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The Journal of Immunology, 1998, 161: 1132-1139.
Copyright © 1998 by The American Association of Immunologists

Surrogate Light Chain Production During B Cell Differentiation: Differential Intracellular Versus Cell Surface Expression1

Yui-Hsi Wang*, Jun Nomura*,{ddagger}, Ona Marie Faye-Petersen{dagger} and Max D. Cooper2,*,{dagger},{ddagger}

* Division of Developmental and Clinical Immunology; Departments of Medicine, Pediatrics, Microbiology, and {dagger} Pathology; and {ddagger} Howard Hughes Medical Institute, University of Alabama, Birmingham, AL 35924


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of the surrogate light ({psi}L) chain genes encoding the VpreB and {lambda}5/14.1 proteins is restricted to B-lineage cells. Pro-B and pre-B cells produce {psi}L chains, but whether both employ these as cell surface receptor components remains enigmatic. Recombinant human VpreB protein was used to generate a large panel of monoclonal anti-VpreB Abs to examine this issue. Native {psi}L chain proteins within pro-B cells as well as those serving as receptor components on pre-B cells were precipitated by 16 of the 26 anti-VpreB Abs. Surrogate light chains were easily detected on pre-B cell lines, whereas these anti-VpreB Abs reacted with pro-B cell lines only after plasma membrane permeabilization. The subpopulation of normal bone marrow cells bearing pre-B receptors included large and small pre-B cells exclusively, although pro-B cells also contained intracellular VpreB. VpreB proteins were not detected on or within B cells in bone marrow or the circulation, but a subpopulation of B cells in germinal centers was found to express the VpreB proteins intracellularly. Surrogate L chains are thus intermittently produced during human B-lineage differentiation, while their role as receptor components appears limited to the pre-B cell stage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
B-lineage differentiation is characterized by a preferential order of rearrangement for the variable (V), diversity (D), and joining (J) gene segments encoding the Ig H chains and L chains. D->JH gene rearrangements are initiated in progenitor B (pro-B) cells, and subsequent V->DJH rearrangements allow precursor B (pre-B) cells to express µH chains (1, 2), most of which undergo degradation in the endoplasmic reticulum (3). Typically, although not invariably (4, 5), VL->JL rearrangements of {kappa} or {lambda} L chain genes occur later to allow cell surface IgM expression by immature B cells. Regulated expression of other B-lineage-specific genes, including mb1 (Ig{alpha}, CD79{alpha}), B29 (Igß, CD79ß), VpreB, and {lambda}5 (mouse)/14.1(human), is essential for the progression of cells along this developmental pathway (6, 7, 8, 9, 10).

During the pre-B cell stage, the VpreB- and {lambda}5/14.1-encoded {psi}L chain proteins may associate with µH chains and Ig{alpha}/ß heterodimers to form a pre-B receptor complex (11, 12, 13, 14, 15, 16, 17, 18, 19). The single VpreB gene in humans encodes two polypeptide products of approximately 16 and 18 kDa (11, 20, 21, 22). The {lambda}5/14.1 gene, which shares sequence homology with both the J region and the Ig {lambda}L chain constant region, encodes a 22-kDa protein (11, 15, 22, 23, 24). Expression of the VpreB and {lambda}5/14.1 genes, which does not require their rearrangement, is initiated during the pro-B stage, continues through the pre-B cell stage, and is extinguished at the immature B cell stage (8, 9, 19, 22). The VpreB and {lambda}5/14.1 {psi}L chain proteins are noncovalently associated, and this complex can be covalently linked to the µH chains to allow expression of the composite {psi}L chain/µH chain/Ig{alpha}ß receptors on pre-B cell lines (16, 19) and on normal pre-B cells (18). An essential role for this receptor in B cell development is indicated by the arrest that occurs at the pro-B cell stage in differentiation when one of the pre-B receptor genes or the recombinase activating genes, RAG-1 and RAG-2, is deleted (25, 26, 27, 28, 29). The pro-B cell arrest in RAG-/- mice can be alleviated by a µH chain transgene (30, 31). Despite compelling evidence indicating that pre-B receptors provide an important checkpoint in B-lineage development, receptor expression on the cell surface has nevertheless been difficult to elucidate, being variably reported to occur early, late, or never on pre-B cells (18, 19, 32, 33, 34, 35).

Whether {psi}L chain proteins are expressed on pro-B cells has also been difficult to resolve. Murine pro-B cell lines have been reported to express {psi}L chain proteins on the cell surface in association with 45-, 65-, and 130-kDa proteins, collectively termed surrogate heavy ({psi}H) chains (36). Immunofluorescence evidence has also been reported for the expression of {psi}H chain/{psi}L chain receptors on human pro-B cells (37, 38), and one anti-VpreB Ab identified putative {psi}L chain/{psi}H chain receptors on both pro-B and pre-B cell lines (39). Conversely, other Abs that identify {psi}L chain proteins in association with 40-, 60-, and 98-kDa proteins within pro-B cells failed to identify these on the cell surface (19, 39, 40).

