Silencing of B Cell Receptor Signals in Human Naive B Cells
Niklas Feldhahn,1, 2 Ines Schwering,2 Sanggyu Lee,4 Maria Wartenberg,3
Florian Klein,1, 2 Hui Wang,1, 2 Guolin Zhou,4 San Ming Wang,4
Janet D. Rowley,4 Jürgen Hescheler,3 Martin Krönke,1 Klaus Rajewsky,5
Ralf Küppers,2 and Markus Müschen1, 2
1Institute
for Medical Microbiology, Immunology and Hygiene, 2Institute for Genetics, and 3Institute for
Neurophysiology, University of Cologne, 50931 Köln, Germany
4Department of Medicine, University of Chicago, Chicago, IL 60637
5Center for Blood Research, Harvard Medical School, Boston, MA 02115
Abstract
Key words: B cell receptor • IL-4 • ITIM • memory B cells • SAGE
Introduction
Signal transduction pathways initiated through the B cell receptor (BCR)* determine the fate of B cells within a context of BCR-affinity to antigen, expression levels of stimulatory or inhibitory coreceptors and the differentiation stage
of B cells (1). Whereas BCR engagement by self-antigen in
Address correspondence to Dr. Markus Müschen, Emmy-NoetherGroup at the Institute for Genetics, University of Cologne, Room III.10,
Weyertal 121, 50931 Köln, Germany. Phone: 49-221-470-3408; Fax:
49-221-470-5170; E-mail: [email protected]
*Abbreviations used in this paper: BCR, B cell receptor; GC, germinal
center; IgSF, Ig superfamily; ITAM, immunoreceptor tyrosine-based activation motif; HSC, hematopoietic stem/progenitor cell; ITIM, immunoreceptor tyrosine-based inhibitory motif; MAP, mitogen-activated
protein; PKC, protein kinase C; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SAGE, serial analysis of gene expression.
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immature bone marrow B cells induces receptor editing,
deletion, or inactivation (anergy), BCR cross-linking in
mature B cells initiates a signaling cascade that ultimately
confers positive selection, proliferation, and differentiation.
Early B cell differentiation is defined by a sequence of Ig
gene rearrangements determining the configuration of the
(pre)-BCR. The recombination machinery first targets DH
and JH gene segments at the pro-B cell stage followed by
VH-DHJH gene rearrangement in pre-B cells, which subsequently express a pre-BCR, composed of Ig heavy and
surrogate light chains, on their cell surface. Expression of
Vpre-B and -5 surrogate light chain genes precedes the expression of V-J or V-J light chain gene rearrangements at the immature B cell stage, at which, for the first
time, a BCR is expressed and the cells enter the peripheral
J. Exp. Med.  The Rockefeller University Press • 0022-1007/2002/11/1291/15 $5.00
Volume 196, Number 10, November 18, 2002 1291–1305
http://www.jem.org/cgi/doi/10.1084/jem.20020881
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To identify changes in the regulation of B cell receptor (BCR) signals during the development
of human B cells, we generated genome-wide gene expression profiles using the serial analysis
of gene expression (SAGE) technique for CD34 hematopoietic stem cells (HSCs), pre-B cells,
naive, germinal center (GC), and memory B cells. Comparing these SAGE profiles, genes encoding positive regulators of BCR signaling were expressed at consistently lower levels in naive
B cells than in all other B cell subsets. Conversely, a large group of inhibitory signaling molecules, mostly belonging to the immunoglobulin superfamily (IgSF), were specifically or predominantly expressed in naive B cells. The quantitative differences observed by SAGE were
corroborated by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR)
and flow cytometry. In a functional assay, we show that down-regulation of inhibitory IgSF receptors and increased responsiveness to BCR stimulation in memory as compared with naive B
cells at least partly results from interleukin (IL)-4 receptor signaling. Conversely, activation or
impairment of the inhibitory IgSF receptor LIRB1 affected BCR-dependent Ca2 mobilization
only in naive but not memory B cells. Thus, LIRB1 and IL-4 may represent components of two
nonoverlapping gene expression programs in naive and memory B cells, respectively: in naive B
cells, a large group of inhibitory IgSF receptors can elevate the BCR signaling threshold to prevent these cells from premature activation and clonal expansion before GC-dependent affinity
maturation. In memory B cells, facilitated responsiveness upon reencounter of the immunizing
antigen may result from amplification of BCR signals at virtually all levels of signal transduction.
Materials and Methods
Isolation of Human Hematopoietic Stem Cells, Pre-B Cells, and Mature B Cell Subsets. HSCs and pre-B cells were purified from
bone marrow and umbilical cord blood. Purification of bone
marrow CD34 HSC was described (7). Cord blood HSC were
isolated using anti-CD34 immunomagnetic beads (Miltenyi Biotec). For enrichment of pre-B cells, mononuclear cells were isolated from four bone marrow samples (Poietics) and from 28 um-
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bilical cord blood samples (according to the principle of informed
consent) by Ficoll density gradient centrifugation. T cells and
myeloid cells were depleted using anti-CD3 and anti-CD15 immunomagnetic beads (Dynal). Among the remaining cells, immature CD10lowCD19CD20 B cells and CD138 plasma cells
were depleted using an anti-CD20 IgG1 antibody (BD Biosciences) together with anti-IgG1 beads and anti-CD138 beads
(Miltenyi Biotec), respectively (8). Thereafter, pre-B cells were
enriched using anti-CD19 immunomagnetic multisort-beads
(Miltenyi Biotec). The beads were released from the CD19 cells
enzymatically. The purified cells were subsequently labeled by a
mouse anti-CD10 IgG1 antibody (CALLA; BD Biosciences) and
separated using anti–mouse IgG1 beads (Miltenyi Biotec).
IgDCD19CD27 naive B cells and CD19CD27 memory B
cells were isolated from peripheral blood using anti-CD19 and
anti-CD27 immunomagnetic beads (Miltenyi Biotec) as described (9) and from seven tonsilectomy specimens. For enrichment of tonsillar memory B cells, CD27lowCD38 GC B cells
were depleted using an anti-CD38 PE antibody (BD Biosciences)
together with anti-PE microbeads (Miltenyi Biotec). Tonsillar
CD77 GC B cells were isolated as described previously (9) using
a rat anti-CD77 IgM antibody (BD Biosciences) together with a
mouse anti–rat IgM IgG1 antibody (Serotec) and anti–mouse
IgG1 microbeads (Miltenyi Biotec). Only cell purifications of a
purity 90% were included in the SAGE analysis.
Verification of B Cell Subset Purification. The identity of the
purified B cell subsets was verified genotypically and phenotypically. The genotype of purified preB cells and mature B cell subsets (naive and memory B cells) was assessed by PCR amplification of rearranged VH1 genes from genomic DNA and
subsequent cloning and sequencing of the PCR products as described (10). In addition, the phenotype of the purified HSC,
pre-B cells, naive B cells, GC B cells, and memory B cells was assessed by semiquantitative RT-PCR at the mRNA level using
IgH C
-, IgH C1-, Ig C-, Ig C-, VpreB1-, 5-, and 2Mspecific primers (Integrated DNA Technologies). To distinguish
“mature” from germline C1 transcripts, primers were chosen for
a fragment between the JH and the hinge region of the constant
region (11). The phenotype of purified preB cells was also verified by flow cytometry using CD10- and CD19-specific antibodies for pre-B cells, CD20-, and CD27-specific antibodies for naive and memory B cells, CD38- and CD77-specific antibodies for
GC B cells, respectively (FITC- and PE-conjugated antibodies
from BD Biosciences).
