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Studies on B Cell Precursors

Notch-1 and Notch-2 exhibit unique patterns of expression in human B-lineage cells

Abstract

The Notch genes encode a conserved family of receptors that influence developmental fate in many species. Prior studies have indicated that Notch-1 and Notch-2 signaling influence the development of hematopoietic stems cells and thymocytes, but little is known regarding Notch expression and function in B-lineage cells. We analyzed the expression of Notch receptors and Notch ligands in human B-lineage cells and bone marrow (BM) stromal cells. Notch-1 mRNA and protein is expressed throughout normal B cell development and in leukemic B-lineage cells. In contrast, Notch-2 expression is limited to pre-B cells expressing low levels of surface μ. The Notch ligand Delta is expressed in BM B-lineage cells. The Notch ligand Jagged-1 is not expressed in B-lineage cells, but is expressed in BM stromal cells. These results suggest a model wherein lateral signaling between Notch and Delta on B-lineage cells and/or Notch/Jagged-1 interactions between B-lineage cells and BM stromal cells may regulate human B cell development.

Introduction

The Notch family of receptors and their DSL (Delta, Serrate, Lag-2) family of ligands mediate cell fate decisions in myogenesis, neurogenesis, adipogenesis, boundary formation, wing development, eye development and lymphohematopoiesis in organisms ranging from drosophila to humans (reviewed in Refs 123). A potential common denominator of Notch function in these diverse cell fate decisions appears to be that Notch signaling maintains cells in an undifferentiated state; most likely by preventing cells from responding to other developmental cues. Notch signaling initiates with the ligand-induced generation of an intracytoplasmic Notch (ICN) cleavage product, followed by nuclear translocation of ICN and transcriptional activation or repression of a large number of target genes.1

The human genome contains four Notch receptor genes (Notch-1, -2, -3 and -4) and at least three DSL ligand genes (Jagged-1, Jagged-2 and Delta).45678 Human Notch-1 was originally identified at the breakpoint of the t(7;9)(q34;q34.3) chromosomal translocation in a subset of T cell acute lymphoblastic leukemias (ALL).9 Expression of Notch-1 mRNA was subsequently found in human CD34+ hematopoietic stem cells.10

Notch signaling exerts complex effects on mammalian hematopoietic stem cells (reviewed in Ref. 3). For example, truncated/constitutively active forms of Notch-1 and Notch-2 inhibit murine myeloid cell development in response to G-CSF and GM-CSF, respectively.11 Co-culture of the murine 32D myeloid cell line with Jagged-1+ stromal cells inhibits G-CSF-induced differentiation.6 Furthermore, murine LinSca-1+c-kit+ cells (expressing Notch-1 and Notch-2) cultured on 3T3 cells transfected with human Jagged-1, or with beads bearing the extracellular domain of Jagged-1, exhibited an increase in primitive hematopoietic precursors.12 Recent studies with human hematopoietic stem cells suggest that maintenance of precursors in the presence of a Notch signal may reflect enhanced progression through the G0/G1 phase of the cell cycle.13

The role of Notch signaling in thymocyte development has been investigated (reviewed in Refs 1415). Constitutively active Notch was originally reported to favor development of CD8+ over CD4+ thymocytes16 and TCRαβ+ over TCRγδ+ thymocytes.17 More recent studies suggest that Notch-1 signaling may render thymocytes more resistant to apoptotic signals.1819 A physiologic role for Notch signaling in normal murine thymocyte development is reinforced by the finding that Jagged-1 and Jagged-2 are expressed on thymic epithelial cells.20

In contrast to the extensive analysis of Notch function in thymocyte development, much less is known regarding the role of Notch in mammalian B cell development. A recent study demonstrated that retroviral infection of constitutively active Notch-1 into murine bone marrow (BM) hematopoietic cells led to a precocious development of CD4+/CD8+ T-lineage cells in BM, with a concomitant suppression of B cell development.21 However, because B cell development was suppressed in this model of constitutive Notch signaling, it was not possible to determine the expression and function of Notch in cells developing and expanding into the B-lineage. In the current study, we examined the expression of Notch and Notch ligands in human fetal BM B-lineage cells and normal human BM stromal cells using RT-PCR and flow cytometry. We show that Notch is expressed in normal and leukemic B-lineage cells and Notch ligands are expressed by B-lineage cells and BM stromal cells. These results suggest a potentially complex role for Notch signaling in regulating the proliferation and/or differentiation of human B-lineage cells.

