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The Journal of Immunology, 2005, 175: 358-366.
Copyright © 2005 by The American Association of Immunologists

The Unique Region of Surrogate Light Chain Component {lambda}5 Is a Heavy Chain-Specific Regulator of Precursor B Cell Receptor Signaling1

F. Betul Guloglu2,*,{dagger}, Ewa Bajor2,*,{dagger}, Brendan P. Smith*,{dagger} and Christopher A. J. Roman3,*

* Department of Microbiology and Immunology and {dagger} Program of Molecular and Cellular Biology, School of Graduate Studies, State University of New York, Downstate Medical Center at Brooklyn, Brooklyn, NY 11203


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Signals transduced by precursor-BCRs (pre-BCRs) composed of Ig µ heavy chains (HCs) and the surrogate L chain components {lambda}5 and VpreB are critical for B cell development. A conserved unique region (UR) of {lambda}5 was shown to activate pre-BCR complexes in transformed cells and to engage putative ligands, but its contribution to pre-B cell development is not known. It is also not clear why the {lambda}-like sequences in {lambda}5 are used to select HCs that will associate mainly with {kappa} L chains. In this study, we show that, in transformed and primary mouse B cell progenitors, receptors containing full-length HCs and lacking the {lambda}5UR were expressed at higher surface levels, but exhibited reduced activity compared with normal pre-BCRs in supporting developmental changes that accompany the progenitor to pre-B cell transition in primary cell culture systems and in the bone marrow in vivo. In contrast, deletion of the {lambda}5UR did not change net signaling output by the Dµ-pre-BCR, a developmentally defective receptor that exhibited impaired activity in the primary cell culture system. Moreover, the {lambda}-like sequences in {lambda}5 were more accommodating than {kappa} in supporting surface expression and signaling by the different HCs. These results show that the {lambda}5UR is important, although not essential, for surrogate L chain-dependent receptor signaling in primary cells, and furthermore may help allow discrimination of signaling competency between normal and Dµ-pre-BCRs. That the {lambda}-like portion of {lambda}5 in the absence of the UR was nondiscriminatory suggests that the {lambda}5UR focuses pre-BCR-dependent selection on the HC V region.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Developmental progression of B cells in vivo depends on the functional rearrangement of IgH and L chain genes and the assembly of the resultant proteins into signaling competent receptor complexes (1, 2). To differentiate into a precursor (pre)4-B cell (fraction C and D (3); large and small pre-BII (4)), the heavy chain (HC) newly synthesized by the progenitor (pro)-B cell (fractions A–C (3); pro-B and pre-BI (4)) normally must form a receptor complex with the surrogate L chain (SLC) components {lambda}5 and VpreB, known as the pre-BCR (reviewed in Refs. 2 and 5). Pre-BCR signaling is critical for the selection of pre-B cell clones that have productively rearranged an IgH gene, resulting in proliferation, repression of SLC gene expression, and the stimulation of L chain rearrangement once cells come out of cycle. Genetic deficiency of pre-BCR components results in partial to complete B cell deficiency, due to developmental arrest at the pro-B cell stage in mice and humans. The incomplete block of B cell development in {lambda}5-deficient mice has been largely attributed to either rare, precocious, and productive L chain rearrangements (6, 7) or rare HC clones that do not require the SLC complex for signaling (so-called "SLC-independent HCs") (8, 9); in contrast, developing human B cells expressing HCs with this property appear to be deleted (10). However, the general observation is that HCs that fail to form a pre-BCR complex do not contribute to the Ig repertoire of the adult mature conventional B cell pool (11), because the vast majority of HCs depend on the SLC for receptor biosynthesis and signaling (so-called SLC-dependent HCs). This process selects B cells expressing HCs that are most suitable for pairing with L chains and thus fundamentally shapes the preimmune Ig repertoire.

Because of the critical role of the pre-BCR in development and structural differences from the mature BCR, the mechanism of pre-BCR signaling and the role of the SLC in that process have been the focus of intensive study. Unlike Ig L chains of BCRs, {lambda}5 and VpreB proteins are invariant, products of separate genes, and each contain unique and evolutionarily conserved non-Ig sequences (unique regions; URs) that append the Ig sequences. These URs interrupt the SLC complex at what would be the equivalent of the VL CDR3 region of an Ig L chain, which would participate in Ag binding (12). Because of these differences, a key issue is whether or to what degree pre-BCR signal activation is ligand-dependent, like the BCR, and/or ligand-independent, like the pre-TCR (13). These mechanisms are not necessarily mutually exclusive. If ligand-dependent, it is possible other cell types that express putative pre-BCR ligands may be involved in regulating pre-BCR activation.

A related but distinct question is how the SLC participates in pre-BCR signaling, whether it be through ligand dependent or independent means. For SLC-dependent HCs, the SLC is required to allow their release from the endoplasmic reticulum so that the resulting pre-BCR complexes localize to the cell surface where signaling molecules are engaged (e.g., Refs. 14 and 15). Moreover, there is growing evidence that the SLC as part of the pre-BCR has an additional role in actively triggering signal transduction at the cell surface. Unlike the BCR, a significant fraction of pre-BCR complexes appear to constitutively localize to lipid rafts in the absence of extrinsic cross-linking, a property shared by the pre-TCR (13, 16). Consistent with this, pre-BCR complexes expressed in Abelson-transformed cell lines activate phosphorylation of Ig{alpha}, an indicator of pre-BCR signaling, in the absence of auxiliary cells or cell-extrinsic cross-linking, and this property required the UR of {lambda}5 (17). A correlative property was that wild-type (wt) pre-BCR complexes were efficiently internalized and aggregated, in contrast to those lacking the {lambda}5UR; rather, the mutant complexes were present at higher levels than wt on the cell surface. These data show that the {lambda}5UR can have an active role in cell autonomous pre-BCR signaling. Whether a cell-autonomous ligand was involved remains an open question. Putative pre-BCR ligands have been identified that specifically interact with the {lambda}5UR and which mediate pre-BCR cross-linking, namely galectin-1 in humans and heparan sulfate in mice (18, 19). However, the functional significance of these interactions in vivo has not yet been established, and it is controversial whether pre-BCR aggregation occurs and is necessary for signaling in primary B cells (20).

