By
§
§
From the * Basel Institute for Immunology, CH-4005 Basel, Switzerland; Centro de Investigaciones
Biologicas, E-28006 Madrid, Spain; and § Department of Medicine, Alcala University, E-28871
Madrid, Spain
CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells,
which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and
terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for
DHJH and VJ
, V
J
K(de) and V
K(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B
lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged,
L germline cycling pre-B I cells
CD34
VpreB+µH chain+ (pre-B receptor+)
RAG-1
TdT
, VHDHJH-rearranged,
L germline, cycling pre-B II cells
CD34
VpreB
, intracytoplasmic µH chain+ (pre-B receptor
) RAG-1+/
TdT
, VHDHJH-rearranged, mainly
L
germline cycling pre-B II cells
CD34
VpreB
intracytoplasmic µH chain+, RAG-1+TdT
,
VHDHJH-rearranged, V
J
-rearranged, IgM
, resting pre-B II cells CD34+VpreB
, sIgM+,
RAG-1+TdT
, VHDHJH- and V
J
-rearranged IgM+ immature B cells
CD34
, CD10
,
sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage
cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.
The differential expression of genes as proteins on the
surface and inside of B lymphocyte precursors has allowed a definition and separation of subpopulations of
bone marrow cells on their way to mature, antigen-reactive
B cells. In the mouse, these markers include the tyrosine
kinase c-kit (CD117), the In mice and humans, the surrogate L chain has been
found on the surface of two distinct early precursor populations. On the earlier cell, the surrogate L chain is associated
with a complex of glycoproteins; on the later cells, together
with µH chains, it forms the pre-B receptors (11). In
this paper, we show that a new mAb specific for human
VpreB (16) detects these two early precursor B cell populations in human bone marrow.
Single-cell PCR analyses of the status of rearrangements
in both alleles of the IgH and L chain gene loci have previously allowed to order the different B lineage subpopulations of mouse bone marrow in their developmental pathway (17). In this paper, we use the same technique to
analyze the configurations of the IgH and L chain alleles in
single cells of human B lineage subpopulations. This allows
us for the first time to order these human precursors in
their developmental pathway in bone marrow.
Cells
Human heparinized bone marrow was obtained by iliac crest
aspiration from 53 individuals of different ages (between 9-mo
and 76-yr old), healthy or free of hematological diseases, according to institutional guidelines established by the ethical committees at Kantonsspital and Kinderspital in Basel, Switzerland.
Bone marrow cells were isolated by Ficoll-Hypaque gradient
(density = 1.077 g/ml) (Pharmacia, Uppsala, Sweden).
Dendritic cells were separated and cultured as described previously (18, 19).
Antibodies, Immunofluorescence, and Flow
Cytometric Analysis
mAbs.
Surface staining of bone marrow cells was performed
using the following antibodies: FITC-labeled anti-CD3 and antiCD4; biotin (BIO)-labeled anti-CD8; FITC-labeled anti-CD10
(CALLA), anti-CD13, anti-CD14, and anti-CD16; FITC-, PE-
or BIO-labeled anti-CD19; FITC- or PE-labeled anti-CD34;
FITC-labeled anti-CD56 and anti-CD57 (Becton Dickinson & Co., Mountain View, CA); BIO-labeled anti-CD40 (Caltag Laboratories, San Francisco, CA); FITC-labeled anti-CD33 (Dakopatts, Glostrup, Denmark); FITC-labeled anti-TdT (Immunotech, Marseille, France); and nonconjugated anti-VpreB (mouse
IgM; SL688; 16).
Polyclonal Antibodies.
Polyclonal FITC-conjugated rabbit anti-
human IgM F(ab)2 and polyclonal PE-conjugated rabbit anti-human
chain of the IL-2 receptor
(CD25), CD19, B220 (CD45), leukosialin (CD43), BP-1, components of the rearrangement machinery, i.e., terminal
deoxynucleotidyl transferase (TdT)1, recombination activating gene (RAG)-1 and RAG-2, µ heavy (H) chains and
light (L) chains, and the components of the surrogate L
chain, VpreB and
5 (1). In humans, markers include
CD10, CD19, CD34, TdT, RAG-1 and RAG-2, the µH
and L chains, as well as the surrogate L chains, (7).
