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IMMUNOBIOLOGY
From the Immunobiology and Cancer Program, Oklahoma
Medical Research Foundation, Oklahoma City, OK; and the Department of
Internal Medicine and Molecular Science, Graduate School of Medicine,
Osaka University, Japan.
Recently, a collection of surface markers was exploited to isolate
viable Lin The humoral immune system is replenished throughout
life with B cells produced by the bone marrow. This process is being
increasingly understood in terms of selective gene expression with
transcription factors playing a major role in determining how
hematopoietic stem cells adopt one of 8 major fates. However, much
remains to be learned about the timing of events and how they are
influenced by positive and negative extracellular cues provided by the
microenvironment. Multiparameter flow cytometry and cell sorting allows
isolation of rare populations of bone marrow cells and it is important
to order them in a differentiation sequence that culminates with the
production of B lymphocytes. Expression of the enzyme terminal deoxynucleotidyl transferase (TdT) is thought to be an early, lymphocyte-specific event with functional consequences.1
TdT contributes to the diversity of antibody combining sites by adding non-germline-encoded nucleotides at sites of V-DJ segment joining during immunoglobulin gene recombination.2,3 We recently developed a means to identify and sort TdT+ bone marrow
cells lacking surface markers associated with any of the mature blood
cell lineages (Lin Some of the early pro-B cell characteristics are similar to ones
previously used by another group to isolate "common lymphoid progenitors" (CLP).6 At least 20% of
Lin Mice
Antibodies
Reagents Recombinant mouse IL-7 was purchased from Endogen (Woburn, MA). Recombinant mouse stem cell factor (SCF), Flt-2/Flk-3 ligand (FL), and transforming growth factor- (TGF-) were purchased from R&D
Systems (Minneapolis, MN). 1, 3, 5[10]-Estratriene-3, 17-diol
(-estradiol) was purchased from Sigma Chemical (St Louis, MO).
Epidermal growth factor (EGF) was purchased from Upstate Biotechnology (Lake Placid, NY). Mouse interferon (IFN)- and rat IFN- were purchased from Life Technologies (Rockville, MD). Limitin-human immunoglobulin (IgG1) heavy-chain
constant region fusion protein was prepared as previously
described.11
Isolation of lineage-negative early pro-B cells Bone marrow cells were collected from 4 to 10 mice and suspended with phosphate-buffered saline (PBS) without Ca++ or Mg++ (PBS ) and supplemented with 3%
heat-inactivated fetal calf serum (staining wash). Cells were incubated
with antibodies to lineage markers (Gr-1 and Mac-1 for myeloid cells,
anti-CD19 and anti-CD45R for B lineage cells, and Ter-119 for erythroid
cells) for 30 minutes. Then cells were washed and incubated with BioMag
goat-antirat IgG-coated magnetic beads (Polysciences, Warrington, PA)
for 15 minutes. Cells attached to beads were removed with a magnetic separator. Recovered cells were further incubated with new magnetic beads and subjected to a second round of magnetic separation. These
enriched lineage marker-negative bone marrow cells were then stained
with a cocktail of labeled antibodies to the lineage markers
(FITC-conjugated anti-CD3, anti-CD8, anti-Gr-1, PE-conjugated Ter-119
and anti-CD45R) and APC-conjugated anti-c-kit antibody. In some
experiments, PE-labeled anti-FL antibody and biotinylated anti-IL-7R antibody were also used. In this case, PE-labeled Ter-119 was eliminated and FITC-conjugated anti-CD45R was used instead
of PE-conjugated antibody. Streptavidin-RED613 (Life Technologies) was
used as the secondary reagent for biotinylated anti-IL-7R. Stained
cells were subjected to sorting on a MoFlo (Cytomation, Fort Collins,
CO). When reanalyzed after sorting, lineage-negative cells were
generally more than 95% pure; that is, less than 5% had even slightly
above background levels of staining. Subsets of these Lin
cells were usually more than 90% pure when sorted according to absence, low density, or high density of c-kit. The
IL-7R+ cells were relatively homogenous, but the
IL-7R preparations included some cells with low, but
above threshold staining.
