(D) Comparison of the IgG concentration in culture supernatants between paired CD11c+ B cells and CD11c- B cells in each batch from different GD patients. fT3) in all enrolled GD patients. The correlation analyses above were performed using the Spearman correlation test. Image_4.tif (340K) GUID:?349AD713-4DE7-43E1-A960-AC438C13CF1C Supplementary Figure?S5: Global gating strategy for analysis of CD11c expression distribution. After gating the single cells and lymphocytes, B cells were circled as CD19+ cells for further analysis. Then, B cells were divided into 13 subsets according to the expression of IgD, CD27, CD38, and CD138. The B-cell subsets are as follows: Q1-na?ve B cells (CD27-IgD+), Q2-unswitched memory B cells (CD27+IgD+), Q3-switched memory B cells (CD27+IgD-), Q4-double negative memory B cells (CD27-IgD-), Q5-na?ve mature B Dihydroethidium cells (CD38-IgD+), Q6-activated na?ve mature B cells (CD38+IgD+), Q7-early memory mature B cells/germinal center B cells (CD38+IgD-/CD38highIgD-), Q8-resting memory B cells (CD38-IgD-), Q9-transitional B cells (CD38-CD27+), Q10-plasmablasts (CD38+CD27+), Q11-transitional-like B cells (CD38+CD27-), Q12-memory B-cell precursors (CD38-CD27-), and plasma cells (CD138+). CD11c+ B cells were circled in the above B-cell subsets. Image_5.tif (1.8M) GUID:?C8FEF229-0421-4684-B87A-E4F12CDA7F13 Supplementary Figure?S6: Global gating strategy for comparing immune marker expression between CD11c+ and CD11c- B cells. CD19+ B cells were gated for the following analysis, and CD11c+/high and CD11c- B cells were circled to analyze the frequency and MFI of the positive subpopulation among all immunomarkers, including CD27, CD38, IgD, CD138, T-bet, CXCR5, CXCR3, CD32, and CD21. Image_6.tif (516K) GUID:?D9518308-0074-48CE-8827-385636D457BA Supplementary Physique?S7: IgG production of B cells stimulated with different concentrations of R848. Total B cells from GD patients were stimulated with a concentration gradient of R848 (a TLR7/8 agonist). The culture supernatants were collected on day 9 and measured by ELISA. Data are presented as the mean SD and were assessed by ANOVA. The 0.1, 1, and 10 g/ml groups were compared with the 0 g/ml group, and the results are marked above the histogram bars. 0.05 was considered statistically significant. ns, not significant; ****0.0001. Image_7.tif (1.0M) GUID:?E8FD86E7-B6E6-425C-9329-064CA79456E8 Supplementary Table?S1: Antibodies used in flow cytometry analysis. Table_1.docx (14K) GUID:?9729443A-ECD9-4120-B3E0-D52B9C928642 Supplementary Table?S2: Antibodies used in multiplex immunofluorescence staining. Table_2.docx (14K) GUID:?04BC5C56-8FBB-40A8-A995-0DBBC0302A7E Supplementary Table?S3: The lower limits of detection (LLOD) of all cytokines in the Luminex liquid suspension chip. Table_3.docx (14K) GUID:?A0465B2F-FBA4-4DBA-9427-242ACCE7054F Data Availability StatementThe initial datasets analyzed in the current study are available from the corresponding author on affordable request. Dihydroethidium Abstract Graves disease (GD) is usually a common Dihydroethidium autoimmune disorder with an elevation in pathogenic autoantibodies, specifically anti-thyrotropin receptor antibodies (TRAbs), which are secreted by autoreactive B cells. To date, there has been little research on self-reactive B cells in GD. In the current study, we reported that a unique B-cell subset, CD11c+ B cells, was expanded in the peripheral blood (PB) of GD patients, as detected by flow cytometry. The frequency of CD11c+ B cells was positively correlated with serum TRAb levels. The flow cytometry data showed that CD11c expression was higher in a variety of B-cell subsets and that CD11c+ Dihydroethidium B cells presented a distinct immunophenotype compared to paired CD11c- B cells. Immunohistochemical and immunofluorescence staining indicated the presence of CD11c+CD19+ B cells in lymphocyte infiltration areas of the GD thyroid. Flow cytometric analysis of PB and fine-needle aspiration (FNA) samples showed that compared to PB CD11c+ B cells, CD11c+ B cells in the thyroid accumulated and further differentiated. We found that CD11c+ B cells from the PB of GD patients were induced to differentiate into autoreactive antibody-secreting cells (ASCs) capable of secreting TRAbs the CXCR3-CXCL10 axis. In conclusion, our study decided that CD11c+ B cells were involved in the pathogenesis of GD in multiple ways and might represent a promising immunotherapeutic target in the future. Keywords: CD11c+ B cells, TRAb, Graves disease, cytokines, CXCR3-CXCL10 Introduction Graves disease (GD), as the most common cause of persistent Dihydroethidium hyperthyroidism in adults (1), is an organ-specific autoimmune disease characterized by diffuse goiter and an elevation in anti-thyrotropin receptor PRDM1 antibodies (TRAbs). Some patients also present with extrathyroidal complications, such as Graves orbitopathy (GO), and untreated hyperthyroidism is related to increased risks of osteoporosis (2), fracture (3), stroke (4), and cardiovascular events (5, 6). For the past 70.