Materials and methods

Isolation of human B-lineage cells and FACS sorting

Human fetal BM B-lineage cells and stromal cells were isolated as described previously.22 Briefly, single cell suspensions were layered on to Ficoll–Hypaque cushions and mononuclear interface cells were collected following room temperature centrifugation at 1000 r.p.m. for 30 min. The mononuclear cells were washed, plated on plastic tissue culture dishes in RPMI-1640 supplemented with 10% heat-inactivated FBS and penicillin/streptomycin (Gibco-BRL, Gaithersburg, MD, USA), and incubated for 2 h at 37°C in 5% CO2. The adherent cells were expanded to confluence, passaged three times, and maintained in X-VIVO 10 serum-free medium (Bio-Whittaker, Walkersville, MD, USA) supplemented with 2 mM L-glutamine prior to use.22

The adherence-depleted mononuclear cells were stained with anti-CD34, anti-CD19, and anti-μ mAbs as previously described.23 In most experiments the sample was split into two halves. One half was stained with anti-CD34-FITC (HPCA-2; Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA, USA) and anti-CD19-biotin revealed with streptavidin-PE (Molecular Probes, Eugene, OR, USA). The other half was stained with anti-CD19-biotin (revealed with streptavidin-PE) and anti-μ-FITC. The anti-CD19 and anti-μ conjugates have been previously described.24 In some experiments the entire sample was stained with anti-CD34-FITC, anti-CD19-allophycocyanin (25C1, BDIS) and anti-μ-biotin revealed with streptavidin-PE. The two staining/sorting strategies yielded equivalent results. Cells with lymphoid light scatter characteristics were sorted into the following B-lineage compartments using a FACSVantage (BDIS): CD34+CD19+μ pro-B cells, CD34CD19+μ early pre-B cells, CD34CD19+μlo late pre-B cells and CD34CD19+μhi immature B cells.23

In experiments involving Western blot analysis, total fetal BM B cell precursors were isolated by magnetic bead depletion of non-B-lineage cells and surface μhi immature B cells, as described previously.22 CD19+ cells were thawed and sorted from cryopreserved tonsilar mononuclear cells. B-lineage ALL cells were thawed from cryopreserved BM samples.

The use of all human tissue was approved by the Institutional Review Board: Human Subjects Committee of the University of Minnesota.

Isolation of murine B-lineage cells

BM from C57BL/6 mice was prepared by flushing femurs with a 23-gauge needle to generate a single cell suspension. Spleen and thymus were disaggregated in cold PBS using a mesh screen. Red cells were removed from the single cell suspensions by incubation with hemolytic buffer comprised of 0.15 M NH4Cl, 1.0 M NaHCO3 and 0.1 M Na2EDTA. Spleen and BM cells were incubated with antibodies against murine CD19 and CD3 or CD19 alone, respectively, and FACS purified using a FACSVantage. PE-conjugated anti-CD19 and FITC-conjugated anti-CD3 were obtained from BD- Pharmingen (San Diego, CA, USA).

RT-PCR and Southern analysis

Total RNA was extracted from 5 × 104 cells of each sorted fraction using Tri-Reagent (Molecular Research Center, Cincinnati, OH, USA). Oligo-dT primed cDNA was synthesized as follows: 10 μl of RNA was incubated with 1.6 μl of 5 × RT buffer (Gibco-BRL), 0.5 μl of DNAse I (Boehringer Mannheim, Indianapolis, IN, USA) and 0.5 μl of Rnasin (Promega, Madison, WI, USA) at 37°C for 1 h, followed by heat inactivation at 75°C. To this reaction was added 24 μl 5 × RT buffer, 2 μl 0.1 M DTT, 1 μl of Oligo-dT, 1 μl of 10 mM dNTPs, and 1 μl of RT (Gibco-BRL). Following 1 h incubation at 42°C and 10 min heat inactivation at 65°C, 1 μl of this preparation was used for each PCR reaction.