SAGE Analysis. cDNA-synthesis, SAGE analysis, cloning,
and sequencing of SAGE concatemers was performed according
to Velculescu et al. (6). The UniGene reference database (March
2001) was obtained at http://www.sagenet.org/SAGEDatabases/
unigene.htm. A total of 306,000 SAGE tags were collected for
the five SAGE profiles. 106,000 tags were analyzed from the
HSC library, 110,000 for pre-B cells, and each 30,000 tags for
naive, GC, and memory B cells. All SAGE libraries were normalized to 100,000 tags.
Controls for the Accuracy of SAGE Library Construction.
RNA-degradation and incomplete digestion of 3cDNAs by the
so-called tagging enzyme NlaIII may interfere with the quantitative representation of expressed genes in SAGE libraries. RNA
degradation within 3 regions would result in the underrepresentation of genes, whose last NlaIII recognition site (CATG) is particularly far from the poly(dA) tail. The average distance between
the last CATG site and the poly(dA) tail is 250 bp (6). To search
for a potential bias against SAGE tags derived from 5 sequences,
we selected 10 housekeeping genes, whose extreme 3 CATG site
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blood as naive B cells. The specificity and structure of the
BCR is further modified by somatic hypermutation and
class switch recombination during the affinity maturation
process within germinal centers (GCs), in which GC B
cells are destined to die by apoptosis unless they are rescued
through BCR-dependent survival signals upon antigen
cross-linking. GC B cells expressing a BCR of high affinity
to their cognate antigen subsequently differentiate into
memory B cells or antibody-secreting plasma cells. Notably, signaling through the BCR not only determines the
fate of a B cell at developmental checkpoints within the
bone marrow and in GCs. Also mature B cells depend on
the presence of a functional BCR, which continuously delivers a “maintenance” signal (2).
The BCR signaling cascade is initiated by rapid phosphorylation of tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) of the BCR coreceptors Ig and Ig upon antigen cross-linking. The
proximal signal transduction mainly involves three different
protein tyrosine kinase (PTK) activities including src-family PTKs, SYK, and BTK (3). In addition to kinases, several
phosphatases (SHP1, SHP2, CD45, SHIP) and linker proteins (BLNK, GRB2, SHC, NCK) also regulate BCR signal transduction. BLNK was recently shown to act as a
scaffolding protein, which mediates the interaction between SYK and the downstream signaling molecules VAV
(4) and PLC. The latter can hydrolyse PIP2 to IP3 and diacylglycerol, which increases the levels of free cytoplasmic
Ca2 and result in subsequent protein kinase C (PKC) and
mitogen-activated protein (MAP) kinase activation, which
ultimately initiates functional B cell responses including
proliferation, isotype switching and antibody secretion.
However, the downstream propagation of activation signals
may be attenuated by inhibitory receptor molecules bearing one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Many of these inhibitory receptors belong to the Ig superfamily (IgSF), which includes surface
molecules such as CD5, CD22, and FcRII/ CD32 (5).
To identify changes in the regulation of BCR-dependent activation signals at checkpoints during normal human
B cell development, we analyzed and compared genomewide gene expression profiles from human bone marrow
hematopoietic stem cell (HSC), bone marrow pre-B cells,
naive B cells, GC B cells, and memory B cells. These gene
expression profiles were generated using the serial analysis
of gene expression (SAGE) technique, which allows for the
genome-wide quantitative analysis of any expressed mRNA
in a given cell population (6).
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memory B cells. OD measurements did not reveal a significant
difference between the two populations. In addition, we repeated
semiquantitative RT-PCR for five genes for 200 and 1,000 cells,
which were sorted into reaction tubes by a FACStar™ 440 cell
sorter (BD Biosciences). RNA isolation, cDNA synthesis and
PCR amplification was done in two independent experiments.
The RT-PCR analysis was performed in 30 (for 200 cells) and 26
(for 1,000 cells) cycles for the BAM32, BLNK, SIgLec5, LIRB1,
and COX6B genes (not shown). The quantitative proportions
were similar as in the RT-PCR analysis, which was normalized
for equal mRNA amounts (see Fig. 3). Thus, mRNA contents in
naive versus memory B cells appear to be similar and do not significantly interfere with quantitative accuracy of SAGE- and RTPCR data.
Clustering Analysis of SAGE Data. For graphic representation of SAGE data, SAGE tags derived from known transcripts
were arranged in functional clusters including positive and inhibitory BCR signaling molecules. The SAGE data were sorted
based on the ratio of their frequency in memory B cells and naive
B cells. For transformation and graphic representation of SAGE
tag counts, the Cluster and Treeview software was used (kindly
provided by Dr. Michael B. Eisen, Berkeley, CA, at http://
rana.lbl.gov/).
Assessment of Expression of BCR Signaling-related Surface Molecules. To verify differential expression of molecules related to
BCR-signaling in naive and memory B cells at the protein level,
we analyzed a panel of 10 positive and 10 negative regulators of
BCR signaling by flow cytometry. Therefore, naive and memory
B cells were purified from peripheral blood from 12 donors.
Anti-CD5 PE, anti-CD19 PE, anti-CD21 PE, anti-CD22 FITC,
anti-CD24 FITC, anti-CD31 FITC, anti-CD32/ FcRII FITC,
anti-CD40 PE, anti-CD66 FITC, anti-CD72 FITC, anti-CD74
FITC, anti-CD79a/ Ig PE, anti-LIRB1 FITC, anti-CD100
FITC, anti-CD124/ IL-4R PE, anti-CD130/gp130 PE, antiCD150/SLAM PE, biotinylated anti-CD153/CD30 ligand, antiPD1 PE, and streptavidin PE were from BD Biosciences. Goat
anti-BCMA IgG and donkey anti–goat IgG FITC are from Santa
Cruz Biotechnology, Inc.
IL-4–dependent Regulation of BCR Signaling-associated Genes in
Naive and Memory B Cells. Naive and memory B cells were purified from peripheral blood as described above and cultured at
37C either in RPMI medium (including 10% fetal calf serum)
alone, or with 1 ng/ml recombinant human IL-4 (Genzyme) or
50 g of a neutralizing anti–IL-4R antibody (Genzyme)/ml,
which was added after 8 h preincubation with IL-4. The cells
were cultured at a density of 5 105 cells/100 l/well in 96well plates. After 48 h, the cells were subjected to RNA isolation
and subsequent semiquantitative RT-PCR analysis for COX6,
BLK, BTK, BLNK, SYK, LIRB1, LIRB2, LIRB5, SIgLec5,
SIgLec8, CD66, CSK, SHIP, and SHP1 at 28 cycles and for
LAIR1 at 32 cycles.
Effect of LIRB1 Signaling on BCR-dependent Ca2 Mobilization in
Naive and Memory B Cells. To address directly how LIRB1 (an
inhibitory IgSF receptor found prominently expressed on naive B
cells) can affect responsiveness of the BCR to antigen in naive
and memory B cells, BCR-dependent Ca2 mobilization was
studied. To this end, peripheral blood naive and memory B cells
were purified from four healthy donors as described above and
cultured in medium, which had been conditioned for 24 h by
PBMCs at a density of 106 PBMCs/ml. PBMCs were stimulated
with 1 g LPS/ml to induce secretion of soluble MHC class I
molecules (13), which act as natural ligand of LIRB1 (14). To
study the effect of LIRB1 on BCR signals, naive and memory B
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was more than 450 bp distant from the poly(dA) tail. We compared the tag counts for these genes with the tag counts in 55 published SAGE libraries (at http://www.ncbi.nlm.nih.gov/SAGE/
index.cgi?cmdtagsearch). In the SAGE libraries described here,
tags for these genes were found at frequencies close to the average
of 55 reference SAGE libraries, which argues against bias of quantitative representation introduced by RNA degradation.
