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*CYSTEINE
*L-SERINE
The Journal of Immunology, 2004, 173: 6564-6573.
Copyright © 2004 by The American Association of Immunologists

The Solvent-Inaccessible Cys67 Residue of HLA-B27 Contributes to T Cell Recognition of HLA-B27/Peptide Complexes1

Heiner Appel2,3,*, Wolfgang Kuon3,*, Maren Kuhne*, Martin Hülsmeyer{dagger}, Simon Kollnberger§, Stefanie Kuhlmann*, Elisabeth Weiss, Martin Zeitz*, Kai Wucherpfennig||, Paul Bowness§ and Joachim Sieper*,{ddagger}

* Division of Gastroenterology, Infectiology and Rheumatology, Charité Berlin, Campus Benjamin Franklin, {dagger} Institute for Chemistry/Crystallography, Free University Berlin, and {ddagger} Deutsches Rheumaforschungszentrum Berlin, Berlin, Germany; § Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; Institute for Anthropology and Human Genetics, Ludwig-Maximillians-Universität Munich, Munich, Germany; and || Department of Cancer Immunology and AIDS, Dana Farber Cancer Institute and Department of Neurology, Harvard Medical School, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Crystallographic studies have suggested that the cysteine at position 67 (Cys67) in the B pocket of the MHC molecule HLA-B*2705 is of importance for peptide binding, and biophysical studies have documented altered thermodynamic stability of the molecule when Cys67 was mutated to serine (Ser67). In this study, we used HLA-B27.Cys67 and HLA-B27.Ser67 tetramers with defined T cell epitopes to determine the contribution of this polymorphic, solvent-inaccessible MHC residue to T cell recognition. We generated these HLA-B27 tetramers using immunodominant viral peptides with high binding affinity to HLA-B27 and cartilage-derived peptides with lower affinity. We demonstrate that the yield of refolding of HLA-B27.Ser67 molecules was higher than for HLA-B27.Cys67 molecules and strongly dependent on the affinity of the peptide. T cell recognition did not differ between HLA-B27.Cys67 and HLA.B27.Ser67 tetramers for the viral peptides that were investigated. However, an aggrecan peptide-specific T cell line derived from an HLA-B27 transgenic BALB/c mouse bound significantly stronger to the HLA-B27.Cys67 tetramer than to the HLA-B27.Ser67 tetramer. Modeling studies of the molecular structure suggest the loss of a SH ... {pi} hydrogen bond with the Cys->Ser substitution in the HLA-B27 H chain which reduces the stability of the HLA-B27/peptide complex. These results demonstrate that a solvent-inaccessible residue in the B pocket of HLA-B27 can affect TCR binding in a peptide-dependent fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ankylosing spondylitis (AS)4 is a frequent chronic rheumatic disorder with strong association to HLA-B27 (1). Mainly based on this HLA association, it has been postulated to be a T cell-driven disease, but the molecular basis for the pathogenesis remains an open question. The principal known function of HLA-B27 is the binding of antigenic peptides and the presentation of such peptides to CD8+ T lymphocytes. Therefore, one main hypothesis advanced for the association between HLA-B27 and spondyloarthropathies is the arthritogenic peptide theory. It states that particular subtypes of HLA-B27, due to unique properties of the peptide binding site, bind certain arthritogenic peptide(s) that are recognized by CD8-positive T cells (2, 3, 4, 5) and cause local immunopathology (6). Therefore, the identification of HLA-B27-restricted T cell epitopes in HLA-B27-associated diseases is of ongoing interest (3, 5, 7, 8, 9, 10) and the use of HLA-B27 tetramers is an attractive method to trace these cells (11).

Mature MHC class I molecules are cell surface glycoproteins consisting of an H chain, noncovalently bound {beta}2-microglobulin, and a peptide (12). Crystallographic studies show that the H chain of MHC class I molecules forms a peptide-binding groove with six pockets termed A to F for Ag presentation (13). The Cys67 residue of HLA-B27 is an integral part of the B pocket, which determines the specificity of the MHC molecule to peptides with arginine at position 2 (pR2) (14, 15, 16). Comparative studies of HLA-A and -B alleles indicated that position 67 in the {alpha}1 domain is highly polymorphic suggesting that this residue is relevant for peptide binding (17, 18). A cysteine is present at position 67 in all subtypes of HLA-B27 (B*2701–2725), with the exception of the rare subtypes B*2718 (67-Ser) and B*2723 (67-Phe) (19, 20). The crystal structure of HLA-B27 suggests that the Cys67 contributes to the specificity of the critical B pocket of the binding site together with the highly conserved Glu45 and Thr24 residues. Additionally, Cys67 allows the formation of homodimeric and oligomeric HLA-B27 H chain complexes (21, 22, 23). It has been suggested that polymorphisms within the MHC class I peptide binding groove influence peptide presentation and T cell repertoire selection (16, 24, 25, 26, 27, 28, 29). Most recently the power of a single H chain residue on peptide presentation was nicely demonstrated in a crystallographic study (10). In the nondisease-associated allele HLA-B*2709 which features histidine 116 in the F pocket, the central pR5 residue of the bound viral peptide points toward the solvent. In contrast, in the disease-associated allele B*2705 pR5 from the same peptide points into the binding groove and forms a salt bridge with the subtype-specific aspartate 116. This unusual interaction results in a drastically altered peptide conformation in B*2705 compared with that in HLA-B*2709.