L and anti-
L F(ab)2 were purchased from Dakopatts (Glostrup,
Denmark).
L and anti-
L
were purchased from Tago Diagnostics, Inc. (Burlingame, CA).
Streptavidin. Streptavidin-PE was purchased from Southern Biotechnology Associates. Streptavidin TRICOLOR was obtained from Caltag Laboratories.
Cell Surface Staining and Cell Sort
Three-color immunofluorescence analysis and sort were used
for identification of the different B cell precursor populations. Cell-surface immunofluorescence, flow cytometric analysis, and cell sort were performed as described on a FACStar Plus® or
FACSVantage® (Becton and Dickinson) at 4°C (20) within 4-8 h
from aspiration of the material (21). Only cells exhibiting low
forward angle and low right angle light scattering properties (lymphoid gate) were analyzed and, when needed, sorted. All the
shown percentages refer to the total cells within this gate. To
identify pre-B I precursors, bone marrow cells were stained with
BIO-labeled anti-CD19 or anti-CD10 (and streptavidin TRICOLOR) and PE-labeled anti-CD34 antibodies. These cells
could be sorted to be stained intracellularly (see below) with either FITC-labeled anti-human IgM or FITC-labeled anti-TdT
antibodies after fixation and permeabilization. To evaluate VpreB
expression on the surface of these cells, bone marrow samples were stained with BIO-labeled anti-CD19 or anti-CD10, FITClabeled anti-CD34, and purified anti-VpreB, followed by incubation with streptavidin TRICOLOR and PE-labeled anti-mouse
IgM. Using this procedure for staining, we were also able to sort
CD34VpreB+ cells and evaluate, in this population (pre-B II,
VpreB+), the cytoplasmic expression of µH chain or nuclear expression of TdT, as well as the surface expression of
L or
L
chains.
Furthermore, to identify large and small pre-B II VpreB populations, bone marrow samples were stained with BIO-labeled
anti-CD19 or anti-CD10, purified anti-VpreB, and FITC-labeled
anti-
L and -
L chains (followed by streptavidin TRICOLOR
and PE-labeled anti-mouse IgM). These cells were also discriminated by size on the side forward scatter profile. The two populations could be restained intracellularly for cytoplasmic µH chain
and TdT expression (see Fig. 1).
Immature B cells could be discriminated by staining with
FITC-labeled anti-CD10, PE-labeled anti-L and
L-chains, and
BIO-labeled anti-IgD, followed by incubation with streptavidin
TRICOLOR. Single cells were sorted using the FACStar Plus®
equipped with an automatic cell deposition unit, directed into polycarbonat 96-well plates (Biozym Diagnostik GmbH, Oldendorf, Germany) containing 10 µl of PCR buffer (three times
concentrated) or 5 µl of PBS. The plates were immediately frozen on dry ice and then stored at
70°C until use.
DNA and cDNA Preparation
To prepare DNA (17), 2 µl of proteinase K (5 mg/ml; Boehringer Mannheim GmbH, Mannheim, Germany) were added to
the plates containing PCR buffer. Samples were then overlaid
with PCR oil (Fluka, Buchs, Switzerland) and incubated for 1 h
at 55°C. Proteinase K was subsequently inactivated for 10 min at
95°C. The plates were then stored at 20°C.
To prepare cDNA, single cells in PBS were heated up to 65°C for 2 min, and reverse transcription (RT) mix was added and incubated at 37°C for 1 h (Ten Boekel, E., F. Melchers, and A. Rolink, manuscript in preparation). The enzyme was inactivated for 2 min at 95°C.
Between 24 and 40 cells of each sorted population from three different bone marrows, from donors who were 9 mo to 13 yr old, were analyzed as follows.
