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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 713-723
Loss of c-kit Accompanies B-Lineage Commitment and Acquisition of CD45R
by Most Murine B-Lymphocyte Precursors
By
Kimberly J. Payne,
Kay L. Medina, and
Paul W. Kincade
From the Oklahoma Medical Research Foundation, Immunobiology and
Cancer Program, Oklahoma City, OK.
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ABSTRACT |
Using surface markers, we identified two bone marrow (BM) subsets
enriched for TdT+ cells on the brink of CD45R
acquisition. These two populations, Lin c-kitLo and
Linc-kit, consisting of 35.4% and 7.4%,
respectively, TdT+ cells, generated B-lineage cells in
overnight cultures. Approximately half of the c-kitLo
B-lineage precursors were bipotential, yielding myeloid and lymphoid progeny, whereas most that were c-kit gave rise only to
lymphocytes. Analysis of B-lineage progression during a finite culture
period showed that the most mature precursors were concentrated in the
Linc-kit population. Moreover, a majority
of the earliest CD45R+ pro-B cells in BM, identified as
CD45R+ CD43+ BP-1
CD25 natural killer (NK)1.1
sIgM, were also c-kit. These
c-kit cells, like their c-kitLo
counterparts, expressed TdT, proliferated in response to interleukin (IL)-7, and generated sIgM+ cells. These data suggest
that TdT expression is initiated as c-kit downregulation begins in
Lin cells, with progressive loss of c-kit during
B-lineage differentiation. CD45R expression is initiated during the
transition from c-kitLo to c-kit with many
cells losing c-kit before acquiring CD45R. The ability to isolate
highly enriched populations of viable CD45R precursors
will be instrumental in characterizing the earliest B-lineage cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
IN THE ADULT MOUSE, self-renewing
hematopoietic stem cells (HSC) reside in bone marrow (BM) in which they
generate all blood lineages, including B lymphocytes (for review see
Morrison et al1). Enrichment of murine HSC and
multipotential progenitor activity has been achieved in sorted BM cells
that lack surface markers of the various blood lineages
(Lin) but express the receptor for stem cell factor,
c-kit (c-kit+).2 Recently, common lymphoid
progenitors (CLP) that can generate T- and B-lymphoid and natural
killer (NK), but not myeloid cells, were identified.3 This
population has no, or limited, capacity for self-renewal. CLP resemble
stem cells in being Lin, but they lack Thy-1 and express
the receptor for interleukin (IL)-7 as well as low levels of c-kit and
Sca-1. B-lineage cells can be isolated from BM on the basis of
CD45R4,5 and CD196 expression, but
Lin precursor populations poised to enter the
B-lymphocyte lineage are poorly resolved.
The stages of B-lineage differentiation in which sequential heavy- and
light-chain gene rearrangements occur have been designated the pro-B
and pre-B stages, respectively (for review see Loffert et
al7). Using combinations of cell surface and intracellular differentiation markers, three schemes for B-cell development have been
proposed.8-10 Although these models define intermediate stages of maturation, the earliest stages of B-lineage commitment are
not well characterized.
Park and Osmond8 used expression of terminal
deoxynucleotide transferase (TdT) and cytoplasmic µ heavy chains
(cµ), in addition to CD45R to identify early pro-B cells
(TdT+ CD45R cµ),
intermediate pro-B cells (TdT+ CD45R+
cµ), late pro-B cells (TdT
CD45R+ cµ), and pre-B cells
(TdT CD45R+ cµ+) in murine
BM. TdT adds nontemplated nucleotides at junctions of gene segments
during immunoglobulin heavy-chain gene rearrangement,11,12 whereas cµ is the protein product of the productively rearranged immunoglobulin heavy-chain gene. This model is clearly indicative of
differentiation within the B lineage and identifies a putative population of Lin B-lymphocyte precursors. However,
assessment of TdT and cµ expression requires cell fixation and
permeabilization, precluding subsequent exploration of cellular
function. Thus, the fate of TdT+ CD45R
cµ early pro-B cells is unknown.
Hardy et al9 characterized pro-B cells by coexpression of
CD45R and CD43. Populations presumed to be developmentally sequential pro-B subsets were resolved on the basis of successive expression of
heat-stable antigen (HSA, CD24) and BP-1. In this model, Fraction A
(CD45R+CD43+CD24
BP-1) B-lineage precursors give rise to
Fraction B (CD45R+CD43+CD24+
BP-1) and then Fraction C
(CD45R+CD43+CD24+
BP-1+).9 After a report that Fraction A
contained cells expressing an NK cell marker,6 Hardy et
al9 more extensively characterized this population and
identified a possible CD45R precursor to Fraction A
that expressed AA4.1.13 From AA4.1+
CD43+ CD24 BM, Hardy et al9
resolved fraction A0
(CD45RCD4+), fraction A1
(CD45R+CD4+), and fraction A2
(CD45R+CD4). These were postulated to
represent sequential populations, primarily on the basis of progressive
expression of transcripts for B-lineage-associated
genes.13 However, the lineage potential of Fraction
A0 and verification of the successive nature of these populations have not been reported.
