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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3203-3209
Four-Color Flow Cytometric Investigation of Terminal Deoxynucleotidyl
Transferase-Positive Lymphoid Precursors in Pediatric Bone Marrow:
CD79a Expression Precedes CD19 in Early B-Cell Ontogeny
By
Michael N. Dworzak,
Gerhard Fritsch,
Gertraud Fröschl,
Dieter Printz, and
Helmut Gadner
From the Children's Cancer Research Institute, St Anna Kinderspital,
Vienna, Austria.
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ABSTRACT |
Terminal deoxynucleotidyl transferase (TdT)-positive cells in human
bone marrow (BM) are a phenotypically inhomogeneous population of
precursor cells. In their majority, these TdT+ cells are
unambiguously committed to the B lineage, as evidenced by CD19
expression. However, TdT+ precursors that lack CD19 also
exist and these may encompass a differentiation potential for the B as
well as for other lineages. Because recent data suggested that CD19
expression is not the earliest differentiation event in B-cell
ontogeny, we sought to reevaluate TdT+ lymphoid
precursors in pediatric BM to define the phenotypic denominator of
B-lineage affiliation upstream of CD19. Using four-color flow
cytometry, we focused on the assessment of the CD79a antigen, which is
highly B-cell specific and which may also be expressed very early in
B-cell ontogeny. We found that a majority of TdT+ cells
coexpressed CD19 and CD79a in addition to CD10 and CD34, whereas, in
all investigated samples, some TdT+ precursors lacked
CD19 but expressed CD79a, which suggestively indicates also their
B-lineage affiliation. In contrast to the CD19+
precursors, which were usually CD10hi and
CD79b+, these CD19 CD79a+
putative B-cell precursors preferentially expressed CD10 at low levels
and were CD79b+ in only 41%. About 17% of these
TdT+CD19CD79a+ precursors
also coexpressed CD33 and CD7, but not myeloperoxidase, CD14, or
cytoplasmic CD3, which is discussed in the light of cellular activation
rather than lineage promiscuity. Our data confirm that the earliest
differentiation stages of B cells can be dissected upon expression of
the lineage antigens CD79a and CD19 and imply that CD79a is earlier
expressed than CD19. This raises the chance to follow the sequential
events heralding B-cell commitment in the most immature precursors by
correlating phenotypic and genetic differentiation markers.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THE SEQUENCE OF antigen acquisition
during early B-cell development has been a recurrent issue of
investigation. From studies of neoplastic cells it was initially
suggested that CD19 is the first B-cell antigen to appear on terminal
deoxynucleotidyl transferase (TdT)-positive lymphoid progenitors, thus
heralding lineage-commitment.1,2 Investigations of normal
human bone marrow (BM) led to the conclusion that, on physiologic
TdT+/CD34+ precursors, CD19 and CD10 appear
essentially at the same time.3-5 Bright CD10 expression was
recognized typical for these earliest B-cell precursors
(BCPs).3,4 More recent publications documented that the
proportions of CD10+ cells exceeded those of
CD19+ cells in CD34+/TdT+ precursor
populations.6-9 CD19+ B cells could be grown
from such CD10+CD19 progenitors derived
from fetal liver as well as from adult BM, which corroborates the
current concept that CD10 is the upstream marker in early B-cell
ontogeny.10,11 However, CD10 is not B-cell
specific,12 and it has been documented that the
CD10+CD19 progenitor pool harbors also
cloning potential for T, natural killer (NK), and
dendritic cells.11 Quantitative relations of these
different capacities have remained elusive, not at least due to the in
vitro culture conditions.
In an independent approach aimed at the assessment of CD99 expression
in B-cell development, we have recently observed that early
(CD34+/TdT+) lymphoid precursors in the BM can
be dissected according to their levels of CD10
expression.13 Aside from a major
CD10hiCD19+ population, a CD10lo
subset was found that only partially coexpressed CD19. The
CD10loCD19 cells were considered to
correspond to the CD10+CD19 progenitors
claimed in the above-mentioned studies. We speculated that, due to
their uniformity in high CD99 expression and in light scattering
patterns and due to their regeneration kinetics in parallel with B-cell
outgrowth after chemotherapy, the majority of the CD10lo
progenitors may be committed to the B lineage, irrespective of CD19
expression.14 To test this hypothesis, we reevaluated in the present study the marker coexpression patterns of TdT+
progenitors in BM. Special interest was dedicated to the analysis of
CD79a expression in these cells, because this marker has been claimed
to be of extraordinary B-lineage specificity.15-17 Our data
document for the first time that many of those TdT+
progenitors that lack CD19 express CD79a in addition to CD10, which
corroborates the concept that these cells are committed to the B
lineage and which implies that CD79a precedes CD19 in the earliest
differentiation events of B-cell ontogeny.
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MATERIALS AND METHODS |
Sample description.
