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Antibody Repertoire and Gene Expression
Profile: Implications for Different
Developmental and Functional Traits of Splenic
and Peritoneal B-1 Lymphocytes
Karsten Kretschmer, Anke Jungebloud, Jana Stopkowicz, Britta
Stoermann, Reinhard Hoffmann and Siegfried Weiss
J Immunol 2003; 171:1192-1201; ;
doi: 10.4049/jimmunol.171.3.1192
http://www.jimmunol.org/content/171/3/1192
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Copyright © 2003 by The American Association of
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Supplementary
Material
The Journal of Immunology
Antibody Repertoire and Gene Expression Profile: Implications
for Different Developmental and Functional Traits of Splenic
and Peritoneal B-1 Lymphocytes1
Karsten Kretschmer,2* Anke Jungebloud,* Jana Stopkowicz,* Britta Stoermann,*
Reinhard Hoffmann,3† and Siegfried Weiss*
T
he mature preimmune B cell compartment of adult mice
consists of at least four subpopulations, each of which
exhibits specific developmental and functional characteristics: recirculating follicular B cells (or B-2 cells) representing the
major B cell subpopulation of lymphoid follicles, marginal zone
(MZ)4 B cells enriched in the MZ surrounding the splenic lymphoid follicles, and B-1 lymphocytes, which can be subdivided
into B-1a cells expressing CD5 and the CD5⫺ B-1b sister population (1, 2).
Physiologically, B-1a cells are more dependent on signals via
the B cell receptor (BCR) than are B-2 cells (2). In agreement, the
generation and/or maintenance of B-1a cells is affected when signals via the BCR are dampened due to natural mutations or gene
knockouts of BCR components, whereas inactivation of negative
regulators of the BCR complex increases B-1a cell numbers. Consistently, in transgenic (Tg) mice in which B cells display a high
BCR density, primarily B-1a cells are found (3–5).
The development of B-1a cells is also influenced by the specificity of their Ag receptors. Expression of a Tg BCR comprising a
B-2-associated specificity results in a decrease or even loss of B-1a
*Molecular Immunology, German Research Center for Biotechnology, Braunschweig, Germany; and †Basel Institute for Immunology, Basel, Switzerland
Received for publication December 23, 2002. Accepted for publication June 2, 2003.
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.
1
This work was partly supported by grants from Deutsche Forschungsgemeinschaft,
Bundesministerium für Bildung und Forschung, and niedersächsisches Ministerium
für Wissenschaft und Kultur. Part of the expression analysis was performed at the
Basel Institute for Immunology, which was founded and supported by F. HoffmannLaRoche, Ltd.
2
Address correspondence and reprint requests to Dr. Karsten Kretschmer, Molecular
Immunology, German Research Center for Biotechnology, Mascheroder Weg 1,
D-38124 Braunschweig, Germany. E-mail address: [email protected]
3
Current address: Department of Bacteriology, Max-von-Pettenkofer-Institut, Pettenkoferstrasse 9a, D-80336 Munich, Germany.
4
Abbreviations used in this paper: MZ, marginal zone; BCR, B cell receptor; Tg,
transgene/transgenic; BrMRBC, bromelain-treated mouse RBC; BM, bone marrow;
CDR3, complementarity determining region 3; PtC, phosphatidylcholine; N, nontemplated; P, palindromic.
Copyright © 2003 by The American Association of Immunologists, Inc.
cells, whereas Tg expression of a B-1a-derived BCR leads to an
increase of B-1a cells (2). The requirement for continuous BCR
signaling and characteristic features of the Ab repertoire distinguishes B-1a cells from other B cell subsets (6 –10). Such characteristics are paralleled by specific differences in cellular functions.
B-1a cells produce the majority of natural Abs that play a nonredundant role in providing early protection against various pathogens (2, 11).
Although B-1a cells represent a minor B cell subpopulation in
the spleen of normal mice, their absolute number is similar to the
number of B-1a cells found in the peritoneum (12). Both B-1a
compartments appear to differ in the frequencies of cells specific
for bromelain-treated mouse RBCs (BrMRBCs) and ssDNA (13).
The molecular basis for such differences is still unclear, because a
comprehensive molecular analysis of the VHDJH repertoire of
splenic B-1a cells has not been performed so far.
Except for differences in the expression levels of surface IgM
and B220 (14) and differential expression of Mac-1 (12), the presently known surface marker phenotype of splenic and peritoneal
B-1a populations is identical. Only a few molecular differences
between these two B-1a subpopulations have been described. Peritoneal B-1a cells constitutively exhibit nuclear ptyrSTAT3, whereas
splenic B-1a cells do not (15). CCR2 and CCR3 transcripts have been
found in splenic, but not in peritoneal B-1a cells (16).
The acquisition of high surface IgM levels on splenic VH11V␬9
Tg B-1 cells after i.p. adoptive transfer suggests that the phenotypical and physiological differences between splenic and peritoneal B-1a cells are at least partially influenced by the specific environment (14). Such differences could be due to local
environmental influences on migrating B-1 cells. Alternatively, the
developmental program of resident B-1 cells could determine the
phenotype. In this regard, a remaining question is whether an exchange takes place between B-1a cells of the spleen and peritoneum. The potential of peritoneal B-1a cells to migrate in response
to B lymphocyte chemokine (B lymphocyte chemokine/CXC chemokine ligand 13) has been demonstrated recently: B-1a cells of
CXC chemokine ligand 13-deficient mice failed to home to the
body cavities (17). In the same study, a significant degree of exchange of peritoneal B-1a cells between parabiosed partners was
0022-1767/03/$02.00
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In L2 mice, a high expression level of the transgenic ␭2315 L chain results in nearly complete exclusion of endogenous L chains
and a predominance of B-1a cells. In this study, we show that splenic and peritoneal B-1a cells differ considerably in their Ab
repertoire and gene expression profile. Splenic B-1a cells exhibit a more diversified repertoire under L chain limitation. Despite
oligoclonal overlaps between both B-1a compartments, some B cell receptor specificities are clearly restricted to the peritoneum.
