Efficacy of HLA-DRB1∗03:01 and H2E transgenic mouse strains to correlate pathogenic thyroglobulin epitopes for autoimmune thyroiditis

Abstract

Thyroglobulin (Tg), a homodimer of 660 kD comprising 2748 amino acids, is the largest autoantigen known. The prevalence of autoimmune thyroid disease, including Hashimoto's thyroiditis and Graves' disease, has provided the impetus for identifying pathogenic T cell epitopes from human Tg over two decades. With no known dominant epitopes, the search has long been a challenge for investigators. After identifying HLA-DRB1∗03:01 (HLA-DR3) and H2E(b) as susceptibility alleles for Tg-induced experimental autoimmune thyroiditis in transgenic mouse strains, we searched for naturally processed T cell epitopes with MHC class II-binding motif anchors and tested the selected peptides for pathogenicity in these mice. The thyroiditogenicity of one peptide, hTg2079, was confirmed in DR3 transgenic mice and corroborated in clinical studies. In H2E(b)-expressing transgenic mice, we identified three T cell epitopes from mouse Tg, mTg179, mTg409 and mTg2342, based on homology to epitopes hTg179, hTg410 and hTg2344, respectively, which we and others have found stimulatory or pathogenic in both DR3- and H2E-expressing mice. The high homology among these peptides with shared presentation by DR3, H2E(b) and H2E(k) molecules led us to examine the binding pocket residues of these class II molecules. Their similar binding characteristics help explain the pathogenic capacity of these T cell epitopes. Our approach of using appropriate human and murine MHC class II transgenic mice, combined with the synthesis and testing of potential pathogenic Tg peptides predicted from computational models of MHC-binding motifs, should continue to provide insights into human autoimmune thyroid disease.

Fig. 1

Depletion of CD4⁺CD25⁺ regulatory T cells exacerbates mTg-induced, but not hTg-induced, thyroiditis. DR3⁺ AE⁰ mice were given 1 mg anti-CD25 i.v. on days −14, −10, immunized i.v. with 40 µg mTg or 100 µg hTg + 20 µg LPS on days 0, 7 and sacrificed on day 28 or 35.

Fig. 2

H2E MHC class II-derived peptide Eα52-68 blocks presentation of an mTg peptide by H2A^b. (A) B220-gated APC from A⁺E⁻ (left) or A⁺E⁺ (right) spleen cells were FACS analyzed for expression of Eα52-68/A^b (mAb Y-Ae, thick line), or total A^b (mAb Y-3P, dotted line) as a control. The dashed line shows background fluorescence. (B) A⁺E⁻ mice were immunized with peptide mTg1677 in CFA and lymph nodes were removed on day 10. On day of culture, irradiated spleen cells were incubated for 1 h with or without Eα52-68 (1° incubation, 10 µg/ml) and washed. The spleen cells were subsequently incubated for another hour with mTg1677 (2° incubation, 10 µg/ml) and washed. These pulsed APC were then added to cultures (4×10⁵/well) along with mTg1677-primed LNC from immunized mice (6×10⁵/well) for 5 days. Proliferation was assessed by [³H]thymidine incorporation (background mean±SE cpm: 1200±160). *, P<0.01 (Modified and reproduced by permission from Brown et al. [11]. Copyright 2008. The American Association of Immunologists, Inc.).

Fig. 3

Proliferation of hTg-primed mouse spleen cells to hTg-derived DR3 peptides. DR3⁺DQ8⁺ Ab⁰/NOD (A) or DR3⁺ AE⁰ (B) mice were immunized with 100 µg hTg followed 3 hours later by 20 µg LPS (days 0, 7). When mice were sacrificed 4–5 weeks later, spleen cells (6×10⁵/well) were cultured for 4 days with hTg (40 µg/ml) or hTg peptides (5 µg/ml). Proliferation was assessed by [³H]thymidine incorporation (background mean±SE cpm: A, 7280±400; B, 13320±4120).

Fig. 4

mTg179 is pathogenic for DR3⁺ AE⁰ mice and causes a higher incidence and more severe thyroid destruction after CD4⁺CD25⁺ regulatory T cell depletion. Mice were given 0.5 mg anti-CD25 i.v. on days −14, −10, immunized i.v. with 100 µg mTg179 in CFA on days 0, 7 and sacrificed on day 29.