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The Journal of Immunology, 2006, 177: 7913-7922.
Copyright © 2006 by The American Association of Immunologists, Inc.

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 Maturation1

Ezequiel M. Fuentes-Pananá, Gregory Bannish, Fredrick G. Karnell, John F. Treml and John G. Monroe2

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The individual contribution of Ig{alpha} and Igbeta for BCR-triggered fates is unclear. Prior evidence supports conflicting ideas concerning unique as well as redundant functions for these proteins in the context of BCR/pre-BCR signaling. Part of this ambiguity may reflect the recent appreciation that Ig{alpha} and Igbeta participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Ig{alpha} and Igbeta under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or two copies of the cytoplasmic domains of either Ig{alpha} or Igbeta and Ig{alpha}/Igbeta heterodimers with targeted Tyr->Phe modifications. The ability of these proteins to act as surrogate receptors and trigger early bone marrow and peripheral B cell maturation was tested in RAG2–/– primary pro-B cell lines and in gene transfer experiments in the µMT mouse model. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of the Ig{alpha}/Igbeta ITAM domain are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We found that the ligand-independent tonic signal is sufficient to drive development into mature follicular B cells and both Ig{alpha} and Igbeta chains supported formation of this population. In contrast, neither marginal zone nor B1 mature B cell subsets develop from bone marrow precursors under conditions where only tonic signals are generated.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The BCR is a complex composed of a ligand-binding unit formed by the Ig H and L chains and a signaling unit consisting of a heterodimer of Ig{alpha} (CD79a) and Igbeta (CD79b) (1, 2, 3). Formation of a mature immunocompetent B cell involves an ordered process whose steps are defined by the sequential expression and assembly of the individual components of the BCR (4, 5, 6, 7). Continued maturation and survival of mature B cells is dependent upon the ability of the pre-BCR and mature BCR to generate signals through the Ig{alpha}/Igbeta heterodimer at critical checkpoints during developmental progression (2). Studies support the existence of two mechanisms of generating signals through Ig{alpha}/Igbeta complexes. The first is Ag dependent, requires BCR aggregation, and is associated with effector cell generation from mature B cells and negative selection of immature B cells (8, 9). The second mechanism is Ag independent and although less well-biochemically characterized, is argued to be relevant for pre-BCR-dependent maturation (10, 11) and for the BCR-dependent survival of peripheral B cells (12, 13, 14). These latter signals are often referred to as ligand-independent or tonic signals to distinguish them from those triggered as a consequence of ligand (Ag)-induced receptor oligomerization (15, 16, 17, 18).

The mechanism by which the receptor initiates tonic signaling is currently unclear with some evidence supporting the need for receptor oligomerization (16, 17, 18). Because the pre-BCR lacks the Ag-binding domain, it has been reported that positive selection at the pro-B to pre-B transition might be dependent on pre-BCR self-interactions or pre-BCR interactions with nonpolymorphic ligands present on the surrounding stromal cells in the bone marrow (16, 17, 18). These studies targeted the surrogate L chain (SLC)3 protein {lambda}5 as the structure responsible for such interactions. Because pre-B cells can be generated in genetic knockouts of the SLC proteins and signaling competent pre-BCRs have been observed in cells lacking SLC (19, 20, 21), it can be argued that such mechanisms of pre-BCR aggregation are not essential to initiate tonic signaling and to trigger developmental progression at the pro-B to pre-B transition. In contrast, we have found that the cytoplasmic domains of Ig{alpha} and Igbeta in the absence of extracellular or transmembrane domains are sufficient to trigger B cell development to transitional stages supporting a model where an unligated receptor is sufficient for this function. Accordingly, fluorescence resonance energy transfer studies support that the BCR is present in a monomeric form in resting B cells (22) and Src tyrosine kinases are found in close association with the unligated receptor (23, 24, 25).

Both Ag-dependent and -independent (tonic) signals require specific motifs in the cytoplasmic tails of Ig{alpha} and Igbeta known as ITAMs (11, 25, 26, 27). Tyrosine residues within the ITAM domains are phosphorylated by protein tyrosine kinases and then act as docking sites where a higher order signaling complex is assembled (28, 29, 30, 31, 32, 33, 34, 35, 36, 37). Ig{alpha} contains two additional tyrosines flanking the ITAM domain, positions 176 and 204, which are also phosphorylated upon receptor-ligand engagement and participate in the formation of the signalosome (32, 33, 38). Signaling downstream of the Ig{alpha} and Igbeta complex leads to activation of multiple signaling pathways resulting in changes in patterns of gene expression and cytoskeleton reorganization (39, 40, 41).

Ig{alpha} and Igbeta are evolutionarily conserved proteins that fulfill a similar function in mice, birds, and humans. The requirement for two highly homologous proteins containing almost identical ITAMs is presently unclear, and studies to define the specific roles of these proteins during B cell development have yielded conflicting results. Early studies using immunoprecipitation and binding assays from cultured cell lines suggested a specific role for Ig{alpha} in binding fyn kinase and the adapter protein B cell linker (BLNK) (32, 33, 38, 42, 43). However, more recent studies using recombinant mice expressing a sole copy of either Ig{alpha} or Igbeta or copies of both proteins where one of the ITAMs is nonfunctional due to introduced mutations showed that each protein is sufficient to trigger development to the immature B cell stage (44, 45, 46, 47, 48, 49, 50, 51, 52). In these studies, a clear decrease in the efficiency of formation of pre-B and immature B cell bone marrow populations was found with BCRs associated with only Igbeta relative to those associated with only Ig{alpha} or wild-type (WT) receptors, suggesting an increased potency for Ig{alpha} over Igbeta (44, 45, 46, 47, 48, 49, 50, 51, 52). Accordingly, developing B cells carrying Ig{alpha} or Igbeta alone tend to compensate by adjusting receptor expression levels at the plasma membrane (48, 50). Nevertheless, the sufficiency of receptors composed of only a functional Ig{alpha} or Igbeta chain to form pre-B and immature B cell populations in vivo argues that both proteins fulfill overlapping functions at least during early stages of development. Thus, the need for these separate ITAM proteins remains unclear, perhaps reflecting differences in the need for Ag-dependent and -independent pre-BCR/BCR signals during development.

