Presence of Epstein-Barr virus-infected B lymphocytes with thyrotropin receptor antibodies on their surface in Graves' disease patients and in healthy individuals

Abstract

Graves' disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein-Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves' disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves' disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves' disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves' disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves' disease.

Figure 1.

FCM pattern of EBER1 and TRAbs in cultured PBMCs. We confirmed the background for each fluorescence by fluorescence minus one (FMO) control [22,23] 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 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.