The goals of the present studies were to determine whether these pre-BCR and SLC properties were important for pre-BCR signaling output in primary pro-B cells, and whether it was evident with diverse SLC-dependent HC clones that are known to either positively or negatively select B cells at the pre-B stage in vivo. An example of the latter is the variant mouse HC Dµ, which forms pre-BCR complexes with the SLC (21, 22, 23) but fails to support development (24, 25). The mechanisms that underlie the signaling defect of these variant pre-BCRs are not completely understood. In addition, although a focus has been the {lambda}5UR, it is not apparent why the remaining Ig sequences in {lambda}5 are {lambda}-like rather than {kappa}-like, given that pre-BCR-selected HCs will, for the most part, associate with {kappa} L chains. To address these issues, we have used primary pro-B cell culture systems (26) and adoptive transfer approaches to probe the structural requirements for pre-BCR components in pre-BCR signaling. Using these strategies, we show that the {lambda}5UR selectively enhances pre-BCR signaling in a manner dependent on HC structure that allowed the best discrimination of signaling competency between normal vs Dµ pre-BCRs. In contrast, {lambda}5 Ig sequences were more accommodating of HC structure than {kappa} in enabling surface expression and signaling by different HCs. Therefore, diverse components of the pre-BCR differentially regulate pre-BCR homeostasis and signaling in a manner sensitive to the structural attributes of the IgH clonotype.


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

The creation of cDNAs encoding mouse µ HC 17.2.25, human µ HC TG.SA, {lambda}1, {lambda}5, JC{lambda}5, and JC{kappa} have been described previously (21). Briefly, JC{lambda}5 was created by deleting the leader and UR of {lambda}5 and replacing them with the leader of {lambda}1; JC{kappa} was made by replacing the leader and V{kappa} sequence of a MOPC21{kappa} L chain with the leader of {lambda}1. The cDNAs were subcloned into either MiG (27), a murine retroviral construct that contains the marker gene GFP linked to the cDNA of interest via an internal ribosome entry site, or to MihCD4{Delta}, a retroviral plasmid that contains the marker gene hCD4{Delta} similarly linked via an internal ribosome entry site. MihCD4{Delta} was created by replacing GFP in MiG with the hCD4{Delta} sequence from pMACS 4.1 (Miltenyi Biotec). hCD4{Delta} is an inactive allele of human CD4 that lacks the cytoplasmic domain necessary for signal transduction but which can still be expressed on the cell surface and detected by flow cytometry.

Cells and in vitro cell culture

The v-abl-transformed Rag1–/–{lambda}5–/– pro-B cell line was established by standard methods; the Rag1–/– line 1–2 was described previously (Ref. 28 , and references therein). Fresh primary IL-7-dependent pro-B cell cultures were established by harvesting and plating total bone marrow of 4- to 6-wk-old Rag1–/– (29), {lambda}5–/– ({lambda}5T; Jackson Laboratories) (30), and Rag1–/–{lambda}5–/– mice (kept under specific pathogen-free conditions) in RPMI 1640 (Invitrogen Life Technologies) supplemented with FBS (Invitrogen Life Technologies), antibiotics, and rIL-7 (100 U/ml; Cell Science) seeded at a density of 0.5–2 x 106 cell/ml, and maintained in culture for 2 days before retroviral infections (26, 31).

Retroviral infections

Retrovirus-containing supernatants were produced by cotransfection of HEK293 cells with the retroviral plasmids plus p{psi}ECO encoding ecotropic helper functions (32). V-abl-transformed and primary cells were spin-infected with recovered supernatants (33). IL-7-dependent pro-B cells were kept in culture for 2 days after plating before spin-infection. Double infections of v-abl-transformed cells were done by simultaneous coinfection. Briefly, viral supernatants and polybrene were added to 0.5–2 x 106 bone marrow cells per well in 12- or 24-well plates, followed by centrifugation at 2500 rpm at 25°C for 1.5 h. Supernatants were then replaced with fresh medium supplemented with 100 U/ml IL-7 after infection. For double infections, the spin infection was repeated twice with 1 day apart between infections. Double infections of primary IL-7-dependent pro-B cells were done by first infecting cells with the surrogate or conventional L chain ((S)LC) virus, then splitting the infected cells into separate wells for infection with each HC virus. Cells were analyzed 2–4 days after infection by flow cytometry as described in the figures. Similar results were obtained by the reverse infection order.

