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The Journal of Immunology, 2000, 164: 4586-4593.
Copyright © 2000 by The American Association of Immunologists

A VH11V{kappa}9 B Cell Antigen Receptor Drives Generation of CD5+ B Cells Both In Vivo and In Vitro1

Michael J. Chumley*,{dagger}, Joseph M. Dal Porto*, Susumu Kawaguchi{ddagger}, John C. Cambier*,{dagger}, David Nemazee§ and Richard R. Hardy2

* Division of Basic Sciences, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206; {dagger} Department of Immunology, University of Colorado Health Sciences Center, Denver, CO 80206; {ddagger} Department of Microbiology and Immunology, Shimane Medical University, Izumo, Shimane, Japan, § Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037; and Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
B lymphocytes can be divided into different subpopulations, some with distinctive activation requirements and probably mediating specialized functions, based on surface phenotype and/or anatomical location, but the origins of most of these populations remain poorly understood. B cells constrained by transgenesis to produce an Ag receptor derived from a conventional (B-2) type cell develop a B-2 phenotype, whereas cells from mice carrying a B-1-derived receptor acquire the B-1 phenotype. In this study transgenic enforced expression of a B cell receptor (µ/{kappa}) originally isolated from a CD5+ (B-1a) B cell generates B-1 phenotype cells in bone marrow cultures that show a distinctive B-1 function, survival in culture. Despite their autoreactivity, we find no evidence for receptor editing or that the paucity of B-2 cells is the result of tolerance-induced selection. Finally, Ca2+ mobilization studies reveal a difference between transgenic B-1 cells in spleen and peritoneal cavity, with cells in spleen much more responsive to anti-B cell receptor cross-linking. We discuss these results in terms of specificity vs lineage models for generation of distinctive B cell subpopulations.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Based on their characteristic features and anatomic location, B cells in the adult mouse can be divided into several subsets, such as B-1, B-2, marginal zone, transitional, germinal center, and memory, but the role of surface Ig in this differentiation is poorly understood (1, 2, 3, 4, 5, 6, 7, 8, 9). The majority of B cells in the spleen and lymph nodes are referred to as B-2, a population of small, long-lived, resting cells that expresses high levels of IgD and lacks detectable expression of the pan-T cell surface glycoprotein CD5. In contrast, the B-1 subset comprises about 5% of B cells in spleen, has lower levels of IgD, and is CD5+. In the adult animal these cells are enriched in certain anatomic locations, such as the peritoneal cavity and lamina propria (10), and show increased frequency of {lambda} light chain usage (11). In neonatal spleen, B-1 cells are relatively more abundant (12). Besides their phenotypic distinctions, B-1 cells are somewhat larger than B-2 cells and, when tested in vitro, differ from B-2 cells in their responsiveness to a number of stimuli (13, 14, 15, 16). Studies indicate that B-1 cells also contribute disproportionately to the levels of serum IgM (17, 18, 19).

Comparison of expressed Ag receptors of B-1 and B-2 cells (BCR)3 suggests that these cell types use a partly overlapping repertoire of gene segments, but may differ in Ag specificity. Many B-1 cells appear to have germline-encoded self-reactive BCR (20, 21, 22, 23, 24, 25, 26), a property that may be specifically selected (27, 28, 29, 30, 31, 32). Although B-1 cells are subject to tolerance (25, 33, 34, 35), their threshold for tolerance induction may differ from that of B-2 cells, which are highly tolerance sensitive even at low binding affinities (36, 37, 38).

Phosphatidylcholine (PtC)-reactive Ab specificities are highly represented in the normal B-1 cell population, where they represent 5–10% of the total. They were initially identified as conferring reactivity to bromelain-treated mouse RBC, and such Abs predominantly use VH11 or VH12 genes with restricted CDR3 structure (30, 32, 39, 40, 41, 42). Interestingly, in B-1 cells VH11 and VH12 heavy chains pair predominantly with V{kappa}9 and V{kappa}4 light chains, respectively.