Importantly, B cell development in transgenic mice expressing only one intact chain, either Ig{alpha} or Igbeta, was arrested at the immature stage, illustrating the need for receptor-mediated signals to form and/or maintain mature B cell populations. Given the recent appreciation of both tonic and ligand-dependent signaling through pre-BCR and BCR complexes, it is possible that ligand-dependent and -independent mechanisms of receptor signaling differ in their importance at different stages of development and in their requirement of Ig{alpha} and Igbeta. Therefore, we considered that the conflicting evidence regarding the specificity of Ig{alpha} and Igbeta function might be explained by the fact that these proteins act differently during the ligand-independent phase of development in the bone marrow, compared with ligand-dependent development in peripheral lymphoid organs. Thus, Ig{alpha} and Igbeta may fulfill both individual as well as distinct roles at different points of development.

The present study sought to evaluate the importance of the aggregation-independent pre-BCR/BCR tonic signaling for bone marrow and peripheral B cell maturation and to define the individual requirement for Ig{alpha} and Igbeta under conditions where only the tonic signaling of the receptor was operative. In this regard, we have enforced expression in B cells of chimeric proteins containing one or two functional copies of either Ig{alpha} or Igbeta. These proteins act as surrogate pre-BCR/BCRs, and their ability to trigger developmental progression was compared. We considered that the threshold for a functional activity mediated by the pre-BCR/BCR might only be reached when two functional copies of either the Ig{alpha} or Igbeta ITAM domains are expressed together, and therefore the specificity conferred by these proteins can only be observed in these conditions. We report here that tonic signaling is sufficient to drive B cell development to the mature stage and that tonic signaling-driven B cells develop into mature follicular (FO) populations but fail to form marginal zone (MZ) and B1 populations. We also observe that either Ig{alpha} and Igbeta chains are sufficient to provide the positive signal to form FO B cells when two copies of their cytoplasmic chains are present.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

C57BL/6 (WT), µMT, and RAG2–/– mice were bred and maintained under pathogen-free conditions at the University of Pennsylvania, and experimental procedures in these animals were performed according to local and National Institutes of Health guidelines.

Oligonucleotides and construction of MAHB variants

Construction of MAHB and ITAMmut and their cloning into the MIGR1 retroviral vector have been previously described (11). Either MAHB or ITAMmut served as templates to synthesize all the variants used in this study. As explained below, the oligonucleotides used to make MAHB variants were as follows: Ig{alpha}-Kpn-S, 5'-CGCGGGTACCAGGAAACGGTGGCAAAATGAG-3'; Ig{alpha}-Eco-A, 5'-CGGCGAATTCATGGCTTTTCCAGCTGGGC-3'; Igbeta-Bam-S2, 5'-CGCGGATCCGACAAGGATGACGGCAAGGCT-3'; Igbeta-Xba-A, 5'-GCGAGATCTAGATTCCTGGCCTGGATGCTC-3'; HA-A2, 5'-GCGCGAATTCAAGCGTAGTCTGGGACGTCGTATGGGTA-3'; Myr-S2, 5'-GCCGGAATTCCACCATGGGCTGTGTCTGCAGCT-3'; Ig{alpha}Y176S, 5'-ATGCCAGATGACTTTGAAGATGAA-3'; Ig{alpha}Y176, 5'-TTCATCTTCAAAGTCATCTGGCA-3'; Ig{alpha}Y204S, 5'-CTCCAGGGCACCTTTCAGGATGTGGGC-3'; and Ig{alpha}Y204A, 5'-GCCCACATCCTGAAAGGTGCCCTGGAG-3'.

As previously described (11), each domain of parental MAHB and ITAM genes is flanked by unique restriction sites. To synthesize the MAmutB, an EcoRI-KpnI fragment containing MAH was excised from MAHB and replaced with the homologous sequence derived from the ITAMmut. Similarly, to synthesize MAHBmut, the KpnI-EcoRI site encoding the Igbeta cytoplasmic domain from MAHB was replaced by the homologous sequence from ITAMmut.

MBHB. To subclone MBHB into MIGR1, we first deleted a NotI-SmaI fragment from vector pBluescript SK (Stratagene), end filled, and religated to form SK{Delta}NS. An MAHB EcoRI fragment was ligated into the EcoRI site of SK{Delta}NS to form SK{Delta}NSEcoEcoMAHB. The Ig{alpha} domain of MAHB was then excised with BamHI and HindIII. An Igbeta cytoplasmic PCR fragment was synthesized from MAHB using IgbetaBamS2 and IgbetaXba-A oligonucleotides, cut with BamHI and XbaI, purified, and ligated into the BamHI and HindIII sites from SK{Delta}NSEcoEcoMAHB to form SK{Delta}NSEcoEcoMBHB. MBHB was excised with EcoRI and ligated into MIGR1 at the EcoRI site.

MBH. The MBH sequence was amplified by PCR using MBHB template and MyrS2 and HA-A2 oligonucleotides. The resulting fragment was cut with EcoRI and ligated into MIGR1.

MAH. An MAH fragment was amplified by PCR from MAHB using MyrS2 and HA-A2 oligonucleotides. The MAH fragment was digested with EcoRI, purified, and ligated into MIGR1.