The capacity of peritoneal B-1a cells to enter the splenic B-1a compartment was found to be very limited. Gene expression profiling
revealed genes up-regulated in splenic B-1a cells that are involved in mediating specialized first-line-of-defense effector functions
and interaction with T cells. Thus, splenic and peritoneal B-1a cells differ not only in their developmental program but also in
functional properties. The Journal of Immunology, 2003, 171: 1192–1201.
The Journal of Immunology
Materials and Methods
Mice
L2 mice (3), backcrossed on the BALB/c background for 20 –25 generations, non-Tg littermates, and IE␬T mice (20) were used at 8 –10 wk of age.
L2 mice of the IgHb allotype were obtained by crossing L2 mice of the
IgHa allotype to CB20 mice. RAG-1-deficient mice on BALB/c background were obtained from The Jackson Laboratory (Bar Harbor, ME).
Flow cytometry and cell sorting
Single-cell suspensions were obtained by flushing the peritoneal cavity and
spleen with IMDM. Blood obtained by cardiac puncture was collected in
PBS containing heparin (50 U/ml) followed by separation on Ficoll gradients. For each sorting experiment, pools of cells from three to five mice
were used unless stated otherwise.
mAb against CD72, CD80, VCAM-1, CD1, IgMa (DS-1), IgMb (AF678), CD5 (53-7.3), and B220 (RA3-6B2) were obtained from BD PharMingen (San Diego, CA). The Ab to CD19 (1D3) was homemade following
standard protocols. Flow cytometry was conducted using a FACSCalibur
and CellQuest software (BD Biosciences, Mountain View, CA). Cell sorting was performed on a MOFLO (Cytomation, Fort Collins, CO) or a
FACSVantage (BD Biosciences).
Phosphatidylcholine (PtC) liposomes were prepared using a protocol
essentially identical with methods described previously (8). PtC liposome
specificity was confirmed by IgH chain sequence analysis of sorted PtC⫹
peritoneal B-1a cells from normal BALB/c mice (8).
Amplification of VHDJH␮ chain transcripts, sequencing, and
sequence analysis
Amplification, cloning, and sequencing of IgH ␮ chain transcripts from
bulk sorted IgM⫹CD5⫹ cells was done as described previously (19). For
single-cell RT-PCR, the Qiagen (Valencia, CA) OneStep RT-PCR kit was
used. B cells were first bulk sorted and then sorted again to deposit single
cells into strips of eight PCR tubes containing 15 ␮l of RNase-free water
plus the supplied buffer and 5 U of RNAsin. This two-step sorting process
ensures deposition of single cells by exclusion of doublets. As a negative
control, one of the eight tubes did not receive a cell but was otherwise
processed like tubes containing single cells. Samples were freeze-thawed
three times (⫺20°C/65°C) and put on ice, and a mix containing RNase-free
water, dNTPs at 400 ␮M each, 1.7 ␮l of Qiagen OneStep RT-PCR enzyme
mix, the oligonucleotides VHcons and C␮1 (VHcons, 5⬘-GAGGTG
CAGCTGCAGGAGTCTGG-3⬘; C␮1, 5⬘-ATGGCCACCAGATTCTTAT
CAGA-3⬘) (both at 0.4 ␮M), and the supplied buffer were added to a final
volume of 30 ␮l. Reverse transcription was performed at 50°C for 45 min
followed by 95°C for 15 min. PCR conditions were as follows: 94°C for
15 s, 50°C for 1 min, 68°C for 1 min for 10 cycles, followed by 94°C for
15 s, 50°C for 1 min, 72°C for 30 s for 15 cycles, with final extension at
72°C for 15 min.
For the second amplification, 1 ␮l of first-round RT-PCR product was
added to a 20-␮l mix containing each dNTP at 200 ␮M, 1.5 U of HotStarTaq (Qiagen), the supplied buffer, and the VHcons oligonucleotide in
combination with an internal constant region primer (C␮2, 5⬘-CATTTGG
GAAGGACTGA-3⬘) each at 0.2 ␮M. Conditions for the second round of
amplification were as follows: 95°C for 15 s, 50°C for 30 s, and 72°C for
30 s. PCR was conducted for 35 cycles followed by incubation at 72°C for
10 min. PCR products were gel purified and then sequenced directly using
the oligonucleotide C␮2.
Generation of cRNA and microarray hybridization
All of the experiments were performed in triplicate. For each replicate
experiment, cells were isolated from different pools of mice, with the exception of B-2 cells, which were isolated from the same pool of mice. A
total of 150,000 cells of each B cell subpopulation was sorted directly into
TRIzol. RNA amplification and hybridization of Affymetrix GeneChip
Probe arrays were done as previously described (21). Affymetrix U74Av2
high-density oligonucleotide arrays, interrogating 12,488 transcripts, were
hybridized, washed, stained, and scanned according to the manufacturer’s
specifications. Scanned raw data images were processed with Affymetrix
GeneChip, version 4.0, software. Arrays were normalized to correct for
variations in fluorescence intensities using average difference values and
the DNA chip analyzer software (dChip) according to the author’s instructions (22). Then, the hybridization background was calculated, and all gene
expression values below 61 were set to 61 to eliminate low-level signals.
Assessment of differentially expressed genes was done by a nonparametric,
permutation-based method using the program significance analysis of microarrays (SAM) (23). To control for multiple testing, a false discovery rate
(24) was calculated as the percentage of false-positive genes among all
genes detected as differentially expressed. The q value used by SAM is
similar to the familiar p value, but adapted to the analysis of a large number
of genes, and represents the lowest false discovery rate at which the gene
is called significant. Genes were considered to be differentially expressed
if they were detected at a false discovery rate of 0.15%, which was the
highest false discovery rate by which the number of falsely called genes
was ⬍1. Additionally, only transcripts that were scored as positive (perfect
match intensity ⬎ mismatch intensity), showing a change of at least 2-fold
and a difference of at least 200 average difference units, were considered to
be differentially expressed. Cluster analysis of either all transcripts present
on the Affymetrix U74Av2 high-density oligonucleotide arrays or exclusively differentially expressed genes was performed according to Eisen et
al. (25).