BM samples were obtained from 9 children: n = 5 were BM
donors for transplantation (2 years and 7 months, 3 years and 7 months, 8 years and 11 months, 12 years and 4 months, and 12 years and 11 months), n = 1 was a boy (3 years and 10 months old) after autologous
peripheral stem cell transplantation for neuroblastoma without current
evidence of disease, n = 1 was a girl (1 year and 10 months old)
regenerating after chemotherapy for Langerhans cell histiocytosis (LCH)
not involving BM and without signs of disease-associated hematological
dysfunction, n = 1 was a boy (2 months old) suffering from
neuroblastoma without BM infiltration, and n = 1 was a girl (4 years
and 1 month old) with bcr/abl-positive common acute lymphoblastic
leukemia (ALL) that was in remission at the time of immunologic
investigation (polymerase chain reaction-negative for bcr/abl). Data
concerning these patients were obtained from the Austrian study centers
of the international Berlin-Frankfurt-Münster (BFM) study group,
of the international LCH trials, and of the national neuroblastoma
study NB-94.
Antibodies.
Fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-,
allophycocyanin (APC)-labeled, or unconjugated pure monoclonal
antibodies (MoAbs) were used: CD3 (UCHT1-PE), CD10 (SS2/36-PE), CD14
(TÜK4-PE), CD19 (HD37-PE, pure), CD33 (WM54-PE), CD79a
(HM57-PE, pure), and CD79b (SN8 pure) were all from Dako (Glostrup,
Denmark); CD3 (SK7-APC), CD19 (SJ25C1-APC), and CD34 (HPCA2-PE, -APC)
were from Becton Dickinson (BD; Sunnyvale, CA); CD7 (3A1-RD1-PE) was
from Coulter Immunology (Krefeld, Germany); antihuman TdT (H
Tdt-1,3,4-FITC) was from Immunotech (Marseille, France); and antihuman
myeloperoxidase (H-43-5-PE) was from An der Grub (Kaumberg, Austria).
Murine irrelevant isotype-matched fluorescent MoAbs were purchased from
Dako and BD.
Immunofluorescence staining procedure.
Mononuclear cells (MNCs) were isolated from the BM specimens by density
gradient centrifugation and washed twice in RPMI 1640 medium containing
2% fetal calf serum (FCS; both from GIBCO, Paisley, UK) before
immunofluorescence labeling. Staining protocols used four-color
investigations in all experiments, except for the cell sorting (see
below). All antibodies were used at concentrations titrated for optimal
staining. Cellular permeabilization (for the investigation of CD79a,
CD79b, TdT, myeloperoxidase, and cytoplasmic CD3) was usually performed
as the first step of a five-step labeling cascade, because CD79a
detection was performed using preferentially the unconjugated HM-57
MoAb. In brief, 5 to 10 × 105 MNCs per analysis were
permeabilized as recently described using a commercially available
formaldehyde-based erythrocyte lysing solution (BD) supplemented with
Triton-X100 (0.015%).14 The concentration of the latter
had been titrated for optimal permeabilization together with a good
preservation of light scattering and fluorescence properties. Next, the
cells were incubated with the unconjugated MoAbs and then with
biotinylated rabbit F(ab )2 antimouse Ig antibodies (from Dako). To exclude nonspecific staining via unsaturated binding sites of the second step reagent, this was followed by incubation with
unlabeled irrelevant murine IgG1 (MOPC21; Sigma, St Louis, MO). Without washing, the directly labeled MoAbs were then added simultaneously and together with streptavidin-Peridinin Chlorophyll Protein (PerCP; BD). Murine irrelevant isotype-matched fluorescent MoAbs (from Dako as well as from BD) were included in the staining protocols as negative controls. Antibody incubations were performed at
4°C in the dark over 30 minutes and were followed by washing steps
using phosphate-buffered saline (0.13 mol/L NaCl, 7 mmol/L Na2HPO4, 3 mmol/L
NaH2PO4) with 2% FCS. Before flow cytometry, cell suspensions were passed through a 30-µm nylon mesh (Swiss Silk
Bolting Manufacturing Co, Zurich, Switzerland).
Flow cytometry.
We used a FACSCalibur (BD) equipped with an Argon laser tuned to 488 nm
and a diode laser with 635nm emission. Calibration beads (Calibrite;
BD) were routinely used for monitoring and optimizing the instrument
settings. Data were acquired with the CELL Quest software (BD). Forward
light scattering (FSC), transformed orthogonal light scattering
(T-SSC), and fluorescence signals (FL-1-FITC, FL-2-PE, FL-3-PerCP, and
FL-4-APC) were stored in listmode data files. Samples were first
analyzed conventionally (30,000 cells), and then gated acquisition was
performed using the same material for a better definition of the small
TdT+ subset of MNCs. Live gates were set in the appropriate
fluorescence versus SSC correlations and gated events acquired to
maximum yield (mean, 8,550; range, 1,750 to 30,000; n = 9). All data
were analyzed using PAINT-A-GATE software (BD).