Analysis of surface marker expression and cell growth

Western analysis of extracts from primary and transformed cells were performed with mouse IgM-specific Ab (Jackson ImmunoResearch Laboratories) as described previously (21). The following Abs (directed against mouse Ags except where noted) were used to stain cells for flow cytometry by standard protocols: anti-CD19-TRI; anti-IgM-PE, and anti-CD4-Tri from Caltag Laboratories; anti-pre-BCR-biotin (SL156), anti-{lambda}5-biotin (LM34), anti-CD2-PE, anti-{kappa}-PE, anti-B220-Tri, and streptavidin-PE from BD Pharmingen; and anti- {lambda}1-PE and anti-human IgM-PE from Southern Biotechnology Associates. The VpreB Ab is an affinity purified and biotinylated chicken IgY directed against a peptide within the C-terminal tail of mouse VpreB1 (BioGenes). The values shown for CD2 induction are the percentage of CD19+GFP+ cells that were also CD2+ 4 days after infection. For calculating the fold change in relative growth, the fold change in the percentage of CD19+GFP+ cells in the cultures after 24 h (double infections) or 48 h (single infections) of growth was divided by the fold change in the percentage of CD19+GFP-negative cells in the same culture over the same time period. The fold change in CD19+GFP-negative cells in each sample was defined as 1 (no change in relative growth rate) in the bar graphs: in both CD2 regulation and growth properties, HC-only or (S)LC-only infected cells behaved the same as MiG (empty control)-infected cells.

Adoptive transfers

Bone marrow from 6- to 8-wk-old {lambda}5T mice treated with 5-fluorouracil was harvested, cultured, and then infected with retroviral supernatants prepared as described above according to established protocols (27). Approximately 1 x 105 infected cells were transferred by tail vein injection into lethally irradiated 6- to 8-wk-old syngeneic wt recipients. Cells were harvested from lymphoid organs 6–8 wk posttransfer. Data shown were from mice that contained >0.5 x 103 B220+GFP+ cells per 105 events collected. All procedures in this study involving animal subjects were reviewed and approved by the Institutional Animal Care and Use Committee.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The non-Ig UR of {lambda}5 limits pre-BCR surface expression with structurally diverse HCs in Abelson-transformed and primary pro-B cells

Our previous studies in a nonlymphoid system showed that clonotypically diverse pre-BCR complexes that lacked the {lambda}5UR were expressed on the cell surface at much higher levels than the wt pre-BCR, rather at levels comparable to the mature BCR (21). This was evident with different engineered {lambda}5 mutants in which the UR was deleted, including JC{lambda}5, in which the leader of {lambda}1 replaced the leader and UR of {lambda}5, and JC{kappa}, structurally similar to JC{lambda}5 but with J{kappa} and C{kappa} regions (21). More recent studies by Ohnishi and Melchers (17) showed that the {lambda}5UR similarly controlled pre-BCR homeostasis and was required for pre-BCR activation in an Abelson-transformed {lambda}5–/– cell line expressing an endogenous HC. However, it is not known whether that HC could support normal B cell development in vivo and thus whether its properties with {lambda}5 mutants were representative of positively selected HCs. Therefore, we tested whether these properties were evident with structurally diverse, SLC-dependent HC clonotypes for which the biological properties are known. Both normal µ HCs used in this study can form complexes with the SLC, {lambda}1, and {kappa} LCs, and support B cell development in vivo (mouse 17.2.25 (34) and human TG.SA (35)). The mouse HC will be referred to as "µ," and the human HC, when specified, will be referred to as "T" in the figure legends. The truncated HC Dµ (300-19P), which forms pre-BCR complexes but cannot support normal B cell development in vivo (25), was used as an example of a SLC-dependent but signaling defective pre-BCR.

The wt and mutant pre-BCR expression was analyzed in Rag1–/–{lambda}5–/– v-abl-transformed pro-B cell lines retrovirally transduced with the mouse µ HC and wt or mutant {lambda}5 genes linked to marker genes (GFP or hCD4{Delta}; see Materials and Methods). Little to no µ surface expression was detected on Rag1–/–{lambda}5–/– cells that expressed only the marker gene or the full-length mouse µ HC (Fig. 1A, columns 1 and 2; IgM stain); as expected, the same cells were also negative for pre-BCR and VpreB surface staining. The pre-BCR mAb is reported to recognize an unspecified conformational epitope dependent on {lambda}5 and µ (Ref. 36 , Fig. 1A, and data not shown), whereas the VpreB Ab recognizes the C-terminal UR tail of VpreB. The positive control showed that Rag1–/–{lambda}5–/– cells transduced with both the µ HC and wt {lambda}5 displayed pre-BCR complexes on the surface, with detectable surface expression of µ, the pre-BCR epitope, and endogenous VpreB (Fig. 1A, column 3). In contrast, cells expressing µ with the truncated JC{lambda}5 allele that lacked the {lambda}5UR (21) had ~5-fold higher surface pre-BCR levels than wt as detected with the IgM, pre-BCR, and {lambda}5 Abs (Fig. 1A, column 4, and data not shown). The VpreB stain also suggested increased surface levels by deleting the {lambda}5UR, but it cannot be excluded that the {lambda}5UR deletion better exposed the VpreBUR epitope recognized by that Ab (Fig. 1A, column 4), which may also account for the low staining intensity of wt pre-BCR complexes with this Ab relative to the IgM and pre-BCR stains. JC{kappa} behaved similarly to JC{lambda}5 in this assay as measured by changes in µ HC and VpreB surface expression (Fig. 1A, column 5). Consistent with the µ surface staining by flow cytometry, Western analysis of µ protein in the infected cells showed highest amounts of Golgi-modified mature HCs in JC{lambda}5- and JC{kappa}-expressing cells and lower amounts with wt {lambda}5; these species were not detected in HC-only infected cells (Fig. 1C, and data not shown). These results indicate that the {lambda}5UR restricts the levels of pre-BCR surface expression in pro-B cells and that {lambda}5 sequences per se are not required for cooperation with VpreB to form HC receptor complexes on B cells.



