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Major Differences in Antigen-Processing Correlate with a Single Arg⁷¹Lys Substitution in HLA-DR Molecules Predisposing to Rheumatoid Arthritis and with Their Selective Interactions with 70-kDa Heat Shock Protein Chaperones¹

Sabine Roth^*, Nicholas Willcox, Rita Rzepka^*, Matthias P. Mayer and Inga Melchers^2,*

^* Clinical Research Unit for Rheumatology and Institute of Biochemistry and Molecular Biology, Albert Ludwigs University, Freiburg, Germany; and Neurosciences Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom

	Abstract

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Several HLA-DR alleles are genetically associated with rheumatoid arthritis. DRB1*0401 predominates in Northern Europe and has a characteristic ⁷⁰QKRAA motif. This sequence contacts bound peptides and the TCR. Further interactions have been suggested with additional proteins during Ag loading. We explored the much stronger processing/presentation of full-length recombinant human acetylcholine receptor subunit to a specific T cell clone by APC from DRB1*0401⁺ than *0408⁺ donors. Using DR*04 transfectants, we show that this difference results largely from the single Lys⁷¹Arg interchange (04010408), which scarcely affects epitope binding, rather than from any other associated polymorphism. Furthermore, we proved our recombinant polypeptides to contain the Escherichia coli 70-kDa heat shock protein molecule DnaK and its requirement for efficient processing and presentation of the epitope by DRB1*0401⁺ cells. According to a recent report, 70-kDa heat shock protein chaperones preferentially bind to the QKRAA, rather than the QRRAA, motif. Variations between the shared epitope motifs QKRAA and QRRAA are emphasized by underlining. We propose that such interactions enhance the intracellular epitope loading of *0401 molecules. They may thus broaden immune responses to pathogens and at least partially explain the distinct contributions of DRB1*0401 and other alleles to disease predisposition.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Immune responses to proteins require processing by APCs and the subsequent presentation of peptides to specific T cells by class I or class II molecules of the MHC, the HLA complex in Homo sapiens (1). Most autoimmune diseases show HLA associations, implying a key role for autoepitope presentation (2). Rheumatoid arthritis (RA)³ is an unusual and particularly interesting example, as it is genetically associated with the shared epitope (3), a motif of five amino acids at positions 70–74 in the middle of the helix of the HLA-DR -chain (QKRAA, QRRAA, and RRRAA). DRB1*0401 with QKRAA is a common allele, especially in the Northern European population (4, 5). QRRAA occurs in subtypes of several DR serotypes, e. g., DR1 (DRB1*0101), DR4 (DRB1*0404, DRB1*0405, DRB1*0408), and DR6 (DRB1*1402); RRRAA occurs in DR10 (DRB1*1001). Shared epitope-positive DR molecules may differ in detail functionally, because of other sequence differences elsewhere in the peptide binding groove (6).

The QKRAA motif has also been detected in microbial molecules, notably chaperones related to the Escherichia coli DnaJ protein (7, 8). The many related DnaJ proteins, defined by a common J domain of 70–80 aa (9), cooperate with members of the 70-kDa heat shock protein (Hsp70) family of chaperones in generating a machinery involved in protein folding and transport (10). Many, but not all, DnaJ proteins contain a QKRAA motif within the J domain that is essential for interaction with Hsp70 proteins (11). Alterations in this motif (QKAAA, QARAA, QAAAA) in the E. coli DnaJ lower its affinity for the E. coli Hsp70 homolog DnaK (12). Variations between the shared epitope motifs are emphasized by underlining. In addition, a peptide containing this motif competes with DnaJ for binding to DnaK (13). Auger et al. (14, 15) reported that the QKRAA motif also mediates binding between QKRAA-positive DR -chains and certain Hsp70 molecules, e.g., DnaK and the mammalian constitutive Hsc70, but the specificity of this binding was subsequently questioned (16).

The CD4⁺ T cell clone PM-A derives from the thymus of a patient with myasthenia gravis (DR3, DRB1*0408). It was raised against recombinant human acetylcholine receptor (AChR) subunit and recognizes a natural epitope in the 144–163 region of this Ag presented by DR4 molecules with Gly⁸⁶, rather than Val⁸⁶ (17, 18). In contrast with most other DR4-restricted T cells, which absolutely require the autologous Lys⁷¹ or Arg⁷¹, PM-A T cells recognize short peptides very well on both the autologous 0408 (Arg⁷¹) and the heterologous 0401 (Lys⁷¹), both of which bind these peptides well (6, 19). Surprisingly, however, 0401⁺ PBMC present longer polypeptides and whole AChR much better than those with the autologous 0408 (20).

