Maturation Development and Peripheral B Cell Signaling for Bone Marrow B Cell (Cd79b)-Mediated Tonic Β (Cd79a)- and Ig Α Analy

Analysis of the Individual Contributions of Ig α (CD79a)- and Igβ (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell This information is current as Maturation of September 28, 2021. Ezequiel M. Fuentes-Pananá, Gregory Bannish, Fredrick G. Karnell, John F. Treml and John G. Monroe J Immunol 2006; 177:7913-7922; ; doi: 10.4049/jimmunol.177.11.7913 Downloaded from http://www.jimmunol.org/content/177/11/7913

References This article cites 97 articles, 55 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/177/11/7913.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Analysis of the Individual Contributions of Ig␣ (CD79a)- and Ig␤ (CD79b)-Mediated Tonic Signaling for Bone Marrow B Cell Development and Peripheral B Cell Maturation1

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

The individual contribution of Ig␣ and Ig␤ 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␣ and Ig␤ participate in both Ag-independent (tonic) and Ag-dependent signaling. The present study undertook defining the individual requirement for Ig␣ and Ig␤ under conditions where only ligand-independent tonic signaling was operative. In this regard, we have constructed chimeric proteins containing one or Downloaded from two copies of the cytoplasmic domains of either Ig␣ or Ig␤ and Ig␣/Ig␤ heterodimers with targeted Tyr3 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 ␮MT 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␣/Ig␤ 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 http://www.jimmunol.org/ follicular B cells and both Ig␣ and Ig␤ 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. The Journal of Immunology, 2006, 177: 7913–7922.

he BCR is a complex composed of a ligand-binding unit The mechanism by which the receptor initiates tonic signaling is formed by the Ig H and L chains and a signaling unit currently unclear with some evidence supporting the need for re- T consisting of a heterodimer of Ig␣ (CD79a) and Ig␤ ceptor oligomerization (16–18). Because the pre-BCR lacks the (CD79b) (1–3). Formation of a mature immunocompetent B cell Ag-binding domain, it has been reported that positive selection at by guest on September 28, 2021 involves an ordered process whose steps are defined by the se- the pro-B to pre-B transition might be dependent on pre-BCR self- quential expression and assembly of the individual components of interactions or pre-BCR interactions with nonpolymorphic ligands the BCR (4–7). Continued maturation and survival of mature B present on the surrounding stromal cells in the bone marrow (16– cells is dependent upon the ability of the pre-BCR and mature 18). These studies targeted the surrogate L chain (SLC)3 protein ␭5 ␣ ␤ BCR to generate signals through the Ig /Ig heterodimer at crit- as the structure responsible for such interactions. Because pre-B ical checkpoints during developmental progression (2). Studies cells can be generated in genetic knockouts of the SLC proteins support the existence of two mechanisms of generating signals and signaling competent pre-BCRs have been observed in cells through Ig␣/Ig␤ complexes. The first is Ag dependent, requires lacking SLC (19–21), it can be argued that such mechanisms of BCR aggregation, and is associated with effector cell generation pre-BCR aggregation are not essential to initiate tonic signaling from mature B cells and negative selection of immature B cells (8, 9). The second mechanism is Ag independent and although less and to trigger developmental progression at the pro-B to pre-B well-biochemically characterized, is argued to be relevant for pre- transition. In contrast, we have found that the cytoplasmic domains ␣ ␤ BCR-dependent maturation (10, 11) and for the BCR-dependent of Ig and Ig in the absence of extracellular or transmembrane survival of peripheral B cells (12–14). These latter signals are of- domains are sufficient to trigger B cell development to transitional ten referred to as ligand-independent or tonic signals to distinguish stages supporting a model where an unligated receptor is sufficient them from those triggered as a consequence of ligand (Ag)-in- for this function. Accordingly, fluorescence resonance energy duced receptor oligomerization (15–18). 