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Stromal Cell-Independent Maturation of IL-7-Responsive Pro-B Cells¹

Robert J. Ray^2,*,, Angela Stoddart^*,, Jacqueline L. Pennycook^*,, H. Ozgur Huner^*, Caren Furlonger^*, Gillian E. Wu^*, and Christopher J. Paige^*,

^* The Ontario Cancer Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The proliferation, survival, and differentiation of B cell progenitors in primary hematopoietic tissues depends on extracellular signals produced by stromal cells within the microenvironment. IL-7 is a stromal-derived growth factor that plays a crucial role in B lineage development. We have shown that in the presence of IL-7, pro-B cells proliferate and differentiate to a stage in which they are responsive to stromal cells and LPS, leading to terminally differentiated IgM-secreting plasma cells. In this report, we examine in detail the role of stromal cells in the transition from the IL-7-responsive pro-B cell stage to the mature LPS-responsive B cell stage. We demonstrate that this transition fails to occur, even in the presence of stromal cells and LPS, if constant exposure to IL-7 is maintained. The transition from the large pro-B cell stage to the small cµ⁺ pre-B cell stage occurs independent of stromal cells. Moreover, the "stromal cell-dependent" maturation that occurs subsequent to the expression of surface IgM leading to responsiveness to B cell mitogens can also be accomplished in the absence of stromal cells if pre-B cells are cultured in proximity to each other or at high cell concentrations. Together these results suggest that stromal cells mediate B cell differentiation by providing the necessary growth requirements (i.e., IL-7) to sustain the development of pre-B cells. The progeny of these pre-B cells can then differentiate through as yet unidentified homotypic interactions, leading to the production of LPS-responsive B cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells of the hematopoietic system are dependent upon their microenvironment to both commit and progress through a specific cell lineage. B lymphocytes develop in the liver during fetal life and in the bone marrow of adult animals (1). In vivo, B cell progenitors are found in close association with several heterogeneous populations of stromal cells, suggesting that stromal cells support the differentiation of these progenitors (2, 3). It is currently unclear whether stromal cells merely provide the necessary requirements for the growth and survival of B cell progenitors or play an inductive role in regulating lymphopoiesis. Bone marrow stroma is composed of a number of diverse cell types, including adventitial reticular cells, fibroblasts or endothelial-like cells, macrophages, and adipocytes (4, 5). Due to the heterogeneity of the bone marrow, the nature of the interaction between lymphocytes and their environment has been difficult to study.

In vitro systems involving long-term bone marrow cultures have been used effectively to study the microenvironmental interactions of hematopoietic precursors (6, 7, 8, 9). Much of our understanding of the biology underlying stromal cell support of B lymphopoiesis is derived from the study of murine stromal cell lines (10). From these studies, it has become apparent that stromal cells provide a variety of signals to lymphocyte progenitors via both cell-cell interactions and the secretion of soluble mediators (10, 11, 12). However, stromal cell lines vary in their ability to promote the proliferation and differentiation of B cell progenitors (13, 14, 15, 16, 17, 18). In vitro culture assays using S17 stromal cells have permitted us to study several discrete stages of B cell development (19). Uncommitted progenitors from both day 10 yolk sac and day 12 fetal liver have been shown to be dependent on S17 stromal cells for their growth and differentiation into the B lineage in vitro (20, 21, 22). In these cultures, growth factors have been identified (IL-11, MGF, FL, IL-7) that can replace the function of S17 stromal cells in mediating the commitment and maturation of IL-7-responsive pro-B cells from uncommitted progenitors (23, 24). Pro-B cells are predominantly large, cycling cells that are in the process of recombining Ig heavy chain genes and express B220, CD43, BP-1, and 5 (25, 26, 27, 28). These IL-7-responsive cells subsequently give rise to small, noncycling pre-B cells that are CD43^-, BP-1^-, and cytoplasmic µ⁺ (cµ+)³ cells that undergo rearrangement at the light chain locus (29, 26, 27, 28). The events that regulate the transition to an immature surface IgM⁺ (sIgM⁺) cell and subsequent stages are currently unknown, although stromal cells appear to play a key role in regulating these events (15, 30, 31, 32, 33).

In vivo, the development of mature B lymphocytes has been shown to be dependent on IL-7 signaling (34, 35, 36, 37). IL-7 was originally identified as a soluble growth factor with lymphopoietic activity (38), and was the first cytokine to be identified and cloned from a stromal cell line (39). IL-7 has been shown to be required for both the proliferation and differentiation of committed B cell progenitors in vitro (32, 40, 41). However, IL-7-responsive cells require stromal cells to mediate their differentiation to mature B cells, which are responsive to the B cell mitogen, LPS (40). The requirements for the transition from an IL-7-responsive pro-B cell to a functionally mature sIgM⁺ B cell remain largely unknown. Previous studies have shown that the removal of IL-7 from cultures containing IL-7 and stromal cell-dependent B cell clones results in the induction of V_H to DJ_H and V_L to J_L rearrangements (42, 43). Although some cells become sIgM⁺ and phenotypically mature, very few (<1:3000) of these cells respond to LPS, further suggesting that stromal cells are required to mediate the transition to a functionally mature stage of B cell development (44).

The precise role of stromal cells in mediating the maturation of B cell progenitors is currently unclear. We therefore set out to determine the specific stages in which stromal cells interact with B cell progenitors to mediate their differentiation to the LPS-responsive stage. We show that the maturation of pro-B cells cocultured with S17 + LPS is influenced by the presence or absence of IL-7. Upon removal of IL-7, pro-B cells differentiate into small cµ⁺ pre-B cells that contain a greater number of rearrangements at the locus. This transition is independent of stromal cells. Moreover, in the continued presence of IL-7, pro-B cells fail to differentiate into small pre-B cells and fail to undergo stromal cell-dependent maturation. Cells that become surface µ⁺ (sµ⁺) in our culture system can mature to the LPS-responsive stage if they interact with stromal cells in a contact-dependent manner. The nature of the stromal cell-mediated signal(s) that influence differentiation to the mitogen-responsive stage remain to be elucidated. Interestingly, we have found that the "stromal cell-dependent" maturational event can also be accomplished by culturing IL-7-responsive pro-B cells in proximity to each other or at a high cell density. These results raise the possibility that stromal cells mediate lymphopoiesis by providing the necessary growth factors (such as IL-7) that regulate the growth and differentiation of B cells that, at the appropriate stage, mature further through homotypic interactions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the animal colony of the Wellesley Hospital Research Institute. Timed pregnancies were established by mating mice overnight and observing vaginal plugs the following morning on day 0. Pregnant females were killed by cervical dislocation on day 15 of gestation.

