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Available online http://arthritis-research.com/content/6/6/R521 Research article Open Access Vol 6 No 6 Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis Heiner Appel1, Wolfgang Kuon1, Maren Kuhne1, Peihua Wu1,2, Stefanie Kuhlmann1, Simon Kollnberger3, Andreas Thiel2, Paul Bowness3 and Joachim Sieper1,2 1Charite Berlin, Campus Benjamin Franklin, Department for Gastroenterology, Infectiology and Rheumatology, Berlin, Germany Rheumaforschungszentrum Berlin, Germany 3MRC HIU, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK 2Deutsches Corresponding author: Heiner Appel, heinerappel@yahoo.com Received: 7 Apr 2004 Revisions requested: 13 May 2004 Revisions received: 8 Jun 2004 Accepted: 2 Jul 2004 Published: 23 Sep 2004 Arthritis Res Ther 2004, 6:R521-R534 (DOI 10.1186/ar1221) © 2004 Appel et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is cited. http://arthritis-research.com/content/6/6/R521 Abstract Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein– Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (CtReA). Four of six HLA-B27+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02–0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigenspecific CD8+ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8+ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8+ T cells could be increased by stimulating CD8+ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8+ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27+ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional. Keywords: HLA-B27, T cells, tetramers, reactive arthritis Introduction Chlamydia-triggered reactive arthritis (Ct-ReA) is strongly associated with HLA-B27 like other spondylarthropathies, and especially ankylosing spondylitis [1,2]. ReA occurs 1 to 4 weeks after urogenital infection with Chlamydia trachomatis or gastroenteral infection with enterobacteria such as Yersinia enterocolitica [3]. After acute onset, most patients have a self-limiting course, but up to 20% suffer from a disease duration of more than 1 year [4]. Of HLAB27+-reactive arthritis patients, 20–40% move on to anky- losing spondylitis after 10–20 years, suggesting that the ReA-associated bacteria can cause ankylosing spondylitis [5] and that immune mechanisms triggering the disease are induced by T cell responses to microbial antigens. The main hypothesis advanced for the association between HLA-B27 and spondylarthropathies is the arthritogenic peptide theory. It states that some HLA-B27 subtype alleles, owing to their unique amino acid residues, bind a specific arthritogenic peptide that is recognized by CD8+ T cells [6-9]. Recently we and several other groups have BIMAS = BioInformatics and Molecular Analysis Section; BSA = bovine serum albumin; Ct-ReA = Chlamydia-triggered reactive arthritis; DC = dendritic cell; EBV = Epstein–Barr virus; FACS = fluorescence-activated cell sorting; IFN-γ = interferon-γ ; IL = interleukin; MACS = magnetic activated cell sorting; MNC = mononuclear cell; MOMP = major outer membrane protein; PBS = phosphate-buffered saline; PE = phycoerythrin. R521 Arthritis Research & Therapy Vol 6 No 6 Appel et al. reported on Chlamydia-specific CD8+ T cells capable of lysing target cells primed with Chlamydia antigens [10-12]. CD8+ T cell responses in spondylarthropathies other than Ct-ReA have also been described [13-15]. Recently a new method for antigen-specific T cell recognition has been established by using multimerized MHC/peptide molecules [16]. These molecules are called tetramers because they contain four soluble and biotinylated MHC molecules linked to labelled streptavidin that specifically bind with high avidity to T cell receptors. In comparison with intracellular cytokine staining, the major advantage of tetramer technology is the identification of antigen-specific T cells independently of their cytokine secretion profile, the possibility of sorting unstimulated T cells and of having a tool for