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 (23–25). Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104 Both Ag-dependent and -independent (tonic) signals require spe- Received for publication June 28, 2006. Accepted for publication September 8, 2006. cific motifs in the cytoplasmic tails of Ig␣ and Ig␤ known as ITAMs The costs of publication of this article were defrayed in part by the payment of page (11, 25–27). Tyrosine residues within the ITAM domains are phos- charges. This article must therefore be hereby marked advertisement in accordance phorylated by protein tyrosine kinases and then act as docking sites with 18 U.S.C. Section 1734 solely to indicate this fact. where a higher order signaling complex is assembled (28–37). Ig␣ 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.). 2 Address correspondence and reprint requests to Dr. John G. Monroe, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Biomedical Research Building 3 Abbreviations used in this paper: SLC, surrogate L chain; BLNK, B cell linker; MZ, II/III, Room 311, Philadelphia, PA 19104. E-mail address: [email protected] marginal zone; FO, follicular; WT, wild type; HA, hemagglutinin.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 7914 Ig␣ AND Ig␤ TONIC SIGNALING contains two additional tyrosines flanking the ITAM domain, posi- tions but fail to form marginal zone (MZ) and B1 populations. We tions 176 and 204, which are also phosphorylated upon receptor-li- also observe that either Ig␣ and Ig␤ chains are sufficient to provide gand engagement and participate in the formation of the signalosome the positive signal to form FO B cells when two copies of their (32, 33, 38). Signaling downstream of the Ig␣ and Ig␤ complex leads cytoplasmic chains are present. to activation of multiple signaling pathways resulting in changes in patterns of gene expression and cytoskeleton reorganization (39–41). Materials and Methods Ig␣ and Ig␤ are evolutionarily conserved proteins that fulfill a Animals similar function in mice, birds, and humans. The requirement for C57BL/6 (WT), ␮MT, and RAG2Ϫ/Ϫ mice were bred and maintained un- two highly homologous proteins containing almost identical der pathogen-free conditions at the University of Pennsylvania, and exper- ITAMs is presently unclear, and studies to define the specific roles imental procedures in these animals were performed according to local and of these proteins during B cell development have yielded conflict- National Institutes of Health guidelines. ing results. Early studies using immunoprecipitation and binding Oligonucleotides and construction of MAHB variants assays from cultured cell lines suggested a specific role for Ig␣ in binding fyn kinase and the adapter protein B cell linker (BLNK) Construction of MAHB and ITAMmut and their cloning into the MIGR1 retroviral vector have been previously described (11). Either MAHB or (32, 33, 38, 42, 43). However, more recent studies using recom- ITAMmut served as templates to synthesize all the variants used in this binant mice expressing a sole copy of either Ig␣ or Ig␤ or copies study. As explained below, the oligonucleotides used to make MAHB of both proteins where one of the ITAMs is nonfunctional due to variants were as follows: Ig␣-Kpn-S, 5Ј-CGCGGGTACCAGGAAACG Ј ␣ Ј introduced mutations showed that each protein is sufficient to trig- GTGGCAAAATGAG-3 ;Ig -Eco-A, 5 -CGGCGAATTCATGGCTTT TCCAGCTGGGC-3Ј;Ig␤-Bam-S2, 5Ј-CGCGGATCCGACAAGGAT Downloaded from ger development to the immature B cell stage (44–52). In these GACGGCAAGGCT-3Ј;Ig␤-Xba-A, 5Ј-GCGAGATCTAGATTCCTG studies, a clear decrease in the efficiency of formation of pre-B and GCCTGGATGCTC-3Ј;HA-A2,5Ј-GCGCGAATTCAAGCGTAGTCTG immature B cell bone marrow populations was found with BCRs GGACGTCGTATGGGTA-3Ј; Myr-S2, 5Ј-GCCGGAATTCCACCATGG associated with only Ig␤ relative to those associated with only Ig␣ GCTGTGTCTGCAGCT-3Ј;Ig␣Y176S, 5Ј-ATGCCAGATGACTTTGAA GATGAA-3Ј;Ig␣Y176, 5Ј-TTCATCTTCAAAGTCATCTGGCA-3Ј; or wild-type (WT) receptors, suggesting an increased potency for ␣ Ј Ј ␣ ␣ ␤ Ig Y204S, 5 -CTCCAGGGCACCTTTCAGGATGTGGGC-3 ; and Ig Y204A, Ig over Ig (44–52). Accordingly, developing B cells carrying 5Ј-GCCCACATCCTGAAAGGTGCCCTGGAG-3Ј.

