In this work, we studied the mechanisms underlying the increased susceptibility to EBV infection of memory B cells from tonsils as opposed to memory B cells from other lymphoid sites. Our experimental data indicate that (i) high-level expression of α5β1 integrin is unique to memory B cells from the NALT, and EBV indeed uses β1 integrin as a cofactor to attach to memory B cells; (ii) triggering the α5β1 integrin signaling pathway is a key event for EBV entry and thus for tonsillar B cells' susceptibility to in vitro infection; and (iii) inducing expression of β1 integrin by activating memory B cells with CD40L plus anti-IgM antibody increases the susceptibility of memory B cells to EBV infection. We concluded that memory B cells from tonsils exhibit increased susceptibility to EBV infection, by virtue of their high basal activation status reflecting their high level of β1 integrin expression, which mediates increased attachment and entry of the virus, compared to memory B cells from other lymphatic tissues where B-cell-activating factors or antigen is less abundant.
The starting points of our hypothesis were the observations that tonsillar memory B cells are particularly susceptible to ex vivo
EBV infection (9
) and that Janz et al. recently reported the possible existence of additional receptors for EBV on B cells, besides CD21 and HLA class II molecules (14
). Thus, we hypothesized that memory B cells from the NALT exhibit specific properties rendering them highly susceptible to EBV infection, and we assumed that memory B cells primed in the tonsils would keep their susceptibility to EBV infection when circulating in the blood. To address this hypothesis, we employed the unique CD19+
phenotype of NALT-originating memory B cells (4
) to compare the relative infection susceptibilities of NALT-originating memory B cells in distinct lymphoid compartments and memory B cells selected at other lymphoid sites. Although NALT-originating (CD62L+
) memory B cells exhibit a higher level of susceptibility to EBV infection irrespective of whether they are isolated directly from tonsils, peripheral blood, or mesenteric lymph nodes, we excluded the possibility that CD62L itself functions as a coreceptor for EBV by using neutralizing antibodies against CD62. We concluded that CD62L phenotypically marks a population of memory B cells that have been selected in the NALT and have retained or obtained receptors required for an efficient infection with EBV.
Recently, it was found that the interaction of an EBV envelope protein, BMRF-2, with α5
integrin plays a role in the infection of polarized epithelial cells (38
; R. Speck, unpublished data), which do not express the known B-cell receptors CD21 and HLA class II molecules, both of which are crucial for EBV entry. Here we found that memory B cells from the NALT express α5
integrin at least 10-fold more than their counterparts from peripheral blood or mesenteric lymph nodes. Notably, we identified two distinct populations, defined as α5
cells, and found most tonsillar memory B cells expressing large amounts of α5
integrin and most peripheral blood or mesenteric lymph node memory B cells expressing small amounts of α5
integrin. Given that blocking and saturating α5
integrin by use of antibodies and the recombinant extracellular domain of β1
integrin, respectively, resulted in a clear decrease of EBV entry into memory B cells, β1
integrin has a key role as a cofactor in the viral entry process. Notably, memory B cells from distinct lymphatic tissues express equal amounts of the cell surface molecules CD21 and HLA class II (9
), which constitute the EBV receptor complex: this consequently does not explain the distinct susceptibilities to EBV infection. In contrast, the level of α5
integrin expression discriminates between degrees of susceptibility of memory B cells to EBV infection.
As we show here, the correlation of susceptibility to EBV infection with the α5
integrin expression level is mediated by binding of EBV's structural protein BMRF-2 to α5
integrin. Furthermore, we show that each step of β1
integrin-mediated signaling leading to the activation of the downstream targets (20
) is crucial for EBV entry into B cells. Very importantly, activation of memory B cells from peripheral blood with CD40L plus anti-IgM resulted in a vigorous upregulation of β1
integrin expression. Thereby, the susceptibility of peripheral blood memory B cells to infection by EBV, i.e., of memory B cells which were not from the NALT, was increased. Stimulation of the B-cell receptor results in increases of the expression levels of integrin, whereas CD40L, which binds to CD40 on B cells and, in addition, to α5
), may activate the downstream integrin signaling pathway and in turn may lead to an increased uptake of EBV via actin cytoskeleton reorganizations. Thus, these data demonstrate clearly that the susceptibility of memory B cells to EBV infection is greatly dependent on their activation status and the resulting high expression levels of β1
integrin. In fact, in a primary immune reaction, the CD40L expressed on activated T cells has a key role in subsequent activation of B cells by binding to CD40 (35
). Thus, the events described above may be operative during primary or secondary immune responses in the NALT, explaining its marked susceptibility to EBV infection and its central role in EBV pathogenesis. Whether the interaction of EBV's essential envelope protein gH with αv
integrin, which very recently was shown to trigger epithelial cell fusion by EBV (7
), plays a role in B-cell infection remains to be investigated.
We propose a model in which EBV takes advantage of targeting activated CD62L+
memory B cells which preferentially reside in or home to NALT and, following circulation, are likely to home back to NALT. We have previously shown that naïve B cells from distinct tissues are equally highly susceptible to EBV infection ex vivo
). Here we report that naïve B cells from distinct tissues essentially express the α5
phenotype, in contrast to memory B cells expressing the α5
phenotype preferentially when originating from NALT. Our data speak in favor of a direct infection of tonsillar memory B cells with EBV which, in addition to primary infection of naïve (33
) and GC B cells (2
), contributes to a more efficient establishment of EBV persistence. In NALT, the memory B cells may encounter recall antigen and subsequently either propagate EBV as a latent infection in progeny cells or undergo differentiation into plasma cells, resulting in lytic replication of EBV, which then can be transmitted via saliva to new susceptible hosts. Triggering of CD40 and the B-cell receptor mediated by exposure to microorganisms may be involved in this process, as such triggering increased the proportion of cells expressing high levels of β1
integrin. NALT is readily accessible to a wide variety of antigens, explaining why memory B cells generated within and homing back to the NALT are much more readily found in an activated, i.e., high-level β1
integrin-expressing, state than remote lymphoid organs, which are not accessible by antigen or pathogens directly but only following initial immune recognition by sentinel immune cells such as dendritic cells. The idea that EBV establishes persistence by direct infection of memory B cells was also proposed by Kurth et al., who examined EBV-infected B cells in infectious mononucleosis (15
). Our findings give a detailed insight of EBV B-cell infection and biology.