PCR Analysis
PCR amplification of DH segments, Ig gene and
-deleting
element (
-de) rearrangements was carried out in two rounds, using degenerate oligonucleotide primers (as listed in Table 1a) to detect as many complementary sequences as possible. Nested
primers (5
and/or 3
end) were used when needed, as shown in
Table 1 a. The primers used to detect DH-JH rearrangements were
designed to be complementary with the D element upstream of
heptamer-nonamer sequences of the genes available on DNA sequence databases, since not all the DH genes are known. Hence,
they were complementary to the members of 3 (DLR, DXP, and
DQ52) of the 7 known families within the major DH gene locus, and
they are expected to detect 10 of the 18 published sequences (22).
They were also complementary to all the known DH genes within
the minor locus (23). In detail, D primer 1 is complementary to
DLR1, DLR2, DLR3, DLR4, and DLR5 genes. D primer 2 is
complementary to DLR5, DLR6, DXP1, DXP
1, DXP4, D22/
12, D21/7, D23/7, D21/9, D21/10, and D21/0.5 genes. D
primer 3 is complementary to the DQ52 gene sequence. With
these primers, two thirds of the published sequences can be expected to be detectable.
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The four primers for the V genes (Table 1a) were designed
according to the consensus sequence derived from the alignments of the V
sequences of the 32 potentially functional genes and of
the 16 genes with only minor defects (24). Within the two sets of
primers, one was complementary to the sequences of the genes
belonging to V
I and III families, in the sense that the primer had
a complete concordance in sequence for 14 genes, only a 1-bp
difference for 12 and a 2-bp difference for another 5 genes. The
other set of primers was complementary to the genes in V
II and
IV families, again in the sense that the sequence had a complete
homology with six genes, only 2 bp difference with 6 and 4 bp
difference (all in the 5
region of the primer) with only three
genes. No primers to detect families V, VI, and VII were designed since they are all nonfunctional. With such primers, at least
90% of the V
genes that are potentially functional or have only
minor sequence defects can be expected to be detectable.
The PCR amplification conditions were as described by Ten Boekel et al. (17). PCR amplification of cDNA samples was carried out using the primers listed in Table 1b with the same strategy described above. The lengths of the different RTPCR products of the DNA and RNA analyses are listed in Table 2a and b, respectively. All the primer pairs are spanning intron sequences, allowing the distinction between expressed RNA from genomic DNA. Moreover, the primers used to detect B29 cDNA were also able to detect a splicing variant that lacks the entire exon III of the gene (25).
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PCR Product Sequencing
The PCR products, obtained with VpreB-specific primers, were sequenced using the Ready Reaction Dye Determinator (PerkinElmer Corp.,Norwalk, CT) and an automated DNA sequencer (model 373; Applied Biosystems, Inc., Foster City, CA).
Intracellular Staining for FACS® Analysis
Cytoplasmic Staining. For testing cytoplasmic µH protein expression, cells were fixed for 10 min in ice-cold 4% paraformaldehyde (dissolved in PBS; Fluka Chemie AG, Buchs, Switzerland) and then permeabilized with 0.2% Tween 20 (dissolved in PBS; Fluka Chemie) for 20 min at 37°C.
Subsequent staining and washing procedures, as well as FACS® analyses, were performed in the same way as described for surface staining of cells, using polyclonal FITC-conjugated rabbit anti- human IgM F(ab)2 (Dakopatts).Nuclear Staining. After performing the surface staining, as described before, the samples could be analyzed for the expression of the nuclear enzyme TdT (26).
The cells were fixed in 1 ml of an ice-cold solution containing 1% paraformaldehyde (dissolved in PBS), and were incubated for 2 min on ice. They were then permeabilized in a 1.5-vol of icecold absolute methanol and incubated for 20 min on ice. After washing, cells were resuspended in an appropriate volume of FCS (as a blocking serum) for 15 min, and a mixture of three FITCconjugated, anti-TdT mAbs (Immunotech) was added at a final concentration of 1:20 for 30 min on ice. FACS® analysis was performed as described before.Cell Cycle Analysis
Sorted bone marrow cells were fixed in 70% ethanol at 4°C overnight. Fixed cells were resuspended in RNAse A (0.5 mg/ml; Boehringer Mannheim), and were incubated for 30 min, at 37°C. 1 mg/ml pepsin was then added and incubated for 15 min at 37°C to prepare nuclei. Finally, ethidium bromide (0.02 mg/ml in 0.2 M Tris, pH 8-8.5, and 0.5% BSA) was added, mixed, and the nuclei were incubated for another 15 min at room temperature. DNA content was analyzed using a FACScan® instrument equipped with the Doublet Discriminator Module.
