Figures & data
Table 1. Clinical data, EBV infection status and TRAbs(+) cells%.
Figure 1. FCM pattern of EBER1 and TRAbs in cultured PBMCs. We confirmed the background for each fluorescence by fluorescence minus one (FMO) control [Citation22,Citation23] as the negative controls using cultured PBMCs from a patient (A–D). There was no leaking of fluorescence but was weak non-specific fluorescence with the APC staining. We did not take the weak APC fluorescence as the positive results because these were difficult to distinguish from non-specific fluorescence. Right two panels are the representative data of healthy control (E), and patient (F). TRAbs(+) EBV(+) DP cells appeared in the right upper quadrant. (A) non-stain control. (B) background without APC. (C) background without Alexa Fluor 488. (D) full stain. (E) dot-plot of a healthy control. (F) dot-plot of a patient. Abbreviations: APC, allophycocyanin; DP, double-positive; EBER, EBV-encoded small RNA; EBV, Epstein–Barr virus, human herpesvirus 4; FCM, flow-cytometry; FMO, fluorescence minus one; TRAbs, thyrotropin receptor antibodies.
![Figure 1. FCM pattern of EBER1 and TRAbs in cultured PBMCs. We confirmed the background for each fluorescence by fluorescence minus one (FMO) control [Citation22,Citation23] as the negative controls using cultured PBMCs from a patient (A–D). There was no leaking of fluorescence but was weak non-specific fluorescence with the APC staining. We did not take the weak APC fluorescence as the positive results because these were difficult to distinguish from non-specific fluorescence. Right two panels are the representative data of healthy control (E), and patient (F). TRAbs(+) EBV(+) DP cells appeared in the right upper quadrant. (A) non-stain control. (B) background without APC. (C) background without Alexa Fluor 488. (D) full stain. (E) dot-plot of a healthy control. (F) dot-plot of a patient. Abbreviations: APC, allophycocyanin; DP, double-positive; EBER, EBV-encoded small RNA; EBV, Epstein–Barr virus, human herpesvirus 4; FCM, flow-cytometry; FMO, fluorescence minus one; TRAbs, thyrotropin receptor antibodies.](/cms/asset/2da377c8-5221-4add-925d-3564caf4fe45/iaut_a_879863_f0001_b.jpg)
Figure 2. Images of TRAbs(+) EBV(+) double-positive cells from a healthy control (A) and a Graves’ disease patient (B). Cell surface TRAbs were stained as red spots of APC. The granular signals of Alexa Fluor 488 showed EBV encoded small RNA (EBER) 1 in the nuclear structures. The Alexa Fluor 488 signal was also observed in the cytoplasm, and a deep blue DAPI signal represented the cell nucleus. Abbreviations: APC, allophycocyanin; DAPI, 4′, 6-diamidino-2-phenylindole; EBER, EBV-encoded small RNA; EBV, Epstein–Barr virus, human herpesvirus 4; TRAbs, thyrotropin receptor antibodies.
Figure 3. Effects of short-term culture of PBMCs (Figures do not represent the accurate cell frequency). After short-term culture, EBV(−) lymphocytes decreased and only EBV(+) cells increased, because EBV(+) cells were immortalized by persistent EBV. DP cells increased with the expansion of EBV(+) cells. However, the percentage of TRAbs(+) cells in the PBMCs was regarded as the same as that in the noncultured PBMCs. (A) The case (most healthy controls) with low influence of genetic susceptibility. In the case with low genetic influence, the frequencies of TRAbs(+) cells in original PBMCs and thus cultured PBMCs are low for the case to be healthy. (B) The case (most patients) with high influence of genetic susceptibility. Under the high influence of genetic susceptibility, the frequencies of TRAbs(+) cells both in original PBMCs and cultured PBMCs are high. Abbreviations: DP, double-positive; EBV, Epstein–Barr virus, human herpesvirus 4; PBMC, peripheral blood mononuclear cells; TRAbs, thyrotropin receptor antibodies.
Figure 4. The hypothesis of EBV-related pathogenesis in Graves’ disease: EBV may have the potential to stimulate TRAbs production in host B cells. EBV-infected memory B cell with TRAbs on its surface could be terminally differentiated to plasma cell (possibly through EBV-reactivation) and produces excessive TRAbs that are the causative antibodies of Graves’ disease. Abbreviations: EBV, Epstein–Barr virus, human herpesvirus 4; TRAbs, thyrotropin receptor antibodies.