Using the murine macrophage-like cell line P388.D1 transfected with HLA-DR - and -chains (17), we show here that 1) APCs differing only by Lys⁷¹Arg (QKRAAQRRAA) handle the same autoantigen preparation very differently; and 2) the efficiency of QKRAA-positive murine and human APCs is enhanced by DnaK, the E. coli Hsp70 chaperone, in the Ag preparation. These data imply that Hsp70 chaperones selectively facilitate the peptide loading of certain MHC class II molecules.

	Materials and Methods

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Ag-presenting cells

The mouse macrophage-like cell line P388.D1 was transfected to express human HLA-DRA*0101 and DRB1*0401 (Lys⁷¹) or *0408 (Arg⁷¹) genes (17). The latter was derived by site-directed mutagenesis from the 0404 sequence (Arg⁷¹) by changing its Val⁸⁶Gly⁸⁶. The published sequence of DRB1*0408 (aa 36–123) is identical with that of 0401 apart from this Lys⁷¹Arg⁷¹ interchange (21). TP cells were grown as previously described (17). Lines were cloned when necessary and were selected for similar DR expression, which was checked routinely before and after each experiment. For Ag presentation assays they were untreated, fixed with glutaraldehyde (0.05%, 30 s, 21°C), or treated with mitomycin C (50 µg/ml, 45 min, 37°C; Roche, Indianapolis, IN). Human PBMC were T cell-depleted by rosetting with SRBC and irradiated (30 Gy). In some experiments TP cells were pretreated with murine IFN- (100 U/ml, 48 h; BD PharMingen, Heidelberg, Germany).

T cells

The generation, maintenance. and specificity of the human CD4⁺ T cell clone PM-A and the murine T cell hybridoma 3DO were previously described (6, 18, 19, 20, 22).

Antigens

The recombinant polypeptide r1–437 was expressed in E. coli. It was purified as previously described (23). The synthetic peptide p144–163 was used at 10 µg/ml. OVA and superantigen (staphylococcal enterotoxin B (SEB)) were purchased from Sigma-Aldrich (St. Louis, MO).

Presentation assays

Fixed TP cells were pulsed with p144–163 for 150 min at 37°C, washed, and plated at 3 x 10⁴ cells/culture in flat-bottom microculture plates (Nunc, Wiesbaden, Germany) together with the same number of PM-A T cells in RPMI complete medium (17) supplemented with 5% human A⁺ serum. Viable TP cells were used at concentrations ranging from 2.5 x 10³ to 1 x 10⁵ cells/culture and were incubated with 5 x 10⁴ PM-A cells/culture and 1.7 µg/ml r1–437. Alternatively, the Ag r1–437 was titrated from 1.6–10,000 ng/ml in the presence of 5 x 10⁴ TP cells. For stimulation of the OVA-specific 3DO T cells, TP cells were used untreated or were induced by IFN-, treated with mitomycin C, and plated at 1 x 10⁵ cells/culture together with 3DO T cells (5 x 10⁴ cells/culture) and SEB (1 µg/ml) or OVA (titrated from 1 mg/ml to 30 µg/ml). Supernatants were taken after 2 days or 24 h (3DO experiments only). All cultures were set up at least in triplicate.

T cell response assays

T cell cytokines (human IFN- or TNF-, or murine IL-2) released into culture supernatants were quantified by ELISAs (BD PharMingen). Proliferation was assayed by [³H]thymidine incorporation. Briefly, 1 µCi [³H]thymidine (sp. act., 185 GBq/mmol; Amersham Life Science, Little Chalfont, U.K.) was added after 72 h; plates were harvested 18 h later (1295-001 Cell Harvester; Wallac, Turku, Finland). The incorporated radioactivity was assessed on a Betaplate counter (Wallac).

