IL-9-Induced Expansion of B-1b Cells
Restores Numbers but Not Function of B-1
Lymphocytes in xid Mice
This information is current as
of June 14, 2017.
Laurent Knoops, Jamila Louahed and Jean-Christophe
Renauld
J Immunol 2004; 172:6101-6106; ;
doi: 10.4049/jimmunol.172.10.6101
http://www.jimmunol.org/content/172/10/6101
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References
The Journal of Immunology
IL-9-Induced Expansion of B-1b Cells Restores Numbers but
Not Function of B-1 Lymphocytes in xid Mice1
Laurent Knoops, Jamila Louahed, and Jean-Christophe Renauld2
T
he B-1 lymphocytes were originally identified by their
expression of CD5 (1) and represent the main B cell population in the peritoneal and pleural cavities of mice (1,
2). They can also be distinguished from the conventional B (B-2)
cells as IgMhighIgDlowCD23⫺CD43⫹ and Mac-1(CD11b)⫹. B-1
cells produce most of the natural Abs of the IgM class in the serum
(3) and contribute significantly to the IgA-producing plasma cells
in the lamina propria of the gut (4). The B-1 repertoire is biased,
as illustrated by the overrepresentation of antiphosphatidylcholine
(PtC)3 specificities (5, 6). They recognize common bacterial Ags,
such as phosphorylcholine (PC), as well as self-Ags, such as PtC
or membrane proteins on erythrocytes and thymocytes (7), and are
involved in Ab responses to certain type 2 T-independent Ags (8,
9). They therefore are related to innate immunity and provide a
degree of serological protection against a range of microorganisms
before the specific immunization that accompanies microbial
pathogenesis (10).
Beside CD5⫹ B-1 cells, another population of peritoneal B cells
was identified with the same surface phenotype as B-1 cells, except
for the absence of CD5 expression (3, 11, 12). CD5⫹ B-1 cells
were referred to as B-1a lymphocytes and CD5⫺ as B-1b lymphocytes or CD5 “sister” B cell population (11). The B-1b sister population was described as closely related to B-1a cells based on
phenotype, tissue distribution, function, and development (11).
Ludwig Institute for Cancer Research and Experimental Medicine Unit, Université de
Louvain, Brussels, Belgium
Received for publication October 28, 2003. Accepted for publication March 12, 2004.
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 supported in part by the Belgian Federal Service for Scientific,
Technical and Cultural Affairs and the Actions de Recherche Concertées of the Communauté Française de Belgique. L.K. is a Research Fellow with the Fonds National
de la Recherche Scientifique, Belgium.
2
Address correspondence and reprint requests to Dr. Jean-Christophe Renauld, Ludwig Institute for Cancer Research and Experimental Medicine unit, Université de
Louvain, Avenue Hippocrate 74, 1200 Brussels, Belgium. E-mail address:
[email protected]
3
Abbreviations used in this paper: PtC, phosphatidylcholine; PC, phosphorylcholine;
TNP, trinitrophenyl; Br, bromelain.
Copyright © 2004 by The American Association of Immunologists, Inc.
Cell transfer experiments indicated that both populations have the
capacity for self-replenishment (11), are able to secrete high levels
of IgM (3), and are able to bind PtC liposomes (11). Both lineages
also generate IgA plasma cells in the lamina propria (4) and have
a defective response to B cell receptor cross-linking or CD19dependent signaling, as compared with B-2 lymphocytes (13).
However, the two lineages differ by their temporal appearance during
ontogeny and by the better capacity of adult bone marrow to reconstitute the B-1b population (11, 14). Moreover, these populations were
differentially affected by splenectomy, which decreased the B-1a lymphocyte population, but not the B-1b cells (15), and by gene targeting
of the G␣i2, PD-1, or ␮ H chain genes (16 –18). An age-dependent
specific increase of the B-1b subpopulation in leaky SCID mice (19)
and in xid mice (20) was also previously shown.
Mice expressing the X-linked immune defect (xid) were originally described in the CBA/N strain (21). This defect was associated with a missense mutation in the Bruton’s tyrosine kinase, one
of the components of the B cell signal transduction pathway (22).
By contrast with the human X-linked agammaglobulinemia syndrome, where mutation of this gene causes a near complete absence of mature peripheral B cells (1% of normal) and severe
hypogammaglobulinemia of all Ig isotypes (23), xid mice have a
less severe B cell depletion. These mice have a one-half to a onethird of the conventional follicular B cell numbers (B-2 cells), but
show a severe reduction of the B-1 cell subset (24). Functionally,
xid mice have reduced levels of natural IgM and fail to respond to
type 2 T-independent Ags (25).
Because IL-9 administration or overexpression induces a B-1
lymphocyte expansion in vivo (26), we wondered whether IL-9
overexpression could compensate the xid mutation. Interestingly,
IL-9 restored a B-1 population in xid mice, but exclusively with the
B-1b surface phenotype. xid-B-1b cells however failed to restore
classical functions of B-1a cells, indicating that this xid sister population is functionally distinct from the B-1a cell subset.