Incomplete NlaIII digestion would result in the generation of
SAGE tags that are aberrantly derived from upstream CATG sites
instead of the extreme 3 CATG site. To address this issue, we
amplified cDNA fragments of the GAPDH and EF1 genes for the
SAGE libraries for naive and memory B cells. Primers were chosen so that cleavage by NlaIII would result in the loss of the 5
primer binding site for GAPDH but not for EF1. Consistent with
high efficiency of NlaIII digestion, either no or only very small
amounts of PCR product were obtained for GAPDH while amplification of EF1 fragments yielded abundant amplification products (unpublished data).
Selection of BCR-related Signaling Molecules. In a comprehensive search for positive and negative BCR-related signaling molecules in PubMed, UniGene (http://www.ncbi.nlm.nih.gov/UniGene/) and OMIM (http://www.ncbi.nlm.nih.gov/entrez/
query.fcgi?dbOMIM), we collected 211 genes, for which a role
in positive (129) or negative (82) regulation of BCR-dependent
signals was shown. Based on their UniGene-ID, 148 (97 positive
and 51 inhibitory signaling molecules) of these genes could be retrieved from at least one of the five SAGE libraries.
Verification of Quantitative Accuracy of SAGE Data. To corroborate
quantitative differences in gene expression among the five populations as determined by SAGE, semiquantitative RT-PCR analysis was performed for a set of 41 selected genes. For verification
of SAGE data, unrelated cDNAs from HSC and pre-B cells purified from umbilical cord blood (see above), naive, and memory B
cells from peripheral blood were used as template. To specifically
address quantitative differences in the expression of positive and
inhibitory BCR signaling molecules in naive and memory B cells,
we performed semiquantitative RT-PCR verification for 22
genes implicated in propagation and 19 genes involved in attenuation of BCR signals, respectively. RT-PCR was done for 18,
20, 24, 28, 30, or 32 cycles as indicated and normalized for the
COX6B gene encoding the cytochrome c oxidase subunit VI and
the 2M gene encoding 2-microglobulin.
Effect of mRNA Content per Cell on the Interpretation of SAGE
Data. For all SAGE profiles, expression levels of genes are given
as tags per 100,000. This is based on the assumption that the relative contribution of a gene to the transcriptome of a cell population accurately reflects its expression level. However, an alternative viewpoint would be that gene expression levels should be
given as absolute number of transcripts per cell. It is critical in the
latter but not in the former case that the overall amounts of
mRNA are similar in the cell populations compared by SAGE
and semiquantitative RT-PCR. For instance, B lymphoblasts
such as centroblasts within GCs may double their size and
mRNA content as compared with their resting precursors (12).
Thus, up-regulation of positive regulators of BCR signals in blastoid GC as compared with resting naive B cells as observed by
SAGE (see Fig. 2, A and B) should be even higher if individual
cells are analyzed. On the other hand, the quantitative difference
of the expression of inhibitory BCR signaling molecules between
individual resting naive or blastoid GC B cells would be lower
than suggested by SAGE. As for size and mRNA content of
memory versus naive B cells no pertinent information was available, total RNA and mRNA was isolated from 106 naive and 106
Results and Discussion
Verification of the Identity of Purified B Cell Subsets.
CD34 HSCs (reference 7) and CD10 CD19 pre-B cells
(Fig. 1 B) were purified from human bone marrow,
CD19CD27 naive and CD19CD27 memory B cells
were purified from peripheral blood and CD38CD77
GC B cells from tonsils and analyzed by flow cytometry
(Fig. 1 B). The identity of the purified subsets was further
supported by the analysis of their genotype (Table I). As
opposed to memory B cells, Ig VH1-gene rearrangements
amplified from pre-B cells and naive B cells were virtually
devoid of somatic mutations. Consistent with ongoing se-
Figure 1. Genotype and phenotype
of purified B cell subsets. Bone marrow
pre-B cells (CD10 CD19), peripheral
blood naive B cells (CD19 CD27),
tonsillar GC B cells (CD20 CD77),
and peripheral blood memory B cells
(CD19 CD27) were purified as described in Materials and Methods. The
mRNA expression of C
, C1, C,
and VpreB was analyzed by semiquantitative RT-PCR analysis (A). The identity of the purified subsets was further
verified by flow cytometry (B): FACS®
plots for preB cells (CD10 CD19),
naive (CD20 CD27), GC (CD20
CD77), and memory B cells (CD20
CD27; from top to bottom) are given.
Table I. Sequence Analysis of VH1-Gene Rearrangements in
Purified B Cell Subsets
VH1-gene
rearrangements
Among 10 clones
In-frame
Out-of-frame
Pseudogene
Potentially functional
Average mutation
frequency (102 bp)
Pre-B cells
Naive
B cells
Memory
B cells
3
7
3
2
7
3
0
7
8
2
0
8
0.4
0.6
7.7
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lection for the expression of a functional Ig heavy chain in
bone marrow pre-B cells, nonproductive VH1-gene rearrangements (either due to loss of reading frame or rearrangement of a pseudogene of the VH1 family) were overrepresented in the isolated pre-B cell population (Table I).
Prior to the SAGE analysis, specific fragments of the C
-,
C1-, C, and VpreB genes were amplified from pre-B
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cells were cultured for 24 h in the presence or absence of an antagonistic (clone HP-F1; reference 14) or agonistic (clone GVI/
75; BD Biosciences; reference 15) LIRB1 antibody cross-linked
by goat anti–mouse IgG serum (Jackson ImmunoResearch Laboratories). HP-F1 was a gift from Dr. Miguel López-Botet, Universitat Pompeu Fabra, Barcelona, Spain. After the preincubation,
cells were washed and stained with Fluo-3 dye (Calbiochem) for
30 min. Changes of cytosolic Ca2 were measured by laser scans
using confocal microscopy (16). After 30 s of measurement, antihuman IgM F(ab)2 and anti–human IgG IgM F(ab)2 fragments (Jackson ImmunoResearch Laboratories) were added to
naive and memory B cells, respectively. Cytosolic Ca2 concentrations were calculated as described previously (17). As a negative
control, purified B cell populations were also treated with an antiCD3 antibody (BD Biosciences), which induces Ca2 mobilization
in T but not B cells. For statistical analysis, area under curve values
were calculated and compared using Fisher’s exact test. P 0.05 was
considered statistically significant.
Effect of IL-4R Signaling on BCR-dependent Ca2 Mobilization in
Memory B Cells. As treatment of naive B cells with IL-4 had no
effect on the expression of genes related to BCR signaling (see
above; see Fig. 5), modulation of BCR signals by IL-4 was studied in memory B cells only. Memory B cells from four healthy
donors were purified and cultured in supernatant from LPS-stimulated PBMCs for 24 h in the presence or absence of human recombinant IL-4 or an inhibitory anti–IL-4R antibody (Genzyme). Changes of cytosolic Ca2 concentrations upon BCR
engagement were measured and analyzed as described above.
