Download Cells: A Multiple Time Point Analysis Chronic Lymphocytic

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Lack of Intraclonal Diversification in Ig Heavy
and Light Chain V Region Genes Expressed by
CD5+IgM+ Chronic Lymphocytic Leukemia B
Cells: A Multiple Time Point Analysis
Edward W. Schettino, Andrea Cerutti, Nicholas Chiorazzi and
Paolo Casali
J Immunol 1998; 160:820-830; ;
http://www.jimmunol.org/content/160/2/820
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Copyright © 1998 by The American Association of
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References
Lack of Intraclonal Diversification in Ig Heavy and Light
Chain V Region Genes Expressed by CD51IgM1 Chronic
Lymphocytic Leukemia B Cells: A Multiple Time Point
Analysis1
Edward W. Schettino,* Andrea Cerutti,* Nicholas Chiorazzi,† and Paolo Casali2*
C
hronic lymphocytic leukemia (CLL)3 is the most frequent form of adult leukemia in Western societies, accounting for 30% of all leukemias. In the recently proposed definition of the disease, the malignant B cells are surface
CD51 (1, 2), express mainly IgM, and are thought to represent the
neoplastic expansion of a single clone that replaces the normal
polyclonal B cell population in the peripheral blood (3– 8). CLL B
cells putatively arise as a transformant of B-1a lymphocytes. In
healthy humans, B-1a cells are committed mainly to the production
of IgM, and account for 5 to 30% of the normal circulating, tonsillar, and splenic B lymphocytes (9 –11). They also account for
the majority of B cells in the fetus and neonate (10, 12, 13). B-1a
cells have long been thought to be primordial elements that rearrange only a restricted selection of V(D)J genes, and that lack the
machinery for Ig gene hypermutation. Consistent with this view,
*Division of Molecular Immunology, Department of Pathology, Cornell University Medical College, and The Immunology Program, Cornell University Graduate School of Medical Sciences, New York, NY 10021; and †Department of
Medicine, North Shore University Hospital and New York University School of
Medicine, Manhasset, NY 11030
Received for publication June 10, 1997. Accepted for publication September
26, 1997.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by U.S. Public Health Service Grants CA 68541 and
AR 40908 to P.C., and AI 10811 to N.C.
2
Address correspondence and reprint requests to Dr. Paolo Casali, Division of
Molecular Immunology, Department of Pathology, Cornell University Medical
College, 1300 York Avenue, New York, NY 10021-4896.
3
Abbreviations used in this paper: CLL, chronic lymphocytic leukemia; CDR,
complementarity-determining region; FR, framework region; H chain, heavy
chain; L chain, light chain; PMN, polymorphonuclear cell; R, replacement (mutation); S, silent (mutation).
Copyright © 1998 by The American Association of Immunologists
gained mainly from studies in the mouse (14, 15), CLL B cells
have been thought to express a restricted set of Ig V(D)J genes, in
general, in unmutated configuration (14, 15).
Recent findings from our laboratory, however, have shown that
human B-1a cells can express different V(D)J genes in mutated
configuration to encode for naturally occurring Abs and autoantibodies (16 –20). Many of these somatically mutated Abs and autoantibodies display traces of an Ag-driven selection process that
includes preferential segregation of somatic point mutations yielding an amino acid replacement (R mutations) within the complementarity-determining regions (CDRs), and various degrees of intraclonal diversification, as assessed by variation in the frequency
and distribution of somatic point mutations among colinear Ig
V(D)J gene sequences, that is different transcripts from the same
clonotype. Thus, human B-1a lymphocytes rearrange different
V(D)J genes, display the machinery necessary for somatic hypermutation, and can undergo a process of Ag-driven selection, somatic diversification, and, possibly, affinity maturation.
