Germinal Center B Cells Latently Infected with Epstein-Barr Virus Proliferate Extensively but Do Not Increase in Number

Abstract

In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively. Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute number is limited either by the immune response or by the availability of an essential survival factor. We suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism unless verified in vivo.

Full text

JOURNAL OF VIROLOGY, Jan. 2010, p. 1158–1168 Vol. 84, No. 2
0022-538X/10/$12.00 doi:10.1128/JVI.01780-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Germinal Center B Cells Latently Infected with Epstein-Barr Virus
Proliferate Extensively but Do Not Increase in Number
䌤
Jill E. Roughan,† Charles Torgbor,‡ and David A. Thorley-Lawson*
Department of Pathology, Tufts University School of Medicine, Jaharis Building, Boston, Massachusetts 02111
Received 22 August 2009/Accepted 22 October 2009
In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells
undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively.
Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal
center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data
fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell
survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division
without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that
EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute
number is limited either by the immune response or by the availability of an essential survival factor. We
suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute
infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not
correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made
regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism
unless verified in vivo.
Epstein-Barr virus (EBV) is a human herpesvirus that es-
tablishes a lifetime persistent infection in ⬎90% of all adults
(reviewed in references 21 and 51). The virus persists in rest-
ing, latently infected memory B cells that circulate in the pe-
riphery and lymph nodes (5; reviewed in references 53 and 55).
In these cells the virus is quiescent, at least at the level of viral
protein expression (23). This lack of viral proteins is presum-
ably a major reason why these cells are able to persist in the
face of a healthy immune response. The other property for
which EBV is well known is its ability to infect resting B cells
and drive them to become activated proliferating lymphoblasts
through the expression of nine latent proteins and several
untranslated RNAs (reviewed in reference 45). The latter in-
clude a large number of microRNAs (miRNAs) (7, 12, 17, 44).
The current model of Epstein-Barr virus persistence holds
that the virus drives resting B cells to become activated lym-
phoblasts so that they can differentiate through a germinal
center (GC) reaction to become resting memory B cells (55–
57), where the virus persists. The GC is the structure in the
follicles of secondary lymphoid organs where antigen-activated
B cells undergo a T-cell-dependent immune response (1, 35,
37). The production and maintenance of GCs are absolutely
dependent on the expression of the transcription factor bcl-6
(13, 58) and are initiated by the rapid proliferation of antigen-
specific B cells. During this expansion phase, B cells divide
approximately every 6 to 12 h (2, 37) so that 3 ⫾2 founder cells
can create a GC of approximately 10
5
cells in a few days (29,
36, 37). In the classical model of GC development (37), it was
proposed that in the proliferating state, the B cells (centro-
blasts) reside in the dark zone of the follicle and do not express
their antigen receptor while actively undergoing the processes
of somatic hypermutation and immunoglobulin class switching.
These processes are governed by the enzyme AID (activation-
induced cytidine deaminase), whose expression is also a defin-
ing feature of GC B cells (39). After several rounds of division,
the cells move to the light zone, rest, reexpress their surface
immunoglobulin (centrocytes), and compete for antigen bind-
ing and T-cell help. Survival requires that the B cell success-
fully competes for both signals. It should be noted, though, that
the clear-cut distinction between centroblasts and centrocytes
has been called into question (1).
While in the GC, B cells are highly apoptosis prone, and a
failure to receive the requisite survival signals leads rapidly to
death. If the survival signals are received, then there are three
possible fates for the GC cell. It can recapitulate the process by
once more becoming a centroblast, or it can upregulate pro-
survival molecules such as bcl-2 (29, 36) and leave the GC as a
plasma or memory B cell. Which fate the B cell follows de-
pends on the cytokine environment and the relative strength
and endurance of its interaction with antigen and T-helper
cells.
In a previous study we presented direct evidence that EBV-
infected cells transiting from activated lymphoblast to memory
B cells are found to physically reside in tonsil GCs and partic-
ipate in the GC reaction (46). This included the expression of
bcl-6 and AID, defining makers of the GC, and GC-specific
chemokine receptors that would cause them to be retained in
the GC. Moreover, the cells express markers associated with
* Corresponding author. Mailing address: Dept. of Pathology, Jaha-
ris Building, Tufts University School of Medicine, 150 Harrison Ave.,
Boston, MA 02111. Phone: (617) 636-2726. Fax: (617) 636-2990.
E-mail: david.thorley-lawson@tufts.edu.
† Present address: Department of Immunology and Microbial Sci-
ence, The Scripps Research Institute, La Jolla, CA 92037.
‡ Present address: Department of Biochemistry and Biotechnology,
Kwame Nkrumah University of Science and Technology, Kumasi,
Ghana, West Africa.
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Published ahead of print on 4 November 2009.
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