Abstract
Epstein-Barr virus (EBV) persists within the B cell system and, in the blood of healthy virus carriers, is sequestered in the immunoglobulin (Ig)Dneg, CD27+ (“class-switched”) memory B cell compartment. This selective colonisation is apparent even in the blood of infectious mononucleosis (IM) patients undergoing primary EBV infection. Exactly how this is achieved is not understood, one theory being that the virus infects naïve B cells preferentially in vivo and drives them into memory using the physiologic process of germinal centre (GC) transit within lymphoid tissues. Here we have studied the distribution of EBV-infected B cells in tonsil cell preparations from acute IM patients and from chronic carriers. The following subsets of tonsillar B cells were isolated to high purity by FACS sorting and their EBV DNA loads were then determined by quantitative PCR: Naïve (CD38neg, IgD+, CD27neg); Class-switched memory (CD38neg, IgDneg, CD27+); Non-switched memory (CD38neg, IgD+, CD27+); Germinal centre (CD38+); and Plasma cell (CD38hi). In 15 carrier tonsils, the highest EBV load was consistently detected in class-switched memory, followed by significant amounts in non-switched memory. However, viral load in the CD38+ GC subset was usually very low, comparable to that in the naïve B cell fraction. This contrasted with the situation in 7 IM tonsils where overall viral loads were on average 100-fold higher than above. Here most viral DNA was consistently found in the CD38+ fraction, with lower loads in the class-switched memory and the smaller non-class switched memory subsets. We then investigated further the phenotype of the CD38+ population and found that, in IM tonsils, this not only included cells with the CD10 and CD77 markers of germinal centre origin but also other cells lacking these markers. Adopting a different FACS sorting strategy based on CD10, CD77 and IgD staining, we found that the virus was not present at significant levels in either the centrocyte (CD10+ CD77neg) or centroblast (CD77+) fractions. Thus EBV appeared to be concentrated in B cells expressing CD38 as a marker of activation rather than of germinal centre origin; this concurred with the results of immunohistochemical staining for the EBER RNAs showing an extrafollicular distribution of virus-infected cells in these IM tonsils. Our findings suggest that CD38 expression is a useful marker for B cells recently activated by EBV infection in vivo and show that, during primary EBV infection, the tonsillar CD38+ B cells lacking germinal centre markers harbour the highest viral load. If EBV does exploit the germinal centre pathway as an entry route into B cell memory, where primary infection is manifest as IM this pathway must be taken by a very small fraction of infected B cells and/or must have been completed before disease symptoms appear.