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Diversity in the Complementarity-Determining Region 3 (CDR3) of Antibodies from Mice with Evolving Anti-Thyroid-Stimulating Hormone Receptor Antibody Responses

Department of Microbiology and Immunology (E.G., D.M., S.G., B.S.P.), School of Public Health, Epidemiology, and Biostatistics (S.D.), University of Illinois at Chicago, Chicago, Illinois 60612; Laboratoire d’ImmunoGénétique Moléculaire (M.-P.L.), Université Montpellier II, Unité Propre de Recherche, Centre National de la Recherche Scientifique 1142, Institut de Génétique Humaine, 34396 Montpellier, France; and Department of Microbiology (O.M.), Mount Sinai School of Medicine, New York, New York 10029

Address all correspondence and requests for reprints to: Bellur S. Prabhakar, Department of Microbiology and Immunology (M/C 790), Room E-709, Building 935, 835 South Wolcott Avenue, Chicago, Illinois 60612. E-mail: bprabhak{at}uic.edu.

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 T₄ 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.