Cell culture Sorted cells were put into 24-well culture plates (Costar, Cambridge, MA) containing 1 mL X-VIVO15 medium (Biowhittaker, Walkersville, MD) containing 1% detoxified bovine serum albumin (Stem Cell Technologies, Vancouver, BC, Canada), 5 × 105 M
2-mercaptoethanol (2-ME), 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and cytokines as indicated and
cultured at 37°C and 5% CO2 in a humidified atmosphere.
At the end of culture, cells were harvested, cell viability was
determined with a trypan blue dye exclusion method, and then cells were
subjected to flow cytometric analysis. For single-cell sorting
experiments, cells were directly sorted into wells of 96-well U-bottom
tissue culture plates (Costar) containing 100 µL of the same media.
The concentrations of cytokines were IL-7, 1 ng/mL; FL,100 ng/mL; and
SCF, 20 ng/mL.
Colony assay for granulocyte-macrophage progenitors Sorted cells were put into methylcellulose medium containing murine IL-3 (mIL-3), human IL-6 (hIL-6), and murine SCF (mSCF) (MethoCult GF M3534, Stem Cell Technologies) with 200 to 1000 cells plated per dish. After 10 days of culture in a humidified CO2 incubator at 37°C, numbers of colonies were counted with an inverted microscope.Immunofluorescence staining For the analysis of surface antigens, cells were incubated with combinations of labeled antibodies in PBS. They were then washed and incubated with streptavidin-RED613 to detect biotinylated primary antibodies. To characterize TdT+ cells, sorted lineage-negative cells were first stained using PE-conjugated, APC-conjugated, or biotinylated antibodies in combination with streptavidin-RED613. Then cells were fixed with 1% formaldehyde in 1.25 × PBS followed by permeabilization with 70% ethanol. Then, intranuclear TdT was stained using anti-TdT polyclonal rabbit antibody and the FITC-conjugated F(ab')2 fraction of a goat antirabbit IgG antibody. PE-conjugated anti-Ki-67 antibody was included at this step in some experiments. Stained cells were run on a FACScalibur flow cytometer (Becton Dickinson, San Diego, CA) and the data were analyzed with Flojo software (Treestar, San Carlos, CA).In vivo transfer Lineage marker-negative cells from C57BL/6 SJL mice were sorted to isolate the c-kitLo Flk-2/Flt-3+ IL-7R+ fraction. One thousand to 3000 of these early
pro-B cells were injected into RAG1/ mice that had been
given 400 rad irradiation. Spleens and thymuses were harvested from
these mice 2 or 3 weeks after injection and stained for flow
cytometric analysis.
Three cytokines permit survival and differentiation of early lymphocyte precursors Jacobsen and colleagues reported the production of CD45R/B220+ and then CD19+ lymphocytes in serum-free cultures supported only by recombinant cytokines.12 They used magnetic beads and sorting to enrich for c-kit+ Sca-1+ cells with low expression of lineage markers and their focus was on investigating roles played by SCF and Flt-3 ligand. We now report that their stromal cell-free conditions are also ideal for B lymphopoiesis in cultures initiated with the TdT+ cell-enriched Lin
c-kitLo fraction of marrow, that is, early pro-B cells. For
initial experiments, bone marrow cell suspensions were rigorously
depleted of lineage marker-positive (GR-1, CD11b/Mac-1, CD45RA, CD19,
TER119, CD3, and CD8) cells and then separated into 3 fractions on the
basis of c-kit density.4 At least one third of the cells
in the c-kitHi population were myeloid precursors that
proliferated in a colony-forming unit in culture (CFU-c) assay
(as described in "Materials and methods"). However, small numbers
of CD19+ cells were produced during a 7-day culture with
SCF, FL, and IL-7 (Figure 1). Although
essentially no myeloid progenitors were present in the
c-kitLo subset, it produced at least 10 times more (up to
800/input cell) CD19+ B-lineage progeny in culture.
