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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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No tetramer staining of peripheral blood-derived CD8+ T cell lines generated with Chlamydia-infected dendritic cells with nine different HLA-B27/Chlamydia peptide tetramers in the HLA-B27- Chlamydia-triggered reactive arthritis (Ct-ReA) patient no. 9. The same experiment was performed in an HLA-B27- patient with Ct-ReA: even after stimulation with autologous Chlamydia-infected dendritic cells, Chlamydia-peptide-specific CD8+ T cells could not be detected in peripheral blood by HLA-B27/Chlamydia peptide tetramers. Chl, Chlamydia trachomatis.
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Figure 7: No tetramer staining of peripheral blood-derived CD8+ T cell lines generated with Chlamydia-infected dendritic cells with nine different HLA-B27/Chlamydia peptide tetramers in the HLA-B27- Chlamydia-triggered reactive arthritis (Ct-ReA) patient no. 9. The same experiment was performed in an HLA-B27- patient with Ct-ReA: even after stimulation with autologous Chlamydia-infected dendritic cells, Chlamydia-peptide-specific CD8+ T cells could not be detected in peripheral blood by HLA-B27/Chlamydia peptide tetramers. Chl, Chlamydia trachomatis.

Mentions: 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% antigen-specific 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 HLA-B27/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 HLA-B27/EBV peptide tetramer was performed as a positive control. We repeated this procedure in an HLA-B27- patient 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 Chlamydia-infected DCs (patient no. 9; Fig. 7).


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)

No tetramer staining of peripheral blood-derived CD8+ T cell lines generated with Chlamydia-infected dendritic cells with nine different HLA-B27/Chlamydia peptide tetramers in the HLA-B27- Chlamydia-triggered reactive arthritis (Ct-ReA) patient no. 9. The same experiment was performed in an HLA-B27- patient with Ct-ReA: even after stimulation with autologous Chlamydia-infected dendritic cells, Chlamydia-peptide-specific CD8+ T cells could not be detected in peripheral blood by HLA-B27/Chlamydia peptide tetramers. Chl, Chlamydia trachomatis.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: No tetramer staining of peripheral blood-derived CD8+ T cell lines generated with Chlamydia-infected dendritic cells with nine different HLA-B27/Chlamydia peptide tetramers in the HLA-B27- Chlamydia-triggered reactive arthritis (Ct-ReA) patient no. 9. The same experiment was performed in an HLA-B27- patient with Ct-ReA: even after stimulation with autologous Chlamydia-infected dendritic cells, Chlamydia-peptide-specific CD8+ T cells could not be detected in peripheral blood by HLA-B27/Chlamydia peptide tetramers. Chl, Chlamydia trachomatis.
Mentions: 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% antigen-specific 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 HLA-B27/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 HLA-B27/EBV peptide tetramer was performed as a positive control. We repeated this procedure in an HLA-B27- patient 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 Chlamydia-infected DCs (patient no. 9; Fig. 7).

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