Table II. SAGE-tag Counts for Differentiation
Stage–specific Genes
CD34
HSC
CD34
CD164
CD10
TdT
RAG-2
IL-7R
CD23
CD38
BCL-6
AID
CD21
CD27
19
27
2
16
3
2
1
Pre-B cells
2
1
40
173
125
41
2
9
Naive
B cells
4
20
4
2
4
8
GC
B cells
Memory
B cells
17
3
3
3
3
24
27
17
20
17
3
7
3
7
7
3
51
78
Figure 2 (continues on next page)
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cells, naive, and memory B cells by RT-PCR, which was
normalized for 2-microglobulin (Fig. 1 A). As expected, expression of VpreB is confined to pre-B cells, which, in turn,
lack expression of Ig light chains and IgG1 heavy chains.
C1 transcripts were detected in memory but not pre-GC
B cells (Fig. 1 A). The identity of the purified B cell subsets
was also confirmed retrospectively by the expression pattern of a set of subset-specific markers within the SAGElibraries (Table II).
Verification of Quantitative Accuracy of SAGE Profiles. In the
analysis of SAGE profiles for CD34 HSC, pre-B, naive, GC,
and memory B cells, we identified a particular gene expression pattern, which involves positive and negative regulatory BCR signaling molecules (see references in Fig. 2). To
corroborate the quantitative differences in the expression of
BCR signaling molecules as observed by SAGE, semiquantitative RT-PCR was performed for 22 positive and 19
negative regulatory BCR signaling molecules (Fig. 3). For
all 41 genes tested, the amounts of the amplification product mirrored the SAGE tag counts in the libraries for naive
and memory B cells. Moreover, the expression of 10 costimulatory and 10 inhibitory surface molecules implicated
in the propagation or attenuation of BCR-dependent signals was analyzed at the protein level by flow cytometry
(Fig. 4). For all 20 surface molecules, the FACS® data correlated with SAGE tag counts, indicating that the large majority of SAGE data are indeed reproducible by alternative
methods.
Opposing Regulation of BCR Signaling in Naive and Memory B
Cells. In a comprehensive search throughout the five
SAGE profiles and the UniGene database, we identified
cells are similar. Many of the signaling molecules included
in this study are also involved in signaling pathways that are
not related to the BCR. This applies in particular to downstream kinases (e.g., p85, JAK3, p110, LCK, FAK, IKK,
AKT, HPK1, p115, PKC
, PKC, PKA; Fig. 2) and receptors that are not specific for the B lineage, whose intracellular signals may converge with those of the BCR
(SLAM, PDGFR, CD38, CD27, FcRH1, HRH1, CD86,
NGFR, CD36, CD74, CD66, CD31, LAIR1, CD5, CD33,
LLIR, LIRB1, LIRB2, LIRB5, PIR; Fig. 2).
However, mouse mutants for some of these genes exhibit a particular B cell phenotype, and many receptor molecules are involved in either ITAM- or ITIM-dependent
signaling, which allows a prediction of their role in either
amplification or attenuation of BCR-dependent activation
signals. The five SAGE libraries also identified a number of
components of the proximal BCR-signaling complex,
which have an unambiguous function in (pre)-B cells.
These “classical” BCR signaling molecules include BLK,
BLNK, BTK, Ig, Ig, SYK, and CD19, whose expression
levels are consistently lower in naive B cells as compared
with other B cell subsets (Fig. 2 A and B, and Figs. 3 and
4). To the contrary, well characterized inhibitors of BCRinduced activation signals including the protein tyrosine
phosphatases (PTPs) SHP1 and SHIP (19), the PTK CSK,
the PTK-associated ubiquitin ligase CBL, and the ITIM-
Figure 2 (continues on facing page)
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148 molecules involved in positive (97 genes) and negative
(51 genes) regulation of BCR signaling present in at least
one SAGE library. Whereas most of the genes that positively regulate BCR-dependent activation signals are expressed at high levels in pre-B, GC, and memory B cells,
this was not the case for naive B cells (Fig. 2, A and B, and
Figs. 3 and 4). In many cases, expression of positive BCR
signaling molecules was either missing in the SAGE library
for naive B cells or reduced to expression levels as in
CD34 HSC. Conversely, inhibitory molecules were expressed either exclusively or predominantly in naive B cells
(Figs. 2 C, 3, and 4).
Concomitant down-regulation of mediators of BCR-related
activation together with increased expression of inhibitory
molecules in naive B cells (Fig. 2, A–C) suggests that an elevated signaling threshold prevents naive B cells from being
inappropriately activated upon antigen encounter. There
are, however, some exceptions to this seemingly uniform
picture: for instance, IKAROS, which appears to reduce
BCR-dependent B cell activation (18) is expressed at
higher levels in pre-B cells and memory B cells as compared with naive B cells. Also, SAGE tags matching the
LYN gene, which is critical for ITIM-dependent negative
signaling, and the IB gene, encoding an inhibitor of nuclear factor (NF)-B, were found most frequently in pre-B
cells, while expression levels in naive, GC, and memory B
bearing surface receptors CD22, CD32 and CD72 are expressed at high levels in naive B cells but not in pre-B, GC,
or memory B cells (Figs. 2 C, 3, and 4).
Facilitated responsiveness of memory B cells to BCR
engagement was suggested from earlier findings, namely
the expression of the activating coreceptors CD21 (20)
and CD27 (21) together with increased expression levels
of the costimulatory molecules CD80 and CD86 (22) and
in vitro experimentation, which demonstrated that memory cells have higher propensity to undergo activationinduced terminal differentiation than naive B cells (23).
To date, a genome-wide analysis of BCR signaling molecules in pre- and (post-) GC B cells is missing. As shown
in Fig. 2, A and B, sensitization of memory B cells to
BCR-dependent activation signals is not only related to
upregulation of surface molecules such as CD21, CD27,
CD80, and CD86 but involves virtually all levels of intracellular signal transduction including transmembrane receptors, kinases, linker molecules, phosphatases, Ca2 channels
and transcription factors.
Figure 2. Cluster analysis of activating and inhibitory B cell receptor signaling molecules. In a systematic survey of PubMed, UniGene and OMIM databases, 211 BCR-related genes were identified, 148 of which could be retrieved from at least one of the SAGE libraries for CD34 HSC (HSC), pre-B
cells (PBC), naive B cells (NBC), GC B cells (GCB), and memory B cells (MBC). In total, 97 activating (A and B) and 51 inhibitory (C) signaling molecules were identified the five SAGE libraries and listed with their gene names, SAGE tag counts for each library, UniGene ID, a brief description of
their putative function including a reference. It should be noted that because of limited space in many cases only one functional aspect among others has
been included. The SAGE data were sorted based on the ratio of tag counts in memory and naive B cells. For calculation of ratios, a tag count of 0 was
set to 0.5. For graphic representation of SAGE data, tag counts have been transformed using the Cluster and Treeview softwares by M.B. Eisen, in which
red denotes strong and black no or low expression.
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In particular, it was not known that BCR signals in naive B cells can be specifically silenced by the prominent
expression of ITIM-bearing molecules belonging to the
emerging group of inhibitory immunoglobulin superfamily (IgSF) receptors (Figs. 2 C, 3, and 4). In the following,
we describe the expression pattern of inhibitory and stimulatory BCR signaling molecules in naive and memory B
cells more in detail, dividing these molecules into functional groups.