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  		FIGURE 1. The UR of {lambda}5 regulates pre-BCR surface expression in transformed pro-B cells. FACS and Western blot analyses of Rag1–/–{lambda}5–/– (A–C) and Rag1–/–{lambda}5+/+ (D and E) v-Abl-transformed pro-B cells infected with the indicated HC and/or SLC viruses. Histograms (A and B) and Western blots (C and E) are representative of three separate experiments. A and B, Surface staining profiles for the indicated pre-BCR components on GFP+hCD4+-gated cells. A, Cells infected with the mouse µ HC virus; B, cells expressing Dµ. Filled-in histograms are the profile of cells infected with empty GFP and hCD4 viral vectors. Overlays show profiles of cells infected with the viral vectors listed atop each column. A and B, The percentage of cells within the boundary marker for the indicated profile is shown in the upper right corner. C, Anti-Ig µ HC immunoblot of lysates from transformed Rag1–/–{lambda}5–/– cells infected with pre-BCR components, as indicated above each lane. The mature form for each HC is indicated with an *. D, Pre-BCR component surface expression of µ HC (µ) and Dµ-infected Rag1–/–{lambda}5+/+ v-Abl-transformed pro-B cells. Percentage of GFP+ cells that are positive for the marker is indicated. E, Western blot of HCs from samples in D, and primary Rag1–/–{lambda}5+/+ B cells from Fig. 2B.

 
The restrictive property of the {lambda}5UR on surface pre-BCR levels was also evident with Dµ. In the Rag1–/–{lambda}5–/– Abelson cell line, Dµ-pre-BCR complexes with retroviral wt {lambda}5 and endogenous VpreB were expressed at much lower levels on the surface than µ-pre-BCRs, even though both HCs were expressed at comparable levels (Fig. 1, B and C, and data not shown). This was recapitulated in a Rag1–/–{lambda}5+ Abelson line that expressed retroviral Dµ with endogenous {lambda}5 and VpreB (Fig. 1D, compare Dµ and µ columns). In both cases, this corresponded to little to no detectable complex glycosylation of Dµ (Fig. 1, C and E). However, in the Rag1–/–{lambda}5–/– Abelson cells, deletion of the {lambda}5UR dramatically increased Dµ-pre-BCR surface expression and Dµ complex glycosylation (Fig. 1B, compare columns 3 and 4, and C, lanes 3 and 4). Interestingly, unlike the µ-{lambda}5 and µ-JC{lambda}5 pre-BCR complexes, the Dµ-JC{lambda}5 surface complexes appeared to lack VpreB (Fig. 1B, column 4). Another difference was that Dµ was not effectively transported to the cell surface with JC{kappa} (Fig. 1B, column 5, and C, lane 5). We conclude that the {lambda}5UR limits pre-BCR surface expression with structurally diverse HCs and that JC{lambda}5 was more accommodating of HC structure than JC{kappa}.

To address whether the properties of the {lambda}5UR were evident in primary pro-B cells with endogenous HCs, pre-BCR homeostasis was examined in IL-7-dependent, {lambda}5–/– ({lambda}5T; Ref. 30) bone marrow pro-B cells that were infected with either the empty marker retrovirus (MiG), or viruses that expressed the {lambda}5, JC{lambda}5, or JC{kappa} proteins (Fig. 2A). For comparison, a conventional {lambda}1 LC was included in parallel, the results of which will be presented and discussed in later sections. Pre-BCRs with endogenous µ HCs were detected on the surface when these cells virally expressed {lambda}5 (Fig. 2A, top row). In contrast, higher levels of pre-BCR and/or µ HC surface intensity was observed when cells expressed JC{lambda}5 (pre-BCR/IgM stain) or JC{kappa} (IgM stain) compared with the {lambda}5-expressing cells. However, fewer surface µ HC+ cells were observed with JC{kappa} than with JC{lambda}5 (Fig. 2A, bottom). Similar observations were obtained in Rag1–/–{lambda}5–/– primary cells infected with HC and either {lambda}5, JC{lambda}5, or JC{kappa} viruses (Fig. 2B). As expected, steady-state levels of wt pre-BCR complexes were much lower on primary cells than on Abelson lines (compare IgM and pre-BCR stains for µ in Figs. 1, and 2, A–D). However, higher surface µ expression with JC{lambda}5 compared with {lambda}5 was observed with the mouse µ HC, the human µ HC (T), and Dµ (Fig. 2B, rows 2 and 3; and higher pre-BCR expression, Fig. 2, C and D), but as in the Abelson system, JC{kappa} only facilitated surface transport of the mouse µ HC (Fig. 2B, row 4, and D, and data not shown). Differences in HC surface expression could not be attributed to differences in protein expression (Figs. 1E, and 2E). These results show that the {lambda}5UR controls pre-BCR homeostasis in primary cells with endogenous HCs. They also further support the model that, independently from the {lambda}5UR, the Ig sequences of {lambda}5 are less restrictive than {kappa} in accommodating HC structural differences to allow surface expression of resulting complexes.



