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The Journal of Immunology, 2000, 164: 1529-1537.
Copyright © 2000 by The American Association of Immunologists

Multispecific CD4+ T Cell Response to a Single 12-mer Epitope of the Immunodominant Heat-Shock Protein 60 of Yersinia enterocolitica in Yersinia-Triggered Reactive Arthritis: Overlap with the B27-Restricted CD8 Epitope, Functional Properties, and Epitope Presentation by Multiple DR Alleles1

Andreas K. H. Mertz*,{dagger}, Peihua Wu{dagger},{ddagger}, Tiziana Sturniolo§, Dieter Stoll, Martin Rudwaleit{dagger}, Roland Lauster{ddagger}, Jürgen Braun{dagger} and Joachim Sieper2,{dagger},{ddagger}

* Department of Medicine II, University Hospital, Ulm, Germany; {dagger} Free University of Berlin, Berlin, Germany; {ddagger} Department of Medicine IV, Deutsches Rheumaforschungszentrum, Berlin, Germany; § Milan Roche Riserche, Milan, Italy; and Naturwissenschaftliches und Medizinisches Institut, University of Tübingen, Tübingen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Yersinia heat-shock protein 60 (Ye-hsp60) has recently been found to be a dominant CD4 and CD8 T cell Ag in Yersinia-triggered reactive arthritis. The nature of this response with respect to the epitopes recognized and functional characteristics of the T cells is largely unknown. CD4+ T cell clones specific for Ye-hsp60 were raised from synovial fluid mononuclear cells from a patient with Yersinia-triggered reactive arthritis. and their specificity was determined using three recombinant Ye-hsp60 fragments, overlapping 18-mer synthetic peptides as well as truncated peptides. Functional characteristics were assessed by cytokine secretion analysis in culture supernatants after specific antigenic stimulation. Amino acid positions relevant for T cell activation were detected by single alanine substitutions within the epitopes. Fragment II comprising amino acid sequence 182–371 was recognized by the majority of clones. All these clones were specific for peptide 319–342. Th1 clones and IL-10-secreting clones occurred in parallel, sometimes with the same fine specificity. The 12-mer core epitope 322–333 is a degenerate MHC binder and is presented to some T cell clones in a "promiscuous" manner. This epitope is almost identical with a B27-restricted CTL epitope of Ye-hsp60. Cross-reactivity of Ye-hsp60-specific T cell clones with self-hsp60 was not observed. In conclusion, an interesting Ye-hsp60 T cell epitope has been identified and characterized. It remains to be determined whether this epitope is also relevant in other reactive arthritis patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Reactive arthritis (ReA)3 is a T cell-dependent inflammatory disease secondary to a urogenital or gastrointestinal infection (1). About 60% of ReA patients are positive for the MHC-class I Ag HLA-B27. Among the organisms triggering ReA, Chlamydia trachomatis and Yersinia enterocolitica are frequent in western countries. The presence of bacterial Ag (2, 3) or DNA (4) in the joint suggests that local Ag may drive the immune response. The recent description of a Th2-cytokine secretion pattern in ReA (5, 6, 7, 8) suggests that an ineffective Th1 response may contribute to bacterial persistence in vivo, because Th1 cytokines such as IFN-{gamma} and TNF-{alpha} are crucial for the elimination of both Chlamydia (9, 10) and Yersinia (11).

The Yersinia heat-shock protein 60 (Ye-hsp60) has attracted increasing attention as an immunodominant Ag for T cells in ReA and other diseases. First, it has recently been found to be the major bacterial component recognized by synovial Th cells in a series of ReA patients (12). Second, nonamers derived from it stimulate synovial CD8+ T cells from patients with Yersinia-ReA in a HLA-B27-restricted way (13). Third, the Ye-hsp60 could be identified in peripheral blood cells of ReA patients years after onset of the arthritis (2). And finally, Ye-hsp60 belongs to the conserved hsp60 family (14) that has repeatedly been incriminated in induction of autoimmunity (15, 16) and in modulation of established inflammatory conditions (17, 18).

The present study was performed to determine the exact specificity and function of CD4+-T cells derived from the joint of a ReA patient in response to the Ye-hsp60. We identified an immunodominant CD4 T cell epitope within the Ye-hsp60 molecule, which was not restricted by a single DR haplotype and which showed plasticity in its capacitiy to elicit cytokines. Therefore, the response to this epitope could play a dominant role in regulating the pathogenesis of ReA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures, T cell cloning, Ag specificity determination, and HLA class II restriction

Synovial fluid from an 18-year-old male patient (HLA phenotype A2, 24; B27, 40; DR6(13), 52; DQ6; DPw1) suffering from ReA after a preceding diarrhea was obtained by arthrocentesis, performed for therapeutic reasons. The Yersinia-based etiology was confirmed by positive standard serology. Separation of synovial fluid mononuclear cells (SFMC) and lymphocyte proliferation assays were performed as described previously (19). SFMC were then cultured in the presence of Ye-hsp60 (10 µg/ml) for 6 days and cloned by limiting dilution in 20-µl Terasaki plates, at 1 cell/well, in the presence of 100 U/ml rIL-2 (Eurocetus, Frankfurt, Germany), 3 µg/ml PHA (Sigma, Deisenhofen, Germany), and 104 irradiated allogeneic PBMC. Outgrowing T cell clones (TCC) were further expanded as described (12, 20) and then tested for Ag specificity in T cell proliferation assays (19) using irradiated (80 Gy) EBV-transformed autologous B lymphoblastoid cell lines (LCL) (104 T cells and 104 EBV-B-LCL). In some cases, irradiated (40 Gy) autologous PBMC (2 x 104) were used as APC. The HLA-restriction of the TCCs was determined using anti-DR, anti-DQ, and anti-DP mAbs (Becton Dickinson, Heidelberg, Germany; each at 2.5 µg/ml).

For further specification of the HLA-DR restriction, PBMC from donors of the following DR haplotypes were used as APC: CL (DR1,-); JB (DR15 (2),-); JS (DR4,-, 53); MN (DR11 (5),-,52); IR (DR7,-, 53).

Recombinant proteins (three fragments spanning the Ye-hsp60, the whole Chlamydia-hsp60 (CT-hsp60), the Borrelia-hsp60 (Bb-hsp60), and the human-hsp60 (hu-hsp60) were tested at a final optimal concentration of 10 µg/ml as previously tested (12). Peptides were tested at a final concentration of 10 µg/ml. For investigation of DR-restricted presentation, irradiated PBMC were first preincubated with the candidate peptide (10 µg/ml) for 2 h, followed by two extensive washing steps, and TCCs were added subsequently. In addition, peptide titration experiments were performed (for details, see Results).

