Peptide scanning for thyrotropin receptor T-cell epitopes in mice vaccinated with naked DNA

Vaccinating mice with DNA encoding the thyrotropin receptor (TSHR), the major autoantigen in Graves' disease, induces memory T cells that secrete interferon-γ (IFN-γ) in response to TSHR antigen. We used a panel of 29 synthetic TSHR peptides encompassing the ectodomain and three extracellular loops to identify T-cell epitopes after TSHR-DNA vaccination of BALB/c, NOD.H-2h4, and AKR/N mice. These strains were chosen because of their previous use in animal models of thyroid autoimmunity. In initial studies, challenge of splenocytes with TSHR protein induced IFN-γ and tumor necrosis factor-α (TNF-α) production in all three strains of mice. BALB/c mice recognized three peptides, all in the TSHR A subunit. These peptides differed from the four peptides recognized by nonobese diabetic (NOD mice NOD H-2h4). Three of the latter were also in the A subunit. The fourth was within the intervening C peptide region excised on TSHR cleavage into A and B subunits. Because of high and erratic responses in AKR/N mice, their TSHR T-cell epitopes could not be determined. In summary, we report that TSHR DNA vaccination of BALB/c and NOD.H-2h4 mice, with different major histocompatibility complex (MHC) class II genes (I-A^d and I-A^k, respectively), recognize restricted, nonoverlapping TSHR T-cell epitopes, nearly all in the TSHR A subunit.