Follicular dendritic cells display microvesicle-associated LMP1 in reactive germinal centers of EBV+ classic Hodgkin lymphoma

Abstract

Expression of the latent membrane protein-1 (LMP1) of Epstein-Barr virus (EBV) was investigated in 153 cases of EBV+ classic Hodgkin lymphoma (cHL); 120 cases were pediatric patients (< 14 years of age) from Iraq, and 33 cases were adult patients from Italy. We describe for the first time the presence of LMP1 protein in EBV-encoded RNA (EBER)-negative follicular dendritic cells (FDCs) of reactive germinal centers (GC) associated with EBV+ cHL. Presence of LMP1+ GCs was independent of geographic region and age of patients. Variable numbers of reactive GCs were present in 22.2% of cases (34 of 153), whereas LMP1 staining of FDCs was present in about a third of cases (10 of 34) with reactive GC. Most cases with LMP1+ GC were mixed-cellularity (MC) subtype, but some nodular sclerosis (NS) was also present. GC cells with LMP1+ FDCs were surrounded by numerous EBV-infected cells which were positive for EBER, LMP1, and CD30. Double immunolocalization analysis revealed that LMP1 was associated with CD63, an exosomal marker, and with CD21. The possibility is discussed that peri-follicular EBV-infected cells release LMP1 protein, perhaps through exosomes, and that the protein is then captured by FDCs and is presented to EBER-negative GC B cells.

Keywords: Classic Hodgkin lymphoma (cHL); Epstein-Barr virus (EBV); Exosomes and microvesicles; Follicular dendritic cells (FDCs); Latent membrane protein-1 (LMP1); Programmed death ligand 1 (PD-L1).

Fig. 1

a Cervical lymph node of a 26-year-old male with partial involvement by EBV+ cHL NS subtype. Two small CD20+ GCs in the reactive portion of the lymph node (red circle) are intensely stained for LMP1. Stained cells have a dendritic morphology consistent with a FDC origin. In the HL lesion, numerous large cells have HRS morphology and are EBER+. b Cervical lymph node of an 8-year-old boy with CD20+ follicular hyperplasia and with a small focus (HL) of EBV+ cHL. In the area with follicular hyperplasia, a large LMP1+ reactive GC (red circle) is surrounded by numerous LMP1+ cells infected by EBV (higher magnification)

Fig. 2

EBV+ cHL with follicular hyperplasia. EBER+/LMP1+ large cells surround a B cell follicle with FDCs positive for LMP1 and negative for EBER

Fig. 3

Immunostaining for PD-L1 (clone 22C3) in two cases of EBV+ cHL with reactive GCs. Numerous intensely stained cells, probably macrophages, encircle B cell follicles

Fig. 4

Case 1. Numerous LMP1+ large cells surround and partially infiltrate B cell follicles with LMP1+ FDCs. Double staining with immunofluorescence confirmed the presence of peri-follicular EBV-infected cells (LMP1+, red) and of CD21+ reticular FDCs (green). CD63, a marker for exosomes, was present in most stromal cells with reticular morphology inside and outside GCs. A high magnification of GC revealed the presence of double-positive cells (LMP1/CD63) with dendritic morphology

Fig. 5

A GC positive for LMP1 was double stained for CD21 (green) and for CD63 (red). Extensive areas of co-localization of the two signals (yellow) are present, thus suggesting that CD63 protein was associated with FDCs