the antigen-specific detection of T cells in experiments in situ [17]. synovial proliferation against enterobacteria. We also tested the synovial fluid of three patients with rheumatoid arthritis. In addition we used HLA-B27+ and HLA-A2+ blood donors with previous EBV infection for experiments comparing HLA-B27 and HLA-A2 tetramers. The ethical committee of the Benjamin Franklin Medical Centre gave ethical approval for this study. Search for peptide binding affinity The quantification of HLA-B27 binding affinity was conducted with two different programs that analyse HLA-peptide binding motifs, one called SYFPEITHI described by Rammensee and colleagues [23] and the other called BioInformatics and Molecular Analysis Section (BIMAS; http:/ /bimas.dcrt.nih.gov/molbio/hla_bind/). Peptide synthesis In humans, MHC class I tetramers are widely used, and HLA-A2 tetramers in particular are an important tool in tumour immunology [18]. However, the use of HLA-B27 tetramers in HLA-B27-related diseases is rare [10,19]. The rarity of their use might be related to heavy protein aggregation during the refolding procedure of the recombinant HLA-B27 monomer [19,20]. To determine optimised conditions for the refolding procedure of soluble HLA-B27 monomers with bacteria-derived epitopes we first used HLA-B27 tetramers with a well-described HLA-B27restricted viral epitope from Epstein–Barr virus (EBV). We analysed the refolding rate of HLA-B27 monomers and compared our results with refolding gained with an HLA-A2 molecule loaded with a viral epitope from EBV [21]. On the basis of these results we applied the HLA-B27 tetramer technology to specify the HLA-B27-restricted CD8+ T cell response to Chlamydia-derived peptides in patients with Ct-ReA. This is the first report of a systematic use of HLA-B27 tetramers in humans in an HLA-B27-related disease. Methods Patients We analysed six HLA-B27+ and three HLA-B27- patients with ReA after infection with Chlamydia trachomatis (Table 1). We diagnosed ReA if patients had a prior urogenital infection, which was confirmed by the detection of Chlamydia trachomatis in the morning urine by polymerase chain reaction. An additional criterion was the detection of Chlamydia-specific antibodies [6] at the beginning of the disease or highest synovial T cell proliferation against Chlamydia trachomatis [22] in proliferation assays with whole Chlamydia antigen. The results were compared with tetramer staining in six HLA-B27+ healthy blood donors. We also examined synovial T cells from three HLA-B27+ patients with ReA after gastroenteritis and having highest R522 Nonamer peptides were synthesized by standard 9-fluorenyl-methyloxy-carbonyl solid-phase synthesis methods on a Syro-Synthesizer (MultiSyn Tech, Witten, Germany), purified by high-performance liquid chromatography (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 more than 95%. Peptides were dissolved in dimethyl sulphoxide. For T cell stimulation and fluorescence-activated cell sorting (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. FACS analysis of antigen-specific T cells with HLA-B27 tetramers HLA-B27 tetramers were generated as described previously [19], with some modifications. The expression vector pLM1-HLA-B27 was modified by tagging with the BirA recognition sequence as described previously and by mutating the cysteine residue at position 67 to serine. After being refolded, the recombinant protein was concentrated and centrifuged at 13,000 rpm (16,060g; Haereus Biofuge Pico; Kendro Laboratories, Langenselbold, Germany) followed by biotinylation and gel filtration with a Superose 12 column (Pharmacia) on an Äkta Basic system (Pharmacia). Correct folding and biotinylation were analysed by gel filtration (Äkta Basic, Pharmacia) and gel electrophoresis (BioRad). Tetramers were generated by adding phycoerythrin (PE)-labelled streptavidin (Molecular Probes) at a ratio of 1.5:1. We generated HLA-B27 tetramers with the EBV EBNA peptide (residues 258–266) [24]. For