The following oligonucleotides were used for the validation of the arraybased expression of some transcripts in cluster 6 by RT-PCR: SpiC, forward, 5⬘-CAGAGGCAACGCTAACTACTATGG-3⬘, and SpiC, reverse,
5⬘-CAGCTTTCTCCGGATTTTTATGAT-3⬘; NAD, forward, 5⬘-GCAGG
CGTGAACAATGAGAAAAAC-3⬘, and NAD, reverse, 5⬘-CACAAAGC
CTGGGCAAATGACA-3⬘; CCR3, forward, 5⬘-CTCGCTATCCAGAAG
GTGAAGAAG-3⬘, and CCR3, reverse, 5⬘-GTGGAAAAAGAGCCGAA
GGTGTT-3⬘; CD206, forward, 5⬘-TTTTACGAGAAGTTGGGGTCAGG-3⬘,
and CD206, reverse, 5⬘-TTTGGTCAGCTTTGGTTGTAATGG-3⬘; VCAM-1,
forward, 5⬘-TCCCTAATGTGTATCCCTTTGACC-3⬘, and VCAM-1, reverse, 5⬘-AGGCTGCAGTTCCCCATTATTTAG-3⬘; LPL, forward, 5⬘-CGA
GAGGATCCGAGTGAAAG-3⬘, and LPL, reverse, 5⬘-CTAATGCTGGAA
GACCTGCTATG-3⬘; CD5, forward, 5⬘-AGAAAAAGCAGCGTCAGTGG
ATTG-3⬘, CD5, reverse, 5⬘-TCAGGATGGAGGGGATGC-3⬘; and HPRT,
forward, 5⬘-GCTGGTGAAAAGGACCTCTCG-3⬘, and HPRT, reverse,
5⬘-CTTGCGCTCATCTTAGGCTTTG-3⬘.
Results
The predominance of B-1a cells exclusively expressing the Tg
␭2315 L chain in the peripheral B lymphocyte compartments of L2
mice facilitates the comparison of B-1 subpopulations (3). In the
peritoneum, IgMlowIgDhighCD5⫺Mac-I⫺ B-2 cells are completely
absent, whereas the few CD5⫺ B cells are IgMhighIgDlow
CD5⫺Mac-I⫹, thus showing the B-1b surface marker phenotype
(supplementary Fig. 1 and data not shown). Similarly, B-1a cells
represent the major B cell population in the spleen. The remaining
minor population of IgM⫹CD5⫺ B cells exhibit the CD23low/
high
⫺CD21
MZ surface marker phenotype.5
5
K. Kretschmer, A. Jungebloud, J. Stopkowicz, T. Kleinke, R. Hoffman, and S.
Weiss. The selection of marginal zone B cells differs from that of B-1a cells. Submitted for publication.
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observed. Whether the peritoneal and splenic B-1a compartments
are also interrelated by exchange was not addressed in this study,
although a partial loss of peritoneal B-1a cells in splenectomized
mice suggests a role of the spleen for maintenance of such cells
(18).
We have described the mouse line L2, Tg for the ␭2315 L chain
previously (3, 19). The peripheral B lymphocyte compartments of
L2 mice are predominantly made up of IgMhighIgDlowCD5⫹ B-1
cells that exclusively express the Tg ␭2315 L chain (3). Splenic and
peritoneal CD5⫹ B cells of such mice exhibit all characteristic
markers of B-1a cells (3). Consistent with a severe block in B cell
development in bone marrow (BM) of adult L2 mice, C1qRp⫹
(AA4.1, 493) immature B cells are below detection level in the
spleen. The almost exclusive expression of a single L chain restricts the IgH chain repertoire of peritoneal B-1a lymphocytes in
adult L2 mice, as well as of fetal and neonatal liver-derived B
cells. This pronounced oligoclonality, characterized by a high frequency of reoccurring identical amino acid sequences, was attributed to strong positive selection mediated by (auto)Ags (19). We
now extended our analysis of L2 mice by comparing the Ab repertoire and gene expression profile of B-1 cells from different anatomical locations.
1193
1194
DIFFFERENCES BETWEEN SPLENIC AND PERITONEAL B-1a LYMPHOCYTES
The same Ab specificities can be selected in the splenic and
peritoneal B-1a compartment
Increased junctional diversity of splenic B-1a cells
Despite the reoccurrence of identical sequences between splenic
and peritoneal B-1 cells, the CDR3 diversity of peritoneal B-1a
cells was more restricted, because 45.9% did not exhibit nontemplated (N) nucleotides in their VH-D and D-JH junctions compared
with splenic B-1a (23.1%) and peritoneal B-1b cells (11.5%) (Table I). A similar increase of CDR3 diversity was observed in normal mice: 45.8% of IgH chains from peritoneal B-1a cells (n ⫽ 24)
did not exhibit N nucleotides in their VH-D and D-JH junctions,
whereas only 18.8% of splenic B-1a cells exhibited no N nucleotides (Table I).
To allow conclusions about the frequency of individual clones,
we amplified IgH chains of individual IgM⫹CD5⫹ B cells from
spleen, peritoneum, and blood by single-cell RT-PCR (Fig. 2). No
striking differences in the pattern of VH, D, and JH gene segments
engaged in these rearrangements could be detected (data not
shown). However, peritoneal B-1a cells from L2 mice showed a
lower frequency of N nucleotides compared with splenic and
blood-derived B-1a cells (Table I). The slightly lower frequency of
N nucleotides in the splenic and peritoneal B-1a compartment of
L2 mice revealed by the bulk sorting compared with the single-cell
RT-PCR experiment was most likely due to an underestimation,
because in the case of bulk sorting, repeatedly isolated identical
transcripts were counted only once.
