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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4509-4520
RAPID COMMUNICATION
In Vitro Reconstitution of Human B-Cell Ontogeny: From
CD34+ Multipotent Progenitors to Ig-Secreting Cells
By
Anne-Catherine Fluckiger,
Eva Sanz,
Maria Garcia-Lloret,
Thomas Su,
Qian-Lin Hao,
Roberta Kato,
Shirley Quan,
Antonio de la Hera,
Gay M. Crooks,
Owen N. Witte, and
David J. Rawlings
From the Department of Pediatrics, the Jonsson Comprehensive Cancer
Center, the Department of Microbiology and Molecular Genetics, the
Howard Hughes Medical Institute, and the Molecular Biology Institute,
University of California, Los Angeles, Los Angeles, CA; the Department
of Medicine, Universidad de Acala, Acala de Henares, Madrid, Spain; the
Centro de Investigaciones Biologicas, Valazquez, Madrid, Spain; and the
Division of Research Immunology and Bone Marrow Transplantation,
Childrens Hospital Los Angeles, Los Angeles, CA.
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ABSTRACT |
We describe a long-term, in vitro culture system initiated with
CD34+ or CD34+CD38 umbilical
cord blood hematopoietic progenitors that supports normal human
B-lineage development, including the production of mature Ig-secreting
B cells. In the first stage (human B-progenitor long-term culture
[HB-LTC]), CD34+ hematopoietic progenitors are cultured
on the murine stromal cell line, S17, leading to the sustained
production of large numbers of CD10+, CD19+
early B progenitors. Reverse transcriptase-polymerase chain reaction (RT-PCR) and three-parameter flow cytometry for VpreB
(surrogate light chain), cytoplasmic µ chain, and surface IgM
expression were used to characterize the CD19+ B
progenitors present within these cultures. This analysis showed distinct B-lineage subpopulations, including pro-B cells, cycling pre-B
cells, and IgM+, IgD/+ immature B cells.
The limited expansion of IgM+ B cells and the immature
surface phenotype of this population (IgM+,
IgD+, CD10+, CD38+) suggested
that HB-LTC conditions were unable to provide appropriate signals for
further differentiation. A second culture stage was used to determine
if these immature B cells were functionally competent. Purified
CD19+ cells were transferred onto fibroblasts expressing
human CD40-ligand in the presence of IL-10 and IL-4. This lead to cell
proliferation, modulation of the IgM+ cell surface
phenotype to one consistent with an activated mature B cell, secretion
of Ig, and isotype switching. Notably, IgM and IgG producing B cells
were also generated using two-stage cultures established with highly
purified multipotent CD34+CD38
hematopoietic stem cell progenitors. This culture model should permit
detailed in vitro analysis and genetic manipulation of the major
transition points in human B ontogeny, beginning with commitment to the
B lineage and leading to development and activation of mature B cells.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
B LYMPHOPOIESIS is a developmentally
regulated process characterized by expression of specific regulatory
genes, cell surface molecules, and somatic gene rearrangements leading
ultimately to production of Ig-secreting plasma B cells. These events
are controlled through cell-cell interactions, by hematopoietic growth factors, and by signaling through the pre-B- and B-cell antigen receptors. These receptors regulate two key steps during B-lineage development: (1) the pre-B to immature B cell and (2) the naive to
activated B-cell transitions.1-5 Pre-B- and B-cell antigen receptor-dependent signals are essential for Ig chain allelic exclusion
and proliferation of bone marrow pre-B cells and for activation and
proliferation of naive B cells after encounter with antigen in the
peripheral lymphoid tissues. The timing, dosage, and cellular
microenvironmental context of antigen receptor engagement further
modulates these events. Dysregulation of these signals can lead,
alternatively, to humoral immunodeficiencies or to autoimmunity or
malignancy.6-8
The complexity of mammalian bone marrow and lymphoid tissues has
impeded detailed in vivo molecular and phenotypic analysis of events
regulating both primary (bone marrow-dependent) and secondary
(peripheral lymphoid tissue-dependent) B-lineage development. Analysis
of normal and altered human B lymphopoiesis must rely primarily on in
vitro or SCID/Hu in vivo animal models. Whereas progress has been made
in defining human B-lineage developmental populations9-14
and the consequences of inherited B-lineage genetic defects,4,15-18 the evolution of human B-lineage culture
models analogous to those established using murine
cells19-23 has been technically challenging.24
Recently, important progress has been made in sustaining the growth of
immature human B progenitors using primary human bone marrow stroma or
using human or murine stromal cell lines.25-30 As part of
these studies we have developed a stromal cell-dependent culture model
that permits the generation and long-term growth of
CD10+CD19+ human B progenitors derived from
either CD34+CD38+ or more primitive
CD34+CD38 multipotent hematopoietic stem
cell progenitors.31-33
An ideal B-lineage culture model should support B-lymphoid commitment
from multipotent input stem cells and both primary and secondary
B-lymphoid development. A major limitation of the human long-term
B-progenitor culture system we have described and of related murine
B-lineage culture systems has been their limited capacity for expansion
of surface Ig+ (sIg+) B cells and the
generation of mature B-cell populations. Some of the key signals
controlling mature B-lineage development have been modeled in vitro
using fibroblast lines expressing the CD40 ligand (CD40L; reviewed in
Banchereau et al49).34,35 CD40 activation in
association with specific growth factors has been used to promote
mature B-cell survival, proliferation, and differentiation. These
observations suggested that a B-lineage culture system that combined
sequentially the use of a bone marrow stromal cell line and CD40
activation signals might overcome some of the limitations of current in
vitro culture models.
We describe here the establishment of a human B-lineage culture system
that supports the major events in both primary and secondary B-lineage
development. This system uses two in vitro culture stages:
generation of pro-B-cell to immature B-cell populations from
multipotent hematopoietic progenitors using stromal cell support; and
polyclonal expansion and maturation of these immature IgM+IgD+/ B cells into
Ig-producing B cells after CD40 cross-linking. This system
provides a powerful model for use in a range of future studies of both
normal and altered human B lymphopoiesis, including evaluation of the
capacity for B-lineage development after gene transfer into normal or
immunodeficient hematopoietic stem cells.
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MATERIALS AND METHODS |
Cell sources, cytokines, antibodies, and flow cytometry.
Umbilical cord blood, obtained according to the guidelines approved by
the UCLA or Childrens Hospital Los Angeles Committees on Clinical
Investigation (IRB), was placed in sterile tubes containing heparin (10 U/mL), stored at room temperature, and processed within 24 hours. The
murine stromal cell line S17 (kindly provided by Dr K. Dorshkind,
University of California, Los Angeles, CA)36 and murine
fibroblasts stably transfected with a human CD40 ligand (CD40L) cDNA
(kindly provided by Dr J. Banchereau, Schering-Plough, Dardilly,
France)37,38 were maintained as previously described. Human
recombinant cytokines were used at the following concentrations: interleukin-10 (IL-10; 100 ng/mL) and IL-4 (100 ng/mL) (generous gift
from Dr S. Narula, Schering-Plough, Kenilworth, NJ), Flt3 ligand (FL;
50 U/mL), and IL-2 (100 ng/mL; a gift from Amgen Inc, Thousand Oaks,
CA).
Antibodies, including fluorescein isothiocyanate (FITC)- or
phycoerythrin (PE)-conjugated anti-CD19 (Leu 12),
PE-conjugated anti-CD20 (Leu 19), FITC-conjugated anti-CD10 (Calla),
tricolor-conjugated anti-CD38 (OKT10), Simultest anti- / ,
FITC-conjugated anti-major histocompatibility complex
(MHC) class II DR (Becton Dickinson Immunocytometry
Systems, San Jose, CA), FITC-or PE-conjugated anti-IgM
F(ab) 2, PE-conjugated anti-IgD, and FITC-conjugated antimouse IgM (Southern Biotechnology, Birmingham, AL)
were used for phenotypic studies as recommended by the manufacturers.
The biotinylated anti-VpreB monoclonal antibody (MoAb; MAD688) was used
as previously described.39 Two-color flow cytometry
analysis of cultured cells was performed as previously
described,31,32 and all samples were simultaneously
analyzed using isotype control antibodies. For multicolor staining
using indirect staining with the anti-VpreB MoAb, cells were
successively incubated with biotinylated anti-VpreB and spectral-Red
conjugated streptavidin before incubation with other antibodies or cell
permeabilization. For intracellular analysis of µ heavy chains, cells
were permeabilized and fixed by 30 minutes of incubation at room
temperature in Permeafix solution as recommended by the manufacturer
(Ortho Diagnostics Systems, Raritan, NJ). Gating on MAD688-positive
cell populations was determined using analysis with control Ig (or
spectral-Red-conjugated streptavidin) versus anti-/ or anti-IgM.
Control stains were evaluated using for both cell surface analysis and
in analysis of permeabilized cells using VpreB-positive and -negative
control cell lines and CD19+ progenitors. Cell cycle
analysis was determined by measurement of the incorporation of the DNA
fluorochrome propidium iodide (32 µmol/L) as described.40
Enrichment of CD34+ cord blood cells and
establishment of human B-progenitor long-term cultures (HB-LTC).
Low-density mononuclear cells (MNC) from umbilical cord blood were
separated by centrifugation on Ficoll-Hypaque (d = 1.077; Amersham
Pharmacia, Piscataway, NJ) and CD34+ progenitor cells were
isolated using the MACS CD34 Isolation Kit (Miltenyi Biotechnology,
Auburn, CA) as recommended by the manufacturer. Collected adsorbed
cells were typically greater than 95% CD34+ by flow
cytometry. Highly purified CD34+CD38
cord blood cells were defined strictly as those with high CD34 expression and anti-CD38 PE fluorescence less than one half of the
maximum PE fluorescence of the isotype control and were isolated by
cell sorting as previously described.32 S17 stromal cells, resuspended in RPMI1640 (Irvine Sciences, Tustin, CA) supplemented with
2 mmol/L glutamine, 50 mmol/L 2-mercaptoethanol, and 3% defined fetal
calf serum (Hyclone Laboratories, Logan, UT) were plated in tissue culture plates (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) to obtain 50% confluent monolayers within 24 to
48 hours. Cord blood stem cell progenitors were plated on the 50% to
60% confluent S17 monolayers as previously described.31,32 Briefly, CD34+ enriched cells were plated at a density of
0.5 to 2 × 106 cells per 10-cm dish and sorted
CD34+CD38 cells were plated at a density
of 0.5 to 5 × 103 cells/well. Cultures were fed
biweekly, once with the addition of fresh medium and once
by aspiration of 80% of the medium in the culture well and addition of
fresh medium. Some cultures were supplemented with recombinant soluble
FL at a final concentration of 50 U/mL as described
previously.32
Separation of the CD19+ cells from long-term S17
culture and culture on CD40L fibroblasts.
Long-term cultured cells were harvested with or without the adherent
cell monolayer and passed through a 40-µm nylon cell strainer
(Falcon). Dead cells and debris (mostly from the S17 monolayer) were
removed by centrifugation of the single-cell suspension over a
Ficoll-Hypaque cushion and CD19+ B cells were isolated by
using the MiniMacs cell separation system as recommended by the
manufacturer (Miltenyi Biotech). Briefly, cells were incubated with a
anti-CD19 antibody coupled to super-paramagnetic beads before being
passed through a positive selection column (MACS column, or MACS MS
when stromal cells were harvested in association with hematopoietic
cells). Collected adsorbed cells were used for staining and/or
for secondary cultures. Secondary cultures were prepared using either a
newly plated 60% confluent S17 monolayer or CD40L fibroblasts as
previously described.37,38,41 CD40L fibroblasts were
irradiated with 8,000 rad before plating in 96- or 48-well plates at 5 × 103 or 2 × 104 cells/well,
respectively. CD19+ cells were added to CD40L fibroblasts
(0.05 to 5 × 105 cells/well) with cytokines as
indicated in Results. CD19+ cells were transferred to
freshly irradiated CD40L fibroblasts every 5 to 7 days for the duration
of the experiments described.
Ig secretion assay.
Purified CD19+ cells were seeded on CD40L L cells at 5 × 105 cells/mL (or at other cell densities as noted)
in 96-well plates in a final volume of 100 µL with the indicated
cytokines. At days 8 and 15, cells were transferred to freshly
irradiated CD40L fibroblasts with fresh cytokines (final volume, 200 µL) and cultured for an additional 7-day period. Culture supernatant
(180 µL) was collected at day 7, 14, or 21; spun to remove cell
debris; and frozen for further analysis. Enzyme-linked immunoabsorbtion
assay (ELISA) for IgM production was performed in microtiter plates
coated overnight with 1/5,000 dilution of goat antihuman IgM (µ specific; Southern Biotechnology) in carbonate buffer. Plates were
incubated with samples or standards (human Ig standard; ICN
Pharmaceuticals, Aurora, OH) for 4 hours at 4°C and bound antibody
was detected using an alkaline phosphatase-conjugated rabbit antihuman
IgM (1:5,000 dilution; Jackson Laboratories, West Grove,
PA) followed by p-nitrophenyl phosphate as substrate.
Plates were scanned at 409 nm in a multiwell reader (Molecular Devices,
Sunnyvale, CA) and analyzed using Soft Max software
(Molecular Devices). For IgG determinations, goat antihuman IgG
(1:5,000 dilution; Southern Biotechnology) was used as capture
antibody, followed by detection using biotinylated goat antihuman Ig (H
+L chains; 1:5,000 dilution; Southern Biotechnology),
streptavidin-alkaline phosphatase (1:1,000 dilution; Sigma, St Louis,
MO), and substrate. Limits of detection were 2 ng/mL
(IgM) and 0.5 ng/mL (IgG). Results are presented as the average
of  3 determinations. The coefficient of variation between samples
was always less than 10%. Notably, the addition of 50 µmol/L
2-mercaptoethanol supported cell survival and was associated with an
approximately threefold to fivefold increase in detectable Ig in
cultures established with HB-LTC CD19+ progenitors.
cDNA preparation and polymerase chain reaction (PCR).
Cells were collected and RNA extracted using RNAzol (Biotecx, Houston,
TX) or, alternatively, limited numbers of fluorescence-activated cell
sorting (FACS)-sorted cells (103,
102, 101, or single cells) were resuspended in
5 µL of phosphate-buffered saline (PBS). Samples were heated for 2 minutes at 65°C and then chilled on ice before the addition of the
reverse transcriptase (RT) reaction mix (Superscript GIBCO; GIBCO,
Grand Island, NY; Ghia et al12). The RT reaction was
performed by incubation for 1 hour at 37°C, followed by
inactivation of the enzyme for 10 minutes at 95°C. RT-PCR
amplification of VpreB, µ, CD79b (B29), light chain, terminal
deoxynucleotidyl transferase (TdT), and recombination associated gene
(Rag) transcripts were performed in one or two rounds using primer
pairs specific for VpreB, µ, CD79b, , TdT, Rag 1, and G3PDH
(complete primer sequences available from D.J.R. upon request).
RT-PCR amplification of VH families was performed using
primers described previously.42 After two rounds of PCR,
products of the expected size were evaluated by ethidium bromide
staining after separation by agarose gel electrophoresis. Comparison of the reaction products using decreasing numbers of input
CD19+ cells (103, 102, and
101 cells, respectively) were used to semiquantitatively
evaluate the relative expression of the individual VH
families. The limits for the detection of VH messages by
ethidium staining was 100 to 1,000 cells from HB-LTC and 10 to 100 cells from CD40L cultures. This was consistent with the expansion of
IgM+ B cells in the CD40L system. Each PCR reaction
consisted of denaturation cycle at 94°C for 2 minutes, followed by
30 cycles each of 94°C for 30 seconds, 55°C for 40 seconds,
72°C for 60 seconds, and a final extension cycle at 72°C for 7 minutes. The reaction mix for the first round PCR included all the
VH family primers. Second-round PCR was performed using
individual primer pairs and 1 µL of the first-round product.
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RESULTS |
Multiple early B-lineage transcripts are detectable in HB-LTCs.
Long-term culture of human cord blood CD34+ input cells on
the S17 murine bone marrow stromal cell line results in the
emergence of a highly enriched population of
CD34CD19+CD10+ B-lineage
progenitors (HB-LTC).31,32 In the current study, we sought
to determine if these cultures supported B-lineage subpopulations representative of those present in vivo in normal human bone
marrow and, if so, whether these populations retained the
capacity for mature B-lineage development.
HB-LTC progenitors were first evaluated for the expression of key
developmentally regulated B-lineage-specific transcripts, including
TdT, the recombination-activating genes (Rag-1 and -2), surrogate light
chain (VpreB), µ heavy chain, and or light chains. Analysis
of the expression pattern of these transcripts and their protein
products has previously been used to order the pro-B-cell to immature
B-cell developmental sequence in normal human bone
marrow.12 CD19+ B-progenitor cells were
collected from long-term cultures at 8 weeks for RNA extraction and
RT-PCR analysis. Messages for TdT and Rag-1 and -2 (Fig 1) were clearly detected, consistent
with their expression by an early B-progenitor population. Transcripts encoding VpreB, µ heavy chain, and or light chains were also detectable by RT-PCR analysis (Fig 1). The results obtained evaluating bulk CD19+ populations were confirmed by additional
analysis of FACS-purified HB-LTC subpopulations (data not shown).
Together, these results suggested that HB-LTC might support several
distinct B-lineage developmental subpopulations, including early
TdT+ B progenitors, VpreB+ cells, and,
possibly, IgM+ immature B cells.
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| Fig 1.
B progenitors from HB-LTC express mRNA for TdT, Rag,
VpreB, µ, and light chain. RT-PCR was performed using the primer
pairs as described in Materials and Methods with (lane 1) RNA from
nonadherent cells of 8-week-old HB-LTC; (lane 2) RNA from S17 cells
grown in the same conditions without addition of the
CD34+ progenitors; (lane 3) no added RNA; (N) RNA from
the human pre-B-cell line, Nalm-6; or (R) RNA from the human
IgM+ immature B-cell line, Ramos. Lane M contains DNA
size standards. Results are representative of at least three
independent experiments using unrelated HB-LTCs.
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HB-LTC supports the expansion of human pro-B cells, pre-B cells, and
immature IgM+ B cells.
To further characterize the B-progenitor subsets in HB-LTC, nonadherent
cells from multiple 4- to 10-week-old cultures were analyzed by flow
cytometry for expression of CD19 with either the VpreB surrogate light
chain or intracytoplasmic or surface Ig µ chains (cµ and IgM;
Fig 2). The MoAb, MAD688, recognizes both
VpreB+cµ and
VpreB+cµ+ B progenitors and has been used to
characterize these populations in human cell lines and bone
marrow.12,39 VpreB expression was detected as early as 4 weeks in HB-LTC, and 5% to 15% of the CD19+ cells
expressed VpreB by 6 to 8 weeks (Fig 2A).
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| Fig 2.
Multiple B-progenitor cell subsets are represented in
HB-LTC. Nonadherent cells from HB-LTC were collected at 6 to 8 weeks
for FACS analysis. (A) Two-color analysis demonstrating the presence of
HB-LTC CD19+ progenitors expressing surrogate light chain
(VpreB), cytoplasmic µ (Cµ), or surface IgM. (B) Analysis
demonstrating independent expression versus light chains and of
VpreB versus or light chain. (C) Analysis demonstrating
coexpression of IgM and IgD within HB-LTC immature IgM+
B-cell population. (D) Example of three-color analysis of purified
HB-LTC CD19+ progenitors showing staining of
VpreB versus cµ. This analysis showed
VpreBcµIgM (R1)
progenitors; VpreB+cµIgM
(R2) and VpreB+cµ+IgM (R3)
subsets; and VpreBcµ+IgM
and VpreBcµ+IgM+ subsets
(R4; IgM data not shown). Results are representative from more than
eight independent experiments. Numbers indicate the percentage of cells
in each quadrant.
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Rearrangement of the µ heavy chain locus leads initially to
intracytoplasmic expression of µ in early B-cell precursors.
Expression of cµ was found in 8% to 18% of the CD19+
cells (Fig 2A). Successful rearrangements of the or light chain
genes in cµ+ cells leads to surface IgM expression. IgM
expression was detected in 3% to 10% of the
CD19+CD10+ progenitors (3% in Fig 2A). Double
staining demonstrated coexpression of either or light chains
with surface IgM (data not shown). The CD19+ cells
expressing either or light chain did not react with the
anti-VpreB antibody (Fig 2B). Notably, approximately 60% of the
IgM+ cells also coexpressed IgD (Fig 2C). Overall, the
frequency of VpreB+, cµ+, and
IgM+ progenitor cells varied relatively little between
cultures or within individual cultures during weeks 6 through 8. In six
independent cultures, an average of 6% (±2%) VpreB+
cells, 10% (±2.5%) cµ+ cells, and 3.3%
(±0.9%) sµ+ cells were detected.
We used three-color FACS analysis for VpreB, cµ, and surface IgM
chain expression to further delineate the CD19+
subpopulations from several HB-LTCs. Analysis of purified
CD19+ progenitors (>95% CD19+) confirmed the
presence of several distinct B-lineage subpopulations, including a
predominant population of
VpreBCµIgM
cells (74% in this example; Fig 2D and data not shown); smaller numbers of VpreB+ cells consisting of
VpreB+CµIgM and
VpreB+Cµ+IgM subsets
(18%); and a limited number of both
VpreBCµ+IgM and
VpreBCµ+IgM+ cells (8%).
Because pre-B cells comprise the major cycling population in murine and
human bone marrow,12,43 we evaluated the relative size and
cell cycle status of the VpreB+ progenitors present in
HB-LTCs. Analysis according to cell size showed that the
VpreB+ population consisted predominantly of larger cells
(Fig 3A, R2 gate), whereas the
VpreBIgM and IgM+
populations were composed of very small cells (R1 gate). In addition, analysis of DNA content indicated that the VpreB+ cells
contained the majority of CD19+ cells in cell cycle (25%
of VpreB+ were in S/G2/M v 2 % for
VpreB cell population; Fig 3B).
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| Fig 3.
The HB-LTC CD19+ VpreB+
population is composed of predominantly large, cycling cells. B-lineage
cells were separated from 8-week-old HB-LTC by positive
selection using anti-CD19 magnetic beads and stained with anti-VpreB
and anti-IgM antibodies and analyzed by flow cytometry. (A) (Left)
Forward and side scatter analysis of total cell population showing
gated R1 (smallest) or R2 (largest) cell populations. (Right)
VpreB+ and VpreB populations in gates R1
or R2, with numbers indicating the percentages of the total
CD19+ cell population in each quadrant. (B) Cell cycle analysis of
CD19+VpreB+ cells.
CD19+ were stained with the anti-VpreB antibody before
permeabilization and staining with propidium iodide for FACS analysis
of DNA content versus surface VpreB expression (R3 = VpreB and R4 = VpreB+ populations,
respectively). Numbers indicate the relative percentage of cells in
S/G2/M.
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Together, these results demonstrate that HB-LTC results in the
expansion of multiple stages of early B-cell development, including a
majority population of pro-B cells
(CD19+VpreBCµIgM);
large, cycling pre-B cells
(VpreB+CµIgM and
VpreB+Cµ+IgM cells); and a
limited number of relatively quiescent, immature B cells
(VpreBCµ+IgM+IgD+/).
The multiparameter analysis described above also suggested the presence
of transitional subpopulations within these major developmental stages.
As previously reported, CD34+ pro-B cells were not detected
in HB-LTC.31
The continuum of B-lineage differentiation in HB-LTC stops at the
stage of immature IgM+ B cells.
IgM+ cells were undetectable by FACS analysis before 5 weeks of culture on S17 stroma. The relative frequency of
IgM+ cells increased slowly beginning at 5 weeks and
reached a maximum of 3% to 10% between weeks 6 and 8. Extended
culture for up to 12 weeks resulted in no significant further increase
in the fraction of IgM+ B cells (data not shown).
Consistent with these observations, analysis of cell size and cell
cycle demonstrated that the - or -expressing cells were
predominantly within the smaller, quiescent CD19+
population (data not shown). Notably, all the HB-LTC-derived CD19+ cells, including the IgM+,
IgD+ population, coexpressed both CD10 and CD38 (data not
shown and Fig 4C). This phenotype contrasts
with that of circulating naive human peripheral B cells, which are
predominantly composed of IgM+IgD+CD10CD38
cells. Together, this relatively immature surface phenotype and the
limited expansion of IgM+ cells suggested that the S17
stromal line was unable to provide the appropriate developmental
signals required for the expansion and subsequent maturation of the
IgM+ immature B cells generated in these HB-LTCs.
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| Fig 4.
CD40 ligand stimulation promotes the expansion and
maturation of long-term cultured IgM+ B cells. Purified
CD19+ progenitors from 8-week-old HB-LTC were transferred
onto freshly plated S17 stroma or onto irradiated CD40L fibroblasts in
medium supplemented by IL-4 and IL-10 and collected after 8 days in
culture. (A) Wright-Giemsa staining of CD19+ cells
(original magnification × 1,000) demonstrating plamacytoid
appeareance of CD19+ cells in the CD40L system. (B)
(Upper panel) FACS analysis demonstrating increase in
IgM+ B cells after transfer to the CD40L system. (Lower
panel) Results of anti-IgM staining after transfer to S17 versus the
CD40L system using CD19+ cells from 7 independent
HB-LTCs. (C) FACS analysis demonstrating the increase in
IgM+, IgM+IgD+ populations,
the increase in CD23 expression, and the decrease CD10 and CD38
expression in CD19+ progenitors after transfer to the
CD40L system.
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CD40 ligand stimulation promotes the expansion and maturation of
long-term cultured IgM+ B cells.
Analysis of HB-LTC-derived CD19+ progenitors clearly
demonstrated cell surface expression of CD40 (data not shown). Both
fetal BM-derived CD34+CD19 and
CD19+IgM+/ progenitors express CD40 and
proliferate in response to CD40 activation.44-46 CD40
activation is also critical for activation and proliferation of mature
human B cells.47-49 We therefore evaluated whether a
combination of CD40L activation in association with cytokines would
promote the expansion and/or the maturation of HB-LTC B
progenitors. CD19+ cells were isolated from 8-week-old
cultures and transferred onto either irradiated mouse fibroblasts
stably expressing human CD40L37,38 or to fresh S17 stroma,
each in the presence of IL-4 and IL-10 (100 ng/mL). Strikingly, large,
loose aggregates of activated blast-like CD19+ cells in
close association with the CD40L fibroblasts were evident within 5 to 8 days after cell transfer. The majority of these CD19+ cells
exhibited eccentric nuclei, a low nuclear to cytoplasmic ratio, and
increased mitotic figures (Fig 4A). Within 14 days of culture on CD40L
fibroblasts, a predominant population of large plasmacytoid cells were
present. In contrast, cells transferred to S17 with or without
cytokines contained only small lymphoid cells, with a high nuclear to
cytoplasmic ratio without evidence of cellular activation.
FACS analysis showed significant alterations in the surface phenotype
of the CD40L-cultured B cells (Fig 4B and C). In seven independent
cultures established with cord blood progenitors, the frequency
IgM+ B cells increased to an average of greater than
fourfold within 7 to 14 days. This included an increase in the
frequency of both IgM+IgD and
IgM+IgD+ B cells. In the representative
experiment presented in Fig 4C, the total IgM+ population
increased from 4% to 12% and the IgM+IgD+
population increased from 2% to 9%, respectively.
IgM and naive IgM+ B cells upregulate
CD23 expression, and naive B cells also downmodulate CD10 and CD38
expression in response to CD40 cross-linking.45,49-51 The
majority of HB-LTC-derived CD19+ cells, including all the
IgM+ cells, acquired CD23 expression after culture with
CD40L fibroblasts. CD10 and CD38 expression also decreased
significantly in both the IgD and IgD+
populations (Fig 4C). In contrast, CD19+ cells transferred
onto S17 with IL-4 and IL-10 showed no alteration in phenotype, with
the exception of a slight increase in CD23 expression.
We also determined the proliferative capacity of HB-LTC-derived
CD19+ cells after transfer to the CD40L system.
CD19+ cells isolated from 8-week-old HB-LTCs were cultured
with CD40L or S17 stroma with or without added cytokines
(Fig 5A). Whereas culture on CD40L
fibroblasts without added cytokines maintained input CD19+
cell numbers, the addition of IL-4 increased cell recovery fivefold. IL-10 had no significant effect on total cell recovery. The addition of
IL-4 and/or IL-10 to S17 stromal culture did not appreciably affect viable cell recovery. Analysis of thymidine incorporation in
CD19+ cells under these culture conditions yielded similar
results (data not shown).
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| Fig 5.
CD40L culture enhances the proliferation of
HB-LTC-derived CD19+ progenitors. A representative
experiment in which CD19+ progenitors from an 8-week-old
HB-LTC were seeded in microwells (105 cells/well)
containing S17 stromal cells or CD40L fibroblasts in medium containing
no added cytokines, IL-4, IL-10, or both cytokines. (A) Cell recovery
was determined by trypan blue dye exclusion and expressed as the mean
number of viable cells. (B) Cell cycle analysis of CD19+
cells removed from the CD40L system. (Left) Dot plot of IgM versus DNA
content analyzed by propidium iodide staining. (Right) Histogram
illustrating DNA content in the IgM+ (R1) versus the
IgM (R2) populations. Numbers indicate the percentage of
cells in S/G2/M.
|
|
Consistent with these observations, the combination of CD40 activation
and IL-4 supported entry of the B-lineage cells into cell cycle. By day
8, 15% to 35% of cells under these conditions were in S/G2/M phase
(~25% in the experiment shown in Fig 5B). Double staining for
surface IgM and DNA content indicated that both the
IgM and IgM+ CD19+
progenitors were in cell cycle at this time point (with 21% and 23%
of cells in cycle, respectively). By transferring the cells weekly onto
newly irradiated fibroblasts with IL-4 and IL-10, CD19+
cell expansion could be maintained for more than 4 weeks.
HB-LTC-derived IgM+ B cells secrete Ig and undergo
isotype switch in response to CD40 cross-linking and cytokines.
Although our data indicated that both the IgM and
IgM+ HB-LTC populations proliferated and modulated their
surface phenotype in response to CD40 activation, it remained unclear
if these populations had the capacity for more mature B-lineage
functions, including Ig secretion. We therefore evaluated if culture in
the CD40L system lead to Ig secretion.
Table 1 shows a
representative experiment in which CD19+ cells isolated
from a 6-week-old HB-LTC were transferred to CD40L fibroblasts with
cytokines including IL-2, IL-4, and/or IL-10. Strikingly, IgM
production was detected by ELISA within 7 to 14 days in supernatants of
cultures supplemented with both IL-4 and IL-10. Secretion of IgM was
detected using CD19+ progenitors derived from 6 of 6 independent HB-LTCs cultures after transfer onto CD40L-expressing
fibroblasts supplemented with both IL-4 and IL-10 (data not shown). IgM
production peaked after 21 days and subsequently decreased (Table 1 and
data not shown). Using limiting dilution, IgM production (2 to 5 ng/mL) was readily detectable with as few as 103 of
CD19+ input cells. More limited IgM secretion was also
detected in cultures supplemented with only IL-4, with only IL-10, or
with both IL-2 and IL-10.
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|
Table 1.
CD40L Culture Supports Differentiation of the
HB-LTC-Derived CD19+ Cells Into IgM-Producing B
Cells and Leads to Ig Isotype Switching
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Engagement of CD40 on naive B cells activates signals required for Ig
isotype switching and this response is modulated by specific
cytokines.49 We evaluated whether culture of HB-LTC CD19+ cells in the CD40L system could support the events
leading to IgG secretion by this cell population (Table 1). Soluble IgG was clearly detected in supernatants from cells cultured in IL-4 and
IL-10 after 14 days. IgG secretion was significantly less than the
production of IgM (9.7 v 111 ng/mL). IgG production increased eightfold by day 21 and the relative amount of IgG versus IgM also
increased (78 v 176 ng/mL). Limited IgG production (~10-fold less than in cultures containing both cytokines) was also detected using either IL-4 or IL-10 alone.
The concentration of IgM produced in the CD40L system correlated with
the relative percentage of IgM+ cells in the input cell
population (data not shown). To determine the CD19+
population responsible for the majority of Ig production, identical numbers of FACS sorted CD19+IgD and more
mature CD19+IgM+IgD+ cells from a
7-week-old culture were cultured in the CD40L system. Cell expansion
was equivalent for both populations. A small subpopulation of
IgM+ IgD+ cells (5%) was detectable within 8 days in cultures initiated with IgD cells (data not
shown). However, only cultures initiated with the IgD+
cells produced significant amounts of soluble IgM within that time
period (>300 ng/mL; data not shown). These data support the conclusion that expansion and differentiation of the IgM+
B-cell subpopulation from HB-LTCs is responsible for the majority of Ig
production after transfer to the CD40L system.
HB-LTC B progenitors exhibit polyclonal VH family gene
usage before and after transfer to the CD40L system.
The cell expansion observed in the HB-LTC or the CD40L stages of this
culture model could have resulted from either expansion of a limited
number of independent B-cell clones with a high proliferative capacity
or by expansion of multiple independent clones. To broadly distinguish
between these possibilities, we evaluated whether the emergence of
IgM+ B cells in HB-LTC and/or the subsequent
expansion of these cells after transfer to the CD40L system was
associated with the presence of oligoclonal B-cell populations.
VH family gene usage in CD19+ cells from
8-week-old HB-LTC was determined by RT-PCR analysis using primers
specific for the six major human VH families
(VH1 to VH6).42 A portion of the
CD19+ cells from each HB-LTC were also transferred in CD40L
fibroblasts with IL-4 and IL-10 and reevaluated by RT-PCR after 8 days
in culture (Table 2).
View this table:
[in this window]
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|
Table 2.
HB-LTC B Progenitors Exhibit Polyclonal VH
Family Gene Usage Before and After Transfer to the CD40L System
|
|
VH transcripts for all six families (with predominant use
of the VH1, VH3, VH4, and
VH6 families) were detected in HB-LTC B progenitors from
five independent cultures (Table 2). Transfer of these same HB-LTC
CD19+ populations onto CD40L fibroblasts with IL-4 and
IL-10 (and the associated proliferation of IgM+ B cells)
did not significantly alter the relative pattern of VH gene
family usage. Thus, rather than supporting the outgrowth of a limited
subset of oligoclonal B-lineage progenitors, these data strongly
suggest that the HB-LTC system and culture of HB-LTC-derived B
progenitors on CD40L support the expansion and subsequent activation of
polyclonal B-cell populations expressing a diverse repertoire of
VH family genes.
Generation of Ig-secreting IgM+ B cells from highly
purified CD34+CD38 hematopoietic
stem cells.
We evaluated whether HB-LTCs initiated with a highly enriched
hematopoietic stem cell population (composed of
CD34+CD38 cells) were also capable of
generating IgM+ B cells and, if so, whether these cells
could differentiate in the CD40L system. A small subpopulation of
IgM+ cells coexpressing either or light chains were
detectable by 12 weeks in cultures initiated with highly purified
CD34+CD38 cord blood cells
(Fig 6A). The generation IgM+
cells was relatively delayed in cultures initiated with
CD34+CD38 cells compared with cultures
containing CD34+CD38+ input cells (10 to 12 weeks v 5 to 6 weeks). These findings were consistent with our
previous studies demonstrating delayed expansion of this relatively
quiescent population in long-term cultures.32,52 As
previously reported for CD19+ B-progenitor
outgrowth,32,53 addition of FL throughout the culture
period increased the total production of B-lineage progenitors, including immature IgM+ B cells (data not shown).
View larger version (29K):
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| Fig 6.
HB-LTCs initiated with
CD34+CD38 stem cells lead to production of
IgM+ B cells capable of secreting of Ig after transfer to
the CD40L system. HB-LTCs were established in microwells
using FACS-sorted CD34+CD38 cord blood
cells. (A) Total cultured cell populations (adherent and nonadherent
cells) from independent wells were collected at weeks 12 and 13 and
evaluated by FACS after gating on the lymphoid-sized population. (B)
Decreasing numbers CD19+ cells from cultures initiated
with either CD34+CD38+ (0.125 to 2 × 104 CD19+ cells/well) or
CD34+CD38 input cells (5 × 103 CD19+ CD19+ cells/well; 4 independent wells shown) were transferred to CD40L system with IL-4 and
IL-10. Production of IgM was evaluated by ELISA after 14 days. Results
shown are representative of three independent experiments.
|
|
CD19+ B progenitors from HB-LTC initiated with
CD34+CD38 were isolated by FACS sorting
and transferred onto CD40L fibroblasts with IL-4 and IL-10 (5 × 103 cells/well). After 15 days, culture supernatants were
evaluated by ELISA for the presence of Ig. Replicate wells containing
CD19+ cells from three independent
CD34+CD38 cord blood cultures contained
3 to 6 ng/mL of IgM (Fig 6B). This level of IgM production was
comparable to the levels produced using identical numbers of
CD19+ cells isolated from cultures initiated with
CD34+CD38+ progenitors. Notably, IgG production
was also clearly detectable in cultures initiated with
CD34+CD38 cells (20 to 25 ng/mL; Table
1). Interestingly, in this limited set of experiments, the relative
production of IgG versus IgM was significantly higher than in cultures
initiated with CD34+CD38+ input cells.
Together, these observations strongly support the conclusion that this
culture model supports both the events regulating commitment of
hematopoietic stem cells to B-lymphoid development and the sequential
B-lineage developmental events culminating in production of mature
Ig-secreting B cells.
| |
DISCUSSION |
The culture system described in this work meets several key criteria of
an optimal in vitro human B-lineage developmental model
(Fig 7). First, this model supports the
growth of B-lineage progenitor populations with cell surface phenotypes
consistent with most of the major B-lineage developmental
subpopulations present in normal human bone marrow, including pro-B
cells, cycling pre-B cells, and IgM+
IgD+/ immature and naive B cells. Notably, because
the signals generated by the human pre-BCR remain very poorly defined,
the capacity for this culture model to support a cycling pre-B-cell
population may facilitate future studies regarding this receptor
system. Most notably, activation of these long-term cultured
CD19+ progenitors by CD40 cross-linking lead to mature
B-lineage developmental events, including Ig production and isotype
switching. Determination of the expression pattern of developmentally
regulated B-lineage gene products (including VDJ gene rearrangement
status, Rag and TdT expression, and mature B-lineage marker expression)
within individual FACS-sorted cells is essential to fully characterize these populations. However, the demonstration of mature B-lineage function strongly supports the conclusion that this model represents a
physiologically relevant continuum of human B lymphopoiesis.
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| Fig 7.
In vitro culture model of human B lymphopoiesis.
B-lineage developmental subpopulations supported by the two-stage
culture system: HB-LTC followed transfer to CD40L fibroblasts. (Bottom)
Bars approximate the relative cell expansion of the subpopulations
supported in each culture stage.
|
|
Second, this model supports both the commitment of multipotent stem
cells to B-lineage development and the progression of these committed
progenitors into functionally mature B cells. This conclusion is
supported by analysis of HB-LTCs initiated with highly purified
CD34+CD38 cells, a rare, quiescent,
primitive progenitor cell population capable of myeloid, B- and
T-lymphoid, erythroid, and megakaryocitic differentiation.30,32,33,54-56 The mature B cells generated in these cultures are very unlikely to be derived from contaminating IgM+ input cells for several reasons, including the lack of
CD19 expression in the purified
CD34+CD38 input
population32; the demonstrated capacity of single
CD34+CD38 cells to generate both myeloid
and B-lymphoid progenitors in S17 stroma-supported
culture33; the delayed kinetic of
CD19+IgM+/ cell outgrowth; and the
inability of S17 stromal cultures to support long-term expansion of
isolated cord blood IgM+ B cells (Fluckiger and Rawlings;
unpublished data).
Third, a key objective of in vitro culture models is that they support
efficient expansion of polyclonal cell populations representative of
those present in vivo. Analysis of the repertoire of Ig VH
gene expression demonstrated that this model supports expansion and
activation of multiple independent B-lineage clones. In addition,
B-lineage subpopulations are reproducibly generated in sufficient
numbers for relatively extensive phenotypic, molecular, biochemical,
and/or functional analyses. For example, a typical 6-week-old
culture established using CD34+ progenitors yielded
approximately 1 × 108 CD19+
progenitors (including 0.5 to 1 × 106
CD19+IgM+ B cells) and sustained this
production level for several weeks.
Fourth, several groups have used SCID/Hu and related in vivo models to
maintain either immature human B progenitors derived from multipotent
hematopoietic progenitors or mature IgM+ B
cells.57-60 However, the continuum of human B lymphopoiesis present in the culture model described in this work has not yet been
possible with SCID/Hu systems. This in vitro model uses two well-characterized cell lines, recombinant cytokines, and has been
established with a relatively broad group of serum sources. It should
therefore be reproducible in many laboratories. These features are
likely to permit studies that are not readily achievable using a more
complex SCID/Hu system.
The most significant development described in this work is the
combination of HB-LTC with signals leading to mature B-lineage development. This study and previous studies have suggested that bone
marrow stroma-supported cultures do not provide the signals required
for maturation of IgM+ immature B
cells.29,61,62 We demonstrate that a combination of CD40
activation in association with IL-4 and IL-10 is sufficient to promote
the expansion and maturation of CD19+ progenitors derived
from multipotent postfetal hematopoietic progenitors. These signals
lead to modulation of cell surface receptor expression, polyclonal
expansion of IgM+ B cells (including generation of
IgM+IgD+ cells from
IgM+IgD cells), Ig production, and Ig
isotype switching. The HB-LTC-derived IgM+IgD+/ B cells were responsible for
the vast majority of Ig production under these culture conditions.
Transfer to CD40L fibroblasts also led to expansion of the
CD19+IgM population and supported
limited differentiation of this population into
IgM+IgD+ cells. Our observations are consistent
with previous work demonstrating that short-term culture with an
activated CD4+ T-cell line and IL-4 led to generation of
Ig-producing B cells from human fetal bone marrow-derived pre-B
cells.63 The activated T-cell line could be replaced using
a membrane preparation from the same cells. Our data suggest that the
key costimulatory signal in those studies was also most likely provided
by CD40L.
Culture of HB-LTC CD19+ cells in the CD40L system leads to
generation of a population of
CD19+CD10dim/CD38dim/IgM+IgD+/
B cells phenotypically similar to naive tonsillar B cells. The response
of these cells in the CD40L system also closely paralleled the response
of naive B cells under similar conditions.49 Both HB-LTC
CD19+ cells and naive B cells proliferated in response to
IL-4 and secreted significant amounts of IgM and IgG with the addition of IL-10.41,47,48 The addition of IL-10 also resulted in
generation of activated B cells with plamacytoid cell morphology from
HB-LTC-derived CD19+ cells, as previously reported using
naive B cells.64 Finally, adjusting for relative numbers of
IgM+IgD+/ B cells,
HB-LTC-derived B cells secreted similar amounts of total IgM
as naive B cells in response to IL-10 and IL-4 in the presence of CD40
cross-linking (~0.05 to 0.1 ng secreted IgM per IgM+
input cell). Together, these observations indicate that the
HB-LTC-derived immature B-cell population functions analogously to in
vivo postnatally derived naive B cells, further supporting the
conclusion that this model reproduces key events in mature B-cell
development.
In summary, this culture system provides a unique model for the study
and genetic manipulation of the major transition points in human B
ontogeny. Because this model uses postfetal tissues (including both
cord blood and bone marrow), it should also facilitate future analyses
using hematopoietic tissues derived from individuals with disorders
leading to altered B lymphopoiesis as well as normal individuals. Use
of this model should allow experiments not previously possible with
other human cell culture systems, including, eg, the in vitro analysis
of the B-lineage developmental consequences of oncogenic protein
expression and the mechanisms regulating the B-cell antigen receptor
repertoire. Finally, optimization of this model should ultimately
permit the generation of Ig-producing B cells from single multipotent
hematopoietic progenitors.
| |
ACKNOWLEDGMENT |
The authors thank J. Shimaoka and J. White for assistance with
manuscript preparation; B. Ank, the obstetrical nursing staffs of Santa
Monica-UCLA, St. John's, and Sunset Kaiser Permanente Hospitals, and
J. Fraser and members of the UCLA Cord Blood Bank for assistance in
obtaining cord blood; and K. Dorshkind and E. Montecino for helpful
discussion and critical reading of the manuscript.
| |
FOOTNOTES |
Submitted July 7, 1998;
accepted September 29, 1998.
Supported in part by NIH Grants No. CA12800, AR01912, HL54850, and NCI
2CA-14089 and the facilities of the UCLA Jonsson Comprehensive Cancer
Center. A.-C.F. was supported by a Human Frontier Science Program
fellowship. E.S. was recipient of short-term fellowship from the
Comunidad Autonoma de Madrid and was supported in part by Grants No.
SAF96-2010CO2-01 and CAM98-96. G.M.C. is the recipient of a
Translational Research grant from the Leukemia Society of America.
O.N.W. is an Investigator of the Howard Hughes Medical Institute. D.J.R
is recipient of a National Institutes of Health (NIH) Physician
Scientist Award, a McDonnell Scholar Award, and UCLA Child Health
Research, HHMI, and Pennington Scholar Awards.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to David J. Rawlings, MD, Division of
Pediatric Immunology/Rheumatology, UCLA School of Medicine, 22-387 MDCC, 10833 Le Conte Ave, Los Angeles, CA 90095-1752; e-mail:
Drawling{at}pediatrics.medsch.ucla.edu.
| |
REFERENCES |
1.
Rolink A, Melchers F:
Molecular and cellular origins of B lymphocyte diversity.
Cell
66:1081, 1991[Medline]
[Order article via Infotrieve]
2.
Melchers F, Rolink A, Grawunder U, Winkler TH, Karasuyama H, Ghia P, Andersson:
Positive and negative selection events during B lymphopoiesis.
Curr Opin Immunol
7:214, 1995[Medline]
[Order article via Infotrieve]
3.
Burrows PD, Cooper MD:
B cell development and differentiation.
Curr Opin Immunol
9:239, 1997[Medline]
[Order article via Infotrieve]
4.
Fischer A, Malissen B:
Natural and engineered disorders of lymphocyte development.
Science
280:237, 1998[Abstract/Free Full Text]
5.
Neuberger MS:
Antigen receptor signaling gives lymphocytes a long life.
Cell
90:971, 1997[Medline]
[Order article via Infotrieve]
6.
Goodnow CC, Cyster JG, Hartley KSB, Bell SE, Cooke MP, Healy JI, Akkaraju S, Rathmell JC, Pogue SL, Shokat KP:
Self-tolerance checkpoints in B lymphocyte development.
Adv Immunol
59:279, 1995[Medline]
[Order article via Infotrieve]
7.
Goodnow CC:
Balancing immunity and tolerance: Deleting and tuning lymphocyte repertoires.
Proc Natl Acad Sci USA
93:2264, 1996[Abstract/Free Full Text]
8.
Van Parijs L, Abbas AK:
Homeostasis and self-tolerance in the immune system: Turning lymphocytes off.
Science
280:243, 1998[Abstract/Free Full Text]
9.
Loken MR, Shah VO, Dattilio KL, Civin CI:
Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development.
Blood
70:1316, 1987[Abstract/Free Full Text]
10.
LeBien TW, Wormann B, Villablanca JG, Law CL, Steinberg LM, Shah VO, Loken MR:
Multiparameter flow cytometric analysis of human fetal bone marrow B cells.
Leukemia
4:354, 1990[Medline]
[Order article via Infotrieve]
11.
Nunez C, Nishimoto N, Gartland GL, Billips LG, Burrows PD, Kubagawa H, Cooper MD:
B cells are generated throughout life in humans.
J Immunol
156:866, 1996[Abstract]
12.
Ghia P, ten Boekel E, Sanz E, de la Hera A, Rolink A, Melchers F:
Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin HG and L chain gene loci.
J Exp Med
184:2217, 1996[Abstract/Free Full Text]
13.
Paramithiotis E, Cooper MD:
Memory B lymphocytes migrate to bone marrow in humans.
Proc Natl Acad Sci USA
94:208, 1997[Abstract/Free Full Text]
14.
Uckun FM:
Regulation of human B-cell ontogeny.
Blood
76:1908, 1990[Free Full Text]
15.
Conley ME:
X-linked immunodeficiencies.
Curr Opin Genet Dev
4:401, 1994[Medline]
[Order article via Infotrieve]
16.
Rawlings DJ, Witte ON:
Bruton's tyrosine kinase is a key regulator in B cell development.
Immunol Rev
138:105, 1994[Medline]
[Order article via Infotrieve]
17.
Ochs HD, Hollenbaugh D, Aruffo A:
The role of CD40L (gp39)/CD40 in T/B cell interaction and primary immunodeficiency.
Semin Immunol
6:337, 1994[Medline]
[Order article via Infotrieve]
18.
Rosen FS, Cooper MD, Wedgwood RJP:
The primary immunodeficiencies.
N Engl J Med
333:431, 1995[Free Full Text]
19.
Kincade PW:
Experimental models for understanding B lymphocyte formation.
Adv Immunol
41:181, 1987[Medline]
[Order article via Infotrieve]
20.
Kincade PW:
B lymphopoiesis: Global factors, local control.
Proc Natl Acad Sci USA
91:2888, 1994[Free Full Text]
21.
Denis KA, Witte ON:
Long term culture systems for analysis of early B lymphocyte development.
Int Rev Immunol
2:285, 1987[Medline]
[Order article via Infotrieve]
22.
Dorshkind K:
Regulation of hemopoiesis by bone marrow stromal cells and their products.
Annu Rev Immunol
8:111, 1990[Medline]
[Order article via Infotrieve]
23.
Rolink A, Ghia P, Grawunder U, Haasner D, Karasuyama H, Kalberer C, Winkler T, Melchers F:
In-vitro analyses of mechanisms of B-cell development.
Semin Immunol
7:155, 1995[Medline]
[Order article via Infotrieve]
24.
LeBien TW:
Growing human B-cell precursors in vitro: The continuing challenge.
Immunol Today
10:296, 1989[Medline]
[Order article via Infotrieve]
25.
McGinnes K, Letarte M, Paige CJ:
B-lineage colonies from normal, human bone marrow are initiated by B cells and their progenitors.
Blood
77:961, 1991[Abstract/Free Full Text]
26.
Wolf ML, Buckley JA, Goldfarb A, Law C-L, LeBien TW:
Development of a bone marrow culture for maintenance and growth of normal human B cell precursors.
J Immunol
147:3324, 1991[Abstract]
27.
Moreau I, Duvert V, Banchereau J, Saeland S:
Culture of human fetal B-cell precursors on bone marrow stroma maintains highly proliferative CD20dim cells.
Blood
81:1170, 1993[Abstract/Free Full Text]
28.
Ryan DH, Nuccie BL, Ritterman I, Liesveld JL, Abboud CN:
Cytokine regulation of early human lymphopoiesis.
J Immunol
152:5250, 1994[Abstract]
29.
Pribyl JAR, LeBien TW:
Interleukin 7 independent development of human B cells.
Proc Natl Acad Sci USA
93:10348, 1996[Abstract/Free Full Text]
30.
Berardi AC, Meffre E, Pflumio F, Katz A, Vainchenker W, Schiff C, Coulombel L:
Individual CD34+CD38lowCD19CD10 progenitor cells from human cord blood generate B lymphocytes and granulocytes.
Blood
89:3554, 1997[Abstract/Free Full Text]
31.
Rawlings DJ, Quan SG, Kato RM, Witte ON:
Long-term culture system for selective growth of human B-cell progenitors.
Proc Natl Acad Sci USA
92:1570, 1995[Abstract/Free Full Text]
32.
Rawlings DJ, Quan S, Hao Q-L, Thiemann FT, Smogorzewska M, Witte ON, Crooks GM:
Differentiation of human CD34+CD38 cord blood stem cells into B cell progenitors in vitro.
Exp Hematol
25:66, 1997[Medline]
[Order article via Infotrieve]
33.
Hao Q-L, Smogorzewska EM, Barsky LW, Crooks GM:
In vitro identification of single CD34+CD38 cells with both lymphoid and myeloid potential.
Blood
91:4145, 1998[Abstract/Free Full Text]
34.
Liu YJ, Arpin C:
Germinal center development.
Immunol Rev
156:111, 1997[Medline]
[Order article via Infotrieve]
35.
Liu YJ, de Bouteiller O, Fugier-Vivier I:
Mechanisms of selection and differentiation in germinal centers.
Curr Opin Immunol
9:256, 1997[Medline]
[Order article via Infotrieve]
36.
Collins LS, Dorshkind K:
A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis.
J Immunol
138:1082, 1987[Abstract/Free Full Text]
37.
Garrone P, Neidhardt EM, Garcia E, Galibert L, van Kooten C, Banchereau J:
Fas ligation induces apoptosis of CD40-activated human B lymphocytes.
J Exp Med
182:1265, 1995[Abstract/Free Full Text]
38.
Galibert L, Burdin N, de Saint-Vis B, Garrone P, Van Kooten C, Banchereau J, Rousset F:
CD40 and B cell antigen receptor dual triggering of resting B lymphocytes turns on a partial germinal center phenotype.
J Exp Med
183:77, 1996[Abstract/Free Full Text]
39.
Sanz E, de la Hera A:
A novel anti-Vpre-B antibody identifies immunoglobulin-surrogate receptors on the surface of human pro-B cells.
J Exp Med
183:2693, 1996[Abstract/Free Full Text]
40.
Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM, Strober W:
Current Protocols in Immunology. New York, NY, Wiley, 1992.
41.
Banchereau J, Rousset F:
Growing human B lymphocytes in the CD40 system.
Nature
353:678, 1991[Medline]
[Order article via Infotrieve]
42.
Adderson EE, Shackelford PG, Quinn A, Carroll WL:
Restricted Ig H chain V gene usage in the human antibody response to Haemophilus influenzae type b capsular polysaccharide.
J Immunol
147:1667, 1991[Abstract]
43.
Karasuyama H, Rolink A, Melchers F:
Surrogate light chain in B cell development.
Adv Immunol
63:1, 1996[Medline]
[Order article via Infotrieve]
44.
Saeland S, Duvert VR, Pandrau D, Caux C, Durand I, Wrighton N, Wideman J, Lee F, Banchereau J:
Interleukin-7 induces the proliferation of normal human B-cell precursors.
Blood
78:2229, 1991[Abstract/Free Full Text]
45.
Saeland S, Duvert V, Moreau I, Banchereau J:
Human B cell precursors proliferate and express CD23 after CD40 ligation.
J Exp Med
178:113, 1993[Abstract/Free Full Text]
46.
Renard N, Duvert VR, Blanchard D, Banchereau J, Saeland S:
Activated CD4+ T cells induce CD40-dependent proliferation of human B cell precursors.
J Immunol
152:1693, 1994[Abstract]
47.
Banchereau J, de Paoli P, Valle A, Garcia E, Rousset F:
Long-term human B cell lines dependent on interleukin-4 and antibody to CD40.
Science
251:71, 1990
48.
Galibert L, Durand I, Banchereau J, Rousset F:
CD40-activated surface IgD-positive lymphocytes constitute the long term IL-4-dependent proliferating B cell pool.
J Immunol
152:22, 1994[Abstract]
49.
Banchereau J, Bazan F, Blanchard D, Briere F, Galizzi JP, van Kooten C, Liu YJ, Rousset F, Saeland S:
The CD40 antigen and its ligand.
Annu Rev Immunol
12:881, 1994[Medline]
[Order article via Infotrieve]
50.
Arpin CD, Déchanet J, Kooten CV, Merville P, Grouard Gr, Bri re F, Banchereau J, Liu Y-J:
Generation of memory B cells and plasma cells in vitro.
Science
268:720, 1995[Abstract/Free Full Text]
51.
Arpin C, Banchereau J, Liu YJ:
Memory B cells are biased towards terminal differentiation: A strategy that may prevent repertoire freezing.
J Exp Med
186:931, 1997[Abstract/Free Full Text]
52.
Hao QL, Thiemann FT, Petersen D, Smogorzewska EM, Crooks GM:
Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population.
Blood
88:3306, 1996[Abstract/Free Full Text]
53.
Shah AJ, Smogorzewska EM, Hannum C, Crooks GM:
Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38 cells and maintains progenitor cells in vitro.
Blood
87:3563, 1996[Abstract/Free Full Text]
54.
Terstappen LWMM, Huang S, Safford M, Lansdorp PM, Loken MR:
Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38 progenitor cells.
Blood
77:1218, 1991[Abstract/Free Full Text]
55.
Huang S, Terstappen LWMM:
Lymphoid and myeloid differentiation of single human CD34+, HLA-DR+, CD38 hematopoietic stem cells.
Blood
83:1515, 1994[Abstract/Free Full Text]
56.
Hao Q-L, Shah AJ, Thiemann FT, Smogorzewska EM, Crooks GM:
A functional comparison of CD34+CD38 cells in cord blood and bone marrow.
Blood
86:3745, 1995[Abstract/Free Full Text]
57.
McCune JM:
Development and applications of the SCID-hu mouse model.
Semin Immunol
8:187, 1996[Medline]
[Order article via Infotrieve]
58.
Tary-Lehmann M, Saxon A, Lehmann PV:
The human immune system in hu-PBL-SCID mice.
Immunol Today
16:529, 1995[Medline]
[Order article via Infotrieve]
59.
Greiner DL, Hesselton RA, Shultz LD:
SCID mouse models of human stem cell engraftment.
Stem Cells
16:166, 1998[Medline]
[Order article via Infotrieve]
60.
Dick JE, Bhatia M, Gan O, Kapp U, Wang JC:
Assay of human stem cells by repopulation of NOD/SCID mice.
Stem Cells
15:199, 1997
61.
Whitlock CA, Witte ON:
Long term culture of B lymphocytes and their precursors from murine bone marrow.
Proc Natl Acad Sci USA
79:3608, 1982[Abstract/Free Full Text]
62.
Rolink A, Kudo A, Karasuyama H, Kikuchi Y, Melchers F:
Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo.
EMBO J
10:327, 1991[Medline]
[Order article via Infotrieve]
63.
Punnonen J, Aversa G, de Vries JE:
Human pre-B cells differentiate into Ig-secreting plasma cells in the presence of interleukin-4 and activated CD4+ T cells or their membranes.
Blood
82:2781, 1993[Abstract/Free Full Text]
64.
Rousset F, Peyrol S, Garcia E, Vezzio N, Andujar M, Grimaud JA, Banchereau J:
Long-term cultured CD40-activated B lymphocytes differentiate into plasma cells in response to IL-10 but not IL-4.
Int Immunol
7:1243, 1995[Abstract/Free Full Text]
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Top
Abstract
Introduction