Detection of VpreB Protein on Bone Marrow Cells with the mAb 688
Cells from human bone marrow (from 24 donors, 10-mo
to 76-yr old) were examined for the expression of VpreB
protein, by surface staining with mAb 688 in three-color
FACS® analysis initially for VpreB, CD10, and L/
L chains.
After removal of the surface
L/
L+ cells and a gating for
lymphoid cells, cells were stained with CD34-specific antibody, and then sorted as shown in Fig. 1. CD19 expression
could be used instead of CD10 expression to achieve this
separation and enrichment of precursor B cells, since CD10+
are also CD19+ (not shown). Hence, from now on, we call
all CD10+ precursors CD19+CD10+ cells.
The separation shown in Fig. 1 demonstrates that the majority (around two out of three) of VpreB+ CD10+ B lineage cells did express CD34 on their surface (Fig. 1, R2). These triple-positive cells also expressed TdT, but not cytoplasmic µH chains (Fig. 1, R4). Note in the legend of the figure that a separate staining and sorting had to be performed for the R4 analysis.
An analysis by forward and side scatter gating revealed that the R4 population was composed of a mixture of small and large cells. Cell cycle analysis showed that ~40% of them were in S and G2/M phases (Fig. 1, R4).
A small part of the CD10+VpreB+Ig L chain
L chain
cells did not express CD34 on their surface (Fig. 1, R2).
These cells expressed µH chain, which is easily detectable
in the cytoplasm. They did not express TdT (Fig. 1, R5).
This cell population represents ~5% of all CD10+ B lineage cells in the bone marrow. The vast majority of them, by forward scatter profile, were found to be large. More
than 60% of these cells were in S and G2/M phases of the
cell cycle (Fig. 1, R5).
With the remaining CD10+CD34 cells, in marrow that
represented 45-60% of all of the CD10+ B lineage cells,
VpreB could not be detected on the surface (Fig. 1, R3).
This population consisted of 15-20% large and 80-85%
small cells; while 70-90% of the large cells were in S and
G2/M phases, only 3-5% of the small cells were in these
phases of the cell cycle, i.e., resting.
VpreB could also not be detected on the surface of CD10+
CD34 sIgM+
L chain+ or
L chain+ cells, nor on CD10
IgM+, IgD+ (
L chain+ or
L chain+) cells. The former
population consisting of immature B cells represents 20-25%
of the CD10+ B lineage cells in bone marrow. Less than
2% were in S and G2/M phases, i.e., practically all of them
were resting cells. No CD3+, CD4+, CD8+, CD13+,
CD14+, CD16+, CD33+, CD56+, or CD57+ cells showed
detectable expression of VpreB on their surface (all data not
shown).
We conclude that mAb 688, specific for VpreB, detects
VpreB molecules on the surface of all CD34+ and a small
fraction of CD34 cells that also expresses CD19 and
CD10, but not surface (sIgM). These two precursor populations, the first TdT+ and the second TdT
, are analyzed
in more detail below.
Age-related Changes in the Number of B Lineage Precursor Cells in Human Bone Marrow
The representative of the CD19+CD10+ B lineage subpopulations of human bone marrow described above was analyzed in 53 donors who were 10 mo to 76 yr old. The results are presented in Fig. 2 a as the percentage values of cells within the lymphoid compartments, and as the percentage values of cells among the CD19+CD10+ B lineage precursor compartments in bone marrow.
It is evident that the frequency of CD19+CD10+ B lineage cells and the frequency of cells in all identified B precursor compartments, hence also of the VpreB-expressing B lineage precursors, decrease with age. They are highest in young donors who are up to the age of 10 yr old. It appears, however, that the proportions of different B lineage precursors relative to each other remain the same throughout life.