MAHA. An Ig{alpha} fragment was amplified from MAHB using Ig{alpha}-KpnS and Ig{alpha}-EcoA oligonucleotides, and the resulting fragment was digested with KpnI at the 5' end and EcoRI at the 3' end to form 5'-Kpn-Ig{alpha}-EcoRI-3'. Separately, an MAH fragment was excised from MAHB with EcoRI and KpnI to form 5'-EcoRI-MAH-KpnI-3'. These two fragments were ligated into the EcoRI site of MIGR1 in a three-way ligation.

MAnonITAMmutHB. The non-ITAM tyrosine residues of Ig{alpha} (Y176, Y204) were mutated to phenylalanine using PCR mutagenesis and overlap PCR, with the primers Ig{alpha}-ITAMmt-S and Ig{alpha}-ITAMmt-A oligonucleotides. Residues were then excised with EcoRI and KpnI to form 5'-EcoRI-MA(176,204)H-KpnI-3'. The Igbeta domain of MAHB was excised with KpnI and EcoRI to form 5'-KpnI-Igbeta-EcoRI-3'. These two fragments were ligated into the EcoRI site of MIGR1 in a three-way ligation.

Abs and flow cytometry analysis

Flow cytometric analysis was used to analyze the expression levels of markers to define primary pro-B/pre-B and B cells from recipient mice after adoptive transfers. Erythrocyte-depleted samples were incubated with fluorescence-tagged Abs against the development stage markers B220-allophycocyanin, CD43-PE, CD22-PE, CD23-PE, CD23-biotin CD21-PE, AA4.1-allophycocyanin, and CD5-PE (BD Pharmingen), and with anti-hemagglutinin (HA) (clone 3F10; Roche). After staining with CD23-biotin, cells were treated with Streptavidin-Red 670 (Invitrogen Life Technologies) and then fixed in 1% paraformaldehyde. For intracellular staining with anti-HA, cells were fixed first with a 4% formaldehyde solution and then permeabilized with 0.1% saponin. During the pro-B to pre-B in vitro transition, cells were also treated with the fluorescent dye TOPRO-3 iodide to exclude for dead cells, and cells were analyzed without fixation.

Retroviral infection of progenitor-enriched cultures and adoptive transfers

These procedures have been previously described (11, 53). Briefly, bone marrow cells from 5-fluorouracil-treated 6- to 8-wk-old female µMT mice (The Jackson Laboratory) were spin infected with the MIGR1 retroviral vectors. Infections were conducted on days 2 and 3 after harvest in medium containing IL-3 (6 ng/ml), IL-6 (10 ng/ml), stem cell factor (100 ng/ml) (R&D systems), and 5% WEHI-3B-conditioned supernatant. A total of 1 x 106 cells/mouse were injected into lethally irradiated (950 rad) syngeneic mice. Mice were sacrificed 4–6 wk posttransfer, and B cells obtained from bone marrow, spleen, and peritoneal cavity were analyzed by flow cytometry.

Generation and analysis of pro-B cells in culture

Long-term pro-B cell cultures were derived from RAG2–/– (Taconic Farms) mice and were maintained in Iscove’s complete medium (Invitrogen Life Technologies) supplemented with 10% FCS, 1% NEN mix (Invitrogen Life Technologies), 1% OPI mix (Invitrogen Life Technologies), 50 mM 2-ME, and 5% supernatant obtained from IL-7-producing J558L cells as previously described (54, 55). After culture of bone marrow cells in IL-7-containing medium, cells were analyzed daily by flow cytometry for surface expression of developmental markers to assess progression into pro-B cell lines (B220posCD43posBP1posCD22negCD25neg). Consistently, by day 6 of culture, >99% of the cells were pro-B cells and remained in this phenotype for the duration of this study. These RAG2–/– pro-B cell lines were then transduced with retroviral vectors and sorted to obtain pure GFP-expressing populations. The studies depicted here were performed with pro-B cells that have been maintained in IL-7 for less than a year. For analyses of developmental progression, cells were depleted of IL-7 either for 3 days or daily during 4 days to test the kinetics of the pro-B to pre-B transition. Cells were then stained for FACS analysis using Abs against stage-specific differentiation markers as well as TOPRO-3 (Molecular Probes) to identify dead cells. Cells capable of developmental progression were identified by their phenotype (B220posCD43negBP1posCD22posCD25pos).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Two functional ITAMs derived from either Ig{alpha} or Igbeta are required to trigger in vitro development through the pro-B to pre-B transition

We previously developed an experimental model to isolate and study tonic pre-BCR- and BCR-signaling processes (11). This model involves the expression of a chimeric protein containing the cytoplasmic domains of Ig{alpha} and Igbeta separated by an epitope tag from the HA protein. This protein is termed MAHB and is targeted to the inner leaflet of the cytoplasmic membrane by the N-terminal 10 aa of the protein kinase Lck. The ability of MAHB to model ligand- and aggregation-independent functions of the pre-BCR and BCR has been described (6, 11). Because of its ability to isolate the ligand-independent tonic-signaling functions of Ig{alpha}/Igbeta complexes, MAHB provides an opportunity to evaluate the individual contributions of Ig{alpha} and Igbeta tonic signaling for bone marrow and peripheral B cell development.