Two recent studies addressed the influence of Cys67 on the peptide-binding properties of HLA-B27 by performing functional analysis of the interaction between HLA-B27 and peptide. The peptide elution profile from HLA-B27 with mutant Ser67 was compared with nonmutant HLA-B27 molecules. These studies suggested that Cys67 plays a critical role in controlling the thermodynamic stability of soluble HLA-B27 molecules but that the destabilization of this molecule through the Cys->Ser substitution is not accompanied by an alteration of the peptide-binding specificity (25). This observation differs from the results of another study showing that HLA-B27 peptide ligands failed to bind to mutant HLA-B27 molecules with serine at position 67. Instead, this molecule bound peptides not being found in nonmutated HLA-B27 molecules (30). However, the relevance of the latter seems to be more relevant because more than 1000 peptides that are naturally bound to either one or both of these molecules were analyzed compared with four peptides in the first study. It was also shown in that study that TCR binding of HLA-B27/peptide complexes was altered by using alloreactive CTLs in cytotoxicity assays suggesting that the Cys->Ser mutation may weaken the interaction between B pocket and peptide (30). In such cellular systems it is difficult to discriminate between an effect of the Cys->Ser substitution on HLA-B27 assembly/transport vs TCR recognition. Therefore, we used HLA-B27 tetramers to directly probe TCR binding of HLA-B27 Cys67 or Ser67 molecules loaded with defined peptides. We show that the Cys->Ser substitution at residue 67 in the H chain of HLA-B27 affects TCR recognition in a peptide-dependent fashion, even though this MHC residue is located deep within the B pocket of the peptide-binding site.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Blood donors

Peripheral blood was taken from healthy blood donors with a history of EBV infection.

Mice and peptide immunization of mice

BALB/c mice, transgenic for HLA-B2705 and human {beta}2-microglobulin, and nontransgenic BALB/c mice were used for these experiments. They were maintained at the barrier facilities of the Benjamin Franklin Medical Center (Berlin, Germany). The first peptide injection of transgenic female mice with the aggrecan peptide (SRHHAFCFR) was performed s.c. with 100 µg of the peptide emulsified in CFA, while for the following four to five injections, 100 µg of the peptide was emulsified in IFA. The time interval between peptide injections was usually 2 wk and the maximum time from the first injection to the last one was 10–12 wk. One week before the last immunization mice were sacrificed, the spleen was taken and splenocytes were pooled and used for FACS analysis (HLA-B27 tetramer staining or cytokine secretion experiments) and a part of the spleen was used for in situ HLA-B27 tetramer staining. The spleen was embedded in Tissue Tek (Sakura, Zoeterwoude, The Netherlands) and shock-frozen in liquid nitrogen. All experiments with the wild-type and transgenic animals were performed under the guidelines of the animal health ethical committee.

Peptide synthesis

Nonamer peptides were synthesized according to standard F-moc solid phase synthesis methods on a Syro-Synthezier (MultiSyn Tech, Witten, Germany), purified by HPLC (Shimadzu LC-10; Shimadzu Scientific Instruments, Duisburg, Germany) and identified by mass spectroscopy (LCQ, ion trap; Thermoquest, Eberbach, Germany). The purity of the peptides was >95%. Peptides were solubilized in DMSO. For T cell stimulation and FACS analysis of intracellular cytokine staining, the peptides were further diluted with serum-free medium at a concentration of 5 mg/ml and frozen at –80°C. The peptides used in this manuscript are listed in Table I.


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  		Table I. Sequence and binding scores of peptides to HLA-B2705

 
Analysis of HLA-B27 peptide-binding properties

The peptide-binding properties were estimated by using two different epitope prediction programs described by Rammensee et al. (31) and by the BioInformatics and Molecular Analysis Section (BIMAS) (http://bimas.dcrt.nih.gov/molbio/hla_bind/). These programs combine knowledge about MHC class I-binding patterns and functional data. A scoring system for HLA-B27 was used, giving the highest numbers for arginine at anchor position 2, the second highest numbers for amino acids at the auxiliary position 9, and lower numbers for preferred amino acids. Peptides with scores of >27 (31) and >1500 (BIMAS) were regarded as high affinity ligands.

T cell lines

Human peripheral blood cells were stimulated with 5 µg/ml EBV EBNA 258–266 peptide (32) in the presence of 20 U/ml IL-2, 10 ng/ml IL-7, and 10 ng/ml IL-15 in T cell medium containing 10% human serum (PAA Laboratories, Cölbe, Germany), penicillin, and glutamine (Sigma-Aldrich, Taufkirchen, Germany) for 2 wk. Cytokines were added at day 2 and afterward every 3–4 days.

Murine splenocytes primed in vivo with the aggrecan peptide were restimulated in vitro with 10 µg/ml of the respective peptide. The murine splenocytes were cultured at 37°C and 5% CO2, in RPMI 1640 culture medium, supplemented with 10% FCS (Invitrogen Life Technologies, Karlsruhe, Germany), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate and 5 x 10–5M 2-ME (Sigma-Aldrich).