In light of the ability of normal B-1a cells to mutate their expressed Ig genes, we analyzed the sequences of V(D)J genes expressed by three CD51IgM1CLLs for the presence of somatic
point mutations, at multiple and sequential time points. We found
that these leukemic B cells expressed Ig VHDJH and VkJk or VlJl
genes that contained a number of somatic point mutations. These
point mutations did not show any preferential enrichment for R
mutations in the CDRs. In addition, they were absolutely conserved in multiple Ig cDNAs from the same time point and in
different transcripts from multiple time points over a 2-yr period,
and they were not associated with any translocation of the bcl-1 or
bcl-2 proto-oncogenes. Thus, the absence of selection of R mutations together with the lack of V gene mutation over time in
0022-1767/98/$02.00
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To analyze the modalities of clonal expansion of chronic lymphocytic leukemia (CLL) cells, we sequenced at multiple time points
the V(D)J genes expressed by CD51IgM1CLL B cells in three patients. All three V(D)J gene sequences were found to be point
mutated. The mutation frequency in the Ig VH (3.96 3 1022 and 2.41 3 1022 change/bp) and Vk and Vl (6.67 3 1022 and
1.74 3 1022 change/bp) genes of two CLLs (1.19 and 1.32, respectively) was similar, and higher than that in the corresponding
gene segments of the third CLL (1.69; 3.4 3 1023 and 6.67 3 1023 change/bp). In all three CLLs, there was no preferential
representation of nucleotide changes yielding amino acid replacement (R mutations), nor was there any preferential segregation
of R mutations within the Ig V gene complementarity-determining regions. In all three CLLs, the somatic mutations were all
identical in multiple Ig VHDJH transcripts at any given time point, and were all conserved at multiple time points throughout
a 2-yr period. The lack of concentration of R mutations in the complementarity-determining regions and the lack of intraclonal
heterogeneity suggest that Ag may no longer be able to play a significant role in the clonal expansion of these cells. This
conclusion would be strengthened further by the germline configuration of the bcl-1 and bcl-2 proto-oncogenes that are
translocated in neoplastic B cells that display significant traces of intraclonal diversification and Ag-dependent selection, such
as B-prolymphocytic leukemia and low grade follicular non-Hodgkin lymphoma. The Journal of Immunology, 1998, 160:
820 – 830.
The Journal of Immunology
821
CD51IgM1CLL cells suggests that Ag may no longer be capable
of inducing clonal diversification in these leukemic cells.
recognition sequence. PCR amplification of the germline VH6 gene consisted of 30 cycles of denaturation (95°C, 1 min), annealing (65°C, 1 min),
and extension (72°C, 2 min).
Materials and Methods
Analysis of Ig V gene mutations
PBMCs and immunophenotyping
B lymphocytes were enriched from PBMCs by depletion of T cells and
monocytes (21). Enriched B cells were reacted with FITC- or phycoerythrin-labeled mouse mAbs to human CD5, CD19, CD3, CD25, CD23,
CD10, HLA-DR, k Ig, l Ig, IgG, IgM, IgD, or IgA (Coulter Immunology,
Hialeah, FL, and Becton Dickinson Labware, Bedford, MA), in ice-cold
sterile PBS, pH 7.4, containing 1% BSA and 1% human AB serum (Life
Technologies, Gaithersburg, MD). After washing with the same buffer, the
cells were applied to a Becton Dickinson FACScan fluorescence flow cytometer (Becton Dickinson, San Jose, CA) for analysis (21).
PCR amplification, cloning, and sequencing of expressed Ig
V(D)J gene cDNA
Genomic VH segment DNA analysis of CLL 1.19
Genomic DNA was extracted from the polymorphonuclear cells (PMNs) of
patient 1.19, whose CLL B cells were used to generate the expressed 1.19
VH and VL gene sequences. The DNA was subjected to PCR amplification
using the sense VH6 leader primer in conjunction with an antisense 23-bp
primer, consisting of the reverse complement (59-TTTGTGTCTGGGCT
CACACTGACT-39) sequence of the 39 VH6 gene spacer-nonamer signal
Clonality of the CLL B cells
In addition to the single PCR amplification product, the clonality of the
CD51 B cells was assessed by Ig gene rearrangement analysis using a
genomic JH DNA probe on HindIII, EcoRI, and BamHI DNA digests (28).
Briefly, B cell genomic DNA (5 mg) was digested with a fivefold excess of
EcoRI, BamHI, or HindIII (Boehringer Mannheim Corp., Indianapolis, IN)
in appropriate buffer, loaded onto a 0.8% agarose gel (Life Technologies),
and electrophoresed at 22 V for 24 h. Size-fractionated DNA was transferred overnight onto Hybond-N nylon membranes (Amersham Life Sciences, Arlington Heights, IL) and prehybridized at 37°C for 4 h. Membranes were incubated overnight at 37°C in hybridization solution
containing a g-32P-labeled 2.2-kb genomic JH DNA probe (28), and then
washed four times before being autoradiographed at 270°C for 16 to 48 h
with Kodak XAR film (Eastman Kodak Co., Rochester, NY).