Materials and Methods
Mice
Mice were maintained in a specific pathogen-free environment and were
8 –12 wk of age for the experiments. The Tg5 IL-9-transgenic line,
0022-1767/04/$02.00
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Mice expressing the X-linked immunodeficiency (xid) mutation lack functional Bruton’s tyrosine kinase and were shown to be
specifically deficient in peritoneal B-1 lymphocytes. We have previously shown that IL-9, a cytokine produced by TH2 lymphocytes, promotes B-1 cell expansion in vivo. To determine whether IL-9 overexpression might compensate the xid mutation for B-1
lymphocyte development, we crossed xid mice with IL-9-transgenic mice. In this model, IL-9 restored normal numbers of mature
peritoneal B-1 cells that all belonged to the CD5ⴚ B-1b subset. Despite this normal B-1 lymphocyte number, IL-9 failed to restore
classical functions of B-1 cells, namely, the production of natural IgM Abs, the T15 Id Ab response to phosphorylcholine immunization, and the antipolysaccharide humoral response against Streptococcus pneumoniae. By using bromelain-treated RBC, we
showed that the antigenic repertoire of these IL-9-induced B-1b lymphocytes was different from the repertoire of classical CD5ⴙ
B-1a cells, indicating that the lack of B-1 function by B-1b cells is associated with distinct Ag specificities. Taken together, our data
show that B-1b cell development can restore the peritoneal B-1 population in xid mice but that these B-1b cells are functionally
distinct from CD5ⴙ B-1a lymphocytes. The Journal of Immunology, 2004, 172: 6101– 6106.
6102
B-1b LYMPHOCYTE EXPANSION IN IL-9-TRANSGENIC xid MICE
obtained in the FVB/N background and expressing high levels of IL-9 in all
organs, was described previously (27). The CBA/N (xid) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and from the Harlan
Laboratory (Horst, The Netherlands) with similar results. To obtain IL-9transgenic xid mice, we crossed male Tg5 with female CBA/N (xid) mice.
Control male FVB/N mice were crossed with female CBA/N (xid) mice.
We obtained four groups of F1 mice by this procedure that were used for
our experiments: male FVB/xid, with a xid phenotype, female FVB/xid in
which the xid mutation was compensated by a nonmutated X chromosome,
male Tg5/xid, transgenic for IL-9 and having the xid mutation on their
single X chromosome, and female Tg5/xid, transgenic for IL-9 but in
which the xid mutation was compensated by a wild-type copy of the X
chromosome.
Flow cytometry
Peritoneal cells were obtained by washing the peritoneal cavity with 6 ml
of HAFA (Hanks’ medium supplemented with 3% decomplemented FBS
(Perbio, Erembodegen, Belgium) and 0.01 M azide). Cells were counted,
washed, and suspended at 3 ⫻ 106/ml in HAFA. Double staining of cells
was performed with FITC-conjugated anti-IgM (LOMM9) and biotinylated
rat Abs against Mac-1 (M1/70, both provided by H. Bazin, Université de
Louvain, Brussels, Belgium) followed by PE-conjugated streptavidin (BD
Biosciences, Mountain View, CA), PE-conjugated anti-CD5 (BD PharMingen, San Diego, CA), or PE-conjugated anti-CD23 (BD PharMingen).
For other stainings, FITC anti-IgD and biotinylated anti-IgM (LOMD6 and
LOMM9, provided by H. Bazin) followed by PE-conjugated streptavidin
were used. Three-color analysis was performed with FITC-conjugated antiIgM, PE-conjugated anti-CD5, and biotinylated anti-Mac-1 followed by
RED670 (Invitrogen, San Diego, CA)-conjugated streptavidin. After staining, cells were fixed with 1.25% paraformaldehyde, and fluorescence intensity was measured on 104 cells/sample or on 3 ⫻ 104 cells/sample for
the three-color analysis on a FACScan apparatus (BD Biosciences).
Spontaneous IgM and specific Ig production
For the natural IgM level, blood samples were collected by retro-orbital
puncture. Serum was obtained after blood coagulation for 1 h at 37°C and
stored at ⫺20°C before usage. For the ELISA, 96-well plates (Maxisorp;
Nunc, Roskilde, Denmark) were coated overnight at 4°C with 50 ␮l of
anti-IgM (LOMM3, 5 ␮g/ml, provided by H. Bazin) in glycine (20 mM)buffered NaCl (30 mM, pH 9.2). After washing with 0.1 M NaCl plus
0.05% Tween 20, plates were blocked for 1 h at 37°C with 150 ␮l of 1%
BSA in PBS. Plates were washed and 50 ␮l of diluted serum in 1% BSA
in PBS or IgM standards was incubated for 2 h at 37°C. After washing,
plates were incubated with streptavidin-conjugated anti-IgM (LOMM8,
provided by H. Bazin). After 1 h at 37°C and washing, bound Ig was
detected with 50 ␮l of 1Step Turbo tetramethylbenzidine-ELISA (Perbio)
for 15 min. Reaction was stopped with 50 ␮l of 2 M H2SO4 and absorbance
was read at 450 nm.
