Transcriptional Profiling of Antigen-Dependent Murine B Cell
Differentiation and Memory Formation1
Deepta Bhattacharya,2* Ming T. Cheah,* Christopher B. Franco,* Naoki Hosen,†
Christopher L. Pin,‡William C. Sha,§and Irving L. Weissman*
Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly
generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive
and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared
the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice
immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays
several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes,
chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators
specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag
receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary
responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high
frequencies and to detect and rapidly respond to antigenic re-exposure. The Journal of Immunology, 2007, 179: 6808–6819.
of research, it has become clear that the basis for this resistance is
so-called immunological memory, in which Ag-specific B and T
cells are maintained at higher frequencies than in the naive host,
sometimes for the lifetime of the organism. Particular subsets of
these Ag-specific memory B cells can rapidly respond to Ag re-
exposure in a T cell-dependent manner (2, 3), thus generating a
humoral recall response that is more rapid than the primary re-
sponse. Furthermore, the Abs generated in the recall response tend
to be of higher affinity to foreign Ags than those generated in the
early phases of the primary response (4–8). Although there is a
general consensus that the Ag-specific B cell precursor frequency
is substantially increased in recall responses, there is considerable
debate as to the mechanisms by which these increased frequencies
are maintained. Several studies have concluded that Ag must per-
sist to maintain and perpetually activate naive and memory B cells
he acquisition of lifelong resistance to pathogens follow-
ing resolution of the primary infection has been observed
and documented for over 2 millennia (1). After centuries
to maintain protective concentrations of neutralizing Abs (9, 10),
whereas others have demonstrated that neither the immunizing Ag
nor Ag-trapping immune complexes must be present to maintain
memory B cells or long-lived plasma cells (11–14). Coupled with
the findings that memory B cells return to a state of relative qui-
escence after surviving the germinal center reaction (15), the latter
studies imply that B cell-intrinsic differences at least in part un-
derlie the differences between primary and recall responses. How-
ever, the extent to which cell-intrinsic alterations in memory B
cells are responsible for the increased Ag-specific B cell precursor
frequencies and for the global differences between primary and
recall responses is largely unknown.
Following the initial encounter with foreign protein Ags, Ag-
specific naive B cells can differentiate within secondary lymphoid
tissues into short-lived low-affinity Ab-secreting plasma cells or
undergo a rapid proliferative phase known as the germinal center
reaction, a T cell-dependent phase in which somatic hypermutation
of the variable regions and Ig isotype switching occur (16, 17). A
small fraction of these germinal center B cells survives the reaction
(18) and proceeds to form either memory B cells or long-lived
high-affinity Ab-secreting plasma cells, which are radioresistant
and reside primarily within the bone marrow (13, 14, 19). Al-
though the B cells are in the phase of antigenic stimulation, ger-
minal center cells and early memory B cells temporarily lose hom-
ing receptors for lymph nodes and Peyer’s patches, but memory B
cells later regain these homing receptors (L-selectin and integrin
?4?7), and therefore regain the ability to survey these sites of new
Ag deposition and presentation (20). Importantly, the early phases
of mouse Ag-dependent differentiation are accompanied by the
expression of unique combinations of surface markers that allow
for the ready isolation of naive, plasma, and germinal center B
cells. For example, naive follicular B cells, which are the major,
but not exclusive (21, 22), source of precursors for memory B
cells, express high levels of B220, CD23, and IgD, but low levels
of IgM (23). Plasma cells express high levels of syndecan-1 (also
known as CD138) and low levels of B220 (24), whereas germinal
center B cells express high levels of B220 and low levels of
*Institute of Stem Cell Biology and Regenerative Medicine, Stanford Cancer Center,
Stanford University School of Medicine, Stanford, CA 94305;†Osaka University
Graduate School of Medicine, Department of Cancer Stem Cell Biology, Department
of Respiratory Medicine, Allergy and Rheumatic Disease, Osaka, Japan;‡Depart-
ments of Pediatrics and Physiology and Pharmacology, University of Western On-
tario, Children’s Health Research Institute, London, Ontario, Canada; and§Division
of Immunology, Cancer Research Laboratory, Department of Molecular and Cell
Biology, University of California, Berkeley, CA 94720
Received for publication July 10, 2007. Accepted for publication September 6, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health Grant P01DK053074 (to
I.L.W.). D.B. was supported by a fellowship from the Cancer Research Institute and
from the National Institutes of Health (T32AI0729022), C.B.F. was supported by a
predoctoral fellowship from the National Science Foundation, and N.H. was sup-
ported by a fellowship from the Japanese Society of Promotion of Science, Yamada
Memorial Foundation, and Mitsubishi Pharma Research Foundation.
2Address correspondence and reprint requests to Dr. Deepta Bhattacharya, B261
Beckman Center, 279 Campus Drive, Stanford, CA 94305-5323. E-mail address:
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
surface Ig, and bind peanut agglutinin (PNA)3(25). In contrast,
whereas human memory B cells are identifiable through their ex-
pression of CD27 and several recent studies have examined the
global gene expression profiles of these cells (26–28), the isolation
of mouse memory B cells is more challenging in large part due to
the absence of unequivocally characteristic markers. Many mem-
ory B cells have undergone Ig class switching and express isotypes
other than IgM and IgD (16), but perhaps the best method to iden-
tify and purify homogenous preparations of memory B cells is
through immunization with defined Ags and isolation of B cells
specific for the immunizing Ag at time periods long after the ger-
minal center reaction has ceased (29). This is an approach uniquely
available to studies performed with animal models of Ag-depen-
dent B cell differentiation.
We hypothesized that the differences between primary and recall
humoral responses could be explained by distinct abilities of naive
and memory B cells to detect Ags at sites of entry and/or distinct
abilities to generate specific Abs upon antigenic encounter. To
obtain evidence for or against this hypothesis, we compared the
transcriptional programs of highly purified naive follicular, short-
lived plasma, germinal center, and memory B cells. Our data sug-
gest that memory B cells express a transcriptional program that
allows for the maintenance of increased Ag-specific precursor cell
frequencies, but also to efficiently survey peripheral sites of infec-
tion and rapidly obtain T cell help upon antigenic re-exposure.