There has been much debate concerning the origins of B-1 and B-2 cells. Several lines of evidence suggest that B-1 cells are derived during a distinctive fetal/neonatal phase of B lymphopoiesis. For example, in contrast with adult bone marrow (BM) pro-B cells, fetal pro-B cells do not express TdT or precursor lymphocyte regulated myosin-like light chain, show delayed expression of class II molecules (I-A and I-E), and, when transferred to adoptive recipients, mature primarily to the B-1 phenotype (43, 44, 45, 46, 47, 48, 49, 50, 51).

On the other hand, adult BM-derived B cells with a B-2 phenotype can be induced to express a B-1 phenotype when appropriately stimulated (52, 53, 54, 55, 56). Furthermore, B-1 cells appear uniquely sensitive to mutations that alter BCR signaling, implicating such signaling in their generation or survival (3, 26, 57, 58, 59). Such studies have led to the proposal of lineage vs specificity models for B-1 cell generation (60, 61), although some of these differences may now be reconciled by postulating that fetal and adult B cell precursors represent developmental lineages that differ in their expressed Ag receptor repertoires and, hence, their specificities (53, 62, 63). This can occur through the rearrangement process, which is biased in fetal cells compared with that in adult BM precursors as a result of the absence of TdT expression (49, 60, 64, 65, 66), nonrandom V gene recombination, and altered heavy chain-selective mechanisms (63).

However, it remains to be determined whether differences in early stages of B cell development completely account for the fetal-biased generation of B-1 B cells or, instead, whether differences in BCR selection/tolerance also play a role. Therefore, in this report we test whether B cells with a transgenic B-1 BCR experience tolerance in BM differently from transgenic B-2 BCR B cells. Our work was prompted by the general observation that most Ig transgenic mice are deficient in B-1 cells, and that B-1 cells present in such mice usually express endogenous Ig chains (67, 68, 69). In contrast, in two Ig-Tg mice expressing B-1 B cell-derived receptors reactive to PtC, B-1 B cells are abundant and express the transgenic specificity, whereas B-2 cells are less abundant (63, 69). Specifically, we have followed the fate of B cells generated in the BM of VH11/V{kappa}9 Tg mice, comparing it with that in 3-83 µ{delta} (anti-H-2Kk,b) Ig-transgenic mice (70, 71). In this VH11V{kappa}9 transgenic model, we find no evidence for immune tolerance or receptor editing, so that self-reactive B cells are produced and populate a peripheral B-1 pool. These cells show persistence in culture, a function shared with normally generated B-1 B cells. Furthermore, these cells are not anergic, producing large amounts of serum Ig and showing a calcium flux in response to receptor cross-linking.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

3-83 µ{delta} transgenic mice were maintained on the autoantigen-free, nondeleting (H-2Kd) background of B10.D2nSn/J (The Jackson Laboratory, Bar Harbor, ME) or on the autoantigen-containing, deleting background (H-2Kk) of B10.BR (provided by Pippa Marrack, National Jewish Medical and Research Center, Denver, CO). VH11V{kappa}9 transgenic mice, originally on the CB17 background, were backcrossed at least six times on the B10.D2nSn/J background. Mice were housed and bred at the National Jewish Medical and Research Center Biological Research facility and were used at 8–12 wk of age.

Cell culture

BM cells were isolated from aseptically removed femurs and grown in vitro as previously described (72, 73). Briefly, BM cells were depleted of RBCs and cultured at a concentration of 2 x 106 cells/ml in IMDM (Life Technologies, Gaithersburg, MD) supplemented with 10% FCS (Gemini Bio-Products, Calabasas, CA), 5 x 10-5 M 2-ME, 1% sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37°C with 7% CO2. Primary BM cultures were supplemented with 50–100 U/ml of rIL-7, which preferentially expanded IL-7-responsive B cell precursors, and after 5–6 days the cultures contained >98% B220+ cells (72). Where noted, BM cells were incubated at 4°C for 1 h with rat anti-mIgM-biotin (b-7-6) precoupled to M-280 streptavidin magnetic beads (Dynal, Lake Success, NY), and the IgM+ cells were removed magnetically. Purity was assessed by immunofluorescence, and in all cases the IgM- population was >99% pure. Twenty-four-hour and 13-day cultures (secondary cultures) of BM were performed without the addition of IL-7. Splenic cells were depleted of erythrocytes using buffered ammonium chloride. B cells were purified by T cell depletion with anti-Thy-1 mAb and rabbit complement (Cedarlane, Westbury, NY), followed by density separation ({rho} = 1.066) through Percoll (Amersham-Pharmacia, Piscataway, NJ). Purified B cells were cultured as described above. Peritoneal cells were removed by aseptic injection of 10 ml of IMDM (Life Technologies) into the peritoneal cavity followed by withdrawal of the peritoneal exudate. Where noted, 10 µg/ml LPS (Life Technologies), 10 µg/ml rat anti-mIg{kappa} (187.1) mAb (a gift from Dr. J. Cambier, National Jewish Medical and Research Center), 10 µg/ml rat anti-azophenylarsenate (5 Ci) mAb (a gift from Dr. L. Wysocki, National Jewish Medical and Research Center), or 10 µg/ml PtC-containing liposomes (24) were added to the cultures.