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  		FIGURE 2. The UR of {lambda}5 regulates pre-BCR surface expression in primary pro-B cells. FACS analysis of primary bone marrow pro-B cells from Rag+{lambda}5–/– (A) or Rag1–/–{lambda}5–/– mice (B–D) cultured in IL-7, 2 days after infection with the indicated HC and SLC/LC retroviruses. A, Rag1+ {lambda}5–/– IL-7-expanded pro-B cells were infected with the indicated SLC retrovirus and analyzed 2 days later for surface pre-BCR and Igµ expression. B, Surface µ HC expression (IgM stain) of Rag1–/–{lambda}5–/– cells infected with HC (µ or T) and (S)LC retroviruses, as indicated on the top of each column and left side of each row of dot plots, respectively. Cont, corresponding marker-only controls. Percentage of GFP+ cells that are positive for the marker is indicated. N.B. The surface expression of human µ HC TG.SA (T) was detected with a human-specific Ab, and therefore relative staining intensity cannot be directly compared between the µ HCs; it is shown here for illustrative purposes only. C, Pre-BCR and {lambda}1 and (D) VpreB surface expression profiles for the same samples in B. E, Western blot of mouse Ig µ expression from Rag1–/–{lambda}5–/– cells infected with the indicated retroviruses from B–D.

 
IgH clonotype dependence on the {lambda}5UR for pre-BCR signaling in primary pro-B cells in culture

We then determined the relationship of the {lambda}5UR and pre-BCR surface levels to signaling competence in primary cells. A pre-BCR-dependent change that accompanies the pro- to pre-B cell developmental transition in vivo is the acquisition of the surface marker CD2 (37). Pre-BCR expression also enables cells to better proliferate and accumulate at a faster rate than pre-BCR– cells in the same culture during the first 3 days in IL-7 (26, 38). We first explored whether these properties could be used as a way to examine the consequences of wt and mutant pre-BCR expression on IL-7-dependent pro-B cell growth and differentiation in culture.

Retroviral-mediated mouse HC expression in Rag1–/–{lambda}5+ pro-B cells increased the proportion of those cells that expressed CD2 compared with cells expressing the control (MiG) virus and uninfected cells in the same culture (Fig. 3A, top, and B). In this assay, the mouse µ was better able to induce CD2 than the human (Fig. 3B). This effect was dependent on endogenous {lambda}5, because no increase in the proportion of CD2+ HC-infected Rag1–/–{lambda}5–/– cells was observed (Fig. 3A, bottom); this result also confirmed that pre-BCR signaling by the HC clones was SLC-dependent. We then took advantage of this last observation to ask whether simultaneous retroviral expression of both a HC and {lambda}5 could activate pre-BCR signaling in the Rag1–/–{lambda}5–/– cells. Indeed, induction of CD2 expression in the Rag1–/–{lambda}5–/– cells required coexpression of the mouse or human HCs with {lambda}5 (Fig. 3C).



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  		FIGURE 3. IgH clonotype dependence on the {lambda}5UR for pre-BCR signaling in primary pro-B cells in culture. A, Differential induction of CD2 by Dµ vs µ HCs in Rag1–/– pro-B cells. Representative FACS analysis of CD2 surface expression on cells infected with the indicated retroviruses, analyzed 4 days after infection. The percentage of GFP-negative cells that are CD2+ (left), and GFP+ cells that are CD2+ (right) are indicated in the respective upper quadrants. Upper panels, Rag1–/–{lambda}5+ cells; lower panels, Rag1–/–{lambda}5–/– cells. B, Bar graph of CD2 surface expression data from Rag1–/–{lambda}5+ cells. The percentage CD2+GFP+ cells was calculated by defining the total of all GFP+ cells as 100% (n = 15; SE bars shown). C, Bar graph comparing relative fold increase of GFP+ cells infected with control, Dµ (D), mouse HC (µ), and human HC TG.SA (T) viruses compared with uninfected (GFP–) in Rag1–/–{lambda}5+ cells (n = 10 for Dµ and µ; n = 5 for TG.SA). D, Differential induction of CD2 by Dµ vs a normal HC and {lambda}5 vs JC{lambda}5 and other (S)LCs in Rag1–/–{lambda}5–/– pro-B cells. As in A, cells were analyzed for CD2 expression; the proportion of CD2+ cells in each quadrant was normalized to GFP-negative cells to generate the bar graphs. The HC viruses are indicated below each bar; the (S)LC virus is indicated in each panel (n = 7). E, Fold increase in GFP+ cell number compared with GFP– cells by Dµ vs a normal HC and {lambda}5 vs JC{lambda}5 and other LCs in Rag1–/–{lambda}5–/– pro-B cells. Bar graphs are labeled as in C (n = 19 except for JC{kappa}, n = 3; SE bars are shown).