PCR was performed essentially as described previously.23 Each PCR reaction mixture consisted of 39 μl H2O, 5 μl 10 × buffer (Gibco-BRL), 1.5 μl 50 mM MgCl2, 1 μl of 10 mM dNTPs, 1 μl each primer at 20 μM, 2.5 U Taq polymerase (Gibco-BRL), and 1 μl cDNA. Cycle conditions were 95°C for 1 min, annealing at various temperatures for 1 min (see Table 1), and extension at 72°C for 1 min. A final extension at 72°C was performed for 10 min. Annealing temperatures and cycle conditions for each primer pair were optimized such that each PCR was in the linear range of amplification. Table 1 lists the PCR primers, annealing temperature, and cycle number for each gene analyzed.

Table 1  Primers, oligo probes and cycle conditions used in RT-PCR analysis

Ten microliters of each PCR product were separated on a 1.5% agarose gel and transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH, USA). Following standard pre-hybridization the blots were hybridized at 42°C with the oligonucleotide probes listed in Table 1. Probes were labeled with digoxygenin (DIG) using the DIG Oligonucleotide 3′-End Labeling Kit (Boehringer Mannheim) per the manufacturer's instruction. Following washing at 42°C, hybridization signals were revealed using the DIG Luminescent Detection Kit for Nucleic Acids (Boehringer Mannheim) per the manufacturer's instruction.

Western analysis of Notch-1 protein expression

Cells were lysed in RIPA buffer containing 10 mM Tris HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS supplemented with the following protease inhibitors: aprotinin (22 μg/ml), PMSF (1 μg/ml), DTT (1 μM), iodoacetamide (0.01 μM), pepstatin A (1 μg/ml) and leupeptin (1 μg/ml). The protease inhibitors were obtained from Sigma Chemical Co (St Louis, MO, USA). Between 60 and 80 μg of total protein was electrophoresed per lane on a 7.5% SDS-PAGE gel with a 5% stacking gel. Following electrophoresis the proteins were transferred to nitrocellulose membranes and incubated overnight at 4°C in 5% milk dissolved in 1 × TBST. Blots were incubated with 15 μg/ml of rat anti-human Notch-1 mAb 18G25 (produced in the laboratory of S Artavanis-Tsakonas and kindly provided by I Bernstein, Fred Hutchinson Cancer Research Center, Seattle), washed with 1 × TBST for 50 min at room temperature, and probed with a 1:5000 dilution of sheep anti-rat Ig conjugated to horseradish peroxidase (Amersham Life Science, Arlington Heights, IL, USA) in 1 × TBST for 30 min at room temperature. Following a 90 min wash in 1 × TBST at room temperature the blots were developed using enhanced chemiluminescence (Amersham Life Science). Equal protein loading between lanes was confirmed using mouse anti-human β-tubulin detected with sheep anti-mouse Ig conjugated to horseradish peroxidase (Amersham).

For experiments using murine BM, the blots were incubated with rabbit anti-human Notch-1 (kindly provided by J Aster, Brigham and Women's Hospital, Boston) diluted 1:1000 in 5% milk dissolved in 1 × TBST. Blots were counter-stained with donkey anti-rabbit Ig conjugated to horseradish peroxidase (Amersham) and developed by enhanced chemiluminescence as described above. The rabbit anti-human Notch-1 (designated anti-TC) was made against a peptide comprising amino acids 2278–2470 of human Notch-1.26 This antibody cross-reacts with murine Notch-1.26

Flow cytometric analysis of Notch-1 surface expression

BLIN-1 cells, a pre-B ALL cell line, or SUP T1 cells, a T-ALL cell line, were incubated with a 1:20 dilution of rabbit anti-rat Notch-1 extracellular domain (Notch-1EC) IgG (Upstate Biotechnology, Lake Placid, NY, USA) for 20 min on ice. This Ab cross-reacts with mouse and human Notch-1EC. Cells were washed in PBS + 1% calf serum, then incubated with a 1:50 dilution of human adsorbed goat anti-rabbit IgG conjugated to biotin (Southern Biotechnology Associates, Birmingham, AL, USA). The biotinylated antibody was revealed with streptavidin-PE (Molecular Probes). Fetal BM mononuclear cells isolated by Ficoll–Hypaque centrifugation and adherence depletion were stained for Notch-1EC as above, except that the cells were incubated with 50 μg of human IgG for 10 min on ice prior to the addition of the anti-Notch-1EC. The fetal BM cells were also co-stained with anti-CD19 (25C1) that had been conjugated to FITC in our laboratory. Cells were analyzed on a FACSCalibur (BDIS) using CellQuest software.