Refolding of rHLA-B27 molecules

The renaturation of HLA-B27 molecules (HLA-B27 heterotrimers consisting of HLA-B27 H chain, {beta}2-microglobulin, and peptide) was performed at 4°C for 48 h in 200 ml of refolding buffer containing 100 mM Tris, pH 8.3, 400 mM L-arginine, 2 mM EDTA, and 20% glycerol. {beta}2-microglobulin and HLA-B27 H chain in an injection buffer consisting of 3 M guanidine hydrochloride (GuHCl), pH 4.2, 10 mM sodium acetate, and 10 mM EDTA and the respective peptides were added to the refolding buffer. Following refolding, proteins were concentrated in a Vivaflow 50 concentrator (Vivascience, Hannover, Germany) against 50 mM bicine, pH 8.3. Subsequent concentration was performed with a Vivaspin 6 concentrator (cutoff 10,000 Da; Vivascience) down to 1 ml. The yield of HLA-B27.Cys67 and HLA-B27.Ser67 refolding with different peptides (Table I) was analyzed by HPLC gel filtration in PBS, pH 7.4 (Superose 12, Äkta Basic System; Amersham Biosciences, Freiburg, Germany). The product peak was identified by SDS-PAGE at the elution volume of ~13.7 ml. The yield of correctly folded HLA-B27 molecules was determined by integration of the area below the elution profile using the Unicorn software (version 4; Amersham Biosciences). The peak area between 7.8 and 15.9 ml was defined as 100%.

Peaks eluted after 16 ml were excluded from analyzing the percentage of refolded proteins because SDS-PAGE analysis did not detect any proteins in these fractions. The elution profile was analyzed by using Unicorn Software (version 4; Amersham Biosciences). For refolding of an HLA-A2 molecule (a kind gift of Dr. K. H. Lee, Department for Hematology and Oncology, Charité Berlin, Campus Benjamin Franklin), we used an immunodominant HLA-A2-restricted epitope from EBV (Table I).

FACS analysis of CD8+ T cells with HLA-B27 tetramers

HLA-B27 tetramers were generated as previously described (21) with some modifications. The expression vector pLM1-HLA-B27 was modified by tagging a BirA recognition site sequence as previously described (33). We used a wild-type HLA-B27 H chain with cysteine at position 67 and a mutated HLA-B27.Ser67 H chain for generating HLA-B27 molecules (21). For studying TCR binding of T cell epitopes presented by both rHLA-B27 molecules, three peptides were used: 1) EBV EBNA 258–266 (32), 2) an aggrecan-derived peptide (W. Kuon, unpublished data), and 3) a Chlamydia trachomatis-derived peptide no. 138 (11 ; Table I). The tetramers containing the latter peptide were used for control staining experiments. Soluble and refolded HLA-B27/peptide complexes were purified following biotinylation by HPLC gel filtration (Äkta Basic; Amersham Biosciences) and further analyzed by gel electrophoresis (Bio-Rad, Munich, Germany). Tetramers were generated by adding PE-labeled streptavidin (Molecular Probes, Eugene, OR) at a molecular ratio of 1:4. For FACS analysis, fresh mononuclear cells from peripheral blood or splenocytes from HLA-B27 transgenic mice were incubated with a tetramer and allophycocyanin-labeled anti-human CD8 Ab or anti-murine CD8 Ab (BD Pharmingen, San Diego, CA) for 30 min at room temperature followed by washing twice with PBS/2% BSA and incubation with anti-human CD3 Ab (BD Pharmingen) for 30 min at room temperature. Cells were washed twice in PBS/2% BSA and resuspended in annexin V buffer (Molecular Probes) and 2.5 µl of annexin V (Molecular Probes) was added. CD8+ and tetramer-positive T cells were analyzed after gates were set on CD3-positive and annexin V-negative cells.

Analysis was done by using a BD Biosciences (San Jose, CA) FACScan flow cytometer with CellQuest software.

Staining for T cell surface markers, intracellular cytokines, and analysis by flow cytometry

T cells were stained after in vitro stimulation as described before (34). Briefly, cells from whole peripheral blood were washed with PBS/BSA, centrifuged, and stained for the CD8- and CD69-surface markers and for the intracellular cytokine IFN-{gamma}. All stainings were performed in FACS Permeabilizing Solution (BD Biosciences, Heidelberg, Gemany). To avoid nonspecific binding of Abs to FcRs, staining was done in the presence of beriglobin (3 mg/ml; Centeon Pharma, Berlin, Germany). The following Abs were used: anti-human CD8 PerCP (clone Leu-3a; BD Biosciences), anti-CD69 PE (Leu-23; BD Biosciences), and anti-IFN-{gamma} coupled to Cy5 (Amersham Pharmacia Biotech, Freiburg, Germany). Positive cells were subsequently quantified by flow cytometry using a FACSCalibur from BD Biosciences with CellQuest Software. After gating on CD8+ T cells, only cytokine-positive T cells which were also positive for the early activation surface Ag CD69 were counted.

CD8+ T cells were regarded as positive after Ag-specific stimulation as judged by the percentage of CD69/cytokine double-positive cells if the gated CD8+ T cells were positive without background staining (stimulation with anti-CD28 without Ag only) (34). CD69 is an early T cell activation marker and is up-regulated shortly after stimulation with specific Ags. Thus, specificity of intracellular cytokine staining is increased by excluding cytokine+/CD69– T cells (nonspecific staining of the intracellular cytokines) from analysis.

