Monoclonal Antibodies to Thyrotropin Receptor With Thyroid-Stimulating Activity Activate the NF-κB Pathway to Induce...

The A subunit of thyrotropin receptor (TSHR) is thought to be the crucial gene mediating stimulatory autoantibodies in Graves' diease (GD), but it remains unclear what the molecular basis of this pathological antibody response is. Stimulatory TSHR autoantibodies may induce activation of multiple signalling pathways in GD, modulate chemokine exposure and further stimulate immune imbalance. In this study, we prepared TSHR 289 protein by using insect baculovirus expression, adenovirus-expressed TSHR289 immunised mice, and obtained three mouse anti-TSHR monoclonal antibodies (mAbs), 1A4, 7C3 and 22B1, by the hybridoma technique. Flow assay and ELISA tests tested the activity and competitive binding of the mAbs. After mAbs stimulation of human thyrocytes, RT-qPCR and ELISA were used to detect the expression of chemokine; Western blotting detected the expression of CCL19 and the level of phosphorylation of NF-κB. Nanogram concentrations of the IgG mAbs 1A4, 7C3 and 22B1 and their Fab induce TSHR stimulation. TRAb in the serum of GD patients competitively inhibits the binding of HRP-conjugated mAbs to TSHR on the coated plate. Injection of micrograms of 7C3 resulted in elevated serum thyroxine and columnar and papillary hyperplasia of thyroid follicular epithelial cells. All three mAbs induced distinct expression of CCL2, CCL19 and CCL5 by activating canonical and non-canonical NF-κB signalling pathways in human thyrocytes. Collectively, we obtained three mouse anti-TSHR mAbs which provide an improved approach to characterise the molecular basis of this pathological response, and confirmed that stimulating antibodies activate NF-κB, inducing chemokines involved in the autoimmune response.