The first question addressed was whether Ig{alpha} and Igbeta provide distinct functions during the early stages of B cell development or whether the normal coexpression of both proteins reflects a requirement for two ITAMs to achieve a threshold for pre-BCR function. To achieve this goal, MAHB variants containing single or double copies of either Ig{alpha} or Igbeta were constructed (Fig. 1A) and expressed in primary RAG2–/– pro-B cells. Fig. 1B compares the relative expression of fusion protein, as determined by HA levels, with that of GFP expressed from the bicistronic retroviral message. This analysis verifies that the levels of expression of the fusion protein and GFP are proportional. Thus, the expression of fusion protein can be grossly inferred from the GFP levels expressed by the pro-B population. We also found that most of the pro-B cell lines expressed similar levels of fusion protein. Only the MBHB variant was consistently found expressing higher levels of both chimeric and GFP proteins (bottom right panel). The cellular localization of the fusion proteins was addressed by immunofluorescence microscopy using an anti-HA Ab. All fusion proteins used throughout this study were preferentially associated with the plasma membrane (data not shown).


Figure 1
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  		FIGURE 1. Expression of surrogate Ig{alpha}/Igbeta receptors. A, BCR/pre-BCR surrogate receptors were constructed containing one or two copies of either Ig{alpha} or Igbeta cytoplasmic domains. Similar to parental MAHB, these variants are targeted to the cytoplasmic membrane by the Lck myristoylation/palmitoylation site. They were cloned into the MIGR1 retroviral vector and transduced into primary RAG2–/– pro-B cells (B220posCD43posBP1posCD22negCD25neg). Expression of GFP from the MIGR1 bicistronic message was used to sort for chimeric protein-expressing cells. B, Fusion protein expression levels were compared with GFP expression by flow cytometry analysis. Expression of the fusion protein was measured with an Ab against the epitope tag derived from the influenza HA protein.

 
Fig. 2A depicts an analysis of the number and relative frequency of pro-B cells undergoing transition to the pre-B stage following removal of IL-7. Transition is evaluated by the differential expression of CD22 and CD43 (as described in Refs. 5, 54 and 56). The actual number of CD22posCD43neg pre-B cells present at the beginning and end of the experiment is shown. In this figure, pre-B cells are only found in conditions where MAHB is expressed, illustrating the importance of the Ig{alpha}/Igbeta-mediated tonic signal for this transition. We typically observed 40–70% pre-B cells in the MAHBpos cultures after 2–3 days of IL-7 depletion, while the rest of the cells remain in the CD22negCD43pos pro-B stage. A similar pre-B/pro-B cell ratio is found in cultures that express an Ig H chain transgene, indicating a similar efficiency of transition mediated by MAHB and the WT pre-BCR (54). Fig. 2B shows the same analysis for MAHB and variants, where CD43 down-regulation was used to identify pre-B cells (plotted in Fig. 2C). In the experiment depicted in this study (n >10), 68% of the MAHBpos cells in culture are in the pre-B stage, whereas in MAHApos and MBHBpos cultures, pre-B cells represent 51 and 36% of the total population, respectively. We observed transition to the pre-B stage only in cells expressing two ITAMs from either Ig{alpha} or Igbeta. The ability of MAHA and MBHB variants to induce progression through the pro-B to pre-B checkpoint is also illustrated in Fig. 2D, which documents the kinetics of transition during 4 days of IL-7 depletion. In the cultures expressing a single copy of Ig{alpha} and/or Igbeta (only one ITAM), <5% of pre-B cells were found at any time after IL-7 depletion, illustrating the inability of these cells to progress beyond the pro-B stage.


Figure 2
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  		FIGURE 2. In vitro developmental capacity of surrogate pre-BCRs containing single or double copies of Ig{alpha} or Igbeta. Pro-B cell cultures were obtained after culturing RAG2–/– bone marrow-derived cells in the presence of IL-7. Pro-B cell lines expressing surrogate receptors were obtained by MIGR1 retroviral transduction and GFP sorting of a pure transduced population. A, Analysis of pro-B-pre-B progression comparing cell lines transduced with parental MAHB and the MIGR1 empty vector. Pro-B cell cultures were deprived of IL-7 for 3 days, and then cells were stained for subsequent flow cytometric analysis. Panels depict FACS analysis of CD22 vs CD43 expression to determine the frequency of B220pos cells progressing to the pre-B stage (CD22posCD43neg). Pre-B cell numbers before and after the IL-7 depletion are given in the adjacent table. B, The capacity of the single and double copy Ig{alpha}/Igbeta surrogate pre-BCRs to induce the pro-B to pre-B transition was analyzed after sorting for equal levels of GFP expression. Cell lines were depleted of IL-7 for 3 days and then stained for FACS analysis. Panel compares transition to the pre-B stage (CD43neg boxed cells) and GFP expression levels. C, Plot depicting the frequency of pre-B cells in culture after 3 days of IL-7 depletion. D, Analysis of the kinetics of the pro-B to pre-B transition after 4 days in culture without IL-7.

 
In summary, this analysis argues that the threshold for pre-BCR signaling at the pro-B to pre-B transition cannot be achieved with a single ITAM domain. However, two ITAM copies, either in the context of Ig{alpha} or Igbeta sequences, are able to generate the signals to bypass this developmental checkpoint. Importantly, neither MAHA nor MBHB fusion proteins were as efficient as the fusion of both Ig{alpha} and Igbeta domains (MAHB), suggesting that although Ig{alpha} and Igbeta have overlapping functions, the optimal efficiency of the receptor is only achieved when both proteins are present, thereby arguing for yet unknown individual contributions of Ig{alpha} and Igbeta early in development.

