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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-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.
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Figure 1: Gel filtration 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.

Mentions: 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 SDS-PAGE. 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.


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-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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064864&req=5

Figure 1: Gel filtration 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.
Mentions: 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 SDS-PAGE. 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.

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.

Show MeSH
Related in: MedlinePlus