Jagged-1 flow cytometric and Western analysis

Jagged-1 surface staining was conducted using goat anti-rat Jagged-1 kindly provided by Frank Mortari (R&D Systems, Minneapolis, MN, USA). Binding was revealed with rabbit anti-goat FITC (Southern Biotechnology Associates). Cells were analyzed on a FACSCalibur (BDIS) using CellQuest software. For Western blotting, membranes were incubated with 20 μg/ml of the goat anti-rat Jagged-1 antibody, washed and then probed with a 1:10000 dilution of rabbit anti-goat Ig conjugated to horseradish peroxidase (Southern Biotechnology Associates). All other steps were as described above for Notch-1 Western analysis.

Results

Notch receptors are expressed in normal humanB-lineage cells

Notch gene expression was analyzed in specific stages of human B cell development by sorting fetal BM B-lineage cells on the basis of CD34, CD19 and μ surface expression.23 The sorted compartments are shown in Figure 1a. CD34+CD19+μ pro-B cells harbor DJH and some VDJH rearrangements and the vast majority express TdT.2327 CD34CD19+μ early pre-B cells express cytoplasmic μ H chains, but have yet to assemble μ H chains on the cell surface with VpreB and λ5.27 CD34CD19+μlo late pre-B cells are enriched for cells expressing surface μ H chains associated with VpreB and λ5 to form the μ-surrogate light chain (ψLC).2829 The CD34CD19+μhi compartment is comprised of immature B cells that express surface μ H chains associated with κ or λ L chains. Post-sort analysis revealed that the purity of each compartment was greater than 95% (Figure 1b). Each sorted compartment was also analyzed by RT-PCR for the expression of B-lineage associated genes as further confirmation that discrete stages of B cell development were isolated (data not shown).

Figure 1
figure1

 B-lineage compartments isolated on the basis of CD34, CD19 and surface μ expression. (a) Sorting gates on fetal BM cells stained for CD34 vs CD19 and CD19 vs μ. Early pre-B cells were distinguished from pro-B cells on the basis of high CD19 surface expression on the former. (b) Post-sort analysis of cells gated in (a).

Figure 2 shows the results obtained when specific stages of B cell development were analyzed for Notch expression by RT-PCR. Notch-1 transcripts were detected at all stages of human fetal BM B cell development (Figure 2a). Interestingly, Notch-2 transcripts were only detected in the late pre-B compartment of B cell development (Figure 2b). Notch-3 transcripts were not detected in BM B-lineage cells. This pattern of expression was observed in five independently sorted and analyzed fetal BM samples. Notch-1 and Notch-2 transcripts were also detected within the heterogeneous CD34+CD19 hematopoietic stem cell compartment (data not shown), consistent with previous studies.310

Figure 2
figure2

 Notch-1 and Notch-2 are differentially expressed in human fetal BM B-lineage cells. Fetal BM cells FACS-purified as shown in Figure 1 were analyzed for Notch gene expression by RT-PCR followed by Southern blotting. (a) and (b) were separate samples. RT, no reverse transcriptase; H2O, no cDNA template; Fetal BM, fetal BM mononuclear cells.

Human B-lineage cells express Notch-1 protein

The expression of Notch-1 protein in human B-lineage cells was assessed by Western blotting using a rat mAb against the cytoplasmic domain of human Notch-1. Results in Figure 3 show that the pre-B ALL cell line BLIN-1, normal human B cell precursors, tonsil B cells, and leukemic cells from three individual cases of B-lineage ALL all expressed p120 Notch-1. This is the expected size of the portion of the Notch-1 heterodimer containing the cytoplasmic domain, as reported by others.30

Figure 3
figure3

 Notch-1 protein is expressed in normal and leukemic B-lineage cells. Whole cell lysates (80 μg total protein/lane) were separated on a 7% SDS-PAGE gel under reducing conditions and transferred to nitrocellulose for Western blotting. Membranes were probed with a rat anti-human Notch-1 mAb that recognizes the cytoplasmic domain of Notch-1. Membranes were re-probed with mouse anti-human β-tubulin to confirm equal protein loading between lanes.