The relatively low levels at which {psi}L chain proteins are produced by early B-lineage cells is one impediment to resolution of the issue of their cellular distribution. Compounding this problem is the limited availability of well-characterized mAbs that unambiguously identify {psi}L chain proteins. Some of these Abs may identify exposed VpreB or {lambda}5 epitopes on candidate {psi}L chain/{psi}H chain complexes, while others may not (8, 39). In addition, some anti-human {psi}L chain mAbs are IgM Abs (19, 37, 38, 39), and Abs of this isotype often exhibit relatively low binding affinity and multireactivity (41, 42). To readdress the issues of when and where {psi}L chains are expressed, we have generated a large panel of anti-VpreB mAbs of IgG isotype and defined their patterns of cellular reactivity as a function of B-lineage differentiation.


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

The 207 (43), Nalm16 (44), and RS4;11 (45) pro-B cell lines; the 697 (43), Nalm6 (44), and OB5 (46) pre-B cell lines; the Daudi (IgM, {kappa}) (47) and Ramos (IgM, {lambda}) (48) B cell lines; the Jurkat T cell line; the U937 myelomonocytic cell line; and the K562 erythroid cell line were cultured in RPMI 1640 medium supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10% heat-inactivated FCS, 50 µM 2-ME, and 2 mM L-glutamine. Mononuclear cells were isolated from bone marrow and tonsillar cell suspensions by centrifugation on a Ficoll-Hypaque gradient. Bone marrow samples were obtained from kidney donors undergoing rib resection and aborted previable fetuses, 13 to 19 wk gestational age. Heparinized blood samples were obtained from normal donors, and tonsillar tissues were obtained from individuals undergoing tonsillectomy. All tissues were procured in accordance with policies established by an institutional review board for human experimentation.

Antibodies

The SA-DA4.4 ({gamma}1{kappa}) anti-human µ (49), CB3-1 ({gamma}1{kappa}) anti-human Igß (50), the SLC1 ({gamma}1{kappa}) and SLC2 (µ{kappa}) anti-human {psi}L chain (19), and the JS-2 ({gamma}1{kappa}) control (51) mAbs were used in immunoprecipitation studies. Immunofluorescence assays employed the following mAbs: CY-chrome-labeled anti-CD19 (PharMingen, San Diego, CA); FITC-labeled anti-CD34, anti-CD10, and anti-CD45 (Becton Dickinson, Mountain View, CA); FITC-labeled anti-CD38 (AMAC, Westbrook, ME); FITC-labeled anti-TdT (SuperTechs, Bethesda, MD); FITC-labeled anti-c-Kit (ImmunoTech, Marseille, France); FITC-labeled anti-human µH chain; FITC-labeled goat Abs to human IgM; phycoerythrin (PE)3-labeled goat Abs to human IgD, anti-CD19, and PE-conjugated streptavidin and goat Abs to mouse IgG (H+L) (Southern Biotechnology, Birmingham, AL); CY5-labeled anti-VpreB mAb 8 prepared by conjugation of the CY5 dye following the procedure recommended by the manufacturer (Amersham, Arlington Heights, IL); and biotinylated mouse anti-human {kappa}/{lambda}L chain mAbs (a gift from Hiromi Kubagawa, University of Alabama, Birmingham). Anti-VpreB Abs were also biotinylated following procedures described by the manufacturer (Pierce, Rockford, IL).

Production of recombinant human VpreB proteins

A full-length human VpreB DNA was obtained by PCR amplification of the corresponding cDNA derived from a RS4;11 pro-B cell line cDNA library as a template. The upstream 5'-GTAGAGGCATGCCAGCCGGTGCTG-3' and downstream 5'-CTTGAAGCTTTCAAGGGACACGTGT-3' primers were designed to incorporate SphI and HindIII restriction sites. The amplified product was subcloned into the pQE-30 expression vector (Qiagen, Hilden, Germany) using SphI and HindIII restriction sites. The construct was sequenced to confirm the fidelity of the insert and transformed into Escherichia coli (M15 strain). After induction with 0.1 mM isopropyl-ß-D-thiogalacto-pyranoside, the His-tagged VpreB recombinant protein of about 16 kDa was purified from 8 M urea-denatured cell lysates by passage over a nickel column (HiTrap Chelating, Pharmacia, Piscataway, NJ) and subsequent elution with PBS containing 0.5 M imidazole. Human {lambda}5/14.1 recombinant protein of approximately 22 kDa was obtained by the same cloning procedure after PCR amplification of the corresponding cDNA using upstream 5'-ACTGTCGGATCCTCGCAGAGCAGG-3' and downstream 5'-CAGTCAAGCTTCTATGAACATTCT-3' primers designed to incorporate HindIII and BamHI restriction sites.