Cell purification

Cell suspensions were prepared from pooled day 15 fetal livers by passage through a 26-gauge needle; debris was removed by gravity sedimentation on ice for 5 min. The cell suspension was collected and cell viability was determined by trypan blue exclusion. B cell progenitors from day 15 fetal liver cell suspensions were isolated by enriching for B220⁺ cells using 14.8 coated panning plates (2 x 10⁷ cells/4 ml/plate). Panning was performed using Optilux 100-mm plastic petri dishes (Falcon no. 1001, Oxnard, CA). Petri dishes were first coated with mouse anti-rat IgG (10 µg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA) in 0.05 M Tris-Cl, pH 9.5, 0.15 M NaCl at room temperature for a minimum of 1 h. The plates were washed three times in 5% FCS/balanced salt solution, followed by the addition of 4 ml of 1:2 diluted 14.8 hybridoma supernatant. Plates were then incubated overnight at 4°C. The plates were washed three times in 5% FCS/balanced salt solution followed by the addition of cell suspensions at 2 x 10⁷ cells/plate. Cells were incubated at 4°C for 1 h. Adherent cells were recovered by scraping with a plastic scraper (no. 3010; Costar, Cambridge, MA) after carefully washing the plates eight times in 5% FCS/balanced salt solution. The recovered cells were centrifuged at 1250 rpm for 5 min and resuspended in 2 to 3 ml of cold 10% FCS/OptiMEM (Life Technologies, Grand Island, NY). Cell viability was again determined by trypan blue exclusion. Typically, 2 to 3 x 10⁷ cells were recovered per day 15 C57BL/6 fetal liver, of which approximately 1.4% were B220⁺.

Cell culture conditions

Cells were maintained in OptiMEM supplemented with 10% FCS, 50 µM 2-ME, 2.4 g/L NaHCO₃, 100 µg/ml penicillin, 100 µg/ml streptomycin, and the appropriate growth factors at saturating concentrations in a humidified atmosphere of 5% CO₂ at 37°C. B220⁺ day 15 fetal liver cells were cultured (2–5 x 10⁴ cells/well) in a 24-well plate for 4 days in the presence of recombinant human IL-7 (100 U/ml) (Immunex, Seattle, WA) to further enrich for IL-7-responsive pro-B cells (proB_d4-IL7 cells). There was a 6- to 10-fold increase in the number of viable cells cultured under these conditions. IL-7 was removed by washing cells twice with 10 ml of 10% FCS/OptiMEM. The differentiation of proB_d4-IL7 cells to the mitogen-responsive stage was quantitated in a maturation assay by placing 10³ cells in a secondary culture (96-well microtiter plate) containing 10³ irradiated (2000 rad) S17 stromal cells and 15 µg/ml LPS (Salmonella typhosa W0901; Difco, Detroit, MI) (40). IgM secretion was measured 7 days later in an ELISA assay. A total of 10³ S17 stromal cells were determined to be the minimal number of stromal cells required to mediate the maturation of 10³ proB_d4-IL7 cells in liquid cultures.

To determine whether the stromal cell-dependent maturation was contact dependent, S17 stromal cells were separated from proB_d4-IL7 cells in secondary cultures using Nunc TC inserts (Nunc, Dannstadt, Denmark) for flat-bottom 96-well microtiter plates. mAbs and reagents used to target stromal cell-mediated maturation were as follows: anti-CD44 (IM7 or KM114), anti-CD49d (9C10 and R1-2), 0.1 µg/ml anti-µ (33.60) (45), 1 µg/ml anti- (1050-01) (Southern Biotechnology, Birmingham, AL), anti-5 (FS1) (46), anti-CD19 (1D3), anti-CD81 (2F7), anti-CD22 (Cy34.1), and anti-CD40 (3/23). mAbs were purchased from PharMingen (San Diego, CA) without sodium azide and used at 10 µg/ml, unless otherwise stated. PMA (Sigma, St. Louis, MO) and ionomycin (Calbiochem, La Jolla, CA) were used at 1 ng/ml.

ELISA

ELISAs were performed by coating enzyme immunosorbant assay (EIA) plates (Costar; no. 3590) with 5 µg/ml of affinity-purified goat anti-mouse µ-chain Ab (Jackson ImmunoResearch Laboratories) for 30 min at 37°C. Plates were washed twice with cold tap water and blocked for 30 min at 37°C with 5% FCS/PBS followed by an additional eight washes with cold tap water. Ten-fold serial dilutions of culture supernatants in 5% FCS/PBS were added to the plates and incubated for 30 min at 37°C. Plates were washed eight times in cold tap water and a 1:2000 dilution of goat anti-mouse µ-chain conjugated to horseradish peroxidase (Sigma) was added for 30 min at 37°C. Plates were again washed eight times in cold tap water followed by the addition of 50 µl of the substrate consisting of 0.5 mg/ml 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.05 M phosphate-citrate buffer, and 0.03% sodium perborate (Sigma). Plates were further incubated for 30 min at 37°C and the absorbance was read at 405/630 nm. Control experiments revealed that the rat anti-mouse µ-chain mAb, 33.60 (45), did not interfere with the detection of secreted IgM in the ELISA assay.

Proliferation assays

B220⁺ cells from day 15 fetal livers were cultured for 4 days (proB_d4-IL7 cells) in the presence of recombinant human IL-7 (100U/ml). ProB_d4-IL7 cells were subsequently transferred to 96-well microtiter plates (10³ cells/well) containing medium, 10³ irradiated S17 stromal cells, IL-7 (100 U/ml), LPS (15 µg/ml), S17 + LPS, S17 + IL-7 or S17 + LPS + IL-7, and cultured for an additional 4 days. Six hours before the end of culture, wells were pulsed with 1 µCi of [³H]TdR (DuPont, Wilmington, DE). Lysed cells were harvested onto microplate filters, and radioactivity was measured in a scintillation counter (Topcount; Canberra Packard, Downers Grove, IL).