For the T15 Id response, PC was coupled to keyhole limpet hemocyanin
(KLH) and to BSA as described before (28). The AB1–2 hybridoma, secreting mouse IgG1 anti-T15 Id Ab, was obtained from the American Type
Culture Collection (Manassas, VA). The Ab was purified on a protein A
column. For the immunization, mice were injected i.p. with 50 ␮g of PC
coupled to KLH (PC-KLH) in CFA (Difco, Detroit, MI). Sera were obtained at day 15, before an i.p. boost with 50 ␮g of PC-KLH in IFA
(Difco). Sera were collected again at day 30. Control mice received CFA
and IFA alone. Anti-PC response was measured by ELISA, as described
above, by coating the plates with PC-BSA (5 ␮g/ml) and detecting bound
Ig with streptavidin-conjugated anti-␬ L chain (LOMK1, 2.5 ␮g/ml, provided by H. Bazin). Serial dilutions of the sera were applied and the titer
was defined as the dilution that showed an OD of 400% of background, a
value that was in the linear range of the curve. T15 Id IgM response was
measured by ELISA by using the anti-T15 Id Ab AB1–2 as coating reagent
(20 ␮g/ml) and streptavidin-conjugated anti-IgM (10 ␮g/ml, LOMM8) for
the detection. The titer was defined as the dilution that showed an OD of
300% of background.
For the bacterial antipolysaccharide response, we used a protocol described previously (15). Mice were injected i.p. with 5 ␮l of the pneumo-23
vaccine (Aventis Pasteur, Lyon, France) in 200 ␮l of PBS. Pneumo-23
contains polysaccharides of Streptococcus pneumoniae of the 23 capsular
type, each at a concentration of 50 ␮g/ml. A second injection was done at
day 14. Sera were harvested at days 8, 14, and 30. Control mice received
PBS alone. Antipolysaccharide humoral response was detected by ELISA.
Plates were coated with pneumo-23 diluted 1/50 in glycine buffer, and
bound Ig or bound IgM were detected with streptavidin-conjugated antiIg␬ or anti-IgM. The titer was defined as the dilution that showed an OD
of 400% of background.
For the type 2 T-independent response against trinitrophenyl (TNP)Ficoll, mice were injected i.p. with 100 ␮g of TNP-Ficoll, without adjuvant, in PBS, at days 0 and 14. Control mice received PBS alone. Sera were
collected at days 14 and 30. Anti-TNP-Ficoll response was measured by
ELISA. Plates were coated with TNP-Ficoll (10 ␮g/ml) and specific bound
Ig was detected with streptavidin-conjugated anti-Ig␬. The titer was defined as the dilution that showed an OD of 300% of background.
Rosetting with bromelain-treated RBC (Br-RBC)
Enrichment in B lymphocytes specific for Br-RBC was performed as described previously (26, 29, 30). Briefly, nucleated cells from blood of female F1 FVB/xid mice were separated from RBC on a Lymphoprep (Axis,
Oslo, Norway) solution. RBC were treated for 45 min at 37°C with Br (10
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FIGURE 1. B-1 lymphocyte expansion in IL-9-transgenic (Tg) xid mice. Peritoneal cells from groups of five mice from 8 to 10 wk old were harvested
by peritoneal washout and stained with anti-IgM and anti-Mac-1. A, FACS analysis of one representative mouse per group. B, Total peritoneal IgM⫹Mac-1⫹
B-1 cell numbers, with results expressed as mean and SEM. xid ⫹ corresponds to male mice with the xid mutation on their single X chromosome. xid⫺
corresponds to female mice heterozygous for the xid mutation. This B-1 B lymphocyte expansion was confirmed in ⬎20 mice tested.
The Journal of Immunology
xid mutation. As expected, male xid mice showed no detectable
IgM⫹Mac-1⫹ B-1 cell population in the peritoneal cavity. By contrast, as shown in Fig. 1, IL-9-transgenic male xid mice showed
similar numbers of IgM⫹Mac-1⫹ B-1 cells as female heterozygous
F1 mice (between 10 and 15% of total peritoneal cells). B-1 cell
numbers were further increased in IL-9-transgenic female mice
(between 35 and 45% of all peritoneocytes). To confirm that this
population corresponds to B-1 cells, we analyzed the surface markers of peritoneal lymphocytes expanded by IL-9 in xid mice. As
shown in Fig. 2, the population induced by IL-9 was IgMhigh,
IgDlow, Mac-1⫹, and CD23⫺, corresponding to a bona fide B-1
lymphocyte population.
In FVB normal mice, IL-9 induces a preferential, but not specific, expansion of B-1b lymphocytes (26). As expected, in xid
heterozygous female mice, both B-1a and B-1b cell numbers were
increased by IL-9 (3.7-fold for B-1a and 10.4-fold for B-1b cells).