Hybridoma production

After multiple s.c. immunizations with 20 µg of purified VpreB protein, BALB/c mice were boosted with 697 pre-B cells the day before fusion of regional lymph node cells with the Ag8.653 plasmacytoma cell line (52). Hybridomas were cultured in hypoxanthine-aminopterin-thymidine medium for 10 days, and the supernatants were screened for anti-VpreB Abs by ELISA and for reactivity with the recombinant VpreB protein by Western blot analysis. Additional screening included immunofluorescence analysis of reactivity with pre-B and B cell lines. Selected hybridomas were cloned by limiting dilution, and the isotypes of their Ab products were determined by an indirect capture ELISA (Zymed, South San Francisco, CA).

Immunochemical analysis

For Western blot analysis of the anti-VpreB Abs, purified Xenopus CD3{gamma}{delta} (53), human VpreB, and {lambda}5/14.1 recombinant proteins (1 µg each) were separated by SDS-PAGE (13%) and transferred onto nitrocellulose membranes with a Protean II xi Cell apparatus (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat dry milk in PBS plus 0.1% Tween-20 before incubation with test Abs. Washed membranes were incubated with goat Abs to mouse IgG-conjugated horseradish peroxidase (Southern Biotechnology) in blocking solution for 1 h at room temperature and washed again before chemiluminescence detection of Ab-reactive bands with an ECL kit (Amersham).

For analysis of anti-VpreB mAb reactivity with cell surface proteins, viable cells (5 x 107) surface labeled with [125I]sodium iodide (2 mCi; Amersham) by the lactoperoxidase method were lysed in 1% Nonidet P-40 lysis buffer. Cell lysates were successively precleared with rat anti-mouse {kappa}L chain and human Ig-coated Sepharose 4B beads before incubation with beads bearing test or control mAbs. Washed immunoprecipitates were eluted by boiling in Laemmli sample buffer and were resolved by SDS-PAGE using 13% acrylamide. For metabolic protein labeling studies, cells (1–2 x 107) were preincubated in Met- and Cys-free RPMI 1640 medium for 2 h, then labeled with 300 to 500 µCi of both [35S]Met and [35S]Cys for 5 h before harvesting, lysis in 1% Nonidet P-40 lysis buffer, and centrifugation at 10,000 x g for 20 min. After incubation with Sepharose 4B beads coupled with human Ig, the precleared lysates were incubated in plastic wells coated with test or control Abs. Bound materials were eluted with Laemmli sample buffer for analysis by SDS-PAGE and autoradiography. For analysis of VpreB protein expression in tonsillar B cells, 4 x 108 cells were lysed in 1% Nonidet P-40 lysis buffer, and the cell lysates were precleared with protein A and Sepharose 4B beads coupled with human Ig before incubation with protein A beads bearing test or control Abs. Precipitated proteins separated by SDS-PAGE were transferred onto nitrocellulose membrane, which were blocked and incubated with the anti-VpreB mAb 8. After washing, the membrane was probed with goat Abs to mouse IgG-conjugated horseradish peroxidase (Southern Biotechnology) and revealed by SuperSignal ULTRA chemiluminescent substrate (Pierce).

Immunofluorescence

Viable cells incubated with hybridoma supernatant or purified mAb (0.05–0.1 mg/ml) were washed before staining with PE-conjugated goat anti-mouse Ig. For cytoplasmic staining, cells were fixed and permeabilized with 70% ethanol on ice for 1 h, blocked with 2.5% FCS in PBS for 15 min, and then stained indirectly with unlabeled mAbs plus PE-conjugated goat Abs to Ig. In three-color immunofluorescence assays, viable cells from human bone marrow were incubated first with the anti-VpreB Abs, washed, and counterstained with FITC-labeled goat Abs against human IgM, {kappa}/{lambda} L chain, or CD34, and then with CY-labeled anti-CD19. For analysis of cytoplasmic VpreB expression in a subpopulation of bone marrow cells, cells were stained with FITC-labeled mAbs specific for CD10 or µH chain, then counterstained with PE-labeled mAbs against CD19 or biotinylated anti-human {kappa}/{lambda} L chain detected by PE-labeled streptavidin (Southern Biotechnology). After washing, cells were fixed in 2% paraformaldehyde solution at 4°C for 1 h, permeabilized with 0.2% Tween-20 in PBS at 37°C for 15 min, blocked with mouse serum for 10 min, and then stained with Cy5-labeled anti-VpreB or control mAbs. For analysis of surface and cytoplasmic VpreB expression by peripheral B cells, viable cells were stained initially with FITC-labeled anti-CD38 or goat Abs against human IgM and with PE-labeled anti-CD19, then were counterstained on the surface or intracytoplasmically with the Cy5-labeled anti-VpreB mAb 8. Viable tonsillar mononuclear cells were likewise stained first with the FITC-labeled anti-CD38 mAb and PE-labeled goat Abs to human IgD, before cell surface or intracytoplasmic counterstaining with Cy5-labeled anti-VpreB mAb 8. Stained cells were analyzed by flow cytometry using FACScan or FACScalibur instruments (Becton Dickinson).