To extend this structural analysis of Ig{alpha} and Igbeta sequences, we synthesized MAHB variants that carry targeted Tyr->Phe substitutions. The following MAHB variants were constructed (depicted in Fig. 3A): 1) the ITAMmut variant, where all ITAM-associated tyrosines on Ig{alpha} and Igbeta were modified; 2–3) the MAmutHB and MAHBmut variants, where the two ITAM tyrosines of Ig{alpha} or Igbeta were modified, respectively; and 4) the MAnonITAMmutHB variant, where the two non-ITAM tyrosines of Ig{alpha} were modified (Tyr176 and Tyr204). The MIGR1 retrovirus was also used to transduce these MAHB derivatives into RAG2–/– primary pro-B cells. Fig. 3B compares the levels of chimeric protein expression. As can be seen, the levels of expression of chimeric protein and GFP are proportional and there is little detectable difference in the steady state expression levels of the individual variants relative to each other. Fig. 3C depicts an analysis of the pro-B to pre-B transition after IL-7 depletion of sorted pro-B cells expressing equal GFP levels. We observed an ~9-fold decrease of the MAmutHB and MAHBmut variants, relative to the ability of parental MAHB, to trigger the pro-B to pre-B transition. This result further illustrates the importance of two intact ITAM domains to overcome the pro-B to pre-B developmental checkpoint. In contrast, we found a 3-fold decreased efficiency in the variant where both non-Ig{alpha} ITAM tyrosines were modified, arguing that although these tyrosine are important, most of the Ig{alpha} and Igbeta function resides in the ITAM domains. The fraction of CD43neg pre-B cells in each culture is plotted in Fig. 4. We also tested variants of MAHA and MBHB where the second ITAM domain carries Tyr->Phe substitutions (MAHAmut and MBHBmut variants, respectively). Because these variants were also unable to trigger the pro-B to pre-B transition they were not included in the results presented here. However, the inability of these constructs to drive the pro-B to pre-B developmental progression also argues that two functional ITAMs are required for this transition.


Figure 3
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  		FIGURE 3. In vitro developmental capacity of surrogate pre-BCRs containing Ig{alpha}/Igbeta targeted Tyr->Phe substitutions. A, The parental MAHB surrogate receptor was modified to carry the targeted Tyr to Phe modification within the Ig{alpha}/Igbeta ITAM domains or outside the ITAM domain (Ig{alpha} tyrosines 176 and 204). These constructs were then cloned into the MIGR1 retroviral vector and transduced into primary RAG2–/– pro-B cells. B, Fusion protein expression levels (measured with an Ab against the HA tag) were compared with GFP expression by flow cytometry analysis. C, Analysis of the pro-B to pre-B transition was analyzed after depriving the cultures of IL-7 for 3 days. Panels depict FACS analysis of CD43 vs GFP expression levels. The pre-B cell population (CD43neg) is boxed in these panels, and the frequency of this population is plotted in D.

 

Figure 4
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  		FIGURE 4. Development of mature B cells by either Ig{alpha}- or Igbeta-mediated tonic signaling. Bone marrow progenitor cells from µMT mice were transduced either with MAHB or the double Ig{alpha}/Igbeta copy variants MAHA and MBHB. Transduced progenitors were used to reconstitute lethally irradiated syngeneic µMT mice, and 4 wk postreconstitution, spleen cells were analyzed by flow cytometry. Splenocytes were stained for surface expression of AA4.1, CD23, and the pan B cell marker B220 to identify the transitional and mature B cell populations (T1, T2, and M boxed populations, respectively). Cells were gated on the transduced (GFPpos) B220pos B cell population. Normal development in WT C57BL/6 mice is also shown.

 
Tonic signals originated from either Ig{alpha} or Igbeta chains are sufficient to generate a peripheral mature B cell pool

We have previously shown that MAHB is able to provide signals that allow for maturation to the peripheral transitional immature stage in BCR-deficient B cells from µMT and RAG2–/– mice (11). Therefore, we evaluated whether the MAHB variants were also able to induce B cell developmental progression in vivo using the µMT mouse model. The MAHB variants were expressed in bone marrow-derived hemopoietic progenitors that were then transferred into lethally irradiated syngeneic recipients. Four weeks after transfer, recipient mice were sacrificed, and bone marrow and splenic B cells were analyzed by flow cytometry. We did not observe developing B cells in mice transduced with fusion proteins containing only one functional ITAM, either single copy or Tyr->Phe mutant variants (data not shown). B cells expressing these variants remained in the CD43posCD22neg pro-B stage, consistent with their observed in vitro developmental potential (see Figs. 2B and 3C). However, developing B cells were found in bone marrow and spleens of mice reconstituted with B cells expressing the Ig{alpha}/Ig{alpha} and Igbeta/Igbeta fusion proteins. Similar to MAHB, these variants were able to trigger development through the CD23pos transitional 2 (T2) stage in spleen (Fig. 4). B cell populations in spleen can be divided into immature T1 and T2/T3 and mature cells by their differential expression of surface proteins CD23 and AA4.1. Transgenic models where BCRs are assembled expressing only one functional ITAM of either Ig{alpha} or Igbeta are arrested at the T2 to mature transition (44, 45, 46, 47, 49, 51, 52). These studies have helped to identify another developmental checkpoint at the T2/T3 to mature transition, which is also dependent on the capacity of the expressed BCR to transduce signals for positive selection. Unlike the pro-B to pre-B checkpoint driven by the pre-BCR, the receptor at this transition is fully competent to recognize and bind Ags. Additionally, because Ig{alpha} and Igbeta transgenic models are arrested at this transition, it has been speculated that it is not until the T2/T3 stage that Ig{alpha} and Igbeta play nonredundant functions (44, 45, 46, 47, 49, 51, 52). Therefore, it was important to evaluate whether the receptor-mediated basal signal is also sufficient to bypass the T2/T3 to mature checkpoint. Fig. 4 shows the phenotypic analysis of spleen cells derived from the recipient mice 4 wk after adoptive transfer. For this figure, cells were gated on B220pos B cells in the WT C57BL/6 mouse and on B220posGFPpos B cells in the transduced recipient µMT mice, and T1, T2, and mature (M) B cell populations are indicated by marked squares. This analysis shows that the BCR-mediated basal signal is sufficient to drive development into mature B cells (B220posCD23posAA4.1neg), and importantly, both MAHA and MBHB fusion proteins are sufficient to transduce the developmental signal required to form this B cell pool.