By contrast, in xid mice, all the IgM⫹Mac-1⫹ B-1 cells induced by
IL-9 were CD5⫺ (Fig. 3). Thus, in xid mice, IL-9 induces the
expansion of a B-1b lymphocyte population, but has no effect on
the B-1a subset.
mg/ml; Sigma-Aldrich, St. Louis, MO) and washed with PBS. Peritoneocytes from three to seven mice per group were collected in Hanks’ medium
supplemented with 3% decomplemented FBS, washed, and resuspended at
10 ⫻ 106/ml. Nine hundred microliters of cell suspension was incubated
with 100 ␮l of a 1/10 Br-RBC solution and centrifuged at 580 ⫻ g for 5
min. Cells were resuspended at 4°C on a rotor and RBC-bound cells were
harvested on a 40/70% Percoll (Amersham, Arlington Heights, IL) gradient. RBC were lysed with NH4Cl and cells were counted and stained for
cytoflurometry analysis as described above.
In vitro B cell culture
Peritoneocytes were harvested from groups of three to seven mice in
Hanks’ medium supplemented with 3% FCS, washed, and suspended at
5 ⫻ 106/ml. Cells were labeled with FITC-anti-B220 (RA3-3A1, provided
by H. Bazin) and biotinylated Abs against Mac-1 followed by PE-conjugated streptavidin. B-1 cells were sorted as B220⫹Mac1⫹ cells on a FACSCalibur cell sorter and peritoneal B-2 cells as B220⫹Mac1⫺ cells. Cells
were suspended in RPMI 1640 supplemented with 10% FCS, 0.24 mM
asparagine, 1.5 mM glutamine, 0.55 mM arginine, and 50 ␮M 2-ME and
put in sterile 96-well tissue culture plates (105 or 2 ⫻ 104 cells in 200
␮l/well; Nunc). In duplicates, cells were treated with IL-4 (200 U/ml, produced in our laboratory), IL-5 (10 ng/ml), and LPS from Salmonella minnesota (5 ␮g/ml; Sigma-Aldrich) or left in control medium alone. After 7
days, culture supernatants were harvested, and total Ig secretion was measured by ELISA as described above.
Results
IL-9 overexpression restores a B-1 lymphocyte population in
xid mice
Since IL-9 was shown to induce B-1 cell expansion in mice, we
wondered whether this cytokine could compensate for the lack of
B-1 cells in xid mice. We therefore generated IL-9-transgenic xid
mice by crossing female CBA/N (xid) mice with male IL-9-transgenic mice. All male F1 mice inherited the xid mutation and one
allele of the IL-9 transgene. Female F1 mice, in which the xid
mutation was compensated by a nonmutated X chromosome, were
used as control IL-9-transgenic mice. To obtain nontransgenic F1
control mice of the same genetic background, we crossed female
CBA/N (xid) mice with male FVB mice. Male F1 mice inherited
the xid phenotype, while female mice were heterozygous for the
FIGURE 3. All IL-9-induced B-1 lymphocytes are CD5⫺. Peritoneal
cells from 8- to 10-wk-old mice were stained with anti-IgM, anti-Mac-1,
and with or without anti-CD5. IgM⫹Mac-1⫹ B-1 cells were gated and
represented as a histogram plot for the CD5 staining (black line). The gray
histogram represents control fluorescence of CD5 staining. Results of analysis of 3 ⫻ 104 cells are shown for one representative individual from each
group of five mice. Similar results were obtained in two independent
experiments.
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FIGURE 2. Cell surface markers of IL-9-induced B-1 lymphocytes in
xid mice. Peritoneal cells from five mice per group were stained with antiIgM and anti-Mac-1, anti-IgM and anti-CD23, or anti-IgD and anti-IgM.
FACS analysis was performed on lymphocytes, gated as small and nongranular cells. One FACS analysis representative of the other individuals of
the group is shown. Mice used in this experiment were 8 to 10 wk old.
Similar results were obtained in two independent experiments. Tg,
Transgenic.
6103
6104
B-1b LYMPHOCYTE EXPANSION IN IL-9-TRANSGENIC xid MICE
dicate that the failure to reconvey natural IgM concentrations was
not due to a defect in Ig production or differentiation.
IL-9-induced xid B-1b cells do not restore the capacity to mount
a T15 Id nor antipolysaccharide humoral response
FIGURE 4. Natural IgM levels in naive mice. Total IgM was measured
by ELISA in sera from 6- to 8-wk-old naive mice. Results are expressed in
micrograms per milliliter as mean and SEM (n ⫽ 5). Statistical analysis
was performed by an unpaired Student t test. Similar results were obtained
in two independent experiments. Tg, Transgenic.
IL-9-induced xid B-1b cells do not restore natural IgM
production
FIGURE 5. In vitro Ig secretion by B lymphocytes from IL-9-transgenic
xid mice. B-1 and B-2 lymphocytes from IL-9-transgenic xid mice were
sorted by FACS. Two ⫻ 104 cells were stimulated in vitro with or without
LPS, IL-4, and IL-5. After 7 days, total Ig was measured by ELISA in the
supernatant. Results are represented as mean and SEM. This experiment
was reproduced with similar results.