Rolink et al14 designated B-lineage precursor populations
on the basis of gene rearrangement and expression, partially enriching subsets using surface markers. In their model, CD45R
c-kitLo/+ precursors are thought to have immunoglobulin
genes in germline configuration.15,16 These pro-B cells
give rise to CD45R+ c-kit+
CD25 pre-B I cells that initiate heavy-chain gene
rearrangement. Further differentiation yields CD45R+
c-kit CD25+ pre-B II cells that
rearrange light-chain genes. In this model, TdT is lost and cµ
acquired as the cells enter the pre-B II stage.10,14,16-17 However, CD45R precursors isolated on the basis of
c-kit included non-B-lineage (Mac-1+ Gr-1+,
TER-119+, CD3+) cells, almost all of which lack
c-kit.18 Such cells selectively diluted B-lineage precursor
activity measured in the c-kit subset.15
For this reason, details are lacking about when c-kit is downregulated
and how this corresponds to B-lineage commitment.
Lack of resolution of the earliest B-cell precursor has significantly
hampered investigations of the regulatory processes influencing lineage
commitment. Park and Osmond's8 early studies describing a
putative B-lineage precursor population
(CD45RTdT+) likely included such cells
but, because assessment of TdT required fixation, their functional
characteristics could not be examined in tissue
culture.11,12 Therefore, our first goal was to isolate a
population of viable BM cells enriched for those B-lymphoid precursors
poised to enter the B lineage. Subsequent culture of those cells and
concomitant analysis of murine BM showed that many B-lineage cells lose
c-kit earlier than previously believed.
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MATERIALS AND METHODS |
Mice.
BALB/c mice were used for sorting Lin cells and
CD45R+ cells were isolated from C57/BL6 mice. Mice were of
either sex and ranged in age from 5 to 15 weeks. Mice were obtained
from either the Oklahoma Medical Research Foundation Laboratory Animal
Resource Center (Oklahoma City, OK), Charles Rivers Breeding
Laboratories (Wilmington, ME), or Taconic Farms (Germantown, NY).
Animals not bred in our facility were maintained at least 1 week after
shipping before experimentation.
Antibodies.
TER-119 and Gr-1 (RB6-8C5) purchased from PharMingen (San Diego,
CA), and 10× concentrated anti-Mac-1 culture supernatants (M1/70
hybridoma obtained from American Type Culture Collection, Rockville,
MD) were used for enrichment of Lin cells and early
CD45R+ pro-B cells. For enrichment of
Lin cells, 10× concentrated CD45R culture
supernatant (14.8 hybridoma developed in our laboratory4)
was also used. To sort Lin subsets the following
antibodies were used: Mac-1 fluorescein isothiocyanate (FITC) (M1/70)
(Boehringer Mannheim Corp, Indianapolis, IN), CD3 phycoerythrin (PE)
(29B) (GIBCO BRL, Gaithersburg, MD) or CD3 FITC (145-2C11), Gr-1 FITC
(RB6-8C5), CD19 FITC (1D3), CD8 FITC or CD8 PE (53-6.7), TER-119 PE,
CD45R PE (RA3-6B2), and biotinylated c-kit (2B8) shown by Streptavidin
CyChrome (PharMingen). To sort early CD45R+ pro-B cells
CD45R allophycocyanin (APC) or CD45R FITC (RA3-6B2), NK1.1 PE (PK136),
BP-1 PE (6C3) (PharMingen), goat antimouse immunoglobulinM(IgM) PE
(Southern Biotechnology Associates, Birmingham, AL), CD25 PE (PC61 5.3)
(Caltag Laboratories, Burlingame, CA), and biotinylated CD43 (purified
from S7 hybridoma supernatant and biotinylated in our laboratory using
standard protocols, hybridoma purchased from American Type Culture
Collection) were used. APC-labeled antibody to c-kit (2B8) purchased
from PharMingen was added when sorting c-kit+ and
c-kit subsets. The following antibodies were used
for surface staining of cultured cells: CD45R PE or CD45R APC
(RA3-6B2), CD19 FITC, CD19 PE, or biotinylated CD19 (1D3), CD24 FITC
(M1/69), BP-1 PE (6C3), c-kit PE, or c-kit APC (2B8) purchased from
PharMingen and goat antimouse IgM purchased from Southern Biotechnology
Associates. Biotinylated reagents were using either Streptavidin
CyChrome (PharMingen) or Streptavidin-Red613 (GIBCO BRL). Polyclonal
FITC-labeled goat antimouse IgM purchased from Zymed (San Francisco,
CA) was used for cµ staining. For TdT staining, polyclonal rabbit
anti-TdT and goat antirabbit FITC from Supertechs (Bethesda, MD) were
used. Appropriate isotype control antibodies were used at the same
concentration to set gates. Supernatant from the anti-Fc receptor
hybridoma, 2.4G2, (American Type Culture Collection) was used to reduce
nonspecific staining of cultured cells.