FIGURE 1. BCR specificities overlapping between splenic and peritoneal B-1 cells. IgM⫹CD5⫹ B-1a cells from spleen and peritoneum and IgM⫹CD5⫺
B-1b cells from peritoneum were isolated from a single L2 mouse by sorting and used for the amplification of IgH chain transcripts. B-1b cells could be
identified as IgM⫹CD5⫺, because B-2 cells are completely absent (supplementary Fig. 1). Reanalysis revealed a purity of cells of ⬎94%. The CDR3
nucleotide sequence and the corresponding amino acid sequence of IgH chains completely identical in amino acid sequence but recovered from different
B-1 compartments are displayed. Many additional sequences differed only in a single CDR3 amino acid (data not shown). The frequency of plasmids (n),
the VH gene family, the D element, if possible, the JH element, and the loss of JH nucleotides is indicated (VH, D, JH, and ⫺JH). N/P indicates the addition
of N and/or P nucleotides. Boxes indicate triplets that differ in their nucleotide sequence but encode an identical amino acid sequence. n.f., Not found. The
complete sequence data are available from GenBank under accession numbers AY172346 –AY172404 (peritoneal B-1a), AY172405–AY172437 (peritoneal B-1b), and AY172455–AY172509 (splenic B-1a).
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Earlier studies have shown that peritoneal B-1a cells from adult L2
mice exhibit a high frequency of reoccurring identical VHDJH
amino acid sequences (19). We now performed a comparative
analysis of splenic and peritoneal B-1 cells, isolated from a single
L2 mouse. Fifty-two splenic B-1a, 48 peritoneal B-1a, and 26 peritoneal B-1b sequences were established, representing unique rearrangements that were distinguishable by their nucleotide sequences. Some of these IgH chains displayed identical amino acid
sequences, although they were encoded by variant nucleotide sequences (Fig. 1, groups A and C). Thus, such B-1 cells were descendants of different clones. Some identical VH sequences are
shared among the three B-1 populations. In total, 17.3% (9 of 52)
of splenic B-1a-derived sequences were identical with IgH chains
recovered from the peritoneum of the same mouse. Six of these
splenic IgH chain sequences were found in the peritoneal B-1a
compartment (Fig. 1, groups A–F), whereas three were identical
with IgH chains found among peritoneal B-1b cells (Fig. 1, groups
G–I). Such high frequency of overlapping sequences between both
anatomical locations could be the result of independent identical
VHDJH rearrangements of different B-1 cell precursors, possibly
selected by the same (auto)Ag. Consistently, six overlapping IgH
chains did not exhibit complementarity determining region 3
(CDR3) diversification (Fig. 1, groups A, B, D—F, and I), thus
increasing the chance of independent identical rearrangement
events. Alternatively, the single overlapping sequence from the
splenic and the peritoneal B-1a compartment displaying JH nibbling (Fig. 1, group C) might be indicative of migration between
the two locations, because the introduction of CDR3 diversity limits the probability of independent identical rearrangements.
Two identical IgH chain amino acid sequences were shared between splenic and peritoneal B-1a and peritoneal B-1b cells (Fig.
1, groups A and C) indicating that some Ab specificities can be
selected into all three B-1 compartments analyzed, but cross-contaminations when isolating B-1 lymphocytes cannot be totally
excluded.
The Journal of Immunology
1195
Table I. Comparison of N nucleotide insertions
N in D-JH but
not VH-D
N in VH-D but
not D-JH
N in VH-D and
D-JH Junctions
L2 bulk sort and cloning (single mouse)b
B-1a peritoneum (48)c
45.9% (22)
B-1b peritoneum (26)
11.5% (3)
B-1a spleen (52)
23.1% (12)
8.3% (4)
7.7% (2)
7.7% (4)
22.9% (11)
30.8% (8)
25.0% (13)
14.6% (7)
38.5% (10)
40.4% (21)
8.3% (4)
11.5% (3)
3.8% (2)
L2 SC-RT-PCR (pool of three mice)
B-1a peritoneum (54)
61.1% (33)
B-1a spleen (48)
31.3% (15)
B-1a blood (40)
32.5% (13)
5.6% (3)
6.3% (3)
15.0% (6)
20.3% (11)
14.6% (7)
25.0% (10)
7.4% (4)
37.4% (18)
22.5% (9)
5.6% (3)
10.4% (5)
5.0% (2)
BALB/C bulk sort and cloning (pools of three mice)b,d
B-1a peritoneum (24)
45.8% (11)
B-1a spleen (32)
18.8% (6)
8.3% (2)
21.9% (7)
33.3% (8)
18.8% (6)
8.3% (2)
28.1% (9)
4.2% (1)
12.5% (4)
No N in VH-D
and D-JH
D?a
a
Frequency of IgH chain transcripts that could not be placed to a D gene segment.
To avoid any bias due to cloning, identical sequences isolated repeatedly from the bulk-sorting experiment were counted only once.
Number of sequences analyzed is shown in parentheses.
d
These sequence data are available from GenBank under accession numbers AY172858 –AY172883 (peritoneal B-1a cells) and AY172884 –AY172917 (splenic B-1a cells).
b
c
The frequency of PtC-specific B-1a cells differs between the
peritoneum and spleen of L2 mice
Despite the fact that some identical IgH chains can be readily
detected in the spleen, peritoneum, and blood, certain B-1a specificities prevalent in the peritoneal repertoire (VHQ52-DSP2.7/
DST-JH1: 13.0%, 7 of 54 sequences; VHQ52-DSP2.10-JH4: 7.4%,
4 of 54 sequences) (Fig. 2A) were found neither in spleen nor
blood. Because one could argue that the absence of such sequences
is due to the limited sample size, we analyzed L2 mice for the
presence of PtC-specific B cells. In normal mice, this specificity is
only found among B-1a cells expressing IgH and IgL chains that
are restricted to a few IgH chains in combination with certain ␬L
chains (8). Surprisingly, despite the virtually exclusive expression
of the Tg ␭2 L chain, PtC-specific B-1a cells are present in the
peritoneum of L2 mice in substantial numbers (Fig. 2C). Parallel
analysis of IE␬T mice that only produce ␭ L chains, due to a
deletion of the Ig␬ chain intron enhancer (20), showed that PtCspecific B-1a cells can be formed in the absence of ␬ L chains (Fig.