bcl-1 and bcl-2 proto-oncogene configuration in the CLL B
cells
To analyze the configuration of the bcl-1 and bcl-2 proto-oncogenes, the
nylon membranes blotted with DNA from the CLL B cells, and previously
hybridized with the g-32P-labeled JH DNA, were stripped of the JH probe,
according to the manufacturer’s protocol, and then reacted with the following probes: MTC on the HindIII digest for the major translocation
cluster of bcl-1 (29); p94PS on both EcoRI and BamHI digests for the
second break point on bcl-1 (29); and pFL-1 and pFL-2 on HindIII and
BamHI digests for the major and minor break points of bcl-2, respectively
(30, 31). After four washings, the membranes were autoradiographed at
270°C for 16 to 48 h with Kodak XAR film.
Results
Phenotypic analysis and clonality of the CLL B cells
PBMCs were obtained from three male patients (69 to 75 yr of
age: one African-American, 1.19; and two Caucasians, 1.32 and
1.69), who fulfilled the established diagnostic criteria for CLL
(1, 2, 30, 32), ranging from Rai stage I to stage II (Table I).
Direct immunofluorescence analysis of the T cell-depleted PBMCs from each CLL patient showed that virtually all of these
cells were surface IgM1, k1 or l1, CD191, HLA-DR1, and
CD231 (Table I), with less than 1% being CD31 and/or CD101
(data not shown). More than 99% of these CD191 cells expressed surface CD5 at high density (Fig. 1), consistent with a
virtually complete replacement of the PBL by the clonally expanded neoplastic B cells.
The monoclonality of these cell populations was verified by
isolating genomic DNA from each of the CLL B cells and separately digesting it with BamHI, HindIII, and EcoRI for Southern blot hybridization analysis. The reaction of the filter-immobilized DNA with the radiolabeled genomic JH probe
yielded, in each CLL, a hybridization pattern consistent with a
monoclonal Ig gene rearrangement (Fig. 2). Consistent with a
monoclonal B cell composition, only one of the six VH gene
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mRNA was extracted from CLL B cells using the Mini RiboSep Ultra
mRNA Isolation Kit (Becton Dickinson). First strand cDNA, synthesized
using the SuperScript First Strand cDNA Synthesis Kit (Life Technologies)
(16, 17, 22–24), was used as a template (100 ng) for PCR in a volume of
50 ml containing 200 mM of each dNTP, 2.5 U of Taq polymerase (PerkinElmer Cetus Corp., Norwalk, CT), and 10 pmol of each oligonucleotide
primer. Six individual PCR amplifications were performed for the H chain.
Each reaction included a sense leader VH primer, specific for the members
of one of the six VH families, in conjunction with an antisense Cm oligonucleotide primer. Each sense oligonucleotide primer consisted of a degenerate sequence encompassing an area of Ig gene leader region plus an
EcoRI site, as follows: VH1, 59-GGGAATTCATGGACTGGACCTG
GAGG(AG)TC(CT)TCT(GT)C-39;
VH2,
59-GGGAATTCATGGA
CATACT(GT)TG(GT)T(CT)CACGCT(CT)CT(GC)C-39; VH3, 59-GGGA
ATTCATGGAG(CT)TTGGGCTGA(CG)CTGG(CG)TTT(CT)T-39; VH4,
59-GGGAATTCATGAA(AG)CA(TC)CGTGGTTCTT(CT)(AC)T(CT)C
T(CG)C-39; VH5, 59-ATGGGGTCAACCGCCATCCTCGCCCT-39; and
VH6, 59-GGGAATTCATGTCTGTCTCCTTCCTCATCTTCC-39. Due to
the relatedness of the VH7 to the VH1 family (25), the VH7 family members
can be amplified by the VH1 family primer. The H chain antisense oligonucleotide primer consisted of the reverse complement (59-CCGAATTCAGACGAGGGGGAAAAGGGTT-39) of a 21-nucleotide Cm sequence plus
an EcoRI site. The L chains were amplified in five individual PCRs. Each
PCR included a sense leader Vk or Vl primer specific for the members of
the L chain gene families, in combination with an antisense Ck or Cl
oligonucleotide primer. Each sense primer consisted of a degenerate sequence encompassing an area of the Ig gene leader region, as follows: Vl1,
59-ATG(GA)CC(TG)GCT(CT)CCCTCTCCTCCT-39; Vl2– 6, 59-ATG
(AG)C(CT)TGGACCC(CT)(AT)CTC(CT)(TG)(TG)TT-39; Vk1,2, 59AGCTCCTGGG-GCT(GC)CT(AG)(AC)TGCTCT-39; Vk3, 59-TCTCT
TCCTCCTGCTACTCTGGCT-39; and Vk4, 59-ATGGTGTTGCAGACC
CAGGTCTTC-39. The L chain antisense oligonucleotide primers consisted
of the reverse complement of a 23-nucleotide Cl sequence (59-AG
GAGACTCCTCGAAGTTCGGTT-39) or a 23-nucleotide Ck sequence
(59-AGAAGGGCGGTAGACTACTCGTC-39). PCRs for the amplification
of both VH and VL gene cDNA consisted of 30 cycles of denaturation
(95°C, 1 min), annealing (60°C, 1 min), and extension (72°C, 2 min).
