scholarly journals Osteoblast Stimulating Factor-5 Regulates B Lymphopoiesis Via Inhibiting Pre B Cell Proliferation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2926-2926
Author(s):  
Natsuko Fujita ◽  
Kenji Oritani ◽  
Michiko Ichii ◽  
Norimitsu Saitoh ◽  
Kengo Yamawaki ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
B Lymphocyte ◽  
Signal Sequence ◽  
B Lymphopoiesis ◽  
Immature B Cells ◽  
Stimulating Factor

Abstract B lymphopoiesis is a complex and multistep process originated from hematopoietic stem cells (HSC). Recent studies showed that the microenvironment surrounding progenitor cells affects the development of B cells. Soluble factors that bone marrow (BM) stromal cells produce are crucial to the process. For example, SDF1α regulates the survival and homing of HSC and the lineage progenitor cells. IL7 is produced by osteoblasts and promotes the differentiation and proliferation of pre B cells. Although various molecules have been reported for the important roles, there are many proteins, which are secreted from stromal cells and function as a direct regulator of progenitors, remained unknown. In this study, we aimed to find novel secreted or membrane proteins, which modulate B cell differentiation via the microenvironment. We used MS-5 stromal cells to identify the novel secreted regulators because the cell line is known to have the potential to support HSC and B lymphopoiesis. The secreted proteins have signal sequence to pass into endoplasmic reticulum, followed by cell surface expression. Thus, we applied the modified signal sequence trap method (Tashiro et al, Science, 1993). Briefly, the cDNA library from MS-5 was inserted to the HPC4-TF/pEFBOS vector to produce fusion proteins, composed of proteins from cDNA, a HPC4-epitope, and a tissue factor transmembrane. With the screening of each plasmid based on the capacity to express HPC4-epitope on cell surface, we successfully identified 21 secreted or transmembrane proteins, which MS-5 cells produce. Among these proteins, pleiotrophin, proliferin-2 and osteoblast stimulating factor-5 (OSF-5) were selected because of the limited mRNA expression within stromal cell lines. For the functional analysis without the effects on the process of fetus development, we generated transgenic chimera mice (Tg), which produce the indicated protein under the control of kappa chain promoter. As a result, OSF-5, but not pleiotrophin or proliferin-2, Tg showed impaired B lymphocyte development. In OSF-5 chimera mice, the number of B lineage cells was decreased. B220+ cells in the spleen as well as pre B cells and immature B cells in the BM were significantly decreased (B220low CD43low IgM- pre B cells: 2.2 ± 0.2 x 105 cells in OSF-5 Tg vs. 6.7 ± 1.9 x 105 cells in control, B220+ CD43- IgM+ immature B cells: 2.3 ± 0.6 x 105 cells in OSF-5 Tg vs. 5.3 ± 0.8 x 105 cells in control). OSF-5 is widely expressed in mice BM, spleen, thymus, liver, kidney and lung, and the effects on hematopoiesis have never been examined. OSF-5 includes two splicing variants. Variant 1 is a secreted protein and known as aortic carboxypeptidase like protein. Variant 2 is a non-secreted, intracellular protein, known as adipocyte enhancer binding protein. In mice BM, OSF-5 variant 1 is secreted only from stromal cells, while OSF-5 variant 2 is expressed in hematopoietic cells. First, to exclude the cell-intrinsic effects of OSF-5, we ectopically expressed variant 2 in lineage- Sca-1+ c-Kit+ Flt3- HSC and co-cultured them with MS-5. As a result, the generated number of CD19+ B cells was not changed. In contrast, when we knocked down the secreted type of OSF-5 in OP9 stromal cell line to mimic the BM environment, the modified OP9 cells could support the proliferation of pre B cell line, 2E8 more efficiently, compared to control (7.3 ± 1.1 x 105 cells in KD vs. 4.4 ± 0.7 x 105 cells in control). In addition, the knock-down (KD) of variant 1 protein increased the recovered number of CD19+ cells in co-cultures of BM mononuclear cells (6.6 ± 2.8 x 105 cells in KD vs. 4.7 ± 2.8 x 105 cells in control). Finally, we found that colony-forming unit of pre B cells was decreased in the existence of OSF-5 variant 1 (17 ± 8 colonies in variant 1 vs. 198 ± 22 colonies in control). This result indicated that OSF-5 produced by BM stromal cells had a direct effect to inhibit the proliferation of pre B cells. In conclusion, we identified OSF-5 as a BM stromal cell-derived secreted protein, which has an ability to inhibit B lymphopoiesis via regulating the pre B cell proliferation. Our findings could help us to understand molecular regulatory mechanisms of normal B lymphopoiesis as well as causes of B lymphocyte dysregulation, such as change during aging. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