FIGURE 6. Anti-PC and T15 Id humoral response. A, Five mice per
group were immunized i.p. with PC- KLH in CFA and boosted at day 15
with PC-KLH in IFA. Three control mice per group received CFA and IFA
alone. At day 30, serum anti-PC Ig was measured by ELISA as described
in Materials and Methods. B, T15 Id IgM were measured by ELISA at day
15 before the boost immunization. Results are represented as mean and
SEM. Mice were 10 to 12 wk old. This experiment was reproduced three
times with similar results. Tg, Transgenic.
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One of the main functions of B-1 cells is to produce a significant
amount of natural serum IgM, thereby serving as a first line of
defense against systemic viral and bacterial infection (9, 31). Because IL-9-transgenic xid mice have normal numbers of B-1 cells,
we wondered whether this could restore the natural seric IgM
level. As illustrated in Fig. 4, serum IgM concentrations in naive
IL-9-transgenic xid mice were similar to those of nontransgenic xid
mice and were much lower than in control non-xid mice. This
difference cannot be explained by the gender of the F1 mice, because comparable seric IgM concentrations were found in male
and female xid, FVB, and Tg5 mice (data not shown). This experiment indicates that B-1b cells induced by IL-9 in xid mice are not
able to restore normal production of natural IgM.
To demonstrate that these xid B-1b cells were nevertheless able
to secrete Igs, we sorted B-1 (B220⫹Mac-1⫹) and B-2 (B220⫹
Mac-1⫺) lymphocytes from the peritoneal cavity of IL-9-transgenic xid mice and stimulated these cell populations in vitro with
IL-4, IL-5, and LPS. As shown in Fig. 5, xid B-1b cells showed a
similar in vitro Ig production to that of B-2 cells. FACS analysis
of in vitro-activated xid B-1b cells showed that all cells remained
CD5⫺ (data not shown), indicating that the production of Ig was
not associated to a cytokine-driven terminal in vitro differentiation
of this population into B-1a cells. Taken together, these data in-
The humoral response against PC, the immunodominant epitope
found on the surface of a number of microorganisms, is dominated
by the T15 Id. Abs with this Id are specifically produced by the B-1
lymphocyte subset (32) and play an important role in the protection against S. pneumoniae (8, 33). As previously described (34),
xid mice are able to mount an Ig response to PC-KLH, but fail to
produce T15 Id Abs. Although IL-9 expression restored normal
numbers of B-1 cells in xid mice, it failed to reconvey the T15 Id
response (Fig. 6). This difference cannot be explained by the incapacity of male mice to produce T15 Id Abs, because male
FVB/N and male Tg5 mice were able to secrete such Abs (data not
shown). Thus, IL-9-induced B-1b cells were not able to secrete
T15 Id Abs in response to PC immunization.
To test a more physiological model, we investigated whether
IL-9 could restore the immune humoral response of xid mice
against S. pneumoniae polysaccharides contained in the
pneumo-23 vaccine. It was shown before that B-1a lymphocytes
were important for this response (15) and that xid mice failed to
respond (25). As shown in Fig. 7, the IgM primary response to S.
pneumoniae vaccine was absent in male F1 FVB/xid mice and was
not restored by IL-9 overexpression, although IL-9 increased the
The Journal of Immunology
6105
Table I. IL-9 does not increase numbers of anti-Br-RBC xid B-1 cells
Total B-1 cellsa
xid IL-9 Tg
(⫻10⫺3)
Male FVB/xid
Male Tg/xid
Female FVB/xid
Female Tg/xid
⫹
⫹
⫺
⫺
⫺
⫹
⫺
⫹
10.3
1041.5
799.6
4390.0
Rosetting B-1 cellsb
(⫻10⫺3)
1.5
10.0
166.8
319.7
a
Peritoneal cells from three to seven mice per group were harvested by peritoneal
washout, counted, and stained with anti-IgM and anti-Mac-1 to assess the number of
B-1 cells per mouse.
b
Nine million peritoneal cells from different groups of mice were incubated with
Br-RBC. Anti-Br-RBC lymphocytes were enriched by a Percoll gradient centrifugation as described in Materials and Methods, counted, and stained with anti-IgM and
anti-Mac-1 to assess the number of anti-Br-RBC B-1 cells per mouse.
FIGURE 7. Anti-pneumo-23 humoral response. Five mice per group
were immunized i.p. with pneumo-23 in PBS. Three control mice received
PBS alone. Pneumo-23-specific IgM were measured by ELISA after 8
days. Results are represented as mean and SEM. Mice were 10 to 12 wk of
age. Similar results were obtained in two independent experiments. Tg,
Transgenic.
IL-9-induced xid B-1b cells do not share the classical B-1
repertoire
One possible explanation for the absence of a classical B-1 humoral response in the IL-9-transgenic xid mice might be that these
B-1b cells would have similar specificities as B-1a cells but would
fail to secrete Ig because of the xid mutation. If this hypothesis is
correct, surface IgM presented on the IL-9-induced xid B-1b cells
should share the biased repertoire of classical B-1 lymphocytes.