the detection of Chlamydia-peptide-specific CD8+ T cells we used the previously described immunodominant peptides 8, 68, 80, 131, 133, 138, 144, 145, 146, 194, 195 and 196 [10] (Table 2). Peptides 144 and 194 caused heavy aggregation during refolding procedure and were excluded from Available online http://arthritis-research.com/content/6/6/R521 Table 1 Characteristics of patients Patient no. B27 Disease Sex Age (years) Disease duration Chlamydia in urine (PCR) Synovial T cell proliferation Antibodies 1 + ReA Male 18 5 months + Chlamydia trachomatis n.d. 2 + ReA Male 20 2 months + Chlamydia trachomatis n.d. 3 + ReA Male 32 5 months + Chlamydia trachomatis + 4 + ReA Male 34 3 months + Chlamydia trachomatis n.d. 5 + ReA Male 20 1 month + n.d. + 6 + ReA Male 26 1 month + Chlamydia trachomatis + 7 - ReA Male 56 1 month + Chlamydia trachomatis n.d. 8 - ReA Male 43 3 months + Chlamydia trachomatis n.d. 9 - ReA Female 32 6 months + Chlamydia trachomatis n.d. 10 + Chronic ReA Male 20 7 years - Enterobacteria n.d. 11 + ReA Male 47 3 weeks - Enterobacteria n.d. 12 + ReA Female 49 6 months - Enterobacteria n.d. 13 n.d. RA Female 67 10 years n.d. n.d. n.d. 14 n.d. RA Female 49 >1 year n.d. n.d. n.d. 15 n.d. RA Female 62 14 years n.d. n.d. n.d. n.d., not done; PCR, polymerase chain reaction; RA, rheumatoid arthritis; ReA, reactive arthritis. tetramer staining; peptide 146 was excluded because of high background staining in more than 50% of the patients. HLA-A2 monomers with the EBV peptide [21] were generated with an HLA-A2 heavy chain (gift from Dr KH Lee, Berlin, Germany) with the same protocol. For FACS analysis, frozen mononuclear cells (MNCs) from synovial fluid or peripheral blood were incubated with tetramer and PerCP-labelled anti-human CD8 antibody (BD Pharmingen, San Diego, USA) in parallel for 30 min at room temperature (20°C) followed by washing twice with phosphate-buffered saline (PBS)/2% bovine serum albumin (BSA) and incubation with Cy5-labelled anti-human CD3 antibody 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 Alexa 488-labelled Annexin V (Molecular Probes) was added. CD8+ and tetramer-positive T cells were analysed after gates were set on CD3+ and Annexin V-negative cells. Depending on the availability of additional synovial lymphocytes we repeated the staining experiments, which was true for the synovial fluid of patient no. 6. T cell lines from magnetic activated cell sorting (MACS)sorted HLA-B27 tetramer-positive CD8+ T cells Peripheral MNCs were incubated for 30 min with Cy5labelled anti-CD8 antibody (BD) and 5 µg/ml PE-labelled HLA-B27/EBV EBNA (258–266) tetramer at room temperature. Cells were washed twice and incubated for 15 min at 4°C with anti-PE-labelled MACS beads (Miltenyi) at a ratio of 20 µl of beads to 80 µl of cell suspension. Labelled cells were loaded on an LS MACS column (Miltenyi) and eluted after the column had been washed three times with washing buffer including PBS, EDTA and BSA. MACSsorted tetramer-positive and CD8+ T cells were further separated by FACS sorting. Sorted cells (1000) were incubated with 500,000 autologous antigen-presenting cells in the presence of 20 U/ml interleukin (IL)-2, 10 ng/ml IL-7 and 10 ng/ml IL-15 added every 3–4 days. Determination of the refolding rate of recombinant HLAB27 monomers The refolding rate of recombinant HLA-B27 monomer was analysed by gel filtration and by determining the relative amount of soluble HLA-B27 monomer eluted at 13.7 ml in comparison with precipitated protein eluted earlier in a Superose 12 column (Pharmacia). An Akta basic system (Pharmacia) was used. The elution profile was analysed by using Unicorn (version 4) software (Pharmacia). Refolding R523 Arthritis Research & Therapy Vol 6 No 6 Appel et al. Table 2 Sequence and binding scores of peptides to HLA-B27 and HLA-A2 Name of peptide HLA-B27/EBNA (258–266) (EBV) Sequence of peptide Binding scorea RRIYDLIEL 28 Binding scoreb Refolding Reference 2000 ++ [24] HLA-A2/BMLF1 lytic antigen peptide 280–288 (EBV) GLCTLVAML 29 6000 ++ [21] HLA-B27/Influenza NP 383–391 SRYWAIRTR 26 1500 + [32] HLA-B27/Chlamydia peptide 8 NRFSVAYML 26 10,000 ++ [10] HLA-B27/Chlamydia peptide 68 NRAKQVIKL 26 2000 (+) [10] HLA-B27/Chlamydia peptide 80 IRMFKILPL 26 2000 + [10] HLA-B27/Chlamydia peptide 131 KRLAETLAL 26 6000 (+) [10] HLA-B27/Chlamydia peptide 133 IRSSVQNKL 27 2000 (+) [10] HLA-B27/Chlamydia peptide 138 ARKLLLDNL 26 2000 ++ [10] HLA-B27/Chlamydia peptide 144 MRDHTITLL 25 2000 - [10] HLA-B27/Chlamydia peptide 145 DRLALLANL 27 200 + [10] HLA-B27/Chlamydia peptide 146 YRLLLTRVL 25 600 (+) [10] HLA-B27/Chlamydia peptide 194 EREQTLNQL 25 200 - [10] HLA-B27/Chlamydia peptide 195 NRELIQQEL 25 2000 (+) [10] HLA-B27/Chlamydia peptide 196 ERFLAQEQL 27 1000 (+) [10] Refolding rates are designated as follows: ++, more than 75% soluble HLA-B27 monomer in gel-filtration analysis after refolding and biotinylation; +, more than 50% refolding; (+), more than 10% refolding; -, less than 10% refolding. aSYFPEITHI, HG Rammensee, University of Tübingen [23]. bHLA-peptide binding motifs (the BioInformatics and Molecular Analysis Section; http://bimas.dcrt.nih.gov/molbio/hla_bind/). EBV, Epstein–Barr virus. was defined as ++ when more than 75% of proteins loaded on the gel filtration column after refolding, biotinylation and sharp centrifugation was soluble HLAB27 monomer molecule; + for more than 50% soluble HLA-B27 monomer, (+) for more than 10% soluble HLAB27 monomer, and - for less than 10% soluble HLA-B27 monomer (Table 2). FACS analysis of intracellular cytokine staining Intracellular cytokine staining was used after antigen-specific T cell stimulation. Synovial MNCs and peripheral MNCs were stimulated for 6 hours in 1 ml of culture medium with anti-CD28 antibody (1 µg/ml) plus single peptides (10 µg/ml) or without antigenic peptide as a negative control. Brefeldin A was added after 2 hours to stop the stimulation, and cells were harvested after a further 4 hours and then stained with 5 µg/ml anti-CD69-PE antibody (BD Pharmingen) and 1 µg/ml anti-CD8-PerCP (BD Pharmingen). Cells were then fixed in 2% formalin and resuspended in saponin buffer, followed by incubation with 1 µg/ ml Cy5-conjugated anti-human interferon-γ antibody (IFN-γ; BD). Gated CD8+ T cells that were positive for early activation marker CD69 and for intracellular IFN-γ were counted as antigen-specific. Analysis was performed with a BD BioR524 sciences FACScan flow cytometer with CellQuest software. Infection of peripheral-blood-derived dendritic cells in vitro with viable Chlamydia trachomatis CD14+ cells from peripheral blood were incubated for 1 hour with anti-CD14-conjugated magnetic beads (Miltenyi) and sorted by MACS. The purity of separated cells was confirmed by FACS analysis. Cells (500,000) were cultured for 7 days in 24-well plates at 37°C at 5% CO2 in 1 ml of RPMI culture medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and 50 ng/ml granulocyte/ macrophage colony-stimulating factor and 10 ng/ml IL-4 to induce transformation to dendritic cells (DCs). Cells were washed and harvested and incubated for 24 hours with infectious elementary bodies of Chlamydia trachomatis at a ratio of 1:50. DCs were analysed by FACS with the use of anti-CD80, anti-CD86, anti-HLA-DR, anti-CD14 (BD Pharmingen) and anti-Chlamydia trachomatis lipopolysaccharide antibodies (Dako) before and after infection with viable Chlamydia trachomatis. Available online http://arthritis-research.com/content/6/6/R521 Expansion of Chlamydia-specific CD8+ T cells in vitro with Chlamydia-infected peripheral-blood-derived dendritic cells We stimulated CD8+ T cells from peripheral blood with Chlamydia trachomatis-infected peripheral-blood-derived DCs at a ratio of 50:1 in RPMI culture medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Recombinant IL-7 (10 ng/ml) and IL-15 (10 ng/ml) were added on both days 2 and 7. T cells were analysed by FACS on day 14. Results MHC class I tetramer staining with HLA-A2/EBV peptidespecific