Analysis of the role of HLA-DR 4 binding peptides for the activation of CD4 T-cells and the inflammatory process in rheumatoid arthritis

Rheumatoid arthritis represents a chronic inflammatory disease of unknown cause that leads to significant morbidity and invalidity due to the destruction of multiple joints. Although inflammation can be suppressed with various treatments, no therapy is currently available that can stop the progressive course of the disease. Recent experimental evidence suggests that antigen-specific Th1-type T cells regulate inflammation in RA by driving the release of inflammatory mediators by synovial macrophages. The agent(s) responsible for the recruitment of pathogenic T cells into the joint and for the persistent activation of synovial T cells are currently unknown. Whether specific antigens mediate T cell dependent activation of macrophages and inflammation remains an unresolved question. In order to address this question I will investigate three aspects of the antigen-specific T cell response in HLA-DR *0401 positive patients with RA. First, I will study the nature of antigens or peptides that are locally presented by synovial HLA-DR *0401 molecules. Second, I will determine the significance of individual antigenic peptides by analyzing the frequency, state of activation and functional abilities of peptide-specific CD4 + T cells in the peripheral blood and synovial fluid of patients with active and inactive disease. Third, I will test the ability of antigen-specific T cells to regulate the production of inflammatory mediators by macrophages. 1 have recently prepared a series of soluble HLA-DR molecules that will be used for the presentation of naturally processed synovial peptides to T cells from patients with RA. Activation of antigen-specific T cells due to short time stimulation with soluble HLA-DR-peptide complexes will be determined at the single cell level by analyzing secreted cytokines in ELISPOT assays and intracellular IFNg , IL-2 or IL-4 by flow cytometry. Stimulating peptides will be isolated and sequenced. The significance of isolated peptides for RA will then be studied with tetrameric HLA-DR 4-peptide complexes and flow cytometry by enumeration and phenotypic characterization of antigen-specific T cells in patients with RA. Tetrameric HLA-DR 4-peptide complexes will also serve to isolate peptide-specific T cells for functional studies in vitro. These experiments are designed to provide results on antigen-specific T cells from patients with RA that directly reflect the situation in vivo. It is hoped that the data obtained from this project contribute to the understanding of the pathogenesis of RA and provide new rational concepts for more specific therapeutic interventions with the disease process in patients with RA.