Cell sorting.
BM was harvested and suspended in staining wash (phosphate-buffered
saline without Ca2+ or Mg2+
[PBS] containing 3% heat-inactivated fetal bovine
serum [FBS]). For sorts of Lin subsets and early
CD45R+ pro-B cells, whole BM was enriched for desired
populations by immunomagnetic bead depletion. In short, BM was
incubated with purified monoclonal antibodies on ice for 25 minutes,
washed two times, and incubated with goat antirat Ig-coated magnetic
beads (Perseptive Biosystems, Framingham, MA) for 25 minutes at 4°C on a rocking table. Beads were removed from cell-bead suspensions by
four rounds of incubation with a magnetic separator. Enriched BM
suspensions were then incubated simultaneously with primary antibodies
for 25 minutes, washed two times, incubated with secondary-labeled Streptavidin for 25 minutes (to detect the biotinylated antibody), washed once, and resuspended in staining wash. CD45R primary antibodies were added along with secondary antibodies for Lin
sorts to insure that there was little opportunity for capping and
shedding of this surface marker. Cells were kept on ice until sorted on
the FACStarPlus (Becton Dickinson, San Diego, CA) cell
sorter and samples were kept chilled during sorting.
Immunofluorescence staining.
Cells in stromal cell cocultures were harvested with 2 mmol/L EDTA in
PBS. After harvesting, wells were visually examined
for complete removal of adherent cells. Cells in stromal-free cultures
were harvested by washing wells with medium. For surface staining, harvested cells were incubated with 50% 10× concentrated 2.4G2 hybridoma supernatant in 4% rat serum for 15 minutes before staining to prevent nonspecific binding of antibodies. Cells were then incubated
with primary antibodies simultaneously for 20 minutes, washed two
times, incubated with secondary antibody for 20 minutes, washed once,
and resuspended in staining wash. Intranuclear staining for TdT and
cµ was performed as described by Melchers et al.14 For
TdT staining, whole BM or sorted cells were first stained for surface
markers as described above and then fixed with 1% paraformaldehyde in
PBS (pH 7.4) for 5 minutes on ice, or 0.25%
paraformaldehyde in PBS (pH 7.4) overnight. Fixed
cells were permeabilized by incubating with 70% methanol for 30 minutes on ice, and then incubated with goat serum to prevent
nonspecific antibody binding. Cells were washed once and then incubated
with anti-TdT for 45 minutes to 1 hour at room temperature, washed
twice, incubated with secondary antibody for 30 minutes on ice, washed
twice, and resuspended in staining wash for flow cytometric analysis.
Preliminary experiments showed that permeabilization did not affect
surface-marker staining, except in the case of APC fluorochrome-labeled
antibodies. Consequently, PE- or FITC-labeled antibodies were used for
this purpose. For cµ staining, cells were harvested and stained for
surface IgM as described above and fixed with 4% paraformaldehyde in
PBS (pH 7.4) for 10 minutes on ice, or 0.25%
paraformaldehyde in PBS (pH 7.4) overnight. Cells
were then permeabilized by incubating with PBS
containing 0.2% Tween 20 at room temperature for 20 minutes, labeled
by incubating with FITC-labeled anti-IgM at room temperature for 30 minutes, washed two times, and resuspended in staining wash. Gates for
surface, intracellular, and intranuclear staining were set with
appropriate isotype controls. After staining, all samples were kept on
ice until analyzed on the FACScan, FACSCalibur, or
FACStarPlus flow cytometer (Becton Dickinson). Forward and
side scatter were used to gate out larger stromal cells in samples
containing cells grown in stromal cell cocultures.
Cell culture and clonal assays.
All liquid cell cultures used Optimem medium (GIBCO BRL) containing
2.5% (single-cell sorts of Lin cells) or 1.5% (all
other cultures) FBS (HyClone, Logan, UT), 2 mmol/L glutamine, 5 × 105 mol/L 2-mercaptoethanol, 100 U/mL penicillin,
and 100 µg/mL streptomycin. Stromal cells were plated 1 or 2 days
before initiation of stromal cell cocultures at 5,000 cells per well in
24-well plates for bulk cultures and at 500 cells per well in 96-well
plates for clonal assays of single sorted cells. Lin
cells were plated on S17 stromal cells (a generous gift from Dr K. Dorshkind) and CD45R+ cells were plated on ST2 stromal
cells (a generous gift from Dr S-I. Nishikawa) and supplemented with
IL-7 (Endogen, Woburn, MA) at 1 ng/mL. T-25 flasks with a subconfluent
layer (~50% confluency) of ST2 stromal cells were used to assess the
ability of c-kit+ and c-kit early
CD45R+ pro-B cells to generate sIgM+ cells.
