Immune Response of Mice Transgenic for Human Histocompatibility Leukocyte Antigen-DR to Human Thyrotropin Receptor-Extracellular Domain

Abstract

Background: Hyperthyroidism of Graves' disease is caused by auto-antibodies to human thyrotropin receptor (hTSH-R). To elucidate important T-cell epitopes in TSH-R, we studied three models of immunity to TSH-R in mice. Methods: Mice transgenic for histocompatibility leukocyte antigen DR3 or DR2 were immunized with cDNA for hTSH-R-extracellular domain (hTSH-R-ECD), or hTSH-R-ECD protein, or hTSH-R peptide epitopes. Proliferative responses of immunized splenocytes to epitopes derived from the hTSH-ECD sequence, anti-TSH-R antibody responses, serum thyroxine and TSH, and thyroid histology were recorded. Results: DR3 mice responded to genomic immunization with proliferative responses to several epitopes, which increased in intensity and spread to include more epitopes, during a 6-week immunization program. DR2 transgenic mice developed weak proliferative responses. Both types of mice developed anti-TSH-R antibodies measured by enzyme-linked immunosorbent assay or TSH-binding inhibition assay in 16-60% of animals. There was evidence of weak thyroid stimulation in one group of animals. Immunization of DR3 transgenic mice to hTSH-R-ECD protein induced a striking response to an epitope with sequence ISRIYVSIDVTLQQLES (aa78-94). Immunization to peptides derived from the TSH-R-ECD sequence (including aa78-94) caused strong responses to the epitopes, and development of immune responses to several other nonoverlapping epitopes within the hTSH sequence (epitope spreading) and antibodies reacting with hTSH-R. This implies that immunization with hTSH-R epitopes produced immunity to mouse TSH-R. Conclusion: T-cell and B-cell responses to genetic immunization differ in DR3 and DR2 transgenic mice, and there is less genetic control of antibody than of T-cell responses. During both genomic and peptide epitope immunization there was evidence of epitope spreading during the immunization. Several functionally important epitopes are evident, especially aa78-94. However, if similar progressive epitope recruitment occurs in human disease, epitope-based therapy will be difficult to achieve.

B cell epitope spreading: Mechanisms and contribution to autoimmune diseases

“…Immunization with CC2 peptides induces EAC, then DCM [91] Lewis rat model Intermolecular Diabetes (Type 1) Diversification of Abs to include ICA, GADA, IA-2, and IAA [56] Clincal study Intermolecular Graves' disease Ab diversification from thyroglobulin AA2471-2490 to other epitopes [6] NZW rabbit model Intramolecular Intramolecular epitope spreading on TSHR protein molecule [78,79] BALB/c mice model Intramolecular Ab diversification on the TSHR protein molecule [80,81] Transgenic mice model Intramolecular Heymann nephritis Ab spreading to various epitopes on Megalin protein [94] Lewis rat model Intramolecular Multiple sclerosis Ab spreading from MOG peptide 92-106 to intramolecular epitopes [60] RR-EAE mice model Intramolecular PLP Abs spread to target other myelin proteins [52] EAE mice model Intermolecular Pemphigus…”

“…When Balb/c and C57BL/6 mice where immunized with TSH receptor protein (TSHR), the Ab diversified from recognizing only the N-terminus domain to additional epitopes including the TSHR ectodomain [79]. Using more specific methods, Inaba et al immunized breeding stock mice transgenic for HLA-DR3 and HLADR2 with 41 different TSHR peptides [80]. When mice where immunized with peptides AA70-88, AA83-102, or AA105-118 there was an Ab diversification outside of the original peptide epitope presented.…”

The Role of Glutamic or Aspartic Acid in Position Four of the Epitope Binding Motif and Thyrotropin Receptor-Extracellular Domain Epitope Selection in Graves’ Disease

“…We tested our hypothesis on the importance of D/Eϩ in position 4 using data developed in vitro (HLA binding assays), ex vivo in humans with GD (14), and in vivo in TSH-R immunized mice (15). We found significant association between D or E in position four of epitopes derived from TSH-R-ECD and several measures of binding to three HLA-DR molecules or T cell responses.…”

Immunoinformatic analysis of Chinese hamster ovary (CHO) protein contaminants in therapeutic protein formulations

“…Hence, CHO protein sequences homologous or completely identical to human are hardly comforting. There are a number of animal models in which imperfect homology between an antigen and the host-origin protein contributes to the development of antibody responses to the protein (human TSH-R [34], to human diabetes antigens [37]), which can lead to; epitope spreading from the original nonhomologous epitopes to other conserved epitopes has been described [38], this one reason for concern. 4.…”

“…At the epitope level, 1 out of 36 nine-mer predicted epitopes contained in the protein is significantly different (more than 2 amino acids different on the TCR face) from their human counterpart and can still bind to the same MHC allele. Differences between epitopes may lead to recognition of the protein as a foreign antigen, leading to epitope spreading (as has been observed for human TSH-R in mice [34]. The number of dissimilar epitopes required to trigger an immune response is unknown.…”