Ig␣ or Ig␤ alone tend to compensate by adjusting receptor expres- As previously described (11), each domain of parental MAHB and ITAM http://www.jimmunol.org/ sion levels at the plasma membrane (48, 50). Nevertheless, the genes is flanked by unique restriction sites. To synthesize the MAmutB, an sufficiency of receptors composed of only a functional Ig␣ or Ig␤ EcoRI-KpnI fragment containing MAH was excised from MAHB and replaced with the homologous sequence derived from the ITAMmut. Simi- chain to form pre-B and immature B cell populations in vivo ar- larly, to synthesize MAHBmut, the KpnI-EcoRI site encoding the Ig␤ cy- gues that both proteins fulfill overlapping functions at least during toplasmic domain from MAHB was replaced by the homologous sequence early stages of development. Thus, the need for these separate from ITAMmut. ITAM proteins remains unclear, perhaps reflecting differences in MBHB. To subclone MBHB into MIGR1, we first deleted a NotI-SmaI the need for Ag-dependent and -independent pre-BCR/BCR sig- fragment from vector pBluescript SK (Stratagene), end filled, and religated to form SK⌬NS. An MAHB EcoRI fragment was ligated into the EcoRI nals during development. ⌬ ⌬ ␣

site of SK NS to form SK NSEcoEcoMAHB. The Ig domain of MAHB by guest on September 28, 2021 Importantly, B cell development in transgenic mice expressing was then excised with BamHI and HindIII. An Ig␤ cytoplasmic PCR frag- only one intact chain, either Ig␣ or Ig␤, was arrested at the im- ment was synthesized from MAHB using Ig␤BamS2 and Ig␤Xba-A mature stage, illustrating the need for receptor-mediated signals to oligonucleotides, cut with BamHI and XbaI, purified, and ligated into ⌬ form and/or maintain mature B cell populations. Given the recent the BamHI and HindIII sites from SK NSEcoEcoMAHB to form SK⌬NSEcoEcoMBHB. MBHB was excised with EcoRI and ligated into appreciation of both tonic and ligand-dependent signaling through MIGR1 at the EcoRI site. pre-BCR and BCR complexes, it is possible that ligand-dependent MBH. The MBH sequence was amplified by PCR using MBHB template and -independent mechanisms of receptor signaling differ in their and MyrS2 and HA-A2 oligonucleotides. The resulting fragment was cut importance at different stages of development and in their require- with EcoRI and ligated into MIGR1. ment of Ig␣ and Ig␤. Therefore, we considered that the conflicting MAH. An MAH fragment was amplified by PCR from MAHB using evidence regarding the specificity of Ig␣ and Ig␤ function might MyrS2 and HA-A2 oligonucleotides. The MAH fragment was digested be explained by the fact that these proteins act differently during with EcoRI, purified, and ligated into MIGR1. ␣ ␣ the ligand-independent phase of development in the bone marrow, MAHA. An Ig fragment was amplified from MAHB using Ig -KpnS and Ig␣-EcoA oligonucleotides, and the resulting fragment was digested compared with ligand-dependent development in peripheral lym- with KpnI at the 5Ј end and EcoRI at the 3Ј end to form 5Ј-Kpn-Ig␣- phoid organs. Thus, Ig␣ and Ig␤ may fulfill both individual as well EcoRI-3Ј. Separately, an MAH fragment was excised from MAHB with as distinct roles at different points of development. EcoRI and KpnI to form 5Ј-EcoRI-MAH-KpnI-3Ј. These two fragments The present study sought to evaluate the importance of the ag- were ligated into the EcoRI site of MIGR1 in a three-way ligation. ␣ gregation-independent pre-BCR/BCR tonic signaling for bone MAnonITAMmutHB. The non-ITAM tyrosine residues of Ig (Y176, Y204) were mutated to phenylalanine using PCR mutagenesis and overlap marrow and peripheral B cell maturation and to define the indi- PCR, with the primers Ig␣-ITAMmt-S and Ig␣-ITAMmt-A oligonucleo- vidual requirement for Ig␣ and Ig␤ under conditions where only tides. Residues were then excised with EcoRI and KpnI to form 5Ј-EcoRI- the tonic signaling of the receptor was operative. In this regard, we MA(176,204)H-KpnI-3Ј. The Ig␤ domain of MAHB was excised with have enforced expression in B cells of chimeric proteins contain- KpnI and EcoRI to form 5Ј-KpnI-Ig␤-EcoRI-3Ј. These two fragments were ing one or two functional copies of either Ig␣ or Ig␤. These pro- ligated into the EcoRI site of MIGR1 in a three-way ligation. teins act as surrogate pre-BCR/BCRs, and their ability to trigger Abs and flow cytometry analysis developmental progression was compared. We considered that the Flow cytometric analysis was used to analyze the expression levels of threshold for a functional activity mediated by the pre-BCR/BCR markers to define primary pro-B/pre-B and B cells from recipient mice might only be reached when two functional copies of either the Ig␣ after adoptive transfers. Erythrocyte-depleted samples were incubated with or Ig␤ ITAM domains are expressed together, and therefore the fluorescence-tagged Abs against the development stage markers B220- specificity conferred by these proteins can only be observed in allophycocyanin, CD43-PE, CD22-PE, CD23-PE, CD23-biotin CD21-PE, AA4.1-allophycocyanin, and CD5-PE (BD Pharmingen), and with anti- these conditions. We report here that tonic signaling is sufficient to hemagglutinin (HA) (clone 3F10; Roche). After staining with CD23-biotin, drive B cell development to the mature stage and that tonic sig- cells were treated with Streptavidin-Red 670 (Invitrogen Life Technolo- naling-driven B cells develop into mature follicular (FO) popula- gies) and then fixed in 1% paraformaldehyde. For intracellular staining The Journal of Immunology 7915 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 ␮MT 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 ϫ 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 RAG2Ϫ/Ϫ (Taconic Farms) mice and were maintained in Iscove’s complete medium (Invitro- gen Life Technologies) supplemented with 10% FCS, 1% NEN mix (In- Downloaded from vitrogen Life Technologies), 1% OPI mix (Invitrogen Life Technologies), FIGURE 1. Expression of surrogate Ig␣/Ig␤ receptors. A, BCR/pre- 50 mM 2-ME, and 5% supernatant obtained from IL-7-producing J558L BCR surrogate receptors were constructed containing one or two copies cells as previously described (54, 55). After culture of bone marrow cells of either Ig␣ or Ig␤ cytoplasmic domains. Similar to parental MAHB, in IL-7-containing medium, cells were analyzed daily by flow cytometry these variants are targeted to the cytoplasmic membrane by the Lck for surface expression of developmental markers to assess progression into myristoylation/palmitoylation site. They were cloned into the MIGR1 pos pos pos neg neg pro-B cell lines (B220 CD43 BP1 CD22 CD25 ). Consistently, retroviral vector and transduced into primary RAG2Ϫ/Ϫ pro-B cells by day 6 of culture, Ͼ99% of the cells were pro-B cells and remained in