These cultures were not supplemented with IL-7 because removal of IL-7
has been reported to promote differentiation to the sIgM+
state.19 Stromal cell free cultures contained 50% ST2
stromal cell conditioned medium and were supplemented with flt ligand at 100 ng/mL, stem cell factor at 20 ng/mL (R & D Systems, Minneapolis, MN) and IL-7 at 1 ng/mL. Liquid cultures were fed every 3 to 4 days.
Semi-solid agar assays were performed as follows. Sorted
c-kit+ or c-kit early CD45R+
pro-B cells were plated at 4.3 × 103 or 1 × 104 cells per mL in colony-forming unit (CFU) medium
consisting of McCoy's Modified 5A medium containing 15% FBS, 2 mmol/L
L-glutamine, 100 U penicillin/mL, 100 µg streptomycin/mL and 5 × 105 mol/L 2-ME, supplemented with 1 mmol/L
sodium pyruvate solution, 1.5% sodium bicarbonate solution (7.5%),
0.8% minimal essential medium (MEM) essential amino acids
(50×), 0.4% MEM nonessential amino acids (100×), and 1.6%
MEM vitamin solution. Medium was warmed to 37°C, cells were added
to medium and then mixed with a 1/10 volume of boiled 3% bacto agar
(Difco, Ann Arbor, MI) in water, which had cooled to approximately
40°C. Then 1 mL aliquots of medium were quickly plated in 35-mm
tissue culture dishes and allowed to gel for about 20 minutes at room
temperature. After 6 days of incubation at 37°C in a fully
humidified atmosphere of 5% CO2 in air, colonies
(aggregates of greater than 30 cells) were counted using a dissecting microscope.
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RESULTS |
Expression of TdT corresponds to reduced c-kit on
Lin precursors.
We have devised a strategy for sorting viable early B-lineage
precursors on the brink of CD45R acquisition. This strategy permits
functional analysis of sorted precursors in cell culture and
accordingly, we have followed the fate of these cells during in vitro
lineage commitment. Enrichment for CD45R B-lymphoid
precursors was accomplished by sorting Lin cells
whose surface c-kit levels best correlated with TdT expression.
We sorted Lin (Mac-1,
Gr-1,
Ter-119,CD45R,
CD19, CD8,
CD3) cells, thereby removing myeloid, erythroid, and
lymphoid lineage cells that make up about 98% of BM mononuclear cells
(BM MNC). Figure 1 shows the analysis of
this population when costained for surface c-kit and intranuclear TdT.
Lin BM was divided into c-kitHi,
c-kitLo, and c-kit populations (Fig 1B)
that made up 1.4%, 0.56%, and 0.33%, respectively, of BM MNC
(Table 1). Gating on TdT+ cells
within Lin BM (Fig 1C) showed that the majority of
Lin TdT+ cells were c-kitLo
(Fig 1D). Indeed, TdT+ cells made up 35% of the
Linc-kitLo population, giving an
estimated 116-fold enrichment for Lin
TdT+ cells when compared with BM MNC (Table 1). A smaller
fraction of Linc-kit cells were
TdT+ (7.4%). TdT+ cells comprised 5.7% of the
Linc-kitHi population, and were among
cells with the lowest c-kit expression within the subset (Fig 1D, and
Table 1). Linc-kit and
Linc-kitHi subsets had 24-fold and
19-fold enrichments, respectively, for Lin
TdT+ cells when compared with BM MNC (Table 1). Thus,
expression of TdT in the Lin population
corresponded to reduced surface c-kit with the majority of
Lin TdT+ cells being
c-kitLo.
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| Fig 1.
Surface c-kit expression identifies Lin BM
populations enriched for TdT+ cells. Lin
BM, representing approximately 2% of BM mononuclear cells, was sorted
from whole BM depleted of Lin+ cells by magnetic
separation (A), and costained for intranuclear TdT and surface c-kit as
described in Materials and Methods. Within costained Lin
BM, c-kit, c-kitLo, and c-kitHi
populations were identified (B). Lin BM was gated for
TdT+ cells (C). (D) shows expression of c-kit in gated
Lin TdT+ cells. The frequencies of these
subsets in BM are shown in Table 1.
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Linc-kit and
Linc-kitLo subsets generate B-lineage
cells in overnight culture.