2C). The important role of anti-PtC Abs in immunity (26) might
explain the compensation of PtC specificity by ␭ L chains even in
the absence of ␬ L chains.
Although the splenic B cells compartment of L2 mice is dominated by B-1a cells, PtC binders were below detection (at least 60to 70-fold lower frequency) (Fig. 2C). Consistently, the splenic
B-1a compartment of normal mice contains a lower frequency of
B-1a cells specific for BrMRBC than the peritoneum (13). The
epitope recognized on BrMRBCs has been identified as PtC (27).
Evidence of previous Ag encounter
Despite considerable differences in their Ab repertoire, freshly isolated splenic and peritoneal B-1 cells from L2 mice show evidence
of previous antigenic experience. The level of the costimulatory
molecule CD80 is low on B-2 cells of normal mice, whereas
splenic and peritoneal B-1 cells of L2 and control mice show constitutive expression of CD80 (Fig. 3). Similarly, surface levels of
CD86 on splenic and peritoneal B-1 cells of L2 mice were slightly
increased compared with B-2 cells from normal mice (data not
shown).
Limited capacity of peritoneal B-1a cells to enter the splenic
B-1a compartment
In L2 mice, certain B-1a specificities show a clearcut segregation
between the splenic and peritoneal B-1a compartment. Therefore,
the oligoclonal B-1a repertoire in the peritoneum of L2 mice cannot be extrapolated to the splenic compartment of such mice. However, recovery of blood-derived B-1a IgH chain sequences from
splenic and peritoneal B-1a cells from the same pool of mice underlines the capacity of B-1a cells to circulate through the body. It
does not necessarily indicate an exchange between peritoneum and
spleen, especially because all overlapping IgH chain sequences of
splenic B-1a cells (6 of 48 (12.5%) from the bulk sorting and 5 of
52 (9.6%) from the single-cell RT-PCR experiment) did not exhibit N and palindromic (P) nucleotides. Hence, they might be
derived from independent rearrangement events.
Intraperitoneal transfer of L2-derived peritoneal exudates into
RAG1⫺/⫺ mice revealed that peritoneal B-1a cells have the capacity to enter the spleen in substantial numbers under such conditions (Fig. 4A). To analyze the extent of exchange between
spleen and peritoneum under more physiological conditions, reconstitution experiments were performed using peritoneal B-1
cells of L2 mice and a congenic IgH allotype system (Fig. 4B).
Although a substantial population of donor-derived B-1a cells (5–
10%) could be detected in the peritoneum of recipients after i.p.
transfer, such cells were below the detection limit in the spleen,
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Consistent with such restricted CDR3 diversity, 44.4% of IgH
chains analyzed from peritoneal B-1a cells could be summarized
into homology groups with identical or very similar amino acid
sequences (Fig. 2A). In contrast, from spleen and blood, only 18.8
and 15.0% of IgH chains, respectively, could be grouped in such
a way. Thus, the strongly restricted IgH chain repertoire that is
consistently found in peritoneal B-1a cells of L2 mice is less pronounced in spleen and blood.
The peritoneal data set was dominated by three different nucleotide sequences representing 14.8%, all of them encoding the same
amino acid sequence (Fig. 2, A and B, group E). The same IgH
chain amino acid sequence also dominated the data set from spleen
(10.4%) and could be recovered from blood as well (5.0%) (Fig.
2B, group E). Remarkably, both nucleotide sequence variants recovered from spleen and peritoneum show no CDR3 diversification, just like the overlapping sequences identified by the bulk
sorting experiment. Of note, the same VHQ52-DFL16.2-J4H-encoded amino acid sequence dominating the peritoneal repertoire at
a similar frequency (14.4%; 17 of 118 sequences) was previously
described by ourselves, using hybridoma techniques (19). Four
additional IgH chain sequences present in blood could also be
detected either in peritoneum or spleen (Fig. 2B, groups A–D).
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FIGURE 2. Comparative IgH chain
repertoire analysis of B-1a cells from
L2 mice derived from different anatomical locations. A, For single-cell RTPCR, splenic, peritoneal, and blood-derived IgM⫹CD5⫹ B-1a cells were
isolated by two subsequent rounds of
cell sorting. After the first round of sorting, B-1a cells were ⬎98% pure. IgH
chains of B-1a cells from the different
anatomical locations were grouped according to amino acid homology and
identity. The frequency of cells exhibiting the same VHDJH nucleotide sequence is given (Freq). See Fig. 1 for
further explanations. B, IgH chains with
identical amino acid sequences can be
recovered from spleen, peritoneum, and
blood (S, P, and B). C, Analysis of the
peritoneum and spleen for the presence
of PtC-specific B-1a cells. The complete sequence data are available from
GenBank under accession numbers
AF546708 –AF546754 (splenic B-1a),
AF537038–AF537090 (peritoneal B-1a),
and AF547095 (blood-derived B-1a).
DIFFFERENCES BETWEEN SPLENIC AND PERITONEAL B-1a LYMPHOCYTES
The Journal of Immunology
even 6 wk after transfer. As expected, after i.v. injection, donorderived B-1a cells could be detected in both spleen and peritoneum
(Fig. 4B). Transfer of host syngeneic BM into irradiated recipients
revealed that adult BM of L2 mice does not result in the production
of CD5⫹ B-1 cells (3), suggesting that both splenic and peritoneal
B-1a cells from L2 mice originate from early mouse development.
Gene expression profile of splenic and peritoneal B-1 cells from
L2 mice
To further characterize B-1 cells of L2 mice from different anatomical locations, we studied their gene expression profile. To this
end, total RNA was extracted from 1.5 ⫻ 105 sorted B lymphocytes (Fig. 5A) and used for mRNA amplification. The samples
were then hybridized to high-density oligonucleotide arrays
(Affymetrix).
