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

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, κ) (47) and Ramos (IgM, λ) (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 (γ1κ) anti-human μ (49), CB3-1 (γ1κ) anti-human Igβ (50), the SLC1 (γ1κ) and SLC2 (μκ) anti-human ψL chain (19), and the JS-2 (γ1κ) 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 κ/λL 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-β-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 λ5/14.1 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 CD3γδ (53), human VpreB, and λ5/14.1 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 × 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 κL 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 × 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 × 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 × 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, κ/λ 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 κ/λ 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 × 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 β-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 β-actin. The amplified products were resolved by electrophoresis in a 2% agarose gel.

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. 1⇓).

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FIGURE 1.

Examination of anti-VpreB Ab specificity by Western blot analysis. Recombinant Xenopus CD3γδ (lane 1), human VpreB (lane 2), and 14.1/λ5 (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. 1⇑; data not shown). These hybridomas were cloned, and Ig isotype analysis indicated that eight of the Abs were of γ1 isotype, one was γ2a, 15 were γ2b, and two were of the γ3 isotype. All the Abs employed κ 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 ψL chain complex from both cell types, while the anti-VpreB mAbs 17 to 26 did not. In Figure 2⇓A, representative anti-VpreB and the SLC1 mAbs are shown to precipitate the 22- and 18-kDa ψL 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 ψL chain proteins with other pre-B receptor components, μH chains, Igα, and Igβ was observed (Fig. 2⇓B), although most of the ψL chains, μH chains, and Igα/Igβ 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 ψL chain proteins and on the ψL chain components of fully assembled pre-B receptors.

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FIGURE 2.

Analysis of anti-VpreB mAb reactivity with the native ψL 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-ψL chain (lane 8); anti-Igβ (lane 10); and IgG1 control (lane 1) mAbs. Anti-VpreB mAb 9 is a γ1κ mAb, and the other VpreB mAbs illustrated here (no. 2, 8, 11, 12, and 14) are γ2bκ Abs. The ψL 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 Igα/β complex were coprecipitated only in pre-B cells (B).

The 22-kDa λ5/14.1 protein is an invariant feature of the ψL 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-ψL 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 ψL 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. 3⇓ and Table I⇓). 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. 2⇑B 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. 3⇓ and Table I⇓). 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 I⇓), 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.

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. 4⇓A). These cell surface VpreB⁺ cells coexpressed μH chains in corresponding low levels, but were nonreactive with anti-κ and anti-λ 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. 4⇓A).

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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 κ/λ 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. 4⇑B). 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 κ/λ⁺ 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. 5⇓A and data not shown). However, the recent demonstration that RAG-1, RAG-2, and λ5 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. 5⇓B), whereas the CD38⁻ cells and CD38^high 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. 5⇓C). 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. 6⇓). 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 β-actin transcripts in the different subpopulations isolated by FACS. RNA isolated from 1 × 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.