Consistent with our previous study,4 small numbers of
rapidly differentiating lymphocyte precursors were present in a
fraction that completely lacks c-kit. As with stromal cell cocultures,
the time required under defined conditions for generation of
CD19+ cells from Lin c-kitHi
cells was longer than when the cultures were initiated with
Lin c-kitLo/ cells (data not shown).
Indeed, down-regulation of this SCF receptor coincides with loss of
myeloid differentiation potential and progression through the early
steps of lymphopoiesis.4 Defined culture models of this
kind allow detailed investigation of the importance and signals
transmitted via such receptors.
A requirement for IL-7 and its receptor Gene-targeting experiments have established the importance of IL-7 for B lymphopoiesis in mice and expression of the IL-7R must represent an important differentiation milestone.13,14 Therefore, this aspect was investigated under defined culture conditions. At least some CD45R+ cells were produced from Lin
c-kitLo precursors in 1-week cultures established with any
of several cytokine combinations, but CD19+ cells emerged
only when IL-7 was present (Figure 2A).
IL-7 was also needed for production of CD19+ cells from
cultures started with Lin c-kit precursors
(Figure 2B). Additional experiments confirmed that the combination of
SCF, FL, and IL-7 was optimal and almost no cells were recovered from
cultures maintained with IL-7 alone. The Lin
Flk-2/Flt-3+ c-kitLo population was then sorted
into subsets on the basis of IL-7R expression and cultured (Figure
3). The starting
IL-7RLo/ category included 9% to 37%
TdT+ cells, whereas 25% to 72% of the
IL-7R+ fraction expressed TdT. CD19+ cells
were produced when cultures were initiated with
IL-7RLo/ precursors, but less efficiently than in
cultures of IL-7R+ cells. Single Lin
Flk-2/Flt-3+ c-kitLo IL-7R+
cells were then placed in 96 individual culture wells along with SCF,
FL, and IL-7 in 2 experiments. After 11 days, growth was observed in 25 or 29 wells and 20 or 25 of them contained CD19+
lymphocytes, respectively. The cloning efficiency was approximately 6-fold lower in a comparable experiment done with single
Lin Flk-2/Flt-3+ c-kitLo
IL-7RLo/ cells. When considered with the results shown
above, these findings indicate that 3 functional cytokine receptors
cooperate to deliver signals for survival, proliferation, or
differentiation of early lymphocyte precursors, but IL-7 is
particularly important for transition to the CD19+
stage.
Early lymphocyte precursors are directly influenced by negative regulators A variety of evidence suggests that lymphopoiesis in the thymus and bone marrow is limited by sex steroids, but the nature of the target cells remains elusive.15 Lin
c-kitHi cells with the potential for lymphoid or myeloid
differentiation were placed in serum-free, stromal cell-free cultures
containing only SCF, FL, and IL-7 (Figure
4). Although total numbers of
Gr-1+ myeloid progeny were slightly increased by inclusion
of -estradiol in the medium (1.1- to 2.5-fold), production of
CD19+ lymphocytes was almost completely prevented.
Titration experiments indicated that 108 M concentrations
of the hormone were sufficient for maximum suppression (not shown). The
next putative stage of differentiation also appeared to be directly
sensitive to estrogen. That is, the ability of Lin
c-kitLo or Lin c-kit marrow
cells to generate CD19+ lymphocytes in 1-week cultures was
suppressed 78% ± 11% (Table 1) or
92% ± 3%, respectively, by -estradiol. Interestingly, absolute
numbers of CD45R+ CD19 cells were actually
increased in estrogen- containing cultures as compared to control
cultures (Table 1). The same accumulation of CD45R+
CD19 cells (168.2% ± 34.8% of controls) occurred
in estrogen-treated cultures initiated with Lin
c-kitHi precursors. We previously found that estrogen had
no influence on the IL-7-driven clonal proliferation of lymphoid
progenitors in semisolid agar cultures.16 Indeed, the same
hormone preparations found here to be suppressive for early stages of
lymphopoiesis were ineffective in CFU-IL-7 assays (not shown).