The data presented in Fig 1 suggested that the B-lineage precursors
generated from Linc-kitHi stem cells
might begin to downregulate c-kit as they acquire TdT. To
determine which of our precursor populations was able to generate
B-lineage cells most rapidly, we looked for appearance of
CD45R+ and CD19+ cells after intervals of
culture. Though acquired before CD19,20 CD45R is a less
exclusive B-lineage marker than CD19.6 Stromal cell free
cultures were used to ensure that stromal cell debris was not
inadvertently included in lymphocyte light-scatter gates and to
preclude the possibility that stromal adherent B-lineage cells might be
excluded from analysis.
After overnight culture, Linc-kitLo and
Linc-kit, but not
Linc-kitHi precursors, generated cells
that expressed CD45R at low levels (Fig 2). By day 4, progeny from both
Linc-kit and
Linc-kitLo BM subsets included cells
that expressed only CD45R and others that coexpressed CD45R and CD19.
By day 7, a majority of these B-lineage cells coexpressed CD45R and
CD19. A substantial population of cells in
Linc-kitHi cultures began to express
CD45R by day 4, but few double-positive cells were observed even by day
7.
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| Fig 2.
Linc-kitLo and
Linc-kit cells generate B-lineage cells
in overnight culture. Linc-kitHi,
Linc-kitLo, and
Linc-kit cells were sorted from BM and
placed in stromal cell free culture containing IL-7, FL, SCF, and
stromal cell conditioned medium. Cells were harvested at days 1, 4, and
7. Costaining is shown for cells within lymphocyte light scatter and
percentages of cells in each quadrant are given. C-kitHi
and c-kitLo cultures were initiated with equal numbers of
cells, but due to low frequency, half as many cells were used to
initiate c-kit cultures. Representative data are shown
from one of two independent experiments using pooled marrow from 10 mice.
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Thus, we found that Lin cells that are
c-kitLo and c-kit include B-lineage
precursors that acquire CD45R after overnight culture. Linc-kitHi cells were able to generate
the B lineage but required more time to do so. In addition,
heterogeneity with respect to CD19 expression on CD45R+
cells was observed in progeny of both
Linc-kitLo and
Linc-kit populations (Fig 2).
This heterogeneity was most noticeable in the
Linc-kitLo cultures in which B-lineage
cells from day 7 retained a distinct CD19 population
that was not evident in day-7
Linc-kit cultures.
Reduction of c-kit in Lin precursor populations
corresponds to increasing maturity in B-lineage progeny.
The above studies suggested that cells poised to enter the B lineage
were abundant among Linc-kitLo and
Linc-kit cells but not the
Linc-kitHi population. The
c-kitLo precursor population included some cells less
mature than those that were c-kit. However, it was
not clear if a progressive loss of c-kit corresponded to increased
maturity in Lin B-lymphocyte precursors. We reasoned
that the maturity of B-lineage cells produced from cultures in a finite
period of time would reflect the maturity of precursors in each
starting population. To determine the maturity of culture-generated
B-lineage cells, we examined expression of CD24 and BP-1, markers
identified by Hardy et al9 as being successively acquired
by CD45R+ CD43+ pro-B cells during differentiation.
Linc-kitLo and
Linc-kit subsets generated
B-lineage cells that were more mature than those from cultures
initiated with Linc-kitHi cells. In a
representative experiment shown in Fig 3,
all B-lineage cells generated from day-4
Linc-kitHi cultures expressed a Fraction
A (CD24 BP-1) or Fraction B
(CD24+ BP-1) surface phenotype. In
contrast, the majority of those in the Linc-kit cultures, and a smaller
population in Linc-kitLo cultures, had
acquired BP-1, placing them in Fraction C. By day 7, the majority of
B-lineage progeny from both
Linc-kit and
Linc-kitLo cultures had reached Fraction
C. However, almost all of those from
Linc-kitHi precursors were still
BP-1. Furthermore, some of the B-lineage progeny
generated in Linc-kitLo, and more of
those in Linc-kit cultures, had
lost CD43 expression by day 7, whereas all of those within
Linc-kitHi cultures retained this marker
(data not shown). Thus, changing patterns of CD24, BP-1, and CD43, as
well as CD45R and CD19, in cultured cells, show that
Linc-kitLo and
Linc-kit precursors give rise to
more mature B-lineage progeny than
Linc-kitHi cells.
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| Fig 3.
Reduced expression of c-kit by precursor populations
corresponds to increasing maturity of B-lineage progeny . Linc-kitHi,
Linc-kitLo, and
Linc-kit cells were sorted from BM and
placed in stromal cell free culture containing IL-7, FL, SCF, and
stromal cell conditioned medium. Cells were harvested at days 4 and 7. Costaining is shown for CD45R+ CD43+ cells
within lymphocyte light scatter. The lower left quadrant contains cells
in Fraction A, the lower right quadrant cells in Fraction B, and the
upper right quadrant cells in Fraction C as identified by Hardy et
al.9 Percentages of cells in each quadrant are shown.
