In this study we demonstrated that during acute infection with EBV the peripheral blood fills up with latently infected, resting memory B cells to the point where up to 50% of all of the peripheral memory cells may carry EBV. At this time, the number of virus-infected cells is already falling, suggesting that the levels of infected cells may peak at even higher percentages than those reported here. Despite this massive level of infection, the virus maintains a highly stringent regulation, such that it remains restricted to resting memory B cells.
These experiments were carried out to test a prediction of our model that EBV persistence is a circle of latent infection, activation, and differentiation into memory cells, the major site of long-term persistence, followed by sporadic terminal differentiation and release of infectious virus to initiate a new round of infection (Fig. ). In this model, the role of the cellular immune response is to modulate the overall level of infection, not the type or location of infected cells. Consequently, during acute infection, before the immune response is effective, the memory compartment of B cells should fill up with latently infected cells. Our results support this prediction of the model and demonstrate that EBV infection is not indiscriminate in the blood but is tightly restricted to resting memory cells even during acute infection. In a separate study (16
), limiting dilution RT-PCR was used to quantitate the numbers of infected cells expressing viral latent genes in the blood of AIM patients. It was found that >99% of the cells express no detectable latent proteins (16
), consistent with our conclusion here that the cells are resting.
Previous studies reported that 0.01 to 20% of B cells in the blood of AIM patients expressed EBNA (21
). We are unable to reproduce these findings (16
). In our studies, we find EBNA expression only in a very small fraction of B cells (
0.1%), even in patients with very high levels of infected cells (>1%). Our data confirm the earlier studies of Crawford et al. (8
), who showed that the vast majority of infected cells in AIM were EBNA negative. The other estimates are probably incorrect because they were based on an old immunofluorescent staining protocol for EBNA proteins. This assay was notoriously difficult and prone to false positives because it employed low-titer human sera and a large amplification step involving complement. The previous claims that the infected cells are proliferating lymphoblasts (39
) also appear to be incorrect, since we have shown that the cells are not proliferating (this study) and do not express EBNA2 (16
). Again, this is in agreement with the earlier studies of Crawford et al. (8
), who showed that the infected cells in AIM were small lymphocytes not lymphoblasts.
It is well known that AIM patients have highly elevated levels of cytotoxic T cells (CTL) specific for EBV lytic antigens (43
). The rate of decay in the levels of these cells approximates that of the latently infected memory cells in the periphery (6
). The memory cells are not expressing viral proteins (16
); therefore, they are not disappearing through CTL-mediated lysis. We suspect that the latently infected cells are decaying by circulating back to the tonsil to produce infectious virus and die. This would provide the antigenic load to stimulate the CTL response, thus explaining the parallel decay in the levels of CTL to lytic antigens. This would mean that the rapid early decay of infected memory cells is a function of the rate at which they initiate viral replication. This phase is followed by a slower decline that, even by 1 year, had not reached a steady state. This poses a fundamental question about the nature of EBV persistence, namely, does it come to a long-term equilibrium, as is generally believed, or does it never reach a stable equilibrium but continue to decay more and more slowly over time. Previous measurements have not been precise enough (22
) to unequivocally resolve these two outcomes which would involve very different biologies. Clarification of this issue will be an important question to address in the future.
One interpretation of our data is that the specificity of EBV for memory B cells in the blood is not a consequence of regulation by the biology of the virus but the elimination of all other infected cell types by CTL. This interpretation cannot be correct, however, for two reasons. First, immunosuppressed organ transplant patients have impaired CTL function that has been shown previously to result in significantly higher (on average 50-fold) levels of EBV infection (2
) (Fig. ), with no evidence for the emergence of other infected cell types. The virus in the blood remains restricted to the memory B cells. Second, despite active CTL responses in healthy carriers, multiple cell types expressing both latent and lytic genes are readily detected in the tonsil (4
; Laichalk and Thorley-Lawson, submitted). Thus, the CTL response does not seem competent to restrict infection to a specific subset, at least in that particular organ.
One limitation of our study is that patients are only tested when they present to the clinic. It is possible that during the very early stages of the infection, before the onset of symptoms, multiple cell types are infected in the blood. Subsequently, the infection could become restricted to memory cells by the time of onset of symptoms, when we first study the patients. A scenario such as this is seen with MHV68, where initially all cell types are infected but after 6 months the virus becomes restricted to memory B cells (12
). The caveat to direct extrapolation of those studies to EBV is that they were done with the spleen. In the case of EBV, infected cells are found in the spleen, but even in persistent infection, the virus remains in multiple cell types (29
). Only in the blood is specificity for memory cells observed.
A more analogous situation to that seen with the spleen and MHV68 might be the tonsil. Recent immunohistochemical studies have identified directly infected memory and germinal center cells which express EBNA2 and appear to have undergone EBV-driven clonal expansion (27
) in the tonsils of AIM patients. The infection of all B-cell subsets in the tonsils of healthy carriers has also been seen (4
); however, in this case, each subset uses a discrete transcription program and EBNA2 was not detected in the memory or germinal center cells. Rather, these cells express a limited subset of latent genes referred to as the default program (4
). It is conceivable that, during acute infection, all subsets are directly infected, in the tonsil, and that this resolves into the more regulated pattern seen in persistent infection.
The work on AIM tonsils has led to the suggestion that EBV may establish persistent infection in the memory compartment by direct infection (26
). Our model does not preclude this possibility, although no evidence was found for it in healthy carriers (3
). One possibility is that the virus gains access to the memory compartment by direct infection during acute infection but not during persistent infection. Another possibility is that the presence of proliferating clones of directly infected memory and germinal center cells in AIM tonsils has occurred because of the large number of virions and tissue damage present during AIM, allowing direct infection of cells that would not otherwise be exposed to the virus. It was striking that EBV-driven clonal expansion of naive B cells was not seen in the studies of Kurth et al. (28
). This would be predicted by our model, which states that only infected naive B cells in the tonsil can differentiate out of the cell cycle by becoming memory cells. If other cell types, such as memory or germinal center cells, were infected directly they would be stuck as proliferating clones. This would mean that the predominant infected cell types seen in AIM tonsils by immunohistochemistry would be these expanding clones that cannot exit the cell cycle and will eventually be destroyed by cytotoxic T cells.
One major caveat with immunohistochemical approaches to identify infected cells in vivo is that the lower threshold for detection is not known. Thus, it is impossible to know how many infected cells have been missed and how representative the identified cells are. Unfortunately, tonsils from AIM patients are not generally available for study, making it impossible for us to apply our techniques that use quantitative analysis of whole-cell populations. Thus, we cannot assess whether the reported direct infection of memory and germinal center cells in AIM tonsils represents (i) a mechanism for establishing persistent infection or (ii) a deregulated state of infection or (iii) whether these observations are a consequence of technical artifacts.
In conclusion, we have shown that EBV in the peripheral blood remains restricted to the resting memory B-cell subset, even when the compartment is massively overwhelmed by virus-infected cells.