Comparison of gene expression profiles by cluster analysis indicates a close correlation between the corresponding B-1a populations from Tg and normal mice (supplementary Fig. 2). Between
splenic B-1a cells from L2 and normal mice, only 60 transcripts
were found to be at least 5-fold regulated, while 25 were regulated
at least 5-fold between the peritoneal B-1a cells. Approximately
one-third of such genes are ␬ L chain transcripts up-regulated in
the populations of normal mice (data not shown). The few differentially expressed transcripts might reflect the transcriptional regulation of the Tg and/or the higher homogeneity of B-1a cells in L2
mice. Thus, the B-1a cells in Tg L2 mice reflect the normal situation to a large extent.
Between splenic and peritoneal B-1 cells from L2 mice, a total
of 522 genes were identified as differentially expressed at a false
discovery rate of 0.15%. Four hundred and eighteen genes were
regulated by a factor of 2 or more: 262 known genes and 156
expressed sequence tags (Fig. 5C). Six different patterns of differentially expressed genes could be identified by grouping the 262
known genes using self-organizing maps with a 1 ⫻ 6 geometry
(Fig. 5B). Fig. 6 summarizes 50 transcripts of known genes present
in clusters 5 and 6 that are at least 5-fold regulated.
The comparison of the ⬃12,000 expression values by cluster
and dot-plot analysis revealed good correlation between replicate
experiments (supplementary Figs. 2 and 3). To further validate the
array-based gene expression, some transcripts present in cluster 6
(Figs. 5B and 6) were selected according to the putative absence in
peritoneal B-1a cells but expression in splenic B-1a cells and validated by RT-PCR. Fig. 5E shows that the detection of these transcripts by RT-PCR was in concordance with the array results.
The analysis for marker genes with a known expression pattern
revealed that our expression data for CCR3 (Figs. 5E and 6, cluster
6) are concordant with previous observations, whereas CCR2
(cluster 2) does not follow the expression pattern described previously (Fig. 6) (16). CCR2 might indicate a difference between Tg
and normal splenic B-1a cells. Although the array-based analysis
indicated an ⬃5-fold regulation of CD5 between splenic and peritoneal B-1a cells (cluster 6), RT-PCR (Fig. 5E) and FACS analysis
(data not shown) revealed similar expression levels in both B-1a
subsets.
Detailed analysis of the six different gene expression patterns of
B-1 cells from L2 mice (Figs. 5B and 6) demonstrated that only
one gene present in cluster 1 shows ⬎5-fold increase in the peritoneal B-1a cells. In contrast, cluster 2 contains 12 genes that show
FIGURE 4. Limited capacity of peritoneal B-1a cells to enter the splenic B-1a compartment of normal L2 mice. Peritoneal B-1a cells (1–1.5 ⫻ 107)
obtained by peritoneal lavage of L2 mice congenic for the IgHb allotype were transferred into RAG1⫺/⫺ or L2 mice displaying the IgHa allotype. A, B-1a
cells can be identified in the spleen of RAG1⫺/⫺ mice 4 wk after i.p. transfer. B, Representative analysis of IgMb⫹ donor-derived B cells after i.p. or i.v.
transfer. Donor-derived B cells were below detection level even 6 wk after i.p. transfer and at any other time analyzed (2 days to 6 wk after transfer). Similar
results were obtained by transferring IgHa B cells into IgHb recipients. As expected, i.v. transfer results in the presence of donor-derived B-1a cells in
peritoneum and spleen at similar frequencies at any time analyzed (2, 4, and 8 days after transfer). Cells were gated on CD19⫹ cells.
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FIGURE 3. High constitutive expression levels of CD80 on splenic and
peritoneal B-1 cells of L2 and non-Tg littermates. Consistent with the
complete absence of B-2 cells, all IgM⫹ cells in the peritoneum of L2 mice
are CD80⫹, whereas IgMlow B-2 cells in the peritoneum of control mice are
CD80⫺.
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DIFFFERENCES BETWEEN SPLENIC AND PERITONEAL B-1a LYMPHOCYTES
an increase of at least 5-fold in the peritoneal B-1 compartment. Of
particular interest are the following: the chemokine receptor
CCR2; Mac-2, a lectin implicated in cell adhesion; S100A4, which
is involved in cell cycle regulation and intercellular adhesion; and
Annexin A1, which mediates anti-inflammatory responses and inhibits T cell proliferation (supplementary Fig. 4).
The number of 128 genes following the expression pattern of
clusters 3 and 4 indicates a high degree of similarity between
splenic B-1a and peritoneal B-1b cells. Genes present in clusters 3
and 4 are associated with intracellular vesicular trafficking, protein
trafficking, general metabolism, cytoskeleton, proteolysis/peptidolysis, transcriptional regulation, and signal transduction (supplementary Fig. 4). Interestingly, splenic B-1a and peritoneal B-1b
cells express H2-M transcripts (cluster 4; two hits), a molecule
involved in MHC class II presentation, at higher levels than peritoneal B-1a cells. H2-O also belongs to this cluster (data not
shown). Only the arbitrarily set cutoff of at least 5.0-fold regulation
excluded this 4.8-fold-regulated transcript.
Genes potentially involved in mediating specialized functional
characteristics of splenic B-1a cells
Clusters 5 and 6 contain genes that show increased mRNA abundance only in splenic B-1a cells compared with peritoneal B-1
cells (Fig. 5B). Some of these molecules are involved in mediating
chemotaxis, e.g. CCR3, the chemokine orphan receptor, the highand low-affinity receptors for bacterial N-formyl-methionyl peptides (FPR1 and FPR2), and calgranulin A (S100 calcium-binding
protein 8), a chemotaxin to neutrophils. The mRNA expression
levels of molecules involved in Ag uptake and presentation like the
mannose receptor (CD206), CD1, and CD68 are also enhanced.
CD206 is a pattern recognition receptor that is involved in TAPindependent presentation of lipoglycan Ags to T cells via CD1
(28). The high expression levels of CD1 on splenic B-1a cells is
comparable to MZ B cells (data not shown) and might improve the
ability to present such nonclassical Ags. CD68 plays a role in
phagocytic activities, intracellular lysosomal metabolism, and extracellular cell-cell and cell-pathogen interactions.
