Peripheral B cells latently infected with Epstein–Barr
virus display molecular hallmarks of classical
antigen-selected memory B cells
Tatyana A. Souza*, B. David Stollar†, John L. Sullivan‡, Katherine Luzuriaga‡, and David A. Thorley-Lawson*§
*Department of Pathology, Tufts University School of Medicine, 150 Harrison Avenue, Boston, MA 02111;†Department of Biochemistry, Tufts University
School of Medicine, 136 Harrison Avenue, Boston, MA 02111; and‡Department of Pediatrics and Molecular Medicine, University of Massachusetts
Medical School, Biotech II, Suite 318, 373 Plantation Street, Worcester, MA 01605
Communicated by John M. Coffin, Tufts University School of Medicine, Boston, MA, October 26, 2005 (received for review October 6, 2005)
Epstein–Barr virus (EBV) establishes a lifelong persistent infection
within peripheral blood B cells with the surface phenotype of
memory cells. To date there is no proof that these cells have the
genotype of true germinal-center-derived memory B cells. It is
critical to understand the relative contribution of viral mimicry
versus antigen signaling to the production of these cells because
EBV encodes proteins that can affect the surface phenotype of
in the absence of cognate antigen. To address these questions we
have developed a technique to identify single EBV-infected cells in
the peripheral blood and examine their expressed Ig genes. The
genes were all isotype-switched and somatically mutated. Further-
more, the mutations do not cause stop codons and display the
pattern expected for antigen-selected memory cells based on their
frequency, type, and location within the Ig gene. We conclude that
latently infected peripheral blood B cells display the molecular
hallmarks of classical antigen-selected memory B cells. Therefore,
EBV does not disrupt the normal processing of latently infected
cells into memory, and deviations from normal B cell biology are
not tolerated in the infected cells. This article provides definitive
evidence that EBV in the peripheral blood persists in true memory
latent ? mononucleosis ? immunoglobulin ? germinal center
world’s population (1, 2). Although in most cases primary
infection is asymptomatic, when transmitted during adolescence
EBV infection can manifest as acute infectious mononucleosis
(AIM), a self-limiting lymphoproliferative disease (3). Perhaps
the most striking property of EBV in vitro is that it can efficiently
infect resting B lymphocytes and drive them to become contin-
uously proliferating lymphoblasts (4). Because of its growth-
transforming properties, EBV is associated with various neopla-
sias (1, 2). In contrast to its growth-promoting activity in vitro,
we have shown that EBV persists in vivo in resting B lymphocytes
in the peripheral blood (PB) that have the surface phenotype of
memory cells (CD27?, IgD?, CD5?) (5, 6).
To address the paradox between the proliferating infected cells
seen in vitro and the resting cells found in vivo, we have proposed
a model (7, 8) in which naı ¨ve B cells newly activated through EBV
infection are driven to differentiate into long-lived memory cells
through surrogate signaling provided by viral latent proteins. We
have provided evidence that EBV in peripheral memory cells
sustains persistence by down-regulating expression of all of the
latent proteins so that the infected cells are neither immunogenic
the latently infected cells in the periphery are maintained by
homeostasis-driven cell division allowing their numbers to remain
relatively constant over time (10).
Classical long-lived memory B cells arise from a germinal
center (GC) reaction after being selected by cognate antigen and
pstein–Barr virus (EBV) is a human ?-herpesvirus that
establishes a lifelong persistent infection in ?90% of the
receiving help from primed T helper (TH) cells (11, 12). Memory
cells exiting the GC exhibit two cardinal features: class switch
recombination (CSR) in the Ig heavy chain constant region,
which determines the effector function of the secreted antibody
(13), and somatic hypermutations (SHM) in the Ig variable (V)
regions, which diversify the B cell pool and improve the affinity
of the B cell receptor (BCR) for its antigen (14). Thus, GC-
derived memory B cells display a well characterized pattern of
SHM in their Ig V regions that is the result of competition for
antigen in the GC.