pos pos pos neg neg http://www.jimmunol.org/ Ϫ Ϫ (B220 CD43 BP1 CD22 CD25 ). Expression of GFP from the 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 MIGR1 bicistronic message was used to sort for chimeric protein-expressing GFP-expressing populations. The studies depicted here were performed cells. B, Fusion protein expression levels were compared with GFP expression with pro-B cells that have been maintained in IL-7 for less than a year. For by flow cytometry analysis. Expression of the fusion protein was measured analyses of developmental progression, cells were depleted of IL-7 either with an Ab against the epitope tag derived from the influenza HA protein. 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 pro-B cell lines expressed similar levels of fusion protein. Only the were identified by their phenotype (B220posCD43negBP1posCD22posCD25pos). MBHB variant was consistently found expressing higher levels of both chimeric and GFP proteins (bottom right panel). The cellular by guest on September 28, 2021 Results localization of the fusion proteins was addressed by immunofluo- ␣ ␤ Two functional ITAMs derived from either Ig or Ig are rescence microscopy using an anti-HA Ab. All fusion proteins required to trigger in vitro development through the pro-B used throughout this study were preferentially associated with the to pre-B transition plasma membrane (data not shown). We previously developed an experimental model to isolate and Fig. 2A depicts an analysis of the number and relative frequency study tonic pre-BCR- and BCR-signaling processes (11). This of pro-B cells undergoing transition to the pre-B stage following model involves the expression of a chimeric protein containing the removal of IL-7. Transition is evaluated by the differential expres- cytoplasmic domains of Ig␣ and Ig␤ separated by an epitope tag sion of CD22 and CD43 (as described in Refs. 5, 54, and 56). The from the HA protein. This protein is termed MAHB and is targeted actual number of CD22posCD43neg pre-B cells present at the be- to the inner leaflet of the cytoplasmic membrane by the N-terminal ginning and end of the experiment is shown. In this figure, pre-B 10 aa of the protein kinase Lck. The ability of MAHB to model cells are only found in conditions where MAHB is expressed, il- ligand- and aggregation-independent functions of the pre-BCR and lustrating the importance of the Ig␣/Ig␤-mediated tonic signal for BCR has been described (6, 11). Because of its ability to isolate the this transition. We typically observed 40–70% pre-B cells in the ligand-independent tonic-signaling functions of Ig␣/Ig␤ com- MAHBpos cultures after 2–3 days of IL-7 depletion, while the rest plexes, MAHB provides an opportunity to evaluate the individual of the cells remain in the CD22negCD43pos pro-B stage. A similar contributions of Ig␣ and Ig␤ tonic signaling for bone marrow and pre-B/pro-B cell ratio is found in cultures that express an Ig H peripheral B cell development. chain transgene, indicating a similar efficiency of transition medi- The first question addressed was whether Ig␣ and Ig␤ provide ated by MAHB and the WT pre-BCR (54). Fig. 2B shows the same distinct functions during the early stages of B cell development or analysis for MAHB and variants, where CD43 down-regulation whether the normal coexpression of both proteins reflects a re- was used to identify pre-B cells (plotted in Fig. 2C). In the exper- quirement for two ITAMs to achieve a threshold for pre-BCR iment depicted in this study (n Ͼ10), 68% of the MAHBpos cells function. To achieve this goal, MAHB variants containing single in culture are in the pre-B stage, whereas in MAHApos and MB- or double copies of either Ig␣ or Ig␤ were constructed (Fig. 1A) HBpos cultures, pre-B cells represent 51 and 36% of the total pop- and expressed in primary RAG2Ϫ/Ϫ pro-B cells. Fig. 1B compares ulation, respectively. We observed transition to the pre-B stage the relative expression of fusion protein, as determined by HA only in cells expressing two ITAMs from either Ig␣ or Ig␤. The levels, with that of GFP expressed from the bicistronic retroviral ability of MAHA and MBHB variants to induce progression message. This analysis verifies that the levels of expression of the through the pro-B to pre-B checkpoint is also illustrated in Fig. 2D, fusion protein and GFP are proportional. Thus, the expression of which documents the kinetics of transition during 4 days of IL-7 fusion protein can be grossly inferred from the GFP levels ex- depletion. In the cultures expressing a single copy of Ig␣ and/or pressed by the pro-B population. We also found that most of the Ig␤ (only one ITAM), Ͻ5% of pre-B cells were found at any time 7916 Ig␣ AND Ig␤ TONIC SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 2. In vitro developmental capacity of surrogate pre-BCRs containing single or double copies of Ig␣ or Ig␤. Pro-B cell cultures were obtained after culturing RAG2Ϫ/Ϫ 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␣/Ig␤ 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. after IL-7 depletion, illustrating the inability of these cells to tutions. The following MAHB variants were constructed (depicted progress beyond the pro-B stage. in Fig. 3A): 1) the ITAMmut variant, where all ITAM-associated In summary, this analysis argues that the threshold for pre-BCR tyrosines on Ig␣ and Ig␤ were modified; 2–3) the MAmutHB and signaling at the pro-B to pre-B transition cannot be achieved with MAHBmut variants, where the two ITAM tyrosines of Ig␣ or Ig␤ a single ITAM domain. However, two ITAM copies, either in the were modified, respectively; and 4) the MAnonITAMmutHB vari- context of Ig␣ or Ig␤ sequences, are able to generate the signals ant, where the two non-ITAM tyrosines of Ig␣ were modified to bypass this developmental checkpoint. Importantly, neither (Tyr176 and Tyr204). The MIGR1 retrovirus was also used to trans- MAHA nor MBHB fusion proteins were as efficient as the fusion duce these MAHB derivatives into RAG2Ϫ/Ϫ primary pro-B cells. of both Ig␣ and Ig␤ domains (MAHB), suggesting that although Fig. 3B compares the levels of chimeric protein expression. As can Ig␣ and Ig␤ have overlapping functions, the optimal efficiency of be seen, the levels of expression of chimeric protein and GFP are the receptor is only achieved when both proteins are present, proportional and there is little detectable difference in the steady thereby arguing for yet unknown individual contributions of Ig␣ state expression levels of the individual variants relative to each and Ig␤ early in development. other. Fig. 3C depicts an analysis of the pro-B to pre-B transition To extend this structural analysis of Ig␣ and Ig␤ sequences, we after IL-7 depletion of sorted pro-B cells expressing equal GFP synthesized MAHB variants that carry targeted Tyr3Phe substi- levels. We observed an ϳ9-fold decrease of the MAmutHB and The Journal of Immunology 7917 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 3. In vitro developmental capacity of surrogate pre-BCRs containing Ig␣/Ig␤ targeted Tyr3Phe substitutions. A, The parental MAHB surrogate receptor was modified to carry the targeted Tyr to Phe modification within the Ig␣/Ig␤ ITAM domains or outside the ITAM domain (Ig␣ tyrosines 176 and 204). These constructs were then cloned into the MIGR1 retroviral vector and transduced into primary RAG2Ϫ/Ϫ 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.

