The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, H.
Right arrow Articles by Clarke, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, H.
Right arrow Articles by Clarke, S. H.
The Journal of Immunology, 2003, 171: 6381-6388.
Copyright © 2003 by The American Association of Immunologists

Evidence for a Ligand-Mediated Positive Selection Signal in Differentiation to a Mature B Cell 1

Hongsheng Wang and Stephen H. Clarke2

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Positive selection is required for B cell differentiation, as indicated by the requirement for expression of the pre-B cell receptor (pre-BCR) and the BCR at the pre-B and immature B cell stages, respectively. Positive selection mediated by a tonic signal from these receptors is sufficient to drive B cell differentiation beyond the pre-B and immature B cell stages, but it is unclear whether additional positive selection signals are required for differentiation to a mature B-2 cell. We have identified a population of Ig transgenic B cells that differentiatively arrest at a transitional B cell stage in the spleen. They exhibit no evidence of Ag encounter or negative selection and can differentiate to mature B-2 cells in vivo upon weak BCR stimulation or adoptive transfer to irradiated hosts. These data are consistent with a requirement for a ligand-mediated BCR signal for differentiation to a mature B-2 cell.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B cell development follows a set pathway involving sequential expression of the pre-B cell receptor (pre-BCR) 3 and BCR. Pre-BII cells express the pre-BCR upon productive VHDJH rearrangement and the association of H chain with surrogate L chain (reviewed in Ref.1). These cells become immature B cells upon productive VLJL rearrangement and expression of an IgM BCR. Immature B cells are initially IgMlow, IgD-, but quickly become IgMhigh and migrate to the spleen as IgMhigh transitional (T) 1 B cells. In the spleen two schemes for transitional B cell maturation are proposed. In the first, described by Loder et al. (2), T1 cells (IgMhigh, CD21-, CD23-) differentiate to T2 cells (IgMhigh, CD21high, CD23+) before entering the long-lived mature B-2 cell (IgMlow, CD21low, CD23+) population. The second differentiative scheme, described by Allman et al. (3), proposes three transitional populations that are distinguished by the expression of the IgM, AA4, and CD23. In this scheme the IgMhigh, AA4+, CD23- T1 cells differentiate to IgMhigh, AA4+, CD23+ T2 cells and then to IgMlow, AA4+, CD23+ T3 cells before becoming IgMlow, AA4-, CD23+ mature B-2 cells. Completing the mature B cell repertoire are two other functionally and phenotypically distinct populations: marginal zone (MZ) and B-1 B cells.

Negative selection has a well-documented role in shaping the mature B cell repertoire by eliminating autoreactive B cells at multiple points along the differentiative pathway. Some autoreactive B cells arrest in differentiation as immature B cells and undergo receptor editing (4, 5, 6). This process is a major mechanism in shaping the B cell repertoire, as ~25% of bone marrow immature B cells are believed to undergo receptor editing (7). B cells that fail to edit successfully away from their autoreactive specificity undergo apoptosis in the bone marrow, prohibiting further differentiation (4, 8). Other autoreactive B cells exit the bone marrow and either developmentally arrest at a transitional stage in the spleen (9, 10) or become anergic B-2 cells (5, 11, 12).

Positive selection also shapes the mature B cell repertoire. MZ and B-1 cells are Ag selected (13, 14, 15, 16), but whether entry into the B-2 subset, the major population of splenic and lymph node B cells, also requires positive selection is controversial. There is indirect evidence for positive selection in the development of B-2 cells. The inability to make a pre-BCR or BCR or in their ability to signal transduce results in a block in differentiation at the bone marrow pre-B cell stage and also at the splenic transitional B cell stage (2, 17). The nature of this positive selection signal is unclear. Monroe and colleagues (18) have demonstrated that the cytoplasmic domains of Ig{alpha} and Ig{beta} in the absence of Ig are sufficient to drive B cell differentiation to a splenic B cell, arguing that positive selection at the pre-B and immature B cell stages may require only tonic signaling from the pre-BCR and BCR. However, differences in the expressed repertoires between mature and immature/transitional B cells (19, 20, 21) suggest a requirement for ligand-mediated positive selection. Indeed, BCR stimulation of T2 B cells can result in differentiation to B-2-like cells in vitro (22, 23). Ligand-mediated positive selection is also suggested by an analysis of signaling-deficient anti-HEL Tg mice. Nonautoimmune anti-HEL B cells that lack CD45 arrest at a transitional B cell stage, but become mature if HEL is available (24). Similarly, nonautoimmune anti-MHC class I B cells that lack CD19 arrest at a transitional B cell stage, but become mature in CD19-sufficient mice (25).

Our studies of VH12 B cell differentiation have indicated unusually restricted positive selection that focuses differentiation toward specificity for phosphatidylcholine (PtC) and B-1 differentiation (15, 26, 27, 28). Positive selection at the pre-B cell stage allows for the survival of only ~5% of VH12 pre-B cells and enriches for cells with a VHCDR3 of 10 aa and a Gly in the fourth position (designated 10/G4), the VHCDR3 motif of anti-PtC B-1 cells (26). Most L chains are unable to associate (nonpermissive) with 10/G4 VH12 H chains, and thus, most VH12 B cells undergo multiple L chain rearrangements (27). Among splenic VH12 B cells, there is a significant bias for the use of V{kappa}4/5H, the L chain used by PtC-specific VH12 B-1 cells. Surprisingly, this bias is evident even among VH12 B cells that cannot bind PtC and do not differentiate to B-1. We hypothesize that V{kappa}4/5H L chains drive VH12 B cell differentiation to the long-lived mature stage, whereas most other VH12-permissive L chains drive differentiation no further than the short-lived transitional stage. Our analysis of mice carrying a 10/G4 VH12 transgene and a V{kappa}1A transgene (6-1/V{kappa}1A) supports this hypothesis; T1 B cells from these mice use the V{kappa}1A L chain, whereas the more mature CD23+ B cells often use endogenous L chains, particularly V{kappa}4/5H (28). We show in this study that PtC nonbinding VH12 B cells, such as 6-1/V{kappa}1A B cells, terminate differentiation at a transitional stage and provide evidence that their differentiative arrest is due to a failure to receive a ligand-mediated, positive selection signal.