scholarly journals Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2311-2320 ◽  
Author(s):  
FM Lemoine ◽  
S Dedhar ◽  
GM Lima ◽  
CJ Eaves
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Cell Attachment ◽  
Attachment Site ◽  
Receptor Antibodies

Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

scholarly journals Ligand-independent Signaling Functions for the B Lymphocyte Antigen Receptor and Their Role in Positive Selection during B Lymphopoiesis

2001 ◽  
Vol 194 (11) ◽  
pp. 1583-1596 ◽  
Author(s):  
Gregory Bannish ◽  
Ezequiel M. Fuentes-Pananá ◽  
John C. Cambier ◽  
Warren S. Pear ◽  
John G. Monroe
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocyte ◽  
Antigen Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Lymphopoiesis ◽  
Biologically Relevant ◽  
Developmental Progression

Signal transduction through the B cell antigen receptor (BCR) is determined by a balance of positive and negative regulators. This balance is shifted by aggregation that results from binding to extracellular ligand. Aggregation of the BCR is necessary for eliciting negative selection or activation by BCR-expressing B cells. However, ligand-independent signaling through intermediate and mature forms of the BCR has been postulated to regulate B cell development and peripheral homeostasis. To address the importance of ligand-independent BCR signaling functions and their regulation during B cell development, we have designed a model that allows us to isolate the basal signaling functions of immunoglobulin (Ig)α/Igβ-containing BCR complexes from those that are dependent upon ligand-mediated aggregation. In vivo, we find that basal signaling is sufficient to facilitate pro-B → pre-B cell transition and to generate immature/mature peripheral B cells. The ability to generate basal signals and to drive developmental progression were both dependent on plasma membrane association of Igα/Igβ complexes and intact immunoregulatory tyrosine activation motifs (ITAM), thereby establishing a correlation between these processes. We believe that these studies are the first to directly demonstrate biologically relevant basal signaling through the BCR where the ability to interact with both conventional as well as nonconventional extracellular ligands is eliminated.

scholarly journals Apoptosis induced by the antigen receptor and Fas in a variant of the immature B cell line WEHI-231 and in splenic immature B cells

2001 ◽  
Vol 13 (4) ◽  
pp. 581-592 ◽  
Author(s):  
Maoxin Tim Tian ◽  
Chih-Hao Gilbert Chou ◽  
Anthony L. DeFranco
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Antigen Receptor ◽  
Wehi 231 ◽  
Immature B Cells

Genetic and Functional Characterization of Isolated Stromal Cell Lines from the Aorta-Gonado-Mesonephros (AGM)-Region.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2328-2328
Author(s):  
Katja C. Weisel ◽  
Ying Gao ◽  
Jae-Hung Shieh ◽  
Lothar Kanz ◽  
Malcolm A.S. Moore
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stromal Cell Line ◽  
Mes Cells