MAHBmut variants, relative to the ability of parental MAHB, to also able to induce B cell developmental progression in vivo using trigger the pro-B to pre-B transition. This result further illustrates the ␮MT mouse model. The MAHB variants were expressed in the importance of two intact ITAM domains to overcome the bone marrow-derived hemopoietic progenitors that were then trans- pro-B to pre-B developmental checkpoint. In contrast, we found a ferred into lethally irradiated syngeneic recipients. Four weeks after 3-fold decreased efficiency in the variant where both non-Ig␣ transfer, recipient mice were sacrificed, and bone marrow and splenic ITAM tyrosines were modified, arguing that although these ty- B cells were analyzed by flow cytometry. We did not observe rosine are important, most of the Ig␣ and Ig␤ function resides in developing B cells in mice transduced with fusion proteins con- the ITAM domains. The fraction of CD43neg pre-B cells in each taining only one functional ITAM, either single copy or Tyr3Phe culture is plotted in Fig. 4. We also tested variants of MAHA and mutant variants (data not shown). B cells expressing these variants MBHB where the second ITAM domain carries Tyr3Phe substi- remained in the CD43posCD22neg pro-B stage, consistent with tutions (MAHAmut and MBHBmut variants, respectively). Be- their observed in vitro developmental potential (see Figs. 2B and cause these variants were also unable to trigger the pro-B to pre-B 3C). However, developing B cells were found in bone marrow and transition they were not included in the results presented here. spleens of mice reconstituted with B cells expressing the Ig␣/Ig␣ However, the inability of these constructs to drive the pro-B to and Ig␤/Ig␤ fusion proteins. Similar to MAHB, these variants pre-B developmental progression also argues that two functional were able to trigger development through the CD23pos transitional ITAMs are required for this transition. 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 ␣ ␤ Tonic signals originated from either Ig or Ig chains are differential expression of surface proteins CD23 and AA4.1. sufficient to generate a peripheral mature B cell pool Transgenic models where BCRs are assembled expressing only We have previously shown that MAHB is able to provide signals one functional ITAM of either Ig␣ or Ig␤ are arrested at the T2 to that allow for maturation to the peripheral transitional immature mature transition (44–47, 49, 51, 52). These studies have helped stage in BCR-deficient B cells from ␮MT and RAG2Ϫ/Ϫ mice to identify another developmental checkpoint at the T2/T3 to ma- (11). Therefore, we evaluated whether the MAHB variants were ture transition, which is also dependent on the capacity of the 7918 Ig␣ AND Ig␤ TONIC SIGNALING

ment 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. For- mation 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 (57–61). 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 (62–65). Therefore, FIGURE 4. Development of mature B cells by either Ig␣-orIg␤-me- we addressed whether the MAHA and/or MBHB fusion proteins diated tonic signaling. Bone marrow progenitor cells from ␮MT mice were could generate these mature B cell populations. FO and MZ mature transduced either with MAHB or the double Ig␣/Ig␤ copy variants MAHA B cells can be distinguished by their differential expression of Downloaded from and MBHB. Transduced progenitors were used to reconstitute lethally ir- CD21 and CD23 surface markers. Fig. 5A shows a ﬂow cytometric ␮ radiated syngeneic MT mice, and 4 wk postreconstitution, spleen cells analysis of the pattern of expression of these proteins in the mature were analyzed by ﬂow cytometry. Splenocytes were stained for surface B cell population of untreated WT C57BL/6 mice (AA4.1neg gate) expression of AA4.1, CD23, and the pan B cell marker B220 to identify the 3 transitional and mature B cell populations (T1, T2, and M boxed popula- and in transduced MIGR1 C57BL/6, MAHB-, MAHA-, and 3␮ neg pos tions, respectively). Cells were gated on the transduced (GFPpos) B220pos MBHB MT B cells (AA4.1 GFP gate). This analysis