RT-PCR assays

Viable cells isolated from tonsils were stained with FITC-labeled anti-CD38 and PE-labeled anti-IgD mAbs. After washing, 1 x 105 cells of the CD38-IgD-, CD38-IgD+, CD38+IgD-, and CD38+IgD+ subpopulations were sorted into TRIzol reagent (Life Technologies, Grand Island, NY), and total RNA was prepared following procedures described by the manufacturer (Life Technologies). The synthesis of first-strand cDNA was performed for 50 min at 42°C in a total volume of 20 µl using the SuperScript II RT kit (Life Technologies). For each cDNA preparation, a control synthesis reaction was performed without RT to test for genomic DNA contamination. After heat inactivation for 10 min, the cDNA solution (2 µl) was amplified using Taq polymerase (Life Technologies) in a 50-µl volume reaction. The primer for VpreB reverse transcription was 5'-CTTGAAGCTTTCAAGGGACACGTGT-3', and those for PCR amplification were 5'-TGCAGTGGGTTCCATTTCTTCCT-3' and 5'-CCATGTCCTCGGCCCTTGAACC-3'. The ß-actin cDNA were reverse transcribed with oligo(dT)12–18, and primers for PCR amplification were 5'-GCGGGAAATCGTGCGTGACAT-3' and 5'-GTGGACTTGGGAGAGGACTGG-3'. The cDNA samples were amplified for 30 cycles at an annealing temperature of 62°C for VpreB and 58°C for ß-actin. The amplified products were resolved by electrophoresis in a 2% agarose gel.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Production of recombinant human VpreB protein and monoclonal anti-VpreB Abs

A full-length cDNA for human VpreB was amplified by PCR using primers designed to exclude the leader sequence. This amplified VpreB PCR product was inserted into an expression vector designed to add a His tag to the N-terminus. Recombinant human VpreB protein produced in an E. coli expression system was eluted from a nickel column with yields of 20 to 25 mg/L. The recombinant human VpreB protein resembled the native VpreB protein of approximately 16 kDa when resolved by gel electrophoresis (Fig. 1Go).



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  		FIGURE 1. Examination of anti-VpreB Ab specificity by Western blot analysis. Recombinant Xenopus CD3{gamma}{delta} (lane 1), human VpreB (lane 2), and 14.1/{lambda}5 (lane 3) proteins were electrophoresed, transferred onto nitrocellulose filters, and blotted with the anti-VpreB mAb 8. This pattern of selective reactivity with the recombinant VpreB protein was demonstrated for each of the anti-VpreB mAb 1 to 26.

 
To enhance the likelihood of obtaining Abs of relatively high affinity and specificity, we employed an immunization schedule favoring the generation of hybridomas producing IgG Abs. After five immunizations with the recombinant protein at weekly intervals, cells of the µH chain+ 697 pre-B cell line were injected on the day before regional lymph node cells were harvested for fusion with a nonproducer plasmacytoma cell line. When supernatants of the hybridoma clones were screened by an ELISA assay and tested further by Western blot analysis, 26 hybridomas were found to produce Abs with specificity for the recombinant human VpreB proteins (Fig. 1Go; data not shown). These hybridomas were cloned, and Ig isotype analysis indicated that eight of the Abs were of {gamma}1 isotype, one was {gamma}2a, 15 were {gamma}2b, and two were of the {gamma}3 isotype. All the Abs employed {kappa} light chains. Subclones of each hybridoma served as the source of anti-VpreB mAbs employed in subsequent studies.

Reactivity of the anti-VpreB Abs with native VpreB proteins

When examined for reactivity with native VpreB proteins derived from pro-B and pre-B cell lines, the anti-VpreB mAbs 1 to 16 were found to precipitate the metabolically labeled {psi}L chain complex from both cell types, while the anti-VpreB mAbs 17 to 26 did not. In Figure 2GoA, representative anti-VpreB and the SLC1 mAbs are shown to precipitate the 22- and 18-kDa {psi}L chain proteins in Nalm16 pro-B cells; a faint band of approximately 17 kDa can also be seen. Associated µH chains were not detected, since the Ig genes in this pro-B cell line are retained in germline configuration (44). Conversely, when the same panel of anti-VpreB mAbs was used to examine pre-B cell lysates, association of the {psi}L chain proteins with other pre-B receptor components, µH chains, Ig{alpha}, and Igß was observed (Fig. 2GoB), although most of the {psi}L chains, µH chains, and Ig{alpha}/Igß receptor components were unassociated with each other, as noted in previous studies (40, 54, 55). The immunoprecipitating anti-VpreB mAbs 1 to 16 thus recognize native epitopes present on free {psi}L chain proteins and on the {psi}L chain components of fully assembled pre-B receptors.



