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 T(4) levels (LTNT(4)), high TBII and normal T(4) levels (HTNT(4)), and high TBII and high T(4) levels (HTHT(4)). 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 HTHT(4) contained a higher frequency of D gene Q52 compared with sequences from LTNT(4). Furthermore, HTHT(4) sequences also contained higher CDR3 replacement mutations, relative to LTNT(4) and HTNT(4) 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.
Martinez
Osvaldo
O
Department of Microbiology and Immunology (M/C 790), Room E-709, Building 935, 835 South Wolcott Avenue, Chicago, Illinois 60612, USA.
Gangi
Eryn
E
Mordi
David
D
Gupta
Sonal
S
Dorevitch
Samuel
S
Lefranc
Marie-Paule
MP
Prabhakar
Bellur S
BS
D000818:Animals; D001323:Autoantibodies / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D022801:Complementarity Determining Regions / Q000235:genetics*; D018450:Disease Progression; D050438:Genes, Immunoglobulin Heavy Chain; D014644:Genetic Variation; D006111:Graves Disease / Q000097:blood / Q000235:genetics* / Q000276:immunology* / Q000503:physiopathology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007136:Immunoglobulins / Q000276:immunology; D018828:Immunoglobulins, Thyroid-Stimulating; D051379:Mice; D016133:Polymerase Chain Reaction; D013972:Thyrotropin / Q000276:immunology; D013974:Thyroxine / Q000097:blood
D001323:Autoantibodies; D022801:Complementarity Determining Regions; D007143:Immunoglobulin Heavy Chains; D007136:Immunoglobulins; D018828:Immunoglobulins, Thyroid-Stimulating; C094372:thyrotropin-binding inhibitory immunoglobulin; D013972:Thyrotropin; D013974:Thyroxine