Abstract The aorta-gonads-mesonephros (AGM) region autonomously generates adult repopulating hematopoietic stem cells (HSC) in the mouse embryo and provides its own HSC-supportive microenvironment. Stromal cells from adult bone marrow, yolk sac, fetal liver and AGM have been used in coculture systems for analysing growth, maintenance and differentiation of hematopoietic stem cells. We generated >100 cloned stromal cell lines from the AGM of 10.5 dpc mouse embryos. In previous studies, we tested these for support of murine adult and human cord blood (CB) CD34+ cells. We could demonstrate that 25 clones were superior to the MS5 bone marrow stromal cell line in supporting progenitor cell expansion of adult mouse bone marrow both, in 2ndry CFC and CAFC production. In addition we demonstrated that 5 AGM lines promoted in absence of exogenous growth factors the expansion of human CB cells with progenitor (CFC production for at least 5 weeks) and stem cell (repopulation of cocultured cells in NOD/SCID assay) function. Now, we could show that one of the isolated stromal cell lines (AGM-S62) is capable in differentiating undifferentiated murine embryonic stem (mES) cells into cells of the hematopoietic lineage. A sequential coculture of mES-cells with AGM-S62 showed production of CD41+ hematopoietic progenitor cells at day 10 as well as 2ndry CFC and CAFC production of day 10 suspension cells. Hematopoietic cell differentiation was comparable to standard OP9 differentiation assay. With these data, we can describe for the first time, that a stromal cell line other than OP9 can induce hematopoietic differentiation of undifferentiated mES cells. Hematopoietic support occurs independently of M-CSF deficiency, which is the characteristic of OP9 cells, because it is strongly expressed by AGM-S62. To evaluate genes responsible for hematopoietic cell support, we compared a supporting and a non-supporting AGM stromal cell line by microarray analysis. The cell line with hematopoietic support clearly showed a high expression of mesenchymal markers (laminins, thrombospondin-1) as well as characteristic genes for the early vascular smooth muscle phenotype (Eda). Both phenotypes are described for stromal cells with hematopoietic support generated from bone marrow and fetal liver. In addition, the analysed supporting AGM stromal cell line interestingly expressed genes important in early B-cell differentiation (osteoprotegerin, early B-cell factor 1, B-cell stimulating factor 3), which goes in line with data demonstrating early B-cell development in the AGM-region before etablishing of fetal liver hematopoiesis. Further studies will show the significance of single factors found to be expressed in microarray analyses. This unique source of > 100 various cell lines will be of value in elucidating the molecular mechanisms regulating embryonic and adult hematopoiesis in mouse and man.

scholarly journals In vivo and in vitro suppression of primary B lymphocytopoiesis by tumor-derived and recombinant granulocyte colony-stimulating factor

Blood ◽  
1993 ◽  
Vol 82 (7) ◽  
pp. 2062-2068 ◽  
Author(s):  
MY Lee ◽  
KL Fevold ◽  
K Dorshkind ◽  
R Fukunaga ◽  
S Nagata ◽  
...  
Keyword(s):  
B Cell ◽  
Stromal Cell ◽  
In Vivo Studies ◽  
Granulocyte Colony Stimulating Factor ◽  
B Lymphopoiesis ◽  
Colony Stimulating Factor ◽  
Tumor Bearing ◽  
Stimulating Factor

Transplantation of a granulocytosis-inducing murine CE mammary carcinoma into mice suppresses primary B lymphopoiesis in the marrow. The mechanisms of this tumor-induced B-cell suppression were investigated using Whitlock-Witte-type lymphoid cultures. When seeded with normal marrow progenitors, stromal cells of tumor-bearing mice supported the production of B220+ cells as well as did either stomal cells derived from control mice or the stromal cell line S17. Cultured over normal stroma, marrow cells of tumor-bearing mice depleted of adherent cells and B220+ cells generated B220+ cells as effectively as a similar cell population from control mice. However, interleukin-7- responsive progenitors, were completely depleted from the marrow of tumor-bearing mice. When conditioned medium (CM) of cloned CE tumor cells known to produce granulocyte colony-stimulating factor (G-CSF) and macrophage-CSF, or recombinant murine G-CSF was added to the cultures established with S17 cells, B220+ cell production was significantly diminished. Antiserum to murine G-CSF blocked these effects. These in vitro observations were corroborated by the elimination of marrow B220+ cells in mice injected with G-CSF. These in vitro and in vivo studies suggest that G-CSF plays an inhibitory role in primary B lymphopoiesis by blocking stromal cell-mediated differentiation of early B-cell progenitors into phenotypically recognizable B220+ pre-B cells.

scholarly journals Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2612-2617 ◽  
Author(s):  
Y Gunji ◽  
T Sudo ◽  
J Suda ◽  
Y Yamaguchi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Liver Cells ◽  
B Cell Differentiation ◽  
Stromal Cell Line ◽  
Fetal Liver Cells

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

SDF-1 Responsiveness Does Not Correlate With CXCR4 Expression Levels of Developing Human Bone Marrow B Cells

