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Prepublished online as a Blood First Edition Paper on September 19, 2002; DOI 10.1182/blood-2002-06-1828.
IMMUNOBIOLOGY
From the Division of Developmental and Clinical
Immunology, and Departments of Medicine, Pediatrics, Pathology, and
Microbiology, University of Alabama at Birmingham and the Howard Hughes
Medical Institute, Birmingham, AL; and Division of Pediatric
Hematology/Oncology/Bone Marrow Transplantation, Emory University
School of Medicine, Atlanta, GA.
The initial B-cell repertoire is generated by
combinatorial immunoglobulin V(D)J gene segment rearrangements that
occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was
provided by the identification of a human acute lymphoblastic leukemic
cell line that undergoes spontaneous differentiation from pro-B cell to
the pre-B and B-cell stages with concomitant changes in the gene
expression profile that normally occur during B-cell differentiation.
After confirming the clonality of the progressively differentiating
cells, an analysis of immunoglobulin genes and transcripts indicated
that pro-B cell members marked by the same DJ rearrangement generated
daughter B cells with multiple VH and VL gene
segment rearrangements. These findings validate the principle of
intraclonal V(D)J diversification during B-cell generation and define a
manipulable model of human B-cell differentiation.
(Blood. 2003;101:1030-1037) B-cell development normally begins within specialized
microenvironments in the hematopoietic fetal liver and bone marrow. Therein precursor cells undergo proliferation during the course of a
series of differentiation events featuring the ordered rearrangement of
variable (V), diversity (D), and joining (J) gene segments to generate
a diverse antibody repertoire.1 The D B-cell clonality is usually defined operationally by the shared
expression of a unique BCR specificity, although intraclonal antigen
receptor diversity may be generated by several mechanisms. Antibody
diversity can be generated by receptor editing, a process by which the
specificity of an autoreactive BCR is changed thorough a secondary
VL-JL rearrangement at an immature B-cell
stage.14,15 Additional intraclonal BCR diversification can
be generated by point mutations in the variable region during antigen
(Ag)-driven clonal expansion in germinal centers.1,16,17
B-cell malignancies, including follicular B-cell lymphoma, chronic
lymphocytic leukemia, and Hodgkin disease, may exhibit this type of
clonal diversification as a manifestation of their germinal center (GC)
origin.18
It has been proposed that intraclonal diversity may also be generated
as a consequence of the cellular proliferation that accompanies the
normal stepwise V(D)J rearrangement events in pro-B and pre-B
cells.19 According to this hypothesis, a pro-B cell
undergoing the initial DJH rearrangement can give rise to daughter cells that select different VH genes for
subsequent V-DJH rearrangement. In turn, the pre-B cell
progeny would have the opportunity to select different VL
and JL gene segments for VJL pairing, thereby
providing additional intraclonal BCR diversity. Because a clonal model
of early B-lineage differentiation would allow testing of this
hypothesis, we sought a stable cell line that recapitulates this
developmental process. Here, we report the characterization of a human
acute lymphoblastic leukemic (ALL) cell line, EU12, that undergoes
continual B-lineage differentiation from the pro-B to the pre-B and
B-cell stages. Analysis of this clonal model of human B-lineage
differentiation validates the principle of intraclonal V(D)J
recombinatorial diversification during B-cell genesis.