Flow cytometric analysis

ProB_d4-IL7 cells (5 x 10⁵ cells/well) were cultured in 24-well plates in the indicated conditions for 24, 48, or 72 h (irradiated S17 stromal cells were plated at 2 x 10⁴ cells/well). Harvested cells were stained for the expression of surface markers by incubating approximately 10⁵ cells in 200 µl of PBS and 5% FCS with Abs for 20 min at 4°C. Biotin-conjugated Abs were detected by a subsequent incubation with streptavidin phycoerythrin. Cells were washed three times with 5% FCS/PBS and the fluorescence intensity was measured on a FACScan (Becton Dickinson, Mountain View, CA) followed by analysis with Cell Quest version 3.1 software. Cells were stained for cµ expression by fixing the cells in 1% paraformaldehyde for 15 min, followed by two washes in PBS. Cells were incubated with 0.2% Tween-20 in PBS for 15 min at room temperature. Cells were then labeled with goat anti-mouse µ-chain Ab conjugated to FITC (Sigma) for 20 min followed by three washes in 5% FCS + 0.2% Tween-20 in PBS. Live cells were gated according to forward- and side-scatter characteristics and propidium iodide staining. Cells were stained using Abs (PharMingen) to B220 (6B2-PE), BP-1 (6C3-FITC), µ (33.60-FITC) (45), (R8-140-FITC), CD22 (Cy34.1-PE), CD43 (S7-PE), and 5 (FS1-biotin) (46). Isotype-matched controls were used to determine the background level of staining (<1%).

Detection of VJ rearrangements

DNA was isolated by a modified direct PCR lysis method (47). A total of 10³ cells/µl were resuspended in PCR lysis buffer (10 mM Tris, pH 8.3, 1.8 mM MgCl₂, 50 mM KCl, 0.45% Nonidet P-40, 0.45% Tween-20, 60 µg/ml proteinase K), and incubated at 56°C for 1 h. Samples were heated to 90°C for 15 min to inactivate the proteinase and used directly for PCR. VJ rearrangements were amplified using primers hybridizing to the framework 3 region of V elements (Vcon-5': 5'-GGCTGCAA(C/G)TTCAGTGGCAGTGG(A/G)TC(A/T)GG(A/G)AC-3') (48) and to 3' of the J5 element (J 5–3': 5'-TGCCACGTCAACTGATAATGAGCCCTCTC-3') (49). PCRs contained 10 mM Tris, pH 8.3, 50 mM KCl, 1.8 mM MgCl₂, 0.5% Triton X-100, 100 µg/ml BSA (Boehringer Mannheim, Laval, Quebec, Canada), 200 mM each dNTP (Boehringer Mannheim), 0.5 µM each primer (Life Technologies), and 2.5 U of Taq polymerase (Boehringer Mannheim). PCR amplifications were conducted for five cycles after a 10-min hot start at 85°C. To control for the number of cell equivalents used from sample to sample, 0.5-µM -actin primers (-actin 5': 5'-GACATGGAGAAGATCTGGCACCACAC-3' and -actin 3': 5'-CGCACAATCTCACGTTCAG-3') (26) were added during a 10-min pause at 85°C. PCR amplification was permitted to proceed for another 25 cycles of 94°C, 1 min; 60°C, 1 min; 72°C, 2 min; plus 5 s/cycle. A 10-min 72°C primer extension period followed. PCR products were separated on 1.5% agarose gels and transferred onto nylon membranes (N-Hybond; Amersham, Chalfont, U.K.) by overnight Southern transfer. Membranes were probed with [-³²P]-labeled J5-IN 30-mer oligonucleotides (5'-CTCCTAACATGAAAACCTGTGTCTTACACA-3') using T4 polynucleotide polymerase (NEBlabs, Mississauga, Ontario, Canada) according to the manufacturer’s instructions. The J5-IN 30 mer hybridizes 20 bases 3' of the J5 element in the J-C intervening sequence (49). Hybridization was conducted at 42°C for 4 h in 5x SSPE, 5x Denhardt’s solution, and 0.5% SDS. Blots were washed twice in 2x SSC, 0.1% SDS at 42°C for 5 min, followed by two washes in 2x SSC, 0.1% SDS for 20 min at 50°C. Blots were exposed in a PhosphorImager cassette (Molecular Dynamics, Sunnyvale, CA). Imaging and quantification was done using a Molecular Dynamics PhosphorImager and ImageQuant 4.1 software. Blots were stripped by boiling in 0.1% SSC for 10 min and reprobed using an -actin-IN oligonucleotide (5'-GACATGGAGAAGATCTGGCACCACAC-3') (26). VJ5 rearrangements were calculated using the following equation: (VJ5 sample intensity/relative actin sample intensity) and are expressed relative to samples containing 100% S107 cells, which contain both VJ4 and VJ5 rearrangements. A titration standard was obtained by serially diluting S107 cells with MC9 cells, which are germline at the locus (Fig. 4B). Results are representative of nine independent PCRs from three similar experiments. Similar results were observed with when blots were analyzed for J4 rearrangements (data not shown).

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Withdrawal of IL-7 leads to a greater proportion of rearrangements in pre-B cells, independent of stromal cells. A, B220+ B cell progenitors were isolated from day 15 fetal liver and incubated with IL-7 for 0, 2, or 4 days. On day 4 of culture, cells were placed in secondary cultures for an additional 48 h. DNA was prepared and subjected to PCR amplification for the analysis of VJ rearrangements. The PCR products from 104 cell equivalents were analyzed by Southern blotting and probed using an oligonucleotide sequence hybridizing 3' of the J5 element in the J-C intervening sequence. Band intensities were quantified using a PhosphorImager. Amplification of actin was used to control for the number of cell equivalents. VJ5 rearrangements corrected for actin are expressed relative to 104 cell equivalents of S107, which is VJ5 rearranged on one allele. B, The relative VJ5 rearrangements of S107 cells titrated into MC9 cells (germline for rearrangements).

