Pathogenesis of Graves’ Disease Determined Using Single-Cell Sequencing with Thyroid Autoantigen Peptide Stimulation in B Cells

This study reports the use of single-cell RNA sequencing to evaluate B cells in the peripheral blood mononuclear cells (PBMCs) and intrathyroidal blood mononuclear cells of patients with Graves’ disease (GD) undergoing thyroidectomy. These cells were stimulated with overlapping peptides of thyroid autoantigens, including thyroid-stimulating hormone receptor (TSHR), thyroglobulin (Tg), and thyroid peroxidase (TPO). In PBMCs, naive B cells are characterized by <i>IL6</i> and <i>CXCR5</i>, whereas memory B cells express <i>IGHG1</i>, <i>IGHG2</i>, and <i>CD74</i>. <i>HLA-DMA</i>, <i>HLA-DRB1</i>, <i>IGHG</i>, <i>IGHM</i>, <i>CD74</i>, <i>CD79A</i>, and <i>MS4A1</i> expression increased in peptide-stimulated naive and memory B cells compared to those in the controls. Thyroid naive B cells are characterized by <i>CD40</i> and <i>TNFRSF13C</i>, whereas memory B cells express <i>IGHM</i>, <i>CD79A</i>, and <i>MS4A1.</i> Thyroid B cells showed higher <i>DUSP1</i>, <i>DUSP2</i>, <i>CD69</i>, <i>FOSB</i>, <i>RGS1</i>, and immunoglobulin gene expression than control PBMCs and thyroid cells. B-cell receptor analysis revealed frequent <i>IGHV3-23</i> and <i>IGHV4-34</i> usage in controls, whereas <i>IGHV4-34/IGHJ4</i> expression was increased in TSHR-stimulated groups. We concluded that B-cell responses to TSHR, Tg, and TPO differed and that changes in B-cell reactivity also occurred in PBMCs and the thyroid. Additionally, <i>IGHV3-23</i> and <i>IGHV4-34</i> may be associated with autoantibody production in GD.