In situ staining of Ag-specific CD8+ T cells with HLA-B27 tetramers

Sections from murine spleen were taken from HLA-B27 transgenic BALB/c mice immunized with aggrecan peptide (W. Kuon, unpublished data), nonimmunized HLA-B27 transgenic BALB/c mice and BALB/c mice immunized with aggrecan peptide. In situ staining was done with HLA-B27.Cys67 aggrecan peptide-, HLA-B27.Ser67 aggrecan peptide-, HLA-B27.Cys67 Chlamydia peptide no. 138-, HLA-B27.Ser67 Chlamydia peptide no. 138-, HLA-B27.Cys67 EBV peptide-, and HLA-B27-Ser67 EBV peptide tetramers. Tissue from the murine spleen was embedded in Tissue Tek and snap-frozen in liquid nitrogen. Seven-micrometer sections were prepared on a microtom at –15°C; nonfixated sections dried for 2 min at room temperature (RT) with subsequent rehydration in PBS. Sections were further blocked for 20 min in a blocking solution containing 4% milk powder solution, anti-mouse Fc{gamma}R Ab (clone 2.4G2/75; a kind gift of German Rheumatology Research Center, Berlin, Germany), and streptavidin from streptavidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) followed by washing in PBS and incubation in biotin for 15 min (streptavidin/biotin blocking kit; Vector Laboratories). After washing in PBS, HLA-B27 tetramers (50 µg/ml in PBS) were incubated for 1 h at RT; tetramer-binding T cells were further labeled with a goat anti-PE (1:500 in milk powder; Biomeda, Foster City, CA) and detected by Cy3-labeled anti-goat IgG1 Ab (1:300 in PBS; Dianova, Hamburg, Germany). For CD8 staining, sections were incubated with an allophycocyanin-labeled anti-mouse CD8 Ab (1:100 in milk powder; BD Biosciences) for 1 h at RT and detected by a rabbit anti-allophycocyanin Ab (30 min, 1:500; Biozol, Munich, Germany) and a Cy2-labeled anti-rabbit IgG1 Ab (30 min, 1:300; Dianova). Cell nuclei were detected by 4', 6-diamidino-2-phenylindole (DAPI) (1:3000; Roche, Penzberg, Germany).

Sections were analyzed with an immune fluorescence microscope (Olympus, Hamburg, Germany). Pictures were taken with a digital camera (Olympus) and further analyzed by analysis software (Soft Imaging System (SIS, Muenster, Germany). From each spleen, four sections were taken and completely scanned for tetramer-binding CD8+ T cells. For further analysis of HLA-B27 tetramer-stained CD8+ T cells, we performed confocal microscopy. After mounting, the tissue sections were analyzed by confocal laser scanning microscopy using a Leica TCS/DMIRB (Deerfield, IL). Individual scans were analyzed using TCS-NT software and Adobe Photoshop (Adobe Systems, Mountain View, CA).

Staining of Ig-like transcript (ILT)-2 bafcells with HLA-B27.Cys67 and Ser67 tetramers

ILT-2 receptor binds MHC class I molecules (35) and delivers a negative signal that inhibits killing by NK cells and T cells. ILT-2 receptor transfected and nontransfected baf cells (a kind gift from L. Lanier, University of California, San Francisco, CA) were incubated with tetramers at 37°C for 20 min in 50 µl of RPMI 1640 supplemented with 10% FCS. Cells were then washed twice on ice with FACS wash buffer (PBS, 0.5% w/v BSA, 0.02% (w/v) sodium azide) and fixed before FACS analysis. To determine the effect of Cys67->Ser67 substitution on the stability of the HLA-B27 peptide complex, we incubated the HLA-B27.Ser67 aggrecan peptide and HLA-B27.Cys67 aggrecan peptide tetramers at 37°C for 1, 6, and 24 h and performed FACS analysis with ILT-2 transfected cells at a concentration of 1.5 µg/250,000 cells in 50 µl. The first staining at 0 h was performed at room temperature.

Homology modeling of the B pocket of HLA-B27

Based on the structure of HLA-B*2705:peptide m9 (36), the Cys67 residue was mutated manually using the program O (37). Distances and angles were determined with O as well. Fig. 6 was generated by using Molscript (Avatar Software, Stockholm, Sweden) (38).



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  		FIGURE 6. Impact of residue 67 on the coordination of pR2 in the B pocket of HLA-B27. A, Overall topological view into the peptide-binding groove based on HLA-B*2705:m9 (36 ). Secondary structure is depicted in gray, the peptide is shown as a stick representation. Close-up views into the B pockets of B*2705.Cys67 (B) and B*2705.Ser67 (C). Oxygen atoms are shown in red, nitrogen in blue, sulfur in yellow, carbon atoms from the H chain are depicted in gray, from the peptide in green. The pR2 side chain is stabilized by several polar interactions (black dotted lines). A rare SH ... {pi} bond (orange dotted line) between the cysteine and the Arg guanidyl head group contributes to the hydrogen bonding network. Mutation of Cys67 to Ser is proposed to establish an altered network with Ser67OH contacting E63O (blue dotted line).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Refolding of HLA-B27.Cys67 and HLA-B27.Ser67 molecules with peptides of different binding affinity to HLA-B27

Tetramers of HLA-B27/peptide complexes have been made with HLA-B27 H chains in which Cys67 has been substituted with serine, in an effort to reduce aggregation based on disulfide bond formation. It has been assumed that this substitution would not affect TCR binding, given the location of this residue within the B pocket and the rather conservative nature of this substitution. Given the published studies indicating a reduced thermal stability of HLA-B27/peptide complexes with the Cys->Ser substitution, we decided to examine the feasibility of generating HLA-B27 tetramers with the native cysteine at position 67, in particular for peptides with potential relevance to the pathogenesis of AS. With the EBV peptide, a well-described immunodominant peptide (32) with a high binding score to HLA-B2705 (score 28 (31) and score 2000 in BIMAS), the yield of refolding was efficient for both HLA-B27.Cys67 and Ser67 molecules: 88.2% for HLA-B27.Ser67 and 61.8% for HLA-B27.Cys67 (Table I, Fig. 1).