Cell sorting was performed on a FACStar Plus (BD). Using a
CD19/CD10/CD34 staining, CD10+CD34+ cells were
sorted into the CD19+ and CD19
fractions. Correspondence of these cell populations to the
CD79a+TdT+CD19+ or cells
was proven in parallel conventional four-color analyses of the same
material using various MoAb combinations. Cytospin preparations of the
sorted subsets were made on a Shandon Cytospin II (Southern Product
Ltd, Astmoor, UK) at 30g for 10 minutes. Slides were stained
with Pappenheim stain.
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RESULTS |
Analysis of TdT+ precursors in the BM.
The TdT expression in the BM of 9 probands was evaluated after
fixation/permeabilization of MNCs using a triple-pool of FITC-labeled MoAbs (IgG2a and IgG1). Pooled irrelevant isotype-matched FITC MoAbs
were used to correct for background fluorescence in the FITC channel. A
mean of 3.6% of MNCs (on event basis in dot plots) were
TdT+ (range, 0.6% to 13.6%). To assess the proportions of
TdT+ precursors coexpressing CD10, CD19, CD34, and CD79a,
gated TdT+ events were acquired in all 9 experiments. The
background positivity in the fluorescence channels used for
quantification of marker coexpression was also evaluated with
appropriately labeled, irrelevant, isotype-matched control MoAbs. The
proportions of cells positive for CD34, CD10, and CD79a were in the
same range, whereas slightly less TdT+ cells were found to
be CD19+. A mean of 86% (range, 75% to 93%) of
TdT+ precursors coexpressed the stem cell antigen CD34,
87% (range, 66% to 96%) were CD10+, and 84% (range,
65% to 93%) were CD79a+, but only 68% were
CD19+ (range, 48% to 92%). It is noteworthy that, even in
individual BM samples, the proportions of cells positive with CD79a (or
CD10) were always higher than those positive with CD19, regardless of whether they were derived from normal or postchemotherapy donors (Table 1). This implied that CD19 was not
expressed on all CD79a+TdT+ precursors. To
investigate this correlation with respect to the earliest phenotypic
signs of B-lineage commitment, we studied, as a next step,
TdT+ cells that coexpressed CD79a or CD19 (data are
presented in the following section). However, according to the aim of
our study, we did not investigate in detail the few TdT+
cells that lacked signs of B-lineage commitment
(CD79aCD19). Most of the latter
cells were TdTdim, CD34hi and lacked
myeloperoxidase (MPO), but were positive with CD33, thus resembling
myeloid precursors. Some TdTdim precursors (which lacked
CD19 and CD79a) also coexpressed CD7. In another small proportion of
cells, TdT expression was ambiguous by being marginal to the negative
area. These cells stained strongly with CD33 as well as MPO, but lacked
CD34, and were considered to be myelomonocytic.
Analysis of TdT+ precursors with respect to
CD79a and CD19 expression.
CD19 was expressed on only 78% of gated
CD79a+TdT+ precursors (range, 61% to 92%)
from 9 BM specimens. Reversely, de facto, all
CD19+TdT+ BCPs were CD79a+ (mean,
97%; range, 91% to 100%). Next, we assessed the antigen coexpression
patterns of these two putatively B-lineage-committed CD79a+TdT+ precursor subsets as dissected by
CD19 expression. These analyses were also performed on gated
CD79a+TdT+ populations. Quantitative data on
the marker correlations are given in Table
2, and a comprehensive phenotypic analysis is shown in
Fig 1. With respect to CD34
expression, there was no difference between the CD19+ and
CD19 subsets, because almost all
CD79a+TdT+ precursors were uniformly
CD34+. CD10 was also expressed on almost all of the cells,
but not homogeneously, which is in accordance with recent
observations.13,14 Most
CD79a+TdT+ precursors were CD10hi,
and fewer cells expressed the antigen only at low levels
(CD10lo). In coanalyzing the
CD79a+TdT+ cells for CD19 and levels of CD10,
we found that the CD19 precursors preferentially
resided in the CD10lo subset (Fig 1). In detail, 61%
(range, 33% to 78%) of
CD10loCD79a+TdT+ cells, but only
19% (range, 4% to 39%) of CD10hi precursors, lacked
CD19.
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| Fig 1.
Four-color flow cytometric analysis of
TdT+ precursors with respect to expression of CD79a and
CD19. BM MNCs of a healthy girl (2 years and 7 months old) were
prepared from a specimen collected at BM donation for her diseased
sister. Cells were stained with CD19, CD79a, and TdT, together with
either CD10, CD34, CD33, or CD7. Isotype controls were also included in
the experiment. First, 30,000 total events of each individual sample
were acquired. Next, live gates were set in parameter correlations
engaging T-SSC, TdT, and CD79a, and approximately 10,000 TdT+CD79a+ events were acquired from the
same material. Dot plots of relevant parameter correlations
(logarithmic scale, except for T-SSC: linear scale) show aspects of
total MNCs in row A (large plots). The small inserted plots in A
visualize the respective isotype controls for each antigen. The large
dot plots of rows B and C show gated events, whereas the small inserted
plots display the same marker correlations in ungated mode. Only
TdT+ precursors are shown in colors: the subset
characterized by coexpression of CD79a and CD19 is depicted in green,
whereas CD79a+ precursors lacking CD19 are painted red.