DnaK depletion of the r1–437 preparation

DnaK was removed from r1–437 preparations by affinity adsorption to ATP-agarose according to the published protocol (24, 25) with minor modifications. Briefly, ATP-agarose (1 ml; A2767; Sigma-Aldrich) was pretreated (25 mM HEPES/KOH (pH 7.6), 50 mM KCl, 5 mM MgCl₂, 10 mM 2-ME, and 1 mM EDTA) and loaded with the r1–437 preparation (1 ml). After incubation for 30 min at 4°C on an end-over-end mixer, the ATP-agarose was pelleted. The supernatant contained the r1–437 devoid of DnaK. The protein concentration of r1–437 preparations before and after depletion of DnaK was determined with the bicinchoninic acid protein assay reagent (Pierce, Augustin, Germany). In both preparations one identical band at 48 kDa was detected with SDS-PAGE, followed by staining with Coomassie brilliant blue. In addition there were occasionally one (or two) faint band(s) in the original preparations of the size of the DnaK monomer (and dimer), which were no longer detected in the DnaK-depleted preparations (not shown). Ag preparations were stored at -80°C and were used within 2 wk after depletion in functional experiments.

Flow cytometry

HLA-DR-specific mAbs (IOT2a, clone B8.12.2; Dianova, Hamburg, Germany) and Ia-specific mAbs (anti-I-A^d, clone AMS-32.1; BD PharMingen) were used, labeled with FITC or biotin. FITC-conjugated streptavidin was bought from Dianova.

Immunoblots

Various preparations of r1–437 (identical protein concentration of 550 µg/ml) as well as DnaK (0.1 µg/ml) were separated on a 10% SDS-PAGE gel, followed by transfer onto nitrocellulose membrane and immunoblotting with the anti-DnaK mAb (SPA-880, clone 8E2; StressGen, Toronto, Canada) or a rabbit antiserum specific for aa 3–181 of the AChR subunit and HRP-conjugated goat anti-mouse IgG or anti-rabbit IgG (DAKO, Hamburg, Germany), using enhanced chemiluminescence (Pierce) as detailed in the manufacturer’s protocol. The amount of DnaK in the Ag preparation of r1–437 was estimated to be 0.075 µg/ml (before depletion).

Immunoprecipitation

A total of 1 ml of a preparation of r1–437 was incubated with 5 µg/ml anti-DnaK mAb at 4°C for 2 h. Protein A-Sepharose (Pharmacia, Freiburg, Germany) was added (20 µl, 10% in 50 mM Tris (pH 8.0), 150 mM NaCl, 0.1% Nonidet P-40, and 100 µg/ml PMSF) and again incubated at 4°C for 2 h. The pellet was washed extensively with 50 mM Tris (pH 8.0), 500 mM NaCl, and 0.1% Nonidet P-40; resuspended in 10 mM Tris, (pH 8.0); and analyzed by SDS-PAGE and immunoblots. Artificial mixtures of DnaK and the DnaK-depleted preparation of r1–437 were analyzed in the same way.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Comparability of the transfectants TP0401 and TP0408

To study the influence of HLA-DR variants on Ag processing and presentation, we compared mouse macrophage-like P388.D1 cells differing only by a single Lys⁷¹Arg exchange in their transfected DR4 molecules (17). We first established that the two cell lines, TP0401 and TP0408, were quantitatively comparable in these functions: 1) HLA-DR expression (Fig. 1A), 2) costimulatory capacity for T cells in the presence of appropriate superantigens (e.g., SEB; Fig. 1, B and C), and 3) processing and presentation of a 40-kDa protein Ag to a murine T cell hybridoma, 3DO cells, recognizing the chicken OVA-derived peptide p323–339 presented by the murine class II molecule I-A^d. TP cells show no constitutive murine class II expression, but it can be induced by murine IFN-. The levels of both I-A^d and DR molecules on TP0401 and TP0408 were comparable before and after IFN- induction (not shown), as were both the SEB-induced stimulation and the OVA-specific response of the hybridoma (Fig. 1C). In addition, TP cell clones differing in their quantitative expression of HLA-DR were tested for their costimulatory capacities (as in no. 2 above). These parameters correlated strongly (r² > 0.95; three experiments with 11 clones); thus, the SEB response of 3DO cells serves as an additional internal standard in functional experiments (e.g., Fig. 1C). Additional experiments proved that TP0401 and TP0408 can both activate human peripheral blood T lymphocytes (not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Phenotype and function of TP0401 and TP0408 cells. A, Expression of HLA-DR on the surface of TP0401 and TP0408 cells (dotted lines) and P388. D1 cells (solid line) analyzed by flow cytometry using FITC-labeled anti-DR Abs. B, Presentation of a superantigen (SEB) to the murine T cell hybridoma 3DO.P388.D1. TP0401 and TP0408 cells were treated with mitomycin C, seeded at various concentrations into microwells, and incubated together with 8 x 104 T cells/culture and 100 ng/ml SEB. Supernatants were harvested 24 h later and assayed for murine IL-2. C, Presentation of the Ag OVA to 3DO cells specific for OVA p323–339 presented by I-Ad. TP0401 and TP0408 cells were preincubated with murine IFN- (100 U/ml) for 2 days, then cocultured with 3DO T cells (5 x 104/culture) and OVA (1000–60 µg/ml) or SEB (1 µg/ml). Murine IL-2 was quantitated in tissue culture supernatants harvested 24 h later, and the OVA-specific response was expressed as a percentage of that to SEB.