Cells and antibodies
Immunofluorescence analysis and cell sorting
RT-PCR assays Twice-sorted subpopulations of EU12 cells were lysed in TRIzol reagent (Gibco, Grand Island, NY), and total cellular RNA prepared following procedures recommended by the manufacturer. The synthesis of first-strand cDNA was performed as previously described.23 For each cDNA preparation, a control synthesis reaction was performed without reverse transcriptase (RT) to test for genomic DNA (gDNA) contamination. The protocols for polymerase chain reaction (PCR) included denaturing at 94°C for 3 minutes amplification by 30 cycles of 94°C for 1 minute, annealing for 30 seconds at 60°C for interleukin 7 receptor (IL-7R) and mb-1; 65°C for C ; and 72°C for
Bcl-2; and extension at 72°C for 5 minutes. The
primers for PCR amplification were IL-7R,
5'-GTCGCTCTGTTGGTCATCTTG-3' and 5'-TTTTG TCTTCTCTGTGCTGTG-3';
mb-1, 5'-GCTCCCCTAGAGGCAGTTAAGG GC-3' and
5'-AGGGTAACCTCACTGTTAGGCCAGGC-3'; C,
5'-TGGCTGCACCATCTGTC TTCA-3' and 5'-TTGAAGCTCTTTGTGACGGGC-3';
Bcl-2, 5'-TGCACCTGACG CCCTTCAC-3' and
5'-AGACAGCCAGGAGAAATCAAACAG-3'. The protocols and primers used for PCR
amplification of Rag1, Rag2, TdT,
B29, Cµ, and  -actin have been
described.11
DNA blotting gDNA samples from sorted EU12 subpopulations and placenta (germline configuration control) were subjected to restriction enzyme digestion, electrophoresis, transfer, and hybridization with 32P-labeled DNA probes for analysis of Ig HC and LC gene configuration as described.24 DNA probes, including a 3.6 kilobase (kb) BglII JH probe, a HindIII J probe, and an
EcoRI C probe, were used as
described.24
Genomic PCR assay gDNA samples (0.5 µg) from EU12 subpopulations and the control HepG2 cells were used as templates. The protocol for PCR amplification of IgH gene segments involved denaturing at 95°C for 5 minutes, amplifying by 36 cycles of 95°C for 1 minute, annealing at 65°C for 30 seconds and at 72°C for 30 seconds, and extending at 72°C for 5 minutes. The primers for amplification, DXP'1, 5'-ATTACTATGGTTCGGGGAGTT-3'; DXP'1ext, 5'-GGTGAGGTCTGTGTCACT-3'; JH5, 5'-GTCGAACCAGTTGTCACATTG TG-3'; JH6, 5'-ACCTGAGGAGACGGTGACC-3', and PanVHFR1, and PanVHFR3 were as described.25 The PCR products were separated by electrophoresis, transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH). After standard prehybridization, the blots were hybridized at 42°C with oligonucleotide DXP'1 or JH5 probes labeled with digoxigenin (DIG) using the DIG oligonucleotide 3'-end labeling kit (Boehringer Mannheim, Germany) according to the manufacturer's instructions. After washing at 42°C, hybridization signals were revealed using the DIG luminescence detection kit for nucleic acids (Boehringer Mannheim) according to the manufacturer's instructions. To detect the VJ rearrangements in each of the EU12 subpopulations, the protocol of PCR amplification using a set of
V and J primers was used as
described.26
Ig gene sequencing and single-cell PCR assay To determine µHC and LC transcript sequences, cDNA of the
test cells was synthesized for PCR amplification as previously described.23 PanVHFR1 and Cµ primers were
used for amplification of the µHC cDNAs, and the set V
and J primers and PCR protocol for LC cDNA
amplification were performed as above. The PCR products of µHC or
LC transcripts were isolated and subjected to the TA cloning
reaction as described by the manufacturer (Invitrogen, Carlsbad, CA).
Plasmids carrying µHC or LC transcripts were purified from
randomly selected Escherichia coli colonies for sequencing reactions. For analysis of single-cell VH usage, individual
µHC+ EU12 cells were deposited into tubes containing 20 µL PCR buffer with 0.25 mg/mL proteinase K. Reactions were incubated
at 50°C for 1 hour and used as templates in a single-cell PCR assay.