Regulation of the Expression of Constituents of the BCR in
Human B Cell Subsets. In mature B cells, the BCR is
composed of surface Ig, the coreceptors CD19, CD21, and
CD81 and the Ig- and Ig-signaling chains (1). In naive
B cells, mRNA levels of BCR-related molecules are either
moderately (Ig, CD19, CD81; Fig. 2 B) or substantially
(Ig, CD21; Fig. 2 A) lower than in other B cell subsets.
Also, expression levels of Ig genes are lower in naive B
cells as compared with other B cell subsets: in the SAGE library for naive B cells, we found 143 tags matching to the
C
gene (pre-B cells: 826; GC B cells: 329; memory B
cells: 450), 29 tags for V- and C-genes (pre-B cells: 5;
GC B cells: 54; memory B cells: 101) and 199 tags for Cgenes (pre-B cells: 10; GC B cells: 447; memory B cells:
1,337). Instead of Ig- and Ig- light chain genes, pre-B
cells mostly express VpreB (287 tags) and -5 (1,975 tags)
surrogate light chains. Higher levels of Ig gene expression
in memory than in naive B cells is in agreement with a previous study, in which Ig mRNA levels were 3- to 11-fold
higher in memory as compared with naive B cells (24). As
expected, SAGE tags matching to C genes are frequent in
memory (1,929 tags) and GC B cells (217 tags), but virtu1298
B Cell Receptor Signaling in Naive B Cells
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Figure 3. Assessment of quantitative accuracy of SAGE data by RTPCR. To corroborate quantitative differences of gene expression in naive
and memory B cells as observed by SAGE, semiquantitative RT-PCR
analysis was performed for 41 selected genes coding for 22 known positive
(top) and 19 negative (bottom) regulatory molecules of BCR-dependent
activation signals. RT-PCR was performed for 20, 24, 28, and 32 cycles.
The amounts of cDNA were comparable for naive and memory B cells as
determined by photometry and RT-PCR analysis of the COX6B (cytochrome c oxidase subunit VI) and B2M (2-microglobulin) genes. For all
41 genes tested, the amount of the amplification product roughly reflects
the quantitative distribution of SAGE tags in the libraries for naive and
memory B cells. In some instances, no SAGE tag was detected for a given
gene. However, in all these cases, an RT-PCR product was obtained at
least after 28 cycles of amplification, which indicates that sensitivity of
SAGE for low abundance-class genes is inferior to that of RT-PCR.
ally missing in the libraries for naive (13 tags) and pre-B
cells (16 tags).
TNF Receptors May Act as Costimulatory Molecules in GC
and Memory B Cells. The genes downregulated in naive B
cells include members of the TNF receptor superfamily: in
contrast to GC and memory B cells, CD40 (TNFRSF5),
CD27 (TNFRSF7), and NGFR (TNFRSF16) are either
missing or expressed only at low levels in naive B cells (Fig.
2, A and B). While CD40-engagement is known to cooperate with IL-4R- and BCR-dependent signals during the
GC reaction (25), ligation of the “memory-specific” receptor CD27 (21) by CD70 increases responsiveness of memory B cells to BCR signals and induces plasma cell differentiation. Also, three recently identified members of the TNF
receptor superfamily are differentially expressed in naive B
cells compared with GC- and memory B cells: BAFF-R
(TNFRSF13B) and BCMA (B cell maturation antigen;
TNFRSF17) are receptors of the B cell activation factor
BAFF and are expressed in GC and memory B but not naive B cells (Fig. 2, A and B, and Fig. 3). Unlike BCMA
(26), BAFF-R is critical for the development of marginal
zone (i.e., mostly memory) B cells and the T cell–dependent B cell response (27). Although not essential for B cell
activation, BCMA was shown to induce NF-B–activation
and p38 MAP kinase-dependent proliferation in synergism
with the BCR (28). A third receptor for BAFF, termed
TACI, however, is, although at low level, only expressed
in naive B cells (Figs. 2 C and 3) and acts as a negative regulator of BCR-dependent B cell activation (29). CD30
ligand (TNFSF8), an inhibitor of B cell costimulation and
class switch recombination (CSR; reference 30), is expressed in naive B cells but virtually missing in GC- and
memory B cells Figs. 2 C, 3, and 4). In this regard, it is notable that SWAP70, which promotes both B cell activation
and CSR (31), is expressed reciprocally with CD30 ligand
in naive and (post) GC B cells (Fig. 2 B).
Specific Expression of Inhibitory Ig Superfamily Members in
Naive B Cells. Many of the inhibitory receptors, which
we find expressed at high levels in naive B cells belong to
the Ig superfamily (IgSF). The inhibitory IgSF molecules
are predominantly or exclusively expressed by naive B cells
(Fig. 2 C) and typically carry one or more ITIMs within
their cytoplasmic tail. Negative regulatory IgSF molecules
specifically or predominantly expressed in naive B cells include SIgLec5, SIgLec6, and SIgLec8, members of the sialic
acid binding Ig-like lectin-like family, and LIRB1, LIRB2,
and LIRB5, which belong to the B group of leukocyte Iglike receptors (collectively termed CD85, Figs. 3 and 4).
Also, ITIM-bearing IgSF molecules, the lectin-like immunoreceptor LLIR, the paired Ig-like receptor PIR, as well
as CD22, CD31, CD32/FcRII, the biliary glycoprotein
CD66 are expressed by naive B cells and, if at all, only at
reduced levels in GC and memory B cells (Figs. 2 C, 3, and
4). This also applies to the newly identified IgSF molecules
PD1 (32), G6B (33), and CMRF35H (34). Also, ITIMbearing but a member of the C-type lectin family, the
CD72 molecule is highly expressed in naive B cells (Figs. 2
C, 3, and 4). On the other hand, its antagonistic ligand
CD100 can relieve CD72-mediated inhibition of BCRsignals and is predominantly expressed in memory B cells
(Figs. 2 B, 3, and 4). Within the IgSF, a group of Fc receptor homologues was recently identified, which comprises
positive and negative regulatory coreceptors based on
whether they harbor ITAMs or ITIMs within their cytoplasmic tail (35). Like many other inhibitory IgSF receptors, the ITIM-bearing FcRH2/SPAP1 (35) was only
found in naive B cells (Fig. 2 C), whereas its ITAM-carrying homologue, FcRH1 was expressed in GC and memory
but not naive B cells (Fig. 2 A).
Cytokine Receptor Signaling in Naive and Memory B
Cells. The B cell–homing chemokine receptor BLR1,
also termed CXCR5, can cooperate with the BCR by
stimulation of Ca2 influx (36) and is stronger expressed in
GC and memory B cells as compared with their naive precursors (Fig. 2 B). Naive B cells also differ from GC B cells
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Feldhahn et al.
and memory B cells in that they lack expression of the signal transducer for IL-6 (CD130 or gp130) and the receptor
for IL-4 (CD124). gp130 has been implicated in post-GC
development of B cells, as mice expressing a dominant negative form of gp130 exhibit a marked reduction of Ig production (37). Engagement of IL-4R can augment activation signals through the BCR (for a review, see reference
38; see below).
Regulation of BCR-Downstream Linker Molecules and Kinases
in Human B Cell Subsets. A large group of activating
linker molecules and PTKs contributes to propagation of
activation-stimuli within the distal BCR-signaling cascade.
