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* Immunology Unit, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona;
Consejo Superior de Investigaciones Cientificas-Universitat Autònoma de Baracelona, Proteomics Facility, Institut d’Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Cientificas-Institut d’Investigacions Biomèdiques August Pi i Sunyer, Barcelona;
Catalan Institution for Research and Advanced Studies and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona;
Laboratory of Immunobiology for Research and Application to Diagnosis, Blood and Tissue Bank, University Hospital Germans Trias i Pujol, Badalona, and Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Barcelona, Spain; and
¶ Torrey Pines Institute for Molecular Studies, San Diego, CA 92121
Endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express HLA class II (HLA-II) molecules that are presumably involved in the maintenance and regulation of the in situ autoimmune response. HLA-II molecules thus expressed by thyroid cells have the "compact" conformation and are therefore expected to stably bind autologous peptides. Using a new approach to study in situ T cell responses without the characterization of self-reactive T cells and their specificity, we have identified natural HLA-DR-associated peptides in autoimmune organs that will allow finding peptide-specific T cells in situ. This study reports a first analysis of HLA-DR natural ligands from ex vivo Graves’ disease-affected thyroid tissue. Using mass spectrometry, we identified 162 autologous peptides from HLA-DR-expressing cells, including thyroid follicular cells, with some corresponding to predominant molecules of the thyroid colloid. Most interestingly, eight of the peptides were derived from a major autoantigen, thyroglobulin. In vitro binding identified HLA-DR3 as the allele to which one of these peptides likely associates in vivo. Computer modeling and bioinformatics analysis suggested other HLA-DR alleles for binding of other thyroglobulin peptides. Our data demonstrate that although the HLA-DR-associated peptide pool in autoimmune tissue mostly belongs to abundant ubiquitous proteins, peptides from autoantigens are also associated to HLA-DR in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response.
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1 This work was funded by the Spanish Ministry of Education Grants SAF2003-08843-C02-01 and SAF00-0131-C02-01. X.D. was supported by the Spanish MEC/FEDER Grant BIO2003-02848. L.M. is a Formación de Personal Investigador fellow of the Spanish Ministry of Education. I.A. was partially funded by the Eurothymaid Integrated European Project LSHB-CT-2003-503410. C.P. was funded by the Multiple Sclerosis National Research Institute.
2 Address correspondence and reprint requests to Dr. Dolores Jaraquemada, Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Campus de Bellaterra, 08193 Barcelona, Spain. E-mail address: dolores.jaraquemada{at}uab.es
3 Abbreviations used in this paper: HLA-II, HLA class II; AITD, autoimmune thyroid diseases; EAT, experimental autoimmune thyroiditis; EBVP, EBV DNA polymerase; GD, Graves’ disease; HMBP, human myelin basic protein; ITMS, ion trap mass spectroscopy; MHC-II, MHC class II; MS, mass spectroscopy; m/z, mass-to-charge; nESI, nanoelectrospray; rpHPLC, reversed-phase HPLC; TFC, thyroid follicular cells; Tg, thyroglobulin; vMALDI, vacuum MALDI.
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