The few TdT+ cells that lack both CD79a and CD19 are
painted black (see A). All other TdT cells appear grey.
Note that CD19CD79a+TdT+
cells (red) are CD34+, display slightly elevated TdT
expression as well as T-SSC properties, and express CD10 at a lower
level compared with CD19+ precursors (green). Weak CD33
positivity can also be seen on some cells of the former subset, whereas
CD7 expression is very rare in either subset of this sample.
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We analyzed the CD79a+TdT+ precursor subsets,
dissected by CD19 expression, for coexpression of myeloid and T-cell
markers (CD33, MPO, CD14, CD7, and cytoplasmic CD3). As shown in detail
in Table 2, we could hardly recognize expression of these markers on
CD19+ populations. However,
CD19CD79a+TdT+ precursors
showed significant coexpression of CD7 and, in particular, of CD33 (see
Fig 1), but not of MPO, CD14, and CD3. The expression levels of the
former two antigens were usually low.
In three experiments, we also included an MoAb to CD79b. We found that
this antigen was also expressed in the cytoplasm of a proportion of
TdT+ cells (mean, 57%; range, 51% to 65%). Whereas de
facto all CD79b+TdT+ cells were
CD79a+ (mean, 95%; range, 94% to 96%) and
most were CD19+ (mean, 88%; range, 78% to
98%), only a proportion of CD79a+TdT+
precursors were CD79b+ (mean, 69%;
range, 62% to 82%). In detail, the
CD19+CD79a+TdT+ BCPs were mostly
CD79b+ (mean, 85%; range, 78% to
92%), whereas the
CD19CD79a+TdT+ precursors
were CD79b+ in only 41% (range, 35% to 47%).
We found also that the CD19+ and CD19
subsets of the CD79a+TdT+ precursors differed
in their light scattering properties as well as in their morphology
(Fig 2). To prepare cytospin preparations for microscopic analysis, MNCs of one BM sample were stained with CD10,
CD19, and CD34, and the CD10+CD34+ cells were
sorted by flow cytometry into a CD19+ and a
CD19 fraction. CD10 and CD34 were used as surrogate
markers substituting for CD79a and TdT to avoid the cellular fixation
and permeabilization step that is obligatory for CD79a and TdT
detection. We proved the correspondance of these populations to
CD79a+TdT+CD19+ or cells by
parallel four-color analyses, finding that similar proportions (1) of
TdT+CD19 cells were CD10+ or
CD79a+ (23% and 25% of TdT+) and (2) of
CD79a+CD19 cells were CD34+
or TdT+ (74% and 77%). With respect to morphology, the
majority of the CD19+ precursors were of typical small
lymphocyte size (also defined by their very low FSC and T-SSC
properties), had very scant cytoplasm, had relatively dense chromatin,
and had no or inconspicuous nucleoli. In contrast, the
CD19 precursors were a relatively homogeneous
population of intermediately sized, blastoid cells with large nuclei
(in part lobulated, possibly as a cytospin artifact), had evenly
dispersed, relatively fine chromatin with prominent nucleoli, and
displayed a comparatively wider, basophilic cytoplasm and focal
juxtanuclear areas of clearing. In keeping with their morphology, these
CD19 precursors showed elevated FSC and T-SSC
properties as compared with the majority of the
CD19+CD79a+TdT+ cells.
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| Fig 2.
Light scattering properties and morphologic
appearence of CD79+TdT+ precursors that
express or lack CD19. A correlation of the FSC and T-SSC properties of
a BM sample stained with CD19, CD79a, and TdT is shown in dot plot A. TdT+ precursor cells that express CD79a are depicted in
colors: the CD19+ subpopulation of these cells is painted
green, whereas
CD19CD79a+TdT+ cells appear
red. Histograms of the same material, correlating the FSC (B) or the
T-SSC (C) in the x-axis with a relative measure of the cell number
(y-axis) show only gated TdT+CD79a+ cells.
Note that CD19CD79a+TdT+
cells (red) exhibit comparatively higher FSC and SSC properties than
the majority of CD19+ precursors (green). To study the
morphology of these subsets, the same sample was stained with CD10,
CD19, and CD34, and the CD10+CD34+ cells
were sorted into a CD19+ and a CD19
fraction. CD10 and CD34 were used as surrogate antigens substituting
for CD79a and TdT to avoid cellular alterations by the
fixation/permeabilization reagents (the correspondance of these markers
was proven in parallel experiments). Pappenheim-stained cytospin
preparations of sorted cells are shown in (D; CD19+
subset) and (E; CD19 subset). Note that the majority of
CD19+ precursors are small lymphoid cells, whereas
CD19 precursors are intermediately sized blasts with
large nuclei and prominent nucleoli and display a wider and basophilic
cytoplasm with focal juxtanuclear areas of clearing.