Without exception, activating linker molecules identified
in the SAGE libraries, including BLNK, BAM32, GRB2,
SOS1, SHC1, GAB1, GAB2, BRDG1, NCK, and BANK,
were up-regulated in GC and memory as compared with
naive B cells (Fig. 2, A and B). In contrast, inhibitory linker
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Figure 4. FACS® analysis of costimulatory and inhibitory receptor molecules. To assess whether quantitative
differences in mRNA expression as assessed by SAGE and
RT-PCR also translate into protein, the surface expression
of 10 costimulatory (top) and 10 inhibitory (bottom) surface molecules was analyzed by flow cytometry. Histograms for the expression of surface molecules, whose differential expression was identified by SAGE, are shown for
naive (top panel) and memory (bottom panel) B cells.
Numbers indicate the tag counts in 100,000 tags in the respective SAGE library.
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initiated from the BCR through transcriptional activation
of Ig (by OCT2; reference 55), synergism with BTK (by
OBF1; reference 56), and amplification of BCR-dependent anti-apoptotic signals and proliferation-stimuli (by
NF-B; reference 43). BCR engagement and subsequent
NF-B activation are linked by the protooncogene
BCL10 (57), which is expressed at high levels in pre-B and
memory but not naive B cells (Fig. 2 B). In GC and memory B cells, OCT2 was found up-regulated together with
its transcriptional target gene CD36 (Fig. 2 B). Stage-specific expression of CD36 could be meaningful because
based on the dependence of CD36 expression on transcriptional activation by OCT2, it was speculated that
OCT2 could regulate B cell differentiation through CD36
(58). The members of the ETS family of transcription factors, FLI-1 (59), PU.1, and SPI-B (60) have recently been
demonstrated as critical components of BCR-dependent
activation-signaling pathways. FLI-1, PU.1, and SPI-B are
expressed at high or intermediate levels in GC and memory B cells but are missing in the SAGE library for naive B
cells (Fig. 2 A).
Relief of BCR Signal Inhibition by Engagement of IL-4R in
(Post) GC B Cells. Recent data indicate that enhancement of BCR-dependent B cell activation by IL-4 at least
in part results from the relief of inhibitory signals through
negative regulatory receptor molecules (61). In agreement
with these findings, we observe specific expression of
ITIM-bearing inhibitory IgSF receptors on naive B cells
(Figs. 2 C and 3), which also lack expression of IL-4R
(Figs. 2 B, 3, and 4). Conversely, the SAGE libraries for
GC B cells and memory B cells show a correlation between the expression of IL-4R and the absence of ITIMbearing and other inhibitory receptors (Fig. 2 C). IL-4–
dependent changes of gene expression are largely mediated
by STAT6 (38), which we find expressed at higher levels
in GC (17 tags) and memory B cells (33 tags) than in naive
B cells (4 tags).
Thus, antigen-encounter during the GC reaction, upregulation of IL-4R, and initiation of IL-4–dependent signals might result in a far-reaching phenotypic change,
namely transcriptional silencing of a large group of inhibitory receptors that are abundantly expressed in naive B
cells. Whether and to which extent the inverse regulation
of responsiveness to BCR cross-linking in naive and memory B cells results from differential IL-4R signaling was
tested in a cell culture experiment (Fig. 5). Naive and
memory B cells were purified from peripheral blood and
cultured either in medium alone, or with human recombinant IL-4. In another set of experiments, memory B cells
were preincubated with IL-4 and subsequently treated with
a neutralizing anti–IL-4R antibody. After 48 h, the cells
were subjected to RNA isolation and subsequent semiquantitative RT-PCR analysis for mRNA expression of
the positive regulatory BCR signaling molecules BLK,
BTK, BLNK, and SYK, their inhibitors CSK, SHIP, and
SHP1 and negative regulatory IgSF receptors including
LIRB1, LIRB2, LIRB5, SIgLec5, SIgLec8, LAIR1, and
CD66 (Fig. 5).
B Cell Receptor Signaling in Naive B Cells
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molecules including p62DOK (39) and the SRC-like adaptor proteins SLAP1 and SLAP2 (40) inhibit BCR downstream signals, predominantly in naive B cells (Fig. 2 C).
Activating kinase molecules, as far as identified in the
SAGE libraries, are expressed at higher levels in (post) GC
B cells as compared with their naive precursors (Fig. 2, A
and B). PI-3 kinases p85 and p110 have in common that
they may activate AKT/PKB (41) and BTK (42), while activated BTK can induce degradation of the NF-B inhibitor IB by phosphorylation through IKK (43). Only
moderately up-regulated in GC and memory B cells, the
MAP kinase HPK1 (44) and the PTK PYK2 (45) can augment BCR signals, the former through interaction with
BLNK, the latter with BRDG1 (Fig. 2 B). Interestingly,
PYK2-deficient mice lack splenic marginal zones, which
are thought to be mainly composed of memory B cells
(45). Not specific for the B cell lineage, but expressed in
GC and memory B cells (Fig. 2 A), the receptor of platelet
derived growth factor PDGFR can act as a stimulatory
PTK and promote proliferation (46).
Among the inhibitory kinases, the CD45-antagonist
CSK (47) and the PKCs (48) and (49) acting as inhibitors of BTK and SYK, respectively, are expressed at higher
levels in naive B cells as compared with GC and memory B
cells (Fig. 2 C). However, expression levels of LYN, a key
mediator of ITIM-dependent negative signaling is expressed at similar levels throughout all B cell subsets. Specific inhibition of the tyrosine kinase BTK represents another level of BCR-signal attenuation, at which the IBTK
(50) and SAB/SH3BP-5 (51) molecules are acting, mostly
in naive B cells (Fig. 2 C).
Regulation of PTPs during B Cell Development. The PTP
CD45 represents a critical component of the proximal
BCR-signaling cascade (52). Together with its antagonist,
CSK, CD45 sets the threshold for antigen receptor signals
in B and T cells by reversing CSK-mediated phosphorylation
of negative regulatory tyrosine-based motifs in src-family
PTKs (53). As opposed to its antagonist CSK, the CD45
gene is higher expressed in memory B cells, GC B cells,
and pre-B cells as compared with naive B cells (Fig. 2, A
and C). From the five SAGE libraries, multiple SAGE tags
matching to the CD45 gene were retrieved, which most
likely reflects the expression of multiple splice variants of
the CD45 gene. Consistent with the notion of BCR-signal
inhibition in naive B cells, the inhibitory PTPs SHP1,
SHIP, and the recently identified PTP-PEST molecule (54)
are expressed at high levels in naive but not GC or memory
B cells (Fig. 2 C). Another PTP, encoded by the PTEN
gene, inhibits the activation of BTK in B cells (38), is frequently inactivated by somatic mutation in advanced malignancies resulting in uninhibited PTK activity and expressed at higher levels in naive than (post) GC B cells (Fig.