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  		FIGURE 2. Analysis of anti-VpreB mAb reactivity with the native {psi}L chain complex in pro-B and pre-B cell lines. Nonidet P-40 lysates of metabolically labeled Nalm16 pro-B cells (A) and 697 pre-B cells (B) were incubated with anti-µH chain (lane 9); anti-VpreB mAbs 2, 8, 9, 11, 12, and 14 (lanes 2–7); anti-{psi}L chain (lane 8); anti-Igß (lane 10); and IgG1 control (lane 1) mAbs. Anti-VpreB mAb 9 is a {gamma}1{kappa} mAb, and the other VpreB mAbs illustrated here (no. 2, 8, 11, 12, and 14) are {gamma}2b{kappa} Abs. The {psi}L chain complex can be seen in anti-VpreB immunoprecipitates resolved by SDS-PAGE (13% gel) under reducing conditions from pro-B (A) and pre-B (B) cells, while µH chains and the associated Ig{alpha}/ß complex were coprecipitated only in pre-B cells (B).

 
The 22-kDa {lambda}5/14.1 protein is an invariant feature of the {psi}L chain complex found in pro-B and pre-B cell lines (40), but size variability of the presumed VpreB proteins has been noted for the different cell lines (19, 40, 54). In the 697 and OB5 pre-B cell lines, the 16- and 18-kDa proteins are expressed in approximately equal amounts, whereas trace amounts of the smaller molecular form were observed in anti-VpreB precipitates of the Nalm16 pro-B cell line. When lysates of the Nalm16 pro-B cells and of the 697 and Nalm6 pre-B cells were examined by immunoprecipitation with anti-µH chain, anti-VpreB, and anti-{psi}L chain mAbs followed by gel electrophoresis and Western blotting with anti-VpreB mAbs, the anti-VpreB Abs identified the 17- and 18-kDa proteins in Nalm16 cells, the 16- and 18-kDa proteins in 697 cells, and the 18-kDa proteins in Nalm6 cells (data not shown), thus confirming the size variability of native VpreB proteins. Variable background band levels were noted for the anti-VpreB Abs of the different IgG isotypes, and one of the Abs, anti-VpreB8, identified an additional band of about 28 kDa in all the pro-B (Nalm 16, RS4;11) and pre-B (Nalm 6, OB5, 697) cell lines examined. This protein, which was not seen in other cell types, may represent a transient associate with the surrogate light chain complex in pro-B and pre-B cells.

IgG anti-VpreB mAbs identify {psi}L chain receptors on pre-B cell lines, but not on pro-B or B cell lines

Having established the VpreB specificity of this panel of isotype-switched mAbs and their ability to react with the native VpreB proteins in free state and in the pre-B receptor complex, we examined the cellular localization of the VpreB proteins in cell lines representative of different stages in B cell differentiation. When cell surface reactivity for B- and non-B-lineage cell lines was examined in an immunofluorescence assay, the 16 anti-VpreB mAbs that reacted with native VpreB proteins also reacted with the cell surface of µH chain+ pre-B cell lines, but not with pro-B-, B-, or non-B-lineage cell lines (Fig. 3Go and Table IGo). Examination of plasma membrane proteins on pre-B cells that were precipitated by the anti-VpreB mAbs 1 to 16 indicated the same pre-B receptor composition that was evident in pre-B cell lysates (Fig. 2GoB and data not shown). A contrasting pattern of immunofluorescence reactivity was observed when the cells were permeabilized before staining, in that the anti-VpreB mAbs reacted with pro-B and pre-B cell lines alike (Fig. 3Go and Table IGo). When the nonprecipitating anti-VpreB mAbs 17 to 26 were examined for cell surface reactivity with different B-lineage representatives, these mAbs were not reactive with viable pro-B or pre-B cells. However, five were reactive with permeabilized pro-B and pre-B cells (Table IGo), suggesting that these Abs identify VpreB epitopes exposed by mild denaturation.



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  		FIGURE 3. Immunofluorescence analysis of cell surface and intracellular VpreB protein expression by pro-B, pre-B, and B cell lines. Viable and permeabilized Nalm16 pro-B, 697 pre-B, and Daudi B cell lines were examined for anti-VpreB mAb 9 reactivity as described in Materials and Methods. The same reactivity pattern was observed for anti-VpreB mAbs 1 to 16.

 

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  		Table I. Summary of the characterization of the anti-VpreB mAb panela

 
The diversity of this panel of anti-VpreB mAbs was examined further in a competitive inhibition assay of epitope specificity. The ability of the unlabeled anti-VpreB mAbs 1 to 16 to inhibit binding of biotinylated preparations of the anti-VpreB mAbs 2, 8, and 11 to the 697 pre-B cell line was examined, and the results of this competitive criss-cross analysis indicated a spectrum of reactivity patterns. Binding of anti-VpreB mAbs 2 and 8 to 697 pre-B cells was inhibited completely by the anti-VpreB mAbs 4, 5, 9, 11, 13, and 15, but only partially or not at all by the other anti-VpreB mAbs. Although the inhibition patterns for anti-VpreB mAbs 2 and 11 were similar, reciprocal inhibition of binding was not observed for the two Abs. The composite data thus suggest that a variety of VpreB epitopes are recognized by members of this anti-VpreB mAb panel.