A key unresolved issue with our model arises from the
knowledge that EBV latent proteins can have profound effects
on the surface phenotype of infected cells (4) and specifically
that the viral latent membrane proteins LMP1 and LMP2a are
expressed by latently infected GC B lymphocytes in healthy
tonsils (15). These two proteins alone potentially have sufficient
signaling capacity to drive an infected naı ¨ve cell into the memory
B cell compartment in the absence of cognate antigen (16–20).
Such a process should presumably produce a cell that resembles
a memory cell phenotypically but lacks the characteristic pat-
terns of SHM found in antigen-selected, GC-derived memory B
cells. Currently, it is unresolved whether the infected cells in the
PB are only phenotypically memory cells because of the action
of viral latent proteins or are genotypically memory cells pro-
duced solely through surrogate signaling by the virus, or if they
are truly products of an antigen-selected GC process. It is critical
to establish how and to what extent EBV disrupts or regulates
normal B cell function in vivo to fully understand the conundrum
of persistent infection and the mechanism of EBV-associated B
cell lymphoma. In this study we sought to distinguish these
possibilities by isolating single infected CD27?B cells from PB
and analyzing the sequences of their expressed Ig genes for the
presence?absence of SHM and CSR and evidence of antigen
Materials and Methods
Primary Cells. Adolescents (ages 17–24 years) presenting to the
clinic at the University of Massachusetts Amherst Campus
Student Health Services with clinical symptoms consistent with
AIM were recruited as described in ref. 9. These studies were
Conflict of interest statement: No conflicts declared.
Freely available online through the PNAS open access option.
Abbreviations: AID, activation-induced cytidine deaminase; AIM, acute infectious mono-
nucleosis; BCR, B cell receptor; CSR, class switch recombination; EBV, Epstein–Barr virus;
EBER1, EBV-encoded RNA 1; CDR, complementarity-determining region; FWR, framework
region; GC, germinal center; LMP, latent membrane protein; PB, peripheral blood; SHM,
somatic hypermutation; TH, T helper; V, variable; VL, V light; VH, V heavy; R, replacement;
database (accession nos. DQ205136–DQ205184).
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
December 13, 2005 ?
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approved by the Human Studies Committees at the University of
Massachusetts Medical School and Tufts University Medical
School. Thirty AIM patients were tested, whose frequencies of
infected cells ranged from 1 in every 2 memory B cells to 1 in
?100 memory B cells. For this study we chose six patients: four
with high frequencies of infected memory B cells (IM1, 1 in 2;
IM2, 1 in 3; IM3, 1 in 5; IM4, 1 in 7) and two with lower
frequencies (IM5, 1 in 22; IM6, 1 in 43).
B Cell Separations. PB mononuclear cells (2 ? 107cells per ml)
were obtained as described in ref. 9 and stained with the
following antibodies: anti-human CD20 phycoerythrin (BD
Pharmingen) or anti-human CD19 CyChrome (DAKO) and
anti-human CD27 FITC (BD Pharmingen). Single memory
(CD20?CD27?) or naive (CD19?CD27?) B cells were sorted
with a Cytomation MoFlo fluorescence-activated cell sorter into
10 ?l of 1? first-strand buffer (Invitrogen) in 96-well plates,
immediately frozen on dry ice, and stored at ?80°C.
Limiting Dilution Analysis. Limiting dilution analysis was used to
determine the frequency of EBV-infected cells for each AIM
patient as detailed in ref. 21.
cDNA Synthesis. Single-cell cDNA synthesis was performed ac-
cording to the protocol of Wang and Stollar (22), with the
exception of adding 5 pmol EBV-encoded mRNA 1 (EBER1)
RNA-specific primer (AGGACCTACGCTGCCCTAGA) and 5
pmol Ig C?-specific primer (GAGGCTCAGCGGGAAGAC) to
C?, C?, and C? Ig constant chain regions. Eight wells containing
all buffers from the time of sorting, minus the single cells, served
as negative controls.