As expected, fixed TP0401 and TP0408 cells both presented the synthetic peptide p144–163 and stimulated the T cell clone PM-A to produce human IFN- (Table I), although the efficiency of these murine cells was somewhat lower than of human PBMC (not shown). TP0408 cells, carrying the autologous DR allele of the PM-A T cells, were slightly more efficient than TP0401 cells, as we have also observed previously with human PBMC (19, 20). TP cells also stimulated PM-A T cells to produce human TNF- (Table I) and IL-3/GM-CSF (17).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Presentation of the recombinant AChR polypeptide r1–437 to PM-A T cells by TP0401 (Lys71) or TP0408 (Arg71) cells. Viable TP0401 and TP0408 cells were cocultured with the T cell clone PM-A and r1–437. Human IFN- was quantified by ELISA in culture supernatants harvested after 2 days. Untransfected P388.D1 cells did not induce detectable IFN- (not shown). A, Titration of TP cells with a constant concentration of r1–437 (1.7 µg/ml) and PM-A T cells (5 x 104/culture). B, Titration of r1–437 with a constant concentration of APC and PM-A T cells (both 5 x 104/culture). A representative experiment of more than five experiments is shown.

Since this difference between TP0401 and TP0408 affects processing of longer polypeptides (Fig. 2) rather than presentation of short peptides (Table I), it must reflect interactions with other molecules participating in the processing pathway. Therefore, we next tested the Ag preparations used above for the presence of chaperones that discriminate between Lys⁷¹ and Arg⁷¹ (14). By immunoblotting with a specific Ab we detected the E. coli DnaK protein in the preparation of r1–437 (Fig. 3, lanes 1–4). DnaK was also detected in other batches of r1–437 tested in the same way. DnaK and r1–437 bound to each other, as demonstrated by coprecipitation experiments (Fig. 3, lane 5). ATP affinity chromatography (24, 25) was used to deplete the r1–437 preparation specifically of DnaK, which binds quantitatively to ATP-agarose columns simultaneously releasing its bound peptides (Fig. 3, lanes 6–8). Indeed, the removal of DnaK neither depleted the Ag r1–437 (as the protein concentration did not change) nor altered it grossly, as it gave the same band at 48 kDa in Coomassie-stained SDS-PAGE (not shown) and could still be detected with anti-AChR in immunoblots (Fig. 3, lanes 9 and 10). In the original preparation (lane 9) the antiserum detected some small antigenic proteins missing in the DnaK-depleted preparation (lane 10). This suggests either 1) partial depletion of these along with DnaK, 2) instability of such breakdown products in the absence of DnaK, or 3) a minor loading inequality of the lanes. Subtle changes in Ag conformation or stability directly due to the removal of DnaK might be expected and may be part of the mechanism through which Hsp70 molecules enhance the antigenicity of the Ag.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Preparations of r1–437 were analyzed by SDS-PAGE and immunoblotting. a–d, Samples applied to SDS-PAGE; e and f, Abs used for immunoblotting. Lanes 1–4, Anti-DnaK detected purified E. coli DnaK (0.02 µg/lane; lane 2) and DnaK in a preparation of r1–437 (lane 3). Lanes 2 and 3, Anti-DnaK Abs and goat anti-mouse IgG (e+); lanes 1 and 4, only anti-mouse IgG (e-); lane 5, coprecipitation of DnaK and r1–437. Anti-DnaK was used for precipitation. The precipitate was analyzed with an antiserum specific for the recombinant AChR subunit. Lanes 6–10, Depletion of DnaK from a preparation of r1–437 before (lanes 6 and 9) and after depletion of DnaK (lanes 7 and 10) by affinity chromatography on ATP-agarose. The same amount of protein was applied in lanes 6, 7, 9, and 10 (20 µg/lane).