Five to 15% of B-1 cells recognize PtC, an epitope that is unmasked by the proteolytic enzyme Br on RBC (35). B lymphocytes
specific for Br-RBC can be enriched by rosetting and belong to the
FIGURE 8. Anti-TNP-Ficoll humoral response. Ten- to 12-wk-old mice
were immunized twice with TNP-Ficoll in PBS (days 1–15, n ⫽ 3). Two
mice per group received PBS alone. Anti-TNP-Ficoll Ig were measured by
ELISA after 30 days. Results are represented as mean and SEM. This
experiment was reproduced three times with similar results.
Discussion
In this report, we took advantage of the ability of IL-9 to expand
peritoneal B-1 lymphocytes in vivo to try to restore this population
in xid mice. By crossing IL-9-transgenic mice and xid mice, we
could generate xid mice that had normal B-1 cell numbers because
of a specific expansion of B-1b cells. B-1b lymphocytes have been
designated as the sister population of B-1a cells because they share
many characteristics with the latter B cell subset. The anatomical
location, phenotypic characteristics, self-replenishment capability,
and sensitivity to feedback inhibition clearly place them within the
B-1 lineage (14).
Interestingly, IL-9-induced xid B-1b cells, while still able to
secrete Ig in vitro, did not reconvey the functional characteristics
of B-1 cells, namely, natural IgM production, T15 Id Ig response,
and anti-polysaccharide Ab secretion. These observations indicate
that B-1b cells are functionally different from B-1a cells. In addition, we showed that these B-1b cells do not bind Br-RBC, demonstrating that they do not have a biased repertoire such as that of
B-1a cells. Thus, it is likely that the difference in function is related
to different Ag specificities associated with these two populations.
An alternative interpretation of these results is that IL-9-driven
B-1b cells are not representative of native B-1b lymphocytes. This
hypothesis is supported by cell transfer experiments, in which
B-1b cells could restore natural IgM levels (3), and by the observation that at least some B-1b cells appeared to bind to PtC liposomes (11). This raises the hypothesis that at least a subpopulation
of B-1b cells behaves like B-1a cells. However, in line with our
data, splenectomy, that causes a specific loss of the B-1a cell subset, while B-1b cell numbers remain unaffected, was followed by
the incapacity to mount an antipolysaccharide immune response,
which was reversed by the transfer of B-1a cells (15), suggesting
that both populations are not functionally redundant.
The origin of B-1 cells is still a matter of controversy (8, 14).
According to the lineage hypothesis, certain early B cell precursors
are committed to become B-1 cells (36). These progenitors might
be further subdivided in B-1a and B-1b progenitors. By contrast,
the differentiation hypothesis implies that every B cell progenitor
can acquire B-1 characteristics upon positive selection through
surface Ig (8). Experiments supporting this hypothesis exclusively
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response in non-xid mice. Similar results were obtained for the
specific Ig primary response (day 15) and secondary (day 30) response (data not shown). IL-9-induced B-1b cells were thus unable
to restore this antipolysaccharide humoral response.
The specific humoral response to TNP-Ficoll, a classical type 2
T-independent Ag, is mainly mediated by splenic marginal zone B
cells rather than by B-1 cells (8), but is also defective in xid mice
(25). We immunized our different groups of mice with TNP-Ficoll
without adjuvant at days 1 and 15. IL-9-induced B-1b cells did not
restore the capacity to respond to this T-independent Ag, neither
for the day 14 primary response (data not shown) nor for the day
30 secondary response, as shown in Fig. 8.
B-1 subset (26, 30). We applied the same system on our different
groups of mice. As shown in Table I, IL-9 overexpression in xid
mice restored normal numbers of the B-1 cell population but not of
the Br-RBC-specific B-1 lymphocytes. Whereas 20.9% of B-1
cells from control nontransgenic female mice were pulled down by
Br-RBC, ⬍1% of IL-9-transgenic xid B-1 cells were found in the
rosetting fraction. These results indicate that IL-9-induced xid
B-1b cells do not share the typical repertoire of B-1a cells.
6106
B-1b LYMPHOCYTE EXPANSION IN IL-9-TRANSGENIC xid MICE
Acknowledgments
We are grateful to André Tonon for expert technical assistance for FACS
analysis and sorting, to Dr. Guy Warnier for the breeding of mice, and to
Dr. Bernard Lauwerys for helpful discussions.
References
1. Hayakawa, K., R. R. Hardy, D. R. Parks, and L. A. Herzenberg. 1983. The “Ly-1
B” cell subpopulation in normal, immunodefective, and autoimmune mice.
J. Exp. Med. 157:202.
2. Kantor, A. B., and L. A. Herzenberg. 1993. Origin of murine B cell lineages.
Annu. Rev. Immunol. 11:501.
3. Herzenberg, L. A., A. M. Stall, P. A. Lalor, C. Sidman, W. A. Moore, and
D. R. Parks. 1986. The Ly-1 B cell lineage. Immunol. Rev. 93:81.
4. Kroese, F. G., and N. A. Bos. 1999. Peritoneal B-1 cells switch in vivo to IgA and
these IgA antibodies can bind to bacteria of the normal intestinal microflora.
Curr. Top. Microbiol. Immunol. 246:343.