Although late events in B lymphopoiesis can be influenced by estrogen
only via effects on stromal cells, early precursors may be direct
hormone targets.16
B lymphopoiesis was preferentially suppressed when limitin, a new
IFN-like cytokine, was injected into mice.11 This factor dramatically reduced the yield of CD19+ cells in 1-week
stromal cell-free cultures and, in contrast to estrogen, also reduced
numbers of CD45R+ CD19 Transforming growth factor- Comparison of early pro-B cells with other committed precursors on the basis of surface marker expression Lin TdT+ cells were uniformly positive
for CD27, an antigen recently found to be absent from long-term
reconstituting stem cells7 and when a PE-conjugated
antibody was used, more than half of the Lin
TdT+ cells also displayed the AA4.1 antigen20
(Figure 5). Large and medium-sized
Lin TdT+ cells could be distinguished
on the basis of forward light scatter, but we found no differences
between them in terms of these surface markers. Both medium and large
Lin TdT+ cells stained for the Ki-67
proliferation-associated antigen (Figure 5 and data not
shown).21 Ly-6C is a protein of unknown function on some
bone marrow cells and we previously found that CD45R+
Ly-6C+ cells had little potential for differentiation to
CD19+ lymphocytes in culture.5 We now report
that less than 8% of Lin TdT+ cells were
positive for the Ly-6C antigen.
In C57BL/6 mice, 42% to 73% of Lin Heterogeneity was also found with respect to CD4 or the CD16/CD32 Fc Marrow fractions enriched for early B-lineage precursors include T-lymphocyte precursors The emphasis of our studies has been on identifying functional precursors for B-lymphocyte lineage cells within bone marrow and the term "early pro-B cells" seemed appropriate for Lin
c-kitLo Flk-2/Flt-3+ IL-7R+
cells.5 However, some of these surface characteristics
were also used by others to isolate "common lymphoid
progenitors"6 and it was important to learn more about
the differentiation potential of early pro-B cells. Immunodeficient
Rag-1/ mice were injected with Lin
c-kitLo Flk-2/Flt-3+ IL-7R+
cells (Table 2). This subset contained
cells with the potential to reconstitute T- as well as B-cell lineages.
Thus, a population of Lin c-kitLo
Flk-2/Flt-3+ IL-7R+ cells that efficiently
gives rise to CD19+ lymphocytes also includes
T-lineage precursors.
The focus of this study was on a population of bone marrow cells
that lacks lineage-associated surface markers but expresses intracellular TdT. Our previous analysis indicated that they represent important intermediates between multipotential stem cells and functional B-cell precursors. We show here that a fraction enriched for
Lin The serum-free, stromal cell-free culture conditions developed by
Jacobsen and colleagues12 proved ideal for our analysis of
early pro-B cells. Remarkably, the same required cytokines were those
whose receptors were used as sort criteria. That is, the cells we
placed in culture bore mAbs to c-kit, IL-7R Our findings complement and extend previous studies regarding roles
played by IL-7. Jacobsen and colleagues concluded that the cytokine is
important for commitment of multipotential progenitors to lymphocyte
development.12 We showed that cells bearing the IL-7R
differentiated slightly better than those lacking this molecule, and
particularly when assessed at short culture intervals (Figure 3). This
suggests that IL-7R It has become increasingly apparent that negative, as well as positive stimuli, control rates of blood cell production and 3 of the inhibitory agents investigated here are effective in vivo. Castration and androgen receptor abnormalities increase B-cell production, whereas it is selectively suppressed during pregnancy and after treatment with sex steroids.34-37 These observations suggest that steady-state lymphocyte production is under hormonal control, but much remains to be learned about relevant target cells and molecular mechanisms. We previously found that IL-7-responsive pro-B cells are insensitive to estrogen, but that stromal cell cocultures initiated with earlier precursors were suppressed.16 The findings indicated that stromal cells might be induced to produce a soluble negative regulator of B lymphopoiesis. This possibility is still open but we show with stromal