The frequencies of CD19+CD10sIgM+ mature B cells
also decrease within the lymphoid compartment (Fig. 2 b).
Their relative contribution to all CD19+ B lineage cells in
bone marrow, however, increase with age.
RNA Expression of the VpreB, TdT, and RAG-1 Genes in Single Cells of Different B Lineage cells in Human Bone Marrow
Single cells from the subpopulations of CD19+CD10+
CD34+VpreB+, CD19+CD10+CD34VpreB+, the large cycling CD19+CD10+CD34
sIgM
, the small resting CD19+
CD10+CD34
VpreB
sIgM
B lineage cells of bone marrow
from three young donors were separated by FACS®, and
were analyzed by RT-PCR developed for human VpreB,
TdT, and RAG-1. As a control for cDNA integrity, B29
cDNA was amplified (Table 2b). The results of these experiments are summarized in Table 3.
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TdT-specific RNA expression followed the pattern of protein expression that was detected in the earlier experiments (see Fig. 1). It was only expressed in the large majority of all CD34+ B lineage cells.
VpreB expression on the RNA level did not completely
follow the pattern of expression of the VpreB protein. Hence,
while CD34+ and some of the large cycling CD34 B lineage cells expressed the protein, and while the rest of the
large and all the small resting CD34
cells did not, RNA
was not only expressed in the two subpopulations with
VpreB protein, but was expressed in the majority of CD34
large cells, as well as in up to 25% of all the small CD34
CD10+ pre-B cells. The VpreB-specific mRNA expressed in
these cells was reverse transcribed, sequenced, and found to
contain the expected VpreB characteristic sequences. It remains to be determined whether the VpreB RNA in the
VpreB protein-negative cells is functional mRNA.
RAG-1 expression was detectable in most of the CD34+
B-lineage precursors, was suppressed in the majority of
CD34 large cells, and was again detectable in half of the
small CD34
cells. This modulated expression of RAG-1
RNA will be discussed below. We have shown previously
that RAG-1 mRNA is expressed in immature CD34
CD10+ sIgM+ B cells, while it is not expressed in CD34
CD10
sIgM+ mature B cells (21).
PCR Analysis of DHJH and VJ
Rearrangements in
Single CD19+CD10+ B Lineage Cells of Different Bone
Marrow Subpopulations
For the amplification of DHJH-rearranged IgH sequences
of VJ
-rearranged (
-de) L chain gene loci, several primers were designed (see Materials and Methods and Table 1a).
Practically the whole human
L chain gene locus is known,
and that includes orphan gene segments (24). Genes in V
families I, II, III, and IV are expected to contain members
that encode L chain proteins, while those in families V, VI,
and VII are all nonfunctional. The V
J
primer sets used in
our studies are expected to detect at least 90% of the 32 potentially functional V
genes, as well as the 16 nonfunctional genes with minor sequence defects.
In contrast to the L chain locus, the DH segments of the
IgH chain locus are not yet completely known. A major
locus of seven families have been detected within the IgH
chain locus on chromosome 14 (22), while another six
have been described on a minor locus on the same chromosome (23). With the primer sets shown in Table 1a, we
expect to detect approximately two thirds of these DH segments in DHJH-rearranged configurations.
Eventual sequencing of many of the PCR products detected from single cells will be able to evaluate the validity of these expectations.
The results of the PCR analyses are discussed below:
DHJH Rearrangements.A total of 36 single CD19+CD10+ CD34+ B cell precursors from three individuals were analyzed for their status of DHJH rearrangements within the IgH locus. The results, shown in Table 4, show that the majority of these cells have at least one of their IgH alleles in DHJH-rearranged conformation (see also legend to Table 4). In many cells, both alleles should be rearranged, since in most cases, no germline H chain locus was detectable in the PCR analyses. It remains to be analyzed how many of the rearranged H chain loci are VHDHJH rather than DHJH rearranged. If they were VHDHJH rearranged, the observed lack of µH chain expression in this cell population would make it likely that they would be nonproductively rearranged.
We conclude that the majority of CD19+CD10+CD34+ B lineage precursors appear to have their IgH chain alleles no longer in germline configuration, since many of them have at least one allele DHJH rearranged.