Analysis of VpreB expression by B-lineage cells in the bone marrow

To address the issue of which types of early B-lineage cells normally express VpreB as a cell surface receptor component, we employed three-color immunofluorescence together with light scatter analyses to examine the reactivity of fetal and adult bone marrow cells with the anti-VpreB mAbs in conjunction with the CD19 B-lineage marker and other hemopoietic lineage markers. A small subset of the CD19+ cells (3.9 ± 1.5% for fetal and 5.8 ± 2.5% for adult bone marrow samples; n = 6) was detectable by cell surface staining with the anti-VpreB mAbs (Fig. 4GoA). These cell surface VpreB+ cells coexpressed µH chains in corresponding low levels, but were nonreactive with anti-{kappa} and anti-{lambda} L chain Abs. Approximately 20% of the VpreB+ µH chain+ CD19+ cells (21.4 ± 1%; n = 6) expressed the CD34 Ag in relatively low levels, and the remainder were CD34 negative. The VpreB/µH chain-bearing cells were further characterized as TdT-/c-kit-/CD45+/CD38+/CD10+/CD19+ cells (data not shown), which included relatively small and large lymphocytes (Fig. 4GoA).



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  		FIGURE 4. Phenotypic analysis of bone marrow cells expressing cell surface or intracellular VpreB. A, Identification and characterization of cell surface VpreB+ cells. Mononuclear adult bone marrow cells stained with anti-VpreB mAb 8 or control mAbs plus PE-conjugated goat Abs against mouse Ig as the developing reagent were counterstained with CY-labeled anti-CD19 and FITC-labeled goat Abs against human IgM. The histogram in the top right panel indicates the relative size distribution of VpreB+ cells, with the bar demarcating the light scatter characteristics of small lymphocytes. B, Characterization of intracellular VpreB+ cells. Viable fetal bone marrow cells stained with FITC-labeled anti-CD10, then counterstained with PE-labeled anti-CD19 or biotinylated anti-human {kappa}/{lambda} L chain plus PE-labeled streptavidin, were fixed and permeabilized before staining with CY5-labeled anti-VpreB mAb 8 or control mAbs.

 
Cytoplasmic VpreB expression was detectable in 17.5 ± 3.4% (n = 5) of the bone marrow lymphocytes (Fig. 4GoB). The cytoplasmic VpreB+ cells expressed cell surface CD10, one of the earliest hemopoietic differentiation Ags to be expressed by B-lineage cells (56, 57), but approximately 30% of these cytoplasmic Vpre-B+ cells did not express the CD19 B-lineage marker. Finally, none of the {kappa}/{lambda}+ B cells in bone marrow expressed intracellular VpreB. These results thus indicate that VpreB is expressed intracellularly before CD19 expression during B-lineage differentiation, reaches the cell surface together with µH chains in pre-B cell receptors, and is down-regulated with B cell differentiation.

Analysis of VpreB expression by B cells in peripheral lymphoid tissues

To examine whether VpreB is expressed in peripheral B cells, blood mononuclear cells were stained with B-lineage markers and the anti-VpreB mAbs. As anticipated from the analysis of bone marrow B cells, none of the circulating B cells expressed VpreB proteins either on their surface or intracellularly (Fig. 5GoA and data not shown). However, the recent demonstration that RAG-1, RAG-2, and {lambda}5 transcripts are expressed by a subpopulation of germinal center B cells (58, 59) suggested that the VpreB gene might be expressed in this microenvironment. To examine this possibility, tonsillar lymphocytes were isolated for immunofluorescent assessment with the anti-VpreB mAbs. The tonsillar cells did not bear detectable levels of VpreB on their surface, but a minor subpopulation of CD38+ B cells appeared to express VpreB intracellularly at very low levels (Fig. 5GoB), whereas the CD38- cells and CD38high plasma cells did not appear to contain VpreB. To confirm this suggestive evidence for VpreB expression in a subpopulation of CD38+ germinal center B cells, we examined lysates of tonsillar cells and control 697 pre-B cells by immunoprecipitation with Abs against µH chains and VpreB. When the immunoprecipitated proteins were resolved by gel electrophoresis, Western blotting with anti-VpreB mAbs revealed the presence of 16- to 18-kDa VpreB proteins in tonsillar cells at levels lower than those in transformed pre-B cells (Fig. 5GoC). To identify more precisely the tonsillar B cells that may express VpreB, tonsillar lymphocytes were sorted on the basis of their CD38 and IgD expression profiles, and the different subpopulations were examined for expression of VpreB transcripts. This RT-PCR-based analysis indicated that both IgD+ and IgD- members of the CD38+ subpopulation may express VpreB transcripts, whereas IgD+CD38- B cells do not (Fig. 6Go). A subpopulation of germinal center B cells thus may express intracellular VpreB proteins regardless of whether they express cell surface IgD.