In addition, SFMC from seven other Yersinia-triggered ReA-patients (5 male, 2 female; mean age 25 (range, 18–39)) were stimulated with the Yersinia-specific 19-kDa protein (12), the Ye-hsp60, its three recombinant fragments, and overlapping synthetic peptides spanning the complete fragment II at a final concentration of 10 µg/ml over 6 days. Lymphocyte proliferation was measured as previously described (19).

Generation of recombinant proteins

Generation of recombinant Ye-hsp60, CT-hsp60, Bb-hsp60, and hu-hsp60 were prepared as previously (12) described. For the preparation of overlapping recombinant Ye-hsp60 fragments, appropriate parts of the open reading frames were amplified by PCR using the following primers pairs: 5'-GATCG AATTC GCAGC TAAAG ACGTA-3' and 5'-GATCA AGCTT TACAG CTCGT CTTGC AG-3' for Ye-hsp60 amino acids (aa) 1–187, 5'-GATCG AATTC CTGCA AGACG AGCTG-3' and 5'-GATCA AGCTT TAAGC GACAC GCTCT TG-3' for Ye-hsp60 aa 182–371, and 5'- GATCG AATTC GAGCG TGTCG CTAAA C-3' and 5'-GATCT CTAGA TTACA TCATG CCGCC CATG-3' for Ye-hsp60 aa 367–550. The annealing temperature was 60°C, and 30 cycles were performed. The amplified fragments were isolated from agarose gels with a Jet Sorb Kit (Genome, Bad Oeynhausen, Germany) and ligated in frame with the maltose binding protein into the pMal vector (New England Biolabs, Beverly, MA) at the EcoRI/HindIII site or the EcoRI/XbaI site (for Ye-hsp60 aa 367–550). Expression of the cloned genes was induced by 1 mM isopropyl- ß-D-thiogalactoside (Sigma, St. Louis, MO), and the cells were lysed with a French press. Recombinant proteins were affinity-purified on an amylose resin column according to the supplier’s instructions (New England Biolabs). The purified fragments were dialyzed against PBS and analyzed by SDS-PAGE.

Peptide synthesis

Peptides were synthesized by a robotic multiple peptide synthesizer (Syro; MultiSynTech, Bochum, Germany) using an F-moc/tBu solid-phase synthesis strategy (21). Wang resin (p-benzyloxybenzyl alcohol polystyrene) (Novabiochem, Bad Soden, Germany) was used as solid support. Side chain-protected F-moc amino acids were obtained from Senn Chemicals (Dielsdorf, Switzerland) and Novabiochem. Peptides were characterized by reversed-phase HPLC (M480 pump, UVD-320 S diode-array UV-detector, GINA 160 autosampler; Gynkotek, Germering/Munich, Germany) on Nucleosil C18, 100Å, 5 µm (Macherey-Nagel, Düren, Germany) and electrospray mass-spectrometry (ESI-Quattro II; Micromass, Altrincham, U.K.).

HLA typing

Conventional HLA typing was performed by standard serological methods. In addition, cells from the ReA patient were HLA typed at the DRB1 loci by PCR using sequence-specific primers.

Cytokine ELISA

Culture supernatants were harvested after 48 h of T cell stimulation for determination of Ag-induced cytokine secretion, and the concentration of cytokines in the supernatant was measured as described previously (5, 22). Briefly, levels of IFN-{gamma}, TNF-{alpha}, IL-10, and IL-4 were analyzed by sandwich ELISA using purified anti-cytokine mAb (capture) and biotinylated anti-cytokine mAbs (detecting) (PharMingen, San Diego, CA), with a detection level of 6 pg/ml for IL-4 and 20 pg/ml for the other three cytokines.

HLA-DR-restricted T cell epitope prediction

The Ye-hsp60 protein sequence was run on a new T cell epitope prediction program, called TEPITOPE (23, 24). For prediction of HLA-DR binding-peptides derived from the Ye-hsp60 to identify "promiscuous" epitopes, the most frequent HLA-DR-alleles in the Caucasian population (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101 DRB1*1302, and DRB1*1501) were selected, and the prediction threshold was set at 4% (23). Peptide sequences predicted to bind at least three of seven DR molecules were selected.

HLA-DR-peptide binding assays

Peptide binding to detergent-solubilized DR molecules was measured using an ELISA-based high-flux competition assay (25). HLA-DR molecules were isolated from the following human LCLs: DR1 (DRB1*0101) from HOM-2, DR3 (DRB1*0301) from WT49, DR4 (DRB1*0401) from PREISS, DR5 (DRB1*1101) from SWEIG, DR6 (DRB1*1302) from the L cell transfectant L605.2, DR7 (DRB1*0701) from EKR, and DR8 (DRB1*0801) from BM9. DR2 (DRB1*1501) was isolated from the L cell transfectant L466.1. The molecules were affinity purified using the mAb 1-1C4 (26) as described (27).

Peptide competition assays were conducted to measure the ability of unlabeled peptides to compete with a biotinylated indicator peptide for binding to purified DR molecules. The following biotinylated indicator peptides were used: GFKA7 for DR1 and DR7; GIRA2YA4 for DR2; IAYDA5 for DR3; UD4 for DR4 (28); TT830–843 for DR5, and GYRA6L for DR8. An optimal concentration of purified DR molecules (30–100 ng) was added to each well of a 96-well plate along with biotinylated peptide (0.1–5 mM) in binding buffer (25). The biotinylated indicator peptide and DR molecules were incubated with 10-fold dilutions (0.001–100 mM) of the unlabeled competitor peptide (Ye-hsp60 peptide). Following incubation overnight, the peptide/class II complexes were transferred to wells coated with anti-DR L243 (HB 55; American Type Culture Collection, Manassas, VA) mAb. After 2 h incubation at room temperature, the wells were washed with an appropriate buffer (25), and the specific degree of the biotinylated peptide bound to DR molecules was revealed by the addition of alkaline phosphatase-labeled streptavidin and 4-p-nitrophenyl phosphate. The amount of substrate hydrolyzed was assessed with a MC Multiscan ELISA reader (405 nm). To determine relative peptide binding affinity, the "promiscuous" PKYVKQNTLKLAT peptide from influenza hemagglutinin (HA) 307–319 (29) was included in each competition assay. The relative binding of the unlabeled competitor peptides were expressed as inhibitory concentrations (IC50).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cell cloning, efficacy, and gross specificity

The cloning procedure yielded 61 CD4+-TCCs specific for Ye-hsp60. All these TCCs responded also to whole heat-killed Yersinia. Fifty of these TCCs showed specific proliferation to Ye-hsp60 fragment II, spanning the aa residues 182–371, eight were specific for fragment III, spanning the aa residues 367–550, two reacted with fragment II and fragment III, and one was specific for fragment I, comprising the aa residues 1–187 (Fig. 1Go).