In the first-round PCR reaction, a combination of
VH1,2,3-specific oligonucleotides was used as forward
primer, and an oligonucleotide sequence common to JH1,2,4,5
was used as the reverse primer. In the second round of a nested PCR, 5 µL of the first PCR reaction product was used as the template,
individual VH oligonucleotides were used as the forward
primer, and the JH1,4,5 oligonucleotide was used as the
nested reverse primer. The primer sequences have been
described27 and the protocol included first-round
VH gene amplification at 95°C for 5 minutes, 59°C for 4 minutes, and 72°C for 3 minutes (cycle 1); 95°C for 30 seconds,
59°C for 30 seconds, and 72°C for 80 seconds (cycles 2-36); then
extension at 72°C for 5 minutes; and maintenance at 4°C. For
second-round PCR, the protocol for amplification of the first round PCR
product included 95°C for 3 minutes, 61°C for 1 minute, 72°C for
1 minute (cycle 1), 95°C for 1 minute, 61°C for 30 seconds, 72°C
for 50 seconds (cycles 2-40), and extension at 72°C for 5 minutes,
and then maintenance at 4°C. PCR products were separated by
electrophoresis on a 1.5% agarose gel and purified for the sequencing
reaction by using a gel extraction kit (Qiagen, Valencia, CA). Purified
PCR products were sequenced directly using VH-specific
primers. All sequences were analyzed with the DNAplot program
(http://www.dnaplot.de/).
Phenotypic and karyotypic characterization of the EU12 leukemic cell line EU12 was unique among a panel of 11 ALL cell lines in that phenotypic analysis identified cells at multiple stages of B-lineage differentiation. All of the EU12 cells expressed the CD19, CD20, CD22, CD23, CD32, CD33, CD38, CD40, and HLA-DR antigens, but subpopulations of this cell line were found to be either positive or negative for CD34, CD10, and µHC (Figure 1). Differential immunofluorescence analysis indicated that the EU12 cell line is composed of 3 distinctive subpopulations: CD34+sµHC (R1),
CD34sµHC (R2), and
CD34sµHC+ (R3). The latter subpopulation
could be subdivided into sµHClowSLC+ (R3A)
and sµHChighLC+ (R3B) subsets (Figure 1).
Cells in both the R3A and R3B subsets expressed cell surface SLCs, and
the cells in the R3B subset also expressed LCs and  HCs. The
µHClow R3A cells thus bear pre-BCR, whereas the
µHChigh R3B cells have pre-BCR and BCR. IL-7R, barely
detectable on the R1 and R2 subpopulations, was present at higher
levels on the R3A and R3B subsets.
To characterize further the developmental stages of the EU12 cell line,
each subpopulation was purified for intracellular immunofluorescence
analysis (Figure 1). VpreB was found in permeabilized cells of all 3 subpopulations, whereas µHC was observed only in R3 cells and rarely
in R2 cells (< 1%). In contrast, TdT expression was evident in most
R1 and R2 cells, but not in the R3 subpopulation. The composite data
from this analysis indicate that the EU12 cell line contains cells of
pro-B (R1 and R2), pre-B (R3A), and B-cell (R3B) phenotypes, although
the B-cell subpopulation is unusual in its IL-7R expression and
simultaneous expression of surrogate and conventional The distinctive phenotype of EU12 cell line prompted a detailed
cytogenetic analysis, and the results indicated the EU12 cell line is
nearly tetraploid and has complex abnormalities involving chromosomes
2, 3, 4, 5, 6, 7, 11, 12, 17, and 21. Detailed description of its
chromosome complement is as follows: 88, XXYY, +2, del(2)(q13) × 2, del(3)(q26.2), EU12 cells spontaneously undergo pro-B to B-cell differentiation The representation of multiple stages of B-lineage differentiation among the EU12 cells suggested this cell line either is undergoing differentiation or represents a multiclonal mixture of cells. To discern between these possibilities, each subpopulation was purified by 2 rounds of FACS before reculture and phenotypic monitoring. Serial immunofluorescence analysis indicated that isolated cells of pro-B cell phenotype (CD34+µHC) gave rise to cells of
pre-B phenotype (CD34+µHClow) by day 12 and
cells of B-cell phenotype (CD34µHChigh) by
3 weeks in culture. Purified cells of the R2 subset
(CD34µHC) likewise gave rise to cells of
pre-B and B-cell phenotypes, whereas the R3 subpopulation retained
cells of pre-B and B phenotypes (Figure
2A). These results indicated that EU12
cells can undergo continual differentiation from pro-B cells to B
cells. To verify their developmental potential at a clonal level,
single R1 pro-B cells were placed in separate culture wells. Serial
examination of their cellular progeny indicated that individual EU12
cells of pro-B cell phenotype are capable of giving rise to mature
B-cell progeny (Figure 2B), although a longer time interval was
required for this progression when the cultures are initiated with
single cells, and B-cell differentiation was not seen in every subclone over the 10-week observation period. These results document the spontaneous differentiation capabilities of the EU12 cell clone and
suggest variability in the progression of individual cells along the
B-cell differentiation pathway.