and HLA-B27/EBV peptide-specific tetramers To determine optimal conditions for the refolding procedure of soluble HLA-B27 monomers we used HLA-A2 tetramers and HLA-B27 tetramers with well-described HLA-B27 and HLA-A2 restricted viral epitopes from EBV. The binding scores of the two immunodominant EBV peptides to the HLA-A2 [21] (score 29) and HLA-B27 [24] (score 28) receptor were almost identical in the SYFPEITHI program. By generating the HLA-B27 tetramer (Fig. 1a,1c; lanes 1 and 2) and the HLA-A2 tetramer (Fig. 1b,1c; lanes 3 and 4) the percentages of protein aggregates eluted between 7 and 13 ml in gel filtration, and the refolded monomer eluted at 13.7 ml in gel filtration, were also similar. The large peak at 16 ml most probably contained reagents from the biotinylation reaction because we did not detect any proteins with a molecular mass of more than 5 kDa by SDSPAGE. This peak was excluded when the relative amount of soluble HLA-B27 monomers was estimated. In FACS analysis, tetramer-positive antigen-specific T cells could be detected with both tetramers (Fig. 1d), although HLA-A2 tetramers stained with greater intensity (log 0.8 more) than the HLA-B27. On the basis of these results we generated HLA-B27/Chlamydia peptide tetramers. Generation of antigen-specific CD8+ T cell lines after MACS sorting of HLA-B27 tetramer-positive T cells To determine whether tetramer-binding CD8+ T cells could be sorted and further cultured we stained peripheral MNCs with HLA-B27/EBV EBNA (258–266) tetramer. Before MACS, 0.22% of peripheral MNCs were CD8+ and tetramer-positive. After MACS, EBV EBNA (258–266)specific T cells were enriched to 41.4%. MACS-sorted cells were further separated by FACS sorting and cultured for 4 weeks in the presence of IL-2. The purity of antigenspecific CD8+ was increased to 95.0%, as shown by tetramer staining (Fig. 2a). In parallel we performed intracellular cytokine staining after peptide-specific stimulation of peripheral MNCs and of the tetramer-sorted T cell line after 4 weeks of culture. In comparison with HLA-B27 tetramer staining, only 68.3% of these antigen-specific T cells were detected by intracellular cytokine staining of IFN-γ (Fig. 2b). Generating HLA-B27/Chlamydia peptide tetramers The generation of HLA-B27 tetramers with Chlamydiaderived peptides strongly indicated that the yield of refolded and soluble HLA-B27/Chlamydia peptide monomers depended on the binding affinity of the peptide for HLA-B27. Gel-filtration analysis showed that Chlamydia peptide 133 (Table 2; binding score 25 in [23]) (Fig. 3a) induced significantly more protein aggregation, seen by protein elution between 7 and 13 ml, than Chlamydia peptide 8 (Table 2; binding score 26 in [23] but 10,000 in BIMAS) (Fig. 3b). In SDS-PAGE analysis the large quantity of aggregated proteins is also shown by numerous bands of higher molecular mass (Fig. 3a). After the addition of streptavidin, the major band with biotinylated HLA-B27 molecule could be captured to become a tetramer (Fig. 3a,3b; SDS-PAGE). This phenomenon of protein aggregation depending on the affinity between peptide and HLAB27 could also be observed with the other Chlamydiaderived peptides. The refolding rate of all HLA-B27 tetramers used in this manuscript are summarized in Table 2. HLA-B27/Chlamydia peptide tetramer staining of synovial T cells On the basis of our recently identified Chlamydia-derived immunodominant peptides in Ct-ReA [10] we successfully synthesized nine HLA-B27 Chlamydia peptide tetramers and used them to stain MNCs from the synovial fluid of nine patients (six HLA-B27+, three HLA-B27-) with Ct-ReA. Four of the six HLA-B27+ patients had a specific T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The results of tetramer staining in all patients are summarized in Table 3; HLA-B27/Chlamydia peptide 195 tetramer bound to the synovial T cells of three (patient nos 2, 3 and 5) of these four patients. Two patients (nos 5 and 6) showed a T cell response to Chlamydia peptide 