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  		FIGURE 1. Schematic representation of recombinant Ye-hsp60, its recombinant fragments, and location of the epitopes. A, Overview. B, Fragment II and the epitope is shown in more detail. C, The peptide epitope p54/55 from fragment II is shown in more detail. The numbers below and beside the fragments represent amino acid residues. The number of clones recognizing each fragment is also indicated. A single letter code is used for the amino acids.

 
Fine specificity of TCCs

Eighty-nine 18-mer peptides, overlapping by 12 aa and covering the whole Ye-hsp60, were employed for initial epitope characterization. The clones responded preferentially to fragment II. Twenty-one of the 50 TCCs specific for fragment II could be grown sufficiently for epitope analysis. Fifteen of the 21 TCCs proliferated specifically to peptide p54319–336, and the other six of the 21 TCCs responded to the adjacent peptide p55325–342 (Fig. 1Go).

Next, truncated peptide variants were constructed to identify the minimal length recognized. Five of the 15 clones specific for p54 and one of the six TCCs specific for p55 were characterized as shown in Table IGo. The 12-mer peptide p54-6b322–333 was the minimal epitope common to all clones responding to p54. Four types of TCCs could then be identified on the basis of a stepwise truncation of peptide 322–336 by 1 aa at the N terminus. The first type (clones 69 and 92) recognized the 10-mer p54-6g322–331; the second type (clones 83 and 85) recognized the 11-mer p54-6h323–333; the third type of clone (clone 98) could be stimulated by p55-8324–337; and the fourth type (clone 39) recognized p55-9325–336. Further truncations of the two latter peptides at the C terminus were not tested. Stepwise truncations at the C terminus of p54-6b322–333 were of minor relevance and influenced only proliferation of clone 98.


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  		Table I. Identification of peptide length and critical amino acid positions for recognition of T cell clones specific for the Ye-hsp60-derived peptide epitope p541

 
The clones responding to fragment III were also investigated for their fine specificity (Table IIGo). Two antigenic regions were identified on fragment III: one clone reacted with peptides p64 and p65 spanning 379–402, and six clones reacted with peptide p76451–468. The truncated variants p65-21385–395 and p65-22386–396 seemed to be the minimal epitope for clone 68 and the 15-mer peptide p76-27454–468 emerged as the minimal epitope for clone 55, because removal of the N-terminal R454 and Q455 abrogated its proliferation.


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  		Table II. Sequence alignments of Ye-hsp60-derived peptide epitopes with corresponding regions of Bb-hsp60, CT-hsp60, and Hu-hsp60 and cross-recognition1

 
The single clone specific for fragment I could not be further cultured and was not further investigated.

Effect of single alanine substitutions within peptide p54-6b322–333 on T cell activation

Substitution of single positions by alanine was performed for the 12-mer peptide p54-6b to determine the aa residues that are crucial for stimulation of the TCCs, as shown in Table IGo for the three different types of TCCs (69/92, 83/85, and 98). Different determinants were identified. Interestingly, for the second type only two residues (K327 and D328) of the 10-mer peptide proved essential.

Cross-recognition of other hsp60s and hsp-derived peptides by TCCs

Cross-recognition of the Bb-hsp60, CT-hsp60, and hu-hsp60 by the TCCs was assessed using whole recombinant proteins as well as hsp60-derived 24-mer peptides homologous to the Ye-hsp60 epitopes (Table IIGo) to circumvent possible "crypticity" of these epitopes. In the case of the Yersinia peptides p54 and p55, cross-reactivity was observed only with the homologous Bb-hsp60-derived 24-mer peptide but not with the CT-hsp60 or the hu-hsp60. Interestingly, the whole Bb-hsp60 was also not recognized. This indicates that the Borrelia peptide recognized was not generated under the experimental conditions used, i.e., it behaved as a "cryptic" epitope. The cross-reactivity was restricted to one clone, clone 83. Table IIGo shows the appropriate sequence alignments. Clone 83 needs only K327 and D328 for activation. Both aa are indeed shared by the Borrelia peptide in the appropriate positions. In contrast, clones 92 and 98 needs several other positions for activation, which are not shared between the peptide p54-6b and the Bb-hsp60 peptide.

Surprisingly, the hu-hsp60 peptide and the Chlamydia-hsp60 peptide were not recognized by clone 83 although they either shared the essential aa residues KD (human peptide) or showed a highly conserved substitution (D->E; Chlamydia peptide), which should be of minor relevance (Table IIGo). This observation could be explained if T329 was also relevant for T cell recognition but was overlooked by the alanine substitutions, because the T->A substitution is a conserved hydrophobic one. In both the Chlamydia peptide and the human peptide, a nonconserved substitution (T329->D) is present (hydrophobic aa->negatively charged aa), whereas a relatively conserved T->N (hydrophobic aa->small polar aa) substitution present in the Borrelia peptide might be less important. Extension of the substitution experiments by introducing a negatively charged amino acid (aspartic acid (D)) instead of alanine could detect such a potentially critical aa. In addition, several substitutions of the C-terminal isoleucines present in the human peptide but not in the Borrelia peptide might result in a significant decrease of MHC binding, thus leading to a subsequent failure of T cell recognition.

There was no cross-reactivity of the fragment III peptide p76451–468 with the corresponding peptides from the other hsp60s. Although no essential aa residues were determined for this peptide, the substantial differences in the sequence between the Ye-hsp60-derived peptide and the corresponding peptide from the hu-hsp60, the CT-hsp60, or the Bb-hsp60 could easily explain this failure (Table IIGo). In contrast, clone 68 specific for the second fragment III epitope responded both to the whole CT-hsp60 and to its derived 24-mer peptide. The sequence alignment in Table IIGo shows a high degree of homology between the putative core epitope region of the Yersinia-p76 and the corresponding Chlamydia peptide in comparison to the borrelial and the human ones.

HLA restriction and "promiscuous" epitope recognition by TCC

We tested the effect of anti-DR-, anti-DP-, and anti-DQ-specific mAbs on proliferation and cytokine production on the TCCs. These experiments revealed that HLA-DR was the restricting class II molecule for all clones investigated. By using APC homozygous for various DR alleles from donors, effective epitope presentation to clone 92 could be shown for all DR alleles investigated, i.e., DR1, DR2, DR4, and/or DR53, DR5, and/or DR52, DR7, and/or DR53. (Fig. 2Go). Dose-response experiments identified autologous APC as the best presenters, particularly in the critical peptide concentration range between 1 µg/ml and 0.1 µg/ml (data not shown). In contrast, clone 69 recognized the peptide epitope only in the context of autologous DR6-positive APC, but not if presented by DR7,53- and DR4,53-positive APC (DR1-, DR2-, and DR5- positive APC were not tested) (Fig. 2Go).



