It is assumed to exert multiple functions including packaging of

It is assumed to exert multiple functions including packaging of pre-mRNA, regulation of alternative splicing, and nucleo-cytoplasmic transport of mRNA 7. HnRNP-A2 appears to be ubiquitously expressed, although the level of expression may greatly vary between different tissues. Interestingly,

hnRNP-A2 is overexpressed in RA synovial tissue, where it is detectable not only in the nucleus but also in the cytoplasm of macrophages and fibroblast-like synoviocytes 8. Autoantibodies 5-Fluoracil datasheet to hnRNP-A2 (which are also known as anti-RA33 Ab) are present in approximately 30% of RA patients 9, but also in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease 9. Remarkably, however, epitope recognition was found to differ between the three disorders 10. Furthermore, also T cells from peripheral blood and synovial fluid of RA patients were found to

react to hnRNP-A2, in about 60% of the patients 8. Interestingly, autoimmunity to hnRNP-A2 has been observed in TNF-transgenic (Tg) mice 11, which develop arthritis spontaneously, and is a dominant immunological event in pristane-induced arthritis in rats 12. Altogether, the results suggest that this protein is an important and potentially pathogenic autoantigen in animal models of arthritis and in RA. Thus, it was the aim of the present study to characterize putative pathogenic T-cell epitopes of hnRNP-A2. To achieve this goal, we started with a comprehensive investigation of MHC binders among a library Bortezomib mw heptaminol of 15-mer peptides spanning the entire human hnRNP-A2 protein. Peptides of this length can bind directly to MHC class II molecules on the cell surface

of APC where they can stimulate peptide-specific T cells. This method allows the analysis of all possible determinants regardless of whether the peptide is dominant or cryptic following natural processing. Then, to identify hnRNP-A2-specific T-cell epitopes in patients with RA, we used a sensitive IFN-γ ELISPOT assay, which detects in vivo-generated antigen-specific T cells in a low frequency range 13. The data obtained were confirmed in proliferation assays and reveal the presence of an immunodominant T-cell epitope associated to active RA. We synthesized 280 15-mer peptides overlapping by 13 or 14 amino acids and spanning the whole hnRNP-A2 sequence. These peptides were tested by competitive ELISA for binding to the RA-associated DR*0101 and DR*0401 molecules. The results obtained show that most epitopes binding to either DR*0101 or DR*0401 were localized in the N-terminal half (first 170 amino acids) of the hnRNP-A2 sequence (Fig. 1). Presence of an MHC epitope was revealed by 4–7 consecutive binding peptides. Frequently, many more consecutive peptides were binding, indicating overlapping epitopes. Six major determinants were found to bind to both DR*0101 and DR*0401: peptides no.

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