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  		FIGURE 1. Refolding of HLA-B27.Ser67 and HLA-B27.Cys67 molecules. Gel filtration analysis of refolded and biotinylated HLA-B27.Cys67 (upper three panels) and HLA-B27.Ser67 (lower three panels) molecules loaded with aggrecan peptide (left panels), EBV EBNA 258–266 peptide (middle panels), and Chlamydia peptide no. 138 (right panels).

 
Refolding was also observed with the aggrecan peptide which has lower peptide binding scores in both computer algorithms (score 20 (31), score 1000 in BIMAS). The yield of refolded protein was ~17% with the HLA-B27.Cys.67 H chain (Fig. 1) and 38.4% with the mutated HLA-B27.Ser67 H chain (38.4%). As a control for FACS analysis and in situ tetramer staining we also generated an HLA-B27 tetramer with a C. trachomatis-derived peptide (peptide no. 138, (11)) with a binding score (score 26 in Ref.31), score 2000 in BIMAS) intermediate between EBV peptide and aggrecan peptide. These data demonstrated that HLA-B27 H chains with the native Cys67 could be refolded with {beta}2-microglobulin and peptide, even though the yield was lower than for the mutated HLA-B27 Ser67 H chain due to a higher extent of aggregation (Fig. 1).

The reliability of the refolding assay and its correlation to peptide-binding affinity was further documented by refolding assays with an HLA-A2 molecule, which is widely used in MHC class I tetramer technology, and an immunodominant peptide from EBV (39) with high-binding affinity (29 (31) and 6000 (BIMAS)) (Table I). The yield of refolded MHC molecule was 92.9%. Another HLA-B27-restricted viral peptide with a lower binding score (influenza virus NP 383–391, binding score 26 (31), 1500 (BIMAS)) gave a lower yield of refolding (52.1%). Refolding with a peptide from collagen II, C34 (P. Atagunduz, unpublished data) revealed a low yield of refolded HLA-B27 molecules supporting the low binding score given by one computer algorithm (BIMAS).

Staining of ILT-2-transfected baf cells with HLA-B27.Cys67 and Ser67 tetramers

We used a biological test to address the question of whether serine for cysteine mutation changes the stability of HLA-B27 molecules and affects HLA-B27 binding to ILT-2-transfected baf cells (35). ILT-2-transfected baf cells were incubated with HLA-B27.Cys67 and Ser67 tetramers. Binding of both HLA-B27 tetramers refolded with EBV and aggrecan peptide was documented by FACS staining indicating that all refolded complexes are structurally intact HLA-B27/peptide molecules linked to streptavidin (tetramers) (data not shown). We next studied the stability of these complexes after different periods of time upon heating to 37°C. The staining intensity with the HLA-B27.Ser67 aggrecan peptide tetramer was already significantly reduced after 1 h of preincubation at 37°C compared with the staining at 0 h at room temperature and further decreased depending on the preincubation time (Fig. 2). The ILT-2 staining with HLA-B27.Cys67 aggrecan peptide tetramers also demonstrates the presence of active HLA-B27/peptide molecules because homodimeric HLA-B27 molecules would not bind to ILT-2 receptors.



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  		FIGURE 2. Staining of ILT-2-transfected and -untransfected (filled plots) baf cells with HLA-B27.Cys67 and Ser67 tetramers refolded with the aggrecan peptide. After preincubation at 37°C for 1, 6, and 24 h FACS staining with HLA-B27.Ser67 aggrecan peptide tetramer and HLA-B27.Cys67 aggrecan peptide tetramer was performed and compared with FACS staining with these molecules at 0 h at room temperature. Already after 1 h, a significant decrease in staining intensity could be observed with the HLA-B27.Ser67 aggrecan peptide tetramer.

 
FACS analysis of HLA-B27-restricted CD8+ T cells with HLA-B27.Cys67 and HLA-B27.Ser67 tetramers loaded with aggrecan peptide

In this experiment we addressed the question of whether the substitution of serine for cysteine at position 67 in the HLA-B27 H chain influences T cell recognition. HLA-B27 transgenic mice were immunized with the aggrecan peptide and intracellular cytokine (IFN-{gamma}) staining was performed on splenocytes after 6 h of in vitro aggrecan peptide-specific restimulation. IFN-{gamma}-secreting CD8+ T cells (0.35%) could be detected as being aggrecan peptide specific (Fig. 3, day 1, upper panels). Stimulation with anti-CD28 or control peptide (Chlamydia-derived peptide no. 138) did not induce any IFN-{gamma}-secreting T cells.



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  		FIGURE 3. HLA-B27 tetramer staining of aggrecan peptide-specific CD8+ T cells. Aggrecan peptide-specific CD8+ T cell detection with both HLA-B27.Cys67 (0.16%) and HLA-B27.Ser67 (0.12%) tetramers in splenocytes from HLA-B27 transgenic BALB/c mice immunized with aggrecan peptide (day 1, lower FACS histograms). The specificity of staining was confirmed by intracellular cytokine staining (0.35%) (day 1, upper FACS histograms). By using a short-term T cell line specific for aggrecan peptide the difference between the two HLA-B27 tetramers becomes obvious: 4.13% Ag-specific T cells were detected by staining with HLA-B27.Cys67 tetramer while only 0.86% were stained with HLA-B27.Ser67 tetramer (day 7, lower FACS histograms). The specificity of Ag-specific T cell detection was confirmed by intracellular cytokine staining (6.53% IFN-{gamma}-secreting CD8+ T cells after peptide-specific stimulation) (day 7, upper FACS histograms).