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  		FIGURE 2. Proliferation of clone 92 and clone 69 to epitope 322–333 in the context of allogenous PBMC as APC of the various DR haplotypes as indicated. In the case of DR1-homozygous APC, EBV-B-LCL were used. Anti-DR mAb almost completely abolished the proliferation. Clone 69 was tested only with DR7,53- and DR4,53-allogenous PBMC in addition to autologous PBMC. a, EBV-B-LCL were used as APC.

 
"Promiscuous" T cell epitope prediction

The TEPITOPE program confirmed that peptides from the 24-mer peptide p54/55319–342 would bind to DR1, DR3, DR4, DR2b, and DR8 with a threshold of 3% and to DR6 with a 4% threshold and that the homologous peptides derived from the Bb-hsp60, the CT-hsp60, and the hu-hsp60 had no significant affinity to DR6. The same "promiscuous" DR-binding properties were predicted for the truncated epitopes, i.e., the 15-mer p54-6 (RVVINKDTTIIIDGV) and the 12-mer p54-6b (RVVINKDTTIII).

A degenerate MHC binding was also predicted for p76/77451–474, with a 1% threshold to DR3, a 2% threshold to DR1 and DR4, a 3% threshold to DR5 and DR6, and a 4% threshold to DR7.

HLA-DR binding assays

The competitive binding assays employing the eight most common DR alleles and the 12-mer epitope p54-6b322–333 (RVVINKDTTIII) revealed only moderate binding affinity for the DR6 molecule as well as to DR5 and DR3, compared with the promiscuous reference peptide from HA307–319. The IC50 was 30 µM for DR3 and DR6 and 60 µM for DR5. DR7 showed the highest affinity for peptide p54-6b with an IC50 of 6 µM. Therefore, the overall binding affinity of the Ye-hsp60 epitope is 10-fold lower than for the strong binder HA307–319. All other DR molecules bound p54-6b with even lower affinity.

There was almost no correlation between the proliferative response to the p54-6b in the context of different DR haplotypes and the peptide’s affinity to these MHC haplotypes. Interactions within the trimolecular complex (MHC/peptide/TCR) beyond the affinity of MHC peptide alone may be responsible for T cell recognition.

HLA binding assays with the epitopes of hsp60-fragment III (p64 and p76/77) were not performed.

Comparison of the Ye-hsp 60 epitope 322–333 with other "promiscuous" epitopes

We compared the predicted HLA-DR binding frame of the "promiscuous" Ye-hsp60 epitope p54-6b, which consists of the nonamer VINKDTTII324–332, with other microbial epitopes "promiscuous" for HLA-DR (Table IIIGo). Sequence alignment with these other T cell epitopes shows that the motif required for binding several DR molecules has similarities (30, 31, 32, 33). The residues crucial for DR binding are position 1, 6, and 9. All the epitopes shown here including the Ye-hsp60324–332 contain a hydrophobic residue (V324 for the Ye-hsp60) at position 1, a small hydrophobic residue (T329 for the Ye-hsp60) at position 6, and an aliphatic residue (I332 for the Ye-hsp60) at position 9.


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  		Table III. Promiscuous T cell epitopes with DR binding motifs1

 
Cytokine profiles upon stimulation of T cell clones with Ye-hsp60 peptides

The cytokine profile was determined for clones 69, 92, 83, and 98, which are all specific for p54-6b, and clone 55, being specific for p76452–468. The data for clone 69 and clone 92 are shown in Fig. 3Go, and the data for clone 55 is shown in Table IVGo. IFN-{gamma} is the prominent cytokine and closely reflects the magnitude of proliferation in response to the particular Ag. TNF-{alpha} is frequently secreted by all clones, except clone 55, in parallel with the level of activation as measured by proliferation.



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  		FIGURE 3. Cytokine secretion by clone 92 and clone 69 in response to various peptides. IFN-{gamma}, IL-10, and TNF-{alpha} were measured in the supernatant by ELISA. The numbers in parenthesis indicate amino acid residues. Values are the mean of three measurements. [322–333]8A is 322–333 with a single alanine substitution at peptide position 10. #, The SI of clone 92 after stimulation with peptide [322–333]8A was reduced by 50%.

 

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  		Table IV. Cytokine secretion of clone 551

 
In contrast, the amount of IL-10 secretion varied among the clones. Clone 92 (Fig. 3Go), clone 98, and clone 83 (data not shown) were potent IL-10 producers with IFN-{gamma}:IL-10 ratios ranging from 10:1 to 5:1, depending on the stimulating peptide Ag. In contrast, clone 69, although recognizing the same peptide (Table IGo), failed to secrete substantial quantities of IL-10 (Fig. 3Go). Using the technique of intracellular cytokine staining of the TCCs with subsequent quantification of positive cells by flow cytometry, we could show that IL-10 was secreted by T cells and not by the non-T cells in the population of APC used for this experiment (data not shown). Moreover, the amount of IL-10 secretion differs not only among the clones but also for a single clone after stimulation with Ags of different length or composition: especially the relatively long Ye-hsp60 fragment II favored the production of IL-10 (clone 92 is shown as an example).

Only 1 clone (clone 55) secreted a reasonable amount of IL-4 together with IFN-{gamma} and IL-10 (Table IVGo).

Comparison of the major Ye-hsp60 Th cell epitope 322–333 with the CTL epitope

When the sequence of the major CD4 T cell epitope p54-6b, RVVINKDTTIII322–333, identified in the present study was compared with the sequence of the recently discovered HLA-B27-restricted CTL epitope, KRVVINKDT321–329, which is also derived from the Ye-hsp60 (13), an almost complete overlap could be observed with a difference of only 1 aa at the N-terminal end. It should be emphasized that the T cells used in both investigations were derived from unrelated ReA patients.

Proliferation of other Yersinia-triggered ReA patients to Ye-hsp60-fragment II peptides

We tested SFMC derived from seven other patients for proliferation to various Yersinia-derived Ags and Ye-hsp60-fragment II-derived overlapping 18-mer peptides. All patients showed a good response to whole Yersinia, to the Yersinia-specific 19-kDa protein (data not shown), to the complete Ye-hsp60, and to the three overlapping Ye-hsp60 fragments. As shown in Fig. 4Go, the highest stimulation index (SI) could be seen in response to fragment II in the majority of patients (four of seven patients). However, we could not detect a proliferative response to any of the peptides (SI < 2), including p54/55, except in patient 7. This patient (DR1; DR2), with the highest SI (72.6) of all patients tested in response to fragment II, showed a slight proliferation to peptide p41241–258 (SI = 6.1).