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

6-1 and 6-1/V{kappa}4 Tg mice have been previously described (10, 15). V{kappa}1A Tg mice were described previously (28) and provided by Dr. M. Weigert (Princeton University, Princeton, NJ). These mice contain a V{kappa}1A-J{kappa}1 rearrangement inserted by homologous recombination into an endogenous J{kappa} locus. These knock-in mice also contain a duplication of the V{kappa}1A-J{kappa}1-C{kappa} locus on the same locus as the knock-in. Recombinase-activating gene-1 (RAG-1)-/- mice on a C57BL/6 background were purchased from The Jackson Laboratory (Bar Harbor, ME). All strains, with the exception of RAG-1-/- mice, were bred and maintained in our pathogen-free facility at University of North Carolina by backcrossing to C.B17 mice. Offspring carrying transgenes were identified by tail genomic DNA as previously described (10, 15, 28).

Flow cytometry

Cells were prepared and stained as previously reported (29). mAbs specific for mouse B220, IgM, IgMa, IgMb, CD43, CD40, CD44, CD80, CD86, CD5, CD19, CD23, CD21/CD35, heat-stable Ag (HSA), AA4.1, and MHC class II (I-Ab) were obtained from BD PharMingen (San Diego, CA) and were either directly conjugated to FITC, R-PE, and allophycocyanin or biotinylated. Biotinylated Abs were revealed with streptavidin-conjugated PerCP (BD PharMingen). Cells were analyzed using a FACSCalibur (BD Biosciences, Mountain View, CA). Data were analyzed by WinMDI software (The Scripps Institute, La Jolla, CA).

For cell-sorting experiments, spleen cells were stained with FITC-labeled anti-B220 and sorted on a MoFlo high-speed sorter (Cytomation, Fort Collins, Co). Sorted populations were >95% pure. The cells were then used for Western blotting.

5-Bromo-2'-deoxyuridine (BrdU) labeling

Dividing cells were labeled with BrdU in vivo as described previously (10). Briefly, BrdU (Sigma-Aldrich, St. Louis, MO) was administered in drinking water continuously for 1–6 days. At each time point, mice were sacrificed, and spleen cells were isolated for staining with anti-IgM-allophycocyanin and anti-B220-PE. Cells were then fixed, permeabilized, treated with DNase (Sigma-Aldrich), and stained with anti-BrdU-FITC (BD Biosciences). The fraction of BrdU-labeled B cells was determined by flow cytometry.

Ab secretion and proliferation assays

Spleen cells from non-Tg, non-Tg that had been irradiated with 500 rad 14 days earlier (Ir-d14), and 6-1/V{kappa}1A/RAG-1-/- mice were purified through a B cell purification column (Accurate Chemical & Scientific Corp., Westbury, NY) according to manufacturer’s instruction. For the proliferation assay, 1 x 105 cells/well in 96-well plates were cultured in triplicate in complete RPMI 1640 medium supplemented with 10% FCS. Goat F(ab')2 anti-mouse µ (10 µg/ml) (Southern Biotechnology Associates, Birmingham, AL), LPS (20 µg/ml) (Sigma-Aldrich), monoclonal anti-CD40 Ab (0.5 µg/ml; Southern Biotechnology Associates), and IL-4 (10 ng/ml; R&D Systems, Minneapolis, MN) were added to cultures for 4 days. On day 3, [3H]thymidine was added at 1 µCi/well and incubated for 16 h. The cells were then harvested and analyzed using a liquid scintillation counter (Beckman, Fullerton, CA). For Ab secretion assay, 2 x 105 cells/well were cultured under the same conditions for 6 days. A µ-chain-specific ELISA was used to measure the secreted IgM in culture supernatant as described previously (29).

Western blot analysis

Splenic B220+ cells from non-Tg mice, Ir-d14, and 6-1/V{kappa}1A/RAG-1-/- mice were purified by cell sorting. Three million cells were stimulated with 20 µg/ml goat F(ab')2 anti-mouse µ for 3 min at 37°C. The cells were then lysed in buffer containing 20 mM Tris (pH 8.0); 150 mM NaCl; 1 mM PMSF; 1 µg/ml each of aprotinin, {alpha}1-antitrypsin, and leupeptin; 10 mM tetrasodium pyrophosphate; 2 mM sodium orthovanadate; and 1% Nonidet P-40. Lysates were cleared by centrifugation and subjected to SDS-PAGE using 10% gels. Fractionated proteins were transferred to nitrocellulose membranes and blocked with 4% BSA. The blots were probed with the mAb Ab-2 recognizing the phosphotyrosine residues (Oncogene Research Products, Boston, MA), followed by an HRP-conjugated anti-rat IgG1 Ab. Immunoreactive proteins were visualized with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Anti-Ig{beta} treatment and adoptive transfer assay

6-1/V{kappa}1A/RAG-1-/- mice were injected i.p. with 10 or 100 µg of either anti-Ig{beta} (HM79) or an irrelevant hamster (HM) IgG Ab (Southern Biotechnology Associates). Bone marrow and splenic cells were analyzed by flow cytometry on day 2.

For adoptive transfer experiments, C.B17 or RAG-1-/- mice were irradiated with 500 rad 1 day before i.v. injection of 3 x 107 spleen cells or 6 x 106 bone marrow cells from 6-1/V{kappa}1A/RAG-1-/- mice. Mice that received spleen cells were sacrificed on day 3, and the spleen cells of donor origin (IgMa+) were analyzed by flow cytometry. Donor B cells from recipients that received bone marrow cells were analyzed at wk 3 and 4. Serum IgM levels were assayed by ELISA as previously described (29).

For cell survival assays, splenic cells from mice that had been pretreated with 10 µg of anti-Ig{beta} or HM IgG for 3 days were cultured in RPMI 1640 medium with 10% FCS for 3 days. Cells were stained for apoptotic cells with anti-IgM-APC, annexin V-FITC (BD PharMingen) and propidium iodide at different time points and analyzed by flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Non-PtC-binding VH12 B cells are predominantly transitional in 6-1 Tg mice.