 
When tetramer staining was done with the same murine splenocytes, 0.16% tetramer-binding CD8+ T cells (gates were set on CD3+ T cells) could be detected with the HLA-B27.Cys.67 tetramers while 0.12% bound to HLA-B27.Ser67 tetramers with a lower shift in the histogram (Fig. 3, day 1, lower left panels). The experiment was repeated in two other equally treated mice resulting in 0.12 and 0.10% HLA-B27.Cys67 aggrecan peptide tetramer-positive CD8+ T cells and 0.10% and 0.12% HLA-B27.Ser67 aggrecan tetramer-positive CD8+ T cells, respectively. The same staining was repeated after these T cells had been stimulated in vitro with the aggrecan peptide for 7 days. By using intracellular cytokine staining, 6.53% of aggrecan-specific IFN-{gamma}-secreting CD8+ T cells could be detected (Fig. 3, day 7, upper panels). Anti-CD28 Abs alone or the control peptide did not induce any T cell stimulation. Comparison of Cys67 and Ser67 HLA-B27 tetramers with this aggrecan peptide-specific T cell line revealed animportant result (Fig. 3, day 7, lower right panel): while HLA-B27.Ser67 tetramers detected only a minority of aggrecan peptide-specific CD8+ T cells (0.86%) with a low shift in the histogram, HLA-B27.Cys67 tetramers detected 4.13% aggrecan peptide-specific CD8+ T cells, a percentage of CD8+ T cells similar to that detected by intracellular cytokine staining. The numbers for the other in vitro-expanded splenocytes of two other mice were 0.64% and 1.35% HLA-B27.Ser67 aggrecan peptide tetramer-positive CD8+ T cells and 4.32% and 34.6% HLA-B27.Cys67 aggrecan peptide tetramer-positive CD8+ T cells, respectively. When these tetramers were used in nonimmunized HLA-B27 transgenic BALB/c mice and in immunized wild-type BALB/c mice, no HLA-B27 tetramer staining was observed (data not shown).

FACS analysis of HLA-B27-restricted CD8+ T cells with HLA-B27.Cys67 and HLA-B27.Ser67 tetramers loaded with a high affinity immunodominant EBV peptide

We repeated the above-mentioned experiments with EBV EBNA 258–266-specific CD8+ T cells to determine whether T cell recognition of the MHC/peptide complex is also altered for peptides with a higher affinity. Peripheral blood was taken from an HLA-B27+ blood donor with previous EBV infection. Intracellular cytokine staining revealed 0.84% IFN-{gamma}-secreting CD8+ T cells after 6 h of EBV EBNA 258–266 peptide-specific in vitro stimulation (Fig. 4, day 1, upper panels). FACS staining with the HLA-B27.Cys67 and HLA-B27.Ser67 tetramers showed a comparable result of Ag-specific T cell detection (Fig. 4, day 1, lower panels). The same experiment was repeated with an EBV peptide-specific T cell line (Fig. 4, day 14, lower panels). HLA-B27.Cys67 and HLA-B27.Ser67 tetramers again detected similar numbers of EBV-specific CD8+ T cells, 76.69% tetramer binding CD8+ T cells with the HLA-B27.Cys67 tetramer, and 79.89% with the HLA-B27.Ser67 tetramer. The finding that similar populations of CD8+ T cells were labeled with both tetramers was reproducible in multiple experiments with the shown T cell line, and also with other T cell lines of the same specificity (data not shown). The specificity of tetramer staining was confirmed by intracellular cytokine staining of IFN-{gamma}-secreting and CD69+ T cells (41.14%) after peptide-specific T cell stimulation (Fig. 4, day 14, upper panels).



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  		FIGURE 4. HLA-B27 tetramer staining of EBV peptide-specific CD8+ T cells. EBV EBNA 258–266 peptide-specific CD8+ T cell detection with both HLA-B27.Cys67 (0.46%) and HLA-B27.Ser67 (0.44%) tetramers in an HLA-B27+ blood donor with previous EBV infection (day 1, lower FACS histograms). The specificity of staining was confirmed by intracellular cytokine staining (0.84% of CD8+ T cells) (day 1, upper FACS histograms). By using a short-term T cell line specific for this EBV peptide no difference between the two HLA-B27 tetramers was seen in FACS analysis: 76.69% Ag-specific T cells were detected by staining with HLA-B27.Cys67 tetramer while 79.89% were stained with HLA-B27.Ser67 tetramer (day 14, lower FACS histograms). The specificity of Ag-specific T cell detection was confirmed by intracellular cytokine staining (41.14% IFN-{gamma}-secreting CD8+ T cells after peptide-specific T cell stimulation) (day 14, upper FACS histograms).

 
In situ tetramer staining of HLA-B27-restricted CD8+ T cells in the spleen of HLA-B27 transgenic BALB/c mice

To compare T cell recognition of both HLA-B27 tetramers loaded with the aggrecan peptide in situ, we examined snap-frozen spleens (40) from aggrecan-peptide immunized HLA-B27 transgenic BALB/c mice, nonimmunized HLA-B27 transgenic BALB/c mice and aggrecan peptide-immunized and nonimmunized BALB/c mice. From FACS analysis of tetramer staining of murine splenocytes a frequency of up to 0.3% tetramer-positive T cells among CD8+ T cells in the spleen was expected.