133 as detected by tetramer staining, and one (patient no. 3) had a T cell response to Chlamydia peptide 68. The results of three patients are illustrated in Figs 4 and 5. Figure 4a shows that T cells specific for Chlamydia peptides 195 and 68 were detected with HLA-B27/Chlamydia peptide tetramers in patient no. 3: 0.09% of CD8+ T cells were positive for peptide 195 and 0.06% were positive for peptide 68. All other HLA-B27 tetramers with Chlamydiaderived peptides such as peptide 138 were negative (data not shown). In patient no. 2 we detected 0.06% HLA-B27/ Chlamydia peptide 195 tetramer-positive T cells (Fig. 4b). All other HLA-B27 tetramers such as HLA-B27/Chlamydia peptide 138 were negative (data not shown). We did not analyse the cytokine secretion profile of CD8+ T cells in response to chlamydial peptides in these two patients. The example of patient no. 6 is shown in Fig. 5a, with 0.02% of HLA-B27/Chlamydia peptide 133 tetramer binding to R525 Arthritis Research & Therapy Vol 6 No 6 Appel et al. Figure 1 lanesfiltration Gel 1 and 2) and gel electrophoresis of refolded HLA-A2/EBV peptide monomers (a; c, lanes 3 and 4) and HLA-B27/EBV peptide monomers (b; c, lanes 1 and 2). In both experiments the amount of protein aggregation is low, indicated by small amounts of eluted proteins between 7 and 13 ml. The peaks eluted at 13.7 ml contained the soluble MHC monomer. The gel in (c) shows that both eluted monomers are highly purified (lanes 1 and 3) and that most soluble monomers bind to streptavidin if added (lanes 2 and 4). (d) Antigen-specific T cells could be detected with both tetramers, although HLA-A2 tetramers stained with greater intensity (log 0.8 more) than HLA-B27. A280, absorption at 280 nm; asterisks, protein aggregates; SA, streptavidin. R526 Available online http://arthritis-research.com/content/6/6/R521 Figure 2 HLA-B27/EBV tetramers and use of magnetic beads to enrich for HLA-B27-restricted tetramer-positive CD8+ T cells cells. In peripheral blood from an HLA-B27+ blood donor, 0.22% EBV EBNA (258–266) peptide-specific T cells were detected with phycoerythrin (PE)-labelled HLA-B27 tetramer (a) and 0.66% of CD8+ T cells by performing intracellular cytokine staining of IFN-γ-secreting cells (b). After magnetic activated cell sorting with anti-PE magnetic beads, antigen-specific T cells were enriched to 41.4% (a). These cells were further purified by fluorescence-activated cell sorting (not shown) and cultured for 4 weeks. After 4 weeks of culturing in the presence of interleukin-2, 95% of the T cell line were antigen-specific as shown by HLA-B27 tetramer staining (a), whereas only 68.3% of antigen-specific T cells could be identified by intracellular cytokine staining (b). R527 Arthritis Research & Therapy Vol 6 No 6 Appel et al. Figure 3 Gel dia peptide filtration8and (b) gel electrophoresis of refolded HLA-B27/Chlamydia peptide monomers loaded with Chlamydia peptide 133 (a) and with Chlamydia peptide 8 (b). The peaks eluted at 13.7 ml contained the soluble MHC monomer. The amount of refolded HLA-B27 monomer with Chlamydia peptide 8 (b) was higher than that of peptide 133 (a) with less protein aggregation (proteins eluted between 7 and 13 ml), indicating that peptide 8 has a higher binding affinity for HLA-B27. The SDS-PAGE in (a) and (b) shows that both eluted monomers are the major protein in the eluted fraction and that most soluble monomers bind to streptavidin if added. In both experiments the amount of protein aggregation was higher than refolding of HLA-B27 with a viral epitope, indicated by a greater amount of eluted proteins between 7 and 13 ml (Fig. 1). A280, absorption at 280 nm; asterisks, protein aggregates; SA, streptavidin. CD8+ T cells but no binding to any of the other HLA-B27 Chlamydia peptide tetramers. In patient no. 6 we were able to repeat this experiment and obtained a similar result, with 0.02% of tetramer binding to CD8+ T cells. To confirm the specificity of the T cell response to peptide 133, two