B cell population. Normal development in WT C57BL/6 mice is also showed that tonic signaling through MAHB or either of the double http://www.jimmunol.org/ shown. Ig␣ or Ig␤ 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␣-orIg␤-speciﬁc sequences. We were expressed BCR to transduce signals for positive selection. Unlike unable to detect MZ B cells (CD21highCD23low-neg) in spleens of the pro-B to pre-B checkpoint driven by the pre-BCR, the receptor mice reconstituted with parental MAHB or any of the variants at this transition is fully competent to recognize and bind Ags. tested. The MIGR13C57BL/6 control indicates that retroviral in- Additionally, because Ig␣ and Ig␤ transgenic models are arrested sertion does not affect formation of the MZ B cell pool and there- at this transition, it has been speculated that it is not until the T2/T3 fore argues that the lack of this pool in the fusion protein-express-

stage that Ig␣ and Ig␤ play nonredundant functions (44–47, 49, ing B cells is most likely due to lack of a competent differentiation by guest on September 28, 2021 51, 52). Therefore, it was important to evaluate whether the re- signal. ceptor-mediated basal signal is also sufficient to bypass the T2/T3 In addition to the analysis of the splenic B cell compartment, to mature checkpoint. Fig. 4 shows the phenotypic analysis of peritoneal cavity cells were taken from recipient mice and stained spleen cells derived from the recipient mice 4 wk after adoptive for detection of CD5 and B220 proteins. Fig. 5B shows that transfer. For this figure, cells were gated on B220pos B cells in the CD5pos and CD5neg B1 B cell populations were present in the WT WT C57BL/6 mouse and on B220posGFPpos B cells in the trans- mouse reconstituted with MIGR1 empty vector, while no B220pos duced recipient ␮MT mice, and T1, T2, and mature (M) B cell B cells were found in peritoneal cavities of mice reconstituted with populations are indicated by marked squares. This analysis shows B cells expressing any of the surrogate receptors. In summary, that the BCR-mediated basal signal is sufficient to drive develop- these data argue that the MAHB-mediated tonic signal is only

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 ␮MT mice. Recipient MIGR13C57BL/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. The Journal of Immunology 7919 sufficient to trigger development into FO mature B cells and through development did not have the time to undergo additional that this maturation signal seems to be independent of either homeostatic processes that characterize repopulation of lymphoid Ig␣ or Ig␤ unique sequences. Importantly, this study does not organs and culminate with the filling of empty biological niches. address whether a B1 population could be generated in similar We considered that an early analysis of the adoptive transfers circumstances using B cells of fetal origin. would allow for a more direct evaluation of the actual capacity of modified receptors to trigger developmental progression. Discussion We have previously developed a model that allows us to isolate ␣ ␤ and study the Ig␣/Ig␤-derived signals that are not dependent on Redundant Ig and Ig activities might help to form a more receptor aggregation and that can be originated outside of the lipid stable BCR signalosome raft membrane compartment (tonic signaling) (11, 54, 55). In this Our experimental approach of expressing dual copies of either Ig␣ study, we address the importance of the ligand-independent tonic or Ig␤ did not discern a unique role for either signaling protein signaling for the pre-BCR-dependent bone marrow and BCR-de- during the ligand-independent stages of B cell maturation. In fact, pendent peripheral B cell maturation and the specific requirement these data support that the minimal functional receptor requires for the Ig␣ and Ig␤ chains for this tonic receptor activity. Bio- two ITAM domains and that either Ig␣ or Ig␤ sequences can pro- chemical and genetic studies of the specific individual roles for Ig␣ vide the context in which the signals for positive selection and and Ig␤ in BCR signaling and B cell development have produced developmental progression are generated. However, we consis- contradictory results. Because such studies of the specificity of Ig␣ tently observed fewer developing B cells driven by either Ig␣ or and Ig␤ were evaluated within receptors competent for ligand- Ig␤ only sequences in our in vitro and in vivo analyses when Downloaded from induced aggregation, we considered that the specificity conferred compared with parental MAHB. Therefore, although our results by these proteins might reflect a differential requirement for ligand argue for Ig␣ and Ig␤ fulfilling overlapping and thus redundant binding during development, and therefore we designed this study functions, optimal receptor function only occurs when both pro- to examine this question under conditions where only tonic sig- teins are present, indicating that these proteins also perform unique naling was operative. Moreover, we tested surrogate receptors with activities. An overlapping of Ig␣ and Ig␤ functions is well-illus-