2 C).
Role of Transcription Factors in the Regulation of BCR Signals. The genes up-regulated in memory and GC B cells
include classical transcriptional activators of Ig genes including OCT2, OBF1, NF-B1, and NF-B2 (Fig. 2, A
and B). These genes are involved in autocatalytic loops
In naive B cells, presence or absence of IL-4 did not affect the expression of positive or negative regulatory BCR
signaling molecules. In peripheral blood memory B cells,
however, complete deprivation from IL-4 in cell culture
medium for 48 h resulted in a concomitant decrease of
mRNA levels of BLK, BTK, BLNK, and SYK with markedly increased expression levels of inhibitory IgSF receptors
(Fig. 5) as compared with expression levels in ex vivo analyzed peripheral blood memory B cells (for comparison, see
amplification products in Fig. 3, at 32 cycles for LAIR1 and
28 cycles for the other genes studied here). Loss of positive
and gain of negative regulatory BCR signaling molecules
upon withdrawal of IL-4 in peripheral blood memory B
cells indicates that gene regulation through IL-4R requires
continuous presence of its ligand. However, serum levels of
IL-4 are low in healthy individuals (i.e., 0.1 pg/ml; reference 38), which suggests that peripheral blood memory B
cells are able to respond also in the presence of low concentrations of IL-4. It is indeed conceivable, that memory
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Figure 5. Regulation of inhibitory IgSF receptors in memory B cells
by IL-4. Naive and memory B cells were purified from peripheral blood
and cultured either in medium alone, or with IL-4. In another set of experiments, memory B cells were cultured in the presence of a neutralizing
anti–IL-4R antibody, which was added after 8 h of preincubation with
IL-4. The left and center panels show amplification products of semiquantitative RT-PCR for positive regulatory PTKs (BLK, BTK, SYK),
the linker molecule BLNK, the negative regulatory PTK CSK, the inhibitory PTPs SHIP and SHP1, and the inhibitory IgSF receptors LIRB1,
LIRB2, LIRB5, SIgLec5, SIgLec8, and CD66. In the right panel, the genomic loci of these genes are indicated.
B cells have acquired higher responsiveness to IL-4 (e.g.,
by up-regulation of IL-4R) during maturation within GCs,
in which IL-4–producing TH2-cells are highly concentrated (62). Treatment of memory B cells with IL-4 at high
concentrations induced a slight increase of mRNA levels of
BLK, BTK, BLNK, and SYK but a marked reduction of
mRNA levels of the inhibitory IgSF receptors LIRB1,
LIRB2, LIRB5, SIgLec5, SIgLec8, LAIR1, and CD66. IL-4
treatment did not affect expression levels of other inhibitory BCR signaling molecules in memory B cells including
CSK, SHIP, and SHP1, which are expressed at constitutively lower levels in memory than in naive B cells (Fig. 5).
Consistent with low-level expression of IL-4R in naive B
cells (Figs. 2 A, 3, and 4), IL-4 can induce transcriptional
repression of inhibitory IgSF receptors in memory but not
naive B cells. Inhibition of IL-4R–dependent signals by a
neutralizing antibody, which was added after preincubation
with high concentrations of IL-4, only slightly reduced
mRNA levels of the positive mediators of BCR signaling
BLK, BTK, BLNK, and SYK and had no effect on expression levels of their inhibitors CSK, SHIP, and SHP1.
However, inhibitory IgSF receptors including LIRB1,
LIRB2, LIRB5, SIgLec5, SIgLec8, LAIR1, and CD66 were
markedly up-regulated in memory B cells in the presence
of the neutralizing anti–IL-4R antibody and reached similar expression levels as in naive B cells. Thus, down-regulation of inhibitory IgSF receptors in memory B cells largely
depends on IL-4R engagement.
Clustering of IgSF Genes Overexpressed in Naive B Cells to a
Region on Chromosome 19. As expression levels of other
negative regulatory BCR signaling molecules such as CSK,
SHIP, and SHP1 remain stable in the presence or absence
of IL-4R signaling, transcriptional repression induced by
IL-4 in memory B cells seems to be specific for inhibitory
IgSF receptors. In this regard, it is notable that the genes
coding for the ITIM-bearing IgSF receptors studied here
and also many inhibitory killer cell Ig-like receptors are arranged in a cluster within a 2.9 Mbp region on chromosome 19 (19q13.2–q13.4; see Fig. 5). This cluster of genes
also includes the inhibitory IgSF receptors CD22, GP6 and
CD33, which are highly expressed in naive but not GC
and memory B cells (Figs. 2 C, 3, and 4). Therefore, it is
tempting to speculate that this cluster on chromosome 19
harbors a large number of IgSF genes sharing a common
mechanism of transcriptional regulation in that they can be
silenced by IL-4R–dependent signals.
Differential responsiveness to IL-4 in naive and memory
B cells suggests that human B cells may acquire sensitivity
to IL-4 during the GC reaction, while IL-4R–dependent
signaling itself facilitates transduction of signals initiated
from the BCR. Further studies are needed to identify other
mediators of the relief of the “inhibition-phenotype” in
human B cells after antigen-encounter in the GC.
Regulation of BCR Responsiveness by IL-4R Signals in
Memory B Cells. Having shown that IL-4 can down-regulate inhibitory IgSF receptors in memory but not naive B
cells (Fig. 5), we studied the effect of IL-4R signals on
BCR-dependent Ca2 mobilization in memory B cells. Af-
ter preincubation of memory B cells in PBMC-conditioned medium in the presence or absence of human recombinant IL-4 or an inhibitory anti–IL-4R antibody,
memory B cells were challenged with anti–human IgG IgM F(ab)2 fragments and Ca2 flux was measured. Addition of IL-4 beyond physiological concentrations had no
effect on BCR responsiveness of memory B cells (Fig. 6
C). However, inhibition of IL-4R resulted in a decrease
of the peak size and a rapid decline of the calcium signal,
which suggests that integrity of BCR responsiveness in
memory B cells requires signals through the IL-4R (Fig. 6
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B Cell Receptor Signaling in Naive B Cells
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Figure 6. Regulation of BCR-dependent Ca2 mobilization by
LIRB1 and IL-4 in naive and memory B cells. Naive (A) and memory (B)
B cells (B) were preincubated for 24 h in medium, which had been conditioned by LPS-stimulated PBMCs, in the presence of either an antagonistic (light gray curve, LIRB1 HP-F1) or an agonistic antibody to
LIRB1 cross-linked by goat anti–mouse IgG serum (dark gray curve,
LIRB1 GVI/ 75-GAM) or no antibody (black curve, none). Naive and
memory B cells were stimulated with anti–human IgM F(ab)2 and anti–
human IgG IgM F(ab)2 fragments, respectively, at the indicated times
(arrows) and changes of intracellular Ca2 concentrations in response to
BCR engagement were measured by confocal microscopy. In another set
of experiments (C), memory B cells were cultured in supernatants from
LPS-stimulated PBMCs for 24 h in the presence of human recombinant
IL-4 (light gray curve, IL-4), an inhibitory anti–IL-4R antibody (dark
gray curve, IL-4R) or no further reagents (black curve, none). For
each experiment, cells from four donors were purified and separately analyzed, yielding similar results. For quantitation, area under curve (AUC)
values were calculated. Statistically significant differences from controls
(black curves; none) with P 0.05 were determined using Fisher’s exact
test and indicated by asterisks.
C). This was expected, as inhibition of IL-4R signaling resulted in reexpression of inhibitory IgSF molecules in
memory B cells (Fig. 5). The failure of supraphysiological
IL-4 concentrations to further augment BCR-dependent
Ca2 mobilization in memory B cells suggests that already
low IL-4 concentrations (e.g., as in human serum) may be
sufficient to maintain full BCR responsiveness to antigen.
