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

Abstract

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.

Clonal relationships between thyroid‐stimulating hormone receptor‐stimulating antibodies illustrate the effect of hypermutation on antibody function

“…Moreover, another panel of TSAb mAbs 13 were also restricted to usage of IGHD52 and IGHJ2 gene segments. In another recent study, diversity in the CDR3 regions of the anti‐TSHR antibodies was studied at the population level of serum antibodies in mice induced to develop hyperthyroidism by correlating with thyroid function 29 . By subcloning and sequencing PCR products from splenic B cells derived from individual groups of immune animals with differing levels of anti‐TSHR antibodies and serum T4 levels, the authors were able to statistically correlate usage at a greater frequency of IGHDQ52 and IgHJ1/IgHJ4 gene segments with hyperthyroidism 29 .…”

“…29 By subcloning and sequencing PCR products from splenic B cells derived from individual groups of immune animals with differing levels of anti-TSHR antibodies and serum T4 levels, the authors were able to statistically correlate usage at a greater frequency of IGHDQ52 and IgHJ1/IgHJ4 gene segments with hyperthyroidism. 29 All these studies show usage of IGHDQ52, including the results described in this study with KSAb1 and KSAb2 TSAbs. Thus, the IgHDQ52 gene segment may be an important genetic element in the repertoire for the development of stimulating antibodies in mice immunized with the human TSHR.…”

Immunoglobulins or Antibodies: IMGT® Bridging Genes, Structures and Functions

“…Thus the annotation of the IG and TR loci [ 78 , 322 , 323 , 324 , 325 , 326 , 327 ] are key to the study and comparison of the expressed adaptive immune repertoires, in normal or pathological situations. The IMGT standardized IG and TR genes and alleles in different species [ 328 , 329 , 330 , 331 , 332 , 333 , 334 , 335 , 336 , 337 , 338 , 339 , 340 , 341 , 342 , 343 , 344 , 345 , 346 , 347 , 348 , 349 , 350 , 351 , 352 , 353 , 354 , 355 , 356 , 357 , 358 , 359 , 360 , 361 , 362 ] offer a unique opportunity for comparison of immune responses and of potential applications in veterinary and human medicine.…”

Euthyroid Graves’ orbitopathy and incidental papillary thyroid microcarcinoma

“…[36][37][38] Thyroid-blocking antibodies are part of the TBII assay, though this assay is blind to the stimulatory or inhibitory nature of each anti-TSH-receptor antibody it measures. In the future, as more specific assays become commercially available, hopefully we will be able to elicit even more information about the biochemical basis for euthyroid GO.…”