B220⁺ proB_d4-IL7 cells were stained for the surface expression of µ with 33.60-FITC mAbs (45) and sorted into sµ^- (>99%) and sµ⁺ (<1%) fractions on a FACStar^Plus Cell Sorter (Becton Dickinson). Sorted cells were cultured in limiting dilutions (32 replicates per cell concentration) in a 96-well plate with either LPS, S17 + LPS, or S17 + LPS + IL-7 for 14 days. The concentration of secreted IgM in the culture supernatant was determined by ELISA. The fraction of wells that were negative for IgM production was plotted against the number of initial cells per well using a least-squares regression line. A linear regression of the logarithm of the percentage of negative cultures with linearly increasing numbers of plated cells indicates, according to Poisson’s distribution, that the clonable progenitor is limiting in the assay. The frequency of B cell progenitors was determined as the number of initial cells per well in which 37% of the wells were nonresponding (i.e., negative for IgM production) using the Poisson distribution.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The stromal cell-mediated differentiation of proB_d4-IL7 cells is influenced by the continued presence of IL-7

We have reported previously that the differentiation of B cell progenitors from early fetal liver is dependent upon the presence of stromal cells (40, 50, 51). To determine the specific developmental stages in which stromal cells influence the differentiation of B cell progenitors, we examined the interactions between IL-7-responsive pro-B cells from B220⁺-enriched day 15 fetal liver and the stromal cell line, S17 (52). Fetal liver is the predominant site of lymphopoiesis in the developing fetus during this gestational period and is a ready source of B cell progenitors that have not undergone significant rearrangements (49). Previous studies in our laboratory demonstrated that B cell progenitors require an IL-7-responsive phase to differentiate to a stage in which they can interact with stromal cells to achieve a state of mitogen responsiveness (40, 53, 54). B220⁺ (a B lineage isoform of CD45) cells were therefore enriched from day 15 fetal livers and cultured for 4 days in the presence of saturating concentrations of IL-7 to obtain a relatively homogeneous population of IL-7-responsive B cell progenitors (proB_d4-IL7 cells). This population expressed surface markers characteristic of the late pro-B cell stage of development (i.e., B220⁺CD19⁺CD43⁺HSA⁺BP-1⁺ and 5⁺) but was negative for mature B lineage surface markers such as IgM and class II MHC (data not shown). B220⁺ fetal liver-derived progenitors cultured in the presence of IL-7 for 4 days increased in cell number ninefold, whereas viable cells were not obtained from cultures containing medium alone.

Cells responsive to IL-7 differentiate into IgM-secreting plasma cells when transferred to irradiated stromal cells in the presence of LPS (40). In these studies, maturation to the LPS-responsive stage may have been dependent on signals provided by stromal cells and/or the removal of IL-7. To investigate the influence of S17 stromal cells in mediating the differentiation of B cell progenitors in the continued presence of IL-7, we transferred proB_d4-IL7 cells to secondary cultures containing irradiated S17 cells in the presence or absence of IL-7 and LPS for an additional 4 days (Fig. 1). ProB_d4-IL7 cells showed a slight increase (4.9-fold) in the incorporation of [³H]TdR after 4 days of culture with S17 cells compared with control cultures containing medium alone (Fig. 1A). The incorporation of [³H]TdR was used as a convenient assay to measure the proliferation of proB_d4-IL7 cells in these cultures since it also correlates with an increase in cell number. The proliferation of proB_d4-IL7 cells cocultured with S17 stromal cells was not due to the presence of IL-7 because S17 stromal cells do not produce IL-7 mRNA (55). In contrast, cultures containing proB_d4-IL7 cells stimulated with IL-7 had considerable proliferative responses. Progenitors cultured with S17 + LPS + IL-7 showed a significant increase (>32-fold) in thymidine incorporation compared with cultures containing medium alone and a 6.6-fold increase compared with cultures containing only S17 cells. The proliferation of proB_d4-IL7 cells in cultures containing S17 + LPS + IL-7 was likely due to their response to IL-7 because the [³H]TdR incorporation observed in S17 + LPS or LPS-containing cultures was identical to that observed for cultures containing S17 or medium, respectively.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. The response of IL-7-responsive B cell progenitors under various conditions. A, B220+ B cell progenitors from day 15 C57BL/6 fetal livers were cultured in the presence of IL-7 for 4 days (proBd4-IL7 cells). ProBd4-IL7 cells were harvested and cultured (103 cells/well) in the indicated conditions for an additional 4 days. DNA synthesis was measured by pulsing cultures with [3H]TdR (1 µCi) 6 h before harvesting. Results are expressed as the mean of five replicates ± SD and are representative of three independent experiments. B, Parallel proBd4-IL7 cell cultures were incubated for 7 days followed by the detection of secreted IgM in an ELISA. Results are expressed as the mean ± SEM from 12 pooled experiments.

Cultures of proB_d4-IL7 cells stimulated with S17 + LPS + IL-7 contained more cells than cultures stimulated with S17 + LPS as indicated by the sixfold increase in [³H]thymidine incorporation (Fig. 1A). We therefore expected to observe a corresponding increase in the amount of secreted IgM in the maturation assay (Fig. 1B). Contrary to expectation, we detected equivalent amounts of secreted IgM in both cultures containing S17 + LPS + IL-7 and S17 + LPS. The data suggest that the additional pro-B cells found in cultures containing S17 + LPS + IL-7 did not differentiate into mature B cells even though S17 + LPS were present. A titration of proB_d4-IL7 cells revealed that the quantity of secreted IgM increases as a function of the initial number of cells cultured in the presence of S17 + LPS, demonstrating that our assay was not limiting (see Fig. 8A). A similar titration was also observed in cultures containing S17 + LPS + IL-7 (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 8. ProBd4-IL7 cells differentiate into LPS-responsive B cells in the absence of S17 stromal cells. A, ProBd4-IL7 cells were cultured with LPS, S17 + LPS, or S17 + LPS+IL-7 (data not shown) for 7 days followed by the detection of secreted IgM in an ELISA. ProBd4-IL7 cells transferred to IL-7, S17, or medium were negative for IgM production. B, A total of 103 proBd4-IL7 cells were similarly cultured in flat-bottom, U-bottom, or V-bottom tissue culture plates for 7 days. Results are expressed as the mean ± SEM from three pooled experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. Sµ+ cells arising in IL-7-stimulated cultures require stromal cells to respond to LPS. A, B220+ day 15 fetal liver cells were cultured in the presence of IL-7 for 4 days. Cells were sorted into sµ- (circles) and sµ+ (squares) fractions (99 vs <1%, respectively) and subsequently cultured in limiting dilutions with LPS, S17 + LPS (open symbol), or S17 + LPS+IL-7 (closed symbol) for 14 days followed by the detection of secreted IgM in an ELISA. The fraction of wells negative for IgM production was plotted against the number of initial cells cultured per well. B, The frequency of proBd4-IL7 cells that gave rise to functionally mature B cells was determined as the number of initial cells per well where 37% (arrow) of the wells were negative for IgM production.