Materials and Methods
All animal procedures were approved by the International Animal Care and
Use Committee and Stanford University’s Administrative Panel on Labo-
ratory Animal Care. C57BL/Ka-Thy1.2 and Bmi1-deficient (30) mice were
derived and maintained in our laboratory. Mist1-deficient mice were de-
rived as previously described (31).
B cell purification
B cell purification was performed previously (32). To reiterate, male
C57BL6/Ka mice (4–8 wk old) were immunized i.p. with 100 ?g of hy-
droxy-3-nitrophenylacetyl (NP)-chicken ?-globulin (CGG) (Biosearch
Technologies) precipitated in 10% aluminum potassium sulfate (Sigma-
Aldrich). Splenic plasma cells were harvested 7 days after immunization,
germinal center B cells were sorted 14 days after immunization, and mem-
ory B cells were harvested 10–12 wk postimmunization. Naive cells were
harvested from spleens of unimmunized mice. Splenocytes were first
stained with unconjugated rat anti-mouse CD138 (BD Pharmingen) for
plasma cells, biotinylated PNA for germinal center cells, or purified anti-
Ig? for memory cells, and enriched using magnetically conjugated anti-rat
or streptavidin beads (Miltenyi Biotec), followed by separation on AutoMacs
columns (Miltenyi Biotec). The following Abs were used to stain cells as
appropriate before FACS: anti-rat PE-Texas Red (Caltag Laboratories),
anti-mouse Ig? FITC (BD Pharmingen), NP-PE (Biosearch Technologies),
Cy5PE-conjugated lineage Abs (anti-mouse CD3, CD4, CD8, CD11b,
Gr-1, and Ter119; eBiosciences), anti-mouse IgM allophycocyanin (BD
Cy7PE (eBiosciences), anti-IgD FITC (eBiosciences), and anti-IgD biotin
(eBiosciences). Cells were double sorted on a BD-FACS Aria directly into
TRIzol (Invitrogen Life Technologies) before RNA amplification.
Throughout the procedure, cells were maintained on ice and in 0.01%
B cell RNA processing and amplification
RNA isolation and amplification were performed in previous studies (32).
Briefly, RNA was isolated from cells using TRIzol and linear polyacryl-
amide (Ambion), according to manufacturer’s instructions (32). RNA was
subjected to two rounds of amplification using Arcturus RiboAmp kits.
After the second round of cDNA synthesis, Affymetrix IVT Labeling kits
were used to generate biotin-labeled cRNA. Fragmented cRNA (10 ?g)
was hybridized to Affymetrix Mouse Genome 430 2.0 microarrays.
Background subtraction and quantiles-based normalization were performed on
previously obtained raw data (32) using the GCRMA algorithm (33), available
from www.bioconductor.org. Normalized data sets for all transcripts with un-
logged expression values above 20 were compared using the 2-class unpaired
test with 100 permutations and the k-nearest neighbor imputer of Significance
Analysis of Microarrays version 3.0 (34). All raw .CEL dataset files are avail-
able at www.ncbi.nlm.nih.gov/geo (accession GSE4142).
All cDNAs were cloned upstream of the internal ribosomal entry site (IRES)
in murine stell cell virus (MSCV)-IRES-GFP (35). Klf2 cDNA was a gift from
M. Jain (Harvard University, Boston, MA), Klf3 cDNA was a gift from
M. Crossley (University of Sydney, Sydney, Australia), Klf9 was a gift
from R. Simmen (University of Arkansas, Little Rock, AR), and Ski cDNA
was a gift from K. Luo (University of California, Berkeley, CA). NF-?B1 and
RelA retroviral constructs have been described previously (35), and NF-?B1-
RelA fusion proteins were generated by insertion of 10 copies of an in-frame
SerGly4cassette downstream of NF-?B1 and upstream of RelA.
Phoenix-eco cells (a gift from G. Nolan, Stanford University, Stanford,
CA) were plated to 60–80% confluency and transfected using calcium
phosphate coprecipitation with 10 ?g of control MSCV-IRES-GFP or
MSCV-cDNA-IRES-GFP constructs. Medium was changed 6 h after trans-
fection, and retroviral supernatant was harvested 48 h after transfection.
Splenocytes were harvested from C57BL/Ka mice layered over a His-
topaque 1077 gradient (Sigma-Aldrich), and spun for 20 min, room tem-
perature, at 2000 ? g. The interface was collected, washed, and stained
with purified Abs against CD3 (2C11), CD4 (GK1.5), CD8 (53-6.7), CD5
(53-7.3), Gr-1 (8C5), CD11b (M1/70), Ter119, CD138 (281-2; BD Pharm-
ingen), Ly77 (GL7; BD Pharmingen), and subsaturating doses of CD21/
CD35 (7G6; BD Pharmingen). Stained splenocytes were depleted using
sheep anti-rat Dynalbeads (Invitrogen Life Technologies), and the remain-
ing cells were cultured for 24 h in freshly prepared B cell medium (RPMI
1640 (Invitrogen Life Technologies) ? 10% FCS (Omega Scientific) ? 50
?M 2-ME ? 10 mM HEPES ? 10 ?g/ml anti-IgM F(ab?)2(Jackson
ImmunoResearch Laboratories) ? 10 ?g/ml anti-CD40 (1C11)) at 2 ? 106
cells/ml/well of a 24-well plate. Cells were then resuspended in 2.5 ml of
retroviral supernatant containing 4 ?g/ml Polybrene (Sigma-Aldrich),
plated in 1 well of a 6-well plate, and spun at 2000 ? g at room temperature
for 1.5 h. Cells were washed and resuspended in 2 ml of B cell medium
plated at 1 ml/well of a 12-well plate. LPS stimulations and infections were
performed, as previously described (35). For division-tracking experi-
ments, purified splenic B cells were resuspended at 107cells/ml in PBS and
labeled with 1 ?M CellTrace Far Red DDAO-SE (Invitrogen Life Tech-
nologies) for 15 min at 37°C, according to manufacturer’s instructions,
before stimulation with anti-IgM and anti-CD40. For annexin V experi-
ments, cells were resuspended at 107cells/ml in annexin V-binding buffer
(10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2), stained with 20 ?l/ml
biotin-conjugated annexin V (Invitrogen Life Technologies) for 15 min at
4°C, washed, stained for 5 min with 1 ?l/107cells streptavidin Qdot 605
(Invitrogen Life Technologies), washed twice, and resuspended in annexin
V-binding buffer containing 200 ng/ml 4?,6-diamidino-2-phenylindole
Mice were immunized i.p. with 100 ?g of NP-CGG precipitated in 10%
aluminum potassium sulfate as before. NP-specific ELISAs were per-
formed with serum obtained 1 wk after immunization on high-protein-
binding 96-well plates coated with 5 ?g of NP-BSA (Biosearch Technol-
ogies). Wells were developed with anti-mouse IgG-HRP (Southern
Biotechnology Associates), followed by 1 mg/ml ABTS reagent (Sigma-
Aldrich), and the reactions were stopped by the addition of 0.1% sodium
azide. Absorbance was read at a wavelength of 405 nm.