After depletion of DnaK, r1–437 evoked substantially lower IFN- responses by T cells, with either TP0401 cells or human DRB1*0401⁺ PBMC as APC (Fig. 4, A–C), even though the same amount of Ag still was available for processing. Moreover, its presentation was consistently reconstituted by adding purified E. coli DnaK at certain concentrations, with an optimum at levels similar to those detected in the original, undepleted, preparation (Fig. 4, C–G). The same effects were observed when we measured proliferative responses of the PM-A T cell clone using PBMC as APC (Fig. 5). IFN- production and proliferation, assayed in the same experiment, also correlated well. Presentation by DRB1*0408⁺ human PBMC (Fig. 5) and TP0408 cells (not shown) was scarcely influenced by these treatments. In additional controls, DnaK alone clearly did not stimulate the PM-A cells (Figs. 4 and 5) (26), nor did it alter the SEB response or the Ag-specific response of 3DO cells (Fig. 6).

View larger version (42K): [in this window] [in a new window]   FIGURE 4. Effects of DnaK on the presentation of r1–437 by DRB1*0401-positive cells. APCs were irradiated PBMC prepared from an HLA-DRB1*0401+ donor () or TP0401 (). APCs (5 x 104 cells/culture) were incubated with PM-A T cells (5 x 104 cells/culture) and the additions indicated. Supernatants were harvested 48 h later and assayed for human IFN-. The responses were normalized to the levels of IFN- obtained in group B. The results shown are from one experiment representative of three experiments performed.

View larger version (24K): [in this window] [in a new window]   FIGURE 5. The presence of DnaK preferentially enhances the presentation of DRB1*0401-positive cells. APCs were irradiated PBMC prepared from HLA-DRB1*0401-positive () or DRB1*0408-positive () donors. APCs (5 x 104 cells/culture) were incubated with PM-A cells (5 x 104 cells/culture) and the additions indicated. The T cell response was measured by [3H]thymidine incorporation. The results shown are from one experiment representative of three experiments performed

View larger version (27K): [in this window] [in a new window]   FIGURE 6. DnaK does not interfere with the response of 3DO cells to a superantigen or its specific Ag. The experiment was performed as described in Fig. 1C. In addition, all groups were cultured either in the absence () or in the presence of DnaK (500 ng/culture; ).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Successful presentation of an Ag by nascent MHC class II molecules usually requires its uptake and transport into the endosomal compartment and its enzymatic degradation there. With the help of HLA-DM and HLA-DO (H2-M and H2-O in the mouse), the processed epitope replaces the class II-associated invariant chain peptide in the class II binding groove. In TP cells autologous murine class II molecules are usually only present at low levels, if at all. If the same applies to the invariant chain, H2-M and H2-O, then it seems unlikely that they participate in Ag presentation by transfected HLA-DR molecules. However, even after induction with IFN-, the difference between TP0401 and TP0408 was still as great as that without induction (not shown), again arguing that it did not depend on these molecules.

The recombinant AChR polypeptide r1–437 was purified from E. coli inclusion bodies. It is therefore not surprising that it was contaminated with bacterial DnaK. We have already shown the presence of E. coli Hsp60 in some preparations of r1–437, recognized by another specific T cell clone (26). Since r1–437 also includes the four transmembrane segments, which are rich in hydrophobic residues, it has a high number of potential DnaK binding sites (27). Furthermore, we have detected the human Hsc70 molecule in preparations of purified native human AChR (not shown) that are also processed/presented much better to PM-A by 0401⁺ than 0408⁺ APC (20).

Our results strongly suggest that APC take up r1–437, much of it complexed with DnaK (Fig. 3). Such complexes reformed rapidly when DnaK was added back to DnaK-depleted r1–437 (not shown). Hsp70 molecules may protect antigenic epitopes from destruction, favor suitable proteolytic trimming, and/or greatly enhance presentation to T cells (14, 28, 29). In our experiments the E. coli Hsp70 homolog DnaK might have performed these proposed functions instead of, or in combination with, endogenous Hsp70 molecules such as Hsc70 in the APC. However, in the processing of r1–437 there must, in addition, be a selective interaction directly between DnaK and 0401; by contrast, the presentation of short synthetic peptides to this T cell actually appears better with 0408 (Table I) (19). Probably, therefore, DnaK and 0401 interact in endosomal compartments during loading of the epitope into the DR peptide binding groove. Indeed, DnaK can evidently bind both r1–437 and 0401, almost certainly in separate sites. Alternatively, although less likely, binding of DnaK to 0401 alone might provide danger signals (30) and/or enhance peptide presentation to T cells independent of any effect on intracellular processing/presentation of whole proteins. Whether endogenous Hsc70 indeed performs the same function in vitro or in vivo needs to be tested in additional experiments.