The vast majority of the CD19+CD10+CD34 cells express µH chains in their cytoplasm (Fig. 2). Hence, they
are expected to be productively VHDJH rearranged on at
least one allele. Therefore, we did not analyze their IgH
gene loci by PCR.
Our results confirm previous suggestions of the order of
human B cell subpopulations during development in bone
marrow. The analyses of the conformations of the IgH
gene loci show that CD19+CD10+CD34+TdT+ cells are
DHJH-rearranged precursors of the CD19+CD10+CD34
TdT
cells.
CD19+CD10+ B lineage cells of
bone marrow were separated by FACS® into single cells of
the subpopulations of CD34+VpreB+, CD34VpreB+, large
CD34
VpreB
, small CD34
VpreB
, sIgM+ immature, sIgM+
sIgD+, and sIg+
L+ or
L+ mature cells. Ig
L chain gene
loci of between 20 and 25 single cells of the different populations from the bone marrow of two donors were analyzed for their conformations (Table 4b).
It is evident that the vast majority of the DHJH-rearranged CD19+CD10+CD34+ precursors have their L
chain loci in germline configuration.
L chain gene rearrangements are detectable in a large part of all cells, i.e., in
at least 60% of all CD34
small cells, and from there on, as
expected, in 80-95% of all immature and mature sIg+ B cells.
Interestingly,
L+ sIg+ B cells appear to have less
L chain
gene loci in germline configuration, and have more of
them deleted.
We conclude from these analyses that the majority of all
L chain gene rearrangements occur at the transition from
VpreB
large CD34
to VpreB
small CD34
cells. Hence,
the largest subpopulation of B lineage cells in the human bone
marrow, the CD19+ CD10+CD34
VpreB
TdT
RAG-l+,
cyto-plasmic µH chain+, small, resting sIg
precursor B
cell has, to a large part, its Ig
L chain gene loci in V
J
rearranged conformation, but does not yet express them as
proteins so that the cells would become sIg+. Collectively,
these PCR analyses allow us to order CD19+ CD10+ B
lineage subpopulations in human bone marrow in the following sequence: CD34+TdT+RAG-l+VpreB+ cells are DHJHrearranged precursors of large, VpreB+ and VpreB
, CD34
,
TdT
, RAG-1
, productively VHDJH-rearranged cells which,
in turn, are precursors of small, VpreB
, CD34
, TdT
,
RAG-1+, VHDJH-rearranged cells, which, finally, are followed by sIgM+ immature and sIgM+ sIgD+ mature B cells.
The analysis of the status of the IgH and L chain gene
loci in single cells of B lineage subpopulations in the bone
marrow of mice has been instrumental in ordering these
subpopulations along a pathway of B lymphocyte development (17, 27). This ordering has been guided by the fact
that rearrangements in the Ig loci occur in stepwise fashion,
so that DH segments are rearranged first to JH segments,
then VH segments are rearranged to DHJH segments, and finally, V and V
segments are rearranged to J
and J
segments, respectively (28). Rearrangements in the
L chain
locus appear often to precede those in the
L chain locus,
and can be terminated by rearrangements into
L chain gene-deleting elements (29, 30). Stepwise, ordered rearrangements in the Ig gene loci are also characteristic for human B lymphocyte development.
Single-cell PCR analyses of the status of the Ig H and L
chain loci of B lineage subpopulations in human bone marrow performed in this paper allow a similar ordering of human B lineage subpopulations, characterized by the expression of CD19, CD10, CD34, VpreB, TdT, RAG-1, and by
their cell cycle status. Comparisons with B cell development in the bone marrow of mice allow the following conclusions:
(a) Human CD19+CD10+CD34+TdT+RAG-l+ VpreB+
µH chain cycling cells are comparable to murine B220+
CD19+TdT+RAG-1/2+VpreB+/
5+c-kit+, µH chain
CD25
cycling pre-B I cells. Both have DHJH-rearranged
Ig H chain loci. Mouse pre-B I cells often have both alleles
DHJH rearranged. Their Ig L chain loci remain in germline
configuration. In analogy to murine pre-B I cells, we will
call them human pre-B I cells.