5. Hardy, R. R., C. E. Carmack, S. A. Shinton, R. J. Ribblet, and K. Hayakawa.
1989. A single VH gene is used predominantly in anti-BrMRBC hybridomas
derived from purified Ly-1 B cells: definition of the VH11 family. J. Immunol.
142:3643.
6. Rothstein, T. L. 2002. Cutting edge commentary: two B-1 or not to be one.
J. Immunol. 168:4257.
7. Fagarasan, S., and T. Honjo. 2000. T-Independent immune response: new aspects
of B cell biology. Science 290:89.
8. Berland, R., and H. H. Wortis. 2002. Origins and functions of B-1 cells with notes
on the role of CD5. Annu. Rev. Immunol. 20:253.
9. Hayakawa, K., and R. R. Hardy. 2000. Development and function of B-1 cells.
Curr. Opin. Immunol. 12:346.
10. Martin, F., and J. F. Kearney. 2001. B1 cells: similarities and differences with
other B cell subsets. Curr. Opin. Immunol. 13:195.
11. Stall, A. M., S. Adams, L. A. Herzenberg, and A. B. Kantor. 1992. Characteristics
and development of the murine B-1b (Ly-1 B sister) cell population. Ann. NY
Acad. Sci. 651:33.
12. Kantor, A. B., A. M. Stall, S. Adams, L. A. Herzenberg, and L. A. Herzenberg.
1992. Differential development of progenitor activity for three B-cell lineages.
Proc. Natl. Acad. Sci. USA 89:3320.
13. Sen, G., H. J. Wu, G. Bikah, C. Venkataraman, D. A. Robertson, E. C. Snow, and
S. Bondada. 2002. Defective CD19-dependent signaling in B-1a and B-1b B
lymphocyte subpopulations. Mol. Immunol. 39:57.
14. Herzenberg, L. A. 2000. B-1 cells: the lineage question revisited. Immunol. Rev.
175:9.
15. Wardemann, H., T. Boehm, N. Dear, and R. Carsetti. 2002. B-1a B cells that link
the innate and adaptive immune responses are lacking in the absence of the
spleen. J. Exp. Med. 195:771.
16. Dalwadi, H., B. Wei, M. Schrage, T. T. Su, D. J. Rawlings, and J. Braun. 2003.
B cell developmental requirement for the ␥␣2 gene. J. Immunol. 170:1707.
17. Nishimura, H., N. Minato, T. Nakano, and T. Honjo. 1998. Immunological studies on PD-1 deficient mice: implication of PD-1 as a negative regulator for B cell
responses. Int. Immunol. 10:1563.
18. Zou, X., C. Ayling, J. Xian, T. A. Piper, P. J. Barker, and M. Bruggemann. 2001.
Truncation of the ␮ heavy chain alters BCR signalling and allows recruitment of
CD5⫹ B cells. Int. Immunol. 13:1489.
19. Hinkley, K. S., R. J. Chiasson, T. K. Prior, and J. E. Riggs. 2002. Age-dependent
increase of peritoneal B-1b B cells in SCID mice. Immunology 105:196.
20. Riggs, J., K. Howell, B. Matechin, R. Matlack, A. Pennello, and R. Chiasson.
2003. X-chromosome-linked immune-deficient mice have B-1b cells. Immunology 108:440.
21. Scher, I., A. Ahmed, D. M. Strong, A. D. Steinberg, and W. E. Paul. 1975.
X-linked B-lymphocyte defect in CBA/N mice. I. Studies of the function and
composition of spleen cells. J. Exp. Med. 141:788.
22. Rawlings, D. J., D. C. Saffran, S. Tsukada, D. A. Largaespada, J. C. Grimaldi,
L. Cohen, R. N. Mohr, J. F. Bazan, M. Howard, N. G. Copeland, et al. 1993.
Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID
mice. Science 261:358.
23. Gauld, S. B., J. M. Dal Porto, and J. C. Cambier. 2002. B cell antigen receptor
signaling: roles in cell development and disease. Science 296:1641.
24. Hayakawa, K., R. R. Hardy, and L. A. Herzenberg. 1986. Peritoneal Ly-1 B cells:
genetic control, autoantibody production, increased ␭ light chain expression. Eur.
J. Immunol. 16:450.
25. Scher, I. 1982. The CBA/N mouse strain: an experimental model illustrating the
influence of the X-chromosome on immunity. Adv. Immunol. 33:1.
26. Vink, A., G. Warnier, F. Brombacher, and J.-C. Renauld. 1999. IL-9-induced in
vivo expansion of the B-1 lymphocyte population. J. Exp. Med. 189:1413.
27. Renauld, J.-C., N. van der Lugt, A. Vink, M. van Roon, C. Godfraind,
G. Warnier, H. Merz, A. Feller, A. Berns, and J. Van Snick. 1994. Thymic
lymphomas in interleukin 9 transgenic mice. Oncogene 9:1327.
28. Vogel, L. A., T. L. Lester, V. H. Van Cleave, and D. W. Metzger. 1996. Inhibition of murine B1 lymphocytes by Interleukin-12. Eur. J. Immunol. 26:219.
29. Cunningham, A. J. 1974. Large numbers of cells in normal mice produce antibody components of isologous erythrocytes. Nature 252:749.
