Simultaneous detection of the two main proliferation driving EBV encoded proteins, EBNA-2 and LMP-1 in single B cells

Epstein Barr virus (EBV) is carried by almost all adults, mostly without clinical manifestations. Latent virus infection of B lymphocytes induces activation and proliferation that can be demonstrated in vitro. In healthy individuals, generation of EBV induced malignant proliferation is avoided by continuous immunological surveillance. The proliferation inducing set of the virally encoded genes is expressed exclusively in B cells in a defined differentiation window. It comprises nine EBV encoded nuclear proteins, EBNA 1–6, and three cell membrane associated proteins, LMP-1, 2A and 2B, designated as latency Type III. Outside this window the expression of the viral genes is limited. Healthy carriers harbor a low number of B lymphocytes in which the viral genome is either silent or expresses one virally encoded protein, EBNA-1, latency Type I. In addition, EBV genome carrying B cells can lack either EBNA-2 or LMP-1, latency Type IIa or Type IIb respectively. These cells have no inherent proliferation capacity. Detection of both EBNA-2 and LMP-1 can identify B cells with growth potential. We devised therefore a method for their simultaneous detection in cytospin deposited cell populations. Simultaneous detection of EBNA-2 and LMP-1 was reported earlier in tissues derived from infectious mononucleosis (IM), postransplantation lymphoproliferative disorders (PTLD) and from “humanized” mice infected with EBV.We show for the first time the occurrence of Type IIa and Type IIb cells in cord blood lymphocyte populations infected with EBV in vitro. Further, we confirm the variation of EBNA-2 and LMP-1 expression in several Type III lines and that they vary independently in individual cells. We visualize that in Type III LCL, induced for plasmacytoid differentiation by IL-21 treatment, EBV protein expression changes to Type IIa (EBNA-2 negative LMP-1 positive). We also show that when the proliferation of EBV infected cord blood lymphocyte culture is inhibited by the immunomodulator, PSK, the majority of the cells express latency Type IIa pattern. These results show that by modifying the differentiation state, the proliferating EBV positive B cells can be “curbed”. Type IIa expression is a characteristic for EBV positive Reed–Sternberg (R/S) cells in EBV positive Hodgkin's lymphomas. For survival and proliferation, the R/S cells require the contribution of the in vivo microenvironment. Consequently, Type IIa lines could not be established from Hodgkin's lymphoma in vitro. We propose that these experimental cultures can be exploited for study of the Type IIa cells.

► Immunofluorescence method for simultaneous detection of EBNA-2 and LMP-1
► Detection of only LMP-1 or only EBNA-2 positive cells in EBV infected cord blood culture
► PSK induced LTB4 inhibit B cell proliferation in EBV infected cord blood culture.