Stromal cells are not necessary for the differentiation of pro-B cells to the small pre-B cell stage

It has been reported previously that B lineage clones, which are dependent on stromal cells + IL-7, differentiate upon the removal of IL-7 or stromal cells (56, 57, 58). Consequently, cells lose surface membrane proteins characteristic of pro-B cells including c-Kit, 5, CD43, and BP-1 and gain the expression of CD25 and sIgM. However, only a small fraction of the sIgM⁺ cells (<1:3000) become reactive to LPS, suggesting that these cells require additional maturational events that enable them to become mitogen responsive (42, 44, 58). To determine the precise stage of B cell development that stromal cells are required to mediate differentiation events, we examined the maturation of proB_d4-IL7 cells to the small pre-B cell stage in the presence or absence of stromal cells by flow cytometry (Fig. 3). ProB_d4-IL7 cells kept for an additional 48 h in the presence of IL-7 alone expressed B220, 5, and BP-1, but did not express sµ or . The majority of these cells were also large in size as measured by their forward light-scattering characteristics. In contrast, when IL-7 was removed from culture and replaced by medium, S17, or S17 + LPS, proB_d4-IL7 cells rapidly (within 24 h, data not shown) decreased in size and lost the surface expression of 5 and BP-1 (Fig. 3). Moreover, we observed an increase in the proportion of cells expressing CD2 and cµ compared with cultures containing IL-7. This surface phenotype is characteristic of small, resting pre-B cells (26, 28, 59). However, proB_d4-IL7 cells cocultured with S17 + IL-7 maintained a pro-B cell phenotype. These results suggest that differentiation to the small pre-B cell stage is less likely to occur in the presence of stromal cells and IL-7. Apparently, it is the removal of IL-7, rather than the presence of S17 stromal cells, that drives the pro-B cell to pre-B cell transition.

View larger version (62K): [in this window] [in a new window]   FIGURE 3. IL-7 maintains proBd4-IL7 cells in a phenotypically immature state, even in the presence of stromal cells. B220+ B cell progenitors from day 15 fetal liver were cultured in the presence of IL-7 for 4 days. Cells were harvested, washed, and cultured in the indicated conditions for an additional 48 h. Cells were stained using FITC or phycoerythrin-conjugated Abs as described in Materials and Methods. Cultures containing IL-7 resulted in a more than threefold expansion of viable cells (Table I). The absolute number of 5- cells recovered from cultures at the end of 48 h was as follows: IL-7 (3.2 x 104), media (1.1 x 105), S17 (4.1 x 105), S17 + LPS (4.4 x 105), and S17 + IL-7 (8.2 x 104). Isotype controls were >99% negative.

Immature sµ⁺ B cells are dependent upon stromal cells to mature to the LPS-responsive stage

IL-7 plays a significant role in maintaining pro-B cells in a stromal cell-independent state. However, even in the absence of stromal cells, sµ⁺ cells consistently arise at a low frequency (1%) in IL-7-stimulated pro-B cell cultures (see Fig. 3). To determine whether these sµ⁺ cells were competent to make a mitogen response, we cultured sorted sµ⁺ and sµ^- proB_d4-IL7 cells in limiting dilutions with LPS (Fig. 2B). We found that the frequency of progenitors generating IgM-secreting progeny in the presence of LPS was only 3-fold greater in the sµ⁺ population than in the sµ^- population. However, the number of LPS-responsive cells was still 16-fold less than the frequency of progenitors (sµ⁺ or sµ^-) that will mature when cocultured with S17 + LPS (Fig. 2B). These results suggest that the majority of sµ⁺ that arise in IL-7-stimulated cultures remain refractory to LPS until they encounter differentiation signals provided by S17 stromal cells.

To determine whether this stromal cell-mediated maturation was contact dependent, we separated S17 cells from proB_d4-IL7 cells using a transwell insert (Fig. 5). This culture system permits the diffusion of soluble factors through the Anopore membrane but prevents contact between the stromal cells and proB_d4-IL7 cells. When contact with S17 in the presence of LPS was permitted, there was significant IgM secretion in the culture supernatant compared with cultures containing LPS alone. However, in cultures containing the transwell, the amount of secreted IgM was significantly reduced. This result demonstrates that small pre-B cells require contact with stromal cells to differentiate to a stage of LPS responsiveness. It was possible that stromal cell contact was necessary for the induction of secreted growth factors that mediate the pre-B cell to mature B cell transition. To assess this possibility, we cultured irradiated 18.81 pro-B cells or 70Z/3 pre-B cells with either S17 + LPS or LPS alone in the lower chamber of cultures containing primary proB_d4-IL7 cells in the upper chamber. The incubation of B lineage cell lines with S17 cells did not overcome the requirement for the pre-B stromal cell contact (data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Stromal cell contact is necessary for the generation of IgM-secreting cells. ProBd4-IL7 cells (103 cells/well) were cultured with irradiated S17 stromal cells (103 cells/well) and LPS in direct contact or separated from each other using a transwell insert. IgM secretion was quantitated 7 days later in an ELISA. Results are expressed as the mean of 12 replicates ± SEM pooled from three independent experiments.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Maturation of an IL-7-responsive pro-B cell population occurs within 48 h when cocultured with S17 cells. ProBd4-IL7 cells were cultured on irradiated S17 stromal cells for the indicated length of time and then transferred to secondary cultures containing LPS. IgM secretion was quantitated 7 days later as the mean ± SEM in an ELISA. Control cultures containing proBd4-IL7 cells were incubated with LPS for the indicated length of time followed by an additional 7 days in secondary cultures containing LPS.