C-kitHi and c-kitLo cultures were initiated
with equal numbers of cells, but because of low frequency, half as many
cells were used to initiate c-kit cultures.
Representative data are shown from one of two independent experiments
using pooled marrow from 10 mice. Similar trends in maturity were
observed in numerous experiments with cells harvested at time points
from day 2 to 7.
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If a uniform cohort of precursors representing the same degree of
lineage commitment and maturity were present in any of our three
starting populations, it might be expected to move synchronously through subsequent differentiation stages in culture. The
Linc-kit subset was the most
homogeneous in this respect, with a majority of B-lineage progeny
reaching the most mature phenotype more quickly than in the other
precursor populations. Yet, even in this population, some cells took
longer than others to acquire CD19 (Fig 2) or reach the Fraction C
stage (Fig 3). Linc-kitHi cultures
produced the most immature B-lineage cells at all time points. However,
as shown in Fig 3, these precursors included both Fraction A and
Fraction B cells at days 4 and 7. In some experiments, a small
population of Fraction C cells were also present in day-7 cultures
initiated with these cells (Fig 3). The
Linc-kitLo population was the most
heterogeneous with respect to maturity. At both day 4 and day 7 some
B-lineage progeny from Linc-kitLo
cultures clearly overlapped those from the less mature
Linc-kitHi and the more mature
Linc-kit precursors. However,
many B-lineage cells generated in
Linc-kitLo cultures seemed to represent
intermediates in maturity. These cells only reached Fraction C by day
7, unlike the progeny of Linc-kit
cells. Furthermore, they had not progressed beyond Fraction A or
Fraction B by day 4. These data reflect a trend in maturity observed in
numerous experiments in which we assessed expression of CD19 and BP-1
at time points from days 2 to 7 (data not shown).
Therefore, although all precursor populations exhibited heterogeneity,
the majority of B-lineage cells produced in
Linc-kit cultures reached the
more mature phenotype sooner than those generated in
Linc-kitLo or
Linc-kitHi cultures. Likewise, the
majority of B-lineage progeny from
Linc-kitLo cultures attained the more
mature phenotype before those produced by
Linc-kitHi cultures. This pattern is
generally consistent with a progressive loss of c-kit corresponding to
increased maturity in Lin B-lineage precursors,
though downregulation of c-kit is not precisely synchronized with
degree of maturity.
Reduction of c-kit in precursor populations corresponds to increased
lymphoid-lineage restriction.
During the progression from stem cell to B lineage, there is a
progressive loss of the potential to generate the other hematopoietic cells. The above studies suggested that heterogeneity in maturity existed in each of our Lin B-lineage precursor
populations. As a measure of the relative distribution of more and less
mature B-lineage precursors, we examined the frequencies of bipotential
(lymphoid/myeloid) and lymphoid-restricted cells within our precursor populations.
The Linc-kitHi population, which is
enriched for stem cells and multipotential progenitors, has been shown
to generate B-lineage cells in bulk coculture with stromal cells and
IL-7.21 We found that these cultures also supported
differentiation of myeloid cells. Single cells from each of our
Lin precursor populations were sorted directly onto
stromal cells supplemented with IL-7 and assessed for growth at 10 to
13 days. Randomly selected clones were stained for expression of CD45R, CD19, and Mac-1. This allowed determination of cloning frequencies for
Lin precursors capable of generating only B-lineage
(CD45R+ CD19+) cells, only myeloid-lineage
(Mac-1+) cells, or cells of both lineages. Results from
these experiments are shown in Table 2.
The three precursor populations had similar total cloning frequencies
(ranging from 1/3.1 to 1/4.7). Cells capable of giving rise to the B
lineage, including lymphoid-only cells and bipotential B-lineage
precursors, were more frequent in the
Linc-kitLo population (1/17) than in the
Linc-kit subset (1/34) or the
Linc-kitHi subset (1/100). Among
clonable Linc-kitLo B-lineage
precursors, bipotential cells and lymphoid-only cells were equally
common (each type of precursor represented 10% of clonable cells). In
contrast, clonable Linc-kit
B-lineage precursors were much more frequently lymphoid-only (7.6% of
cells that cloned) than bipotential (1.5% of cells that cloned). Both
types of B-lineage precursors were rare in the
Linc-kitHi population, though
Linc-kitHi BM generated B-lineage cells
readily in bulk cultures (Fig 2). Myeloid-only precursors were common
in all three subsets, though their frequency was highest in
Linc-kitHi cells.