 
Similar effects were observed when changes in relative growth were monitored as a readout for pre-BCR signaling (Fig. 3D). The relative growth of Rag1–/–{lambda}5+ pro-B cells infected with mouse or human HCs was 3- to 4-fold greater than pre-BCR– cells in the same culture (Fig. 3D). Similarly, Rag1–/–{lambda}5–/– cells coinfected with HC and {lambda}5 retroviruses outgrew single- and uninfected cells (Fig. 3E). Controls indicated that Rag1–/–{lambda}5–/– CD19+ cells infected with GFP only, HC, or {lambda}5 viruses alone, had the same relative growth rate as uninfected CD19+ cells in the same cultures (Fig. 3E), confirming the pre-BCR dependency of these changes. Thus, pre-BCR signaling, as measured by both CD2 up-regulation and relative growth, can be reconstituted in genetically pre-BCR-deficient pro-B cells by retroviral expression of the missing pre-BCR component(s). Using these readouts in the primary Rag1–/–{lambda}5–/– cell culture system, we tested the activity of mutant pre-BCR complexes that lacked different {lambda}5 sequences. JC{lambda}5-pre-BCR complexes with mouse and human µ HCs were able to promote CD2 induction and confer a growth advantage to Rag1–/–{lambda}5–/– pro-B cells, although at a reduced level (≥50% less) than the corresponding wt pre-BCR complexes (Fig. 3, C and D). Interestingly, subtle differences in pre-BCR activity were observed between the mouse and human HCs. Pre-BCRs with the human µ T were measurably less active and more affected by the {lambda}5UR deletion than the mouse µ. This could not be attributed to differences in pre-BCR expression, because VpreB surface expression dependent on each HC was similar for both HCs (Fig. 2D). More dramatic differences were observed with JC{kappa}, which was less active than JC{lambda}5 with the mouse µ HC, and not active with the human µ HC in this assay. This is consistent with the observation that JC{kappa} could not form surface complexes with the human HC (Fig. 2B). These results indicate that the {lambda}5UR is important but not essential for activation of pre-BCR-dependent developmental changes in pro-B cells. They also show that surface receptor levels do not directly correlate with receptor activity. Finally, they reiterate the observation that the Ig regions of {lambda}5 are optimal for pre-BCR surface expression and signaling.

The Dµ-pre-BCR behaved differently in these assays than the normal µ HC pre-BCRs in several respects. In vivo experiments using Dµ-transgenic Rag2–/– mice showed that Dµ-pre-B cells were defective compared with normal pre-B cells in up-regulating CD2 expression and stimulating proliferation (25). Similarly, in the Rag1–/–{lambda}5+ primary pro-B cell system, Dµ expression led to a detectable but greatly reduced up-regulation of CD2 and only a slight increase in relative growth rate when compared with the normal HC (Fig. 3, A, B, and D). This was paralleled in Rag1–/–{lambda}5–/– pro-B cells in which coinfection with Dµ and {lambda}5 retroviruses led to a modest but significantly decreased ability to induce CD2 and proliferate relative to normal pre-BCR complexes; nevertheless, the low activity observed required both Dµ and {lambda}5 (Fig. 3, C and E). Thus, the impaired ability of the Dµ-pre-BCR complex to up-regulate CD2 expression and promote growth was evident in this system as it was in vivo.

Interestingly, Dµ-JC{lambda}5 complexes were at least the same if not more active than Dµ-{lambda}5 complexes, in marked contrast to complexes with normal HCs (Fig. 3, D and E). Given that Dµ-pre-BCR complexes were expressed on the cell surface at much lower levels than normal pre-BCRs, we hypothesized that the increase in Dµ-surface expression with JC{lambda}5 may have compensated for the loss of the {lambda}5UR, assuming the {lambda}5UR always enhances pre-BCR signaling regardless of HC structure and despite the absence of VpreB. In support of this idea, coexpression of a conventional {lambda}1 LC, which can form surface complexes with Dµ HCs (Fig. 2, B and C, and Ref. 21), led to activation of pre-BCR-dependent changes in growth and CD2 induction with Dµ and at higher levels than with the SLC. In contrast, JC{kappa} was inactive with Dµ, which correlated with its inability to transport this HC to the cell surface compared with JC{lambda}5 (Figs. 1, and 2B, and Ref. 21). Moreover, depending on the assay system, differences in pre-BCR signaling between Dµ and the full-length HCs were either much less pronounced or even nearly indistinguishable with {lambda}1 and JC{lambda}5 than with wt {lambda}5 (Fig. 3, D and E). This is especially noteworthy considering Dµ-{lambda}1 complexes were present on the surface at lower levels than the corresponding µ-{lambda}1 and T-{lambda}1 complexes (Fig. 2B, row 5, and C). We conclude that the relative contribution and functions of the {lambda}5UR and VpreB to pre-BCR signaling are dependent on the HC clonotype. Importantly, this indicates that the {lambda}5UR allows better discrimination of normal vs Dµ HCs by amplifying the differences in their relative pre-BCR activity.

Another property revealed by the analysis of {lambda}1 with the normal HCs was that {lambda}1 and {lambda}5/VpreB complexes had similar activity in CD2 induction, but the {lambda}1-HC complexes were less active than the corresponding pre-BCRs in the proliferation assay. Although these results confirm that HC-LC complexes can support signaling normally dependent on the pre-BCR, in this system the pre-BCR itself was best suited to promote the proliferation and accumulation of pre-B cells.

The non-Ig domain of {lambda}5 expedites but is not required for pre-BCR-dependent development in primary pro-B cells in vivo

Although the primary cell culture experiments showed that the {lambda}5UR-deleted pre-BCRs retained pre-BCR signaling activity, the remaining activity may be insufficient to support pre-B cell development in vivo, for example, if ligand-mediated/cell non-autonomous activities were still required. Thus, to ascertain the functional contribution of the {lambda}5UR to pre-BCR signaling in vivo and its relationship to the properties revealed in cell culture, we used an adoptive transfer approach. As a proof of principle, we first evaluated whether the {lambda}5–/–-B cell deficiency could be corrected by retroviral expression of {lambda}5 in {lambda}5-deficient hemopoietic cells. {lambda}5–/– adult bone marrow cells were infected with control, {lambda}5, or JC{lambda}5 viruses and adoptively transferred to lethally irradiated hosts. FACS analysis of bone marrow cells from the recipients indicated that the fraction of small pre-B and more mature B cells (GFP+B220+CD43–CD2+) within the B220+GFP+ population increased on average from 20% in the MiG-infected/{lambda}5–/– reconstituted chimaeras to 50% in the {lambda}5-infected/{lambda}5–/– reconstituted mice (Fig. 4, A and B). Similarly, there was an increase in the proportion of GFP+ splenocytes that were IgM+B220+ in recipient mice reconstituted with {lambda}5-infected/{lambda}5–/– cells compared with those hosting MiG-infected/{lambda}5–/– cells (Fig. 4C). Therefore, the proportion of both pre-B/B cells and splenic IgM+ B cells increased with retroviral expression of {lambda}5, consistent with a rescue of the developmental defect caused by the endogenous {lambda}5 deficiency.