Previous studies have demonstrated that progression through the B cell lineage requires signaling through both the pre-BCR and BCR (64, 65). Signaling through the pre-BCR promotes the differentiation of pro-B cells to the small, cµ⁺ pre-B cell stage (64, 65). We have attempted to block the interaction between proB_d4-IL7 cells and S17 cells in our culture system using mAbs directed against surface Ags associated with BCR signaling (Fig. 7). Anti-CD44 (IM7) (66, 67) and anti-VLA-4 mAbs (68, 69) have been shown to block B cell progenitor-stromal cell interactions in Whitlock-Witte cultures, preventing the subsequent maturation of these progenitors. However, we found that these Abs had no effect on B cell maturation in our culture system. These results suggest that the critical interaction mediated by these integrins occurs before the small pre-B cell stage. Strikingly, we did not observe secreted IgM in cultures containing mAbs recognizing µ heavy chains or light chains (Fig. 7). Similar results were obtained with PMA + ionomycin, which mimics the downstream effects of BCR signaling (70). Moreover, the amount of IgM secretion detected in S17 + LPS-stimulated proB_d4-IL7 cell cultures containing anti-µ, anti- Abs or those containing PMA + ionomycin was substantially less than that observed in cultures containing LPS alone.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. The generation of IgM-secreting cells can be inhibited by Abs against CD19 or the BCR. ProBd4-IL7 cells were cultured with S17 + LPS in the presence of mAbs recognizing the indicated surface markers or with PMA + ionomycin (Io). IgM secretion was measured as the mean ± SEM of two to four pooled experiments 7 days later in an ELISA and is expressed relative to the IgM secreted by cultures containing S17 + LPS. Conditions that were statistically different (two-tailed Student’s t test) from both S17 + LPS and their isotype controls are indicated by an (*) as follows: CD49d (p < 0.001), µ (p < 0.001), (p < 0.02), PMA + Io (p < 0.002), CD19 (p < 0.002).

ProB_d4-IL7 cells cultured in proximity to each other differentiate into LPS-responsive B cells in the absence of stromal cells

We found that proB_d4-IL7 cells cultured at a high cell density (10⁴ cells) generate IgM-secreting cells in response to LPS in the absence of stromal cells (Fig. 8A), whereas cultures containing 5- to 10-fold fewer proB_d4-IL7 cells produced 200-fold less IgM. This observation suggests that at high proB_d4-IL7 cell concentrations, stromal cells are not required to mediate the proB_d4-IL7 cell to mature B cell transition. Although >99% of the proB_d4-IL7 cells express surface markers characteristic of pro-B cells, we considered the possibility that rare stromal cells may be present in sufficient numbers at the high cell concentrations to mediate this interaction. Alternatively, B cell progenitors themselves at high cell concentrations may mediate B-B interactions that replace the requirement for stromal cells. To address this issue, proB_d4-IL7 cells were cocultured in flat-bottom, U-bottom, and V-bottom plates with LPS or S17 + LPS (Fig. 8B). ProB_d4-IL7 cells cultured in U-bottom or V-bottom plates (10³ cells/well) in the presence of LPS generated significantly greater amounts of IgM (100-fold) compared with proB_d4-IL7 cells incubated in flat-bottom plates with LPS. Moreover, the supernatants from U- or V-bottom plate cultures stimulated with LPS contained equivalent amounts of secreted IgM as cultures that contained S17 + LPS. Significant amounts of secreted IgM were detected in the supernatant of U- or V-bottom plate cultures containing as few as 500 proB_d4-IL7 cells stimulated with LPS alone (data not shown). A titration of S17 cells in flat-bottom plates containing 10³ pro-B cells stimulated with LPS revealed that there was substantially less IgM secreted in cultures containing fewer than 10³ S17 cells, and essentially no IgM secretion was detected in cultures containing 100 S17 cells (data not shown). Therefore, the maturation of proB_d4-IL7 cells observed in U- or V-bottom plate cultures containing LPS in the absence of S17 cells was unlikely to be due to contaminating stromal cells. These results suggest that proB_d4-IL7 cells cultured in proximity to each other do not require stromal cells to mediate the transition from a pre-B cell to a mature B cell generating secreted IgM in response to LPS stimulation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stromal cells are known to promote the proliferation and differentiation of B lymphocytes. However, many stromal cell lines mediate only the early proliferative stages of B lymphopoiesis, and fail to support differentiation beyond the pre-B cell stage (10, 13, 72, 73). Similar to primary stroma (30), S17 stromal cells promote the differentiation of IL-7-responsive B cell progenitors to a sIgM⁺ stage in which these mature B cells are responsive to LPS in both liquid and soft agar cultures (40, 19). In this report, we have shown that IL-7-responsive pro-B cells will proliferate in response to saturating concentrations of IL-7, even if provided with an environment suitable for supporting differentiation to the mature LPS-responsive stage. IL-7-responsive B cells found in cultures containing S17 + LPS + IL-7 are similar to the proB_d4-IL7 cells present at the initiation of the maturation assay. Therefore, there is a greater number of IL-7-responsive B cell progenitors in cultures containing S17 + LPS + IL-7 that have the potential to further differentiate in the presence of stromal cells compared with cultures that are not stimulated with IL-7. However, these additional IL-7-responsive pro-B cells fail to undergo stromal cell-mediated maturational events leading to the stage of LPS responsiveness. These results suggest that IL-7 regulates the development of B cell progenitors in vivo by selectively increasing the pool of IL-7-responsive pro-B cells. Pro-B cells that become unresponsive to IL-7 stimulation proceed to the small pre-B cell stage and become capable of the further differentiation in the presence of stromal cells. Previous studies have described the differentiation of stromal cell + IL-7-dependent pro-B cell clones to the pre-B cell stage when IL-7 is removed from these cultures (42, 44). We have found that this pro-B to pre-B cell transition is independent of whether or not stromal cells are present (Fig. 3). Within 48 h of removing IL-7 from these cultures, cells become smaller and lose the expression of several surface markers characteristic of pro-B cells such as 5 and BP-1. Moreover, we also observe an increase in the proportion of CD2⁺ and cµ⁺ cells upon the removal of IL-7, indicating that an increase in pre-B cell differentiation has occurred. Therefore, IL-7 negatively regulates B cell differentiation by maintaining cells in a proliferative, pro-B cell state, independent of stromal cells.

The possibility that IL-7 regulates the expression of 5 and BP-1 is suggested by a significant decrease in surface expression of these markers, already obvious 24 h after IL-7 withdrawal, even though total cell numbers remain approximately the same (Fig. 3, data not shown). Essentially all pro-B cells (>95%) stimulated with IL-7 express 5 as determined by surface staining with FS1 mAbs. However, sµ⁺ cells are not detected in this population despite the fact that approximately 18 to 27% of these 5⁺ cells express cµ. Previous studies have shown that surrogate light chain (5/V_preB) is expressed on the surface of pro-B cells before the formation of cµ in a complex with a surrogate heavy chain (gp130) (46, 74). However, it remains unclear whether the gp130 protein is expressed past the large pro-B cell stage. In vivo, B cell progenitors that produce a functional pre-BCR proceed to the small pre-B cell stage as a result of the pre-BCR. Our failure to observe µ at the cell surface in IL-7-stimulated cultures may be due to the fact that the majority of µ-chains found in fetal liver-derived cµ⁺ pro-B cells cultured fail to associate with the surrogate light chain. Recent studies have described an early c-Kit⁺cµ⁺ pro-B cell population in the bone marrow of normal mice in which only half of the cµ-chains expressed have the capacity to form a pre-BCR (75). Moreover, fetal-associated µ heavy chains have been identified that permit pre-B cell proliferation in fetal liver, despite their failure to efficiently associate with a surrogate light chain (76). It is therefore possible that IL-7 promotes the proliferation of a small population of pro-B cells that have rearranged their heavy chain locus but fail to generate a functional pre-BCR. Upon the removal of IL-7, a greater proportion of proB_d4-IL7 cells express cµ (Fig. 3). Presumably, those that express a functional pre-BCR at the cell surface exit the cell cycle and differentiate into small pre-B cells.