These data showed that loss of c-kit corresponds with increasing
lymphoid restriction in Lin B-lineage precursors.
Although more mature lymphoid-committed cells represented about half of
the Linc-kitLo B-lineage precursors,
they comprised most of those that were Linc-kit. This was consistent
with our observations that precursors giving rise to the most mature
B-lineage progeny in a finite culture period were concentrated in the
Linc-kit subset (Fig 2 and 3).
Taken together, our data suggest a model of B-lineage differentiation
in which TdT expression begins as c-kit is downregulated in
Linc-kitHi cells, with progressive
loss of c-kit corresponding to increasing maturity and lymphoid
commitment (see Fig 7 below).
Many of the earliest CD45R+ cells generated in
culture lack c-kit.
B-lineage precursors form an expanding pool of cells within BM and we
would expect to see a much larger population of
c-kit TdT+ precursors than those found
in the Lin category. Both the distribution of
TdT+ cells (Table 1) and the frequencies of
lymphoid-committed cells (Table 2) suggest that the majority of
CD45R lymphoid-committed B-lineage precursors are
contained within the Linc-kitLo subset
of BM. Because both the Linc-kitLo and
Linc-kit populations contained
B-lineage precursors on the verge of acquiring CD45R, we wondered if
acquisition of CD45R might be occurring at the transition from
c-kitLo to c-kit. If this were the case,
then most of the earliest lymphoid-committed, c-kit
B-lineage precursors would have been eliminated from our stringently gated Lin population because they had begun to
express CD45R.
Expression of c-kit has been thought to be a feature of early B-lineage
precursors and pro-B cells.10,14-17 However, we showed that
progressive loss of c-kit corresponded to increased maturity (Fig 3)
and lymphoid commitment (Table 2) in Lin B-lineage
precursors. This raised the interesting question of whether c-kit is
lost from many B-lineage cells before or during CD45R acquisition, much
earlier than previously believed. Therefore, we examined c-kit on early
CD45R+ cells generated in culture from
Lin precursor populations
(Fig 4).
Linc-kitHi cultures did not generate
CD45R+ cells until day 3 of culture and all of them were
CD19 (Fig 2 and data not shown). About two thirds of
these cells totally lacked c-kit (Fig 4) and the remainder expressed
the marker at low levels. Thus, the majority of newly generated
B-lineage cells from cultures initiated with very early
Linc-kitHi precursors no longer
expressed c-kit.
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| Fig 4.
Many newly generated CD45R+ cells lack
c-kit . Linc-kitHi,
Linc-kitLo, and
Linc-kit cells were sorted from BM and
placed in stromal cell free culture containing IL-7, FL, SCF, and
stromal cell conditioned medium. Cells were harvested at days 1 to 3 and percentages of CD45R+ CD19 cells
expressing c-kit at each time point generated from each starting
population are represented in the bar graph. (CD45R+
cells were found to express c-kit at low levels.) Data reflect
means±SE from three independent experiments with pooled marrow from 8 or 10 mice. The asterisks indicate that no B-lineage cells were
generated before day 3 in cultures initiated with c-kit+
cells.
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Cultures initiated with Lin cells that were
c-kit or c-kitLo produced
CD45R+ CD19 cells by day 1 (Fig 2) and
CD45R+ CD19+ cells by day 2 (data not
shown). However, more of the cells in Linc-kit than in
Linc-kitLo cultures coexpressed CD19 at
each time point (Fig 2 and data not shown). Of those CD45R+
CD19 progeny generated at day 1, approximately one
third from Linc-kitLo and two thirds
from Linc-kit precursors
completely lacked c-kit (Fig 2). At time points beyond day 1, we gated
on CD45R+ CD19 cells to insure that we
were assessing the earliest B-lineage cells and found that
approximately two thirds of them were c-kit (Fig 4)
. The more mature CD45R+ CD19+ populations were
similar in this respect (data not shown). It is interesting to note
that a third of the B-lineage progeny of Linc-kit precursors expressed
c-kit at low levels (Fig 4), apparently reacquiring this marker along
with CD45R.
In summary, two thirds of the earliest CD45R+
CD19 B-lineage cells generated in culture had
already lost, or were losing, c-kit as they acquired CD45R, whereas the
remaining third expressed c-kit at low levels. A similar pattern of
c-kit expression was observed on the more mature CD45R+
CD19+ B-lineage precursors. Thus, it appears that many
B-lineage precursors acquired CD45R during the transition from
c-kitLo to c-kit. However, these surface
marker changes were not perfectly synchronized; some cells lost c-kit
before CD45R was acquired and others afterwards. A fraction of
B-lineage cells generated from
Linc-kit cultures reacquired
c-kit, raising questions about modulation of this marker in the pro-B compartment.