Cells were double sorted directly into TRIzol reagent, and RNA was pre-
pared, as described above. cDNA was synthesized using the Superscript III
kit (Invitrogen Life Technologies), according to manufacturer’s instruc-
tions, using random hexamers. Amplifications were performed using
3Abbreviations used in this paper: PNA, peanut agglutinin; AID, activation-induced
deaminase; CGG, chicken ?-globulin; DAPI, 4?,6-diamidino-2-phenylindole; IRES,
internal ribosomal entry site; MSCV, murine stem cell virus; NP, hydroxy-3-nitro-
phenylacetyl; S1P1, sphingosine-1 phosphate receptor 1.
6809The Journal of Immunology
SYBR Green PCR core reagents (Applied Biosystems), and ?100 cell
equivalents and transcript levels were quantified using an ABI 7000 Se-
quence Detection System (Applied Biosystems). Primer sequences are as
follows: ?-actin, 5?-GTCTGAGGCCTCCCTTTTT-3? and 5?-GGGAGAC
CAAAGCCTTCATA-3?; Aicda, 5?-GGGAAAGTGGCATTCACCTA-3?
and 5?-GAACCCAATTCTGGCTGTGT-3?; Bcl2, 5?-CCTGGCTGTCTC
CAGATGGAGCATGTTGT-3? and 5?-CGGCTGTTCAGGAACTCTTC-
3?; Bcl-xL, 5?-ATCGTGGCCTTTTTCTCCTT-3? and 5?-TGCAATCCGA
AAGAGGTGGAGGGAACACCT-3?; Irf4, 5?-CTGAGTGGCTGTATGC
CAGA-3? and 5?-ATCAGCAATGGGAAAGTTCG-3?; Jun, 5?-TAACAG
TGGGTGCCAACTCA-3? and 5?-CGCAACCAGTCAAGTTCTCA-3?;
Klf2, 5?-GCCTGTGGGTTCGCTATAAA-3? and 5?-TTTCCCACTTGGG
ATTCGACGGGAAGGACA-3?; Mist1, 5?-AGCTGTTGTCCCTCTGT
GCT-3? and 5?-GATGGAGGTGAGGAGGATCA-3?; Prdm1, 5?-CCCCT
CATCGGTGAAGTCTA-3? and 5?-CTGGAATAGATCCGCCAAAA-3?;
Ski, 5?-AAAAGCCCTCCGCTCTAGTC-3? and 5?-GACGTCAGGGCT
Memory B cells are more similar to naive than germinal center
Naive follicular B cells from unimmunized mice or short-lived
plasma, germinal center, and memory B cells from mice immu-
nized with alum-precipitated NP-CGG were sorted, and microar-
ray RNA hybridizations were performed in previous studies in
which we compared the transcriptional profiles of self-renewing
memory B and T lymphocytes with those of hemopoietic stem
cells (32). Additional examples of gating strategies and postsort
purities are shown in Fig. 1. In all cases, propidium iodide?non-
viable cells were excluded from the sorts. Purities following the
first sort were typically 80–90%, and these cells were again sorted
directly into lysis buffer before RNA amplification to ensure ho-
mogeneity of the starting population. Only isotype-switched
IgM?IgD?memory B cells were considered in this study because
the background frequencies of NP-binding B cells in this subpopu-
lation in unimmunized mice were very low, usually ?5% of that
observed in immunized animals (Fig. 1, bottom row). Consistent
with previous descriptions (36), these cells were uniformly
CD38high, whereas only ?15% of germinal center B cells ex-
pressed CD38 (data not shown). Typically, we were able to isolate
500–2000 NP-specific memory B cells per spleen. These Ag-spe-
cific memory B cells were almost exclusively of the IgG1 isotype
(data not shown). Adoptive transfer of 750–1000 of these memory
B cells alongside primed T cells into RAG2?/?mice, followed by
immunization with NP-CGG, led to clearly measurable recall
plasma, germinal center, and memory B cells can
be highly purified from wild-type mice. Gating
strategies and purities of the starting populations
are shown in the left panels. Plasma cells, germi-
nal center cells, and memory B cells were har-
vested from spleens 7, 14, or ?70 days, respec-
tively, after immunization with 100 ?g of NP-
CGG. Unimmunized mice were injected with
only alum. Cells were analyzed after the first sort,
shown in the right panels, and sorted again di-
rectly into TRIzol to ensure further purity. Plasma
and germinal center B cells were enriched using
magnetic enrichment of syndecan-1?or PNA-
binding cells, respectively, before FACS.
