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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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HLA-B27/Chlamydia peptide tetramer staining and intracellular cytokine staining in patient no. 6. When synovial T cells where stained with HLA-B27 Chlamydia peptide tetramers, only HLA-B27/Chlamydia peptide 133 was positive for CD8+ T cells (0.02%). All other HLA-B27 tetramers with Chlamydia peptides did not bind synovial T cells in this patient (not shown). The specificity of this staining could be confirmed by an increased quantity of tetramer-positive T cells after peptide-specific T cell stimulation (0.22%) after 1 week of peptide-specific stimulation loaded on synovial mononuclear cells (a) and Chlamydia peptide 133-specific induction of IFN-γ production in intracellular cytokine staining (b).
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Figure 5: HLA-B27/Chlamydia peptide tetramer staining and intracellular cytokine staining in patient no. 6. When synovial T cells where stained with HLA-B27 Chlamydia peptide tetramers, only HLA-B27/Chlamydia peptide 133 was positive for CD8+ T cells (0.02%). All other HLA-B27 tetramers with Chlamydia peptides did not bind synovial T cells in this patient (not shown). The specificity of this staining could be confirmed by an increased quantity of tetramer-positive T cells after peptide-specific T cell stimulation (0.22%) after 1 week of peptide-specific stimulation loaded on synovial mononuclear cells (a) and Chlamydia peptide 133-specific induction of IFN-γ production in intracellular cytokine staining (b).

Mentions: The results of three patients are illustrated in Figs 4 and 5. Figure 4a shows that T cells specific for Chlamydia peptides 195 and 68 were detected with HLA-B27/Chlamydia peptide tetramers in patient no. 3: 0.09% of CD8+ T cells were positive for peptide 195 and 0.06% were positive for peptide 68. All other HLA-B27 tetramers with Chlamydia-derived peptides such as peptide 138 were negative (data not shown). In patient no. 2 we detected 0.06% HLA-B27/Chlamydia peptide 195 tetramer-positive T cells (Fig. 4b). All other HLA-B27 tetramers such as HLA-B27/Chlamydia peptide 138 were negative (data not shown). We did not analyse the cytokine secretion profile of CD8+ T cells in response to chlamydial peptides in these two patients. The example of patient no. 6 is shown in Fig. 5a, with 0.02% of HLA-B27/Chlamydia peptide 133 tetramer binding to CD8+ T cells but no binding to any of the other HLA-B27 Chlamydia peptide tetramers. In patient no. 6 we were able to repeat this experiment and obtained a similar result, with 0.02% of tetramer binding to CD8+ T cells.


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)

HLA-B27/Chlamydia peptide tetramer staining and intracellular cytokine staining in patient no. 6. When synovial T cells where stained with HLA-B27 Chlamydia peptide tetramers, only HLA-B27/Chlamydia peptide 133 was positive for CD8+ T cells (0.02%). All other HLA-B27 tetramers with Chlamydia peptides did not bind synovial T cells in this patient (not shown). The specificity of this staining could be confirmed by an increased quantity of tetramer-positive T cells after peptide-specific T cell stimulation (0.22%) after 1 week of peptide-specific stimulation loaded on synovial mononuclear cells (a) and Chlamydia peptide 133-specific induction of IFN-γ production in intracellular cytokine staining (b).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1064864&req=5

Figure 5: HLA-B27/Chlamydia peptide tetramer staining and intracellular cytokine staining in patient no. 6. When synovial T cells where stained with HLA-B27 Chlamydia peptide tetramers, only HLA-B27/Chlamydia peptide 133 was positive for CD8+ T cells (0.02%). All other HLA-B27 tetramers with Chlamydia peptides did not bind synovial T cells in this patient (not shown). The specificity of this staining could be confirmed by an increased quantity of tetramer-positive T cells after peptide-specific T cell stimulation (0.22%) after 1 week of peptide-specific stimulation loaded on synovial mononuclear cells (a) and Chlamydia peptide 133-specific induction of IFN-γ production in intracellular cytokine staining (b).
Mentions: The results of three patients are illustrated in Figs 4 and 5. Figure 4a shows that T cells specific for Chlamydia peptides 195 and 68 were detected with HLA-B27/Chlamydia peptide tetramers in patient no. 3: 0.09% of CD8+ T cells were positive for peptide 195 and 0.06% were positive for peptide 68. All other HLA-B27 tetramers with Chlamydia-derived peptides such as peptide 138 were negative (data not shown). In patient no. 2 we detected 0.06% HLA-B27/Chlamydia peptide 195 tetramer-positive T cells (Fig. 4b). All other HLA-B27 tetramers such as HLA-B27/Chlamydia peptide 138 were negative (data not shown). We did not analyse the cytokine secretion profile of CD8+ T cells in response to chlamydial peptides in these two patients. The example of patient no. 6 is shown in Fig. 5a, with 0.02% of HLA-B27/Chlamydia peptide 133 tetramer binding to CD8+ T cells but no binding to any of the other HLA-B27 Chlamydia peptide tetramers. In patient no. 6 we were able to repeat this experiment and obtained a similar result, with 0.02% of tetramer binding to CD8+ T cells.

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