In order to elucidate the role of anti-thyroglobulin (Tg) autoantibodies (A-Ab) in experimental autoimmune thyroiditis (EAT), a kinetic study was conducted in EAT-susceptible (CBA/J) and non-susceptible (C57BL/6) strains of mice. From day 0 to 70 post-Tg immunization, titers of A-Ab to Tg and to the linear 5-10-kDa Tg tryptic fragment inducing EAT as well as anti-idiotypic A-Ab representing the internal image of the thyroidogeneic antigen were measured. EAT onset, development and recovery correlate with the presence of two subsets of A-Ab only in susceptible strains of mice. First, with the presence of anti-Tg A-Ab to one determinant borne by the linear 5-10-kDa Tg tryptic fragment, and second with the presence of anti-idiotypic A-Ab specific for the monoclonal anti-Tg A-Ab (3B8G9) paratope which binds to Tg determinant inducing EAT.

These data collect the advance made in the last few years in our laboratory in defining one epitope from the thyroglobulin (Tg) molecule (660 KDa) inducing Experimental Autoimmune thyroiditis (EAT) in CBA/J mice. We achieved the characterization of one EAT-inducer Tg peptide by combining "in vitro" biochemical and immunological approaches and "in vivo" studies. Since T cells recognize degraded forms of the antigen and since endogenous antigens preferentially activate class I-restricted T cells, we hypothesized that one cytotoxic T cell hybridoma, named HTC2, which prevents further EAT induction in mice injected with Tg would be specific for one EAT inducer peptide. In order to identify one Tg epitope inducing EAT, enzymatic treatment of the protein by trypsin, HPLC purification and sequence analysis were performed. Simultaneously, tryptic digests were used to pulse CBA/J macrophages and tested for their ability to be recognized by HTC2 cells. Lastly, when digests were recognized by HTC2 cells their capacity to induce EAT in CBA/J mice was evaluated. To further assess the pathogenicity of the sequenced Tg peptide, one synthetic peptide was made and its capacity to induce EAT verified. By this procedure we identified for the first time one 40 amino-acid peptide from human thyroglobulin inducing EAT in CBA/J mice.

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