(b) Human CD19+CD10+CD34TdT
RAG-1
VpreB+,
µH chain+, pre-B receptor+ cycling cells are comparable
to murine B220+CD19+TdT
RAG-1
/2
c-kit
CD25+
VpreB+/
5+ µH chain+ pre-B receptor+ large pre-B II cells.
They are productively VHDJH rearranged in the Ig H chain
locus, while their L chain gene loci remain in germline
configuration. In analogy with mouse pre-B cells, we will
call them large, pre-B receptor+ pre-B II cells.
(c) Human CD19+CD10+CD34TdT
RAG-1
VpreB
,
µH chain+ pre-B receptor
cycling cells are comparable
to murine B220+CD19+TdT
RAG-1
/2
c-kit
CD25+
VpreB
/
5
, pre-B receptor
, µH chain+ sIgM
large pre-B
II cells. Their H chain locus is productively VHDJH rearranged, while their Ig L chain loci are largely in germline configuration. In analogy with mouse pre-B II cells, we
will call them large pre-B receptor
pre-B II cells.
(d ) Human CD19+CD10+CD34TdT
RAG-1+VpreB
,
µH chain+ sIgM
small resting cells are comparable to murine B220+CD19+TdT
RAG-1+/2+c-kit
CD25+VpreB
/
5
, µH chain+ small resting pre-B II cells. Both have a
majority of their IgL chain gene loci in VLJL-rearranged
configurations, but do not yet express L chains on their
surface. In analogy with mouse pre-B cells, we will call
them human small resting pre-B II cells.
(e) Human CD19+CD10+sIgM+ immature B cells are
comparable to murine immature B cells, and human mature CD19+CD10sIgM+sIgD+ B cells are comparable to
murine mature B cells. The RT-PCR analyses of the expression of the RAG-1 genes in different B lineage subpopulations of human bone marrow show the same striking
changes that have been observed in mouse B lineage subpopulations (31). It indicates that the same up- and downregulation of the rearrangement machinery that might be
part of the mechanisms effecting allelic exclusion of the H
chain alleles appear to be operative in the human. It appears
that human and mouse B cell subpopulations in the bone marrow resemble each other, not only in their status of Ig
gene rearrangements of TdT and RAG protein expression
and pre-B receptor expression, but their relative amounts in
the different compartments are also comparable.
The relative proportions of the CD19+CD10+ B lineage
compartments remain the same throughout life, although
they decrease in frequencies. This suggests that while the
flow from early to late B cell precursors decreases with age,
it continues from the same precursors through the same intermediates during life. The relative proportion of the mature, CD19+CD10sIgM+ B cells increases over the CD19+
CD10+ B lineage cells with age, suggesting that the contribution of recirculating B cells in bone marrow increases
with age. These results confirm earlier findings by Nuñez
et al. (32), which showed that B cells are continuously generated through life though the relative frequencies of precursor B cells in human bone marrow decrease with age.
A few differences between human and mouse B lineage
cells are apparent. First, a small but significant portion of
the large, pre-B receptor expressing and pre-B receptor-
negative human pre-B II cells have L chain gene loci in
VLJL-rearranged configurations. It cannot be excluded that
this difference is a consequence of the way the bone marrow samples are collected. Murine bone marrow cells are
prepared on ice within 15 min after the death of the animals, while human marrow is aspirated but then kept for
considerable periods of time before staining with specific
antibodies and FACS® separation procedures are initiated.
This longer time ex vivo may allow the progression along
the pathways of differentiation of a human precursor B cell
and, hence, allow occasionally L chain rearrangements in
large pre-B II cells, after ex vivo isolation.