Differential gene expression profiles in the EU12 subpopulations Cells representative of each definable stage in EU12 differentiation were purified by 2 rounds of FACS before RT-PCR analysis of their gene expression profile (Figure 3). As anticipated from its protein expression profile (Figure 1), the TdT lymphoid cell-specific gene was down-regulated in the R3A subset and extinguished in the R3B subset. Rag1 was found to follow a similar expression pattern, whereas Rag2 transcripts were expressed in all of the subpopulations, as were transcripts for the mb1, B29,5,
and VpreB B-lineage genes. In keeping with results of the
immunofluorescence analysis (Figure 1), IL-7R and
Cµ transcripts were not detected in the R1 pro-B cells,
but were expressed in subpopulations representing later stages in
differentiation. Interestingly, Bcl-2 transcription was
noticeably down-regulated in the R3B B-cell subpopulation, possibly
suggesting that the most mature EU12 cells are more susceptible to
programmed cell death. These data support the conclusion that the EU12
cells undergo many of the changes in gene expression that
characterize normal B-cell differentiation.
Analysis of Ig HC and LC gene configuration in the EU12 cells Southern blot analysis was initially used to characterize the Ig gene rearrangement status at the different EU12 differentiation stages. Restriction enzyme-digested gDNA samples of each EU12 subpopulation, and of placenta as a germline control, were hybridized with JH, C, and C probes (Figure 4
A-B; data not shown). Germline
JH-containing fragments were not detectable in the
unfractionated EU12 cells; instead, 2 rearranged bands were observed, a
dominant 6.2-kb band and a minor 12-kb band (Figure 4A, lane 2). Of
these, the R1 and R2 subpopulations contained only the 6.2-kb band
(Figure 4A, lanes 3 and 4), whereas the µHC+ R3
subpopulation contained both bands of similar intensity (Figure 4A,
lane 5). These findings, together with a karyotypic analysis demonstrating 2 copies of chromosome 14 (data not shown), suggest that
the least differentiated R1 cells of EU12 contain 2 very similar
JH rearrangements, but are incapable of producing µHCs because the rearrangements either are incomplete DJH or
nonproductive VDJH. The occurrence of an additional
rearrangement event on one HC allele in the R3 cells coincides with the
expression of µHC.