Single-Cell PCR. EBER1 PCR, as described in ref. 21, was used to
detect infected PB cells, because it is expressed in ?90% of them
(T.A.S., unpublished observations). Ig gene RT-PCR was per-
formed according to the protocol of Wang and Stollar (22) by
using the high fidelity hot-start Pfu-Turbo polymerase (Strat-
agene). PCR products were visualized by 2% agarose gel elec-
trophoresis. Ig V genes from infected cells were amplified with
similar or better efficiency than from uninfected B cells. PCR
efficiency was 38% for V heavy (VH) chains (32 amplified from
85 infected cells) and 33% for V light (VL) chains (17 amplified
from 48 infected cells) compared with 25% for VHchains from
uninfected cells (16 amplified from 64 cells).
Analysis of Amplified Ig V Genes. V gene PCR products were
excised from agarose gel, and DNA was extracted by using the
QIAquick gel extraction kit (Qiagen). DNA was sent for se-
quencing to the Tufts University Core Facility with correspond-
ing constant region primers. Sequences were aligned by using the
VBase database and the IMGT database [the international
ImMunoGeneTics information system at http:??imgt.cines.fr
(23)]. The probability that an excess or scarcity of R mutations
in VH chain complementarity-determining region (CDR) or
framework region (FWR) were due to chance alone was calcu-
lated by using the multinomial distribution model (24). A total
of 294 bp (FR1 through FR3) from each of the 32 VH gene
sequences and 288 bp (FR1 through beginning of CDR3) from
each of 17 VLgene sequences were analyzed for frequency and
characteristics of the mutations. Mutations in the primer binding
regions were disregarded. Only base substitutions were counted.
SHM hotspots, DGYW (AGT?G?CT?AT), and its reverse
complement, WRCH (AT?AG?C?ACT), were located in all
sequences and quantitated. Sequences determined in this study
can be found in the GenBank database under accession nos.
PB B Cells Latently Infected with EBV Are Genotypically Memory Cells.
True memory B cells have functional antigen receptors on their
surface expressed from Ig genes that have undergone SHM.
Therefore, to establish whether or not EBV persists in true
PB and tested for these characteristics. We isolated single cells
from six AIM patients by FACS using the pan-B cell marker
CD20 and the memory B cell marker CD27 (25). The EBV-
infected cells were then identified by using real-time RT-PCR to
screen for expression of the abundant EBER1, which is ex-
pressed by nearly all of the latently infected cells in the PB
(T.A.S., unpublished observations). The Ig genes from the
EBER1-positive cells were then amplified (Fig. 1A). The result-
ing 32 VH regions were sequenced, and the sequences were
aligned to their closest germ-line counterparts by using the
VBase database and the IMGT database [the international
memory B cells. (A) Amplification of Ig V regions from 11 single cells. Each cell
should result in the 400-bp (arrow) amplification of one heavy chain and one
light chain. (The asterisks denote cells that were also EBER1-positive.) (B)
Sequence alignment of an amplified EBV?VHgene to its germ-line counter-
part from AIM patient 4. The codon numbering is according to CHOTHIA. The
location of the CDRs is indicated by solid lines above the sequences. R muta-
tions are in uppercase, and S mutations are in lowercase. SHM hotspots are in
bold. The location of the forward primer is underlined.
www.pnas.org?cgi?doi?10.1073?pnas.0509311102Souza et al.
ImMunoGeneTics information system at http:??imgt.cines.fr
(23)], which contain all of the known human polymorphic Ig
variants. Fig. 1B shows a VH sequence alignment from AIM
patient 4, and Table 1 lists the VDJ gene segment and constant
region usage for the 32 sequences. All 32 VHsequences were
mutated and in-frame and did not contain any stop codons, as
expected for genotypically normal memory B cells that require
a functional Ig molecule to be expressed on the surface. All
sequences also had unique VDJ joints (data not shown). One
sequence had a 3-bp deletion, which can occur during the SHM
process (26, 27). Mutations in the Ig genes from cells latently
infected with EBV ranged in number from 4 to 40 per sequence
with an overall mutation frequency of 6.0% (Table 2), which is
similar to previous reports for human memory B cells (28, 29).