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 2990-2998 ◽  
Author(s):  
Marek Honczarenko ◽  
Raymond S. Douglas ◽  
Clarissa Mathias ◽  
Benhur Lee ◽  
Mariusz Z. Ratajczak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cxcr4 Expression ◽  
B Lymphopoiesis ◽  
Cell Stage ◽  
Immature B Cells ◽  
Stromal Cell Derived Factor ◽  
And Migration ◽  
And Function

Abstract Chemokines and their receptors are broadly expressed in different tissues and are involved in diverse biologic processes. Gene inactivation studies have shown that both stromal cell derived factor-1 (SDF-1) and chemokine receptor 4 (CXCR4) are essential for B lymphopoiesis. However, it is not yet clear by which mechanisms B lymphopoiesis is affected. In the present study, we have examined CXCR4 expression and function on primary B cells representing sequential stages of development (eg, pro-B, pre-B, immature, and mature B cells) in fetal and adult bone marrow. The expression of CXCR4 was observed to be sinusoidal. Expression was highest on pre-B cells, decreased as cells developed into immature B cells, and then increased again upon transition to the mature B-cell stage. The corresponding ligand SDF-1 was shown to trigger vigorous cell signaling and migration responses, which are restricted to early lineage B cells. The responsiveness to SDF-1 was markedly decreased for immature and mature B cells despite relatively high levels of CXCR4 expression. Thus, the diminished responsiveness to SDF-1 by more mature B cells was determined to be disproportionate to the level of CXCR4 expression. These findings raise the possibility that CXCR4 function is differentially controlled during B lymphopoiesis and may be relevant to the compartmentalization of B-cell precursors in the bone marrow.

scholarly journals Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2612-2617 ◽  
Author(s):  
Y Gunji ◽  
T Sudo ◽  
J Suda ◽  
Y Yamaguchi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Liver Cells ◽  
B Cell Differentiation ◽  
Stromal Cell Line ◽  
Fetal Liver Cells

Abstract We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

scholarly journals The FLK2/FLT3 ligand synergizes with interleukin-7 in promoting stromal- cell-independent expansion and differentiation of human fetal pro-B cells in vitro

Blood ◽  
1996 ◽  
Vol 87 (5) ◽  
pp. 1881-1890 ◽  
Author(s):  
R Namikawa ◽  
MO Muench ◽  
JE de Vries ◽  
MG Roncarolo
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cell Growth ◽  
B Cell ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Mouse Bone Marrow ◽  
Flt3 Ligand ◽  
Cell Growth And Differentiation

Abstract The effects of a novel cytokine FLK2/FLT3 ligand (FL) on human fetal bone marrow-derived CD34+CD19+ pro-B cells were analyzed in a stromal- cell-independent, serum-deprived culture system. FL, like interleukin-3 (IL-3), synergized with IL-7 in promoting pro-B cell growth, and differentiation of these cells into CD34-CD19+clgM+slgM- pre-B cells, whereas a small proportion of these cells even differentiate into more mature slgM+ B cells. In contrast, KIT ligand (KL) and granulocyte- macrophage colony-stimulating factor (GM-CSF) were ineffective in promoting IL-7-dependent pro-B cell growth and differentiation. Maximal levels of pro-B cell expansion, generally resulting in 15- to 30-fold increases in cellularity, were obtained in cultures supplemented with optimal doses of FL + IL-7 + IL-3. The addition of mouse bone marrow stromal cells further enhanced the proliferation and differentiation of pro-B cells obtained in the presence of these three cytokines. Under these conditions, cultures could be maintained for more than 4 weeks, and in general 40- to 50-fold increases in cell numbers were observed by 3 weeks of culture. The percentages of clgM+ and slgM+ B cells increased 1.5- to 3-fold and 2-fold, respectively, suggesting that stromal cells may provide additional costimulatory signals for human B- cell growth and differentiation that are different from IL-7, IL-3, and FL. Collectively, our results indicate that FL, in contrast to KL, strongly promotes long-term expansion and differentiation of human pro- B cells in the presence of IL-7 or in combination of IL-7 and IL-3, which is a novel property of this hematopoietic growth factor.

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