Discovering Thyroglobulin T-cell epitopes by Inducing Thyroiditis in Humanized Mice Expressing Human DR3

Presentation Number: OR28-4
Date of Presentation: June 16th, 2013

Cheuk Wun Li*1, Francesca Menconi1, Roman Osman1, Erlinda Concepcion1 and Yaron Tomer2
1Mount Sinai School of Medicine, New York, NY, 2Icahn School of Medicine at Mount Sinai and James J. Peters VA Medical Center, New York, NY

Abstract

Autoimmune thyroid diseases (AITD) result from the interaction between genetic factors and environmental triggers. Among known AITD susceptibility genes, the HLA gene locus is key. Our lab has previously shown that an HLA-DR variant that contains arginine at position 74 of the DRβ1 chain (DRb1-Arg74) is the specific MHC variant conferring risk for AITD while the presence of glutamine at position 74 is protective. We hypothesized that the DRb1-Arg74 peptide binding pocket can present pathogenic thyroglobulin (Tg) peptides more effectively thereby triggering AITD. Indeed, we identified 5 Tg peptides that bound specifically to the DRb1-Arg74 pocket in vitro. To test in vivo whether these Tg peptides can bind specifically to the DRb1-Arg74 pocket and stimulate T-cell responses, we used “humanized” mice expressing human DR3 that are capable of presenting antigens within the human DRb1-Arg74 peptide binding pocket, which serve as an excellent model to identify human thyroglobulin (hTg) T-cell epitopes that bind to this pocket.

We induced experimental autoimmune thyroiditis (EAT) by immunizing the DR3 transgenic mice (both on C57BL/6 background and on NOD background which is more susceptible to EAT) subcutaneously with hTg in CFA on days 0 and 7, and sacrificed them on day 21. We then assessed T-cell responses to the 5 hTg peptides that bind in vitro to DRb1-Arg74 (Tg1951, Tg1571, Tg202, Tg726, Tg2098) using CFSE test of proliferation and evaluating their cytokine responses. In addition, we confirmed the development of EAT by determining anti-thyroglobulin antibody levels in the serum and looking for thyroidal lymphocytic infiltration.

 Two of 3 NOD mice and 9 out of 21 C57BL/6 mice immunized with hTg showed T-cell responses to hTg or hTg peptides. The T-cell response was accompanied by high levels in anti-thyroglobulin antibody levels and strong cytokine response. Thyroid infiltration was also observed in the immunized NOD mice. Peptide Tg2098 showed the strongest T-cell responses in mice that developed EAT while the 4 other peptides showed lower responses.

 Our studies showed that all 5 peptides that bind in vitro to HLA-DRb1-Arg74 also stimulate T-cell responses, although to a varying degree. We conclude that these 5 Tg peptides are likely to be T-cell epitopes, among which Tg2098 appears to be the major one. Our findings set the stage to designing inhibitors of binding of these hTg epitopes to HLA-DRb1-Arg74 as a potential novel therapeutic modality in AITD.

 

Nothing to Disclose: CWL, FM, RO, EC, YT