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  		FIGURE 4. The {lambda}5UR expedites but is not required for pre-BCR-dependent development in primary pro-B cells in vivo. {lambda}5–/– bone marrow was infected with the indicated retrovirus and adoptively transferred to lethally irradiated syngeneic hosts. Cells from the indicated lymphoid organs were analyzed by FACS for GFP and lymphocyte marker expression 6–8 wk after transfer. A, Shown are representative dot plots of GFP+ cells within the lymphocyte gate (as defined by scatter) for CD2, CD43, B220, and/or IgM expression. Top panel, bone marrow cells; lower panel, splenocytes. B, Bar graphs showing the relative proportion of bone marrow B220+GFP+ pro- and more mature B cells as defined by CD2 and CD43 expression in the bone marrow. C, Bar graph showing the relative proportion of GFP+ splenocytes that are also IgM+B220+ cells. "N" is the number of individual host mice from at least three separate infections/adoptive transfers that received {lambda}5–/– cells infected with the indicated retrovirus and that carried >0.5 x 103 GFP+ cells per 105 events collected. SE bars are shown.

 
By comparison, there was also a greater proportion of pre-B cells and IgM+ cells in mice hosting {lambda}5–/– cells transduced with the JC{lambda}5 virus compared with the MiG control, but on average less than with wt {lambda}5. We conclude that pre-BCRs lacking the UR of {lambda}5 can promote B cell development and accumulation in vivo, although with a modestly reduced efficiency compared with wt {lambda}5. This is consistent with the partial but not complete inactivation of pre-BCR signaling by deleting the UR that was observed in cell culture. However, a caveat to these experiments is that retroviral {lambda}5 expression is not down-regulated at the pre-B stage in the adoptive transfer system, and any influence of ectopic {lambda}5 expression on late pre-B/mature B cells cannot be ruled out.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Experimental evidence supports the model that SLC-dependent regulation of pre-BCR signaling provides a mechanism to positively select B cells that express HCs with the best likelihood of requiring conventional LCs for signaling and thus B cells that can be regulated by Ag (1, 11, 39). Similarly, there is indication that {lambda}5 can help shape that HC repertoire by restricting VH CDR3 length (40). Collectively, our data support these observations and indicate that {lambda}5 is, in fact, endowed with discrete structural determinants that are differentially sensitive to HC structure. Most prominent is the {lambda}5UR, to which multiple activities have recently been linked, including the regulation of SLC/pre-BCR homeostasis and signal activation, possibly via ligands (6, 17, 18, 19, 21). Our data support the model that the {lambda}5UR-associated activities can be important for activating developmental and proliferative signaling pathways in primary B cells, but are not essential for signaling per se. However, an important qualification is that the degree of {lambda}5UR-dependent enhancement of pre-BCR signaling may depend on the structure of the component HC. In this way, the {lambda}5UR could provide an additional selective mechanism to help discriminate between normal and a particular subclass of defective HCs like Dµ.

However, that mutant pre-BCRs lacking only the {lambda}5UR (µHC-VpreB/JC{lambda}5) or the {lambda}5UR and VpreB (Dµ-JC{lambda}5) still retained partial activity has several ramifications concerning the mechanism and regulation of pre-BCR signaling and repertoire selection. Clearly not inactive, pre-BCR complexes lacking the {lambda}5UR may therefore be more akin to HC-LC complexes that can support pre-B development in the genetic absence of the SLC but at reduced efficiency or giving rise to pre-B cells with somewhat altered surface marker expression (6, 41). This may be reflected in the reduced activity of µ-{lambda}1 complexes compared with wt pre-BCRs in the in vitro system. Our data would suggest that the {lambda}5UR is particularly important for activating proliferation pathways because the efficiency of CD2 induction by the µ-{lambda}1 and µ-SLC complexes was nearly indistinguishable, whereas µ-SLC stimulated proliferation better than µ-{lambda}1 (Fig. 3, C and E).

If deleting the {lambda}5UR changes the parameters of pre-BCR signaling competency rather than inactivate it completely, one possibility is that a different population of HCs than normal, rather than no HCs or only SLC-independent HCs, could be able to support pre-BCR signaling and positively select pre-B cells under conditions of {lambda}5UR deletion in vivo, such as in the adoptive transfer system. In this scenario, the repertoire of HC polymorphisms would include HC clonotypes that could somehow structurally compensate for the {lambda}5UR deletion and allow pre-B development in the absence of the {lambda}5UR. This model would account for the relatively modest difference in {lambda}5 and JC{lambda}5 activity as measured by the support of pre-B cell development in the adoptive transfer system in vivo in which the repertoire of endogenous HCs was available for selection, compared with greater differences revealed when pre-BCR activity was compared with individual HC clonotypes in culture. According to this model, reduced activity of JC{lambda}5 is a function of the fewer naturally occurring HCs in the repertoire that could yield "normal" levels of pre-BCR signaling in the absence of {lambda}5UR. Alternatively, the reduced activity of JC{lambda}5 in vivo may not be reflective of a repertoire shift per se, but rather simply reflect overall reduced pre-BCR signaling by the same HC repertoire, for example, by not activating proliferative/survival pathways that allow normal levels of small pre-B cell accumulation. It is also possible that both mechanisms are in play; these models can be tested by comparing the HC repertoire in {lambda}5UR-deficient vs {lambda}5-deficient and normal mice.