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DISCUSSION |
Recent evidence suggests that expression of CD19 is not the first
differentiation event in human B-cell ontogeny. B cells have been grown
from progenitors expressing CD10 but lacking CD19,10,11 and
activation of the IgH locus, a prerequisite for rearrangement, has been
shown to precede CD19 expression.18 However, CD10 is not
restricted to B cells,12 and T, NK, and dendritic cells have also been cloned from CD10+CD19
progenitors.11 Hence, the human precursor population in
which B-lineage commitment first occurs is still phenotypically ill defined. Therefore, we sought to subcharacterize TdT+
progenitors using CD79a, which seemed a good candidate antigen to
specifically delineate early B-lineage affiliation. The CD79 antigen is
a heterodimer containing one subunit each of the MB-1 (CD79a) and B29
(CD79b) molecules that are noncovalently associated with Igs on the
surface membrane of mature B cells.19,20 By linking the
antigen-recognition structure to intracellular protein tyrosin kinases,
CD79 exerts a key role in signal transduction and cellular activation
of B cells.21 Unique in function, the antigen has been
claimed to be completely B-cell specific.15-17,22,23 Specificity was related particularly to the protein level, because a
few myeloid and T-cell lines were found to contain CD79a/CD79b RNA
transcripts, but no antigen.17 To date, the only
observation challenging the B-cell specificity of CD79 was a report on
positivity in paraffin sections of cases of acute promyelocytic
leukemias.24 However, these data were not corroborated by
investigations of the Ig rearrangement status of these leukemias. In
another study dealing with acute myeloid leukemias, 5 of 77 cases were
found to be CD79+, all of which showed Ig rearrangements
and were therefore considered biphenotypic.16 CD79 showed
the highest correlation with molecular data and, in this regard, was
found to be superior to other B-cell antigens such as CD19 or CD22.
Along with the latter finding, the CD79 heterodimer chains
seem therefore the most specific markers of B-lineage commitment
currently known. Expression of both the CD79a and CD79b genes starts
early in B-cell development, suggestively even before Ig gene
rearrangement, as indicated by studies in the mouse and by experiments
with three human cell lines with germline Ig genes.17,23,25
In the mouse, both the CD79a and CD79b genes have been shown to be
expressed on the RNA level before upregulation of CD19 in the earliest
BM B-lineage cells.26 However, such data on human BCPs are
lacking. Only one recent study dealt with the CD79 expression in normal
human BM.15 It showed that the antigen is expressed, before
cytoplasmic µ-chains, already on TdT+ BCPs, but concluded
that CD79 may not be expressed as early as CD19, although evidence for
the latter issue was weak.
In the present study, we focused on the analysis of CD79a, which has
been considered to be expressed earlier in human BCPs than
CD79b.27 We found that many TdT+ precursors in
the BM that lack CD19 express CD79a, which strongly indicates B-lineage
commitment independently of CD19. Conspicuously, it may also indicate
that CD79a precedes CD19 in human B-cell ontogeny. Alternatively, the
existence of TdT+CD79a+CD10+
progenitors that are CD19+ or CD19 might
reflect parallel pathways of differentiation, as postulated for B
lymphopoiesis.28 However, due to (1) phenotypic patterns indicative of a phenotypic drift
(CD19CD10loCD34+ > CD19+CD10loCD34+ > CD19+CD10hiCD34+) in early
BCPs,13,14 (2) characteristic patterns of CD99 expression in these populations,13 (3) a sequential evolution of
CD19+ B cells from CD19CD10+
progenitors,10,11 and (4) other (molecular) signs heralding definitive B-lineage commitment before CD19 expression,18
we find the first interpretation more likely. By examining the
molecular status of the Ig genes in these immature B-cell stages, as
phenotypically dissected on the basis of CD79a and CD19 expression, it
should now be possible to order the sequence of molecular
differentiation events of early B cells upstream to what has recently
been published.18,29 This may also formally prove that
CD79a precedes CD19 in B-cell ontogeny.
It is noteworthy that we document also that CD10 is not homogeneously
expressed on TdT+ precursors. Given the proposed new
concept of phenotypic maturation steps in early BCPs, our data may
indicate that CD10 is expressed first at low levels in the suggestively
most immature BCPs
(TdT+CD79a+CD19), followed
by gradual upregulation to high levels in more differentiated TdT+CD19+ BCPs. In the classical studies on
early B-cell differentiation, CD10 was found to be expressed first at
high levels, followed by downregulation along with loss of expression
of TdT and CD34.3,4 However, these studies only considered
the numerically more prominent (and suggestively more mature)
CD10hiCD19+TdT+ BCPs, whereas the
CD10loTdT+ putative BCPs, which are
characterized by CD79a expression rather than by CD19-positivity, have
elapsed description not at least due to their paucity. Hence, our data
do not principally reverse the established patterns of CD10 expression
in early B-cell development, but rather add a detail upfront. With
respect to this proposed sequence of phenotypic maturation steps, we
found also that most CD19+CD79a+TdT+ cells expressed
CD79b, whereas the latter antigen was positive in only a proportion of
the CD79a+TdT+ BCPs that lacked CD19.