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  		FIGURE 5. Immunofluorescence analysis of VpreB expression by B cells in peripheral lymphoid tissues. Cell surface and intracellular VpreB expression by B cells in the circulation (A) and in tonsils (B). Viable cells were stained with PE-labeled anti-IgM Ab (A) or FITC-labeled anti-CD38 mAb (B), then counterstained with CY5-labeled anti-VpreB8 or an irrelevant control mAb for analysis of cell surface expression (left panel) or cytoplasmic VpreB expression after cell fixation and permeabilization (right panel). The minor shift in the VpreB staining pattern for CD38+ tonsillar B cells was confirmed in four additional experiments. C, Identification of VpreB proteins in tonsillar cells. Nonidet P-40 lysates of pre-B 697 and tonsillar cells were precipitated with anti-µH chain (lanes 2 and 5), anti-VpreB (lanes 3 and 6), and control IgG (lanes 1 and 4) mAbs. Immunoprecipitates (lanes 1–6) and recombinant VpreB protein (lane 7) were separated by SDS-PAGE, and VpreB proteins were identified by the anti-VpreB mAb 8 in a Western blot.

 


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  		FIGURE 6. RT-PCR analysis of VpreB expression in tonsillar B cell subpopulations. A, Viable tonsillar cells stained with FITC-labeled anti-CD38 and PE-labeled anti-IgD mAbs were sorted into CD38-IgD-, CD38-IgD+, CD38+IgD+, and CD38+IgD- subpopulations. B, RT-PCR analysis of VpreB and ß-actin transcripts in the different subpopulations isolated by FACS. RNA isolated from 1 x 105 cells of each subpopulation and from Nalm16 pro-B cells were used to synthesize the cDNA for PCR amplification. PCR products (30 cycles of amplification) were electrophoresed on agarose gel and stained with ethidium bromide. Lane 1, Control (no reverse transcriptase, Nalm16 pro-B cells); lane 2, Nalm16 pro-B cells; lane 3, CD38-IgD- cells; lane 4, CD38-IgD+ cells; lane 5, CD38+IgD+ cells; lane 6, CD38+IgD- B cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Surrogate L chain genes and their protein products have been shown to be expressed throughout the early stages of B-lineage differentiation, but the issue of when the {psi}L chains are expressed as components of surface receptors has been a confounding one (8, 9). In these studies we generated a large panel of IgG anti-VpreB mAbs against exposed epitopes on native intracellular and extracellular VpreB proteins to examine this question. These anti-VpreB Abs identified cell surface receptors composed of {psi}L chains, µH chains, Ig{alpha}, and Igß on pre-B cell lines, but none could identify cell surface components on pro-B and B cell lines. After cell fixation and permeabilization, however, the anti-VpreB mAbs identified the {psi}L chain complexes within both pro-B and pre-B cell lines, although not within B cell lines. In addition, a relative abundance of free VpreB/{lambda}5/14.1 complexes was identified in pre-B cell lysates, in keeping with prior evidence indicating inefficient assembly of the pre-B receptors (19, 40, 54, 55) and catabolism of µH chains retained within the endoplasmic reticulum of pre-B cells (3, 60).

Pro-B cell lines have been reported to express {psi}L chain proteins together with {psi}H chains to form pro-B cell receptors (34, 36, 37, 38, 39) or not to express {psi}L chains on their cell surface (19, 40). Differences in epitope specificity or multireactivity of low affinity anti-{psi}L chain mAbs could contribute to these discordant results. In this regard, three IgM anti-VpreB mAbs that stained human pro-B cells did not precipitate native {psi}L chain proteins (37, 38). Another IgM anti-VpreB Ab, the use of which suggested the presence of a {psi}H chain/{psi}L chain complex on human pro-B and the presence of both {psi}H chain/{psi}L chain and µH chain/{psi}L chain complexes on pre-B cell lines (39, 61), failed to precipitate {psi}L chain proteins in association with the candidate {psi}H chain (39). In addition to the possibility of Ab cross-reactivity, the discordant results could reflect a failure of some anti-VpreB mAbs, such as those reported here, to recognize VpreB epitopes uniquely exposed on {psi}H chain/{psi}L chain complexes. However, this possibility is rendered unlikely by the finding that 16 anti-VpreB mAbs in the present panel together with the four previously described SLC mAbs (19, 40) all recognize free VpreB proteins, VpreB coupled to the {lambda}5/14.1 protein, and a variety of VpreB epitopes exposed on the pre-B cell receptor complex. The availability of this large panel of anti-VpreB Abs will allow the comparative analysis needed to resolve these specificity issues.

