Effective Inhibition of Thyroid Antigen Presentation Using Retro-Inverso Peptides in Experimental Autoimmune Thyroiditis: A Pathway Toward Immune Therapies of Thyroid Autoimmunity

Abstract

Background: Autoimmune thyroid diseases (AITD) represent the most common autoimmune diseases. However, current therapies focus on relieving the symptoms instead of curing AITD, and new therapies to reverse the autoimmune attack on the thyroid are needed. HLA-DRβ1-Arg74 is the key HLA class II allele that triggers AITD by presenting pathogenic thyroglobulin (Tg) peptides that activate thyroid self-reactive T cells. We hypothesized that blocking the presentation of Tg peptides to T cells within the HLA-DRβ1-Arg74 peptide binding cleft could reverse the autoimmune response to the thyroid in AITD. Methods: The approach we used to block Tg peptide presentation within HLA-DRβ1-Arg74 is to design retro-inverso D-amino acid (RID) peptides that have high affinity to the HLA-DRβ1-Arg74 peptide binding pocket. Results: By using computational approaches and molecular dynamics simulations, we designed two RID peptides, RT-15 and VT-15, that blocked peptide binding to recombinant HLA-DRβ1-Arg74 molecule, as well as T cell activation in vitro. Furthermore, RT-15 and VT-15 blocked in vivo T cell activation by thyroglobulin in humanized NOD-DR3 mice induced with experimental autoimmune thyroiditis. Conclusions: In summary, we discovered two RID peptides that block thyroglobulin peptide binding to HLA-DRβ1-Arg74 and their presentation to T cells in AITD. These findings set the stage for a personalized medicine therapeutic approach for AITD patients who carry the DRβ1-Arg74 allele. This antigen-specific therapeutic strategy can potentially be extended to other autoimmune diseases.

Keywords: Graves'disease; HLA-DR3; Hashimoto's thyroiditis; autoimmune thyroiditis; peptides.

FIG. 1.

The fractional contributions of the residues of Tg.726 (gray) and Tg.202 (black) to the interaction energy with DR3. The composite sequence at the top represents the sequence with the better fractional contribution to binding (RT-15) [see results section “Construction of RID peptides (RT-15 and VT-15) that block Tg/Tg.2098 binding to the HLA-DRB1-Arg74 pocket”]. Tg, thyroglobulin.

FIG. 2.

Dose response of RT-15 and VT-15 blocking biotinylated APO peptide binding to recombinant HLA-DRβ1-Arg74 protein in vitro. Ten micromolars biotinylated APO peptide was incubated with 0.012 mg/mL HLA-DRβ1-Arg74 protein, with increasing concentrations (0–80 μM) of RT-15 and VT-15. RT-15 blocked APO binding to DRβ1-Arg74 in a dose-dependent manner, with a peak that plateaued at 20 μM (A). Similarly, VT-15 blocked the binding dose dependently, with a peak that plateaued at 40 μM (B).

FIG. 3.

Effect of RT-15 and VT-15 on hTg- and Tg.2098-induced T cell proliferation in the EAT model. Humanized NOD-DR3 mice were induced with EAT, and splenocytes were isolated and incubated with Tg/Tg.2098, with or without RID peptides RT-15 and VT-15. RT-15 blocked hTg- and Tg.2098-induced T cell proliferation significantly (A). VT-15 only blocked Tg.2098-induced T cell proliferation, but not hTg (B). Anti-CD3/CD28 beads, positive control. **p-Exact <0.01; ***p-exact <0.001. EAT, experimental autoimmune thyroiditis; hTg, human thyroglobulin; NC, negative control peptide; RID, retro-inverso D-amino acid.

FIG. 4.

Effect of RT-15 and VT-15 on hTg- and Tg.2098-induced T cell activation in the EAT model as measured by IFNγ production. Splenocytes were isolated from humanized NOD-DR3 mice induced with EAT and incubated with Tg/Tg.2098, with or without RID peptides RT-15 and VT-15. RT-15 significantly reduced IFNγ production induced by hTg and Tg.2098 (A), while VT-15 only reduced hTg-induced IFNγ production (B). Anti-CD3/CD28 beads, positive control. *p-Exact <0.05; ***p-exact <0.001. IFNγ, interferon gamma.

FIG. 5.

Treatment of EAT in vivo with RID peptide cocktail. Humanized NOD-DR3 mice were pretreated with a mix of RID peptide cocktail RT-15 and VT-15, or PBS as control, before immunization with Tg to induce EAT. Upon sacrifice, splenocytes were isolated and incubated with Tg and Tg.2098. Mice injected with RID peptide cocktail (white bar) showed less hTg-induced T cell proliferation compared with PBS control (black bar), although not statistically significant (p-exact = 0.152). Similarly, splenocytes isolated from mice pretreated with D-peptide cocktail showed less T cell proliferation in response to Tg.2098, compared with PBS control, although not statistically significant (p-exact = 0.094). PBS, phosphate-buffered saline.