Second, VpreB gene expression on mRNA level persists
into a part of the large, pre-B receptor-negative and small,
resting human pre-B II cells, while that is not so in the murine counterparts. Again, a longer period ex vivo before
staining and FACS® might allow cells to change their surface phenotype, downregulate VpreB protein expression but
keep VpreB mRNA intact. Also, it has been observed in human pre-B cells that stability of surrogate L chain protein
differs in different precursor subpopulations (33). Different
laboratories have developed different mAbs specific for different determinants on surrogate L chain expressed on either human or mouse B lineage cells (11, 12, 15). Three
patterns of expressions on different B cell subpopulations of
the marrow have been detected. Lassoued et al. (11) detected surrogate L chain only on CD19+CD10+TdT,
pre-B receptor+ (presumably large) cells, which we would
call pre-B II cells. They have concluded that these cells are
the immediate precursors of sIgM+ immature B cells, although they have not offered a placement of the pre-B
receptor
cytoplasmic µH chain+ precursors, which, according to their studies, comprise as many as 80% of all
CD19+CD10+ B lineage precursors in bone marrow. We
would propose that these pre-B receptor
cytoplasmic µH
chain+TdT
precursors are, in majority, VLJL-rearranged
small pre-B II-like cells. They might be the nonproductively VLJL-rearranged portion of the immature cells and,
hence, one of two direct products of the large, pre-B receptor-positive and -negative pre-B II cells (Fig. 3, Alternative II). This remains to be elucidated by sequencing of
individual V
J
rearrangements in these cells.
It is also possible that they are, at least in part, productively VLJL rearranged, but not (yet) capable of expressing
L chain protein. Such VLJL-rearranged,
L chain-negative
cells have been found in the mouse in the case of the 70Z/3
cell line, which is inducible to
L chain expression by stimulation with lipopolysaccharides (34). If this is the case,
they are precursors of the sIgM+ immature B cells and,
hence, in between large pre-B II cells and immature sIgM+
B cells (Fig. 3, Alternative I).
For murine precursors B cells, Winkler et al. (15). have detected two subpopulations in bone marrow that express surrogate L chain on the surface (15). One is the pro-/preB I cell population, which expresses surrogate L chain together with a complex of glycoproteins (35), and the other is a population of large, cycling pre-B II cells, which is the progeny of the pre-B I cells and the precursors of large, pre-B receptor-negative and small, resting pre-B II cells. Guelpa-Fonlupt et al. (12) have observed the same pattern of surface expression of surrogate L chain in human bone marrow. Their antibody, however, also stained sIgM+ immature B cells. The VpreB-specific mAb 688 (16) used in this paper detects surrogate L chains on human pre-B I and large pre-B II, but not on small pre-B II and not on immature sIgM+ B cells, and hence follows the pattern of murine surrogate L chain expression on the surface. Since our RT-PCR analyses did not detect VpreB mRNA in immature sIgM+ B cells, and since our VpreB mRNA-positive small pre-B II cells did not express detectable levels of VpreB protein, we see no reason to expect that VpreB expression could be carried as far as into the sIgM+ immature B cells population.
The differential expression of CD34, TdT, RAG-1, and
VpreB and differences in cell-cycle status, as well as single
cell PCR analyses of the status of rearrangements in the
IgH and L chain loci, have allowed us to order cellular
development of B cell precursors in human bone marrow
as it has been done in the mouse. These analyses are expected to be helpful in an understanding of the cellular
targets of dysregulation leading to autoimmune diseases,
immunodeficiencies, and neoplasia in the human B lymphocyte lineage.
Address correspondence to Dr. Fritz Melchers, Basel Institute for Immunology, Grenzacherstrasse 487, CH4005 Basel, Switzerland.
Received for publication 8 July 1996
The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland. E. Sanz was supported by contracts from the CSIC and grant CAM92/126, and A. de la Hera was supported by grants SAF-93-0925 and SAF-96-0201 from the CICY.We thank Drs. Rod Ceredig and Thomas Winkler for critical reading of this manuscript. We are grateful to Marcus Dessing for his outstanding skill at the FACS® sorter and his extraordinary help during long, unusual hours. We thank Prof. A. Gratwohl, Dr. E. Signer, and Dr. U. Ramenghi for providing the bone marrow samples and Prof. F. Caligaris Cappio for continuous encouragement and discussions. We gratefully acknowledge Ms. Nadia Straube's technical experience in DNA sequencing.
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