Studies on experimental autoimmune thyroiditis in mice and rats with thyroglobulin and thyroglobulin peptides

Balasa, Balaji (1995) Studies on experimental autoimmune thyroiditis in mice and rats with thyroglobulin and thyroglobulin peptides. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Experimental autoimmune thyroiditis (EAT) induced in mice or rats with thyroglobulin (Tg) emulsified in complete Freund's adjuvant (CFA) has been a model system to understand the immunopathogenesis of Hashimoto's thyroiditis (HT). I have demonstrated, by the immunotargeting approach, that adjuvant-free challenge of mice with small doses of mTg conjugated to monoclonal antibodies (MAbs) specific for class II MHC (I-Ak) expressed on APC, induces an mTg-specific IgG response in CBA (H-2k) but not in B6 (H-2b) mice. Priming of CBA mice with mTg conjugated to an irrelevant MAb (control) did not elicit an autoimmune response. Despite the induction of an mTg-specific IgG response, mononuclear cell infiltration of the thyroid was not detected in CBA mice thus indicating a clear divergence in the requirements for autoantibody (AAb) production and the requirements for disease. These findings may help elucidate the role of various APC subsets in autoimmunity and facilitate study of the initial events that trigger autoreactivity outside a CFA-induced granuloma site. -- Investigation in rats of the immunopathogenicity of a 17mer homologous (rat) Tg peptide, rTgP1 (2495-2511), revealed that TgP1-priming of F334, WKY and WF rats elicits lymph node cell (LNC) proliferative responses to the peptide in vitro without concomitant specific primary IgG responses. LNC proliferative assays demonstrated that specific CD4⁺ T cells recognize TgP1 in the context of class II MHC and that TgP1 contains cryptic T cell epitope(s). Strong TgP1-specific IgG responses were not observed despite the presence of EAT in rats. These results provide the first evidence that a self-Tg peptide, TgP1, is immunopathogenic in rats. In contrast to 17mer TgP1, examination in rats of the immunopathogenicity of an 18mer rTg peptide, rTgP2 (2695-2713) revealed that TgP2- priming of rats induced specific LNC proliferative responses in F344, WKY but not in WF rats, although concomitant specific IgG responses were elicited in all three rat strains. Thyroiditis was readily induced in F344 rats both by direct challenge with TgP2 and by adoptive transfer with TgP2- specific T cells (CD4⁺, CD8⁻, TCR α/β⁺) indicating that peptide-induced rat EAT is a CD4⁺ T cell-mediated disease. Specific T cells recognize TgP2 in the context of class II molecules in an MHC-unrestricted fashion and TgP2 contains non-dominant T cell determinants. The TgP2-specific IgG (day 28) readily binds intact rTg and such binding could be abrogated by free peptide indicating the absence of "determinant spreading". These results indicate that TgP2 is pathogenic in rats but differs in its immunogenicity from that of TgP1. -- Class II rat thyrocytes (IFN-γ treated), to address their putative APC function in TgP2-mediated EAT, were examined by testing their ability to present endogenous Tg epitope to the TgP2-specific cloned T cells. Both unpulsed or peptide pulsed class II+ thyrocytes failed to activate the hybridoma (assessed by IL-2 release) suggesting that thyrocytes by themselves do not function as APC in vitro. The findings that both glutaraldehyde fixation and irradiation of spleenocytes abolished their capacity to present TgP2 leading to activation of the 8F7-5 hybridoma raised the possibility that thyrocytes may be deficient in the expression of certain costimulatory molecules. These findings have implications for understanding Ag-presentation in thyroid autoimmunity. -- Examination of the relative serological immunodominance of the pathogenic TgP1 and TgP2 peptides revealed contrasting findings. Priming of F344 rats either with homologous or heterologous Tgs did not elicit TgP1-specific IgG responses indicating the non-dominance of TgP1. In contrast, homologous Tg-priming elicited TgP2-specific IgG response. The immunogenicity of the TgP2 epitope(s) on heterologous Tgs varied dramatically, being highest on bovine Tg, intermediate on mouse Tg and undetectable on human and porcine Tgs although peptide-specific IgG readily cross-reacted with Tgs of various species. Epitope analysis of heterologous Tg-primed sera revealed that TgP2-reactivity is directed to distinct B epitopes within TgP2. These data provide the first evidence in EAT that variable immunodominance may partially explain why distinct Tg epitopes are recognized by MAbs in various species/Tg combinations and may have implications in serological screening of pathogenic Tg epitopes.

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