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  		FIGURE 4. Proliferation of SFMC of seven Yersinia-triggered ReA patients to Ye-hsp60 fragments and overlapping 18-mer peptides, spanning the entire fragment II. *, None of the peptides stimulated SFMC of patients 1–6 (SI < 2). §, Only in patient 7, a single peptide p41241–258 was recognized.

 
The discrepancy between a good response to the whole fragment II and failure to respond to peptides derived from this protein indicates that stimulation of mononuclear cells with peptides might not be sensitive enough for epitope identification.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hsp60s are dominant Ags in the immune response to bacteria, despite or maybe because of their extraordinary high sequence conservation throughout the eukaryotic and prokaryotic kingdom (34). This has also been demonstrated for the two ReA-associated bacteria Yersinia and C. trachomatis. In a murine model of acute Yersinia infection, the Ye-hsp is an immunodominant T cell Ag with protective properties as shown in both adoptive transfer and vaccination experiments (35, 36). However, it seems to be also a relevant Ag in more chronic immune responses such as in ReA. We could recently show that the Ye-hsp60 is preferentially seen by both CD4+ and CD8+ T cells derived from synovial fluid of patients with Yersinia-induced ReA (12, 13). Synovial T cell responses to CT-hsp60 have also been described in Chlamydia-induced ReA (37), and the immune response to CT-hsp60 have been implicated in the immunopathology of trachoma of the eye (38). Furthermore, in ReA, Ye-hsp60 and CT-hsp60 are even preferentially expressed in the joint (39) or peripheral blood (2), compared with other bacteria-derived proteins such as membrane proteins. This has also be shown for Chlamydia in vitro under experimental conditions mimicking bacterial persistence (40). Thus, although bacterial hsp60 seems to be a preferential target of the acute immune response, its strong expression during bacterial persistence suggests that it might facilitate the bacterial survival of the host’s immune response.

In the present study, we provide a detailed analysis of the CD4+ T cell response to the Ye-hsp60 in one patient with ReA. We were able to identify one major epitope. A small antigenic region comprising the 24-aa 319–342 present on an Ag 550 aa long elicited the majority of TCCs in our study. This finding suggests that this part of the protein is central for induction of an immune response. We could also show that this peptide is presented by different HLA-DR alleles, underlining its possible importance beyond the single patient described here. This main epitope, in addition to the two others discovered in our study, differ from those identified in a mouse model of Yersinia infection (41), probably because of the interspecies MHC class II binding differences. Epitopes might also differ in acute infections from those in more chronic immune responses, such as in ReA.

In the context of ReA, an obvious question is whether the immune response to the up-regulated hsp60 is not effective enough for the elimination of bacteria, leading to bacterial persistence. A Th1 response is crucial for successfully fighting microbes such as Yersinia and Chlamydia, and their elimination is inhibited by IL-10 (9, 10, 11). We have previously reported that a relative high amount of IL-4 and IL-10 and/or a low secretion of IFN-{gamma} and TNF-{alpha} occurs in the joint (5, 6) or peripheral blood (42) of ReA patients, suggesting that such an ineffective Th1 response contributes to bacterial persistence. Therefore, the analysis of the cytokine secretion pattern of the TCCs upon stimulation with the immunodominant epitope was of great interest.

Among the inhibitory cytokines, IL-10 was secreted in considerable amount by several clones, while only one IL-4-secreting clone was identified (Table IVGo). Therefore, Ye-hsp60-specific IL-10 secretion might permit bacterial persistence and could be an explanation for the up-regulation of Ye-hsp60 in chronic inflammations such as ReA. We did not find TCCs secreting only IL-10, formerly described as Tr1 clones (43). However, it can be assumed that IL-10 produced together with IFN-{gamma} and TNF-{alpha} acts also as an effective inhibitor of an antibacterial immune response. One interesting observation should be emphasized: stimulation of virtually all clones investigated with the whole Ye-hsp60 protein fragment II182–371 except clone 69 gave rise to high levels of IL-10, compared with stimulation by the short specific peptide epitope (Fig. 3Go). These results demonstrate that Ag-specific T cells with both high and low IL-10 production are present in the joint. The actual balance of these two types of cells could ultimately give rise to clearance or persistence of Yersinia. The relative contribution of these types of T cells for the in vivo situation has to be addressed in future studies.

In the context of the Ye-hsp60 T cell response, cross-reactivity has attracted a lot of interest in recent years. The hsp60s are highly conserved throughout evolution with an aa sequence identity of up to 50% between prokaryotic and eukaryotic species (14). The homology among enterobacteria can be as high as 90%. Cross-reactivity between Yersinia and other ReA-associated bacteria such as C. trachomatis might help to explain why such diverse bacteria induce a similar disease. However, in our study no cross-reactivity between the dominant Ye-hsp60 epitope 322–333 and the corresponding epitope from the CT-hsp60 was found. The identification of the aa critical for T cell recognition within the 12-mer epitope helps to explain why some hsp60s are recognized but not others. Indeed, the critical residues were not identical on both the Ye-hsp60 and the CT-hsp60. However, these residues were shared by the homologous hsp60 peptide from Borrelia—a microbe not associated with ReA—which was readily recognized by this clone. This epitope was most probably a "cryptic" one, because no T cell activation was seen after stimulation with the whole Bb-hsp60 protein. A T cell response directed to CT-hsp60 has recently been described in a patient with Chlamydia-induced ReA (37). However, epitope analysis of one clone also revealed no cross-reactivity with either the hu-hsp60 or other enterobacterial hsp60s.

We also identified one clone (clone 55) specific for the fragment III epitope p76451–468 that produced a relatively high amount of the Th2 cytokine IL-4 in addition to IL-10 (Table IVGo), a finding that could be confirmed by intracellular cytokine staining (data not shown). However, compared with IL-10, IL-4-secreting cells seemed to represent a minority. The experiments with the single alanine-substituted peptide variants are too limited to draw final conclusions regarding the effects of the so called "altered peptide ligands" to the functional outcome of the clones, a concept that has been extensively studied in recent years (44, 45). Substitutions with the tiny hydrophobic alanine, as used in our study, may result in effects different from those observed after substitution with positively or negatively charged amino acids. Nonetheless, substitution of T329 by alanine at position 8 lead to a 50% reduction of the proliferation and to a 80% reduction in the secretion of the proinflammatory cytokines IFN-{gamma} and TNF-{alpha}, while only slightly decreasing the secretion of IL-10, resulting in an markedly reduced IFN-{gamma}:IL-10 ratio (Fig. 3Go).

