influences on peripheral tolerance were demonstrated by Luhder et al. who showed that particular class II molecules protected from diabetes by stimulating the development of populations of T cells that regulated diabetogenic T cells (13).

In conclusion, we have measured the affinity of class II molecules divergently associated with Goodpasture's disease for peptides representing the entire sequence of {alpha}3(IV)NC1 and deduced ways in which the class II molecules could influence presentation of {alpha}3(IV)NC1 to CD4 T cells, both when expressed in isolation and in combinations associated with different degrees of susceptibility to Goodpasture's disease. Our data have enabled us to raise a hypothesis for the mechanism by which class II molecules influence susceptibility to Goodpasture's disease. It will be possible to test this hypothesis when the specificities of patients' autoreactive T cells are determined.


    Acknowledgments
 
Work supported by grants from the National Kidney Research Foundation (UK) and the Medical Research Council.


    Abbreviations
 
{alpha}3(IV)NC1 NC1 domain of the {alpha}3 chain of type (IV) collagen
APC antigen-presenting cell
DR15a and DRB15b are used for class II molecules composed of the gene products of DRA with DRB5*0101 and DRA with DRB1*1501 respectively. The nomenclature echoes the widely used terms DR2a and DR2b but denotes the DR15 subspecificity of DR2

    Notes
 
Transmitting editor: L. Simpson

Received 7 December 1999, accepted 13 April 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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