VCAM-1 (CD106) (Figs. 5, E and F, and 6; two hits) mediates
adhesion of leukocytes but is also supposed to be a costimulatory
molecule for T cells (29). VCAM-1 expression has been described
for BM stromal cells, follicular dendritic cells, and stimulated endothelial cells (30). Additional molecules mediating adhesive and cellcell communication functions that were found to be up-regulated in
splenic B-1a cells are procollagen XIV, Ig superfamily 4, and ficolin.
CD82 (31) and CD72 (32) are two up-regulated molecules that
have established negative regulatory functions. CD82 may also
play an important role in MHC class II maturation. Furthermore,
the gene knockout suggests that Ly-6E has an inhibitory role in
regulation of T cell activation, whereas cross-linking of the Ly-6E
molecule on B cells provides a signal for proliferation (33).
Four (Deltex homolog 1, pim-2, NF-␬BI⑀, NFATc1) and five
(c-mer; nuclear receptor subfamily 1, group H, member 3; cellular
repressor of E1A-stimulated genes; calcyclin; Spi-C) molecules
involved in regulation of transcription and signal transduction are
present in cluster 5 and 6, respectively. Deltex homolog 1 is a regulator of Notch signaling, and NF-␬BI⑀ might be involved in the
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FIGURE 5. Gene expression profiles of splenic and peritoneal B-1 cells. A, Purification of splenic and peritoneal B-1 cells from L2 mice by FACS
sorting. B, Identification of gene expression patterns by self-organizing maps. Expression levels of each gene were normalized so that the mean ⫽ 0, and
SD ⫽ 1. Black and red lines indicate expression level means and SDs, respectively. C, Hierarchical cluster analysis of 262 genes and 156 expressed
sequence tags with a change of at least 2-fold. Shown are normalized expression levels below (green), near to (black), and above (red) the mean. D,
Hierarchical cluster analysis of ⬃700 transcripts differentially expressed between splenic and peritoneal B-1 cells from L2 mice and splenic B-2 cells from
normal mice with a change of at least 2-fold. E, Analysis of gene expression by RT-PCR. B-1a cells from spleen and peritoneum of L2 mice were sorted
to high purity (⬎98.5%) and used for RT-PCR. LPL, Lipoprotein lipase; NAD, hydroxyprostaglandindehydrogenase 15. F, The expression levels of CD72,
CD1, and VCAM-1 on mRNA level revealed by gene expression profiling correlate with differential surface expression.
The Journal of Immunology
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FIGURE 6. Differentially expressed genes among B-1 subsets from L2 mice. The unique Affymetrix probe set identifier (Affy_id), the mean average
difference value, and the SD of the three replicate experiments, as well as a description of the particular gene are shown. SD was calculated as implemented
in Excel (Microsoft, Redmond, WA). Only at least 5-fold-regulated genes, comparing the populations expressing the lowest and highest levels, are
displayed. Clusters 1– 6 (C1–C6) relate to the groups displayed in Fig. 5B. The two right columns show the mean average difference values and SDs of
the corresponding transcripts of splenic CD5⫺IgM⫹ B-2 cells from normal mice.
regulation of cell adhesion, immune and inflammatory responses, apoptosis, differentiation, and growth. Pim-2 is expressed in hemopoietic tissues, and leukemia and lymphoma cell lines, and is potentially
involved in proliferation. NFATc1 has been described to regulate activation, proliferation, and differentiation of T and B lymphocytes.
The protooncogene mer, which is involved in the regulation of cell
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DIFFFERENCES BETWEEN SPLENIC AND PERITONEAL B-1a LYMPHOCYTES
growth and maintenance, has been described in numerous neoplastic
B and T cell lines, but not in normal B cells. Calcyclin expression is
induced by growth factors and overexpressed in myeloid leukemias.
The array-based strong up-regulation of the Ig J chain in splenic B-1a
cells is consistent with the ⬃40-fold higher frequency of IgM-secreting splenic B-1a cells compared with peritoneal B-1a cells (13).
An additional comparison of the expression data from Tg B-1
cell with splenic B-2 cells of normal mice revealed ⬃700 at least
2-fold-regulated transcripts. Approximately 90 ␬ L chains transcripts were detected only in the B-2 population from normal mice
(Fig. 5D). Similarly, CD9 was found to be up-regulated in B-1
cells and thus was detected at the predicted levels (1). A more
extensive comparison of B-1 and B-2 cells is displayed in Fig. 6.
Taken together, many of the molecules that are specifically upregulated in the splenic B-1 subpopulation compared with peritoneal B-1 and splenic B-2 cells might facilitate the function of
splenic B-1a cells as a first line of defense and link them to cognate
T cell-mediated immune responses.
Recent data on the functional abilities of peritoneal B-1a and
splenic MZ B cells indicate that they provide a first line of defense
against pathogens to ensure rapid development of short-term responses to a limited number of conserved Ags (1, 2). Our data
suggest that splenic B-1a cells, like peritoneal B-1a and MZ B
cells, represent a distinct B-1 cell subpopulation with unique functional characteristics.
In contrast to conventional Ig and IgH chain Tg mice that have
widely been used previously to study various aspects of B-1 cell
development, the restriction of the Ab repertoire by Tg L chain
allows the analysis of IgH chain repertoire generation (19). The
combined analysis of the Ab repertoire, gene expression profile,
and homing properties of B-1 cells from spleen and peritoneum
was facilitated by the several characteristics of the Tg mouse line
used in this study. First, the almost exclusive expression of only a
Tg L chain in L2 mice restricts the IgH chain repertoire to an
analyzable degree. Second, the predominance of B-1a cells in L2
mice facilitates the isolation of appropriate cell numbers, which
especially holds true for the splenic B-1a compartment. Third, L2
mice allow the analysis of the full potential of B-1 cells in the
absence of B-2 cells.