Regulation of BCR Responsiveness by the IgSF Receptor
LIRB1 in Naive and Memory B Cells. As IL-4 can repress
inhibitory IgSF receptors in memory but not naive B cells,
we next investigated the direct consequences of IgSF receptor signaling on the responsiveness of the BCR in both
B cell subsets. As an example for inhibitory IgSF receptors,
we chose LIRB1, which is most prominently expressed in
naive B cells (45 tags; Figs. 2 C, 3, and 4) but missing in the
SAGE profiles for GC and memory B cells (Figs. 2 C, 3,
and 4). To determine how LIRB1 can modify BCR responsiveness, changes of cytoplasmic Ca2 concentration in
response to BCR engagement were measured. To this end,
naive and memory B cells were preincubated in supernatants conditioned by LPS-stimulated PBMCs in the presence or absence of agonistic (clone GVI/75 cross-linked by
goat anti–mouse serum) or antagonistic (clone HP-F1 noncross-linked) antibodies against LIRB1. After LPS treatment, PBMCs release high levels of soluble MHC class I
molecules, which can act as natural ligand for LIRB1 (13,
14). After preincubation, naive and memory B cells were
stimulated with anti–human IgM F(ab)2 and anti–human
IgG IgM F(ab)2 fragments, respectively, and changes of
intracellular Ca2 concentrations in response to BCR engagement were measured by confocal microscopy. Whereas treatment of naive and memory B cells with an anti-CD3 antibody
had no effect on intracellular Ca2 levels (not shown), stimulation with anti–human IgM F(ab)2 and anti–human IgG IgM F(ab)2 fragments increased the concentration of
cytoplasmic Ca2 in naive and memory B cells (Fig. 6).
While stimulation (GAM-cross-linked GVI/75 antibody)
and inhibition (noncross-linked HP-F1 antibody) of LIRB1
significantly affected the peak amplitude and duration of the
calcium signal in naive B cells (Fig. 6 A), agonistic and antagonistic antibodies had no significant effect on BCRdependent Ca2 mobilization in memory B cells (Fig. 6 B).
As expected, the overall signal intensity of BCR engagement was significantly higher in memory (peak [Ca2]i
concentration 220 nmol/L) than in naive B cells (peak
[Ca2]i concentration 140 nmol/L). Also, steady-state levels of cytoplasmic Ca2 were higher in memory (100
nmol/L) as compared with naive B cells (50 nmol/L). Interestingly, occupancy of LIRB1 by the inhibitory antibody
raised Ca2 mobilization in response to BCR engagement to
similar levels as in memory B cells (Fig. 6, A and B). That
inhibition of LIRB1 alone could nearly restore BCRresponsiveness in naive B cells in vitro does not rule out an
important contribution of other inhibitory IgSF receptors
identified in this study to silencing of BCR signals in vivo.
This is supported by a number of animal models, in which
inactivation of a single gene similarly resulted in profound
changes of BCR responsiveness (63–66). That other inhibitory
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Feldhahn et al.
in memory B cells has distinct intrinsic signaling properties
is further supported by recent data on a burst-enhancing
role of the membrane spanning region of IgG in memory
B cells (69). Earlier findings suggested that, unlike IgM
(three cytoplasmic residues), the cytoplasmic tail of IgG1
(28 residues) as such can contribute to signal transduction
through the BCR, presumably involving a tyrosine-based
motif within C1 (70).
Although both naive and memory B cells critically depend on continuous survival signals from a functional
BCR (2), we show in a genome-wide gene expression
analysis that BCR-dependent signals in naive and memory
B cells differ fundamentally from each other. Conceivably,
these differences may owe to some extent to IL-4–dependent
ablation of negative regulatory signals from inhibitory receptor
molecules. However, IL-4R signals, although required for
transcriptional suppression of inhibitory IgSF receptors
and for integrity of BCR-dependent Ca2 mobilization in
memory B cells, had no effect on their naive precursors.
Therefore, the mechanism establishing restraint of BCR
signals in naive B cells and its relief upon antigen-encounter
within the GC remains elusive and awaits further investigation.
We would like to thank Stefanie Jauch for excellent technical assistance and Dr. Miguel López-Botet, Universitat Pompeu Fabra, Barcelona, Spain, for kindly providing us with the HP-F1 antibody.
H. Wang is supported by the DAAD Bioscience Program, M.
Müschen is supported by the Cancer Research Institute, New
York, NY, through the Tumor Immunology Program. This work
was supported by the Deutsche Forschungsgemeinschaft through
the Emmy-Noether-Programm (to M. Müschen; MU1616/2-1 and
MU1616/2-2), Harold and Lelia Y. Mathers Foundation (S.M.
Wang and J.D. Rowley), SFB 502 and a Heisenberg award to R.
Küppers, by the Köln Fortune Program/Faculty of Medicine, University of Cologne, and the German José Carreras Leukemia Foundation (grant to M. Müschen).
Submitted: 30 May 2002
Revised: 12 August 2002
Accepted: 13 September 2002
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IgSF receptors, which were found specifically expressed in
naive B cells by SAGE, RT-PCR and flow cytometry
(Figs. 2 C, 3, and 4), have a similar effect on BCR signals
in naive B cells appears likely but remains to be established.
Conversely, engagement of LIRB1 by the agonistic crosslinked antibody further suppressed and shortened the calcium signal in naive B cells initiated by BCR ligation (Fig.
6 A). None of these effects could be seen in memory B
cells (Fig. 6 B), which is consistent with absence of LIRB1
expression in memory B cells (Fig. 2 C, 3, and 4).
We conclude that IL-4 is essential to down-regulate inhibitory IgSF receptors in antigen-experienced B cells. As
inhibitory IgSF receptors can indeed silence BCR signals,
their repression by IL-4 is required to maintain an intense
signaling capacity and responsiveness of the BCR to antigen.
Concluding Remarks. Unlike naive or memory B cells,
pre- and GC B cells exhibit a particular propensity to apoptosis in that they enter a readily initiated apoptosis program
unless they are rescued by survival signals from the (pre)BCR. Intensified activation signaling in the surviving pre
and GC B cells can, therefore, be an effect of selection.
Neither naive nor memory B cells are directly involved in
selection processes. In the case of naive and memory B
cells, the distinct gene expression pattern of activating and
inhibitory BCR signaling molecules is probably largely B
cell autonomous and marks a constitutive difference between the two subsets.
In naive B cells, attenuation of BCR signals (e.g., by inhibitory IgSF receptors) may prevent these cells from premature activation upon antigen encounter or represent a
safeguard against expansion of autoreactive clones. Conversely, amplification of BCR signals may contribute to facilitated responsiveness to BCR engagement and longevity
of memory B cells (67). Sustained expression of activating
signaling molecules initiated from the BCR seems to be a
characteristic of memory B cells as the expression levels of
activating BCR signaling molecules in memory B cells also
tend to be higher than those in pre-B or GC B cells (Fig. 2,
A and B). High expression levels of costimulatory molecules may reflect activation in pre-B and GC B cells and
sensitization and increased responsiveness to antigen in
memory B cells.
A constitutively active signaling machinery in memory
B cells may not only increase responsiveness to but also
confer independence from BCR cross-linking by antigen.
Indeed, the capacity to persist independently from the immunizing antigen was thought to define “true immunological memory” (68) as opposed to survival within a chronic
immune response, in which the immunizing antigen can
be retained and presented for extended periods of time by
follicular dendritic cells within GCs. To ensure persistence
of B cell memory, perpetuation of a B cell–autonomous
“maintenance signal” would be required in the former but
not in the latter case. Recent data suggest that the continuous presence of the immunizing antigen is indeed dispensable for the survival of memory B cells provided that a
functional BCR is expressed on the cell surface to initiate
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