A cross-reactive T cell response against bacterial and self-hsp60 has also been implicated in the induction of autoimmunity, as discussed previously (15, 16, 46, 47) and has even been described in Yersinia-induced ReA (48). It has recently been reported that an autoimmune response against self-hsp60 could be induced in mice by simultaneous immunization with both the self-hsp 60 and the CT-hsp60 but not with either of these molecules alone, suggesting that under certain circumstances autoimmunity against self-hsp60 with subsequent immunopathology can indeed occur (49). However, in contrast to animal models the evidence is less convincing in humans (17, 34). Rather, van Eden and colleagues suggest that a cross-reactive T cell response against self-hsp60 might be a relevant mechanism for down-regulating an autoimmune response by release of regulatory cytokines (17, 18, 50, 51). In our study, we could not detect a cross-reaction between human and Ye-hsp60 epitopes. However, in contrast to rheumatoid arthritis or experimental animal models such as adjuvant arthritis (46, 47), immunopathology in ReA is most likely caused by a bacteria-specific immune response and not by autoimmunity. Our results indicate that the T cell response to the hsp60 is flexible enough to react with different cytokine patterns, even without cross-reactivity with the human counterpart.

After we had identified peptide p54322–333 as a relevant CD4 T cell epitope in the complete Ye-hsp60 molecule, it was a great surprise that it almost completely overlapped with the Ye-hsp60 nonamer KRVVINKDT321–329, which was recently described by us to be an immunodominant HLA-B27-restricted epitope for the cytotoxic CD8+ T cell response in different patients with Yersinia-induced ReA. These findings are in accordance with earlier reports showing that CD4 T cell epitopes are often similar to CD8 epitopes, either covalently linked to or containing the CTL epitope (52, 53). In particular, this apparent overlap of epitopes for both the CD4 and the CD8 T cell responses might indicate a major immunogenic region within the Ye-hsp60 molecule that might have relevance beyond this particular patient.

Although peptide p54/55 could be presented to our T cell clones by different HLA-DR haplotypes, it is not clear whether this epitope is also seen by other patients with Yersinia-triggered ReA. Using whole SFMC instead of T cell lines and clones, lymphocyte proliferation assays were not sensitive enough for epitope mapping as described by others (33). This might be due to the relatively low peptide-specific T cell frequency among bulk SFMC and strengthens the current need for labor-intensive T cell cloning. More sensitive flow cytometric methods as a new technology for T cell epitope mapping (54) circumventing T cell cloning might be helpful in the future.

At present, there is no proof that the immunodominant peptides identified by T cell clones are relevant for the pathogenesis of ReA. Final confirmation would come from preventing or curing the disease using such peptides for vaccination or induction of Ag-specific tolerance. However, for this purpose identification of immunodominant epitopes is a first and essential step.

In summary, we could define a multiclonal and multispecific CD4+ T cell response to a single epitope derived from Ye-hsp60 with no cross-reactivity to CT-hsp60 or hu-hsp60 in a patient with Yersinia-induced ReA. The promiscuity of this peptide indicates that it might also be important for the immune response of individuals with different MHC class II types. IL-10 secretion was prominent, but differed widely, dependent on the clones and the peptides tested. Future studies applying Ag-specific cytokine expression of nonseparated T cells will clarify the relative importance of Ag-specific IL-10 secretion in ReA (55). The cytokine secretion pattern could play a role for bacterial persistence in ReA and could be a target for future T cell manipulation in patients with ReA and related diseases. It is currently a subject of debate whether inflammation in ReA is the consequence of hypersensitivity to bacterial Ag (56), which could therapeutically be down-regulated by Th2 cytokines, or alternatively, the consequence of an ineffective Th1 response necessary for effective elimination of bacteria. Our data favor the latter possibility.


   Acknowledgments
 
We thank Dr. Avrion Mitchison for critical review of the manuscript.


   Footnotes
 
1 This work was supported by a grant by the Deutsche Forschungsgemeinschaft (SI 620/6-1). Back

2 Address correspondence and reprint requests to Dr. Joachim Sieper, Department of Medicine IV—Rheumatology, University Hospital/FU Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. E-mail address: Back

3 Abbreviations used in this paper: ReA, reactive arthritis; Ye-hsp60, Yersinia enterocolitica heat shock protein 60; SFMC, synovial fluid mononuclear cells; TCC, T cell clone; LCL, lymphoblastoid cell line; CT-hsp60, Chlamydia trachomatis heat-shock protein 60; Bb-hsp60, Borrelia burgdorferi heat-shock protein 60; hu-hsp60, human heat-shock protein 60; HA, influenza hemagglutinin; SI, stimulation index. Back

