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Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.

Appel H, Kuon W, Kuhne M, Wu P, Kuhlmann S, Kollnberger S, Thiel A, Bowness P, Sieper J - Arthritis Res. Ther. (2004)

Bottom Line: Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Charite Berlin, Campus Benjamin Franklin, Department for Gastroenterology, Infectiology and Rheumatology, Berlin, Germany. heinerappel@yahoo.com

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 (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.

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Gel filtration and 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.
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Figure 3: Gel filtration and 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.

Mentions: The generation of HLA-B27 tetramers with Chlamydia-derived 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 HLA-B27 could also be observed with the other Chlamydia-derived peptides. The refolding rate of all HLA-B27 tetramers used in this manuscript are summarized in Table 2.


Use of HLA-B27 tetramers to identify low-frequency antigen-specific T cells in Chlamydia-triggered reactive arthritis.

Appel H, Kuon W, Kuhne M, Wu P, Kuhlmann S, Kollnberger S, Thiel A, Bowness P, Sieper J - Arthritis Res. Ther. (2004)

Gel filtration and 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.
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Related In: Results  -  Collection

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Figure 3: Gel filtration and 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.
Mentions: The generation of HLA-B27 tetramers with Chlamydia-derived 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 HLA-B27 could also be observed with the other Chlamydia-derived peptides. The refolding rate of all HLA-B27 tetramers used in this manuscript are summarized in Table 2.

Bottom Line: Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Charite Berlin, Campus Benjamin Franklin, Department for Gastroenterology, Infectiology and Rheumatology, Berlin, Germany. heinerappel@yahoo.com

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 (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.

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Related in: MedlinePlus