In aggrecan peptide-immunized HLA-B27 transgenic BALB/c mice, we identified aggrecan peptide-specific CD8+ T cells in these in situ staining experiments. As shown in Fig. 5 tetramer-binding cells could be detected when HLA-B27.Cys67 tetramers with the aggrecan peptide were used (Fig. 5B), but not when HLA-B27.Ser67 tetramers with the aggrecan peptide were used (not shown). When double staining with anti-CD8 Ab (green) and HLA-B27.Cys67 tetramer (red) was performed, double-positive cells could be detected (yellow) (Fig. 5C). In this experiment, all tetramer-positive T cells were also CD8+ T cells. Using the HLA-B27.Ser67 tetramer with the aggrecan peptide did not provide any in situ detection of aggrecan peptide-specific CD8+ T cells (not shown). HLA-B27.Cys67 tetramers with Chlamydia-derived peptide no. 138 and with EBV peptide did not reveal in situ staining in the same mice (data not shown). To confirm the specificity of our results, these experiments were repeated in nonimmunized HLA-B27 transgenic BALB/c mice and aggrecan peptide-immunized BALB/c mice (data not shown). In none of these mice did HLA-B27.Cys67 tetramers loaded with the aggrecan peptide bind to splenocytes (data not shown). These experiments demonstrate that in the case of in situ detection, T cell recognition of Ag-specific CD8+ cells with HLA-B27.Ser67 tetramers is completely abolished.



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  		FIGURE 5. In situ tetramer staining of CD8+ murine splenocytes with HLA-B27.Cys67 tetramer analyzed by confocal microscopy. In situ staining of CD8+ T cells (A). In situ HLA-B27 tetramer staining with HLA-B27.Cys67 aggrecan peptide tetramer (B). Double staining of CD8+ T cells with labeled HLA-B27.Cys67 aggrecan peptide tetramer (C). The experiment was performed in a spleen of an HLA-B27 transgenic BALB/c mice immunized with the aggrecan peptide.

 
Impact of Cys67 for the coordination of pR2 in the B pocket

Various crystal structures of HLA-B*2705 demonstrate that the B pocket is sterically and electrostatically ideally suited to accommodate an arginine side chain (Fig. 6). Its hydrophobic part forms van der Waals contacts to Tyr7 and Ile66, while the charged guanidyl moiety is stabilized by a bifurcated salt bridge to Glu45 and a hydrogen bond to Thr24. The side chain of Cys67 (Fig. 6B) points directly toward the pR2 guanidyl group and fulfills the geometric requirements to qualify as SH – {pi} hydrogen bond ({omega} – angleSH... {pi} < 25° [20°] and distance S... {pi} < 4.0 Å [3.5 Å]) (41). This type of hydrogen bond is rarely found and is suggested to be weaker than classical hydrogen bonds (41). Mutation of Cys67 to Ser67 abrogates this interaction (Fig. 6B) as an intact OH ... {pi} hydrogen bond should not be longer than 3.8 Å (41), but is actually found to be 4.1 Å due to the shorter C-O bond distance in the serine side chain. Instead, by only a small reorientation of roughly 25°, the Ser67 hydroyl group can engage in a standard hydrogen bond (2.8 Å) to the backbone oxygen of Glu63. Based on the hard soft acid base concept, this interaction is clearly favored over a weak OH ... {pi} hydrogen bond.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By using HLA-B27 tetramer technology we demonstrate that a substitution of residue Cys67 to Ser67 in the B pocket of the HLA-B27 H chain affects T cell recognition in a peptide-dependent fashion.

The alteration of peptide presentation and reduction of T cell recognition as a consequence of Ser67 substitution is currently a matter of discussion (25, 30). Our results indicate that an alteration of T cell recognition by HLA-B27.Ser67/peptide complexes is present which is strongly dependent on the peptide bound to the HLA-B27 molecule. This conclusion is based on refolding of HLA-B27 molecules with Cys67 and Ser67 H chains with different peptides and the use of such molecules for tetramer staining of peptide-specific CD8+ T cells by in situ and FACS analysis.

In our studies, we used a human aggrecan-derived peptide (W. Kuon, unpublished data) to immunize HLA-B27 transgenic BALB/c mice to obtain CD8+ T cells with the respective specificity. FACS staining with HLA-B27.Ser67 and HLA-B27.Cys67 tetramers of murine CD8+ T cell lines, which were generated in vitro from splenocytes of these mice, revealed significantly reduced numbers of tetramer-positive CD8+ T cells when HLA-B27.Ser67 tetramers were used (0.86% vs 4.13% with HLA-B27.Cys67). By in situ staining, TCR binding with HLA-B27.Ser67 tetramers was, in contrast to HLA-B27.Cys67 tetramers, not detectable. These results indicate that presentation of the aggrecan peptide to CD8+ T cells is fundamentally altered when cysteine is substituted by serine at residue 67. We could not perform the same experiment with HLA-B27.Ser67 transgenic mice to see whether this difference would be the same. However, we would have expected the same result. In contrast, by using a well-described immunodominant EBV-derived peptide, Ag presentation through HLA-B27.Ser67 was not significantly different from HLA-B27.Cys67 because HLA-B27 tetramer staining with both molecules revealed almost identical numbers of Ag-specific CD8+ T cells.