Naive follicular and Ag-specific
6810 GLOBAL EXPRESSION ANALYSIS OF B CELL MEMORY
responses, whereas detectable, but smaller recall responses could
be observed by transfer of as few as 150 memory B cells (32).
cRNA was amplified from these four B cell populations and
hybridized to Affymetrix 430 2.0 Arrays, as previously de-
scribed (32). Background subtraction and normalization of the
resulting data were performed using the GCRMA algorithm
(33). Genes with median expression values ?20 in all four B
cell populations were not considered to be differentially ex-
pressed because we have been unable to consistently obtain
RT-PCR products for transcripts at these levels (our unpub-
lished observations). Significance analysis of microarrays (34)
was then used for pairwise comparisons of all transcripts with
median expression values ?20 in at least one of the B cell
populations, and genes differentially expressed by at least
2-fold and with a q value of ?10 were identified. Quantitative
RT-PCR analysis was performed on 12 genes (Aicda, Bcl2,
Bcl6, Bcl-xL, Bmi1, Irf4, Jun, Klf2, Klf3, Mist1, Prdm1, and Ski)
to confirm the relative expression levels obtained by microarray
analysis. These data revealed no false positives or negatives
with respect to significant gene expression differences between
the four populations, but did show that fold changes were on
average underestimated by 1.5-fold in the microarray experi-
ments relative to quantitative RT-PCR (data not shown), con-
sistent with previous observations (32). Interestingly, naive and
memory B cells showed differential expression of only 2904 of
a total of 45,101 transcripts analyzed, demonstrating a ?94%
overlap in their transcriptional profiles (Supplemental Table I).4
In contrast, the transcriptional profile of germinal center B cells
showed 85% overlap with that of memory B cells (Supplemen-
tal Table II). These data demonstrate that, consistent with stud-
ies performed on human CD27?tonsillar B cells (26), mouse
memory B cells acquire a transcriptional profile that is more
similar to that of naive B cells than that of germinal center B
cells. Nevertheless, clear cell-intrinsic differences do exist be-
tween naive and memory B cells that may explain the changes
in the kinetics and magnitude between primary and recall re-
sponses to Ag.
Importantly, the expression of a number of genes induced by Ag
receptor signaling (37), such as Spred2, Umpk, Pigr, Slc31a1, and
Tgf?3, remained relatively low (less than a value of 100) in both
naive and memory B cells (Supplemental Table VII). Conversely,
the expression of a number of genes repressed by Ag receptor
signaling (37), such as Hck, Ptch1, Acp5, Hist1h1c, and Adrbk1,
remained relatively high (greater than a value of 100) in both naive
and memory B cells (Supplemental Table VII). These data dem-
onstrate that the process of purification using Ig-specific reagents
did not meaningfully alter the endogenous gene expression profiles
of these B cell subsets.
Although our data are largely consistent with previous experi-
ments, several notable differences were apparent. Whereas human
memory B cells express the proapoptotic molecules Bik and Fas at
levels similar to those in germinal center B cells (26), mouse mem-
ory B cells expressed lower relative levels of these molecules,
more similar to those in naive B cells than in germinal center B
cells (Supplemental Table VII). In addition, unlike human memory
B cells (26), mouse memory B cells did not express detectable
levels of IL-2R? or CD27 (Supplemental Table VII). Moreover,
c-jun expression was very low in mouse, but not human, naive, and
memory B cells (26, 28).
Transcriptional regulators influence memory B cell formation
and plasma cell function
To identify factors that are involved in the generation of memory
B cells from the germinal center reaction and in distinguishing
primary and recall B cell responses, we assigned functional cate-
gories, based on Gene Ontology classifications (38) and evalua-
tions of the relevant literature, to selected genes that were differ-
entially expressed during Ag-dependent B cell differentiation.
Because much of the work to date regarding cell-intrinsic control
of B cell responses has focused on transcriptional regulators, we
first examined this functional category of genes in the heatmap
shown in Fig. 2. Numerical fold changes between the different
populations and absolute expression values are listed in Supple-
mental Tables I-VI and Supplemental Table VII, respectively. Im-
portantly, factors that have been demonstrated to influence the ger-
minal center reaction, such as Bcl6 (39) and Bach2 (40), as well as
genes that regulate plasma cell fate decisions, such as Prdm1 (also
known as Blimp-1) (41, 42) and Xbp1 (43), were differentially
expressed by the appropriate lineages (Fig. 2). Similar to human
memory B cells generated in vitro (28), the levels of Bcl6 tran-
scripts were lower in mouse memory B cells relative to both ger-
minal center and naive B cells (Fig. 2 and Supplemental Table
VII). These data are consistent with recent studies that demonstrate
an inhibitory role of Bcl6 in memory formation (28), but are in-
consistent with other studies that suggest Bcl6 expression pro-
motes self-renewal and memory formation (44, 45). Consistent
with previous studies (46), we found that Irf4 transcript levels were
very highly expressed in plasma cells, but not in germinal center B
cells (Fig. 2 and Supplemental Table VII), despite the observation
that Irf4 is required for efficient Ig isotype switching (47). It is
possible that the 3–10% of germinal center cells that do express
Irf4 (46) are the only ones destined for isotype switching.
Relatively few transcription factors were uniquely expressed in
naive B cells; however, a number of transcription factors were
commonly enriched in naive and memory B cells, such as Ets1,
Klf2, Klf3, Hhex, FoxP1, and Notch2 (Fig. 2). Given the prolonged
lifespans and proliferative quiescence of both naive and mem-
ory B cells (15, 48, 49), these factors may play roles in the
prevention of apoptosis and/or in the prevention of cellular di-
vision. Indeed, such a functional role has been proposed for
Klf2 in the survival and maintenance of quiescence in naive and
memory T cells (50, 51).
A number of transcriptional regulators were specifically up-reg-
ulated in memory B cells, including Klf9 (also known as Bteb1),
Ski, Mll3, Pml, Tcf4, and Bmi1. Interestingly, several of these
factors were also enriched in hemopoietic stem cells relative to
their more differentiated progeny, suggesting that they may play
a functional role in the maintenance and self-renewal of mem-
ory B cells (32).
Although transgenic expression of Bcl2 can significantly en-
hance the proportion of memory B cells that survives the germinal
center reaction, it does not provide absolute protection (18, 52).