6-1 mice carry a 10/G4 VH12 transgene and generate large numbers of PtC-specific splenic and peritoneal B-1 cells that stain brightly with PtC-containing liposomes (PtCbri) (15). The remaining B cells stain with liposomes with either intermediate intensity (PtCint) or do not stain (PtCneg), and are not B-1 (27). Previously, we considered many of the PtCint and PtCneg cells B-2 based on the expression of CD23, but because some transitional B cells are now known to also express CD23 (2, 3), we re-examined the phenotype of these cells. Fig. 1 and Table I show that PtCint and PtCneg cells include CD23- T1 cells and MZ B cells, but few CD23+ T2 and B-2 cells, as defined by Loder et al. (2). The few cells that fall within the B-2 cell gate have atypically high IgM levels. The majority of CD23+ PtCint and PtCneg cells are CD21low. This phenotype suggests that these cells are intermediate to T1 and T2, as defined by Loder et al. (2). These data indicate that most PtCneg and PtCint B cells in these mice are transitional, suggesting that they are unable to become long-lived B-2 cells.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 1. FACS analysis of PtCneg and PtCint B cells in 6-1 mice. Spleen cells from 6-1 and non-Tg littermate mice were stained for IgM, CD23, and CD21 expression and for liposome binding. All histograms are gated on lymphocytes according to forward and side light scatter. The gates for each subset are based on the analysis of non-Tg mice. The percentage of B cells in each subset is given. Data are representative of three independent experiments.

 

View this table:
[in this window]
[in a new window]
  		Table I. B cell subsets in the spleens of 6-1/V{kappa}1A Tg micea

 
6-1/V{kappa}1A B cells arrest at a transitional stage in the spleen

To more precisely define the developmental arrest, we examined the B cells from 6-1/V{kappa}1A Tg mice. 6-1/V{kappa}1A B cells do not bind PtC and are not selected into the B-1 subset (27, 28). The more mature CD23+ cells in these mice often express endogenous L chains, whereas the less mature CD23- cells predominantly use the V{kappa}1A L chain (28). 6-1/V{kappa}1A/RAG-1-/- mice were generated to eliminate the effect of endogenous L chain expression on 6-1 B cell development. 6-1/V{kappa}1A/RAG-1-/- mice have similar numbers of bone marrow IgMa+, B220low immature B cells as 6-1 mice (Fig. 2A and data not shown). Strikingly, however, 6-1/V{kappa}1A/RAG-1-/- mice lack recirculating IgMa+, B220high B cells in the bone marrow (Fig. 2A). The pro-B and pre-B populations in 6-1/V{kappa}1A/RAG-1-/- mice are small, as they are in 6-1/V{kappa}4 Tg mice (Fig. 2A), presumably because the presence of H and L chain transgenes accelerates passage through the pre-B cell stage.



View larger version (46K):
[in this window]
[in a new window]
  		FIGURE 2. B cell development in 6-1/V{kappa}1A/RAG-1-/- mice. A, Bone marrow cells from the indicated mice were stained for B220, IgMa or IgMb, and CD43, and gated on lymphocytes (top row) and IgM- cells (bottom row). B cell subsets were defined as follows: pro/pre-BI, B220+CD43high; pre-BII, B220+CD43low; immature B cells, IgM+B220low; and recirculating B cells, IgM+B220high. The percentage of B cells in each category is given. B, Spleen cells from non-Tg and 6-1/V{kappa}1A/RAG-1-/- mice were analyzed for B cell subsets as described in Fig. 1. The gates for each subset were determined from non-Tg mice. The percentage of B cells falling within each gate is given. *, Cells with a T2 phenotype according to Allman et al. (3 ). A and B are representative of the analysis of five or more mice for each group.

 
In the spleen B cell numbers are approximately six times lower in 6-1/V{kappa}1A/RAG-1-/- mice than in non-Tg and approximately five times lower than in 6-1 mice (p < 0.0001; Table I). The surface IgM level on 6-1/V{kappa}1A/RAG-1-/- splenic B cells is high, but within the normal range (Fig. 2B), and based on CD23 and CD21 expression, both T1 and the population seen in 6-1 mice that are intermediate to T1 and T2, as defined by Loder et al. (2), are present (Fig. 2B and Table I). The majority of the CD23+ and CD23- cells are also AA4+ (Fig. 2B). The CD23-, AA4+ cells correspond to T1 cells, and the CD23+, AA4+ cells correspond to T2 cells, as defined by Allman et al. (3). The level of AA4 on the CD23+ cells is lower than that on the CD23- cells, in agreement with the original description of T1 and T2 (3). Thus, we conclude that the splenic 6-1/V{kappa}1A B cells that differentiate belong to the T1 and T2 subsets as defined by Allman (3). These cells will be referred to as T1 and T2 to conform with this nomenclature. These cells express normal levels of CD19 and are HSAhigh and CD5- (Fig. 3A). Consistent with non-Tg transitional cells, 6-1/V{kappa}1A B cells have a rapid turnover rate, suggesting a half-life of ~2.5 days (Fig. 3C). These mice lack detectable splenic B-2, and MZ B cells (Fig. 2B and Table I) and peritoneal B-1 cells (data not shown), and their lymph nodes have few B cells, with those present having either a T1 or T2 phenotype (data not shown). Thus, 6-1/V{kappa}1A B cells appear to differentiatively arrest as CD23+, AA4+ T2 cells soon after exiting the bone marrow. There is no detectable serum IgM in these mice (data not shown), indicating that 6-1/V{kappa}1A B cells do not differentiate to plasma cells.



View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 3. Phenotype and turnover rate of 6-1/V{kappa}1A B cells. Differentiation marker (A) and activation marker (B) expression is compared among 6-1, non-Tg, and 6-1/V{kappa}1A/RAG-1-/- B cells. Each histogram is representative of the analysis of three or more mice of each genotype. Histograms are gated on B220+, IgMhigh transitional B cells, except for 6-1 mice, in which total IgM+ B cells were analyzed. A and B are representative of the analysis of four or more mice for each group. C, The turnover rates of splenic 6-1/V{kappa}1A/RAG-1-/- and non-Tg transitional B cells (B220+, IgMhigh) were determined by BrdU incorporation. The percentages of BrdU+ B cells on days 1.6, 3.6, and 5.6 were determined. Each symbol represents an individual mouse. The dotted lines illustrate the time at which 50% of B cells are BrdU+.

