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 HLA-B27
+-reactive arthritis patients, 20–40% move on to ankylosing 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 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].
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-B27-restricted 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 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.
Table 1
Characteristics of patients
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. |
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
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 (16,060
g; 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 (Bio-Rad). 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 tetramer staining; peptide 146 was excluded because of high background staining in more than 50% of the patients.
Table 2
Sequence and binding scores of peptides to HLA-B27 and HLA-A2
HLA-B27/EBNA (258–266) (EBV) | RRIYDLIEL | 28 | 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] |
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 Cy5-labelled 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. MACS-sorted 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 HLA-B27 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 was defined as ++ when more than 75% of proteins loaded on the gel filtration column after refolding, biotinylation and sharp centrifugation was soluble HLA-B27 monomer molecule; + for more than 50% soluble HLA-B27 monomer, (+) for more than 10% soluble HLA-B27 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 Biosciences 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.
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.
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 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.
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 HLA-B27 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.
Here we have also demonstrated another useful property of HLA-B27 tetramers as a 'proof of principle'. We sorted antigen-specific tetramer-positive CD8
+ T cells and generated highly specific T cell lines. After 4 weeks of non-specific stimulation, 95% of T cells were antigen-specific, which could be detected by tetramers but not by intracellular cytokine staining. The latter experiments detected only 68.3% IFN-γ secreting CD8
+ T cells after antigen-specific stimulation. These results show clearly that HLA-B27 tetramers have the advantage of detecting antigen-specific T cells independently of their cytokine-secreting profile. Tetramers are also capable of detecting resting antigen-specific T cells, which probably constitute most non-IFN-γ-secreting CD8
+ T cells of the T cell line in Fig.
4B (CD69
- and IFN-γ
-).
Using HLA-B27-restricted
Chlamydia peptides with higher binding scores, previously defined as immunodominant in Ct-ReA [
10], we have generated tetramers and identified
Chlamydia-peptide-specific T cell responses in four of six patients with Ct-ReA. These experiments suggest that
Chlamydia peptides 195 and 133 are immunologically important epitopes, because we could detect CD8
+ T cells with such specificity in three and two, respectively, out of six HLA-B27
+ patients. We identified these antigen-specific T cells at a frequency of 0.02–0.09% in the synovial fluid of these patients, which is concordant with previous results [
10]. The low frequency of
Chlamydia-peptide-specific CD8
+ T cells detected with HLA-B27 tetramers sometimes makes discrimination from non-specific staining difficult. We confirmed our tetramer staining result in one patient by expansion of peptide-specific CD8
+ T cells followed by tetramer staining with increased amounts of tetramer-binding CD8
+ T cells; even more importantly, peptide-specific CD8
+ T cells were also detected by intracellular IFN-γ staining after peptide-specific stimulation. However, for future experiments it would be useful to confirm such findings with antigen-specific T cell expansion as shown here (Figs
2,
5a and
6a) and also in collaboration with other authors [
28].
To underline further the specificity of HLA-B27 tetramer staining we performed peptide-specific expansion of CD8
+ T cells specific for
Chlamydia. For this, we generated
Chlamydia-infected DCs, which are assumed to be excellent antigen-presenting cells for both CD4
+ and CD8
+ T cells [
29], for
Chlamydia-antigen-specific CD8
+ T cell stimulation; we obtained antigen-specific T cell expansion. This
Chlamydia-specific CD8
+ T cell line showed an increased response to HLA-B27/
Chlamydia peptides 8, 68, 133, 138, 195 and 196 tetramers and a weaker response to the other HLA-B27/
Chlamydia peptide tetramers. The generation of CD8
+ T cell lines with
Chlamydia-infected DCs has recently been described, but without defining the MHC restriction and peptide specificity of such T cells [
30].
Because we could not detect any
Chlamydia-peptide-specific CD8
+ T cells from peripheral blood with either method (tetramer staining and intracellular cytokine staining) without prior stimulation, we assume that the frequency of CD8
+ T cells with such specificity in the peripheral blood is below the sensitivity of both methods. In contrast, low frequencies of
Chlamydia-derived peptide-specific CD8
+ T cells in the peripheral blood were observed by another group by using HLA-A2 tetramers [
31]. These researchers detected
Chlamydia trachomatis major outer membrane protein (MOMP) 258 peptide-specific and MOMP 249 peptide-specific CD8
+ T cells in patients with acute urogenital tract infection. They found 0.01–0.2% MOMP-specific CD8
+ T cells in the peripheral blood of these individuals with acute infection, who had no clinical symptoms of Ct-ReA.