Flow cytometry

mAbs used for mouse cell staining included anti-B220, RA3–6B2; anti-CD23, B3B4; anti-CD43, S7; anti-CD5, 53-7; anti-IgMa, RS3.1; anti-Id for 3-83, 54.1 (70); and anti-Id for VH11, RidA (33). Fluorescent reagents were either purchased (PharMingen, San Diego, CA) or labeled as previously described (4). Polyclonal FITC-anti-Ig {lambda} light chain was purchased from Southern Biotechnology (Birmingham, AL). Biotinylated Abs were visualized with streptavidin-Tri-Color (Caltag, South San Francisco, CA) for three-color analysis on a FACSCalibur (Becton Dickinson, San Jose, CA) or with Texas Red-avidin for four-color analysis on a dual laser dye laser FACStarPlus (Becton Dickinson). Forward and side scatter gates were adjusted to include only nucleated viable cells.

Cell survival

Before all analyses of cell survival, cells were counted on a hemocytometer using trypan blue exclusion, and the frequency of B220+ cells was determined by immunofluorescence. Cultures were initiated at 2 x 106 cells/well in six-well culture plates. After incubation, wells were harvested and counted, and the frequency of B220+ cells was determined by immunofluorescence. Cell survival is reported for B220+ cells as a percentage of their initial numbers.

Carboxyfluorescein diacetate succimidylester (CFSE) labeling

Cells treated with CFSE (Molecular Probes, Eugene, OR) were labeled as previously described (74). Briefly, a stock solution of 5 mM CFSE in DMSO was diluted 1/10 in serum-free IMDM prewarmed to 37°C. B cells were diluted to 50 x 106 cells/ml and incubated with 10 µl of diluted CFSE/ml at 37°C for 10 min. Labeled cells were washed twice with IMDM and placed in culture as described above.

BM chimeras

BM chimeras were prepared as previously described (75), but with the following alterations. Ten- to 12-wk-old donor femurs were removed aseptically, and B cells were isolated as described above. The purified cells were then incubated with M280-streptavidin magnetic beads (Dynal) conjugated to anti-B220-biotin (RA3-6B2) and anti-CD19-biotin (1D3, PharMingen) mAbs for 45 min at 4°C. Bound cells were removed magnetically, and the remaining mixture was assessed for purity (B220-, CD19-) by immunofluorescence. A purity level of >99% B220-, CD19- was usually attained following just one round of isolation, but in some cases the process was repeated to achieve this level of purity. The viable cell concentration following the removal of B220+, CD19+ cells was adjusted to 5 x 107 cells/ml in sterile HBSS (Life Technologies), and 0.1 ml was injected i.v. per irradiated recipient. Recipients were age-matched B10.D2nSn/J mice that received 960 rad whole-body irradiation at least 1 h before BM transfer. Recipient mice were analyzed 4–6 wk after transfer.

Detection of RAG-2 mRNA

mRNA was isolated from 3–5 x 106 freshly isolated or cultured BM cells using the Oligotex Direct mRNA isolation kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). First-strand cDNA synthesis was conducted by random priming of 1 µg of isolated mRNA using a kit (Ready-to-Go, Amersham-Pharmacia) in a volume of 20 µl. PCR amplification of 1 µl of the resulting cDNA was conducted as previously described (72) using primers specific for CD19 and RAG-2. PCR reactions were electrophoresed in 1% agarose, transferred to a Zeta-Probe membrane (Bio-Rad, Richmond, CA), and hybridized overnight with 32P-labeled CD19- and RAG-2-specific probes. Autoradiography was performed as described previously (72).