The IgH chain repertoire analysis was now extended to additional anatomical locations. Despite a comparable pattern of VH,
D, and JH gene segments engaged in functional IgH chains of B-1a
cells from spleen, peritoneum, and blood of L2 mice, the lower
CDR3 diversity in peritoneal B-1a cells is paralleled by a pronounced oligoclonality. A similar difference in CDR3 diversity by
N nucleotides was found in normal mice. The difference in N nucleotides has implications in both the development and function of
such cells. The most reliable indicator for the origin of a CD5⫹ B
cell from fetal sources is the lack of N nucleotides at both VH-D
and D-JH junctions due to the absence of TdT activity during this
period (34). Transfer experiments have revealed that adult BM of
L2 mice is not capable of producing CD5⫹ B cells (3). Therefore,
the differences of CDR3 diversity between splenic and peritoneal
B-1a cells must be due to differences in the generation of these
cells early in mouse development. Thus, peritoneal and splenic
B-1a cells might be generated at different ontogenetic time points,
e.g. before and after initiation of TdT expression.
Given that splenic and peritoneal B-1a cells exhibit a similar
pattern of VH genes, the differences in CDR3 diversity might explain to some extent the differences in frequencies of certain specificities in both B-1a compartments (Fig. 2C and Ref. 13). Hence,
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Discussion
this appears to be consistent with the reduction of phosphorylcholine-specific B-1a cells in mice Tg for TdT (35).
The skewed IgH chain repertoire of splenic compared with peritoneal B-1a cells could reflect selection of a more diverse splenic
B-1a repertoire, recruited from the same pool of mature B-1a cells
from which peritoneal B-1a cells are also derived. Differences in
the selection and maintenance of both B-1a subsets might be indicated by the higher expression level of H2-M and H2-O transcripts in splenic compared with peritoneal B-1a cells of L2 mice.
Recent evidence indicates that the ratio of H2-O and H2-M differs
in dependence on the B cell differentiation stage and influences the
levels of MHC II-peptide complexes (36). In addition, H2-O appears to modify peptide loading of MHC II molecules and drive
Ag presentation toward proteins that have been internalized by
surface Ig (37). Therefore, splenic and peritoneal B-1a cells might
have different Ag-presenting capabilities, which could influence
the interaction with T cells and change selective processes shaping
the Ab repertoire of such B-1 cell subsets.
The Ab repertoire clearly demonstrates the heterogeneity between splenic and peritoneal B-1 cells. Restriction of B-1a specificities to peritoneal cavity and isolation of some oligoclonal IgH
chains at high frequencies from the peritoneum but not spleen is found
on the one hand. In contrast, identical IgH chains from both spleen
and peritoneum were isolated, clearly indicating that the same Ag
specificities can be selected into both B-1a compartments.
The recovery from blood of some splenic and peritoneal IgH
chain sequences underlines the migratory capacity of B-1a cells
(17). The intraperitoneal reconstitution of RAG-1⫺/⫺ mice revealed that peritoneal B-1a cells are in principle capable of entering the spleen. Similarly, i.v. injection of peritoneal B-1a cells
results into the homing of such cells into the splenic compartment
of L2 mice. However, i.p. reconstitution experiments indicate that
exchange between peritoneum and spleen of L2 mice appears to be
very limited even 6 wk after transfer. In agreement, splenic B cells
of VH11V␬9 Tg mice, adoptively transferred into the peritoneal
cavity of normal mice, were not detectable in the spleen after 72 h
(14). Therefore, the overlapping identical IgH chains appear to be
unlikely due to a migratory exchange of dominant B-1a clones, but
might well be derived from different rearrangement events. Consistently, all splenic IgH chain sequences that are also found in
peritoneum do not exhibit N and P nucleotide insertions despite the
fact that only 23.1% (bulk sorting) and 31.3% (single-cell RTPCR) of splenic sequences have no N nucleotides. Therefore, we
also argue that the reduction of B-1a cells in splenectomized mice
observed by Wardemann et al. (18) is not due to the requirement
of B-1a cells to continuously circulate through the spleen, but
rather to a loss of a survival factor generated in the spleen.
Splenic B-1a and peritoneal B-1b cells show comparably high
levels of CDR3 diversity and striking similarities in gene expression profiles. One hundred and nineteen of 262 differentially expressed transcripts of known genes show comparable expression
levels in both B-1 subsets (clusters 1, 3, and 4). Interestingly, all
three overlaps of identical IgH chains between splenic B-1a and
peritoneal B-1b cells show substantial CDR3 diversification by the
addition of N and P nucleotides and/or JH nucleotide loss. This
finding might suggest a migratory exchange between these B-1
subpopulations in L2 mice, obscured in reconstitution experiments
due to the low frequency of peritoneal B-1b cells in L2 mice.
The diverse Ab repertoire and characteristic gene expression
profile might qualify splenic B-1a cells for mediating specialized
functions in first-line-of-defense responses at their particular location. The expression of FPR1 and FPR2 may help B-1a cells to
survey the extracellular environment for the presence of bacteria.
Such chemotactic responses might be followed by the uptake of
The Journal of Immunology
polymannose ligands mediated by CD206 which then could lead to
Ag presentation via CD1 (38). An additional potential linkage between innate and adaptive immune responses might be represented
by the VCAM-1 expression on splenic B-1a cells, supposedly a
costimulatory molecule for T cells. The high expression levels of
the Ig J chain in splenic B-1a cells could be the result of persistent
activation and the high levels of CD72 and CD82 might have important regulatory functions in limiting the response to continuous
antigenic stimulation.
In summary, our combined analysis of the Ab repertoire and
gene expression profile of splenic and peritoneal B-1 cells indicates that differences in gene expression appear to be due to a
different developmental program rather than local environmental
influences on migrating B-1 cells. Splenic and peritoneal B-1a
cells constitute unique populations of B-1 cells, each with different
developmental and functional features.
Acknowledgments
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The expert technical assistance of Susanne zur Lage, Regina Lesch, Maria
Höxter, Lothar Gröbe, Hubertus Kohler, Tracy Hayden, and Stephanie
Thies is gratefully acknowledged. We also thank Thomas Jacobs for advice
on liposome preparation. We also gratefully acknowledge Ulrich Certa at
F. Hoffmann-LaRoche, Ltd., for providing the facility for gene expression
analysis.
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