Several groups have shown that transgenic complementation of RAG-deficient mice with a µ heavy chain permits the progression of developmentally arrested pro-B cells to the small pre-B cell stage (64, 65, 77). Transition to the small pre-B cell stage is associated with decreased expression of 5 transcripts (64, 77, 78). Cells at the small pre-B cell stage also exhibit an increase in the expression of germ-line transcripts concomitant with increased recombination at the locus (61). Pre-B cells from µ/Rag-2^-/- mice were also shown to have substantially decreased proliferative responses to IL-7 (65). These observations suggest that a functionally rearranged heavy chain promotes the differentiation of IL-7-responsive pro-B cells to an IL-7-unresponsive pre-B cell stage. The removal of IL-7 in pro-B cell cultures similarly mediates the transition to the small pre-B cell stage in which rearrangements occur. Although it is possible that stromal cells have a functional role in regulating the availability of IL-7 in vivo, we have shown that the pro-B cell to pre-B cell transition occurs independent of stromal cells in our culture system. A stromal cell-dependent stage of maturation does occur subsequent to the expression of sIgM. Thus, sµ⁺ cells still require stromal cells to generate IgM-secreting cells in response to LPS stimulation.

Studies in which IL-7R^-/- B cell progenitors from Whitlock-Witte cultures were reconstituted with mutant IL-7R-chains via retrovirus-mediated gene transfer have demonstrated that the proliferative signals generated by IL-7 signaling are distinct from those mediating differentiation (41). Mutant IL-7R receptors that abrogated the ability of the IL-7R to induce the proliferation of B cell progenitors were able to promote the differentiation of these cells to the cµ⁺ stage. In contrast, other chimeric IL-7R-chains proficient in stimulating proliferation could not mediate this transition. These studies show that the differentiative function of IL-7 signaling is independent of proliferation but does not rule out the possibility that the proliferative response to IL-7 inhibits further differentiation. It is likely that IL-7-responsive pro-B cells are not at a stage responsive to stromal cell-mediated maturation and only become so following the absence of IL-7 signaling. Alternatively, it is possible that the signals generated through the IL-7 receptor are dominant to stromal cell-mediated signals and thereby prevent the cell from progressing to the next stage of differentiation.

Contrary to previous reports (42, 57, 58), we do not observe an increase in the percentage of ⁺ cells upon the removal of IL-7. Clearly, there is an increase in the proportion of pre-B cells expressing CD2, cµ-, and sµ-chains, as well as an increase in the number of rearrangements when IL-7 is removed from culture. However, there is little difference in terms of the absolute number of sµ⁺ or ⁺ mature B cells compared with cultures containing IL-7, even after 72 h. Cultures stimulated with IL-7 contain a three- to fivefold greater number of viable pro-B cells and consequently contain relatively fewer sµ⁺ cells. Interestingly, the proportion of rearrangements increased in the absence of IL-7, although the proportion of cells expressing at the cell surface did not. It is possible that -rearranged pre-B cells require additional maturation signals that enable the surface expression of . This possibility is in agreement with a previous report that showed a high proportion of stromal cell-dependent pre-B cell colonies with rearranged light chain genes but undetectable mature transcripts (60). Contact between the S17 cells and the B cell progenitors was prevented by culture in semisolid medium. The lack of cell contact may have eliminated the signals required to promote the expression of mature transcripts and subsequent expression of IgM on the cell surface. Alternatively, many of the -rearranged cells we observed in Figure 4 may have been nonproductively rearranged and were therefore unable to express on the cell surface.

We consistently observe approximately 1% sµ⁺ cells continuously arising in cultures containing fetal liver-derived IL-7-responsive pro-B (Fig. 3). Although the frequency of LPS-responsive cells found in the sµ⁺ population is three times that found in the sµ^- population, the sµ⁺ B cells still require stromal cells to differentiate to an LPS-responsive stage (Fig. 2). Therefore, it is likely that the stage of maturation mediated by stromal cells is subsequent to µ being expressed on the cell surface. These observations are consistent with reports from other in vitro culture systems in which the surface deposition of IgM on immature B cells is insufficient to render these cells mitogen responsive (44).

We have previously tried to replace the stromal cell-mediated maturation signal with several known cytokines (IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-11, LIF, steel factor, M-CSF, IGF-1, and TSLP), but have been unsuccessful in overcoming the requirement for stromal cells at this stage of development (19, 40, 54). In this report, we have attempted to identify the molecules involved in this interaction by targeting several adhesion and signaling molecules known to be on the surface of pre-B/immature B cells (Fig. 7). Abs recognizing the cell adhesion molecules CD44 (66, 67) and VLA-4 (68, 69) were shown to completely inhibit the production of lymphoid and myeloid cells in long-term bone marrow culture (8, 9). We propose that the critical CD44 and/or VLA-4 interactions between B cell progenitors and stromal cells occur before the pre-B cell stage because we have failed to detect any inhibition in the generation of IgM-secreting cells in response to LPS in our culture systems (Fig. 7). This is consistent with the observation that the inhibition of lymphopoiesis only occurred when Abs were present during the first week of culture. At this stage, cultures primarily consist of early progenitors (12). Moreover, we could not induce the differentiation of pre-B cells in the presence of hyaluronate (data not shown), which is one of the stromal cell ligands that associate with CD44 (67, 79, 80). Recently, several chimeric mice containing progenitors lacking either the ß1 or 4 integrins of VLA-4 have revealed that B lymphopoiesis from fetal liver progenitors can occur in their absence (81, 82). However, 4^-/- bone marrow progenitors fail to reconstitute the B cell compartment of irradiated recipients. It is possible that VLA-4 is required for the development of B cell progenitors in the bone marrow but not for fetal liver progenitors. However, stromal cells derived from VCAM-1 knockout mice supported the normal development of B cell progenitors in vivo and in vitro, suggesting that the VLA-4/VCAM interaction is not essential for the development of B cells (83).

Abs recognizing the BCR (µ and ) may have prevented the maturation of pre-B cells to the LPS-responsive stage in our culture system by blocking a critical interaction between stromal cells and pre-B cells. Alternatively, these Abs may have induced apoptosis in immature B cells or may have prevented maturation to the plasma cell stage. We favor these latter hypotheses because the level of IgM secretion in cultures containing anti-µ (or anti-) Abs, or in cultures containing PMA + ionomycin, was less than the background levels of IgM observed in cultures containing LPS alone. We also observe that proB_d4-IL7 cells cultured alone in U-bottom plates for 24 h and then transferred to secondary cultures containing LPS in flat-bottom plates mature to the LPS-responsive stage as measured by IgM secretion (data not shown). The same number of proB_d4-IL7 cells cultured in flat-bottom plates with LPS do not mature in the absence of stromal cells and therefore do respond to LPS and secrete IgM. The U-bottom plate enables pre-B cell interactions, and this stage of differentiation occurs within 24 h of cell contact. When the addition of anti-µ and anti- Abs was delayed for 24 h, we still failed to detect significant IgM secretion (data not shown). We propose that these reagents block maturation subsequent to the mature LPS-responsive B cell stage because proB_d4-IL7 cells cultured alone for 24 h in U-bottom plates are sufficient to mediate their maturation into LPS-responsive B cells. Interestingly, B cell maturation could not be rescued in cultures containing IL-4, anti-µ, and anti-CD40 Abs, or combinations of the Abs described in Figure 7 (data not shown).

We have observed partial inhibition of maturation leading to IgM secretion with mAbs recognizing CD19 but not with anti-murine 5, CD81, CD22, or CD40 Abs. CD19 associates with CD21, CD81, and Leu-13 to form a complex involved in regulating the activation threshold of the BCR (71, 84, 85, 86). Recently, it has been shown that the anti-CD19 mAb used in this report (ID3) can stimulate signaling in pre-B cells and synergize with complexes containing µ heavy chain (71, 87, 88). This suggests that CD19 modulates signaling through the pre-BCR. However, we did not observe a decrease in the maturation of pre-B cells cocultured with stromal cells in the presence of Abs recognizing 5 (46) or CD81 (89). This is in contrast to T cell differentiation, in which the 2F7, anti-CD81 mAb blocked the development of CD4⁺CD8⁺ TCRß thymocytes in fetal thymus organ cultures (89). Moreover, fibroblasts transfected with CD81 could support the differentiation of CD4^-CD8^- into CD4⁺CD8⁺ T cells. Our observations therefore suggest that immature B cells differentiating in vitro are sensitive to both BCR and CD19 receptor engagement similar to previous studies, whereas Abs recognizing the pre-BCR fail to have this effect on differentiation (88, 90, 91).

Although stromal cells are required to mediate the transition from an immature B cell stage to the LPS-responsive stage leading to the secretion of IgM, we have also demonstrated that pro-B cells, incubated at high cell density or in proximity (i.e., U-bottom/V-bottom plates) to each other, mature and become mitogen responsive in the absence of stromal cells. It is unlikely that these results can be explained in terms of contaminating stromal cells for several reasons. First, proB_d4-IL7 cells are approximately 99% homogeneous with respect to B220 expression, a B lineage isoform of CD45 that is not expressed on stromal cells (10). Furthermore, experiments in which decreasing numbers of S17 stromal cells were titrated into cultures of 10³ pro-B cells revealed that a minimum of 10³ irradiated S17 were required to mediate the maturation of proB_d4-IL7 cells. However, fewer than 500 pro-B cells cultured in a V-bottom plate differentiated to a mature B cell stage and secreted IgM in response to LPS. The data presented in the U-bottom experiments raise the possibility that pre-B cells can associate with each other to mediate further maturation. It is possible that pre-B cells contain analogous surface proteins to those on stromal cells, which are responsible for mediating the pre-B to mature B cell transition. Alternatively, stromal cells may provide a supportive framework for immature B cells to develop. This scaffold may permit the association of immature B cells that differentiate through, as of yet, unidentified homotypic interactions. The S17 stromal cell-mediated maturation of proB_d4-IL7 cells in our culture system was dependent on cell contact, but this interaction may have only been required to bring sufficient numbers of immature B cells together. In vivo, there is a clustering of small, B220⁺ sIgM⁺ cells within the lumen of sinusoids (2). It has been proposed that these immature B cells accumulate in closed sinusoidal segments for a period of maturation before being released into the bloodstream. It is therefore possible that the final maturation of immature B cells occurs within these sinusoid compartments through homotypic interactions. The proteins involved in mediating the late maturational stages remain to be identified. The finding that pre-B cells can functionally mature in the absence of stromal cells in vitro provides a novel way to effectively identify the molecules involved in mediating the final stages of B cell maturation in primary lymphoid organs.

	Acknowledgments

 
We thank Dr. F. Bertrand and H. Fleming for critically reading the manuscript and are grateful to the members of the laboratories of Dr. C. Paige and Dr. G. Wu for insightful discussions. We acknowledge Dr. S. Gillis (Immunex Corporation) for the recombinant human IL-7 and Dr. K. Dorshkind for the S17 stromal cell line. We also thank Silvia Xie for her technical assistance with cell sorting.

	Footnotes

 
¹ This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada with funds provided by the Canadian Cancer Society and the Terry Fox Marathon of Hope. G.E.W. is a Medical Research Council Scholar. R.J.R. and J.L.P. are supported by Medical Research Council Studentships.

² Address correspondence and reprint requests to Dr. Robert J. Ray, The Ontario Cancer Institute, Room 8-204, 610 University Avenue, Toronto, Ontario, Canada M5G 2 M9. E-mail address:

³ Abbreviations used in this paper: cµ, cytoplasmic µ; sIgM⁺, surface IgM⁺; sµ, surface µ; BCR, B cell receptor; proB_d4-IL7, B220⁺ day 15 fetal liver cells cultured in IL-7 for 4 days.

Received for publication August 4, 1997. Accepted for publication February 19, 1998.

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