The results further suggest that the relationship of different aspects of pre-BCR homeostasis (assembly and steady-state surface levels controlled by export and internalization rates) to signaling are complex and differentially sensitive to HC and SLC structure. First, although µ HC-VpreB/JC{kappa} and Dµ-{lambda}5/VpreB complexes assemble (21), they were not as effectively or at all transported to the cell surface as normal pre-BCRs or corresponding JC{lambda}5 complexes in primary cells, and this correlated to the decrease or absence of signaling by these mutant pre-BCR complexes. One conclusion is that the molecular basis for the Dµ signaling defect is that most of the Dµ-pre-BCR complexes, although assembled, fail to reach the cell surface. This type of pre-BCR signaling defect at the level of surface transport is distinct from another class of HCs, such as many VH81X HCs, that fail to assemble with the SLC complex at all and consequently are not transported to the surface nor signal from internal locations (e.g., Ref. 15). In addition to the mechanism for selection against Dµ by the {lambda}5UR described in this study, it is possible that the {lambda}5UR also restricts pre-BCR formation at the level of HC pairing, and this remains to be determined with full-length HCs that fail to pair with the SLC. In contrast, increased steady-state levels of surface pre-BCRs with normal HCs have been shown to correspond to impaired pre-BCR signaling activity, as was shown with primary pre-B cells lacking the pre-BCR signaling molecule SLP-65/BLNK (8, 42, 43) and pre-BCRs lacking the {lambda}5UR in Abelson-transformed and primary cells (Ref. 17 , and this study). Taken together, the data would therefore imply that Dµ-pre-BCR signaling must be, in some ways, fundamentally distinct from normal HCs because increasing surface Dµ-pre-BCR levels permitted by the {lambda}5UR deletion also allowed increased signaling, compensating for putative {lambda}5UR activity that would otherwise be important for normal HC signaling. It is possible that the absence of VpreB or the structure of Dµ itself in lacking a VH domain changes the biochemical properties of the resulting pre-BCR complexes in a manner distinct from normal HCs that is responsible for the differences in requirements for SLC components for signaling.

In a related vein, it is noteworthy that Dµ-{lambda}1 complexes were nearly or as active as the HC-{lambda}1 complexes in the primary culture system, although mature B cells expressing Dµ-{lambda}1 complexes have not been reported (24). It is possible that structural differences from normal HCs render Dµ-{lambda}1 complexes incapable of providing survival signals at later developmental stages or chronically activate cells leading to attrition, as was reported for a VH-less human H chain disease protein (44). Interestingly, however, the activity does explain an observation by the Bosma group (45) who found selection for in-frame V{lambda}1-J{lambda}1 rearrangements in scid but not Rag-deficient fetal B cell progenitors. Virtually no full-length µ HCs are produced in scid mice due to a defect in V to DJ joining, but DJH joins are still made in early B cell developmental compartments, and about one-third should produce a Dµ protein (e.g., Refs. 46 and 47). Our data provide experimental evidence to support the model proposed, namely, that the development of fetal B cells that expressed Dµ-{lambda}1 complexes was favored. Collectively, these observations illustrate the multiple selective mechanisms that exist at different stages of development to screen B cells for HC functionality.

The ability to systematically test different aspects of pre-BCR function in transformed and primary pro-B cells can be directed to address how different signaling pathways responsible for proliferation and differentiation respond to HC structure and pre-BCR composition (and related properties). Moreover, these systems can be exploited to examine differences in pre-BCR signaling requirements in fetal vs adult progenitors, the possible contributions of pre-BCR ligands and importance of lipid raft localization to those responses, and ultimately what the long-term relative contributions of such properties are on shaping the mature Ig repertoire.


   Acknowledgments
 
We are grateful to Dr. Warren Pear (University of Pennsylvania, Philadelphia, PA) for training in the adoptive transfer strategy, Dr. C. Lange and the Department of Radiology of State University of New York-Downstate Medical Center for mouse irradiation, and Dr. Avitable for consultation on the statistical analyses. We thank Drs. E. Hsu and S. Gottesman (State University of New York, Downstate Medical Center), and L. Eckhardt (Hunter College/City University of New York) for critical reading of the manuscript and discussions.


   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 Supported in part by Research Project Grant 00-269-01-LBC from the American Cancer Society (to C.A.J.R.) and the New York City Council Speaker’s Fund of the New York Academy of Medicine (to C.A.J.R.). Back

2 F.B.G. and E.B. are co-first authors. Back

3 Address correspondence and reprint requests to Dr. Christopher Roman, the Department of Microbiology and Immunology, Morse Institute for Molecular Genetics, State University of New York, Downstate Medical Center at Brooklyn, 450 Clarkson Avenue, Box 44, Brooklyn, New York, 11203. E-mail address: Christopher.Roman{at}Downstate.edu Back

4 Abbreviations used in this paper: pre, precursor; HC, heavy chain; pro, progenitor; SLC, surrogate L chain; UR, unique region; wt, wild type; (S)LC, surrogate and/or conventional L chain. Back

Received for publication December 29, 2004. Accepted for publication April 19, 2005.


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 Materials and Methods
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