No J
VH and VL gene usage in EU12 cells To examine the extent of V(D)J recombinatorial diversification in the EU12 cells, the µHC+ R3 subpopulation of cells was purified and RNA extracted for cDNA synthesis and PCR amplification of µHC transcripts using PanVHFR3 and Cµ primers. The resultant µHC PCR products were cloned and the analysis of sequences obtained from 17 randomly selected clones identified the same DXP'1/JH4 gene segment rearrangement with no nucleotide addition in this D-JH joint region. This analysis also demonstrated the use of multiple VH gene segments by EU12 cells in that the VH3-7 gene segment was found in 12 of the 17 cDNA clones, VH1-8 in 3 cDNA clones, and VH3-11 in the other 2 cDNA clones (Figure 5A). In addition, different nucleotide additions in µHC transcripts using the same VH gene provided further evidence of intraclonal V(D)J diversification.To avoid possible transcriptional bias in this analysis of
VH gene usage, we performed a single-cell genomic PCR assay
of sorted µHC+ EU12 cells. The sequences derived from a
panel of single-cell VH gene PCR products recaptured the 2 major VH3-7 gene rearrangements observed in the earlier
analysis of µHC cDNA clones (Figure 5B). In addition, we identified a
VH2-5 rearrangement with the same DXP'1/JH4
sequence observed in the cDNA clones; however, this rearrangement was
nonfunctional because of a reading frame shift. The resulting
transcript would be targeted for destruction by nonsense-mediated mRNA
decay,28 thereby explaining why VH2-5 gene
segment usage was not observed in the cDNA analysis. Consistent with
the cDNA sequence analysis, 2 patterns of nucleotide additions were
observed in the joint region between VH3-7 and
DXP'1/JH4. The dominant joint region sequence featured a
CCCC nucleotide addition, presumably added during the
VH With this information in hand, genomic PCR assays were performed to
determine more precisely when the V(D)J rearrangements occurred during
the EU12 cell differentiation process. Primers able to discriminate
between DJH and VHDJH
rearrangements were used in this analysis (Figure
6A). In a control experiment, the internal DXP'1 primer was used in conjunction with a downstream JH6 primer, and genomic PCR products of the expected size
were detected in each EU12 subpopulation after hybridization with a JH5-specific probe (Figure 6B). This result was as expected
because the primer combination amplifies both VDJH and
DJH rearrangements. To assay specifically for
DJH rearrangements, a primer based on the sequence upstream
of the germline DXP'1 gene segment (DXP'1 ext) was used in
conjunction with internal JH5 primers and the PCR products
were detected by a specific internal DXP'1 probe. The EU12 cells in
each purified subpopulation were found to contain a DJH
segment rearrangement, albeit at apparently reduced levels in the R3
pre-B/B cell subpopulation (Figure 6C). Using PanVHFR3 and
JH5 primers in an assay that allows definition of the
presence or absence of VDJH rearrangements but
does not identify unique rearrangements, VDJH gene
rearrangement was detected in the R2 subpopulation, and to a much
greater extent in the R3 subpopulation (Figure 6D). The nonfunctional
VH2-5 DXP'1/JH4 rearrangement was not detected
in this assay because the PanVHFR3 primer does not recognize the VH2-5 gene segment. However, using a
VH2-specific primer, a genomic PCR product was detectable
in the R1, R2, and R3 subpopulations (not shown).
In an analysis of the V The prevalent usage of a limited number of VH gene segments
and the occurrence of the same DJH rearrangements over a
2-year interval for EU12 subclones suggested a predisposition of the individual EU12 pro-B cells to undergo particular
VH
These studies define a clonal model of human B-cell development in which the pro-B cell subpopulation, as well as the individual pro-B cells, can spontaneously generate pre-B and B-cell progeny. The B lineage-specific gene expression profiles for each of these subpopulations resemble those observed during normal B-cell differentiation. These characteristics define the EU12 cell line as a novel model that recapitulates many of the central features of the human B-cell differentiation pathway. Mouse pre-B cell hybridomas,2 Abelson murine leukemia
virus (AMuLV)-transformed cell lines,29 human
Epstein-Barr virus-transformed B-cell precursors,24 and
ALL-derived tumor cell lines30 have been used in previous
studies to gain insight into the sequential nature of Ig H and L chain