single and double copies of either Ig␣ or Ig␤, considering that the trated in the analysis of the variant carrying modified Ig␣ non- http://www.jimmunol.org/ physiological receptor’s activity might only be accomplished with ITAM tyrosines (tyrosines 176 and 204). These Ig␣ non-ITAM two ITAM domains and therefore only under these conditions can tyrosines have been implicated in binding to BLNK (32, 33, 38), the specificity of Ig␣ and Ig␤ be revealed. Our data support that an adapter protein whose function is to translocate different sets of ligand-independent tonic signaling is sufficient to generate mature proteins, such as phospholipase C␥2, Vav, and Grb/SOS, from B cell populations of the FO origin but fail to generate MZ and B1 cytoplasm to the forming signaling complex (39–41). Although a mature B cells. Both Ig␣ and Ig␤ cytoplasmic chains were inde- recombinant mouse strain carrying mutated tyrosines 176 and 204 pendently sufficient to form this mature FO B cell population but has not been reported, an equivalent avian Ig␣ mutant exhibited a only when two intact copies of them are present. Importantly, al- defective signaling and developmental activity when expressed though our results argue for overlapping functions for Ig␣ and Ig␤ alone (66). Importantly, normal signaling activity and develop- by guest on September 28, 2021 during the tonic signaling-induced B cell development, neither Ig␣ ment can be re-established when this Ig␣ mutant is expressed to- or Ig␤ sequences alone were as efficient as the surrogate receptor gether with Ig␤, suggesting that Ig␤ sequences also participate in carrying copies of both proteins. Thus, these data argue that opti- bringing BLNK to the signaling complex (66). Similarly, a murine ␣ ␤ ␣ mal receptor efficiency is only achieved when both Ig and Ig receptor with mutated Ig Y176 and Y204 had a decreased but chains are present in the signaling complex, thereby indicating yet detectable ability to bind BLNK, presumably also through Ig␤ unknown individual contributions of Ig␣ and Ig␤ throughout (32). This redundant ability of Ig␣ and Ig␤ to bind BLNK might development. be necessary to support formation of more stable BCR-signaling complexes and explains why the homo-oligomeric Ig␤ surrogate ␣ ␤ Two copies of either Ig or Ig are needed to trigger the pro-B receptor (MBHB variant), although lacking the Ig␣ sequences im- to pre-B transition portant for BLNK binding (Y176 and Y204), still has significant Analysis of developmental progression in RAG2Ϫ/Ϫ primary pro-B developmental capacity in vitro. In this scenario, the ability of Ig␣ cell lines revealed that only fusion proteins containing two functional and Ig␤ to provide different binding sites to the adapter protein ITAMs either from Ig␣ or Ig␤ or one of each are able to transit BLNK might result in assembly of a more stable signalosome. In through the pre-BCR checkpoint. Surrogate receptors containing agreement, our surrogate receptor modified in the non-ITAM ty- only one intact ITAM either from Ig␣ or Ig␤ failed to induce the rosines failed to induce B cell development in vivo, arguing that ␣ ␤ pro-B to pre-B transition. In agreement with these data, in vivo two copies of either Ig Y176 and Y204 or Ig ITAM sequences are gene transfer experiments showed that only B cells expressing two needed to assemble the competent receptor capable of generating intact cytoplasmic domains of either Ig␣ or Ig␤ or an Ig␣/Ig␤ tonic signaling. 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 func- Ligand-independent BCR signals are sufficient to overcome the tional ITAM were able to reach bone marrow and peripheral tran- transitional to mature developmental checkpoint sitional stages (44–47, 49, 51, 52). It is possible that this difference Our analysis of peripheral development argues that the Ig␣/Ig␤- is a reflection of the different capacities of the Ig␣/Ig␤ receptors to derived ligand-independent tonic signal is sufficient to drive aggregate or of differences in the systems and methodology used. development into the mature B cell pool and that this ability is Although we observed ϳ10% of pre-B cells in the primary cul- similarly supported by Ig␣ or Ig␤ sequences. Progression from tures expressing one intact chain of either Ig␣ or Ig␤ (see Figs. 2C transitional to mature B cells is a critical checkpoint during B cell and 3D), this low level of transition seemed insufficient to produce development, as evidenced by the 30-fold difference between the a detectable number of developing B cells in vivo. It is possible numbers of bone marrow transitional B cells produced and the that in our earliest harvesting time, the few B cells progressing number of B cells that successfully enter the mature B cell pool in 7920 Ig␣ AND Ig␤ TONIC SIGNALING spleen (reviewed in Ref. 67). The T2/T3 developmental arrest ob- to LMP2A-driven development, we did not detect CD5 up-regu- served in recombinant mice with targeted deletions of many dif- lation in our in vitro assays of the pro-B to pre-B transition, and ferent signaling proteins, including Ig␣ and Ig␤, highlights the most of the developing splenocytes detected in vivo are CD23pos, importance for BCR signaling at this transition (1, 29, 44, 45, 49, therefore arguing that the surrogate receptors tested here only sup- 52, 68–72). However, it was unclear whether ligand binding plays port maturation into the FO B cell pool. Contrary to MAHB, which a role in inducing the receptor to trigger the signals for positive like the resting pre-BCR/BCR forms nonaggregated structures (54, selection into the mature B cell pool. 55), LMP2A is constitutively found forming clusters intimately Freitas and colleagues (73–75) studied B cell maturation after associated with the lipid raft compartment of the plasma mem- reconstitution with mixtures of B cells containing BCRs with brane (85–88). Therefore, although LMP2A lacks a known ligand, monoclonal and polyclonal specificities. In these studies, the its constitutive aggregation and/or lipid raft localization closely monoclonal cells were eventually lost from the B cell population, resembles an activated receptor. Supporting this argument, arguing that competition within the clonotype was established. LMP2Apos B cells are found to spontaneously