30. Poncet, P., F. Huetz, M.-A. Marcos, and L. Andrade. 1990. All VH11 genes
expressed in peritoneal lymphocytes encode anti-bromelain-treated mouse red
blood cell autoantibodies but other VH gene families contribute to this specificity.
Eur. J. Immunol. 20:1583.
31. Thurnheer, M. C., A. W. Zuercher, J. J. Cebra, and N. A. Bos. 2003. B1 cells
contribute to serum IgM, but not to intestinal IgA, production in gnotobiotic Ig
allotype chimeric mice. J. Immunol. 170:4564.
32. Masmoudi, H., T. Mota-Santos, F. Huetz, A. Coutinho, and P. A. Cazenave.
1990. All T15 Id-positive antibodies (but not the majority of VHT15⫹ antibodies)
are produced by peritoneal CD5⫹ B lymphocytes. Int. Immunol. 2:515.
33. Briles, D. E., M. Nahm, K. Schroer, J. Davie, P. Baker, J. Kearney, and
R. Barletta. 1981. Antiphosphocholine antibodies found in normal mouse serum
are protective against intravenous infection with type 3 Streptococcus pneumoniae. J. Exp. Med. 153:694.
34. Kenny, J. J., G. Guelde, R. T. Fischer, and D. L. Longo. 1994. Induction of
phosphocholine-specific antibodies in X-linked immune deficient mice: in vivo
protection against a Streptococcus pneumoniae challenge. Int. Immunol. 6:561.
35. Mercolino, T. J., L. W. Arnold, L. A. Hawkins, and G. Haughton. 1988. Normal
mouse peritoneum contains a large population of Ly-1⫹ (CD5) B cells that recognize phosphatidyl choline: relationship to cells that secrete hemolytic antibody
specific for autologous erythrocytes. J. Exp. Med. 168:687.
36. Hayakawa, K., R. R. Hardy, and L. A. Herzenberg. 1985. Progenitors for Ly-1 B
cells are distinct from progenitors for other B cells. J. Exp. Med. 161:1554.
37. Cong, Y. Z., E. Rabin, and H. H. Wortis. 1991. Treatment of murine CD5⫺ B
cells with anti-Ig, but not LPS, induces surface CD5: two B-cell activation pathways. Int. Immunol. 3:467.
38. Hayakawa, K., M. Asano, S. A. Shinton, M. Gui, D. Allman, C. L. Stewart,
J. Silver, and R. R. Hardy. 1999. Positive selection of natural autoreactive B cells.
Science 285:113.
39. Hayakawa, K., M. Asano, S. A. Shinton, M. Gui, L. J. Wen, J. Dashoff, and
R. R. Hardy. 2003. Positive selection of anti-Thy-1 autoreactive B-1 cells and
natural serum autoantibody production independent from bone marrow B cell
development. J. Exp. Med. 197:87.
40. Wortis, H. H., and R. Berland. 2001. Cutting edge commentary: origins of B-1
cells. J. Immunol. 166:2163.
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focused on B-1a cells and include the possible acquisition of CD5
expression by in vitro stimulation of splenic B cells with anti-IgM
(37) and the need for an interaction with autoantigen to generate
B-1a cells (38, 39). The absence of B-1 cells in xid mice might thus
result from the fact that the mutation of the Btk kinase attenuates
signals through B cell receptor and, consequently, positive selection by autoantigens (40). Our data clearly show that xid B-1b cells
can differentiate in conditions that do not allow B-1a differentiation and that these B-1b cells have a distinct repertoire from that of
B-1a cells. These xid B-1b cells can differentiate independently
from the positive selection that has been suggested for B-1a cells.
In line with the lineage hypothesis, such cells might differentiate
from a specific B-1b progenitor, as previously suggested from
transfer experiments (12). Alternatively B-1b cells might result
from a common B-1 progenitor that would differentiate into B-1a
cells upon positive selection or into B-1b cells in the absence of
selection. In such a scenario, B-1a cells would include mostly autoreactive B lymphocytes. In contrast to B-1b cells, expression of
CD5, which can act as a negative regulator of B cell receptor
signaling, could be essential to prevent inappropriate activation of
potentially armful B-1a cells.
In wild-type mice, IL-9 overexpression induced an expansion of
both B-1a and B-1b populations. Typically, in IL-9-transgenic female mice shown in Fig. 1, B-1a and B-1b cell numbers were
increased 3.7- and 10.4-fold, respectively, whereas splenic B-2
cells were not affected (data not shown). This differential effect of
IL-9 on the three subsets is compatible with the hypothesis that
IL-9 could act specifically on B-1, but not B-2 progenitors, and its
actual effect on B-1a and B-1b cell numbers would be further modulated by the putative selection process of B-1a cells.
Taken together, our results show that, upon IL-9 expression, a
B-1b cell population differentiates and expands in xid mice, but
does not recapitulate the functions and the repertoire of B-1a cells,
indicating that B-1b cells should not be considered a simple redundant sister population of B-1a cells and result from a unique
ontogenic pathway.