 
6-1/V{kappa}1A B cells do not exhibit evidence of negative selection

To assess whether negative selection can explain the developmental arrest of 6-1/V{kappa}1A B cells, we examined activation marker expression, the functional status of the cells, and the ability of the BCR to transduce signals. Negatively selected B cells exhibit one or more of a variety of features. These include elevated expression of activation markers (30, 31, 32, 33), unresponsiveness to LPS and T cell-derived factors (32, 34, 35, 36), and deficiencies in BCR signaling (31, 34, 36, 37). However, in contrast to negatively selected cells, 6-1/V{kappa}1A B cells express normal levels of multiple activation markers (Fig. 3B) and undergo proliferation (Fig. 4A) and Ab secretion in response to LPS and anti-CD40 plus IL-4 (Fig. 4B) similar to transitional B cells from non-Tg mice. Non-Tg transitional B cells were obtained from mice that had been given a sublethal dose of irradiation 14 days before spleen cell harvesting. Allman et al. (38) have demonstrated that these cells, designated Ir-d14, are predominantly transitional B cells. Our flow cytometric analysis confirmed that the Ir-d14 cells used in this study are predominantly T1 and T2 cells (data not shown). As expected, anti-µ stimulation does not induce proliferation or Ab secretion by 6-1/V{kappa}1A B cells or control Ir-d14 cells, in contrast to mature non-Tg B cells. The pattern and magnitude of protein tyrosine phosphorylation in 6-1/V{kappa}1A B cells are similar to those in non-Tg transitional B cells following BCR cross-linking with F(ab')2 anti-mouse µ Abs (Fig. 4C). The intensity of phosphorylation of some protein bands in 6-1/V{kappa}1A and Ir-d14 B cells is stronger than that in non-Tg B cells (Fig. 4C), probably due to differences in surface IgM levels (Fig. 2). Thus, although negative selection cannot be excluded, 6-1/V{kappa}1A B cells resemble non-Tg transitional B cells and show no evidence of negative selection.



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 4. Analysis of the functional status of 6-1/V{kappa}1A transitional B cells. A, B cell proliferation. Purified splenic B cells from non-Tg, Ir-d14, and 6-1/V{kappa}1A/RAG-1-/- mice were cultured with or without goat (Fab')2 anti-mouse µ, LPS or anti-CD40 plus IL-4 for 2 days, followed by incubation with [3H]thymidine for an additional 16 h. Ir-d14 cells are B cells derived from non-Tg mice that had received sublethal irradiation 14 days earlier. They consist predominantly of T1 and T2 cells (30 ) (data not shown). Data are presented as the mean counts per minute ± SD of triplicate assays. B, Ab secretion. Purified B cells were cultured with or without the stimuli used in A for 6 days, and the level of secreted IgM was determined by ELISA. Data are presented as the mean OD ± SD of triplicate assays. C, Protein tyrosine phosphorylation following BCR ligation. Sorted splenic B220+ B cells were stimulated or not stimulated with goat F(ab')2 anti-mouse µ Abs for 3 min at 37°C. The whole cell lysates were analyzed by immunoblotting with anti-phosphotyrosine mAbs. Each lane represents 3 x 106 cells. The molecular mass is shown on the right. A and B are representative of two separate experiments; C is representative of three separate experiments.

 
6-1/V{kappa}1A B cells can be induced to mature by weak BCR signaling

An alternative explanation for the block in maturation of 6-1/V{kappa}1A B cells is that they lack a positive selection signal. To test this possibility, 6-1/V{kappa}1A/RAG-1-/- mice were injected with anti-BCR Abs. Two days after injection of 10 µg of anti-Ig{beta}, the percentage of splenic CD23+ cells in 6-1/V{kappa}1A/RAG-1-/- spleens increased from 50 to >80%. The levels of IgM, HSA, and AA4 decreased, and CD21 increased on CD23+ cells compared with unstimulated 6-1/V{kappa}1A B cells, indicative of maturation toward B-2 (Fig. 5, A and B). The expression levels of CD40, MHC class II, and CD5 were slightly higher, whereas CD80, CD86, and CD44 were unchanged (Fig. 5B and data not shown). In addition, anti-Ig{beta}-treated 6-1/V{kappa}1A B cells survived longer in culture than untreated 6-1/V{kappa}1A B cells, similar to mature non-Tg B-2 cells (Fig. 6A), and were able to secret IgM Abs in response to IL-4 and anti-CD40 (data not shown). In contrast, increasing the level of injected anti-Ig{beta} to 100 µg induced deletion (Fig. 6B). Thus, these data suggest that weak in vivo BCR stimulation induces maturation of 6-1/V{kappa}1A B cells to B-2.



View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 5. In vivo stimulation of 6-1/V{kappa}1A B cells. A, The effects of anti-Ig{beta} injection on B cells from 6-1/V{kappa}1A/RAG-1-/- mice. 6-1/V{kappa}1A/RAG-1-/- mice were injected i.p. with 10 µg of anti-Ig{beta} mAb or an isotype-matched HM IgG Ab. On day 2, splenic cells were analyzed by flow cytometry. Dot plots are gated on CD19+ cells. *, Cells with a T1 or T2 phenotype according to Allman et al. (3 ). B, Overlays are of B220+, IgM+ B cells from 6-1/V{kappa}1A/RAG-1-/- mice treated with HM IgG (thin line), anti-Ig{beta} mAb (thick line), or non-Tg mice (dashed line). Each histogram is representative of the analysis of three or more mice from each group.

