MON-424 Peptide-based Tolerance Therapy Targeting The Thyrotropin Receptor Induces Regulatory T Cells In HLA-DRB1*03:01 (DR3) Transgenic Mice With Graves’ Disease

Abstract Disclosure: H. Inaba: None. I. Nonaka: None. D. Hashimoto: None. M. Hirono: None. M. Nakata: None. Background: Graves’ disease (GD) is an organ-specific autoimmune disorder characterized by autoantibody-mediated hyperthyroidism. Genetic susceptibility is strongly associated with the HLA-DRB1*03:01 (DR3) allele. The thyrotropin receptor (TSH-R) has been identified as a key autoantigen in GD. Our previous studies have revealed several immunogenic TSH-R peptides, including the immunodominant epitope TSH-R 78-94 (ISRIYVSIDVTLQQLES; p37) (1-3). A mutated variant of p37, referred to as 37m: ISRIYVSIDATLSQLES, exhibits substantially reduced immunogenicity due to specific amino acid substitutions (4). While oral tolerance using thyroglobulin has been demonstrated in experimental autoimmune thyroiditis (5), the therapeutic potential of TSH-R-based peptide immunotherapy remains to be clarified. Objective: To evaluate whether pre-immunization with TSH-R-derived peptides can induce antigen-specific immune tolerance and prevent the development of GD in HLA-DR3 transgenic mice, and to elucidate the underlying immunological mechanisms. Methods: Female HLA-DR3 transgenic mice (5-8 weeks old) were subcutaneously immunized with a recombinant adenovirus encoding human TSH-R (Ad-TSH-R289) to induce GD. Three weeks prior to immunization, mice received subcutaneous pretreatment with p37, 37m, TSH-R 132-150 (a strong binder to HLA-DR3), TSH-R 109-124 (a control peptide) either via a stepwise dose-escalation protocol (0.05-50 μg) or as a single 50 μg dose. In selected groups, anti-CD25 antibody (500 μg) was administered intraperitoneally to deplete regulatory T cells (Tregs). Outcome measures included serum FT4, TSH-R autoantibodies (TRAb), cytokine profiling (IL-2, IL-4, IL-10, IFN-γ), phenotypic analysis of splenic T cell subsets by flow cytometry, TSH-R-specific proliferative responses, and thyroid histopathology. Results: Stepwise administration of p37 markedly suppressed the elevation of FT4 and TRAb and prevented histopathological changes associated with GD. This was accompanied by a significant increase in CD4⁺CD25⁺Foxp3⁺ Tregs, a decrease in CD4⁺PD-1⁺ T cells, an increase in CD8⁺PD-1⁺ T cells, and an elevated Th1/Th2 ratio. Treg frequencies exhibited an inverse correlation with FT4 and TRAb levels. In contrast, peptide 37m demonstrated a limited protective effect. Anti-CD25 antibody-mediated depletion of Tregs led to increased IFN-γ production. Discussion: These findings indicate that stepwise peptide-based immunotherapy using the immunodominant TSH-R peptide p37 effectively induces antigen-specific regulatory T cells and prevents disease onset in HLA-DR3 transgenic mice. This strategy represents a promising antigen-specific therapeutic approach for GD. References: 1. J Clin Endocrinol Metab. 2006;91:2286. 2. Thyroid. 2009;19:11. 3. J Clin Endocrinol Metab. 2010;95:2909. 4. Endocrinology. 2013;154:2234. 5. Endocrinology. 1995;136:3353. Presentation: Monday, July 14, 2025