Essentially all CD79b+TdT+ cells were
CD79a+. Preliminarily (because derived from only 3 experiments), these data indicate that, in human B-cell ontogeny, CD79b
is expressed later than CD79a, but before CD19. This finding contrasts
suggestively with the situation in the mouse, where CD79b expression,
at least on the RNA level, seems to precede that of CD79a and
CD19.26
Surprisingly, we found also that the myeloid and T-cell markers CD33
and CD7 were expressed at low intensity on a proportion of the
CD19CD79a+TdT+ putative
BCPs, which could be regarded as a challenge for the unambiguous
lineage-assignment of this subset. However, cross-lineage expression,
especially of myeloid markers, is a well-recognized phenomenon in
malignant and normal B lymphopoiesis. CD13 and CD33 are frequently
expressed on BCP-ALLs.30,31 Normal immature CD19+ BCPs as well as CD34+/TdT+
lymphoid progenitors in the BM have also been reported to express these
myeloid antigens.7,28,32-35 Likewise, CD7 and other early T-cell antigens lack lineage fidelity, because they may also be expressed on myeloid as well as on B-lineage
precursors,28,36 although expression of CD7 on
CD19CD34+/TdT+ BM
progenitors has primarily been interpreted as evidence for T-lineage
affiliation.8,37-39 Interestingly, cloning experiments proved a T-cell differentiation capacity in the CD34+
progenitor compartment of adult BM40,41 and targeted this
potential to progenitors that expressed as well as that lacked T-cell
markers40,42 and to CD10+Lin
progenitors.11,43 However, there are no absolute numbers in the literature that relate this T-cell differentiation capacity to the
pool of CD34+/TdT+ progenitors devoid of CD19
expression, and CD7 was recently discouraged as a reliable criterion
for an unambiguous delineation of a genuine T-precursor subset in
BM.36,39 Hence, positivity of CD33 and CD7 on some of the
putative very immature BCPs, which are characterized by lack of CD19
and by expression of the highly lineage-specific antigen CD79a, may not
per se preclude B-lineage affiliation. Alternatively, it may just
indicate cellular activation, as has recently been considered for both
antigens, CD33 and CD7.36,44 Lack of expression of the more
specific lineage markers of myeloid and T cells, MPO, CD14, and
cytoplasmic CD3, in the
CD19CD79a+TdT+ putative BCPs
corroborates this interpretation.
In summary, we believe that a subcharacterization of TdT+
progenitors with the CD79a marker should allow to order the earliest differentiation events that herald B-lineage commitment. It may also
permit a more specific dissection of B-lymphoid differentiation capacity in the BM from that of the T-lineage and from multilineage potential.
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FOOTNOTES |
Submitted February 4, 1998;
accepted June 21, 1998.
Address reprint requests to Michael N. Dworzak, MD, Children's Cancer
Research Institute, St Anna Kinderspital, Kinderspitalgasse 6, A-1090
Vienna, Austria; e-mail: Dworzak{at}ccri.univie.ac.at.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
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ACKNOWLEDGMENT |
The authors are grateful to the hemato-oncology, laboratory, and
anesthesia teams of the St Anna Kinderspital for their support and for
providing BM specimens. Our special thanks, in this regard, go to S. Juhasz, R. Kornmüller, and U. Stalze. We are also indebted to J. Regelsberger for providing the clinical data.
| |
REFERENCES |
1.
Nadler LM,
Korsmeyer SJ,
Anderson KC,
Boyd AW,
Slaughenhoupt B,
Park E,
Jensen J,
Coral F,
Mayer RJ,
Sallan SE,
Ritz J,
Schlossman SF:
B cell origin of non-T cell acute lymphoblastic leukemia A model for discrete stages of neoplastic and normal pre-B cell differentiation.
J Clin Invest
74:332,
1984
2.
Hokland P,
Ritz J,
Schlossman SF,
Nadler LM:
Orderly expression of B cell antigens during the in vitro differentiation of nonmalignant human pre-B cells.
J Immunol
135:1746,
1985[Abstract]
3.
Ryan D,
Kossover S,
Mitchell S,
Frantz C,
Hennessy L,
Cohen H:
Subpopulations of common acute lymphoblastic leukemia antigen-positive lymphoid cells in normal bone marrow identified by hematopoietic differentiation antigens.
Blood
68:417,
1986[Abstract/Free Full Text]
4.
Loken MR,
Shah VO,
Dattilio KL,
Civin CI:
Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development.
Blood
70:1316,
1987[Abstract/Free Full Text]
5.
Schmitt C,
Eaves CJ,
Lansdorp PM:
Expression of CD34 on human B cell precursors.
Clin Exp Immunol
85:168,
1991[Medline]
[Order article via Infotrieve]
6.
Saeland S,
Duvert V,
Pandrau D,
Caux C,
Durand I,
Wrighton N,
Wideman J,
Lee F,
Banchereau J:
Interleukin-7 induces the proliferation of normal human B-cell precursors.
Blood
78:2229,
1991[Abstract/Free Full Text]
7.
Saeland S,
Duvert V,
Caux C,
Pandrau D,
Favre C,
Valle A,
Durand I,
Charbord P,
de Vries J,
Banchereau J:
Distribution of surface-membrane molecules on bone marrow and cord blood CD34+ hematopoietic cells.