As a control, we amplified and analyzed 11 VHgenes of CD19?
CD27?naı ¨ve B cells from AIM patient 4. As expected, EBER1
was never detected in these cells, because EBV does not persist
in naı ¨ve B cells, and the Ig genes had an average mutation
frequency of 0.2% (Table 2). This mutation rate in the naı ¨ve B
cell population is similar to what was observed previously (30,
31). This validated that the rate of Pfu introduced errors during
PCR cycling was negligible and that our method for mutation
analysis was adequate. From this finding we can conclude that
the mutations in the VHgenes from the EBV-infected cells were
the result of the SHM process and not due to PCR artifacts or
analysis errors. These results demonstrate that EBV-infected
CD27?B cells express a functional Ig heavy chain and that their
VHregions carry SHMs at a frequency indistinguishable from
normal memory B cells. This finding is definitive evidence that
B cells genotypically, and it excludes the possibility that the
memory cell surface phenotype of the latently infected cells is an
artifact caused by viral gene expression. Furthermore, it suggests
that EBV, through LMP2a signaling in lymphoid tissue, does not
replace the requirement for an intact antigen receptor for the
survival of the latently infected B cells.
EBV?CD27?Memory B Cells Show Characteristics of Positive Antigen
Selection. The results described above show that EBV?CD27?
B cells bear the hallmark, SHM, of true GC-derived memory
cells. The question arises, therefore, whether these cells were
rescued from the GC through the signaling activities of the viral
latent proteins or whether they were positively selected by
cognate antigen. Analysis of somatic mutations can indicate
whether a cell has experienced positive selection. During the
mutation process, base substitutions can occur in the FWR or in
the antigen-binding CDR of the V genes. Base substitutions that
lead to amino acid replacements are selected against in the
FWRs, to preserve the proper Ig fold, but are favored in the
CDRs through the selection process that increases the affinity of
the Ig molecule for its antigen. Thus, by comparing the replace-
ment (R) to silent (S) mutation ratios (R?S ratios) in the FWR
and CDR it is possible to detect a signature of positive antigen
selection. Random mutagenesis is predicted to result in an R?S
ratio of ?3 in the CDRs and ?1.5 in the FWRs (32). An R?S
ratio in the CDRs that is ?3 should occur only through antigen
We computed the number of R and S mutations in the FWRs
and CDRs of the VHgenes from the EBV-infected peripheral B
cells described in the previous section. The mean R?S ratios for
all donors are listed in Table 3. The mean ratio for all of the
EBV?cells was 3.4 in the CDRs and 2.1 in the FWRs, remark-
ably close to published values for productive rearrangements
from normal memory B cells, which are reported to be 3.6 in the
CDR and 1.9 in the FWRs (33). Therefore, the sequences in the
latently infected cells show higher R?S ratios in the CDRs than
in the FWRs (P ? 0.017, one-tailed paired t test), and the ratios
are similar to those published for antigen-selected memory B
cells. We conclude that the R?S ratios we observe in the
expressed VHgenes from the EBV-infected cells are consistent
with their having experienced antigen selection pressure to
preserve a proper protein fold and to increase antigen-binding
To establish the influence of antigen selection on memory cell
formation more definitively, it is necessary to take into account
the intrinsic susceptibility of FWRs and CDRs of VHgenes to
Table 1. VHgene sequences of CD20?CD27?EBV-infected PB
*VHgene nomenclature by Matsuda and colleagues (55 and 56).
‡% is number of mutations per 100 bp.
§AIM patient 1, clone 1.
