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SAT0031 Fine epitope mapping of immunodominant peptide epitopes by affinity matrix technology using yersinia-hsp60-specific synovial cd4+ t-cells in reactive arthritis (rea) patients
  1. A Thiel1,
  2. P Wu2,
  3. D Stauch1,
  4. M Rudwaleit2,
  5. A Radbruch1,
  6. J Sieper2
  1. 1Rheumatology, German Rheumatism Research Center
  2. 2Rheumatology, Benjamin Franklin University Hospital, Berlin, Germany


Background A T cell response to persitent bacterial antigen is present in patients with reactive arthritis (ReA). A better characterisation of this response might lead to new treatments.

Objectives To identify immundominant Yer-hsp60-derived CD4 T cell epitopes in Yersinia-induced reactive arthritis (ReA).

Methods In two patients with Yersinia-induced ReA synovial fluid (SF) T cell response were first investigated after in vitro stimulation with the Yersinia-derived protein hsp60. Fixed cells were stained with antibodies against CD4, CD8, CD69 and against the cytokine IFNgamma;. Patients with reacting CD4+ Th cells were retested and live IFNgamma-secreting cells were isolated with the affinity matrix technology after short term stimulation with Yer-hsp60. Cells were expanded within 2 to 5 weeks and retested with overlapping 18 mer peptides from the Yer-hsp60 protein performing cytometric analysis of fixed cells after short term stimulation with peptided pulsed autologous PBMCs.

Results Live antigen-specific IFNgamma;-secreting CD4+ T cells were enriched to a purity of 80 to 90%. After two weeks in vitro expansion still 80% of the short-term cultivated T cells reacted with IFNgamma-production upon Yer-hsp60 stimulation, while reaction upon stimulation with a irrelevant protein was below 1%. T cells were further expanded antigen-specifically for 3 weeks and their fine specificity was further mapped to one peptide in each patient (AA 139–156 and AA 7–30 of Yer-hsp60/GroEL). Tese peptides was also recognised by the patient’s CD4+ T cells when whole blood or synovial fluid was stimulated for 6 h.

Conclusion This approach allows a sensitive and fast epitope mapping of immunodominant epitopes possibly involved in the pathogenesis of ReA.

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