gene rearrangements during B-lineage differentiation. Although most of
these cell lines represent clonal populations frozen in a particular
stage of B-lineage differentiation, the AMuLV-transformed cell line
300-19 was found to undergo pro-B to B-cell
differentiation.29 However, this proved to be an unstable phenotype because the AMuLV-transformed cell lines extinguish their
Rag1/2 expression with prolonged
cultivation.31 In humans, the search for an in vitro
culture system or cell lines that recapitulate early B-lineage cell
differentiation and self-renewal for cellular and molecular studies has
been an ongoing challenge.32 One leukemic cell line,
BLIN-1, was shown to undergo pre-B to B-cell differentiation at low
frequency,30 and a more recently described cell line, BLIN-3, can progress from the pro-B to the pre-B cell stage in a
stromal cell-dependent manner.33 The EU12 cell line is
unique in maintaining 4 distinguishable subpopulations,
CD34+sµHC The sorted R2 subpopulation can give rise to occasional
CD34lowµHC Another notable departure from the normal B-cell differentiation scheme
was observed for the EU12 cell line. SLC expression is normally
down-regulated to extinguish pre-BCR expression at the late pre-B cell
stage.11 As a consequence, the receptorless pre-B cells
exit the cell cycle and up-regulate their Rag1 and Rag2 expression to undergo V-JL gene
segment rearrangements. EU12 B cells instead were found to coexpress
pre-BCR and BCR, a feature noted previously for other "transitional
pre-B/B" cell lines,34-36 thus questioning why these
transitional B cells fail to extinguish SLC gene expression and the
possible role this failure may have in the leukemogenesis process. A
complex locus control region, including the promoters and 5' enhancer
for the VpreB and B-cell leukemias and lymphomas are considered clonal diseases derived
from a single transformed precursor, based on the analysis of their
karyotypes, glucose-6-phosphate dehydrogenase (G6PD) isoenzymes, and
BCR idiotypes.49-51 However, 15% to 45% of ALLs of B
lineage represent oligoclonal malignancies according to the diversity
of their HC gene expression.52-57 Analysis of these
patients has indicated that the leukemic population may diversify by
generating multiple IgH gene rearrangements during the process of tumor
progression.55-57 Sequence analysis of the IgH genes has
suggested that both VH All of the cells in the clonal EU12 cell line, established from a
childhood B-lineage ALL, apparently contain the same
DXP'1/JH4 rearrangement with no N nucleotide addition in
the DJH join. This suggests that the formative
transformational event may have occurred during fetal life in a pro-B
cell that had undergone D The discovery of sequential rearrangement and expression of Ig HC and
LC genes in the immediate precursors of mammalian B cells led to the
idea that intraclonal V(D)J recombinatorial diversification might occur
during B-cell genesis.19 The present studies provide direct evidence for the validity of the principle of intraclonal V(D)J
diversification, in that individual EU12 pro-B cells marked with a
DXP'1/JH4 gene segment give rise to a diverse repertoire during their progression from the pro-B to B-cell stage. Our analysis of the EU12 cell line model also suggests that
VH
We thank Dr Andrew J. Carroll for performing karyotypic analysis, Dr Larry Gartland for help with flow cytometry, Charlie Mashburn for technical support, and Marsha Flurry and Ann Brookshire for help in preparing the manuscript.
Submitted June 26, 2002; accepted September 8, 2002.
Prepublished online as Blood First Edition Paper, September 19, 2002; DOI 10.1182/blood-2002-06-1828.
Supported by National Institutes of Health grants AI48098, AI42127, and AI39816. M.D.C. is a Howard Hughes Medical Institute Investigator.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Max D. Cooper, University of Alabama at Birmingham, WTI 378, 1824 6th Ave S, Birmingham, AL 35294; e-mail: max.cooper{at}ccc.uab.edu.
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  A. M. Collins, M. Ikutani, D. Puiu, G. A. Buck, A. Nadkarni, and B. Gaeta Partitioning of Rearranged Ig Genes by Mutation Analysis Demonstrates D-D Fusion and V Gene Replacement in the Expressed Human Repertoire J. Immunol., January 1, 2004; 172(1): 340 - 348. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
JH
gene rearrangements in progenitor B (pro-B) cells are followed by
V
/
4, der(4)t(2;4) (q13;q21), inv(5)(q15q33) × 2, del(6)(q23) × 2, t(7;21) (p13;q11) × 2,
pausing to reflect.
Science.
2001;291:1503-1505