form germinal cen- These studies argue that the competitive factor is the clonotype- ter-like structures in the absence of antigenic challenge (89). Thus, specific ligand, and thus ligand interactions are needed to keep this the differential ability of MAHB and LMP2A to favor maturation monoclonal population in the presence of polyclonal competition. into FO and B1 B cells, respectively, might reflect the ability of Although these studies assigned an important role to ligand bind- these proteins to mimic a tonic or an activated signal. ing to maintain a specific B cell clonotype, they do not distinguish Although FO and MZ B cells originate from a common precur- whether competition for ligand binding is established within the sor, the stage in which the developing B cell decides between these transitional B cell pool for positively selecting signals or within the two lineages is not clear. In contrast to FO cells, the generation of Downloaded from mature pool for survival signals. Therefore, two different models MZ and B1 populations depends on self or environmental Ag. It is of development would reconcile our results with Freitas’ studies. well-documented that self-reactive BCRs with affinities for cellular First, ligand binding is only important for maintenance of an ex- molecules such as phosphocholine and phosphatidyl choline pref- isting mature B cell population. Second, ligand-dependent and erentially differentiate into mature MZ and B1 B cells, and trans- -independent mechanisms of BCR tonic signaling coexist at the genic expression of these self-reactive BCRs mainly supports transitional to mature developmental progression. In the latter sce- generation of these populations. These data strongly argue that http://www.jimmunol.org/ nario, although positive selection into the mature pool requires generation of MZ and B1 cells requires positive selection mediated some level of BCR-ligand interactions, these interactions are only by BCR-Ag interactions (62, 65, 77, 90). Accordingly, MZ and B1 limiting in a polyclonal competitive environment. Because matur- cells are present in a semiactivated state in which they are consti- ing B cells in our system did not express receptors capable of tutively secreting Ab. Phosphocholine and phosphatidyl choline ligand binding, a competitive environment is never established, molecules are present in surface of different strains of Streptococ- allowing the B cells to enter into the mature pool. In agreement cus, and natural Abs against these molecules may help to prevent with them, B cells expressing transgenic receptors lacking the H infection by these pathogens (91–97). Taken together, these and chain V region can successfully reconstitute a mature B cell pop- our data support a model where generation of the different mature ulation but failed to do so when mixed with B cells carrying li- B cell populations depends on a different mechanism of BCR sig- by guest on September 28, 2021 gand-binding competent receptors (76). This model will also rec- naling. In this scenario, ligand-independent tonic signaling prefer- oncile the observation that transitional cells can be forced to entially forms FO mature B cells, whereas receptor signaling, trig- differentiate into FO B cells in vitro by addition of low doses of gered by interactions with self-Ags, forms MZ and B1 populations. anti-Ig␤ Ab, which might mimic a low-affinity ligand-receptor in- These mechanisms of receptor signaling presumably evolved to teraction (77). In this scenario, tonic signaling can be originated provide B cells with the wide plasticity of specificities that con- through aggregation-dependent as well as -independent mecha- stitute the innate and acquired humoral immune response. Thus, nisms, and thus pre-BCR/BCR interactions with itself and with mature MZ and B1 B cells require ligand interactions for matura- nonpolymorphic ligands or self-Ag might also be needed at dif- tion, but once they reached this stage they secrete Abs that, al- ferent stages of development. Perhaps these mechanisms ensure though of low specificity, are sufficient to provide the first barrier the development of B cell populations expressing receptors with of defense against infectious agents. On the contrary, FO B cells wide and more restricted specificities. might only require a low level of pre-BCR/BCR activity sufficient to indicate the maturing B cell about the presence of a functional Only FO B cells are found in the pool of mature B cells signaling-competent receptor. However, mature FO B cells can be expressing the Ig␣/Ig␤-mediated tonic signal instructed to generate a more specific immune response that al- The origin of the mature FO, MZ, and B1 B cell populations is though delayed, is more efficient. It is of note that MZ B cell unclear. Adoptive transfer experiments of progenitors derived development has also been proposed to be driven by weaker BCR from fetal liver or adult bone marrow preferentially originate B1 signals than those that lead to FO B cell development (58). This and FO/MZ mature B cells, respectively, supporting the existence discrepancy is presently unclear. of two different developmental pathways separated by ontogeny (78–84). The data presented here argue that the Ig␣/Ig␤-derived ligand-independent tonic signal preferentially, if not exclusively, Acknowledgments induces differentiation into the FO mature B cell compartment. We thank Justina Stadanlick for editorial assistance in the preparation of Although some studies have also found adult-derived B1 cells this manuscript. We also thank Dr. Leslie King for comments in the prep- (78–84), our analysis is inconclusive regarding the capacity of the aration of this study. We also acknowledge Dr. Warren Pear as core di- ligand-independent BCR signal to form B1 B cells. Transgenic rector for a National Cancer Institute-funded retroviral core and the expression of the ITAM containing EBV protein LMP2A results in Abrahmson Cancer Center Flow Cytometry Core. 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 Disclosures interpreted as commitment to the B1 lineage at that stage. Contrary The authors have no financial conflict of interest. The Journal of Immunology 7921

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