Moreover, we did not find Bcl-x (also known as Bcl2l1), the trans-
genic expression of which can protect germinal center B cells (53),
to be differentially regulated among the different B cell populations
(Supplemental Table VII). To identify additional factors that can
impart a competitive advantage to germinal center B cells, we
retrovirally transduced naive splenic B cells stimulated with anti-
IgM and anti-CD40, which mimic certain aspects of the germinal
center reaction (54, 55), with several of the differentially regulated
transcription factors and suspected antiapoptotic genes specifically
enriched in memory B cells relative to germinal center B cells.
Cultures were then analyzed at 2 and 6 days after infection to
4The on-line version of this article contains supplemental material.
6811 The Journal of Immunology
determine whether cells retrovirally transduced with these genes
were overrepresented relative to control virus-transduced cells us-
ing a previously described formula (56). Cells retrovirally trans-
duced with Bcl2, Klf2, Ski, or NF-?B1 (57) showed a clear and
consistent increase in representation at day 6 relative to the starting
percentage at day 2 (Fig. 3A). Retroviral expression of Klf3 and
Klf9 did not measurably enhance the number of Ag receptor and
CD40-engaged B cells in this assay.
To determine the proliferative effects of the genes that led to this
increased representation after infection, we labeled B cells with
Far Red DDAO-SE, a division tracking dye. At 2 and 3 days
postinfection (Fig. 3B, left column, and data not shown), no dif-
ference in the rate of proliferation between control virus-trans-
duced cells and cells transduced with Bcl2, Klf2, Ski, or NF-?B1
was observed. By day 5, however, a significantly higher proportion
of cells transduced with Bcl2, Ski, or NF-?B1 had undergone 7 or
more divisions than cells transduced with a control virus, as mea-
sured by dilution of DDAO signal (Fig. 3B, middle column). In
contrast, little difference was seen in the divisional profile between
control-transduced and Klf2-transduced cells. These data suggest
that whereas these genes may not influence the rate of B cell pro-
liferation at early timepoints in culture, retroviral expression of
Bcl2, Ski, or NF-?B1 can extend the number of divisions that B
cells can undergo in vitro.
transcriptional differences between at least two of the analyzed B cell populations are shown in heatmap form. Functional categories were assigned based
on a combination of Gene Ontology categorization and literature searches. Red represents high relative levels of expression, and blue represents low relative
Cell-intrinsic differences between naive follicular, plasma, germinal center, and memory B cells. Selected genes with statistically significant
6812 GLOBAL EXPRESSION ANALYSIS OF B CELL MEMORY
To determine the antiapoptotic effects of these genes, we stained
cells in a separate experiment 6 days after infection with annexin
V (58) and DAPI to identify preapoptotic and recently apoptotic
cells. Retroviral expression of Bcl2 led to a ?10% reduction in the
percentage of annexin V?cells relative to control virus-transduced
cells, whereas retroviral expression of Klf2 or NF-?B1 led to
?20% reductions in the percentage of annexin V?cells (Fig. 3B,
right column). In contrast, the percentage of annexin V?cells in
Ski-transduced cells was similar to that of control virus-infected
cells (Fig. 3B, right column). Although these reductions in apop-
tosis were modest and thus somewhat surprising in the case of
Bcl2, it is worth noting that GFP is retained for only short periods
of time in apoptotic cells (59) and that these data provide at most
a snapshot of a continuous process. A 10–20% survival advantage
applied continuously over the course of the 6-day experiment
might well contribute to the competitive advantage observed in
cells transduced with Bcl2, Klf2, and NF-?B1. In contrast, retro-
viral expression of Ski appears to impart a competitive advantage
over control virus-transduced cells solely by extending the number
of divisions that B cells can undergo.
provides a competitive advantage to anti-IgM- plus anti-CD40-stimulated B cells. Naive splenic follicular B cells were purified through depletion
of non-B lineage and syndecan-1?, GL-7?, and CD21highcells; stimulated with anti-IgM and anti-CD40; and transduced with the indicated
retrovirus. The percentage of GFP?cells was quantified at 2 and 6 days postinfection. The GFP?advantage was quantified as follows: ((% GFP ?
day 6)(% GFP ? day 2))/((% GFP ? day 2)(% GFP ? day 6)), and normalized to the control virus for each experiment. Representative plots from
one experiment are shown, and the mean ? SEM values are displayed to the right for four independent experiments. B, Retroviral expression of
Bcl2, Ski, and NF-?B1, but not Klf2, extends the number of divisions that B cells undergo. Cells were labeled with Far Red DDAO, stimulated, and
infected as above, and DDAO expression levels in GFP?cells were monitored at 2 and 5 days postinfection. Gray-shaded histograms in the day 2
plots represent control virus-transduced cells, and overlaid unfilled histograms represent cells transduced by the indicated retrovirus. The percentage
of GFP?cells that had undergone 7 or more divisions, calculated by dilution of DDAO signal relative to day 0, is shown in the day 5 plots. In a
separate experiment, cells were stained at day 6 postinfection with annexin V and DAPI to quantify apoptotic cells within the GFP?population. C,
Retroviral expression of NF-?B heterodimers and Bcl2, but not Klf2 or Ski, protects LPS-stimulated B cells. LPS-stimulated splenic follicular B cells
purified as above were transduced with the indicated retroviruses, and GFP?advantage was calculated as above. Mean ? SEM values from three
independent experiments are shown.
B cells use unique mechanisms to survive T-dependent and T-independent responses. A, Retroviral expression of Klf2, Ski, or NF-?B1
6813 The Journal of Immunology
Surprisingly, despite well-described roles for RelA in the
prevention of apoptosis (60), retroviral expression of RelA or a
covalently linked dimer of NF-?B1 and RelA, which behaves
similarly to cotransfections of NF-?B1 and RelA in transcrip-
tional reporter assays (data not shown), did not measurably pro-
tect cells (Fig. 3A), suggesting that NF-?B1-containing dimeric
complexes other than the canonical NF-?B1-RelA heterodimer
may be involved in preventing apoptosis of Ag receptor and
CD40-engaged B cells. In contrast, retroviral expression of the
NF-?B1-RelA heterodimer provided protection to LPS-treated
B cells at a significantly higher level than that provided even by
retroviral expression of Bcl2 (Fig. 3B). Additionally, retroviral
expression of Ski did not affect the survival of LPS-stimulated
cells (Fig. 3B), demonstrating that, as others have also found
(61, 62), B cells use distinct mechanisms to survive T-depen-
dent and T-independent reactions.