BCR-mediated ligand-independent tonic signaling triggers development into FO mature B cells

Mature B cells consist of three anatomically, phenotypically, and functionally distinct populations: FO, MZ, and B1 B cells. Formation and maintenance of each one of these mature B cell populations seem to rely on a qualitatively and/or quantitatively different set of BCR-transduced signals (57, 58, 59, 60, 61). However, whether the type of BCR signaling required is ligand dependent or independent is presently unclear, with some evidence favoring requirement for low-level BCR-ligand interactions (62, 63, 64, 65). Therefore, we addressed whether the MAHA and/or MBHB fusion proteins could generate these mature B cell populations. FO and MZ mature B cells can be distinguished by their differential expression of CD21 and CD23 surface markers. Fig. 5A shows a flow cytometric analysis of the pattern of expression of these proteins in the mature B cell population of untreated WT C57BL/6 mice (AA4.1neg gate) and in transduced MIGR1->C57BL/6, MAHB-, MAHA-, and MBHB->µMT B cells (AA4.1negGFPpos gate). This analysis showed that tonic signaling through MAHB or either of the double Ig{alpha} or Igbeta variants was able to trigger development into FO mature B cells (CD21low-medCD23high) and thus generation of the FO pool is independent of either Ig{alpha}- or Igbeta-specific sequences. We were unable to detect MZ B cells (CD21highCD23low-neg) in spleens of mice reconstituted with parental MAHB or any of the variants tested. The MIGR1->C57BL/6 control indicates that retroviral insertion does not affect formation of the MZ B cell pool and therefore argues that the lack of this pool in the fusion protein-expressing B cells is most likely due to lack of a competent differentiation signal.


Figure 5
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  		FIGURE 5. Analysis of mature B cell populations. A, Splenocytes from adoptively transferred recipient mice were stained for expression of CD21, CD23, and AA4.1 proteins to analyze development into the mature FO, MZ, or B1 B cell populations (boxed populations). Only mature B cells are shown in WT C57BL/6 mouse (AA4.1pos cell gate) and MAHB-, MAHA-, and MBHB-transduced mice (AA4.1posGFPpos cell gate). As a control, the MIGR1 empty vector was transduced into WT C57BL/6 hemopoietic progenitors that then were used to reconstitute lethally irradiated µMT mice. Recipient MIGR1->C57BL/6 splenocytes are also gated in the AA4.1posGFPpos population. B, To address maturation into the B1 population, peritoneal cavity cells were stained for expression of the CD5 B1a marker and B220. The B1 population is boxed.

 
In addition to the analysis of the splenic B cell compartment, peritoneal cavity cells were taken from recipient mice and stained for detection of CD5 and B220 proteins. Fig. 5B shows that CD5pos and CD5neg B1 B cell populations were present in the WT mouse reconstituted with MIGR1 empty vector, while no B220pos B cells were found in peritoneal cavities of mice reconstituted with B cells expressing any of the surrogate receptors. In summary, these data argue that the MAHB-mediated tonic signal is only sufficient to trigger development into FO mature B cells and that this maturation signal seems to be independent of either Ig{alpha} or Igbeta unique sequences. Importantly, this study does not address whether a B1 population could be generated in similar circumstances using B cells of fetal origin.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We have previously developed a model that allows us to isolate and study the Ig{alpha}/Igbeta-derived signals that are not dependent on receptor aggregation and that can be originated outside of the lipid raft membrane compartment (tonic signaling) (11, 54, 55). In this study, we address the importance of the ligand-independent tonic signaling for the pre-BCR-dependent bone marrow and BCR-dependent peripheral B cell maturation and the specific requirement for the Ig{alpha} and Igbeta chains for this tonic receptor activity. Biochemical and genetic studies of the specific individual roles for Ig{alpha} and Igbeta in BCR signaling and B cell development have produced contradictory results. Because such studies of the specificity of Ig{alpha} and Igbeta were evaluated within receptors competent for ligand-induced aggregation, we considered that the specificity conferred by these proteins might reflect a differential requirement for ligand binding during development, and therefore we designed this study to examine this question under conditions where only tonic signaling was operative. Moreover, we tested surrogate receptors with single and double copies of either Ig{alpha} or Igbeta, considering that the physiological receptor’s activity might only be accomplished with two ITAM domains and therefore only under these conditions can the specificity of Ig{alpha} and Igbeta be revealed. Our data support that ligand-independent tonic signaling is sufficient to generate mature B cell populations of the FO origin but fail to generate MZ and B1 mature B cells. Both Ig{alpha} and Igbeta cytoplasmic chains were independently sufficient to form this mature FO B cell population but only when two intact copies of them are present. Importantly, although our results argue for overlapping functions for Ig{alpha} and Igbeta during the tonic signaling-induced B cell development, neither Ig{alpha} or Igbeta sequences alone were as efficient as the surrogate receptor carrying copies of both proteins. Thus, these data argue that optimal receptor efficiency is only achieved when both Ig{alpha} and Igbeta chains are present in the signaling complex, thereby indicating yet unknown individual contributions of Ig{alpha} and Igbeta throughout development.