 


View larger version (30K):
[in this window]
[in a new window]
  		FIGURE 6. 6-1/V{kappa}1A B cell differentiation in vivo and survival in vitro. A, Prolonged survival of anti-Ig{beta}-treated 6-1/V{kappa}1A B cells in vitro. Splenic B cells pooled from two 6-1/V{kappa}1A/RAG-1-/- mice that had been pretreated with 10 µg of anti-Ig{beta} or an irrelevant HM IgG for 3 days were cultured in vitro for 3 days. The percentage of viable IgM+ B cells at different times was determined by flow cytometry. Data from two independent experiments are shown. B, Response of 6-1/V{kappa}1A/RAG-1-/- mice to anti-Ig{beta}. Mice were injected with the indicated amounts of Ab and analyzed for B220 and IgM expression. Histograms are gated on lymphocytes. Each histogram is representative of the analysis of three or more mice for each group. C, Adoptive transfer of 6-1/V{kappa}1A/RAG-1-/- spleen cells into irradiated non-Tg mice. Spleen cells of the recipient mice were analyzed 3 days after transfer. Histograms are overlays of non-Tg (dashed line), 6-1/V{kappa}1A/RAG-1-/- (thin line), and B220+, IgMa+ spleen cells from recipient (thick line) mice. Data are representative of three separate experiments.

 
As an approach to provide Ag for positive selection, 6-1/V{kappa}1A/RAG-1-/- B cells were exposed to a surge of self-Ags by transfer to sublethally irradiated C.B17 mice. Three days after splenic cell transfer, ~10% of input donor B cells were recovered from recipient spleens. Almost all the recovered cells were CD23+ (data not shown), and the majority of these cells expressed lower levels of IgM and HSA and higher levels of CD21, suggesting that they had differentiated to mature B-2 cells (Fig. 6C). The expression levels of CD5 and MHC class II were also higher, suggesting Ag stimulation. Bone marrow cell transfers gave similar results. Three weeks after bone marrow transfer, ~60% of splenic IgMa+ B cells were IgMlow and HSAlow suggesting differentiation to a B-2 cell (data not shown). The expression levels of CD5, CD40, and CD86 were also slightly increased in bone marrow recipients, although CD44 and CD80 were unchanged (data not shown), suggesting that the cells had encountered Ag. Serum IgMa levels remained undetectable as long as 4 wk after bone marrow transfer (data not shown), arguing that the encounter with Ag was insufficient to induce plasma cell differentiation and was more consistent with the induction of B-2 maturation.

As 6-1/V{kappa}1A/RAG-1-/- mice are a mixture of C.B17 and C57BL/6 backgrounds, the observed differentiation of donor B cells could be due to the absence of a negatively selecting Ag of C57BL/6 origin in C.B17 recipient mice. To rule out this possibility, spleen cell transfers were repeated in C57BL/6/RAG-1-/- recipients. These transfers yielded indistinguishable results from those using C.B17 recipients (data not shown). Thus, the absence of a negatively selecting Ag of C57BL/6 origin cannot explain the induced differentiation of donor B cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This analysis demonstrates that most VH12 B cells not selected into the B-1 subset are transitional. As the majority of newly developing VH12 B cells do not bind PtC and are not selected to become B-1 (27), this suggests that most newly developing VH12 B cells are unable to become mature B-2 cells. This is confirmed by the analysis of PtCneg B cells from 6-1/V{kappa}1A/RAG-1-/- mice. Only T1 and T2 cells are present in the spleen and lymph nodes of these mice, and no B cells, B-1 or otherwise, are present in the peritoneum, indicating that developmental arrest of these PtCneg cells occurs at T2 soon after migration to the spleen. This supports the hypothesis that the observed bias in V{kappa}4/5H L chain gene use by mature B cells in 6-1 mice (27) is due to an inability of non-V{kappa}4/5H L chains to support differentiation to the mature B cell stage. This is the third checkpoint in VH12 B cell differentiation, after the pre-BI to pre-BII and the pre-BII to immature B checkpoints (26, 29, 39), that restricts VH12 B cells primarily to the production of H and L chains that form an anti-PtC BCR and drives differentiation to B-1.

The developmental arrest of 6-1/V{kappa}1A B cells could be explained by either negative selection or a lack of positive selection. B cell negative selection induces a wide range of responses. Some negatively selected B cells, such as anti-dsDNA and anti-MHC class I B cells, undergo receptor editing as a means of altering their specificity or are deleted in the bone marrow (5, 40). Other autoreactive B cells that developmentally arrest in the spleen have lower than normal surface IgM levels (30, 41) and in some cases elevated levels of activation markers such as CD44, HSA, and MHC class II, indicating that they have encountered Ag (30). Anergic autoreactive B cells also exhibit evidence of Ag encounter. Although they are long-lived mature B cells, they are unresponsive to a variety of activating stimuli and may have low levels of surface IgM and high levels of activation markers, including CD80, CD86, and MHC class II (31, 32, 36). In addition, some anergic B cells exhibit impaired protein tyrosine phosphorylation of cytoplasmic proteins upon BCR cross-linking (34, 36). Thus, negatively selected B cells that enter the periphery show phenotypic and functional evidence of an encounter with self-Ag. 6-1/V{kappa}1A B cells differ significantly from the B cells in these models. They are not deleted in the bone marrow, but differentiate to a short-lived transitional B cell in the spleen (Figs. 2 and 3). They have high levels of IgM, and their activation markers are not elevated (Figs. 2 and 3). In addition, they respond normally to BCR and non-BCR activation stimuli, and protein phosphorylation upon BCR cross-linking appears normal (Fig. 4). Thus, there is no evidence that these cells have encountered Ag and no support for negative selection as an explanation for the developmental arrest of 6-1/V{kappa}1A B cells.

The more likely explanation for the arrested differentiation of 6-1/V{kappa}1A B cells is a lack of positive selection. This is suggested by the finding that weak BCR stimulation in vivo induces differentiation of 6-1/V{kappa}1A transitional B cells to become mature B-2 cells (Fig. 5). Not only do treated cells resemble mature B-2 cells in phenotype, they also acquire increased resistance to apoptotic cell death in vitro (Fig. 6A). 6-1/V{kappa}1A B cells can also be induced to acquire a more B-2-like phenotype by in vitro stimulation with anti-Ig{beta} or anti-IgM Abs (data not shown). One day after treatment the percentage of 6-1/V{kappa}1A B cells expressing CD23 increases, and the IgM level on these cells decreases. BCR signaling of already negatively selected cells would be expected to reinforce negative selection and prevent further differentiation rather than promote differentiation, further arguing against negative selection.