Exp Hematol
20:24,
1992[Medline]
[Order article via Infotrieve]
8.
Gore SD,
Kastan MB,
Civin CI:
Normal human bone marrow precursors that express terminal deoxynucleotidyl transferase include T-cell precursors and possible lymphoid stem cells.
Blood
77:1681,
1991[Abstract/Free Full Text]
9.
Farahat N,
Lens D,
Zomas A,
Morilla R,
Matutes E,
Catovsky D:
Quantitative flow cytometry can distinguish between normal and leukaemic B-cell precursors.
Br J Haematol
91:640,
1995[Medline]
[Order article via Infotrieve]
10.
Uckun FM,
Ledbetter JA:
Immunobiologic differences between normal and leukemic human B-cells precursors.
Proc Natl Acad Sci USA
85:8603,
1988[Abstract/Free Full Text]
11.
Galy A,
Travis M,
Cen D,
Chen B:
Human T, B, natural killer, and dendritic cells arise from a common bone marrow progenitor cell subset.
Immunity
3:459,
1995[Medline]
[Order article via Infotrieve]
12.
LeBien TW,
McCormack RT:
The common acute lymphoblastic leukemia antigen (CD10)Emancipation from a functional enigma.
Blood
73:625,
1989[Free Full Text]
13.
Dworzak MN,
Fritsch G,
Buchinger P,
Fleischer C,
Printz D,
Zellner A,
Schöllhammer A,
Steiner G,
Ambros PF,
Gadner H:
Flow cytometric assessment of human MIC2 expression in bone marrow thymus, and periperal blood.
Blood
83:415,
1994[Abstract/Free Full Text]
14.
Dworzak MN,
Fritsch G,
Fleischer C,
Printz D,
Fröschl G,
Buchinger P,
Mann G,
Gadner H:
Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow.
Leukemia
11:1266,
1997[Medline]
[Order article via Infotrieve]
15.
Mason DY,
Cordell JL,
Tse AGD,
van Dongen JJM,
van Noesel CJM,
Micklem K,
Pulford KAF,
Valensi F,
Comans-Bitter WM,
Borst J,
Gatter KC:
The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.
J Immunol
147:2474,
1991
16.
Buccheri V,
Mihaljevic B,
Matutes E,
Dyer MJS,
Mason DY,
Catovsky D:
mb-1: A new marker for B-lineage lymphoblastic leukemia.
Blood
82:853,
1993[Abstract/Free Full Text]
17.
Verschuren MCM,
Comans-Bitter WM,
Kapteijn CAC,
Mason DY,
Brouns GS,
Borst J,
Drexler HG,
vanDongen JJM:
Transcription and protein expression of mb-1 and B29 genes in human hematopoietic malignancies and cell lines.
Leukemia
7:1939,
1993[Medline]
[Order article via Infotrieve]
18.
Bertrand FE III,
Billips LG,
Burrows PD,
Gartland GL,
Kubagawa H,
Schroeder HW Jr:
Ig DH gene segment transcription and rearrangement before surface expression of the pan-B-cell marker in normal human bone marrow.
Blood
90:736,
1997[Abstract/Free Full Text]
19.
Reth M,
Hombach J,
Wienands J,
Campbell KS,
Chien N,
Justement LB,
Cambier JC:
The B-cell antigen receptor complex.
Immunol Today
12:196,
1991[Medline]
[Order article via Infotrieve]
20.
Hombach J,
Leclercq L,
Radbruch A,
Rajewsky K,
Reth M:
A novel 34-kd protein co-isolated with the IgM molecule in surface IgM-expressing cells.
EMBO J
7:3451,
1988[Medline]
[Order article via Infotrieve]
21.
Lin J,
Justement LB:
The MB-1/B29 heterodimer couples the B cell antigen receptor to multiple src family protein tyrosine kinases.
J Immunol
149:1548,
1992[Abstract]
22.
Sakaguchi N,
Kashiwamura S,
Kimoto M,
Thalmann P,
Melchers F:
B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties.
EMBO J
7:3457,
1988[Medline]
[Order article via Infotrieve]
23.
Hermanson GG,
Eisenberg D,
Kincade PW,
Wall R:
B29: A member of the immunoglobulin gene superfamily exclusively expressed on B-lineage cells.
Proc Natl Acad Sci USA
85:6890,
1988[Abstract/Free Full Text]
24.
Arber DA,
Jenkins KA,
Slovak ML:
CD79 alpha expression in acute myeloid leukemia. High frequency of expression in acute promyelocytic leukemia.
Am J Pathol
149:1105,
1996[Abstract]
25.
Palacios R,
Samaridis J:
Fetal liver pro-B and pre-B lymphocyte clones: Expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro.
Mol Cell Biol
12:518,
1992[Abstract/Free Full Text]
26.
Li YS,
Wasserman R,
Hayakawa K,
Hardy RR:
Identification of the earliest B lineage stage in mouse bone marrow.