When these well-characterized anti-VpreB mAbs were used to address the issue of when {psi}L chains are expressed during B cell differentiation in vivo, VpreB and µH chains were identified exclusively on a subpopulation of bone marrow CD19+ B-lineage cells lacking {kappa} or {lambda}L chains. The {psi}L chain/µH chain-bearing subpopulation included relatively large and small lymphocytes, none of which contained the nuclear TdT that characterizes pro-B cells (56, 57). While a minor subpopulation of the pre-B receptor-bearing cells expressed low levels of the CD34 Ag, most did not express this early hemopoietic differentiation marker in detectable levels. The characterization of pre-B receptor expression extending from large CD19+/CD34low/TdT- lymphocytes to small postmitotic CD19+/CD34- lymphocytes indicates that the entire spectrum of pre-B cells may express pre-B receptors, although the receptors were not detectable on all pre-B cells. In this regard, sensitive functional assays have indicated pre-B cell receptor expression by µH chain transgenic RAG-2-/- mice even when the receptor levels were below those detectable by immunofluorescence (62). In another study, pre-B receptors were detected on small postmitotic pre-B cells in the peripheral lymphoid compartment of µH chain/bcl-2 transgenic RAG-2-/- mice (31), a finding reminiscent of the pre-B receptor expression by acute lymphocytic leukemias of childhood (63).

B lymphopoiesis is characterized by the sequential acquisition of intracellular and cell surface markers, with the onset of IgH gene transcription and rearrangement being early definitive features of B-lineage commitment. IgH locus transcriptional activity leading to DJH rearrangements has been noted in CD34+CD10+ bone marrow cells before the onset of CD19 expression (64, 65), and we found that whereas the CD10+ population of bone marrow lymphocytes includes virtually all the cells that express VpreB proteins, some of the VpreB-containing CD10+ cells do not express CD19. These observations indicate that {psi}L chain expression also begins before cell surface CD19 expression. Despite this early onset of intracellular expression of {psi}L chain proteins, the present data reinforce previous analyses (18, 19, 40) in questioning the presence of {psi}L chain/{psi}H chain receptors on human pro-B cells. More convincing evidence for {psi}L chain/{psi}H chain receptors has been obtained in studies of murine pro-B cells, where cell surface association of the {psi}L chain complex with proteins of approximately 45, 65, and 130 kDa has been reported (8, 34, 36). The association of {psi}L chain complexes with proteins of approximately 40, 60, and 98 kDa has also been found in human pro-B cells, but these complexes were located in the endoplasmic reticulum where {psi}L chain degradation occurred (40). In this context, it is noteworthy that Ig{alpha}/Igß signal transducing elements have not been identified in the putative {psi}H chain/{psi}L chain receptor complex on murine pro-B cells, although Ig{alpha}/Igß proteins may reach the pro-B cell surface in association with calnexin as a chaperon (66, 67), an observation that may explain the early arrest of pro-B cell development seen in Igß-/- mice (29). The contrasting absence of impaired pro-B development in {lambda}5-deficient mice (26, 68) further militates against a physiologic role for the putative {psi}H chain/{psi}L chain receptor.

In a remarkable recent development, activated B cells in the germinal centers have been found to reexpress precursor B cell genes, including RAG-1, RAG-2, and {lambda}5, and to undergo secondary V(D)J recombinations (57, 58, 69, 70). Consonant with these observations, we identified a small subpopulation of CD38+ B cells in human tonsils that contained intracellular VpreB proteins, although these could not be detected on the cell surface. Their CD38 expression identifies these cytoplasmic VpreB+ cells as germinal center B cells (71), at least some of which expressed conventional B cell receptors on their surface. Our inability to demonstrate VpreB expression on the cell surface in association with H chains is consistent with the observation that conventional L chains preferentially bind to µH chains in cells that produce both surrogate and conventional L chains (19) and disfavors the possibility of a functional role for the {psi}L chains in the secondary V(D)J rearrangements in germinal center B cells.

In conclusion, bone marrow B-lineage cells produce VpreB proteins before the onset of CD19 expression, and these are later expressed as components of the pre-B receptors on both large and small pre-B cells in humans. VpreB expression is extinguished in B cells, only to be re-expressed by activated B cells in the germinal centers. Our findings support the view that despite this intermittent expression pattern, VpreB serves as a functional receptor component primarily, or even exclusively, during the pre-B cell stage in B-lineage differentiation.


   Acknowledgments
 
We thank Drs. Hiromi Kubagawa, John Kearney, Peter Burrows, and Kaïss Lassoued for providing helpful suggestions and reagents, and Ann Brookshire for help in preparing the manuscript.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI30879 and a Howard Hughes Medical Institute Investigatorship (to M.D.C.). Back

2 Address correspondence and reprint requests to Dr. Max D. Cooper, Howard Hughes Medical Institute, University of Alabama, 378 Wallace Tumor Institute, 1824 6th Ave. South, Birmingham, AL 35924-3300. E-mail address: Back

3 Abbreviation used in this paper: PE, phycoerythrin. Back

Received for publication February 6, 1998. Accepted for publication March 26, 1998.


   References
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 Materials and Methods
 Results
 Discussion
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