further experiments were performed in this patient. First, synovial T cells were expanded by Chlamydia peptide 133-specific T cell stimulation for 1 week ex vivo, which revealed 0.22% tetramer-positive CD8+ T cells (Fig. 5a). Second, when FACS analysis of IFN-γ secretion after peptide-specific stimulation was done in the same patient, only peptide 133 induced this cytokine secretion (Fig. 5b), again confirming the specificity of this response. We also analysed CD8+ T cells from peripheral blood of patient nos 2, 3, 5 and 6, who were responders when synovial fluid was tested for HLA-B27/Chlamydia peptide binding, but we could not detect any specific binding (data not shown). The HLA-B27- patients with Ct-ReA and all six R528 HLA-B27+ healthy controls had no HLA-B27-restricted, Chlamydia-peptide-specific T cell response (data not shown). Tetramer staining of synovial T cells from three HLA-B27+ patients with enterobacteria-triggered ReA and from three patients with rheumatoid arthritis revealed no specific staining of CD8+ T cells with 0–0.01% tetramer binding to CD8+ T cells. Expansion of Chlamydia-specific CD8+ T cells after stimulation with Chlamydia-infected dendritic cells Because the frequency of Chlamydia-specific CD8+ T cells in these patients is low in synovial fluid and absent in peripheral blood with both methods (tetramer staining and intracellular cytokine staining), we investigated whether enrichment of these cells could be achieved by short-term stimulation with autologous Chlamydia-infected DCs. By doing this we intended to obtain a higher frequency of tetramer-positive CD8+ T cells, to underline the specificity of tetramer staining. Available online http://arthritis-research.com/content/6/6/R521 Table 3 HLA-B27/Chlamydia peptide tetramer staining of CD8+ T cells from synovial fluid of six HLA-B27+ patients (nos 1–6) and three HLAB27- patients (nos 7–9) with Chlamydia-triggered reactive arthritis Patient no. B27 No. 68 No. 133 No. 195 1 + - - - 2 + - - + 3 + + - + 4 + - - - 5 + - + + 6 + - + - 7 - - - - 8 - - - - 9 - - - - 10 + - - - 11 + - - - 12 + - - - 13 n.d. - - - 14 n.d. - - - 15 n.d. - - - CD8+ T cells from the synovial fluid of three HLA-B27+ patients with enterobacteria-triggered reactive arthritis (patient nos 10–12), three patients with rheumatoid arthritis (patient nos 13–15) were analysed; the peripheral blood of six healthy HLA-B27+ blood donors was also analysed (not shown). Positive results are indicated with +, no staining with -. All other peptides tested negative. n.d., not done. We generated DCs from CD14+ monocytes from peripheral blood of patient no. 5; they were separated by MACS first. After 7 days of cultivation in vitro, the cells turned into DCs, as indicated by the loss of CD14 receptors and the upregulation of HLA-DR, CD80 and CD86 receptors (data not shown). We infected these DCs with viable Chlamydia trachomatis and confirmed infection by using an antiChlamydia trachomatis lipopolysaccharide antibody and by quantification of Chlamydia-positive cells by FACS analysis (data not shown). We revealed at least 41.3% Chlamydiainfected DCs. Peripheral MNCs from the same patient were stimulated with these Chlamydia-infected DCs for 2 weeks in the presence of IL-7 and IL-15. Subsequently, FACS analysis for intracellular cytokine staining for IFN-γ performed after restimulation of this cell line with Chlamydia-infected DCs revealed 0.11% IFN-γ-secreting CD8+ T cells, and stimulation with different peptide pools including the nine relevant peptides revealed between 0.07% and 0.21% antigenspecific IFN-γ-secreting CD8+ T cells (data not shown). When the cell line was analysed with HLA-B27/Chlamydia peptide tetramers we found a similar quantity of expanded CD8+ T cells with significant tetramer staining of CD8+ T cells specific for Chlamydia peptides 8 (0.09%), 68 (0.10%), 133 (0.17%), 138 (0.08%), 195 (0.23%) and 196 (0.06%) (Fig. 6) and a weaker response to the other Chlamydia-derived peptides. HLA-B27 tetramer