Diversity in the complementarity-determining region 3 (CDR3) of antibodies from mice with evolving anti-thyroid-stimulating hormone receptor antibody responses

In a mouse model of autoimmune Graves' disease, stimulatory anti-TSH receptor (TSHR) antibodies (TSAbs) slowly evolve upon repeated immunization with TSHR and lead to hyperthyroidism. Although all immunized mice developed high levels of TSH-binding inhibitory Ig (TBII), only a subset of these mice become hyperthyroid, suggesting that the generation of pathogenic antibodies (Abs) may require affinity maturation. We analyzed the complementarity-determining region 3 (CDR3) of IGHV1 and IGHV5 heavy chains from mice at different stages of disease development. Subcloned CDR3 PCR products were amplified from RNA isolated from enriched splenic B/plasma cells of a control mouse, and mice with low TBII and normal T₄ levels (LTNT₄), high TBII and normal T₄ levels (HTNT₄), and high TBII and high T ₄ levels (HTHT₄). Using statistical analyses, we correlated usage of D and J genes and the amino acid composition and length of and mutations within the CDR3 with different outcomes after TSHR immunization. CDR3 sequences from TSHR-immunized mice contained a higher frequency of D gene SP2.9 relative to control, whereas sequences from HTHT₄ contained a higher frequency of D gene Q52 compared with sequences from LTNT₄. Furthermore, HTHT₄ sequences also contained higher CDR3 replacement mutations, relative to LTNT₄ and HTNT₄ mice, that are indicative of somatic hypermutation. Collectively, our results suggest that higher somatic mutations within the CDR3 may correlate with pathogenic antibodies against the TSHR.