ELISA

Ig concentrations were measured as previously described (76). Briefly, Immulon 2HB polystyrene plastic microtiter plates (Dynex Technologies, Chantilly, VA) were coated with rat anti-mIgM (b-7-6), washed, and blocked. Sera (diluted in PBS supplemented with 1% BSA) were incubated for 3 h at 25°C, and bound Ig was detected with biotinylated rat anti-mIgM (Ak2) or rat a-mIgMa (RS3.1) and streptavidin-peroxidase (Sigma, St. Louis, MO). Standard curves for the assay were generated using COS line D1 supernatant. Absorbance was measured on a Bio-Rad model 2225 ELISA plate reader (Bio-Rad) and analyzed using the Microplate Manager program.

Analysis of calcium mobilization

Splenic and peritoneal B cells were isolated and purified as previously described and subsequently loaded with indo-1/AM (Molecular Probes) for 45 min at 37°C. Cells were washed twice, resuspended at a concentration of 106 cells/ml in IMDM and 5% FCS, and stimulated with F(ab')2 rabbit anti-mouse Ig (Zymed, San Francisco, CA). Mean fluorescence was evaluated using an Ortho model 50H flow cytometer (Ortho, Westwood, MA) and an appended data acquisition system and MultiTime software (Phoenix Flow Systems, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
VH11V{kappa}9 transgenic B cells have a B-1, and 3-83 µ{delta} B cells have a B-2 phenotype

The cell surface marker phenotype of B cells isolated from VH11V{kappa}9, 3-83 µ{delta} Ig-Tg, and nontransgenic mice, as analyzed by flow cytometry, is shown in Fig. 1Go. The majority of B220+ B cells in 3-83 and wild-type spleens have a conventional B-2 phenotype: B220high, CD23+, CD43-, CD5-. In contrast, splenic B cells of the VH11V{kappa}9 mouse have a distinctive B-1 phenotype: B220low, CD23-, CD43+, CD5+. The peritoneal cavity B cell compartment, which in the nontransgenic B10.D2 is primarily composed of B-1 cells, contains ~25-fold fewer B cells in the 3-83 mouse, the majority of which are 3-83 id- (Table IGo). In contrast, the VH11V{kappa}9 Tg mouse peritoneal cavity contains 7-fold more B cells than the nontransgenic littermate, and nearly all are B-1 cells that express the VH11V{kappa}9 receptor.



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  		FIGURE 1. Phenotype of 3-83 and VH11V{kappa}9 Ig-Tg peripheral B cells. Isolated splenic cells were analyzed by flow cytometry for the expression of various cell surface markers. Numbers above each gate represent the percentage of B lymphocyte-gated cells. Cells from nontransgenic, B10. D2 strain mice were analyzed as a control. Plots shown are representative of three 3-83 mice and five VH11V{kappa}9 mice analyzed.

 

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  		Table I. B cell populations in spleen and peritoneal cavity of VH11V{kappa}9- and 3-83µ{delta}-Tg mice1

 
VH11V{kappa}9 Ig-transgenic B cells exhibit enhanced in vitro survival

To further test the phenotype of the transgenic B cells, we measured the ability of isolated VH11V{kappa}9, 3-83, or nontransgenic splenic B cells to survive in vitro, because unlike B-2 cells, B-1 cells survive in vitro for extended periods (77). After 7 days in culture in standard medium containing serum but no added mitogens, >50% of the VH11V{kappa}9+ B cells were viable (Fig. 2GoA), whereas nearly all the 3-83+ or nontransgenic splenic B cells died. The enhanced in vitro survival of the VH11V{kappa}9+ splenic B cells was not associated with proliferation, as CFSE-labeled VH11V{kappa}9+ cells maintained their label during 5 days of culture (Fig. 2GoB). When the mitogen LPS was included in the culture medium, VH11V{kappa}9+ splenic B cells were capable of proliferating, as demonstrated by the loss of CFSE fluorescence. These data demonstrate that VH11V{kappa}9 Tg splenic B cells acquire not only the characteristic cell surface phenotype of B-1 cells, but also a characteristic functional property, cell culture survival, while 3-83+ splenic B cells display a B-2-like phenotype and functional response.