Two copies of either Ig{alpha} or Igbeta are needed to trigger the pro-B to pre-B transition

Analysis of developmental progression in RAG2–/– primary pro-B cell lines revealed that only fusion proteins containing two functional ITAMs either from Ig{alpha} or Igbeta or one of each are able to transit through the pre-BCR checkpoint. Surrogate receptors containing only one intact ITAM either from Ig{alpha} or Igbeta failed to induce the pro-B to pre-B transition. In agreement with these data, in vivo gene transfer experiments showed that only B cells expressing two intact cytoplasmic domains of either Ig{alpha} or Igbeta or an Ig{alpha}/Igbeta protein progress into more developed B cells that populate bone marrow and spleen. Our adoptive transfer results differ from published data in which B cells carrying receptors with only one functional ITAM were able to reach bone marrow and peripheral transitional stages (44, 45, 46, 47, 49, 51, 52). It is possible that this difference is a reflection of the different capacities of the Ig{alpha}/Igbeta receptors to aggregate or of differences in the systems and methodology used. Although we observed ~10% of pre-B cells in the primary cultures expressing one intact chain of either Ig{alpha} or Igbeta (see Figs. 2C and 3D), this low level of transition seemed insufficient to produce a detectable number of developing B cells in vivo. It is possible that in our earliest harvesting time, the few B cells progressing through development did not have the time to undergo additional homeostatic processes that characterize repopulation of lymphoid organs and culminate with the filling of empty biological niches. We considered that an early analysis of the adoptive transfers would allow for a more direct evaluation of the actual capacity of modified receptors to trigger developmental progression.

Redundant Ig{alpha} and Igbeta activities might help to form a more stable BCR signalosome

Our experimental approach of expressing dual copies of either Ig{alpha} or Igbeta did not discern a unique role for either signaling protein during the ligand-independent stages of B cell maturation. In fact, these data support that the minimal functional receptor requires two ITAM domains and that either Ig{alpha} or Igbeta sequences can provide the context in which the signals for positive selection and developmental progression are generated. However, we consistently observed fewer developing B cells driven by either Ig{alpha} or Igbeta only sequences in our in vitro and in vivo analyses when compared with parental MAHB. Therefore, although our results argue for Ig{alpha} and Igbeta fulfilling overlapping and thus redundant functions, optimal receptor function only occurs when both proteins are present, indicating that these proteins also perform unique activities. An overlapping of Ig{alpha} and Igbeta functions is well-illustrated in the analysis of the variant carrying modified Ig{alpha} non-ITAM tyrosines (tyrosines 176 and 204). These Ig{alpha} non-ITAM tyrosines have been implicated in binding to BLNK (32, 33, 38), an adapter protein whose function is to translocate different sets of proteins, such as phospholipase C{gamma}2, Vav, and Grb/SOS, from cytoplasm to the forming signaling complex (39, 40, 41). Although a recombinant mouse strain carrying mutated tyrosines 176 and 204 has not been reported, an equivalent avian Ig{alpha} mutant exhibited a defective signaling and developmental activity when expressed alone (66). Importantly, normal signaling activity and development can be re-established when this Ig{alpha} mutant is expressed together with Igbeta, suggesting that Igbeta sequences also participate in bringing BLNK to the signaling complex (66). Similarly, a murine receptor with mutated Ig{alpha} Y176 and Y204 had a decreased but detectable ability to bind BLNK, presumably also through Igbeta (32). This redundant ability of Ig{alpha} and Igbeta to bind BLNK might be necessary to support formation of more stable BCR-signaling complexes and explains why the homo-oligomeric Igbeta surrogate receptor (MBHB variant), although lacking the Ig{alpha} sequences important for BLNK binding (Y176 and Y204), still has significant developmental capacity in vitro. In this scenario, the ability of Ig{alpha} and Igbeta to provide different binding sites to the adapter protein BLNK might result in assembly of a more stable signalosome. In agreement, our surrogate receptor modified in the non-ITAM tyrosines failed to induce B cell development in vivo, arguing that two copies of either Ig{alpha} Y176 and Y204 or Igbeta ITAM sequences are needed to assemble the competent receptor capable of generating tonic signaling.

Ligand-independent BCR signals are sufficient to overcome the transitional to mature developmental checkpoint

Our analysis of peripheral development argues that the Ig{alpha}/Igbeta-derived ligand-independent tonic signal is sufficient to drive development into the mature B cell pool and that this ability is similarly supported by Ig{alpha} or Igbeta sequences. Progression from transitional to mature B cells is a critical checkpoint during B cell development, as evidenced by the 30-fold difference between the numbers of bone marrow transitional B cells produced and the number of B cells that successfully enter the mature B cell pool in spleen (reviewed in Ref. 67). The T2/T3 developmental arrest observed in recombinant mice with targeted deletions of many different signaling proteins, including Ig{alpha} and Igbeta, highlights the importance for BCR signaling at this transition (1, 29, 44, 45, 49, 52, 68, 69, 70, 71, 72). However, it was unclear whether ligand binding plays a role in inducing the receptor to trigger the signals for positive selection into the mature B cell pool.

Freitas and colleagues (73, 74, 75) studied B cell maturation after reconstitution with mixtures of B cells containing BCRs with monoclonal and polyclonal specificities. In these studies, the monoclonal cells were eventually lost from the B cell population, arguing that competition within the clonotype was established. These studies argue that the competitive factor is the clonotype-specific ligand, and thus ligand interactions are needed to keep this monoclonal population in the presence of polyclonal competition. Although these studies assigned an important role to ligand binding to maintain a specific B cell clonotype, they do not distinguish whether competition for ligand binding is established within the transitional B cell pool for positively selecting signals or within the mature pool for survival signals. Therefore, two different models of development would reconcile our results with Freitas’ studies. First, ligand binding is only important for maintenance of an existing mature B cell population. Second, ligand-dependent and -independent mechanisms of BCR tonic signaling coexist at the transitional to mature developmental progression. In the latter scenario, although positive selection into the mature pool requires some level of BCR-ligand interactions, these interactions are only limiting in a polyclonal competitive environment. Because maturing B cells in our system did not express receptors capable of ligand binding, a competitive environment is never established, allowing the B cells to enter into the mature pool. In agreement with them, B cells expressing transgenic receptors lacking the H chain V region can successfully reconstitute a mature B cell population but failed to do so when mixed with B cells carrying ligand-binding competent receptors (76). This model will also reconcile the observation that transitional cells can be forced to differentiate into FO B cells in vitro by addition of low doses of anti-Igbeta Ab, which might mimic a low-affinity ligand-receptor interaction (77). In this scenario, tonic signaling can be originated through aggregation-dependent as well as -independent mechanisms, and thus pre-BCR/BCR interactions with itself and with nonpolymorphic ligands or self-Ag might also be needed at different stages of development. Perhaps these mechanisms ensure the development of B cell populations expressing receptors with wide and more restricted specificities.