cell-free cultures and highly purified precursors that early pro-B cells are also potential hormone targets. Efforts to identify estrogen-regulated genes and consequences of hormone therapy should include an analysis of this rare cell population. We recently discovered limitin, an IFN-like cytokine that preferentially suppresses B lymphopoiesis.11 This activity has also been described for type I IFN17 and limitin can use the same receptor.11 We demonstrate here that early pro-B cells are directly and similarly suppressed by limitin, type I IFN, and type II IFN. Stromal cells, macrophages, and T lymphocytes are all potential sources of these factors within bone marrow.38-40 However, B-cell precursor populations appear to be normal in type I IFN receptor-mutated mice41 (and data not shown). In contrast to sex steroids, these agents may only influence lymphopoiesis under conditions of trauma, inflammation, or disease. Transforming growth factor- Epidermal growth factor was of interest for several reasons. This is an estrogen-inducible cytokine and one that can elicit estrogen receptor-mediated signaling.46,47 Therefore, involvement of EGF in or mimicking of sex steroid-elicited responses seemed possible. We show here that early pro-B cells are unaffected by EGF. However, EGF may influence components of the bone marrow microenvironment because it promotes growth of bone marrow-derived stromal cells48 as well as fetal thymic architecture49 in culture. This panel of negative regulators included 2 that selectively inhibit
lymphopoiesis, but not myelopoiesis and this direct comparison now
makes it possible to appreciate differences in their
actions.11,16,50 Although limitin blocked formation of
CD45R+ CD19 One important objective of this study was to learn more about the
surface marker characteristics of Lin Several subsets of nonlymphoid progenitors have recently been found
among the Lin Differences in batches of labeled mAbs and mouse strains as well as
other technical reasons complicate comparisons between laboratories
concerning extremely rare subsets of bone marrow cells. The functional
B-cell precursors we refer to as early pro-B cells only partially
resemble cells designated "fraction A0" by Hardy and
colleagues.9 Some, but not all, early pro-B cells display
the C1qRp receptor recognized by the AA4.1 antibody and many, but not
all, expressed the CD4 antigen used by Hardy to define fraction
A0.9 CD4 has long been used to identify the earliest thymic immigrants and cells with T-lineage potential within
bone marrow.53 That is consistent with our finding that early pro-B cell populations include T-lineage precursors. On the other
hand, others found little or no B-lymphocyte lineage potential
associated with CD4+ bone marrow cells.54,55
Furthermore, we found variable, low level expression of CD4 on
CD45R+ CD19 We demonstrated that functional T-lymphocyte lineage progenitors were
present in the Lin Note added in proof: Thurmond and colleagues recently
described transplantation experiments with estrogen receptor
The authors thank Dr Lisa Borghesi for critical reading of the manuscript, as well as Ms Viji Dandapani and Mr Jim Henthorn for cell sorting. The assistance of Ms Shelli Wasson in manuscript preparation is also appreciated.
Submitted October 10, 2000; accepted December 18, 2000.
Supported by grant AI 20069 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Paul W. Kincade, Immunobiology and Cancer Program, Oklahoma Medical Research Foundation, 825 NE 13th St, Oklahoma City, OK 73104.
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L.-S. Lu, J. Tung, N. Baumgarth, O. Herman, M. Gleimer, L. A. Herzenberg, and L. A. Herzenberg Identification of a germ-line pro-B cell subset that distinguishes the fetal/neonatal from the adult B cell development pathway PNAS, March 5, 2002; 99(5): 3007 - 3012. [Abstract] [Full Text] [PDF]
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| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
) and
CD45R+CD19+ (
) cells per input precursor
were calculated as described in the legend to Figure 
) or
Lin
) cells and evaluated at the
indicated intervals. Numbers of CD19+ lymphocytes recovered
per input precursor are shown from one of 3 independent experiments
that gave very similar results.