Transcription factors such as Blimp-1 drive differentiation to
the Ab-secreting plasma cell fate in part through the transcrip-
tional repression of germinal center-associated factors such as
Bcl6 (63). Other factors involved in the endoplasmic reticulum
stress response such as Xbp1 have also been shown to be im-
portant for plasma cell differentiation (43). We identified sev-
eral other transcription factors, including the basic helix-loop-
helix protein 8, more commonly known as Mist1 (64), as having
very similar expression patterns to Blimp-1, Xbp1, and Irf4 (Fig.
2 and Supplemental Figs. 4–6). Because Mist1 is required for
proper granule organization in both pancreatic and gastrointes-
tinal exocrine cells (31, 65), we hypothesized that this gene may
be important for plasma cell generation and/or function. Serum
levels of NP-specific Ab were modestly, but statistically sig-
nificantly decreased in NP-CGG-immunized Mist1?/?animals
relative to wild-type control mice (Fig. 4A). However, no sig-
nificant difference was observed in the numbers of splenic
plasma cells that were generated 7 days after immunization of
Mist1?/?mice (Fig. 4B), suggesting that Mist1 expression may
affect the function, but not the generation of short-lived plasma
Bmi1 is highly expressed in hemopoietic stem cells and is
critical for their self-renewal (66, 67). Because memory B cells
express 13-fold higher levels of Bmi1 than do their germinal
center precursors (Supplemental Fig. 2), we hypothesized that
Bmi1 may also be important for the generation or self-renewal
of memory B cells. To address this issue, Bmi1-deficient (30) or
control littermates were immunized with NP-CGG and spleens
were harvested 4 wk after injection. NP-binding memory B
cells were clearly apparent in Bmi1?/?mice, indicating that
Bmi1 expression is not essential for the formation of memory B
cells (Fig. 5). Although the absolute numbers of memory B cells
were decreased by 10-fold in Bmi1?/?spleens, the total number
of naive B cells from which memory B cells arise was also
decreased by ?10-fold in unimmunized animals relative to
wild-type mice (data not shown). Thus, no B cell-intrinsic re-
quirement for Bmi1 expression exists for the formation of mem-
ory cells. Because, however, Bmi1-deficient animals rarely sur-
vived past 8 wk of age, we were unable to determine whether
Bmi1 is required for the long-term maintenance of the memory
B cell compartment in this study. Future studies using condi-
tional deletions of Bmi1 will most likely be required to address
Unique combinations of homing receptors and costimulatory
molecules are expressed by memory B cells
Germinal center B cells do not have the capacity to home ef-
fectively to secondary lymphoid tissues from the vasculature in
part due to expression of chemotaxis-attenuating molecules
such as Rgs13 and repression of adhesion molecules such as
L-selectin (68–71). In contrast, memory B cells reacquire ex-
pression of several of the receptors involved in both homing and
egress to and from secondary lymphoid tissues such as L-se-
lectin (Fig. 2), consistent with previous observations (20, 68).
Memory B cells also reacquire expression of Edg1 (also known
as sphingosine-1 phosphate receptor 1 (S1P1) (Fig. 2), a mol-
ecule that is critical for lymphocyte egress from the thymus and
secondary lymphoid tissues (72, 73). Interestingly, however,
memory B cells express slightly lower levels of L-selectin than
naive B cells (Figs. 2 and 6B), but comparable levels of S1P1
(Fig. 2). These data suggest that memory B cells may have a
shorter extravascular residence time than their naive counter-
parts. In addition, transcripts for the chemokine receptor CCR6,
normally associated with homing to mucosal sites (74), were
enriched in memory B cells relative to naive follicular, plasma,
and germinal center B cells.
In addition, memory B cells also displayed enhanced expres-
sion levels of certain cell surface markers relative to naive fol-
licular B cells such as the costimulatory molecules CD80 and
CD86 (Fig. 2 and Supplemental Table Ia). The increased ex-
pression of CD80 and CD86 suggests that memory B cells have
enhanced abilities to elicit T cell help upon Ag re-exposure
(75). The above data suggest that humoral recall responses oc-
cur more quickly than primary responses because memory B
cells can rapidly detect foreign Ag by surveying peripheral mu-
cosal sites and, upon encountering foreign Ag, expeditiously
of short-lived plasma cells. Mist1?/?or age-matched wild-type controls
were immunized with NP-CGG, and serum (A) and spleens (B) were har-
vested 7 days later. Serum titers of NP-specific Abs (A) and the total num-
ber of splenic B220lowsyndecan-1?NP?cells (B) are shown. Mean ?
SEM values are shown from a total of 10 Mist1?/?and 9 Mist1?/?mice
analyzed in two independent experiments.
Mist1 expression affects the function, but not the formation
Bmi1?/?or wild-type littermates were immunized with NP-CGG, and
spleens were harvested 4 wk after immunization. Plots show only B220?
Ig??cells, and memory cells are identified as IgD?NP?cells.
Bmi1 is not required for the generation of memory B cells.