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  		FIGURE 2. Enhanced in vitro survival of purified VH11V{kappa}9+ B cells. A, Splenic B cells purified from 3-83 and VH11V{kappa}9 Ig-transgenic mice were cultured for 7 days, and B220+ cell survival was assessed. SEs for six to nine separate experiments are shown. B, VH11V{kappa}9 splenic B cells were prestained with CFSE and cultured in the presence or the absence of LPS (10 µg/ml) for the indicated number of days. Numbers in each histogram represent the median CFSE fluorescence intensity.

 
VH11V{kappa}9 Ig-transgenic precursors in adult BM generate B-1 B cells after transfer

To test whether the B-1 B cells in the VH11V{kappa}9 Tg mouse could be generated from BM, we transferred B220-/CD19- BM cells from 10- to 12-wk-old VH11V{kappa}9 or 3-83 mice into lethally irradiated B10.D2 hosts and then analyzed the phenotype of reconstituted peripheral B cells 4–6 wk later. Donor-derived cells were identifiable by transgene Id (Fig. 3GoA). Recipients of VH11V{kappa}9 and 3-83 cells contained similar numbers of donor-derived splenic B cells, but their cell surface phenotypes differed, with VH11V{kappa}9 BM-reconstituted mice exhibiting a B-1 phenotype and 3-83 BM recipients exhibiting a B-2 phenotype (data not shown). In mixed VH11V{kappa}9/3-83 BM transfers, B cells expressing either the VH11V{kappa}9 BCR or the 3-83 BCR were produced (Fig. 3GoA) and maintained their respective B-1-like and B-2-like phenotypes (Fig. 3GoB), indicating that VH11V{kappa}9+ B-1 cells do not suppress the development of 3-83 B-2 cells and vice versa. Furthermore, splenic B cells from VH11V{kappa}9 BM-reconstituted mice had an extended life span in vitro, whereas B cells of 3-83-reconstituted mice did not (Fig. 3GoC). Thus, in BM chimeras, splenic B cell surface and survival phenotypes recapitulated those of transgenic donor mice.



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  		FIGURE 3. Analysis of splenic B cells in radiation chimeras reconstituted with adult BM of VH11V{kappa}9, 3-83, or mixed (VH11V{kappa}9 and 3-83) Tg mice. Recipients were irradiated B10.D2 mice. Mixed chimeras received an equal dose of both donor BM types. Chimeras were analyzed at 4–6 wk posttransfer. Results are representative of three separate experiments. A, Transgene-encoded BCR expression was analyzed with idiotypic Abs in spleen cells of the following chimeras: VH11V{kappa}9->B10. D2, 3-83 ->B10. D2, and (VH11V{kappa}9 + 3-83)->B10. D2. The binding of the anti-Ids defined two analysis gates, A and B, used below. B, Splenic B cells of the mixed BM chimera (gated as indicated above) were further analyzed for expression of CD43, CD23, and CD5. C, In vitro survival of splenic B cells from the indicated chimeras was determined as described in Fig. 2Go. Error bars represent the SEs from three different chimera experiments totaling seven recipient mice in each transfer group.

 
In vitro differentiation of adult VH11V{kappa}9+ BM generates B cells that have a B-1 phenotype and enhanced survival

