HLA-B27 Tetramers and Antigen-Specific T Cells in Ct-ReA
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 (Ct-ReA). 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 antigen-specific 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.
Chlamydia-triggered reactive arthritis (Ct-ReA) is strongly associated with HLA-B27 like other spondylarthropathies, and especially ankylosing spondylitis. ReA occurs 1 to 4 weeks after urogenital infection with Chlamydia trachomatis or gastroenteral infection with enterobacteria such as Yersinia enterocolitica. After acute onset, most patients have a self-limiting course, but up to 20% suffer from a disease duration of more than 1 year. 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 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. Recently we and several other groups have reported on Chlamydia-specific CD8 T cells capable of lysing target cells primed with Chlamydia antigens. CD8 T cell responses in spondylarthropathies other than Ct-ReA have also been described.
Recently a new method for antigen-specific T cell recognition has been established by using multimerized MHC/peptide molecules. 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.
In humans, MHC class I tetramers are widely used, and HLA-A2 tetramers in particular are an important tool in tumour immunology. However, the use of HLA-B27 tetramers in HLA-B27-related diseases is rare. The rarity of their use might be related to heavy protein aggregation during the refolding procedure of the recombinant HLA-B27 monomer. 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. 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.
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 (Ct-ReA). 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 antigen-specific 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.
Chlamydia-triggered reactive arthritis (Ct-ReA) is strongly associated with HLA-B27 like other spondylarthropathies, and especially ankylosing spondylitis. ReA occurs 1 to 4 weeks after urogenital infection with Chlamydia trachomatis or gastroenteral infection with enterobacteria such as Yersinia enterocolitica. After acute onset, most patients have a self-limiting course, but up to 20% suffer from a disease duration of more than 1 year. 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 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. Recently we and several other groups have reported on Chlamydia-specific CD8 T cells capable of lysing target cells primed with Chlamydia antigens. CD8 T cell responses in spondylarthropathies other than Ct-ReA have also been described.
Recently a new method for antigen-specific T cell recognition has been established by using multimerized MHC/peptide molecules. 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.
In humans, MHC class I tetramers are widely used, and HLA-A2 tetramers in particular are an important tool in tumour immunology. However, the use of HLA-B27 tetramers in HLA-B27-related diseases is rare. The rarity of their use might be related to heavy protein aggregation during the refolding procedure of the recombinant HLA-B27 monomer. 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. 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.