These findings raise the question of whether the differences in T cell recognition can be correlated to different binding modes of pR2 as a consequence of the mutated residue 67 in the B pocket. In fact, a drastic alteration in the peptide presentation mode caused by a single buried and polymorphic residue was recently described (10). However, the situation is different in the situation discussed here. The Cys67->Ser substitution probably leads to the loss of a peculiar SH ... {pi} hydrogen bond and the formation of a new intrahelical interaction (Fig. 6). Even though this alteration leads to destabilization of the HLA-B27/peptide complex as shown by reduced melting temperatures for the HLA-B27.Ser67 variant (25), we do not think that a grossly different peptide conformation is present. The remaining interactions are unchanged, and the lost SH... {pi} bond is comparably weak, suggesting that the pR2 anchor is still securely bound in the B pocket. However, the differences in hydrogen bonding networks may allow more flexibility of the pR2 side chain in the mutated B27 variant, which, in turn, could be transmitted to the protein surface where such a change in mobility is translated into differential T cell recognition. This hypothesis is supported by the fact that differences in T cell recognition between the B27.Cys67 and the B27.Ser67 form are undetectable when the immunodominat EBNA peptide is present. In this case the pR1 residue stabilizes the B27.Ser67 molecule and compensates for the flexibility resulting from the B pocket mutation. The structural explanation of the thermodynamic stabilization effect is a stacking interaction of pR1 with W167 and R62 (42).

In line with this finding is the observation of the peptide repertoire eluted from HLA-B*2705 which shows a preference for arginine (R) at peptide residue P1 (43). Only in those cases, where p1 is not an arginine, the destabilized B pocket becomes "visible" at the protein surface in terms of differences in the T cell response as seen in our study for those complexes loaded with the aggrecan peptide.

Indeed, it has been proposed that the stability of the MHC-peptide complex is more important for T cell binding and T cell stimulation than peptide-binding affinity (44). We next used a biological test to address the question of whether a loss of stability of the HLA-B27/peptide complex could explain our results. HLA-B27 tetramers refolded with Ser67 and Cys67 H chains bound to baf cells transfected with an ILT-2 receptor, which binds to MHC class I molecules (35). After preincubation at 37°C the binding of the HLA-B27.Ser67 aggrecan peptide tetramer was significantly reduced after 1 h suggesting that the staining of ILT-2-transfected cells strongly depends on the stability of the HLA-B27 complex. These findings are in line with the hypothesis that the stability of the molecule might have been altered by the mutation at position 67 of the HLA-B27 H chain (25), which might also be the reason for an abrogation of mAb recognition of HLA-B27 molecules after site-directed mutagenesis of residue 67 Cys to tyrosine (45, 46). Further support is given by recent thermodynamic studies demonstrating for HLA-B*2705 and HLA-B*2709 that a single amino acid exchange at residue 116 can have large effects on the stability of these two HLA-B27 molecules (42).

The critical mechanisms for T cell recognition of MHC/peptide complexes are currently discussed controversially and there is some evidence that structural rearrangements or flexibility during the binding of MHC/peptide complexes to the TCR contribute to T cell activation (47). Our studies also indicate that in addition to structural properties, biophysical properties like thermodynamic stability may also influence T cell recognition of the MHC/peptide complex. More experiments are necessary to elucidate the precise molecular mechanisms by which the Ser67 mutation affects stability and peptide presentation by HLA-B27.

It has been speculated that the HLA-B27 association to AS is not related to Ag presentation but rather to a misfolding of HLA-B27 H chains or triggering of stress responses from the endoplasmatic reticulum (23, 48). In vitro experiments with HLA-B27 H chains revealed the presence of disulfide-linked HLA-B27 homodimers and stimulation of T cell responses by these homodimer molecules (22, 49, 50). We can rule out that tetramer staining in our experiments was caused by HLA-B27 homodimers because we did not detect any CD8+ T cells when HLA-B27.Cys67 tetramers were used with a control peptide. In addition, the presence of peptide-specific CD8+ T cells was confirmed by intracellular cytokine staining for IFN-{gamma}-producing cells after peptide stimulation. Moreover, we confirmed the presence of the peptide in the HLA-B27-binding groove by HPLC excluding that the absence of tetramer staining was caused by the fact that the aggrecan peptide did not bind to the HLA-B27-binding groove (data not shown).

What are the consequences for generating HLA-B27 tetramers based on these observations? The use of HLA-B27 tetramers was first described by studying critical binding sites of HLA-B27-restricted T cell receptors (21). For these experiments, mutant HLA-B27.Ser67 molecules loaded with an immunodominant peptide from influenza virus (51) were successfully used. We have also used HLA-B27.Ser67 tetramers effectively to study HLA-B27-restricted T cell epitopes in C. trachomatis-triggered reactive arthritis (52). Although the yield of refolded HLA-B27 molecules is higher with Ser67 H chain and T cell staining with HLA-B27-Ser67 tetramers is not abrogated for certain peptides, our data indicate that HLA-B27.Cys67 tetramers may permit identification of T cell populations that could be missed when HLA-B27.Ser67 tetramers are used.


   Acknowledgments
 
We thank Sabine Seibert and Ulrike Erben for performing the analysis of tissue sections by confocal microscopy.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Deutsche Forschungsgemeinschaft (DFG): Ap 82/2-1 and Ap 82/2-2 (to H.A. and J.S.), Sonderforschungsbereich 421 Project C1 (to W.K.), Sonderforschungsbereich 633, Klinische Forschergruppe 104, and by the Arthritis Research Campaign U.K. (to S.Ko.) and the Medical Research Council (to P.B.). Back

2 Address correspondence and reprint requests to Dr. Heiner Appel, Charité Berlin, Campus Benjamin Franklin, Department for Gastroenterology, Infectiology, and Rheumatology, Berlin, Germany. E-mail address: heiner.appel{at}charite.de Back

3 H.A. and W.K. contributed equally to this work. Back

4 Abbreviations used in this paper: AS, ankylosing spondylitis; RT, room temperature; ILT, Ig-like transcript. Back

Received for publication June 4, 2004. Accepted for publication August 20, 2004.


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