Only FO B cells are found in the pool of mature B cells expressing the Ig{alpha}/Igbeta-mediated tonic signal

The origin of the mature FO, MZ, and B1 B cell populations is unclear. Adoptive transfer experiments of progenitors derived from fetal liver or adult bone marrow preferentially originate B1 and FO/MZ mature B cells, respectively, supporting the existence of two different developmental pathways separated by ontogeny (78, 79, 80, 81, 82, 83, 84). The data presented here argue that the Ig{alpha}/Igbeta-derived ligand-independent tonic signal preferentially, if not exclusively, induces differentiation into the FO mature B cell compartment. Although some studies have also found adult-derived B1 cells (78, 79, 80, 81, 82, 83, 84), our analysis is inconclusive regarding the capacity of the ligand-independent BCR signal to form B1 B cells. Transgenic expression of the ITAM containing EBV protein LMP2A results in development of B cells with a CD5posCD23neg B1 phenotype (85). CD5 expression in LMP2Apos B cells was observed during in vitro analysis of the pro-B to pre-B transition, an observation that was interpreted as commitment to the B1 lineage at that stage. Contrary to LMP2A-driven development, we did not detect CD5 up-regulation in our in vitro assays of the pro-B to pre-B transition, and most of the developing splenocytes detected in vivo are CD23pos, therefore arguing that the surrogate receptors tested here only support maturation into the FO B cell pool. Contrary to MAHB, which like the resting pre-BCR/BCR forms nonaggregated structures (54, 55), LMP2A is constitutively found forming clusters intimately associated with the lipid raft compartment of the plasma membrane (85, 86, 87, 88). Therefore, although LMP2A lacks a known ligand, its constitutive aggregation and/or lipid raft localization closely resembles an activated receptor. Supporting this argument, LMP2Apos B cells are found to spontaneously form germinal center-like structures in the absence of antigenic challenge (89). Thus, the differential ability of MAHB and LMP2A to favor maturation into FO and B1 B cells, respectively, might reflect the ability of these proteins to mimic a tonic or an activated signal.

Although FO and MZ B cells originate from a common precursor, the stage in which the developing B cell decides between these two lineages is not clear. In contrast to FO cells, the generation of MZ and B1 populations depends on self or environmental Ag. It is well-documented that self-reactive BCRs with affinities for cellular molecules such as phosphocholine and phosphatidyl choline preferentially differentiate into mature MZ and B1 B cells, and transgenic expression of these self-reactive BCRs mainly supports generation of these populations. These data strongly argue that generation of MZ and B1 cells requires positive selection mediated by BCR-Ag interactions (62, 65, 77, 90). Accordingly, MZ and B1 cells are present in a semiactivated state in which they are constitutively secreting Ab. Phosphocholine and phosphatidyl choline molecules are present in surface of different strains of Streptococcus, and natural Abs against these molecules may help to prevent infection by these pathogens (91, 92, 93, 94, 95, 96, 97). Taken together, these and our data support a model where generation of the different mature B cell populations depends on a different mechanism of BCR signaling. In this scenario, ligand-independent tonic signaling preferentially forms FO mature B cells, whereas receptor signaling, triggered by interactions with self-Ags, forms MZ and B1 populations. These mechanisms of receptor signaling presumably evolved to provide B cells with the wide plasticity of specificities that constitute the innate and acquired humoral immune response. Thus, mature MZ and B1 B cells require ligand interactions for maturation, but once they reached this stage they secrete Abs that, although of low specificity, are sufficient to provide the first barrier of defense against infectious agents. On the contrary, FO B cells might only require a low level of pre-BCR/BCR activity sufficient to indicate the maturing B cell about the presence of a functional signaling-competent receptor. However, mature FO B cells can be instructed to generate a more specific immune response that although delayed, is more efficient. It is of note that MZ B cell development has also been proposed to be driven by weaker BCR signals than those that lead to FO B cell development (58). This discrepancy is presently unclear.


   Acknowledgments
 
We thank Justina Stadanlick for editorial assistance in the preparation of this manuscript. We also thank Dr. Leslie King for comments in the preparation of this study. We also acknowledge Dr. Warren Pear as core director for a National Cancer Institute-funded retroviral core and the Abrahmson Cancer Center Flow Cytometry Core.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by funding from the Cancer Research Institute (to E.M.F.-P.) and the National Cancer Institute and National Institute of Allergy and Infectious Diseases (to J.G.M.). Back

2 Address correspondence and reprint requests to Dr. John G. Monroe, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Biomedical Research Building II/III, Room 311, Philadelphia, PA 19104. E-mail address: monroej{at}mail.med.upenn.edu Back

3 Abbreviations used in this paper: SLC, surrogate L chain; BLNK, B cell linker; MZ, marginal zone; FO, follicular; WT, wild type; HA, hemagglutinin. Back

Received for publication June 28, 2006. Accepted for publication September 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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