To probe the differentiation of 3-83 and VH11/V{kappa}9 BM B cells in more detail, BM precursors were grown in IL-7-containing medium, which allows the expansion of Ig transgene-positive cells and, when Ag is present, the investigation of tolerance mechanisms. After 5 days of culture in the presence of IL-7 (primary cultures) B cell numbers increased substantially (Fig. 4GoA), while other cell types were lost. As in the radiation chimeras, essentially all VH11V{kappa}9+ B cells generated in IL-7 cultures were CD5+, CD23-, and CD43+, whereas B cells generated from 3-83 BM lacked expression of CD5 and CD43 (Fig. 4GoB). Unlike in vivo differentiation, the 3-83 B cells generated in vitro did not express CD23. This difference is probably due to the inability of IL-7-driven BM cultures to progress beyond the immature B cell stage, which is normally CD23-. When these cells were replated in the absence of IL-7 the VH11V{kappa}9+ B cells displayed enhanced in vitro survival, similar to that seen with in situ generated cells, whereas the 3-83+ and nontransgenic B cells died rapidly, after an initial 2-day lag (Fig. 4GoC). CFSE analysis again demonstrated that the enhanced recovery of VH11V{kappa}9+ cells was due to prolonged survival, rather than proliferation (data not shown). This observation strongly suggests that the B-1 phenotype can be rapidly acquired by newly developing BM B cells in vitro, provided that they bear a B-1-derived Ag receptor.



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  		FIGURE 4. BM-derived VH11V{kappa}9+ B cells expand in vitro. A, BM precursor cells isolated from 3-83 and VH11V{kappa}9 mice expand when cultured at 2 x 106 cells/ml for 5 days in the presence of IL-7. Results are representative of both VH11V{kappa}9 and 3-83 BM expansion. B, VH11V{kappa}9 BM B cells display a B-1 phenotype following in vitro expansion. Cells were stained with Ab conjugates immediately after culture (day 5) and analyzed by flow cytometry. The fluorescence of gated B220+ cells is shown. C, In vitro survival of BM-derived BCR transgenic B cells upon removal of IL-7. Cells were isolated from the above BM cultures on day 5 and recultured for the indicated number of days in the absence of IL-7. The recovery of B220+ cells is indicated. Error bars represent the SEs for seven experiments.

 
VH11V{kappa}9 Ig-transgenic B cells do not spontaneously edit in the BM

To further assess the susceptibility of VH11V{kappa}9 transgenic B cells to tolerance in the BM, we investigated the level of receptor editing in these cells. First, we measured BM RAG-2 mRNA levels using a semiquantitative RT-PCR assay, normalizing RNA levels using the pan-specific B cell marker gene CD19. As shown in Fig. 5GoA, 3-83 and VH11V{kappa}9 Ig-transgenic BM had low levels of RAG-2 mRNA, whereas a high level was detected in Ag-containing 3-83/H-2Kk mouse BM and control B10.D2 BM. As a second indicator of receptor editing, we measured the frequencies of B cells bearing Ig {lambda} light chains. In agreement with the RAG-2 mRNA analysis, 3-83 and VH11V{kappa}9 Ig-transgenic BM B cells had a low frequency of Ig {lambda}+ cells, while 3-83/H-2Kk mouse BM cells had significantly higher frequencies of Ig {lambda}+ cells (data not shown). Finally, to determine whether VH11V{kappa}9 BM B cells were, in fact, capable of receptor editing, we treated freshly isolated BM cells with anti-{kappa} mAb and again measured RAG-2 mRNA levels using RT-PCR and Ig {lambda} light chain expression. In BM samples from VH11V{kappa}9, 3-83, and nontransgenic control mice, cross-linking the BCR with anti-{kappa} mAb resulted in a detectable induction of RAG-2 (Fig. 5GoB) and elevated Ig {lambda} light chain expression (data not shown), whereas treatment with control rat mAb did not. Similar enhanced RAG-2 and Ig {lambda} light chain expression were seen following coculture with PtC-containing liposomes (data not shown).



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  		FIGURE 5. RT-PCR analysis of RAG-2 expression in BM of Tg mice. Whole BM mRNA from the indicated sources was analyzed as described in Materials and Methods. A, Lack of spontaneous RAG-2 expression in the BM of VH11V{kappa}9 mice. Results using BM from 3-83 Tg mice on the Ag-bearing (H-2Kk) or Ag-free (H-2Kd) backgrounds indicate the affect of autoantigen on RAG expression. Other controls include thymus and the RAG-2-nonexpressing cell line, IIA1.6. Representative results from four experiments are shown. B, VH11V{kappa}9 B cells express RAG-2 in vitro following receptor cross-linking. Whole BM isolated from indicated mice were cultured with anti-{kappa} mAb (10 µg/ml) or a nonspecific anti-ARS mAb (10 µg/ml) for 24 h. RAG-2 analysis was then performed as stated in Materials and Methods. Data are representative of four experiments.