Table 2. VHgene somatic mutation summary
Total no. of
sequences% mutated RangeAverage
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accumulate R mutations. For example, it has been reported that
CDR1 is especially susceptible to R mutations based on codon
composition (34). To account for this, Lossos et al. (24) devel-
oped a program that uses the multinomial distribution and takes
into account the intrinsic sequence bias of individual germ-line
V genes to estimate the influence of positive antigen selection on
the mutation process. This program calculates P values to
quantitate the significance of increased R mutations in the CDR
and decreased R mutations in the FWRs. Using this analysis we
determined that the proportion of EBV-infected cells having
significantly fewer R mutations (P ? 0.05) in FWRs ranged from
60% to 83% for each donor, and the proportion of cells having
significantly more R mutations in CDRs ranged from 20% to
60% (Table 3). The proportion of sequences from EBV-infected
cells that were significant by these criteria was similar to the
proportion of published sequences of antigen-selected memory
B cells that were subjected to the same test (35). That 100% of
memory cells do not meet these criteria reflects the stringent
nature of the analysis. We conclude that the somatic mutations
in the CDRs and FWRs of the expressed Ig genes of EBV-
infected cells show the characteristics of positive antigen selec-
tion to the same extent as do bona fide memory B cells.
The Light Chains of EBV-Infected Cells Are In-Frame and Somatically
Mutated. The conclusion we derived above, that an intact Ig
molecule is expressed by EBV?cells in the PB, was based
entirely on analysis of the heavy chains. However, it was con-
ceivable that the light chains could contain aberrant mutations
that would result in the loss of Ig expression on the cell surface.
To eliminate this possibility, we also amplified and analyzed the
VL chain regions from single CD27?EBV-infected B cells
isolated from the PB. Seventeen sequences were obtained, all of
which were in frame and did not contain stop codons. Both ? and
? light chains were used by the EBV?cells (10 ? genes and 7 ?
genes). This usage is typical for human B cells, where ? has been
reported to be used by 60% of B cells (36). All light chains
carried SHMs, which ranged in number from 0 to 30 per
sequence, with an average rate of 3.6% (Table 4). The ? light
chains carried a lower rate of mutation (2.7%) than did the ?
light chains (4.9%). This finding is consistent with previously
published values that showed lower rates (3%) of mutation in the
? light chains (25). The analysis of the light chains confirms that
EBV-infected cells express an intact Ig receptor on their surface
that has undergone SHM in both heavy and light chains, as is
expected for normal memory B cells.
The Heavy Chains of EBV-Infected Cells Are of Various Isotypes. The
two defining features of GC-derived memory B cells are SHM
and CSR. We have shown above that EBV-infected cells in the
PB express Ig V regions that show the characteristics of antigen-
selected SHM. To address the question of CSR we performed
RT-PCR for the three most prevalent isotypes found on memory
B cells in PB (?, ?, and ?). CSR was detected in EBV?B cells
(Table 1). Each cell expressed one constant region heavy chain
domain, and all three domains were represented, with ? and ?
being most prevalent. These data further confirm that EBV-
infected cells in PB are true memory cells genotypically because
they have undergone CSR and suggest there may be a bias
against viral persistence in IgM bearing memory cells.
Cells Latently Infected with EBV in the PB Do Not Show Evidence of
Aberrant Activation-Induced Cytidine Deaminase (AID) Behavior.
LMP1 has been shown in vitro to turn on AID and CSR (18).
Therefore, we tested whether the nucleotide composition of the
mutations in the expressed Ig genes from the EBV-positive
memory cells was characteristic of GC-produced SHM or aber-
rant AID expression induced by LMP1. Aberrant AID overex-
pression in cells not ready for SHM results in biased mutations
(37–40), with C and G transitions at hotspots being predominant
(70–100% of mutations). As evident in Table 5, all nucleotides
were targeted for SHM, with only slightly more than half of the
mutations occurring at dC and dG bases (57%). Normal SHM
of human VH genes has been reported to cause 55% of the
mutations to occur at dC?dG nucleotides (41). Approximately
60% of all transition mutations occurred at dC and dG bases,
similar to what was published for VH genes from the normal
human adult memory repertoire (63%) (42). Additionally,
?35% of mutations occurred at dC and dG nucleotides within
the WRCH?DGYW hotspots (43), in remarkable agreement
with the reported 35% of mutations in cells that have undergone
antigen selection (44). Thus, we can conclude that SHM in the
Table 3. Analysis for positive antigen selection of single EBV?