Amplified DNA was ligated into pCR II plasmid vectors (Invitrogen Corp.,
San Diego, CA), and dideoxy sequencing was performed using plasmid
dsDNA prepared from selected bacterial clones, as reported (22, 24). Each
V(D)J sequence was derived from the analysis of four to six independent
bacterial isolates. Differences in nucleotide sequences among different recombinant clones were rarely observed, i.e., less than 0.0002 difference/
base, a frequency that is consistent with the error rate of the Taq polymerase. The DNA sequences were analyzed using the BLAST algorithm, as
found in the NCBI World-Wide-Web home page accessed through the
Netscape Navigator. The MacVector v.5.0 sequence analysis software (International Biotechnologies, New Haven, CT) was used to analyze the
current human Ig gene V-BASE database (MRC Centre for Protein Engineering, Cambridge, U.K.).
The number of expected R mutations in the Ig V segment CDRs and FRs
was calculated using the formula R 5 n 3 CDR Rf or FR Rf 3 CDRrel or
FRrel, in which n is the total number of observed mutations, Rf is the
replacement frequency inherent to CDR or FR sequences, and CDRrel and
FRrel are the relative size of the CDRs or FRs (26, 27). The CDR Rf and FR
Rf inherent to the respective progenitor germline genes are as follows:
V6-1, CDR 5 0.7935, FR 5 0.7404; V4-59, CDR 5 0.8218, FR 5 0.7217;
DP-88, CDR 5 0.7801, FR 5 0.7477; 02/012, CDR 5 0.7991, FR 5
0.7533; lv318, CDR 5 0.8128, FR 5 0.7319; and A23, CDR 5 0.7901,
FR 5 0.7463. A binomial probability model was used to evaluate whether
the excess of R mutations in CDRs or their scarcity in the FRs was due to
chance only: p 5 {n!/[k! (n-k)!]} 3 qk 3 (1-q)n-k, in which q is the probability that an R mutation will localize to CDRs or FRs (q 5 CDRrel 3
CDR Rf or FRrel 3 FR Rf), and k is the number of observed R mutations in
the CDRs or FRs (26).
CD51IgM1CLL B CELL Ig V(D)J GENES
822
Table I. CLL patients: medical history and cellular morphology
B Lymphocytes (CD191 Cells)
Surface expression
Ig
chain
Disease
Oncogenes
Analysis
Sex
(age)
Race
Date of
diagnosis
Rai
stage
Morphology
H
L
CD5
(%)
HLA-DR
(%)
CD23
(%)
bcl-1
bcl-2
Date
Sample
no.
1.19
(E.L.)
Male
(69 yr)
AfricanAmerican
July
1989
II
Typical,
homogenous
m
k
99.0
100
100
Germline
Germline
11-21-91
07-10-92
08-20-92
04-14-93
1.19a
1.19b
1.19e
1.19f
1.32
(P.S.)
Male
(69 yr)
Caucasian
Sept.
1989
I
Typical,
homogenous
m
l
99.7
100
100
Germline
Germline
11-20-91
03-25-93
11-24-93
1.32a
1.32c
1.32e
1.69
Male
(H.M.) (75 yr)
Caucasian
Aug.
1990
II
Typical,
homogenous
m
k
99.7
100
100
Germline
Germline
12-11-91
05-05-93
12-08-93
1.69a
1.69d
1.69g
FIGURE 1. