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Blood, Vol. 94 No. 8 (October 15), 1999:
pp. 2637-2646
Genetic Modification of Human B-Cell Development: B-Cell
Development Is Inhibited by the Dominant Negative Helix Loop Helix
Factor Id3
By
Ana C. Jaleco,
Alexander P.A. Stegmann,
Mirjam H.M. Heemskerk,
Franka Couwenberg,
Arjen Q. Bakker,
Kees Weijer, and
Hergen Spits
From the Division of Immunology, The Netherlands Cancer Institute,
Antoni van Leeuwenhoek Huis, Amsterdam, The Netherlands.
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ABSTRACT |
Transgenic and gene targeted mice have contributed greatly to our
understanding of the mechanisms underlying B-cell development. We
describe here a model system that allows us to apply molecular genetic
techniques to the analysis of human B-cell development. We constructed
a retroviral vector with a multiple cloning site connected to a gene
encoding green fluorescent protein by an internal ribosomal entry site.
Human CD34+CD38 fetal liver cells,
cultured overnight in a combination of stem cell factor and
interleukin-7 (IL-7), could be transduced with 30% efficiency. We
ligated the gene encoding the dominant negative helix loop helix (HLH)
factor Id3 that inhibits many enhancing basic HLH transcription factors
into this vector. CD34+CD38 FL cells were
transduced with Id3-IRES-GFP and cultured with the murine stromal cell
line S17. In addition, we cultured the transduced cells in a
reaggregate culture system with an SV-transformed human fibroblast cell
line (SV19). It was observed that overexpression of Id3 inhibited
development of B cells in both culture systems. B-cell development was
arrested at a stage before expression of the IL-7R . The development
of CD34+CD38 cells into
CD14+ myeloid cells in the S17 system was not inhibited
by overexpression of Id3. Moreover, Id3+ cells, although
inhibited in their B-cell development, were still able to develop into
natural killer (NK) cells when cultured in a combination of Flt-3L,
IL-7, and IL-15. These findings confirm the essential role of bHLH
factors in B-cell development and demonstrate the feasibility of
retrovirus-mediated gene transfer as a tool to genetically modify human
B-cell development.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IT IS WELL-ESTABLISHED that the
generation of lineage-committed cells from pluripotent stem cells is
tightly controlled by transcription factors (TFs). Several TFs have
been identified that contribute to the steering of B-cell
differentiation in the mouse. Some of these factors control B-cell
differentiation at a developmental stage before commitment to the
B-cell lineage. For example, the Ikaros factor is essential for
differentiation of lymphoid cells, including B cells.1 TFs
that direct B-cell development include Pax5,2
EBF,3 Sox4,4 and E12 and E47.5,6 These latter factors, encoded by the E2A gene, belong to the family of
basic helix loop helix (bHLH) proteins. This family is particularly interesting, because its members control lineage commitment of different cell types in organisms varying from yeast to
mammals.7 Two highly conserved bipartite domains
characterize these proteins. A motif of basic residues mediates binding
to a consensus E-box (CANNTG) sequence present in promoters or
enhancers of target genes.8,9 The helix-loop-helix domain,
mainly consisting of hydrophobic residues, permits homodimerization or
heterodimerization of these proteins. The involvement of the E2A gene
products in B-cell differentiation in the mouse has been
well-documented. Two groups generated E2A mutant mice and have shown
that a severe block occurs at an early stage of B-cell differentiation
before Ig gene rearrangements.5,6 More recently, Bain et
al10 presented evidence that both E12 and E47 allow
commitment to the B-cell lineage. Two other factors, HEB and E2-2, are
also involved in B-cell development, because mice deficient for these
factors demonstrate perturbations of B-cell development as
well.11 It seems, therefore, that B-cell development is
controlled by the combined dosage of many bHLH factors. Inhibitors of
DNA binding (Id) proteins control transcriptional activity of bHLH
factors. This family of HLH factors comprises 4 members (Id1-4) that
are highly homologous in their HLH domains.12,13 Id
proteins can heterodimerize with bHLH factors, but lack a basic DNA
binding domain, therefore blocking transcriptional activity of bHLH
factors. Sun14 has demonstrated that constitutive
expression of the Id1 gene under control of the B-cell-specific mb-1
promotor impairs mouse B-cell development, and the cells exhibit a
similar phenotype to the one described for the E2A-deficient
mice.5,6 These investigators also documented that Id1 and
Id2 mRNA levels are relatively high in tumor cell lines representative
for early B-cell progenitors and low in cell lines representing late
B-cell precursors.15 More recently, Li et al16
reported that Id1 levels are high in an uncommitted B-cell progenitors
in the bone marrow and sharply downregulate when the cells enter a
B-cell committed stage, whereas at the same time, E12 and E47 are
upregulated. These findings together have led to the hypothesis that
B-cell differentiation is controlled by the relative proportions of E2A
and Id proteins.
Transgenic technology has provided a powerful tool for the study of
lymphoid development in the mouse. We have developed a novel model
system that allows us to apply these molecular genetic techniques to
the analysis of human T-cell development. This system makes use of
retrovirus-mediated gene transfer to introduce genes into T-cell
progenitors. The effects of the introduced genes on T-cell development
are investigated in a fetal thymic organ culture. Using this system, we
documented that overexpression of Id3 in human thymic progenitors by
retroviral delivery resulted in a complete inhibition of T-cell
differentiation and promotion of natural killer (NK) cell
development.17 We demonstrate here that overexpression of
Id3 into uncommitted
CD34+CD38CD10 fetal
liver (FL) cells strongly inhibits their capacity to develop into B
cells upon coculture with the murine stromal cell line S17 or with a
human stromal cell line in a novel reaggregation assay. B-cell
development was inhibited at a very early stage of development before
acquisition of the interleukin-7 receptor  (IL-7R)
chain and CD10.
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MATERIALS AND METHODS |
Construction of the vector.
We have constructed bicistronic vectors with a gene of interest linked
to a downstream internal ribosomal entry site (IRES) and a marker gene
that allow independent translation of the products of both genes in the
transduced target cells.17 The IRES-GFP sequence was
ligated into the LZRS vector, and a polylinker was placed downstream of
the gag and upstream of the IRES sequence. The Id3 coding sequence was
cloned from the pCDNAId3 plasmid (gift of Dr C. Murre, University of
California at San Diego, San Diego, CA) by polymerase chain reaction
(PCR) using oligonucleotide primers with appropriate linkers. The
product was ligated between the Xho I and SnaBI site of
the polylinker from our plasmid LZRS-linker-IRES-GFP to obtain the
retroviral vector LZRS-Id3-IRES-GFP. As a control, we constructed into
the LZRS-linker-IRES-GFP vector a form of murine Id3 with a mutation in
the HLH domain (R72P; kindly provided by Dr G. Kato, Johns Hopkins
University, Baltimore, MD) that has lost the capacity to dimerize with
bHLH factors.18 The HLH domains of human and murine Id3 are
identical. A modified version of GFP (enhanced GFP) was used in this
study and was obtained from Clontech (Palo Alto, CA). A schematic
representation of the vector is shown in
Fig 1. Helper-free recombinant retrovirus
was produced after transfection into a 293T-based amphotropic
retroviral packaging cell line, Phoenix.19
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| Fig 1.
Structure of the LZRS IRES-GFP vector. The important
features of this vector are the polylinker upstream of the IRES
sequence to insert the different genes of interest and the selection
marker GFP downstream of the IRES sequence. The LZRS vector contains a
puromycin resistance gene and the EBNA-1 nuclear retention sequence
outside the LTRs.
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Isolation of FL progenitor cells.
FL tissue was obtained from elective abortions. Gestational age was
determined by measurement of the diameter of the skull and ranged from
14 to 17 weeks. The use of this tissue was approved by the Medical
Ethical Committee of the Netherlands Cancer Institute and was
contingent upon informed consent. Human FL cells were isolated by
gentle disruption of the tissue, followed by density gradient
centrifugation over Ficoll-Hypaque (Lymphoprep; Nycomed Pharma, Oslo,
Norway). Subsequently, CD34+ cells were positively selected
by immunomagnetic cell sorting (varioMACS; Miltenyi, GmbH, Germany), as
described elsewhere.20 The resulting population was stained
with anti-CD10 fluorescein isothiocyanate (FITC; Immunotech, Marseille,
France), anti-CD38 phycoerythrin (PE; Becton Dickinson, San
Jose, CA), and anti-CD34 Tricolor (Immunotech).
CD34+CD38CD10,
CD34+CD38+CD10, and
CD34+CD38+CD10+ cells were then
sorted with a FACstar plus (Becton Dickinson). The purity of the
populations used in this study was greater than 97%.
Reverse transcriptase-PCR (RT-PCR) assays.
RNA was isolated from fluorescence-activated cell sorting (FACS)-sorted
human FL subpopulations using TRIzol reagent (GIBCO BRL, Grand Island,
NY) according to the manufacturer's instructions and reverse
transcribed using a poly-dT15 oligonucleotide (Promega Corp, Leiden, The Netherlands) and 400 U of Moloney murine leukemia virus (M-MuLV) reverse transcriptase (GIBCO) at
37°C for 1 hour. Semiquantitative RT-PCR was performed essentially
as described previously.21 PCR assays were performed in 30 mL reaction volumes using the appro- priate diluted amounts of cDNA
template, 2 mmol/L MgCl2, 0.25 mmol/L each dNTP, 10 pmol/L of each primer, and 0.8 U Taq polymerase (GIBCO) in
1× buffer (10 mmol/L Tris-HCl, pH 8.5, 50 mmol/L KCl). Reaction
conditions were a 5-minute denaturing step at 95°C
followed by 31 cycles of 1 minute at 95°C, 1 minute at 55°C,
and 1 minute at 72°C. PCR products were separated on 1% agarose
gels, stained with ethidium bromide, and analyzed by video-densitometry using the Eagle Eye still video system and Eagle
Sight software (Stratagene, La Jolla, CA). Three separate FL donor
pools were analyzed and each PCR was repeated 4 to 6 times. The E2A-,
E12-, and E47-specific Id1, Id3, RAG-1, and HPRT primers that were used
are as follows: E2A-sense: 5'-AGCACGTTTGGTGGCCTGC-3'; E47-antisense: 5'-AGCACCTCGTCCGTACTGC-3'; E12-antisense:
5'-TCCTCGTCCTCGTCTGGGC-3'; Id1-sense:
5'-CGCTGTCTGTCTGAGCAGAGC-3'; Id1-antisense:
5'-ATCTCGCCGTTGAGGGTGCTG-3'; Id3-sense:
5'-TCGGAACGCAGTCTGGCCATC-3'; Id3-antisense:
5'-CTCGGCTGTCTGGATGGGAAG-3'; RAG-1 sense:
5'-GAACACACTTTGCCTTCTCTTTGG-3'; RAG-1 antisense: 5'-GCTTTGCCTCTTGCTTTCTCGTT-3'; HPRT-sense:
5'-TATGGACAGGACTGAACGTCTTGC-3'; and HPRT-antisense:
5'-GACACAAACATGATTCAAATCCCTGA-3'.
Retroviral transduction of human CD34+ FL cells.
The transduction procedure using recombinant human fibronectin
fragments CH-296 was performed according to Hanenberg et
al.22,23 The sorted
CD34+CD38CD10 FL
cells were cultured overnight in the presence of 10 ng/mL human IL-7
and 20 ng/mL stem cell factor (SCF; both from R&D, Abingdon, UK). The
cells were then transduced by 6 hours of incubation with virus
supernatant in nontissue culture-treated Falcon petri dishes (3 cm;
Becton Dickinson) precoated with 30 mg/mL recombinant human fibronectin
fragment CH-29624 (RetroNectin; Takara, Otsu, Japan). After
transduction, the cells were washed twice. The transduction efficiency
was tested by determining the percentage of GFP+ cells 2 days after transduction using flow cytometry analysis.
Monolayer cell culture.
The monolayer cell culture assays were performed with the murine bone
marrow stromal cell line S17 (a kind gift from Dr David Rawlings, UCLA,
Los Angeles, CA). Two days before their use in coculture experiments,
S17 cells were plated in 96-well flat-bottom plates (3 to 5 × 103 cells per well in 100 µL medium). Monolayer cultures
were initiated by seeding 5 to 10 × 103 FL progenitor
cells to the wells precoated with S17 cells. Half of the culture medium
was replaced by fresh medium once a week. After incubation in Yssel's
medium25 supplemented with 5% fetal calf serum (FCS;
BioWhittaker, Verviers, Belgium), to which we will refer as complete
medium, cells were harvested, stained with PE- or Tricolor-labeled
human specific antibodies, and analyzed by flow cytometry for cell
surface phenotype and GFP expression. In some experiments, part of the
recovered cells was assayed for their capacity to develop into NK cells.
Reaggregation assays.
The reaggregation assay is based on a similar culture system developed
by Anderson et al26 to test the development of mouse T
cells. The human SV19 stromal cell line27 was used in this study. Briefly, 3 to 5 × 104 FL progenitor cells were
mixed with 10 to 15 × 104 stromal cells in a 1.5-mL
Eppendorf tube and spun into a pellet. After removal of the
supernatant, the cell pellet was vortexed in a remaining volume of 2 to
5 µL, gently expelled as a discrete standing drop on the surface of a
nucleopore filter (0.8 µm; Millipore, Cork, Ireland) that was layered
on a gelfoam raft (Upjohn Co, Kalamazoo, MI), and cultured in an
air/liquid interphase with complete medium. Reaggregates were then
harvested and gently dissociated into a homogeneous cell suspension for
further cell surface analysis by flow cytometry.
Differentiation of NK cells.
After a period of 2 weeks of culture of transduced
CD34+CD38CD10 FL
cells on a monolayer of S17 stroma, the cells were harvested, counted,
and tested for their capacity to develop into NK cells, as previously
described.20 Five thousand to 10,000 FL cells were then
further cultured in U-bottom 96-well plates with complete medium in the
presence of 25 U/mL Flt-3 ligand (a kind gift from Dr M.G. Roncarolo,
DNAX, Palo Alto, CA), 10 ng/mL IL-7 (our own source), and 10 ng/mL
IL-15 (Immunex, Seattle, WA). The cultures were maintained in a
humidified atmosphere at 37°C in 5% CO2 for 10 days.
Cells were then stained and their antigenic phenotype was analyzed by
flow cytometry.
Flow cytometry analysis and monoclonal antibodies (MoAbs).
Flow cytometry analysis was performed using a FACScan (Becton
Dickinson). FL cells were incubated for 10 minutes on ice with normal
mouse serum Ig to prevent nonspecific binding of the MoAbs and then
stained with fluorochrome-conjugated MoAbs. The following mouse MoAbs
(FITC-, PE-, or Tricolor-coupled) were used: CD10-Tricolor (Caltag, San
Francisco, CA); CD34-Tricolor, CD19-Tricolor, CD45-Tricolor, anti-c-kit-PE and CD10-FITC were purchased from Immunotech; CD19-PE, CD45RA-PE, CD38-PE, CD20-PE, CD22-PE, CD56-PE, and CD14-PE were obtained from Becton Dickinson. For detection of the IL-7R chain, an
indirect staining was used with anti-IL-7R (Immunotech) and PE-labeled F(ab)2 fragments of a goat antimouse antibody
(Zymed, San Francisco, CA). After staining, cells were washed twice in phosphate-buffered saline (PBS) supplemented with 2% bovine serum albumin (BSA) and 0.01% NaN3 (PBA) and analyzed by flow
cytometry. All steps of the staining procedure were performed on ice,
and appropriate fluorochrome-conjugated, isotype-matched control Igs were used in all experiments.
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RESULTS |
Expression of RAG-1, Id, and bHLH mRNAs in distinct CD34+
FL populations.
Human pluripotent stem cells are enriched in a subpopulation of
CD34+ cells that express no or only low levels of
CD38.28 Differentiation of these cells is accompanied by
upregulation of the CD38 marker.28 Within the
CD34+ compartment, CD10 is expressed on lymphoid
progenitors,29,30 early B-cell precursors,31
and T-cell precursors.32 To investigate the role of bHLH
factors and Id proteins in human B-cell development, we analyzed the
mRNA levels of 2 Id proteins (Id1 and Id3), the E2A gene products (E12
and E47), and RAG-1 in sorted
CD34+CD38CD10,
CD34+CD38+CD10, and
C34+CD38+CD10+ human FL cells by
semiquantitative PCR (Fig 2). We found that RAG-1 mRNA is absent in the
CD34+CD38CD10
immature population, as expected. Low and high levels of RAG-1 mRNA
were observed in
CD34+CD38+CD10 and
CD34+CD38+CD10+ cells,
respectively. E12- and E47-specific messages were present at similar
levels in
CD34+CD38CD10 and
CD34+CD38+CD10 cells and
were 2- to 3-fold higher in the
CD34+CD38+CD10+ population.
Expression of Id1 was high in the
CD34+CD38CD10 subset
and was dramatically decreased with upregulation of CD38 and CD10. Id3
levels decreased 2-fold in the transition from the CD34+CD38CD10 to
CD34+CD38+CD10 stages and
had the highest expression levels in the more mature CD34+CD38+CD10+ population.
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| Fig 2.
Expression levels of HLH, bHLH, and RAG-1 mRNAs on
CD34+ FL-sorted populations. RNA was isolated from
FACS-sorted human FL subpopulations from 3 separate donor pools and
analyzed by RT-PCR. mRNA levels were compared with HPRT expression and
expressed in arbitrary units (HPRT = 1). One representative
experiment from 1 donor pool is shown. All assays were performed 4 to 6 times. Similar data were obtained with analyses of 2 other donor
pools.
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Retroviral transduction of primitive
CD34+CD38 FL hematopoietic progenitor
cells.
It has been demonstrated that CD34+ cells of different
origins can be efficiently transduced after culture in multiple
cytokines. However, CD34+CD38 cells
appear to be difficult to transduce.33 We isolated
CD34+CD38 cells from FL using
Tricolor-labeled anti-CD34 and PE-labeled anti-CD38 as described in
Materials and Methods (Fig 3A). The use of
an anti-CD38 antibody labeled with PE allows for a better depletion of
CD38+ cells than when using an FITC-labeled antibody, due
to the greater sensitivity of the PE fluorochrome (for a discussion,
see Lanier and Recktenwald34). The CD34+ cells,
rigorously depleted of CD38+ cells, were cultured overnight
in a combination of SCF and IL-7 and incubated for 6 hours with
cell-free virus-containing supernatants. Two facilitating reagents were
used, the lipofectin reagent dotap17 and recombinant
fibronectin-derived peptides (Retro- Nectin; Takara).22 Transduction efficiencies in the presence of RetroNectin fragments were
32.7% ± 4.2% (n = 8; a representative experiment is shown in Fig
3B), as determined by measuring GFP expression by flow cytometry.
Transduction efficiencies using dotap were, in general, 2 to 3 times
lower when performed in parallel with fibronectin fragments (results
not shown).
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| Fig 3.
Retroviral transduction of
CD34+CD38CD10 FL cells does
not affect their capacity to develop along the B-cell lineage. The
CD34+CD38CD10 FL population
was isolated (A), transduced with virus harboring the IRES-GFP
sequence, and cultured for 18 days on a stromal cell monolayer (B).
Cell suspensions were stained with the indicated Tricolor- and
PE-labeled antibodies and analyzed on a FACScan. Isotype-matched
control antibodies were used in all experiments.
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Retroviral transduction does not affect the B-cell differentiation
pattern of
CD34+CD38CD10 FL
progenitors.
We have observed that
CD34+CD38CD10 FL
cells developed into B cells after 2 to 3 weeks of culture on a
monolayer of S17 stromal cells, consistent with observations with
CD34+ cord blood cells.35 To determine whether
retroviral transduction per se would affect the capacity of
CD34+CD38CD10 FL
precursors to differentiate along the B-lineage pathway, we sorted this
population (Fig 3A), infected the cells with a retrovirus carrying the
IRES-GFP sequences, and cocultured the transduced cells with S17. As
shown in Fig 3B, 30% of the cells were GFP+ 2 days after
transduction. After 18 days of coculture, 12% of the recovered cells
were positive for GFP, and no significant differences could be detected
in the phenotype of the B cells generated when compared with the
GFP fraction.
Overexpression of Id3 inhibits B-cell development from
CD34+CD38CD10
uncommitted FL precursors.
To investigate whether bHLH factors are involved in human B-cell
development, we overexpressed Id3 in FL hematopoietic progenitors and
monitored the fate of the transduced cells in a B-cell differentiation assay. Purified
CD34+CD38CD10 FL
cells were cultured overnight with a combination of SCF and IL-7 and
subsequently transduced with retrovirus harboring the Id3-IRES-GFP
sequence. After 18 days of culture on a monolayer of the murine S17
stromal cell line, cell numbers were 10- to 12-fold increased and 8%
of the cells expressed the transgene (Fig
4A). The Id3 fraction developed into B cells and
exhibited a phenotype similar to the control transduced cells (Figs 4A
and 3B). Twenty-six percent of the cells were
CD10+CD19+. In striking contrast,
Id3+ cells lacked expression of CD19, demonstrating that
enforced expression of Id3 in
CD34+CD38CD10 FL
cells strongly blocked B-cell differentiation. In this experiment, some
cells were CD10+CD19 (10%). However, we
found that expression of CD10 was variable. In some experiments, we
observed a small percentage of CD10+CD19
cells in the cultures with Id3-transduced
CD34+CD38 cells, whereas in other
experiments these cells were not detectable. We consider it therefore
likely that the small proportion of CD10+ cells in Id3
cultures that we observed in some experiments is an artifact due to
nonspecific staining of the Tricolor labeled anti-CD10. Cells recovered
after culture of the FL progenitors in this culture system also contain
a population of CD14+ cells. As shown in Fig 4B, the
percentage of CD14+ cells present in both
Id3 and Id3+ fractions was identical,
indicating that the inhibitory effect of Id3 in this assay is
restricted to the B-cell lineage.
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| Fig 4.
Overexpression of Id3 inhibits differentiation of B cells
from CD34+CD38CD10 FL
uncommitted precursors but does not affect development into monocytes.
CD34+CD38CD10 FL cells were
sorted, incubated with retroviral supernatant harboring the
Id3-IRES-GFP sequence, and subsequently cocultured with S17 stroma.
After 18 days, the cells were harvested and expression of B-cell (A)
and monocyte (B) antigens was analyzed by flow cytometry. Thirty-two
percent of the cells were GFP+ 2 days after transduction.
A representative experiment of 3 performed is shown.
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It was recently reported that Id2 could induce apoptosis in a myeloid
precursor cell line independent of its capacity to dimerize with bHLH
factors.36 Therefore, we transduced
CD34+CD38 FL cells with a mutant of Id3,
 Id3, that has no capacity to dimerize with bHLH factors as
E4718 and tested the B-cell precursor activity of these
cells. In these experiments, we performed a more extensive phenotypic
analysis to characterize the Id3-arrested developmental stage in more
detail. Figure 5 shows that
Id3-transduced cells developed into CD19+ cells,
whereas, in contrast, Id3-transduced cells fail to develop into
CD19+ B cells. The Id3-transduced cells were somewhat
less efficient in developing into B cells compared with the
untransduced cells in the same sample, suggesting an Id3 effect
independent of the dimerization domain. However, the inhibition by Id3
is much stronger than that of Id3, indicating that Id3 blocks B-cell
development mainly by sequestering pertinent bHLH factors. Further
analysis of the cells that were blocked by Id3 showed that Id3 also
inhibited the appearance of IL-7R+ cells. It is also
shown that the proportion of
c-kit+CD45RA cells in the Id3 cultures
(30%) was higher than that in the control or Id3 cultures (9% to
12%). The percentages of c-kitCD45RA+
cells in the control and Id3 cultures were higher than in the Id3
culture, but there was no difference in the proportions of c-kitlowCD45RA+ in the Id3 and Id3 cultures.
In the untransduced cultures, a higher proportion of
ckitlowCD45RA+ was found. This could mean that
a proportion of ckitlowCD45RA+ cells in the
untransduced cultures is derived from precursors that are difficult to
transduce. We also analyzed expression of CD38. It is shown in Fig 5
that the control and untransduced cell populations contain a population
of CD38high cells. Part of those CD38high cells
react with an anti-CD19 MoAb. The anti-CD19 MoAb used in this staining
was Tricolor labeled and stained less efficiently than the PE-labeled
antibody used in the CD10/CD19 staining. Nonetheless, one can observe
in the Id3 cultures a strong reduction of these CD38high
cells and almost no CD38highCD19+ cells. Figure
5 also shows that Id overexpression does not affect the percentage of
CD34+ cells. Although the percentage of
CD34+CD38high cells in the Id3-transduced cells
is slightly lower than in the untransduced cells, a similar percentage
of CD34+CD38high cells was present in the
Id3-transduced cells. It seems, therefore, that Id3 inhibits B-cell
development at a
CD34+c-kit+/lowCD45RA±IL-7RCD38highCD10CD19
stage. The phenotype of these cells may correspond with that of an
early lymphoid precursor, as proposed by Ryan et al.30
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| Fig 5.
Id3 inhibits B-cell development at an early stage of
development. The inhibition by Id3 is much stronger than by a mutant of
Id3 that is unable to dimerize with bHLH factors.
CD34+CD38CD10 FL cells were
sorted, transduced either with Id3-IRES-GFP or with Did3-IRES-GFP, and
subsequently cocultured with S17 stroma. After 14 days, the cultures
were analyzed with the indicated antibodies. This experiment is
representative of 3 similar experiments.
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Id3+ FL cells recovered after 2 weeks of coculture with
S17 stroma can develop into NK cells.
As shown in the previous experiments, the Id3+ population
recovered after culture on a S17 stromal cell monolayer did not contain any CD19+ B cells, whereas generation of cells expressing
CD14 was not affected. To determine whether the Id3+
population contains precursor cells that had retained the capacity to
develop into the NK cell lineage, they were cultured in a cytokine combination containing IL-15, as previously described.20
Control cultures were established with cells recovered after culture of the IRES-GFP-transduced population with S17, as shown in Fig 3B. We
observed that the cell numbers increased 3- to 4-fold and the levels of
GFP expression were maintained during the 2-week culture period
(Fig 6). As expected, no B cells developed
from the Id3+ population, whereas 57% of the
CD19+ cells were found in the control GFP progeny (Fig 6).
This observation indicates that the culture conditions used in this
experiment supported the proliferation and/or maturation of B-cell
precursors present after culture of FL progenitors on a monolayer of
S17 stroma. Most importantly, we observed that CD56+ NK
cells could be detected in the progeny of both the
Id3 and the Id3+ fractions (Fig 6). The
absolute numbers of CD56+ Id3+ cells were 25%
higher than the numbers of CD56+ cells found in the control
cultures (Table 1).
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| Fig 6.
Both Id3 and Id3+ FL cells
recovered after culture on S17 stroma retain the capacity to develop
into NK cells. Cells harvested after coculture of transduced FL cells
on a monolayer of S17 stroma were further cultured in a cytokine
combination consisting of Flt-3L, IL-7, and IL-15. Ten days later, cell
suspensions were analyzed by flow cytometry for cell surface expression
of CD19 and CD56 antigens. The markers were set to exclude greater than
98% of cells stained with irrelevant control antibodies.
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Table 1.
Absolute Numbers of CD56+ NK Cells
Upon Sequential Culture of Transduced CD34+
CD38 FL Cells Upon S17 and Cytokines
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Inhibition of human B-cell differentiation by overexpression of Id3
is not due to intrinsic characteristics of the in vitro model used.
It is possible that the inhibitory effect on human B-cell
differentiation observed when FL progenitors overexpressing Id3 were
cocultured with the murine stromal cell line S17 was peculiar for this
in vitro model. To investigate this point, we explored a different
culture system that will be described in detail elsewhere (Jaleco et
al, manuscript submitted) that efficiently supports B-cell
development from uncommitted FL hematopoietic cells.
CD34+CD38CD10 FL
cells, transduced with the IRES-GFP or the Id3-IRES-GFP sequences, expressed similar levels of GFP at the onset of the culture (28% and
30%, respectively). The transduced cells were reaggregated with the
human SV19 stromal cell line and cultured for 2 to 3 weeks. In the
control culture, 12% of the cells recovered expressed high levels of
GFP, and the proportion of CD10+CD19+ B cells
present was identical in both the GFP+ and the
GFP fractions (73% and 79%, respectively;
Fig 7, left panel). In contrast, only 1%
of the FL cells transduced with Id3-IRES-GFP had high expression of
Id3, whereas 2% expressed low levels and the remaining cells were
Id3 (Fig 7, right panel). These fractions were
analyzed separately for expression of CD10 and CD19, and we found that
the proportion of CD10+CD19+ cells decreased
dramatically with increase in GFP expression (91%, 79%, and 28% in
the Id3, Id3low, and Id3high
populations, respectively; Fig 7, right panel). This was not due to
enhanced proliferation and differentiation of the transduced cells into
other cell lineages. We determined the absolute numbers of
Id3-GFPhighCD10+CD19+ cells
recovered at the end of the culture period and compared these with the
numbers of GFPhigh cells recovered from the cultures with
control, GFP-transduced cells (Table 2).
This analysis showed that the numbers of
CD10+CD19+ cells found in the samples
expressing high levels of Id3 were 18-fold lower than those recovered
in the controls, confirming the strong inhibition of B-cell
differentiation upon overexpression of Id3 on FL progenitors (Table 2).
Although not shown, CD19+ cells were also generated in a
reaggregate culture with CD34+CD38 FL
cells transduced with Id3, whereas precursors overexpressing Id3,
tested in parallel, generated much lower numbers of CD19+
cells.
View larger version (30K):
[in this window]
[in a new window]
| Fig 7.
B-cell differentiation of Id3-transduced FL progenitors
is strongly blocked but not completely inhibited in a reaggregation
culture with human stromal cells.
CD34+CD38CD10 FL cells were
sorted, transduced with the IRES-GFP or Id3-IRES-GFP vectors, and
reaggregated with the SV19 human stromal cell line for 2 to 3 weeks.
After the culture period, cells were harvested, stained with the
indicated antibodies, and analyzed by flow cytometry. Quadrant limits
defining positive and negative cells were set up using cells stained
with isotype-matched control antibodies. Thirty percent and 28% of the
cells were GFP+ 2 days after transduction in control- and
Id3-transduced samples, respectively. The results shown are
representative of 2 similar experiments.
|
|
| |
DISCUSSION |
In this report, we show that enforced expression of the dominant
negative HLH Id3 factor in human FL progenitor cells severely impairs
B-cell differentiation at an early stage.
CD34+CD38CD10 FL
progenitors were transduced with Id3, followed by coculture with a
monolayer of murine S17 stroma. The Id3+ cells recovered
after 2 weeks of culture did not express the B-cell specific antigen
CD19, in contrast to the Id3 cells and the cells
generated in cultures with the control GFP- or with Id3-transduced
CD34+CD38CD10 cells.
The effect of Id3 in the S17 system was B-cell-specific, because
development of CD14+ myeloid cells was not affected.
Moreover, Id3-GFP+ cells that accumulate in these cultures
were able to develop into NK cells in the presence of Flt-3L, IL-7, and
IL-15, confirming our previous findings that Id3-overexpression does
not affect NK cell development.17 We also observed a strong
inhibition of B-cell development in the reaggregate culture system,
indicating that the inhibition of B-cell development by Id3 is not
peculiar for the S17 culture system. It was recently reported that the related HLH factor Id2 could induce apoptosis in a myeloid precursor cell line independent of its dimerization motif.36 The
observation that a mutant of Id3 that is unable to dimerize with bHLH
factors18 is not affecting B-cell development in the S17
system indicates that inhibition of B-cell development is based mainly
on its ability to sequester bHLH factors. This observation strongly
suggests that E12 and E47, which are sequestered by Id3, are also
essential for human B-cell development.
In the S17 assays (Fig 5), no IL-7R+ cells were
detected, and the appearance of these cells was also inhibited in the
reaggregation assay (results not shown). Examination by FACS of the
phenotype of cells harvested from the S17 cultures that fell outside of the life gate and that are supposedly dying cells failed to provide evidence for the presence of dying IL-7R+ cells in the
cultures with Id3-transduced cells, indicating a truly developmental
block before acquisition of the IL-7R chain. A detailed analysis of
the developmental stage that is inhibited by Id3 indicated that
progression is arrested at the
CD34+c-kitdimCD45RA±
IL-7RCD38highCD10CD19
stage. In a model proposed by Ryan et al,30 cells with this phenotype represent early lymphoid progenitors. According to Ryan et
al,30 these cells already express RAG-1. Unfortunately, S17 cells do not induce RAG-1 expression in human
CD34+CD38 precursors,37 and
we were therefore unable to verify whether overexpression of Id3
results in inhibition of RAG-1 expression. Our finding that Id3
inhibits development in the S17 system at an early progenitor stage
could indicate that bHLH factors control induction of B-cell
commitment. Indeed, data in the mouse support the notion that E12 and
E47 are required for B-cell lineage commitment.10 In
E2A-deficient mice, B-cell development was arrested at a stage in which
cells express low levels of B220, and CD43 have germline µ0 transcripts and express IL-7R mRNA, but do not
express RAG or have IgH gene rearrangements.5,6,10
Interestingly, IL-7R+ cells could not be detected in the
B-cell differentiation cultures with Id3-transduced cells, suggesting
that the developmental block of human B-cell development in the S17
system imposed by Id3 overexpression could be earlier than that of E2A
deficiency. The discrepancy in IL-7R expression in Id3 transduced
S17 cultures and E2A-deficient mice could be due to the fact that in
the mouse this was analyzed in vivo, and in human, it was analyzed in
an in vitro assay. It is also possible that these
discrepancies are due to species differences. Alternatively, our
observations may mean that bHLH factors different from E12 and E47 are
required for generation of IL7R+ precursor cells. It is
interesting to note that CD34+ cells expressing Id3 develop
normally into NK cells. This combined with the observation that Id3
inhibits appearance of IL-7R+ cells suggests that NK
development can proceed without traversing an IL-7R+ stage.
Previously, we have shown that overexpression of Id3 inhibits
generation of T cells and promotes development of NK cells from CD34+ FL cells.17 Thus, Id3 inhibits
development of both T and B cells but not of NK cells. One
interpretation of these observations is that NK cells branch before T
and B cells, implying that a common T/B-cell progenitor exists in the
FL that is unable to develop into NK cells. Although this possibility
cannot be formally excluded, this seems unlikely for several reasons. T
and NK cells are much more similar to each other than to B cells with
respect to expression of cell surface markers and function (discussed in Lanier et al38 and Spits et al39). In
addition, it has been demonstrated that the murine thymus contains
progenitor cells that can develop into T and NK cells but not into B
cells.40 What could then be the reason that Id3
overexpression inhibits T- and B-cell but not NK cell development? It
seems most logical to assume that this is due to an effect of Id3
overexpression on a process common to T and B cells. Gene
rearrangements of their respective receptors are the most conspicuous
common feature of T and B cells and are essential for the development
and function of both cell types. It is possible that bHLH factors are
contributing to control these gene rearrangements. Some observations in
the mouse support this notion. When E47 is overexpressed in a
pre-T-cell line, RAG expression and gene rearrangements at the IgH
locus are induced.41 However, in the S17 system, inhibition
of human B-cell development by Id3 occurs independently from RAG
expression and IgH gene rearrangements, which should imply that bHLH
factors are required for B-cell differentiation independently of their possible effects on IgH gene rearrangements.
It has been shown that the levels of the E12 and E47 mRNA increase and
Id1 and Id2 decrease in cell lines representative for subsequent stages
of B-cell differentiation, suggesting that the balance of activating
bHLH factors and dominant negative Id proteins drive B-cell
differentiation. Recently, Li et al16 identified subpopulations of early B-cell progenitors based on expression of AA4.1
and B220 and absence of CD24. The earliest progenitor (fraction Ao) is
AA1+ and B220 and can differentiate into
multiple hematopoietic lineages. Upon appearance of B220,
µ0 mRNA is strongly upregulated and RAG1/2 mRNA become
detectable. These cells (fraction A1) are developmentally committed to the B-cell lineage but have not yet initiated IgH gene
rearrangements. In the mouse, Id1 expression is high in a subset of
CD43+CD24B220 cells
(fraction Ao) and decreases when these cells acquire germline Ig
transcripts and commit to the B-cell lineage (fractions A1 and A2).16,42 E12 and E47 are relatively low in
fraction Ao cells and increase upon progression to fraction
A1 and A2 cells. Similarly, we observed the
highest levels of E12 and E47 in the more mature population of
CD34+CD38+CD10+ FL cells that
includes pro-B and pre-B cells.30 In addition, Id1
expression was high in uncommitted
CD34+CD38CD10 cells
and was decreased dramati- cally in the more mature
CD34+CD38+CD10 and
CD34+CD38+CD10+ populations. These
data are consistent with findings in the mouse system.15 It
is interesting to note that Id1 has been shown to inhibit RAG-1
activation, suggesting that the relative ratio of E2A to Id1 is
possibly critical for expression of the RAG-1 gene in the B-cell
lineage and consequently for the onset of the B-cell developmental
pathway.14 However, Id3 mRNA expression, although
decreasing when the cells transit from
CD34+CD38 into
CD34+CD38+ cells, increases upon further
differentiation. The increasing levels of Id3 when cells differentiate
from CD10 into CD10+ would present an
apparent paradox, because enforced expression of Id3 in
CD10 cells inhibited B-cell differentiation.
However, it is possible that upregulation of Id3 drives downregulation
of RAG expression when pre-B cells proliferate.43 On the
other hand, one may assume that upregulation of E2A expression and
probably also of other bHLH factors in the CD10+ subset is
sufficient to neutralize the negative effects of Id3. It thus becomes
important to further investigate the expression levels and functional
relevance of other factors previously implicated in mouse B-cell
differentiation, such as E2-2 and HEB.
This report demonstrates the feasibility of retrovirus-mediated gene
transfer as a way to genetically modify human B-cell differentiation in
vitro. This technology was applied here and in previous
reports17,44 to investigate the role of bHLH factors in
T-cell, B-cell, and NK cell development and can be used to elucidate
the role of other transcription factors in human lymphoid cell development.
| |
ACKNOWLEDGMENT |
The authors thank the staff of the Bloemenhovekliniek in Heemstede (The
Netherlands) for their cooperation in obtaining fetal tissue. We also
thank E. Noteboom for expert technical assistance with flow cytometry.
We are grateful to D. Rawlings for the S17 cell line, to G. Nolan for
the LZRS vector, to M.G. Roncarolo for her gift of recombinant Flt-3L,
to G. Kato for the gift of Id3, to Takara Shuzo Co, Ltd for the
CH-296 fibronectin fragment, and to Immunex for recombinant IL-15. We
thank Dr A. Kruisbeek for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted November 19, 1998; accepted June 8, 1999.
A.C.J. was supported by a PhD fellowship from Junta Nacional De
Investigação Científica e Tecnológica,
Lisbon, Portugal.
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 Hergen Spits, PhD, Division of Immunology,
The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam,
The Netherlands; e-mail: hergen{at}nki.nl.
| |
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R. Schotte, M.-C. Rissoan, N. Bendriss-Vermare, J.-M. Bridon, T. Duhen, K. Weijer, F. Briere, and H. Spits
The transcription factor Spi-B is expressed in plasmacytoid DC precursors and inhibits T-, B-, and NK-cell development
Blood,
February 1, 2003;
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[Abstract]
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M. I. D. Rossi, T. Yokota, K. L. Medina, K. P. Garrett, P. C. Comp, A. H. Schipul Jr, and P. W. Kincade
B lymphopoiesis is active throughout human life, but there are developmental age-related changes
Blood,
January 15, 2003;
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576 - 584.
[Abstract]
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S. Becker-Herman, F. Lantner, and I. Shachar
Id2 Negatively Regulates B Cell Differentiation in the Spleen
J. Immunol.,
June 1, 2002;
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[Abstract]
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K. Weijer, C. H. Uittenbogaart, A. Voordouw, F. Couwenberg, J. Seppen, B. Blom, F. A. Vyth-Dreese, and H. Spits
Intrathymic and extrathymic development of human plasmacytoid dendritic cell precursors in vivo
Blood,
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[Abstract]
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A. C. Jaleco, H. Neves, E. Hooijberg, P. Gameiro, N. Clode, M. Haury, D. Henrique, and L. Parreira
Differential Effects of Notch Ligands Delta-1 and Jagged-1 in Human Lymphoid Differentiation
J. Exp. Med.,
October 1, 2001;
194(7):
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[Abstract]
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A. Ruiz-Vela, F. Serrano, M. A. Gonzalez, J. L. Abad, A. Bernad, M. Maki, and C. Martinez-A
Transplanted Long-Term Cultured Pre-Bi Cells Expressing Calpastatin Are Resistant to B Cell Receptor-Induced Apoptosis
J. Exp. Med.,
August 6, 2001;
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[Abstract]
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C. Beger, L. N. Pierce, M. Kruger, E. G. Marcusson, J. M. Robbins, P. Welcsh, P. J. Welch, K. Welte, M.-C. King, J. R. Barber, et al.
Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach
PNAS,
January 2, 2001;
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[Abstract]
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H. Spits, F. Couwenberg, A. Q. Bakker, K. Weijer, and C. H. Uittenbogaart
Id2 and Id3 Inhibit Development of Cd34+ Stem Cells into Predendritic Cell (Pre-Dc)2 but Not into Pre-Dc1: Evidence for a Lymphoid Origin of Pre-Dc2
J. Exp. Med.,
December 18, 2000;
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[Abstract]
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TOP
ABSTRACT
INTRODUCTION