CD27?PB B cells
Mean% P ? 0.05†
R?SFWR*R?SCDR* FWRCDRFWR ? CDR
*R?S ratios were calculated by dividing the number of R mutations by the
number of S mutations in either FWR or CDR regions. For sequences where
the denominator was 0, 1 was used to obtain a ratio value.
†P values were calculated by using the multinomial distribution model (24).
Percent of VH sequences having significantly fewer R mutations in FWR
(PFWR ? 0.05) and percent of VH sequences having significantly more R
mutations in CDR (PCDR? 0.05) are shown. All VHsequences with PCDR? 0.05
also had PFWR? 0.05. The last column shows percent of VHsequences with
PFWR? 0.05 also having PCDR? 0.05.
‡Mean R?SCDRis significantly greater than mean R?SFWR(one-tailed Student’s
paired t test, P ? 0.017).
Table 4. V?and V?gene sequences of CD20?CD27?EBV-infected
Means (n ? 17)
*V?and V?gene nomenclature in refs. 59 and 60, respectively.
no good match found.
‡VHgene sequences, where available, are listed in Table 1.
www.pnas.org?cgi?doi?10.1073?pnas.0509311102Souza et al.
expressed Ig genes from EBV-infected memory B cells is as
expected for normal GC-derived memory B cells and does not
display characteristics of aberrant AID targeting caused by EBV
In this article we present definitive evidence that cells latently
antigen-selected memory B cells. In our previous studies we
proposed that EBV persists in memory B cells based solely on
their surface phenotype, CD20?CD27?IgD?CD5?(6). How-
ever, EBV is a transforming virus, and its latent proteins can
have a profound effect on the surface phenotype of infected cells
caused by the presence of the virus rather than a consequence
of true memory B cell differentiation, and thus it was unknown
whether EBV persisted in true memory B cells. We have now
shown that these latently infected cells express Igs that are
isotype-switched and carry SHMs. Furthermore, these muta-
tions do not cause stop codons and display the signature of
positive antigen selection based on their frequency, type, and
location within the Ig gene. Thus, the EBV-infected cells in the
PB are indistinguishable from normal memory B cells. Because
we find no genotypic aberrations in the EBV-infected memory
B cells, we may further conclude that viral latent proteins are
prevented from rescuing defective B cells, and any deviations
from normal B cell biology are not tolerated in the latently
infected peripheral B cells.
Typically, an antigen-activated B cell must enter a follicle to
form a GC before becoming a memory cell (11, 12). While in the
GC, cells express AID and undergo SHM and CSR. To survive
and exit as memory cells they must bind antigen, receive signals
from antigen-specific THcells, and down-regulate expression of
the GC master transcription factor bcl-6 (45). LMP2a expression
is sufficient to cause B cells to form GCs and sustain SHM in the
mucosal lymphoid tissue of transgenic mice lacking a BCR (17).
LMP2a can also rescue B cells from apoptosis (16) and augment
a weak BCR signal (46). LMP1, on the other hand, can consti-
tutively replace T cell help, turn on AID and CSR in vitro, and
down-regulate bcl-6 (18–20). Therefore, LMP1 and LMP2a
together could provide all of the necessary signals to cause
infected naı ¨ve B cells to enter, survive in, and exit from the GC
to become circulating memory cells without any requirement for
signaling by cognate antigen or THcells. However, if this were
true, the cells would not display the SHM signature of antigen-
selected memory B cells that we have observed. Why then are
LMP1 and 2a expressed in infected GC cells (15)? The answer
may lie in the fact that LMP2a, although it cannot provide a
growth signal, can augment a weak BCR signal (46), and LMP1
provides constitutive T cell help. During an immune response
be the stage at which cells latently infected with EBV would need
to encounter antigen and would explain why these cells show the
hallmarks of antigen selection. Because LMP2a can augment
weak BCR signals, its role may be to ensure the survival and
BCR as antigen becomes limiting. Ultimately surviving GC cells
exit as plasma or memory cells after a short or long exposure,
respectively, to TH cells (47). The role of LMP1 could be to
provide a long-lived THcell signal to ensure that the latently
infected GC cells preferentially differentiate into memory cells.