Received for publication June 7, 1999. Accepted for publication November 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Keat, A.. 1983. Reiter’s syndrome and reactive arthritis in perspective. N. Engl. J. Med. 309:1606.[Medline]
  2. Granfors, K., R. Merilahti-Palo, R. Luukkainen, T. Möttönen, R. Lahesmaa, P. Probst, E. Märker-Hermann, P. Toivanen. 1998. Persistence of Yersinia antigens in peripheral blood cells from patients with Yersinia enterocolitica O:3 infection with or without reactive arthritis. Arthritis Rheum. 41:855.[Medline]
  3. Granfors, K., S. Jalkanen, R. von Essen, R. Lahesmaa-Rantala, O. Isomäki, K. Pekkola-Heino, R. Merilahti-Palo, R. Saario, H. Isomäki, A. Toivanen. 1989. Yersinia antigens in synovial-fluid cells from patients with reactive arthritis. N. Engl. J. Med. 320:216.[Abstract]
  4. Bas, S., R. Griffais, T. K. Kvien, A. Glennas, K. Melby, T. L. Vischer. 1995. Amplification of plasmid and chromosome Chlamydia DNA in synovial fluid of patients with reactive arthritis and undifferentiated seronegative oligoarthropathies. Arthritis Rheum. 38:1005.[Medline]
  5. Yin, Z., J. Braun, L. Neure, P. Wu, L. Liu, U. Eggens, J. Sieper. 1997. Interleukin 10/interleukin 12 balance is crucial for the regulation of TH2-response in reactive arthritis. Arthritis Rheum. 40:1788.[Medline]
  6. Simon, A. K., E. Seipelt, J. Sieper. 1994. Divergent T-cell cytokine patterns in inflammatory arthritis. Proc. Natl. Acad. Sci. USA 91:8562.[Abstract/Free Full Text]
  7. Sieper, J., G. Kingsley. 1996. Recent advances in the pathogenesis of reactive arthritis. Immunol. Today 17:160.[Medline]
  8. Burmester, G. R., A. Daser, T. Kamradt, A. Krause, N. A. Mitchison, J. Sieper, N. Wolf. 1995. Immunology of reactive arthritides. Annu. Rev. Immunol. 13:229.[Medline]
  9. Yang, X., J. Gartner, L. Zhu, S. Wang, R. C. Brunham. 1999. IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following Chlamydia trachomatis lung infection. J. Immunol. 162:1010.[Abstract/Free Full Text]
  10. Yang, X., K. T. HayGlass, R. C. Brunham. 1996. Genetically determined differences in IL-10 and IFN-{gamma} responses correlate with clearance of Chlamydia trachomatis mouse pneumonitis infection. J. Immunol. 156:4338.[Abstract]
  11. Bohn, E., I. B. Autenrieth.. 1996. IL-12 is essential for restistance against Yersinia enterocolitica by triggering IFN-{gamma} production in NK cells and CD4+ T cells.. J. Immunol. 156:1458.[Abstract]
  12. Mertz, A. K. H., S. Ugrinovic, R. Lauster, P. Wu, U. Böttcher, H. Appel, Z. Yin, E. Schiltz, S. Batsford, C. Schauer-Petrowski, et al 1998. Characterization of the synovial T-cell response to various recombinant Yersinia antigens in Yersinia-triggered reactive arthritis: the hsp60 drive a major immune response. Arthritis Rheum. 41:315.[Medline]
  13. Ugrinovic, S., A. Mertz, P. Wu, J. Braun, J. Sieper. 1997. A single nonamer from the Yersinia 60-kD heat shock protein is the target of HLA-B27-restricted CTL response in Yersinia-induced reactive arthritis. J. Immunol. 159:5715.[Abstract]
  14. Viale, A. M., A. K. Arakaki, F. C. Soncini, R. G. Ferreyra. 1994. Evolutionary relationships among eubacterial groups as inferred from GroEL (chaperonin) sequence comparisons. Int. J. Syst. Bacteriol. 44:527.[Abstract/Free Full Text]
  15. Young, D. B.. 1992. Heat-shock proteins: immunity and autoimmunity. Curr. Opin. Immunol. 4:396.[Medline]
  16. Kaufmann, S. H.. 1990. Heat shock proteins and the immune response. Immunol. Today 11:129.[Medline]
  17. van Eden, W., R. van der Zee, A. G. Paul, B. J. Prakken, U. Wendling, S. M. Anderton, M. H. Wauben. 1998. Do heat shock proteins control the balance of T-cell regulation in inflammatory diseases?. Immunol. Today 19:303.[Medline]
  18. Van Eden, W., S. M. Anderton, R. van der Zee, B. J. Prakken, C. P. M. Broeren, M. H. M. Wauben. 1996. (Altered) self peptides and the regulation of self reactivity in the peripheral T cell pool. Immunol. Rev. 149:55.[Medline]
  19. Sieper, J., G. Kingsley, A. Palacios Boix, C. Pitzalis, J. Treharne, R. Hughes, A. Keat, G. S. Panayi. 1991. Synovial T lymphocyte-specific immune response to Chlamydia trachomatis in Reiter’s disease. Arthritis Rheum. 34:588.[Medline]
  20. Sieper, J., J. Braun, P. Wu, G. Kingsley. 1993. T cells are responsible for the enhanced synovial cellular immune response to triggering antigen in reactive arthritis. Clin. Exp. Immunol. 91:96.[Medline]
  21. Jung, G., A. G. Beck-Sickinger. 1992. Multiple peptide synthesis methods and their applications. Angew. Chem. Int. Ed. Engl. 31:367.
  22. Yin, Z., J. Braun, L. Neure, P. Wu, U. Eggens, A. Krause, T. Kamradt, J. Sieper. 1997. T cell cytokine pattern in the joints of patients with Lyme arthritis and its regulation by cytokines and anticytokines. Arthritis Rheum. 40:69.[Medline]
  23. Hammer, J., T. Sturniolo, F. Sinigaglia. 1997. HLA class II peptide binding specificity and autoimmunity. Adv. Immunol. 66:67.[Medline]
  24. Hammer, J., E. Bono, F. Gallazzi, C. Belunis, Z. Nagy, F. Sinigaglia. 1994. Precise prediction of major histocompatibility complex class II-peptide interaction based on peptide side chain scanning. J. Exp. Med. 180:2353.[Abstract/Free Full Text]
  25. Raddrizzani, L., T. Sturniolo, J. Guenot, E. Bono, F. Gallazzi, Z. A. Nagy, F. Sinigaglia, J. Hammer. 1997. Different mode of peptide interaction enable HLA-DQ and DR molecules to bind diverse peptide repertoires. J. Immunol. 159:703.[Abstract]
  26. Cammarota, G., A. Scheirle, B. Takacs, D. M. Doran, R. Knorr, W. Bannwarth, J. Guardiola, F. Sinigaglia. 1992. Identification of a CD4 binding site on the b2 domain of HLA-DR molecules. Nature 356:799.[Medline]
  27. Sinigaglia, F., P. Romagnoli, M. Guttinger, B. Takacs, J. R. L. Pink. 1992. Selection of T-cell epitopes and vaccine engineering. Methods Enzymol. 203:370.
  28. Hammer, J., F. Gallazzi, E. Bono, R. W. Karr, J. Guenot, P. Valsasnini, Z. A. Nagy, F. Sinigaglia. 1995. Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association. J. Exp. Med. 181:1847.[Abstract/Free Full Text]
  29. Roche, P. A., P. Cresswell. 1990. High-affinity binding of an influenza hemaglutinin-derived peptide to purified HLA-DR. J. Immunol. 144:1849.[Abstract]
