Frequency of proteinase 3 (PR3)-specific autoreactive T cells determined by cytokine flow cytometry in Wegener's granulomatosis

Objective: Previous studies have shown proliferation to Wegener's autoantigen, proteinase 3 (PR3). We tested immunogenicity of PR3-derived peptides and determined frequencies of PR3-specific T cells using cytokine flow cytometry in Wegener's granulomatois (WG). Methods: Peripheral blood T-cell responses were measured after stimulation with previously described PR3-derived peptides. PBMC were stimulated with PR3-derived peptides or control stimuli for up to 10 days. Cells were stained with antibodies against CD4 or CD8, CD69 and intracellular TNF-alpha and analyzed by flow cytometry. PR3-specific T cells were counted as the percentage of CD69⁺TNF-alpha⁺double positive T cells after stimulation. Results: Frequencies of PR3-specific peripheral blood T cells after short-term stimulation (≤0.4%) were lower compared to frequencies of PR3-specific CD4⁺T cells (0.64±0.09%, mean±SEM) and PR3-specific CD8⁺T cells (0.65±0.18%) after 10 days of stimulation in WG. There were no differences of the frequency of PR3-specific T cells between WG and healthy controls after stimulation with other peptides. The frequency of PR3-specific CD8⁺T cells stimulated with a preferentially HLA-A*0201 binding PR3-peptide sequence was higher compared to the frequency of T cells stimulated with a HLA-B*0702 binding PR3-peptide in one WG patient whose HLA type was known (A2B7). Conclusion: Low frequencies of TNF-alpha ⁺PR3-specific T cells can be detected in individual WG patients and controls using cytokine flow cytometry. The pattern and time course of cytokine production in response to PR3 peptides needs to be further elucidated. Additional factors such as the influence of proinflammatory or regulatory T cells might be important for the induction of autoimmunity in WG.