Plasma cell differentiation would be unfavorable for the virus
because it leads to reactivation of EBV (48), preventing the virus
from establishing long-term latency. Therefore, the role of
LMP1 and 2a may be to preferentially direct the survival and
differentiation of the latently infected, antigen-selected GC cells
into the memory compartment.
A less likely explanation for the presence of LMP1 and 2a in
GCs is that they, perhaps in combination with TLR signaling (17,
49), drive the differentiation of infected cells into memory so
precisely that the cells are effectively indistinguishable from
normal memory B cells even though they have never seen
cognate antigen. This outcome could be the result of the SHM
machinery itself. If LMP1 turns on AID in GC B cells, they
would undergo normal SHM and CSR and accrue both phase I
(at C?G nucleotides) and phase 2 (at A?T nucleotides) muta-
tions (50, 51) so that the mutations in the V genes would be
indistinguishable from those in normal memory B cells. The
signature of positive antigen selection could be a result of the
germ-line sequence of the Ig genes and the cytidine deaminase
activity of AID. Recently it has been described that the Ig V
genes have evolved to direct the induction of SHM by AID into
the CDRs and also to minimize R mutations resulting from
direct cytosine deaminations (44). If it is true that a memory B
cell can express the signature of antigen selection without seeing
cognate antigen, it would have profound implications for our
understanding of the GC process and the use of SHM to identify
the origins of B cells. Specifically, it would mean that the pattern
of SHM could no longer be used reliably to identify ‘‘true’’
memory cells. This would not be the first time that EBV studies
in vivo have produced unexpected findings related to B cell
biology. The notion that IgD?memory B cells were not classical
GC-derived memory cells was first suggested by the observation
that they could not sustain persistent EBV infection (5, 6), and
the first evidence that mucosal memory cells could circulate
came from studies of EBV (52).
One of the key observations in this study is that the 32 VHgene
and 17 VL gene sequences from EBV-infected cells did not
contain aberrant mutations or stop codons, and the number of
R mutations in FWRs was consistent with preserving a correct
Ig protein fold. Thus, a proper Ig molecule on the surface of the
infected cells appears to be necessary for their survival. LMP2a
can rescue Ig-less B cells from apoptosis. Because we do not
detect EBV in any Ig-less B cells, LMP2a must not be expressed
very frequently if at all in the infected PB memory B cells. This
finding is consistent with our previous work demonstrating that
viral proteins are not expressed in these cells (9). LMP2a is
present in latently infected GC cells (15) and could rescue these
cells from apoptosis if they had acquired aberrant mutations
during SHM. Such mutations could result in the loss of surface
Ig expression or expression of an autoreactive BCR (16, 53).
However, when the cells exit into the PB memory compartment
the cells would be deleted before entering the periphery. In this
manner EBV ensures that it does not allow aberrant cells to
survive in the periphery and potentially cause harm to the host.
One possible reason why we did not detect cells with stop
codons or aberrant mutations is that such RNAs tend to get
degraded more rapidly (54), making them less abundant and
harder to amplify by RT-PCR. Therefore, we cannot rigorously
exclude the possibility that there are some such cells with
sufficiently low transcript copy numbers that they would be
Table 5. SHM frequency and pattern in single EBV?CD27?PB
Fifty-seven percent of mutations are at dC?dG, 44% of mutations are at
dA?dT, 61% of dC?dG mutations are transitions, and 35% of mutations are at
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