  30. O’Sullivan, D., T. Arrhenius, J. Sidney, M. F. del Guercio, M. Albertson, M. Wall, C. Oseroff, S. Southwood, S. M. Colon, F. C. A. Gaeta, A. Sette. 1991. On the interaction of promiscuous antigenic peptides with different DR alleles: identification of common structural motifs. J. Immunol. 147:2663.[Abstract/Free Full Text]
  31. Panina-Bordignon, P., A. Tan, A. Termijtelen, S. Demotz, G. Corradin, A. Lanzavecchia. 1989. A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells. Eur. J. Immunol. 19:2237.[Medline]
  32. Sinigaglia, F., M. Guttinger, J. Kilgus, D. Doran, H. Matile, H. Etlinger, A. Trzeciak, D. Gillesen, J. Pink. 1988. A malaria T cell epitope recognized in association with most mouse and human MHC class II molecules. Nature 336:778.[Medline]
  33. Contreras, C. E., I. N. Ploton, R. F. Siliciano, C. L. Karp, R. Viscidi, N. Kumar. 1998. Mapping of specific and promiscuous HLA-DR-restricted T-cell epitopes on the Plasmodium falciparum 27-kilodalton sexual stage-specific antigen. Infect. Immun. 66:3579.[Abstract/Free Full Text]
  34. Zügel, U., S. H. E. Kaufmann. 1999. Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin. Microbiol. Rev. 12:19.[Abstract/Free Full Text]
  35. Noll, A., A. Roggenkamp, J. Heesemann, I. B. Autenrieth. 1994. Protective role for heat shock protein-reactive {alpha}ß T cells in murine yersiniosis. Infect. Immun. 62:2784.[Abstract/Free Full Text]
  36. Noll, A., I. B. Autenrieth. 1996. Immunity against Yersinia enterocolitica by vaccination with Yersinia HSP60 immunostimulating complexes or Yersinia HSP60 plus interleukin-12. Infect. Immun. 64:2955.[Abstract]
  37. Deane, D. H. O., R. M. Jecock, J. H. Pearce, J. S. H. Gaston. 1997. Identification and characterization of a DR4-restricted T cell epitope within Chlamydia heat shock protein 60. Clin. Exp. Immunol. 109:439.[Medline]
  38. Morrison, R. P., R. J. Belland, K. Lyng, H. D. Caldwell. 1989. Chlamydia disease pathogenesis: the 57kDa chlamydial hypersensitivity antigen is a stress response protein. J. Exp. Med. 170:1271.[Abstract/Free Full Text]
  39. Gerard, H. C., P. J. Branigan, H. R. Schumacher, A. P. Hudson. 1998. Synovial Chlamydia trachomatis in patients with reactive arthritis/Reiter’s syndrome are viable but show aberrant gene expression. J. Rheumatol. 25:734.[Medline]
  40. Beatty, W. L., G. I. Byrne, R. P. Morrison. 1993. Morphologic and antigenic characterization of interferon {gamma}-mediated persistent Chlamydia trachomatis infection in vitro. Proc. Natl. Acad. Sci. USA 90:3998.[Abstract/Free Full Text]
  41. Noll, A., I. B. Autenrieth. 1996. Yersinia-hsp60-reactive T cells are efficiently stimulated by peptides of 12 and 13 amino acid residues in a MHC class II (I-Ab)-restricted manner. Clin. Exp. Immunol. 105:231.[Medline]
  42. Braun, J., Z. Yin, I. Spiller, S. Siegert, M. Rudwaleit, A. Radbruch, and J. Sieper. A low TNF-{alpha}-secretion of peripheral blood mononuclear cells but no other T helper 1 or 2 cytokines correlates with chronicity in reactive arthritis. Arthritis Rheum. 42:2039.
  43. Groux, H., A. O’Garra, M. Bigler, M. Rouleau, S. Antonenko, J. E. de Vries, M. G. Roncarolo. 1997. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737.[Medline]
  44. Nicholson, L. B., H. Waldner, A. M. Carrizosa, A. Sette, M. Collins, V. K. Kuchroo. 1998. Heteroclitic proliferative responses and changes in cytokine profile induced by altered peptides: Implications for autoimmunity. Proc. Natl. Acad. Sci. USA 95:264.[Abstract/Free Full Text]
  45. Sloan-Lancaster, J., P. M. Allen. 1996. Altered peptide ligand-induced partial T cell activation: molecular mechanism and roles in the T cell biology. Annu. Rev. Immunol. 14:1.[Medline]
  46. Van Eden, W., J. Holoshitz, Z. Nevo, A. Frenkel, A. Klajman, I. R. Cohen. 1985. Arthritis induced by a T-lymphocyte clone that responds to M. tuberculosis and to cartilage proteoglycans. Proc. Natl. Acad. Sci. USA 82:5117.[Abstract/Free Full Text]
  47. Van Eden, W., J. E. R. Thole, R. van der Zee, A. Noordzij, J. D. A. van Embden, E. J. Hensen, I. R. Cohen. 1988. Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis. Nature 331:171.[Medline]
  48. Hermann, E., A. W. Lohse, R. van der Zee, W. van Eden, W. J. Mayet, P. Probst, T. Poralla, K. H. Meyer zum Büschenfelde, B. Fleischer. 1991. Synovial fluid-derived Yersinia-reactive T cells responding to human 65-kDa heat-shock protein and heat-stressed antigen-presenting cells. Eur. J. Immunol. 21:2139.[Medline]
  49. Yi, Y., X. Yang, R. Brunham. 1997. Autoimmunity to heat shock protein 60 and antigen-specific production of interleukin-10. Infect. Immun. 65:1669.[Abstract]
  50. Anderton, S. M., R. van der Zee, B. Prakken, A. Noordzij, W. van Eden. 1995. Activation of T cells recognizing self 60-kD heat shock protein can protect against experimental arthritis. J. Exp. Med. 181:943.[Abstract/Free Full Text]
  51. Elias, D., A. Meilin, V. Ablamunits, O. S. Birk, P. Carmi, S. Könen-Waisman, I. R. Cohen. 1997. Hsp60 peptide therapy of NOD mouse diabetes induces a Th2 cytokine burst and downregulates autoimmunity to various ß-cell antigens. Diabetes 46:758.[Abstract]
  52. Shirai, M., M. Chen, T. Arichi, T. Masaki, M. Nishioka, M. Newman, T. Nakazawa, S. M. Feinstone, J. A. Berzofsky. 1996. Use of intrinsic and extrinsic helper epitopes for in vivo induction of anti-hepatitis C virus cytotoxic T lymphocytes (CTL) with CTL epitope peptide vaccines. J. Infect. Dis. 173:24.[Medline]
  53. Yasutomi, Y., T. J. Palker, M. B. Gardner, B. F. Haynes, N. L. Letvin. 1993. Synthetic peptide in mineral oil adjuvant elicits simian immunodeficiency virus-specific CD8+ cytotoxic T lymphocytes in rhesus monkeys. J. Immunol. 151:5096.[Abstract]
  54. Kern, F., I. P. Surel, C. Brock, B. Freistedt, H. Radtke, A. Scheffold, R. Blasczyk, P. Reinke, J. Schneider-Mergener, A. Radbruch, P. Walden, H. D. Volk. 1998. T-cell epitope mapping by flow cytometry. Nat. Med. 4:975.[Medline]
  55. Waldrop, S. L., C. J. Pitcher, D. M. Peterson, V. C. Maino, L. J. Picker. 1997. Determination of antigen-specific memory/effector CD4+ T cell frequencies by flow cytometry. J. Clin. Invest. 99:1739.[Medline]
  56. Gaston, J. S. H.. 1998. Heat shock proteins as potential targets in the therapy of inflammatory arthritis. Biotherapy 10:197.[Medline]



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