Tonic signaling, which derives from an intact BCR (42) and is sufficient for differentiation to a splenic B cell (18), appears to be intact in 6-1/V{kappa}1A B cells. We have previously shown that the 6-1 H chain and V{kappa}1A L chain can pair and be secreted by cells of a transfected cell line (27), and bone marrow immature and splenic transitional B cells in 6-1/V{kappa}1A/RAG-1-/- mice are IgMhigh (Fig. 2), indicating that this H/L chain pair can generate a high level of BCR expression. That 6-1/V{kappa}1A B cells differentiate to splenic transitional B cells suggests that positive tonic signaling is intact in these cells. Nevertheless, whereas it cannot be excluded that the 6-1/V{kappa}1A BCR is unable to provide a sufficiently strong tonic signal to drive differentiation to the mature B-2 cell stage, its inability would have to be unrelated to its BCR expression level.

The ability of weak BCR stimulation to induce maturation of these B cells in vivo (Fig. 5) suggests that it is more probable that 6-1/V{kappa}1A B cells lack a ligand-mediated positive selection signal. The need for a separate signal at the T2 stage is consistent with the requirement for BCR signal transduction at the transitional B cell stage (17). It is particularly noteworthy that xid B cells deficient in Btk arrest in differentiation at the T2 cell stage (3), suggesting that Btk is necessary for mediating the ligand-mediated positive selection signal. Ligand-mediated positive selection at this stage would account for the differences in V gene use between transitional and mature B cells determined by Levine et al. (19). These findings suggest that certain V gene combinations are favored for maturity, which, based on the results presented in this study, is likely to be due to differences in the ability to bind self-ligands. Moreover, it suggests, as proposed by Levine et al. (19), that some of the cell death at the transitional stage is due to a lack of positive selection, not just negative selection. Ligand-mediated positive selection would ensure that only B cells with a BCR capable of Ag binding and of initiating signal transduction can become long-lived mature B cells. This is also consistent with the model proposed by Freitas and Rocha (43) in which transitional B cells compete with other B cells for survival resources to enter and remain in the mature B cell repertoire (43).

The requirement for ligand-mediated positive selection signal can provide an explanation for the differentiation of 6-1/V{kappa}1A B cells after adoptive transfer to irradiated recipients. Irradiation creates space for recovery of the B cell compartment (38), and this may allow transferred B cells to differentiate to mature B-2 cells more efficiently than in the steady state condition. An alternative possibility is that irradiation provides to B cells new self-Ags or higher concentrations of already available self-Ags that can mediate positive selection. This is suggested by our observation that negative selection of B cells specific for the ribonucleoprotein Sm, a self-Ag targeted in systemic lupus erythematosus, is greatly enhanced in irradiated 2–12H Tg mice compared with nonirradiated mice (unpublished observation) because of the association of Sm with apoptotic cell blebs (46). A third possibility is that irradiation induces cytokine release, which may facilitate T2 differentiation to B-2. We cannot rule out this possibility, but B cell-activating factor of the TNF family, a critical cytokine for mature B cell differentiation and survival (44), alone is unable to induce phenotypic changes in cultured 6-1/V{kappa}1A/RAG-1-/- B cells (data not shown).

The fate of cells that are not positively selected by self-ligand appears to be cell death. However, splenic CD23+ B cells of RAG-sufficient 6-1/V{kappa}1A mice have endogenous V{kappa} rearrangements, and these mice also have a population of splenic PtC-binding B-1 cells expressing an endogenous V{kappa}4/5H L chain (28). The expression levels of RAG-1 and RAG-2 in 6-1/V{kappa}1A immature B cells are lower than those in control immature B cells from non-Tg mice and two other H chain Tg mice (data not shown), ruling out an autoantigen-induced receptor editing mechanism. It is unclear whether endogenous rearrangement is due to secondary rearrangement induced by the absence of ligand-mediated positive selection or to a leakiness of allelic exclusion by the V{kappa}1A transgene. However, V{kappa}1A Tg mice carrying a different H chain transgene show no evidence of endogenous V{kappa} rearrangement (data not shown), suggesting an induction mechanism. Additional experiments will be required to clarify this.

The strength of the ligand-mediated positive selection signal is critical. Too strong a signal induces apoptosis (Fig. 6). In addition, MZ and B-1 cells require self-Ag binding for differentiation (13, 16), and it will be of interest to determine how differences in BCR engagement induces different maturation pathways. The differences may be strictly quantitative, but we have observed, contrary to expectations, that positively selected anti-Sm B-1 cells are better binders than anergic anti-Sm B-2 cells (32, 45). Thus, there may be important qualitative differences that need to be elucidated.


   Acknowledgments
 
We gratefully acknowledge the assistance of the Flow Cytometry Facility at the University of North Carolina. We are also indebted to Dr. Martin Weigert for the V{kappa}1A Tg mice.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI29576 and AI43587 and a grant from the Arthritis Foundation. Back

2 Address correspondence and reprint requests to Dr. Stephen H. Clarke, Department of Microbiology and Immunology, CB#7290, 626 MEJB, University of North Carolina, Chapel Hill, NC 27599. E-mail address: shl{at}med.unc.edu Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; BrdU, 5-bromo-2'-deoxyuridine; CDR3, third complementarity-determining region; HM, hamster; HSA, heat-stable Ag; MZ, marginal zone; PtC, phosphatidylcholine; RAG, recombinase-activating gene. Back