TP0401 cells share the sequence QKRAA with DnaJ, the interaction partner of DnaK in E. coli. In DnaJ, the QKRAA motif is part of an helix as in the DR -chain. Recently, mutations changing the DnaJ ⁶¹QKRAA into ⁶¹QARAA, ⁶¹QKAAA, or ⁶¹QAAAA clearly reduced the affinity between DnaK and DnaJ. This reduction was more pronounced with the Lys⁶² to Ala than with the Arg⁶³ to Ala exchange; both replacements together reduced the interaction even further (12). Interestingly, most T cell clones (at least 12 of the 16 cited in Ref. 6) differentiate absolutely between Lys⁷¹ and Arg⁷¹ in their DR restriction profile; PM-A T cells are highly unusual in tolerating both of them so well. Although considered a conservative difference, the Arg side chain is bulkier and can make several more hydrogen bonds than Lys (31), and it is not surprising that DnaK should distinguish between the two residues. Taken together, these data suggest that the interaction between DnaK and 0401 occurs via the sequence ⁷⁰QKRAA, and that Arg⁷¹ cannot substitute for Lys⁷¹. This interpretation is strongly supported by the data reported by Auger et al. (14), showing binding by DnaK of QKRAA-containing, but not QRRAA-containing, peptides.

After DnaK-depletion of r1–437, there still remained some difference between 0401⁺ and 0408⁺ APC, at least in some experiments (Fig. 5C). This may be due to incomplete DnaK depletion of the Ag or to interactions of r1–437 with the endogenous Hsc70 of the APC. We cannot formally exclude additional Hsp70-independent differences between human 0401⁺ and 0408⁺ APC.

In our experiments DnaK alone had minimal effects on superantigen- or Ag-specific T cell activation ( Figs. 4–6). Its enhancing activity was restricted to the processed AChR epitope recognized by PM-A. Therefore, its abilities to induce cytokines in monocytes (32) or to function as nominal Ag for B and T cell responses (Ref. 33 and our own unpublished observations) were distinct from its chaperoning function in preferential loading of the AChR epitope in 0401⁺ APC.

Both the QKRAA and the QRRAA motifs are strongly associated with RA, but apparently via distinct pathways, since compound heterozygotes, especially 0401/0404, are more susceptible than patients with either allele alone (34). Hence, while the interactions with Hsp70 molecules might not be essential for predisposition, their consequences for the repertoire of epitopes and/or the sensitivity of their recognition may influence the severity or onset of RA. Indeed, Hsp70 proteins and J proteins are also induced in synovial tissue of patients with RA, especially in synovial lining cells belonging to the fibroblast and macrophage lineages (35, 36, 37). Interestingly, too, there are signs of analogous differences in processing within other HLA-DR serotypes (38). In general, these may broaden the repertoire of potential T cell responses to pathogens as well as self Ags.

	Acknowledgments

 
We thank Arne von Bonin (Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany) for the 3DO T cell hybridoma.

	Footnotes

 
¹ This work was supported by grants from the German Academic Exchange Service/British Council (British-German Academic Research Collaboration, to I.M. and N.W.), the Medical Research Council of Great Britain (to N.W.), and the Deutsche Forschungsgemeinschaft (SFB 388 to M.M.; Me 604 to I.M.).

² Address correspondence and reprint requests to Dr. Inga Melchers, Klinische Forschergruppe für Rheumatologie, Universitätsklinikum, Breisacher Strasse 64, 79106 Freiburg, Germany. E-mail address: melchers{at}nz11.ukl.uni-freiburg.de

³ Abbreviations used in this paper: RA, rheumatoid arthritis; AChR, acetylcholine receptor; Hsp70, 70-kDa heat shock protein; SEB, staphylococcal enterotoxin B; TP cell, transfected P388.D1 cell; TP0401 and TP0408, P388.D1 expressing HLA-DRB1*0401 and DRB1*0408, respectively.

Received for publication January 29, 2002. Accepted for publication July 12, 2002.

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