staining with peptides 133 and 195 showed some unusual bright staining, which was also frequently observed with the HLAB27/EBV EBNA (258–266) tetramer and might have been caused by aggregated tetramers. Staining of untreated peripheral MNCs from the same patient did not reveal any tetramer binding (data not shown); staining with an HLAB27/EBV peptide tetramer was performed as a positive control. We repeated this procedure in an HLA-B27patient with Ct-ReA (Fig. 7) and in an HLA-B27+ healthy blood donor (data not shown). In neither case could we observe staining with any of the HLA-B27/Chlamydia peptide tetramers even after stimulation with Chlamydiainfected DCs (patient no. 9; Fig. 7). Discussion The arthritogenic peptide theory states that some HLA-B27 subtype alleles, owing to their unique amino acid residues, bind one or more specific arthritogenic peptides that are recognized by CD8+ T cells [6-9]. To test this theory it is of great importance to establish methods to identify the peptide specificity of such CD8+ T cells in human beings with HLA-B27-associated arthritis. The use of MHC class I R529 Arthritis Research & Therapy Vol 6 No 6 Appel et al. Figure 4 HLA-B27/Chlamydia peptide tetramer-binding synovial T cells in patient nos 2 and 3 3. In patient no. 3, 0.09% Chlamydia peptide 195-specific and 0.06% peptide 68-specific CD8+ T cells were identified. All other HLA-B27 tetramers with Chlamydia-derived peptides such as peptide 138 did not bind synovial T cells in this patient (data not shown). In patient no. 2, 0.06% Chlamydia peptide 195-specific CD8+ T cells were detected by HLAB27 tetramers. All other HLA-B27 tetramers with Chlamydia-derived peptides such as peptide 138 did not bind synovial T cells in this patient (data not shown). Peripheral blood from a blood donor with HLA-B27/EBV EBNA (258–266)-positive CD8+ T cells was used as a control. tetramers to detect antigen-specific CD8+ T cells is well established [16]. However, surprisingly few publications present data with HLA-B27 tetramers. We have reported preliminary experiments with HLA-B27 tetramers in single patients with Ct-ReA [10]. HLA-B27 tetramers were also used to determine critical T cell receptor binding regions in HLA-B27-restricted T cells specific for an immunodominant peptide from influenza virus [19]. The biochemical features of the protein might be the limiting factor for using this molecule as frequently as other MHC class I molecules such as HLA-A2 tetramers. R530 During the refolding process of recombinant HLA-B27, which is expressed in inclusion bodies, significant amounts of aggregated proteins occur [19,20]. The free cysteine residue at position 67 in the HLA-B27 α-chain is chemically highly reactive, causing homodimerization and protein aggregation [19,20,25-27]. It was therefore a reasonable strategy to generate HLA-B27 tetramers by substituting serine for cysteine at position 67 [10,19]. The mutated HLA-B27 heavy chain was also used in these experiments. However, even with the mutated HLA-B27 molecule we experienced significant protein aggregation when HLAB27 molecules were generated with Chlamydia-derived peptides, especially with those with a low binding affinity for HLA-B27. We addressed the question of whether this finding was related to the protocols we used or whether it was specifically related to HLA-B27. We generated an HLA-B27 tetramer with a well-described immunodominant peptide from EBV and compared the results with those for an HLA-A2 molecule also loaded with an immunodominant peptide from EBV having almost the same binding affinity. The refolding rate of both molecules was almost the same, and we obtained comparable results when these molecules were used in FACS analysis. From this we concluded that the use of HLA-B27 tetramers is limited if the binding affinity of a peptide is too low for the molecule to remain stable. We therefore excluded peptides causing heavy protein aggregation and high background staining from further experiments.