Received for publication July 18, 2003. Accepted for publication October 3, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Melchers, F., E. ten Boekel, T. Yamagami, J. Andersson, A. Rolink. 1999. The roles of preB and B cell receptors in the stepwise allelic exclusion of mouse IgH and L chain gene loci. Semin. Immunol. 11:307.[Medline]
  2. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190:75.[Abstract/Free Full Text]
  3. Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton, R. R. Hardy. 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167:6834.[Abstract/Free Full Text]
  4. Hartley, S. B., J. Crosbie, R. Brink, A. B. Kantor, A. Basten, C. C. Goodnow. 1991. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 353:765.[Medline]
  5. Erikson, J., M. Z. Radic, S. A. Camper, R. R. Hardy, C. Carmack, M. Weigert. 1991. Expression of anti-DNA immunoglobulin transgenes in non-autoimmune mice. Nature 349:331.[Medline]
  6. Nemazee, D., K. Buerki. 1989. Clonal deletion of autoreactive B lymphocytes in bone marrow chimeras. Proc. Natl. Acad. Sci. USA 86:8039.[Abstract/Free Full Text]
  7. Casellas, R., T. A. Shih, M. Kleinewietfeld, J. Rakonjac, D. Nemazee, K. Rajewsky, M. C. Nussenzweig. 2001. Contribution of receptor editing to the antibody repertoire. Science 291:1541.[Abstract/Free Full Text]
  8. Nemazee, D. A., K. Burki. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337:562.[Medline]
  9. Mandik-Nayak, L., S. Seo, A. Eaton-Bassiri, D. Allman, R. R. Hardy, J. Erikson. 2000. Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells. J. Immunol. 164:1161.[Abstract/Free Full Text]
  10. Santulli-Marotto, S., M. W. Retter, R. Gee, M. J. Mamula, S. H. Clarke. 1998. Autoreactive B cell regulation: peripheral induction of developmental arrest by lupus-associated autoantigens. Immunity 8:209.[Medline]
  11. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
  12. Tsao, B. P., A. Chow, H. Cheroutre, Y. W. Song, M. E. McGrath, M. Kronenberg. 1993. B cells are anergic in transgenic mice that express IgM anti-DNA antibodies. Eur. J. Immunol. 23:2332.[Medline]
  13. Dammers, P. M., A. Visser, E. R. Popa, P. Nieuwenhuis, F. G. Kroese. 2000. Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. J. Immunol. 165:6156.[Abstract/Free Full Text]
  14. Martin, F., J. F. Kearney. 2000. Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 12:39.[Medline]
  15. Arnold, L. W., C. A. Pennell, S. K. McCray, S. H. Clarke. 1994. Development of B-1 cells: segregation of phosphatidyl choline-specific B cells to the B-1 population occurs after immunoglobulin gene expression. J. Exp. Med. 179:1585.[Abstract/Free Full Text]
  16. Hayakawa, K., M. Asano, S. A. Shinton, M. Gui, D. Allman, C. L. Stewart, J. Silver, R. R. Hardy. 1999. Positive selection of natural autoreactive B cells. Science 285:113.[Abstract/Free Full Text]
  17. Kurosaki, T.. 2000. Functional dissection of BCR signaling pathways. Curr. Opin. Immunol. 12:276.[Medline]
  18. Bannish, G., E. M. Fuentes-Panana, J. C. Cambier, W. S. Pear, J. G. Monroe. 2001. Ligand-independent signaling functions for the B lymphocyte antigen receptor and their role in positive selection during B lymphopoiesis. J. Exp. Med. 194:1583.[Abstract/Free Full Text]
  19. Levine, M. H., A. M. Haberman, D. B. Sant’Angelo, L. G. Hannum, M. P. Cancro, C. A. Janeway, Jr, M. J. Shlomchik. 2000. A B-cell receptor-specific selection step governs immature to mature B cell differentiation. Proc. Natl. Acad. Sci. USA 97:2743.[Abstract/Free Full Text]
  20. Grandien, A., Y. Modigliani, A. Freitas, J. Andersson, A. Coutinho. 1994. Positive and negative selection of antibody repertoires during B-cell differentiation. Immunol. Rev. 137:53.[Medline]
  21. Gu, H., D. Tarlinton, W. Muller, K. Rajewsky, I. Forster. 1991. Most peripheral B cells in mice are ligand selected. J. Exp. Med. 173:1357.[Abstract/Free Full Text]
  22. Su, T. T., D. J. Rawlings. 2002. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J. Immunol. 168:2101.[Abstract/Free Full Text]
  23. Su, T. T., B. Guo, Y. Kawakami, K. Sommer, K. Chae, L. A. Humphries, R. M. Kato, S. Kang, L. Patrone, R. Wall, et al 2002. PKC-{beta} controls I{kappa}B kinase lipid raft recruitment and activation in response to BCR signaling. Nat. Immunol. 3:780.[Medline]
  24. Cyster, J. G., J. I. Healy, K. Kishihara, T. W. Mak, M. L. Thomas, C. C. Goodnow. 1996. Regulation of B-lymphocyte negative and positive selection by tyrosine phosphatase CD45. Nature 381:325.[Medline]
  25. Buhl, A. M., D. Nemazee, J. C. Cambier, R. Rickert, M. Hertz. 2000. B-cell antigen receptor competence regulates B-lymphocyte selection and survival. Immunol. Rev. 176:141.[Medline]
  26. Ye, J., S. K. McCray, S. H. Clarke. 1996. The transition of pre-BI to pre-BII cells is dependent on the VH structure of the µ/surrogate L chain receptor. EMBO J. 15:1524.[Medline]
  27. Tatu, C., J. Ye, L. W. Arnold, S. H. Clarke. 1999. Selection at multiple checkpoints focuses VH12 B cell differentiation toward a single B-1 cell specificity. J. Exp. Med. 190:903.[Abstract/Free Full Text]
  28. Tatu, C., S. H. Clarke. 2000. Selective maturation of VH12 B cells in the spleen enriches for anti-phosphatidyl choline B cells: evidence for receptor editing. Curr. Top. Microbiol. Immunol. 252:77.[Medline]
  29. Wang, H., J. Ye, L. W. Arnold, S. K. McCray, S. H. Clarke. 2001. A VH12 transgenic mouse exhibits defects in pre-B cell development and is unable to make IgM+ B cells. J. Immunol. 167:1254.[Abstract/Free Full Text]
  30. Mandik-Nayak, L., A. Bui, H. Noorchashm, A. Eaton, J. Erikson. 1997. Regulation of anti-double-stranded DNA B cells in nonautoimmune mice: localization to the T-B interface of the splenic follicle. J. Exp. Med. 186:1257.[Abstract/Free Full Text]
  31. Benschop, R. J., K. Aviszus, X. Zhang, T. Manser, J. C. Cambier, L. J. Wysocki. 2001. Activation and anergy in bone marrow B cells of a novel immunoglobulin transgenic mouse that is both hapten specific and autoreactive. Immunity 14:33.[Medline]
  32. Borrero, M., S. H. Clarke. 2002. Low-affinity anti-Smith antigen B cells are regulated by anergy as opposed to developmental arrest or differentiation to B-1. J. Immunol. 168:13.[Abstract/Free Full Text]
  33. Santulli-Marotto, S., Y. Qian, S. Ferguson, S. H. Clarke. 2001. Anti-Sm B cell differentiation in Ig transgenic MRL/Mp-lpr/lpr mice: altered differentiation and an accelerated response. J. Immunol. 166:5292.[Abstract/Free Full Text]
  34. Cooke, M. P., A. W. Heath, K. M. Shokat, Y. Zeng, F. D. Finkelman, P. S. Linsley, M. Howard, C. C. Goodnow. 1994. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179:425.[Abstract/Free Full Text]
  35. Cyster, J. G., C. C. Goodnow. 1995. Protein tyrosine phosphatase 1C negatively regulates antigen receptor signaling in B lymphocytes and determines thresholds for negative selection. Immunity 2:13.[Medline]
  36. Noorchashm, H., A. Bui, H. L. Li, A. Eaton, L. Mandik-Nayak, C. Sokol, K. M. Potts, E. Pure, J. Erikson. 1999. Characterization of anergic anti-DNA B cells: B cell anergy is a T cell-independent and potentially reversible process. Int. Immunol. 11:765.[Abstract/Free Full Text]
  37. Healy, J. I., R. E. Dolmetsch, L. A. Timmerman, J. G. Cyster, M. L. Thomas, G. R. Crabtree, R. S. Lewis, C. C. Goodnow. 1997. Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6:419.[Medline]
  38. Allman, D. M., S. E. Ferguson, V. M. Lentz, M. P. Cancro. 1993. Peripheral B cell maturation. II. Heat-stable antigenhi splenic B cells are an immature developmental intermediate in the production of long-lived marrow-derived B cells. J. Immunol. 151:4431.[Abstract]
  39. Ye, J., S. K. McCray, S. H. Clarke. 1995. The majority of murine VH12-expressing B cells are excluded from the peripheral repertoire in adults. Eur. J. Immunol. 25:2511.[Medline]
  40. Pelanda, R., S. Schwers, E. Sonoda, R. M. Torres, D. Nemazee, K. Rajewsky. 1997. Receptor editing in a transgenic mouse model: site, efficiency, and role in B cell tolerance and antibody diversification. Immunity 7:765.[Medline]
  41. Russell, D. M., Z. Dembic, G. Morahan, J. F. Miller, K. Burki, D. Nemazee. 1991. Peripheral deletion of self-reactive B cells. Nature 354:308.[Medline]
  42. Young, F., B. Ardman, Y. Shinkai, R. Lansford, T. K. Blackwell, M. Mendelsohn, A. Rolink, F. Melchers, F. W. Alt. 1994. Influence of immunoglobulin heavy- and light-chain expression on B-cell differentiation. Genes Dev. 8:1043.[Abstract/Free Full Text]
  43. Freitas, A. A., B. Rocha. 2000. Population biology of lymphocytes: the flight for survival. Annu. Rev. Immunol. 18:83.[Medline]
  44. Mackay, F., J. L. Browning. 2002. BAFF: a fundamental survival factor for B cells. Nat. Rev. Immunol. 2:465.[Medline]
  45. Qian, Y., C. Santiago, M. Borrero, T. F. Tedder, S. H. Clarke. 2001. Lupus-specific antiribonucleoprotein B cell tolerance in nonautoimmune mice is maintained by differentiation to B-1 and governed by B cell receptor signaling thresholds. J. Immunol. 166:2412.[Abstract/Free Full Text]
  46. Qian Y., Wang H., and S. H. Clarke. Impaired clearance of apoptotic cells induces the activation of autoreactive anti-Sm marginal zone and B-1 B cells. J. Immunol. In press.