Immunity
5:527,
1996[Medline]
[Order article via Infotrieve]
27.
Mason DY,
van Noesel CJM,
Cordell JL,
Comans-Bitter WM,
Micklem K,
Tse AGD,
van Lier RAW,
van Dongen JJM:
The B29 and mb-1 polypeptides are differentially expressed during human B cell differentiation.
Eur J Immunol
22:2753,
1992[Medline]
[Order article via Infotrieve]
28.
Uckun FM:
Regulation of human B-cell ontogeny.
Blood
76:1908,
1990[Free Full Text]
29.
Ghia P,
ten Boekel E,
Sanz E,
de la Hera A,
Rolink A,
Melchers F:
Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci.
J Exp Med
184:2217,
1996[Abstract/Free Full Text]
30.
Pui CH,
Behm FG,
Singh B,
Rivera GK,
Schell MJ,
Roberts WM,
Crist WM,
Mirro J Jr:
Myeloid-associated antigen expression lacks prognostic value in childhood acute lymphoblastic leukemia treated with intensive multiagent chemotherapy.
Blood
75:198,
1990[Abstract/Free Full Text]
31.
Wiersma SR,
Ortega J,
Sobel E,
Weinberg KI:
Clinical importance of myeloid-antigen expression in acute lymphoblastic leukemia of childhood.
N Engl J Med
324:800,
1991[Abstract]
32.
Campana D,
Pui C-H:
Detection of minimal residual disease in acute leukemia: Methodologic advances and clinical significance.
Blood
85:1416,
1995[Free Full Text]
33.
Syrjälä M,
Antilla VJ,
Ruutu T,
Jansson SE:
Flow cytometric detection of residual disease in acute leukemia by assaying blasts co-expressing myeloid and lymphatic antigens.
Leukemia
8:1564,
1994[Medline]
[Order article via Infotrieve]
34.
Bradstock KF,
Kerr A,
Kabral A,
Favaloro EJ,
Hewson JW:
Coexpression of p165 myeloid surface antigen and terminal deoxynucleotidyl transferase: A comparison of acute myeloid leukaemia and normal bone marrow cells.
Am J Hematol
23:43,
1986[Medline]
[Order article via Infotrieve]
35.
Adriaansen HJ,
Hooijkaas H,
Kappers-Klunne MC,
Hählen K,
van't Veer MB,
van Dongen JJM:
Double marker analysis for terminal deoxynucleotidyl transferase and myeloid antigens in acute nonlymphocytic leukemia patients and healthy subjects.
Haematol Bloodtransfus
33:41,
1990
36.
Barcena A,
Muench MO,
Galy AHM,
Cupp J,
Roncarolo MG,
Phillips JH,
Spits H:
Phenotypic and functional analysis of T-cell precursors in the human fetal liver and thymus: CD7 expression in the early stages of T- and myeloid-cell development.
Blood
82:3401,
1993[Abstract/Free Full Text]
37.
Van Dongen JJM,
Hooijkaas H,
Comans-Bitter M,
Hählen K,
de Klein A,
van Zanen GE,
van t' Veer MB,
Abels J,
Benner R:
Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes.
J Immunol
135:3144,
1985[Abstract]
38.
Smith RG,
Kitchens SR:
Phenotypic heterogeneity of TDT+ cells in the blood and bone marrow: Implications for surveillance of residual leukemia.
Blood
74:312,
1989[Abstract/Free Full Text]
39.
Terstappen LWMM,
Huang S,
Picker L:
Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow.
Blood
79:666,
1992[Abstract/Free Full Text]
40.
Tjonnfjord GE,
Veiby OP,
Steen R,
Egeland T:
T lymphocyte differentiation in vitro from adult human prethymic CD34+ bone marrow cells.
J Exp Med
177:1531,
1993[Abstract/Free Full Text]
41.
Galy AHM,
Cen D,
Travis M,
Chen S,
Chen BP:
Delineation of T-progenitor cell activity within the CD34+ compartment of adult bone marrow.
Blood
85:2770,
1995[Abstract/Free Full Text]
42.
Mossalayi MD,
Dalloul AH,
Bertho JM,
Lecron JC,
de Laforest PG,
Debre P:
In vitro differentiation and proliferation of purified human thymic and bone marrow CD7+CD2 T-cell precursors.
Exp Hematol
18:326,
1990[Medline]
[Order article via Infotrieve]
43.
Hokland P,
Hokland M,
Daley J,
Ritz J:
Identification and cloning of a prethymic precursor T lymphocyte from a population of common acute lymphoblastic leukemia antigen (CALLA)-positive fetal bone marrow cells.
J Exp Med
165:1749,
1987[Abstract/Free Full Text]
44.
Fritsch G,
Buchinger P,
Printz D,
Dworzak MN,
Strunk S,
Agis H,
Sliutz G,
Wilfing A,
Schulz U,
Gadner H:
Is CD33 a differentiation marker?
Prog Clin Biol Res
389:377,
1994[Medline]
[Order article via Infotrieve]
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Abstract
Introduction
Abstract