6814 GLOBAL EXPRESSION ANALYSIS OF B CELL MEMORY
elicit T cell help through constitutive expression of the costimu-
latory molecules CD80 and CD86. To determine what fraction
of splenic memory B cells exhibits properties consistent with
these hypotheses, we stained these cells for surface expression
of CCR6, CD80, and L-selectin. Splenic memory B cells ex-
pressed uniformly high cell surface levels of both CD80 and
CCR6 and clearly expresssed L-selectin as well, although at
slightly lower levels than naive B cells (Fig. 6A). In contrast,
the expression of CD80 and CCR6 in naive B cells was heter-
ogeneous and weaker than that of memory B cells (Fig. 6A). A
recent study found more heterogeneous expression of CD80 in
memory B cells than we observed in this study, with the CD80?
cells representing cells that had undergone minimal somatic
hypermutation (76). The reason for these differences is not
clear, but may relate to the use of BCR H chain transgenic mice in
their study, which have a relatively high precursor frequency of Ag-
specific B cells, vs wild-type mice used in our study, which may have
a higher selective pressure to expand high-affinity somatically hyper-
mutated B cell clones.
The functional purpose of high levels of CCR6 expression on
splenic memory B cells is unclear, because CCR6 is not re-
quired for homing to the spleen or for proper splenic architec-
ture (74). We hypothesized that the high basal levels of CCR6
expression may allow for splenic memory B cells to migrate to
mucosal sites more rapidly than naive cells through relatively
modest changes in CCR6 surface expression. To test this hy-
pothesis, we compared CCR6 and L-selectin expression levels
on memory B cells isolated from the spleen, Peyer’s patch, and
brachial and axillary lymph nodes. Memory B cells isolated
from the Peyer’s patch expressed significantly higher levels of
CCR6 than their splenic or lymph node counterparts, whereas
memory B cells isolated from the peripheral lymph nodes ex-
pressed considerably less CCR6 than their splenic or Peyer’s
patch counterparts (Fig. 6B). The level of CCR6 expression of
cells express differential levels of CCR6, CD80, and L-selectin. Naive follicular (B220?IgMlowIgDhighIg??NP?) and memory B cells (B220?IgM?
IgD?Ig??NP?) were analyzed for expression of CD80 and CCR6. Mice were immunized 10 wk before analysis. Gray shaded plots represent fluorescence
minus one controls (92), and black unfilled histograms represent cells stained with anti-CCR6, anti-CD80, or anti-L-selectin. B, Differential expression of
CCR6 and L-selectin between memory B cells in spleen, Peyer’s patch, and peripheral lymph nodes. Expression of L-selectin and CCR6 was analyzed on
B220?IgM?IgD?Ig??NP?memory B cells isolated from the spleen, Peyer’s patch, and brachial and axillary lymph nodes isolated from mice immunized
12 wk beforehand with NP-CGG.
Memory B cells simultaneously express molecules associated with both lymph node and mucosal homing. A, Splenic memory and naive B
6815The Journal of Immunology
naive Peyer’s patch B cells was similar to that of Peyer’s patch
memory B cells (data not shown). Thus, the difference in sur-
face expression levels of CCR6 between naive splenic and na-
ive Peyer’s patch B cells appears to be much greater than the
difference between splenic memory and Peyer’s patch memory
Expression of activation-induced deaminase (AID) is maintained
in memory B cells
Consistent with previous studies, AID (77, 78), a gene involved in
both isotype switching and somatic hypermutation, was highly ex-
pressed in germinal centers (Fig. 2). Unexpectedly, although mem-
ory B cells had 9-fold lower transcript levels relative to germinal
center B cells (Fig. 2 and Supplemental Table IIb), they retained
53-fold higher levels of AID expression than did naive follicular B
cells (Supplemental Table Ia), suggesting that they may either be
poised to rapidly undergo additional rounds of isotype switching
and somatic hypermutation upon Ag re-exposure, or in fact are
undergoing low levels of somatic mutation to diversify the affinity
profile of the memory B cell pool. The detection of AID transcripts
in the memory B cell population is unlikely to result solely from
contamination with germinal center B cells, which express low
levels of CD38 (36), because we observed high levels of CD38
expression on ?99% of our sorted memory B cells. In contrast, to
achieve the level of AID expression in memory B cells that we
detected, contamination by germinal center B cells would have had
to approach 10%. Importantly, continued AID expression in hu-
man memory B cells has also been independently observed (28,
79). Thus, conventional memory B cells appear to differ with re-
spect to AID expression relative to the blood-borne NP-binding B
cells that appear at short timepoints after immunization (80). Sim-
ilarly, whereas memory B cells expressed 57-fold lower levels of
the proliferation marker mKi67 than germinal center B cells (Sup-
plemental Table II), they expressed 20-fold higher levels of mKi67
than naive B cells (Supplemental Table I), consistent with their
ability to self renew.
The ability to generate lasting immunity to harmful pathogens
underlies the basis for successful vaccinations. To date, the suc-
cess of such vaccines has depended upon the generation of pro-
tective humoral responses to the immunizing Ags (81). Hu-
moral immunity is thought to consist of two components, the
production of neutralizing Abs by plasma cells (13, 19, 82) and
the maintenance of memory B cells that can generate rapid and
high-affinity recall responses upon re-exposure to the foreign
Ag. In this study, we have examined the cell-intrinsic basis for
the difference between primary and recall Ab responses using
global gene expression analysis. Our data demonstrate that
memory B cells adopt a transcriptional program that may allow
them not only to survive the germinal center reaction, but to
persist long after the germinal center reaction has ceased,
thereby maintaining an increased Ag-specific precursor pool
than that which is observed before the initial response. This
increased Ag-specific B cell precursor frequency alone might be
sufficient to increase the rate at which foreign Ag is detected as
well as the rate at which T cell help is recruited after such
detection (83). Nevertheless, we also found evidence that mem-
ory B cells express high levels of chemotactic molecules gen-
erally associated with trafficking to mucosal sites such as the
Peyer’s patch. Moreover, memory B cells constitutively express
costimulatory molecules that most likely aid in rapidly activat-
ing cognate Th cells.
B cells express a multitude of chemotactic receptors and cell
surface adhesion molecules that allow them to traffic to and from
secondary lymphoid organs through the blood and lymph (84).
