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The Journal of Immunology, 2004, 172: 6101-6106.
Copyright © 2004 by The American Association of Immunologists

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

Ludwig Institute for Cancer Research and Experimental Medicine Unit, Université de Louvain, Brussels, Belgium


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 CD5– 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 CD5+ 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 CD5+ B-1a lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The 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). 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 CD19-dependent 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{alpha}i2, PD-1, or µ H chain genes (16, 17, 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 one-third 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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, 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-9-transgenic 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 x 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 anti-IgM, 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 x 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 µl of anti-IgM (LOMM3, 5 µg/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 µl of 1% BSA in PBS. Plates were washed and 50 µl 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 µl of 1Step Turbo tetramethylbenzidine-ELISA (Perbio) for 15 min. Reaction was stopped with 50 µl 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 µg 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 µg 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 µg/ml) and detecting bound Ig with streptavidin-conjugated anti-{kappa} L chain (LOMK1, 2.5 µg/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 µg/ml) and streptavidin-conjugated anti-IgM (10 µg/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 µl of the pneumo-23 vaccine (Aventis Pasteur, Lyon, France) in 200 µl of PBS. Pneumo-23 contains polysaccharides of Streptococcus pneumoniae of the 23 capsular type, each at a concentration of 50 µg/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 anti-Ig{kappa} 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 µg 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 µg/ml) and specific bound Ig was detected with streptavidin-conjugated anti-Ig{kappa}. 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 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 x 106/ml. Nine hundred microliters of cell suspension was incubated with 100 µl of a 1/10 Br-RBC solution and centrifuged at 580 x 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 x 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 µM 2-ME and put in sterile 96-well tissue culture plates (105 or 2 x 104 cells in 200 µl/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 µg/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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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 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.



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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.

 


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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 anti-IgM 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.

 
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.



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  		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 x 104 cells are shown for one representative individual from each group of five mice. Similar results were obtained in two independent experiments.

 
IL-9-induced xid B-1b cells do not restore natural IgM production

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.



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  		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.

 
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 indicate that the failure to reconvey natural IgM concentrations was not due to a defect in Ig production or differentiation.



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  		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 x 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.

 
IL-9-induced xid B-1b cells do not restore the capacity to mount a T15 Id nor antipolysaccharide humoral response

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.



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  		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.

 
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 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.



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  		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.

 
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.



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  		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.

 
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 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.


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  		Table I. IL-9 does not increase numbers of anti-Br-RBC xid B-1 cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
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 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.


   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.


   Footnotes
 
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. Back

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: Jean-Christophe.Renauld{at}bru.licr.org Back

3 Abbreviations used in this paper: PtC, phosphatidylcholine; PC, phosphorylcholine; TNP, trinitrophenyl; Br, bromelain. Back

Received for publication October 28, 2003. Accepted for publication March 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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