 
BCR cross-linking triggers intracellular Ca2+ mobilization in VH11V{kappa}9+ splenic B cells, but not peritoneal B cells

Given the self-reactivity of the VH11V{kappa}9+ cells (27, 30), we wondered whether the peripheral cells with this BCR were functional or tolerized. Two lines of evidence suggested that they were not anergic. First, the VH11V{kappa}9 mice secreted abundant amounts of this Ab into the serum, indicating that their B cells had effector function (Fig. 6GoA). Second, unlike anergic B cells, spleen cells derived from VH11V{kappa}9 mice had robust Ca2+ mobilization responses upon receptor ligation with F(ab')2 rabbit anti-mouse IgM (Fig. 6GoB). In contrast, peritoneal B cells from these same mice did not respond, suggesting an environmental influence on their function. These data indicate that the VH11V{kappa}9 BCR on splenic B cells is functional and that these B cells are not anergic despite the presence of PtC in the environment.



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  		FIGURE 6. Splenic VH11V{kappa}9+ B cells are not tolerized in the periphery. A, Sera of BCR transgenic mice contain large amounts of transgene-encoded IgM. Serum from 8-wk-old transgenic mice was analyzed for IgMa (transgene encoded) by ELISA. All mice had similar amounts of total serum IgM (850–1500 µg/ml; data not shown). Nontransgenic (NTg) littermates provided a control. B, BCR cross-linking triggers Ca2+ mobilization in VH11V{kappa}9 B cells from spleen, but not peritoneal cavity (PerC). B cells were purified from the spleen and peritoneal cavity, loaded with indo-1, and stimulated with 1.0 µg/ml rabbit anti-mouse Ig (RamIg) F(ab')2. Arrows denote the time when the anti-IgM stimulus was added to the sample.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results show that cells carrying a B-1-derived BCR can be generated from BM precursors if these cells are constrained by transgenesis to produce it, and that cells with such receptors assume a B-1-like phenotype in terms of cell surface markers, in vivo localization, and survival characteristics. In contrast, cells constrained to produce only a B-2-derived receptor do not assume a B-1 phenotype. Interestingly, although the B-1 phenotype appears to be due to autoreactivity, our data suggest that this level does not trigger receptor editing, even in BM-derived immature B cells. Because very weak interactions have been shown to lead to receptor editing in conventional BCR transgenics (37), this indicates that the BCR signaling generating the B-1 phenotype is either extremely weak or else of a different nature compared with the 3-83 anti-class I interaction. BCR autoantigen interactions that induce B cell anergy often are associated with a significant degree of receptor editing (78, 79, 80), suggesting that if BCR:Ag interactions alone determine the response, there is a hierarchy of decreasing signaling thresholds required for the induction of editing, anergy, B-1 selection, and B-2 selection, respectively.

Although the simplest lineage model of B-1 vs B-2 development (61) would not have predicted the results reported here, the clear-cut difference in the abilities of fetal and BM pro-B cells to reconstitute B-1 and B-2 development remain (45, 47). Similarly, these data, while consistent with the ability of the BCR to induce a B-1 phenotype (29), do not obscure the fact that fetal and adult precursors show various distinctions in gene expression (49) and also differ markedly in their ability to give rise to these B cell types. In this study we have only compared the development of BM-derived precursors that carried either B-1-derived or B-2-derived transgenic BCRs. One way to put the present results into context is to consider the role of cell lineage in regulating the preselected BCR repertoire. Several lines of evidence suggest that the VH11 H chain used in this study is normally counterselected in BM compared with fetal precursors due to its poor ability to associate with the surrogate L chain components {lambda}5 and VpreB (63). The particular VH11 and V{kappa}9 transgenes used in our study force B-1 receptor expression in cells that normally disfavor its generation, propagation, and survival, allowing a test of the effect of such a receptor on further B cell development, but obscuring the effect of this receptor on earlier developmental stages. This idea is consistent with the view that B-1 receptors are unusual, germline-encoded specificities that are beneficial (81) and therefore are selected over evolution for particular properties that promote their generation in the fetus, but disfavor their development in the adult (63).