Human B-lineage cells express surface Notch-1 receptors

In order to determine if B-lineage cells express cell surface Notch-1, we analyzed B-lineage cells for expression of the Notch-1 extracellular region (Notch-1EC) by flow cytometry. Analysis of the pre-B ALL cell line BLIN-1 revealed a high level of cell surface Notch-1 expression (Figure 4a), consistent with the readily detectable Notch-1 expression observed by Western analysis (Figure 3). SUP T1 is a T-lineage ALL cell line that bears two copies of a t(7;9) chromosomal translocation between Notch-1 and the TCR-β locus, resulting in expression of a constitutively active ICN. SUP T1 cells have no normal Notch-1 alleles and do not express cell surface Notch-1.30 Consistent with this, the anti-Notch-1EC antibody did not react with the cell surface of SUP T1 cells (Figure 4a). Figure 4b shows that CD19+ normal fetal BM cells expressed a low level of cell surface Notch-1, further confirming that human B- lineage cells express the Notch-1 receptor.

Figure 4
figure4

 CD19+ human fetal BM B-lineage cells express cell surface Notch-1. (a) BLIN-1 and SUP T1 cells were stained with rabbit anti-rat Notch-1EC (solid dark line) or normal rabbit IgG (dotted line) and analyzed by flow cytometry. (b) Adherence-depleted fetal BM mononuclear cells were co-stained with rabbit anti-rat Notch-1EC and mouse anti-human CD19 and analyzed by flow cytometry. CD19+ lymphoid cells defined by light scatter (gate R3) and CD19 expression (gate R4) express low levels of cell surface Notch-1 (far right histogram). The solid dark line is Notch-1EC staining. The dotted line is normal rabbit IgG staining.

Murine BM B-lineage cells express Notch-1 protein

The high degree of inter-species conservation of the Notch pathway, and the finding that Notch-1 is expressed throughout human BM B cell development (Figures 2, 3 and 4), prompted us to examine Notch-1 protein expression in murine B-lineage cells. Murine BM was FACS-purified into CD19+ and CD19 compartments and murine spleen was FACS-purified into CD19+ B-lineage cells and CD3+ T-lineage cells. Post-sort analysis revealed that each fraction was >90% pure (data not shown). Murine thymocytes were used as positive control for Notch-1 expression. As shown in Figure 5, Notch-1 protein was readily detected in murine thymocytes. In addition, CD19+ BM B-lineage cells also expressed Notch-1 protein. Notch-1 protein was detected in CD3+ splenic T cells, but expression in CD19+ splenic B cells was below the level of detection.

Figure 5
figure5

 Murine B-lineage cells express Notch-1 protein. Murine lymphoid populations from BM, spleen, and thymus were lysed, and 80 μg protein was separated on a 7.5% SDS-PAGE gel under reducing conditions and transferred to nitrocellulose for Western blotting. Membranes were probed with rabbit anti-human Notch-1 that cross-reacts the murine Notch-1.26 Membranes were also probed with mouse anti-human β-tubulin (that cross-reacts with murine β-tubulin) to assess protein loading. Murine thymocytes were on a separate blot.

The Notch ligand Delta is expressed by normal human B-lineage cells

The sorted fetal BM B-lineage compartments were analyzed by RT-PCR for the expression of the Notch ligands Jagged-1 and Delta. Jagged-1 transcripts were not detected in BM B-lineage cells (Figure 6). However, Delta was expressed in pro-B (CD34+CD19+), early pre-B (CD19+μ) and late pre-B (CD19+μlo) cells.

Figure 6
figure6

 B-cell precursors express Delta. Fetal BM cells FACS-purified as shown in Figure 1 were analyzed for Notch ligand expression by RT-PCR followed by Southern blotting. VCAM-1 expression was analyzed to ensure there were no stromal cells contaminating the purified B-lineage compartments. RT, no reverse transcriptase; H2O, no cDNA template; Fetal BM, fetal BM mononuclear cells. The expression of VCAM-1 in fetal BM mononuclear cells probably represents rare stromal cells present in this population.