The majority of early CD45R+ pro-B cells in murine
BM are c-kit.
After determining that most early CD45R+ cells generated in
culture were c-kit, we wanted to see if this was
also the case for comparable cells in marrow. We isolated the earliest
CD45R+ pro-B cells that had not progressed to Hardy's
Fraction C or Melchers' Pre-B II, while excluding NK precursors or NK
cells that expressed CD45R. To achieve this, we sorted
CD45R+ CD43+ cells that lacked expression of
BP-1, CD25, NK1.1, and surface IgM (Fig
5A,B). We then assessed expression of c-kit in this population that we
refer to as early CD45R+ pro-B cells. Over three fourths of
these cells lacked c-kit, whereas the remainder expressed the marker at
low levels (Fig 5C and Table 3). This
parallels the pattern observed in early B-lineage cells produced in
culture (Fig 4).
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| Fig 5.
The majority of early CD45R+ pro-B cells in
BM are c-kit. The boxed region of Panel A shows
CD45R+CD43+ cells in lymphoid-enriched BM,
from which BP-1 CD25 NK1.1
sIgM (B) cells were sorted. These gating criteria
allowed isolation of early CD45R+ pro-B cells that were
subsequently stained for surface c-kit, fixed, permeabilized, and
stained for intranuclear TdT. Expression of c-kit (C) and TdT (D) in
this costained population is shown. Panel E shows c-kit expression in
the gated TdT+ cells within CD45R+
CD43+ BP-1 CD25
NK1.1 sIgM cells. The frequencies of
these subsets in BM are shown in Table 3. Means are from two
independent experiments performed on pooled marrow from five or seven
mice.
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Park and Osmond8,22-23 proposed that TdT is an intranuclear
marker of the earliest pro-B cells. Examining TdT+ cells
that express the early CD45R+ pro-B cell-surface phenotype
provides another way of investigating c-kit expression. We
sorted early CD45R+ pro-B cells and then costained for TdT
and c-kit. About three fourths of early CD45R+ pro-B cells
expressed TdT (Fig 5D and Table 3). We gated on the TdT+
cells within the population and found that over two thirds were c-kit (Fig 5E and Table 3). Therefore,
c-kit cells comprised the majority of the
TdT+ early CD45R+ pro-B population.
Of TdT+ early pro-B cells, c-kit+ and
c-kit cells formed 0.48% and 1.2%, respectively,
of BM MNC (Table 3). When Lin TdT+ cells
(Table 1) and early CD45R+ TdT+ pro-B cells
(Table 3) were combined, we found that cells expressing c-kit
(including c-kitHi and c-kitLo cells)
represented 0.76% whereas c-kit cells represented
1.2% of BM MNC. Thus, the c-kit population of early
B-lineage cells is larger, as predicted by the progressive loss of
c-kit with increased maturity in B-lineage precursors.
Both c-kit and c-kit+ cells
contribute to expansion of the pro-B compartment and give rise to B
lymphocytes.
A substantial minority of early CD45R+ pro-B cells either
retained or reacquired c-kit. It is therefore important to ask if this
receptor conferred any special growth or differentiation advantage. We
examined the role of c-kit+ and c-kit
early pro-B cells in expansion of the pro-B compartment by looking at
the ability of these cells to proliferate in culture. Single c-kit+ or c-kit early CD45R+
pro-B cells were sorted directly onto stromal cells supplemented with
IL-7. The average cloning frequency of c-kit+ cells (1/4)
was higher in this assay than that of c-kit cells
(1/21) (Table 4). Using the frequencies of
c-kit+ and c-kit cells in the early
CD45R+ pro-B compartment of BM MNC (Table 3), we calculated
that the frequency of clonable c-kit+ cells was about one
and one half times that of c-kit cells (Table 4).
In semi-solid agar assays, in which only IL-7 was present, the cloning
frequency of the c-kit+ early CD45R+ pro-B
population (1/48) was only about twice that of the
c-kit cells (1/89) (Table 4). When the distribution
of the c-kit+ and c-kit cells within the
early CD45R+ pro-B population of BM MNC was taken into
account (Table 3), we calculated that almost twice as many of the
B-lineage cells clonable in IL-7 alone were c-kit
(Table 4).
These data show that c-kit+ early CD45R+ pro-B
cells had a cloning advantage in the combined presence of IL-7, factors
produced by stromal cells (including SCF, the ligand for c-kit), and
stromal cell contact. However, this advantage was greatly reduced in
semi-solid agar assays in which stromal cells were not present. Given
the abundance and cloning frequency of c-kit cells
within the early CD45R+ pro-B compartment, the contribution
of these cells to the expanding B-cell compartment is likely close to
that of c-kit+ cells.
We next addressed the question of whether c-kit |
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ABSTRACT
INTRODUCTION