Do our results demonstrate that any VH11V{kappa}9+ B cell generated in the BM of normal (nontransgenic) mice will enter the peripheral B-1 B cell pool? Possibly, but one must also consider that differences in the level of expression of Ag receptor can have a striking effect on B cell development (82). The particular VH11-µ transgenic mouse line used in our study, BR1, was selected for its capacity to suppress RAG expression and therefore block endogenous heavy chain rearrangements. This line has a distinctively higher level of µ protein expression compared with normal B cells, probably due to the high transgene copy number (63). A different VH11-µ transgenic line, BR5, shows a lower level of heavy chain expression that may be more physiologic. Therefore, it will be important in future work to perform similar experiments using the BR5 line to generalize our results to normal VH11 development and test whether BCR levels alter selection. Furthermore, considering the distinctions mentioned above that distinguish fetal liver from BM B lymphopoiesis, it will also be important to compare cells isolated from BM with those obtained from fetal liver.

The inability of VH11V{kappa}9+ B cells isolated from the peritoneal cavity to mobilize Ca2+ following BCR ligation suggests an anergic phenotype for these cells. However, VH11V{kappa}9+ splenic B cells are normally responsive to the same stimulus, and the presence of significant levels of VH11V{kappa}9 serum Ig suggests a nonanergic phenotype. We are currently pursuing the idea that differences in the microenvironment between spleen and peritoneal cavity, either Ag availability or cytokine milieu, result in this differential responsiveness.

A key biological response that appears to be controlled by the quality of the BCR signal is B cell survival. Studies of immunological tolerance over the years have clearly shown that in many situations, reactivity to self-tissues radically alters the survival of B cells in the peripheral immune system, leading to outright deletion or shortened lifespan compared with nonautoreactive B cells (76, 83, 84, 85). On the other hand, an insufficiency of BCR-mediated signaling, such as occurs upon the genetic elimination of key BCR coreceptors or molecules in the signaling pathway (Btk, CD45, CD19, Syk), also blocks developmental progression and the acquisition of a long B cell lifespan (3, 58, 59, 86, 87, 88, 89). Furthermore, in B-2 cells, continued BCR expression is required for survival even after recruitment into the long-lived pool (90). The in vivo situation with B-1 cells is more complex, as these cells appear to require Ag for their selection or persistence (or both) and can undergo chronic receptor-driven expansion (62, 69, 91). In the present study we document the extended in vitro survival of B-1 phenotype cells, relative to B-2 cells. Taking advantage of this knowledge to probe B cell differentiation, we find that a long-lived phenotype is rapidly conferred to newly formed, BM B cells expressing a B-1-derived, but not a B-2-derived, receptor. This differential survival in vitro correlates with the B-1 phenotype of the splenic B cells of VH11V{kappa}9 Ig transgenic mouse and the B-2 phenotype of 3-83 Tg mouse B cells. Although it is still unclear how B-1 and B-2 cells of different specificities survive in vitro, it is tempting to speculate that this might point to a general difference in the survival of these two B cell types. This experimental system should be very helpful in the future in determining more precisely how the BCR controls cell survival.


   Acknowledgments
 
We thank D. Russell (National Jewish Medical and Research Center) and D. Melamed (Faculty of Medicine, Department of Immunology, Bat Galim, Haifa, Israel) for technical assistance; V. Kouskoff and R. Benschop for critical review of the manuscript; and W. Townsend (National Jewish Medical and Research Center) and S. Shinton (Fox Chase Cancer Center) for assistance with flow cytometry.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (GM44809 to D.N.; and AI26782 and AI40946 to R.R.H.). Back

2 Address correspondence and reprint requests to Dr. Richard R. Hardy, Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111. Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; PtC, phosphatidylcholine; BM, bone marrow; CFSE, carboxyfluorescein diacetate succimidylester; RAG-2, recombination-activating gene-2. Back

Received for publication December 3, 1999. Accepted for publication February 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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B-1 B cell development.
J. Immunol., September 1, 2006; 177(5): 2749 - 2754.
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