BM stromal cells that support B-cell differentiation express Jagged-1

Our laboratory has developed an in vitro culture system that supports development of IgM+ immature B cells from CD34+CD19 progenitors.2431 This culture system utilizes freshly isolated human BM stromal cells and does not require the addition of exogenous cytokines. These BM stromal cell preparations were analyzed by RT-PCR for Notch ligand expression (Figure 7a). BM stromal cells expressed Jagged-1 but not Delta transcripts, although the latter was expressed in skin fibroblasts and HUVEC. Third passage fetal BM stromal cells were tested for Jagged-1 protein using a goat anti-Jagged-1 antibody raised against the extracellular domain of rat Jagged-1. As shown in Figure 7b, fetal BM stromal cells expressed readily detectable cell surface Jagged-1. Rat fibroblasts were also Jagged 1+, whereas the pre-B ALL cell line BLIN-1 was Jagged-1. Western blotting using this goat anti-rat Jagged-1 reagent revealed a protein of 150–180 kDa (Figure 7c), consistent with the size of human Jagged-1 previously detected by others.612

Figure 7
figure7

 BM stromal cells express cell surface Jagged-1. (a) RT-PCR analysis and Southern blotting for Jagged-1 and Delta. RT, no reverse transcriptase; H2O, no cDNA template; Fetal BM, fetal BM mononuclear cells. (b) Flow cytometric analysis of Jagged-1 protein. Shaded histogram, control staining; open histogram, staining with goat anti-rat Jagged-1. (c) Western blot analysis for Jagged 1 expression. BM stromal cells express the 180 kDa Jagged 1 protein (arrow), while BLIN-1 and JURKAT cells do not.

Discussion

The function of the Notch pathway in lymphopoiesis is beginning to emerge and a role for Notch signaling in thymocyte development has been established (reviewed in Ref. 14). Enforced expression of constitutively active Notch-1 in thymocytes favors the development of TCRα/β cells vs TCRγ/δ cells17 and the development of CD8+ cells vs CD4+ cells.16 One interpretation of these results is that Notch-1 signaling alters the developmental fate of thymocytes mechanistically akin to what has been described in other developmental systems.1 More recent experiments suggest that Notch-1 signaling confers apoptotic resistance to developing thymocytes, which may influence the pool size of thymocyte compartments.1819 Moreover, retrovirus-mediated expression of constitutively active Notch-1 in early lymphoid progenitors promotes thymocyte development at the expense of B cell development in murine BM.21 This suggests that Notch signaling may influence the T- vs B-lineage fate decision, possibly via a dual outcome of CBF-1-mediated induction of HES-1 and/or deltex-mediated suppression of E47 homodimers.14 However, studies in fibroblasts and EBV-transformed B cell lines have suggested that Notch signaling in B-lineage cells may be CBF-1 independent.3233 Pui and colleagues21 reported that Notch-1 was not detected in normal murine BM B-lineage cells encompassing Hardy fractions A-F when analyzed by RT-PCR. In contrast, we have detected Notch-1 protein in murine CD19+ cells from BM and spleen (Figure 5) using a rabbit anti-human Notch-1 antibody26 that is known to cross-react with murine Notch-1. The reason for this discrepancy is unclear.

The current study provides direct evidence that Notch mRNA and protein are expressed in normal and leukemic human B-lineage cells. Notch-1 is expressed at multiple stages of B cell development, while Notch-2 expression is restricted to the late pre-B (CD19+μlo) compartment (Figures 2 and 3). We have also shown that CD19+ normal human BM B-lineage cells express cell surface Notch-1 receptors (Figure 4). This flow cytometric analysis of cell surface Notch-1 at an individual cell level rules out the unlikely possibility that rare non-B-lineage contaminating cells were the source of the Notch signals observed in the RT-PCR and Western blotting experiments.

The ubiquitous expression of Notch-1 in human B cell development suggests that Notch-1 signaling could modify proliferation and differentiation through several developmental checkpoints. However, it seems unlikely that all stages of B cell development constantly undergo Notch activation by virtue of simple ligand binding. Signaling through Notch-1 in B-lineage cells could be regulated by mechanisms other than constitutive cell surface receptor expression. Genetic studies in Drosophila have indicated the existence of Notch modifying genes such as numb and fringe.3435 For example, association of Fringe with Notch-1 impairs Serrate (Jagged)-mediated signaling, but is permissive for Delta-mediated signaling.36 Mammalian homologues of these genes have been identified.3738 In addition, current models of Notch function indicate that generation of ICN must occur in order for a Notch signal to be transduced (reviewed in Ref. 1). A wealth of recent data implicates presenilin-1 and/or its associated γ-secretase activity in the generation of ICN,39404142 although presenilin-1 may not be a universal requirement for Notch signaling.43 Furthermore, TNF-α converting enzyme cleaves a site in the Notch extracellular domain proximal to the transmembrane domain;44 an event that precedes generation of ICN by γ-secretase activity. The role of Notch modifying genes and various enzymes in regulating Notch signaling in B-lineage cells will require further investigation.