Evidence that distinct subsets of B cells express unique combina-
tions of organ-specific homing molecules first came from work
demonstrating that lymphocytes isolated from the Peyer’s patch
adhere more efficiently to high endothelial venules within the Pey-
er’s patch than lymphocytes isolated from axillary and brachial
lymph nodes (85). Moreover, lymphocytes from peripheral lymph
nodes homed more efficiently back to peripheral lymph nodes than
did lymphocytes isolated from the Peyer’s patch after i.v. injection
(85). Further work demonstrated that L-selectin was essential for
homing to peripheral lymph nodes, but not to the Peyer’s patch
(68). Consistent with the importance of L-selectin in extravasation
and homing to peripheral lymphoid tissues, germinal center B cells
lack expression of L-selectin and thus cannot home effectively
upon adoptive transfer (71). Our data demonstrate that memory B
cells that survive the germinal center reaction reacquire expression
of not only L-selectin (20), although to lower levels than is ob-
served in naive B cells, but also of S1P1, a receptor demonstrated
to be critical for lymphoid egress from secondary lymphoid tissues
into the blood (72, 73). Surprisingly, these splenic memory B cells
also express uniformly high levels of receptors associated with
homing to mucosal sites such as CCR6 (74). The simultaneous
expression on memory B cells of receptors important for egress
into the blood as well as for homing to both lymph nodes and the
Peyer’s patch suggests that these cells are poised to rapidly migrate
out of the spleen to potential sites of infection. Although splenic
memory B cells express lower levels of CCR6 than memory B
cells within the Peyer’s patch (Fig. 6B), this difference is relatively
minor when compared with the difference in naive B cells in these
two tissues. Thus, relatively subtle cues or stochastic signals may
direct splenic memory B cells to relocalize to mucosal sites, and
thus, the spleen may act as a repository for migratory memory B
cells. These same subtle cues may not be sufficient to relocalize
naive splenic B cells, which express much lower starting levels of
CCR6. Because recent studies have clearly shown that B cells can
directly take up Ag within minutes of administration without ex-
posure to dendritic cells (86), our data suggest that the facile ability
of memory B cells to home to multiple secondary lymphoid organs
would increase the likelihood that foreign Ags are detected and
responded to rapidly.
Upon antigenic encounter, memory B cells appear poised to
more rapidly respond than naive B cells to foreign pathogens. Con-
sistent with the previous studies performed on human tonsillar
memory B cells (26, 75), murine memory B cells express consti-
tutively high levels of CD80 and CD86. Because both primary and
recall responses to protein Ags depend upon the presence of T cells
(2, 3), the constitutive expression of costimulatory molecules most
likely allows memory B cells to more rapidly obtain T cell help
than naive B cells upon contact with cognate T cells. Moreover,
the constitutive expression of genes such as AID indicates that
memory B cells remain poised to undergo additional rounds of Ig
isotype switching and somatic hypermutation upon antigenic en-
counter. Alternatively, it is also possible that low levels of somatic
hypermutation continue in memory B cells, even after Ag has pre-
sumably been cleared, to diversify the Ab repertoire and guard
against secondary exposure to escape variants. Several recent stud-
ies have also shown that engagement of IgG1 Ag receptors pro-
duces more calcium influx than is produced by engagement of IgM
receptors (37, 87), whereas other reports have shown that human
memory B cells can more easily respond to TLR stimuli than naive
B cells (88), thus providing additional potential molecular mech-
anisms by which recall responses differ from primary responses.
6816 GLOBAL EXPRESSION ANALYSIS OF B CELL MEMORY
Interestingly, we also found differences between naive and
memory B cells with respect to several antiapoptotic genes such as
Bcl2 and Birc6. Through the expression of higher levels of an-
tiapoptotic factors, memory B cells may persist longer than na-
ive B cells, possibly maintaining themselves for life. This hy-
pothesis is consistent with the finding that transgenic Bcl2
expression reduces the rate of B cell turnover (89). Coupled
with our earlier findings that memory lymphocytes may share
certain aspects of a transcriptional program of self-renewal with
hemopoietic stem cells (32), these data suggest that memory B
cells maintain themselves at higher frequencies than their Ag-spe-
cific naive counterparts through a cell-intrinsic transcriptional pro-
gram specifically adapted for this purpose.
Importantly, the expression of a number of factors appears to
be restricted exclusively to memory B cells, suggesting that
memory B cells are a discrete entity and cannot be simply char-
acterized as existing in an intermediate state between germinal
center and naive B cells. It is important to note, however, that
our study focused only on isotype-switched memory B cells. As
previous studies have suggested, memory B cells that continue
to express the IgM isotype may represent a distinct entity alto-
gether (90), a portion of which may be derived from B1 and
marginal zone B cells (21, 22). Indeed, one study has found that
a portion of IgM?Ag-binding memory B cells is localized to
the splenic marginal zone in rats (91). In contrast, our data
suggest that isotype-switched memory B cells are unlikely to be
sessile in nature.
Although a significant amount of work has been performed to
identify factors that regulate plasma and germinal center fate
decisions, relatively little is known about the intrinsic differ-
ences that are important for directing memory B cell differen-
tiation from germinal center precursors. The memory B cell-
enriched genes identified in this study serve as excellent
candidates in directing the transition from germinal center to
memory B cells. For example, two of the factors we found to be
specifically enriched in memory B cells relative to their germi-
nal center precursors, Ski and Klf2, imparted a competitive ad-
vantage to anti-IgM- and anti-CD40-stimulated B cells, sug-
gesting a role in memory B cell differentiation. Thus, our study
establishes a basis for the identification of genes differentially
regulated during Ag-dependent B cell differentiation and for the
assignment of functional roles to these factors.
We thank L. Jerabek for laboratory management, C. Muscat for Ab pro-
duction, J. Dollaga and D. Escoto for animal care, and L. Herzenberg
for helpful suggestions.
Affiliations that might be perceived to have biased this work are as follows:
Irving L. Weissman was a member of the scientific advisory board of
Amgen and owns significant Amgen stock; and Irving L. Weissman co-
founded and consulted for Systemix, is a cofounder and director of Stem
Cells, and recently cofounded Cellerant. All other authors do not have a
conflict of interest.
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6819 The Journal of Immunology