This article has been cited by other articles:


Home page
 JEMHome page
A. Meyer-Bahlburg, S. F. Andrews, K. O.A. Yu, S. A. Porcelli, and D. J. Rawlings
Characterization of a late transitional B cell population highly sensitive to BAFF-mediated homeostatic proliferation
J. Exp. Med., January 21, 2008; 205(1): 155 - 168.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Wang, J. Feng, C.-F. Qi, Z. Li, H. C. Morse III, and S. H. Clarke
Transitional B Cells Lose Their Ability to Receptor Edit but Retain Their Potential for Positive and Negative Selection
J. Immunol., December 1, 2007; 179(11): 7544 - 7552.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. M. Fuentes-Panana, G. Bannish, F. G. Karnell, J. F. Treml, and J. G. Monroe
Analysis of the Individual Contributions of Ig{alpha} (CD79a)- and Igbeta (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation
J. Immunol., December 1, 2006; 177(11): 7913 - 7922.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. William, C. Euler, N. Primarolo, and M. J. Shlomchik
B Cell Tolerance Checkpoints That Restrict Pathways of Antigen-Driven Differentiation
J. Immunol., February 15, 2006; 176(4): 2142 - 2151.
[Abstract] [Full Text] [PDF]

Home page
 Int ImmunolHome page
L. Heltemes-Harris, X. Liu, and T. Manser
An antibody VH gene that promotes marginal zone B cell development and heavy chain allelic inclusion
Int. Immunol., November 1, 2005; 17(11): 1447 - 1461.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. Edry and D. Melamed
Receptor Editing in Positive and Negative Selection of B Lymphopoiesis
J. Immunol., October 1, 2004; 173(7): 4265 - 4271.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
M. P. Cancro and J. F. Kearney
B Cell Positive Selection: Road Map to the Primary Repertoire?
J. Immunol., July 1, 2004; 173(1): 15 - 19.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, H.
Right arrow Articles by Clarke, S. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, H.
Right arrow Articles by Clarke, S. H.

HOME HELP