It is interesting that Notch-2 gene expression is restricted to a compartment of pre-B cells (Figure 2b). The human CD19+μlo compartment is enriched for large and small pre-B cells which express surface μ H chains in association with VpreB and λ5.4546 Cells expressing the pre-BCR undergo selection based on the ability of the μ-H chains to pair with the ψLC.4748 B-lineage cells expressing μ-H chains that fail to pair with the ψLC undergo programmed cell death.49 Human fetal BM surface VpreB+ cells enriched by FACS express Notch-2 by RT-PCR analysis (FE Bertrand et al, unpublished observation). It is therefore conceivable that Notch-2 signaling augments or modulates the cellular response to pre-BCR stimulation. For example, Notch-2 may confer an anti-apoptotic signal to pre-BCR+ cells at a point when these cells are poised to undergo L chain rearrangement. The anti-apoptotic effects of Notch-1 signaling in thymocytes is consistent with this hypothesis.1819

Human BM stromal cells used in the current study are a population of fibroblast-like adventicial reticular cells that support the differentiation of CD34+CD19 progenitors into surface IgM+ immature B cells.2431 These BM stromal cells express cell surface Jagged-1 but do not express Delta (Figure 7). This contrasts with the expression of Delta but not Jagged-1 in pro-B and pre-B cells (Figure 6). These results raise the possibility that lateral signaling between B cell precursors may occur via Notch–Delta interactions. Notch signaling between equivalent cells of equal developmental potential, termed lateral signaling, is a common paradigm in the Notch field that explains how some cells may be signaled to commit to a particular fate, while others are maintained in the original precursor state (see Ref. 1 for review). Such mechanism could possibly play a role in maintaining normal homeostasis of B-lineage developmental compartments. A report showing that Rel/NF-κB can induce Jagged-1 expression on murine splenic B cells also supports a lateral signaling model.50 Signaling between B cell precursors via Notch and Delta may also contribute to other cell–cell interactions described in B-lymphopoiesis.51

In conclusion, we have provided direct evidence that Notch and Notch ligands are expressed in human B-lineage cells and BM stromal cells. Notch signaling in human B-lineage cells could be activated by the Delta ligand on B-lineage cells, or the Jagged-1 ligand on BM stromal cells. These signals may act independently and/or cooperatively to modulate subsequent gene expression, differentiation and apoptotic fate within the B-lineage.

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Acknowledgements

We thank Barbara Ercole for technical assistance, Janet Peller of the University of Minnesota Cancer Center Flow Cytometry Core for cell sorting support, and Diane Hasz and David Largaespada for provision of murine lymphoid tissues/reagents. We deeply appreciate provision of reagents from and discussion with Irv Bernstein (FHCRC, Seattle) and Jon Aster (Brigham and Womens Hospital, Boston). We also thank Sandi Sherman for assistance with the manuscript and members of the LeBien laboratory for helpful discussion. This work was supported by grants R01 CA31685 and R01 CA76055 from the National Institutes of Health, the Graduate School of the University of Minnesota, the Minnesota Medical Foundation, and the Apogee Enterprises Professorship. ASL was the recipient of the Margaret Mitchell Research Scholarship from the American Cancer Society (Minnesota Council). FEB is supported by The Chris P Tkalcevic Foundation for Leukemia Research as a Special Fellow of the Leukemia and Lymphoma Society.

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Bertrand, F., Eckfeldt, C., Lysholm, A. et al. Notch-1 and Notch-2 exhibit unique patterns of expression in human B-lineage cells. Leukemia 14, 2095–2102 (2000). https://doi.org/10.1038/sj.leu.2401942

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Keywords

  • Notch
  • B cell precursors

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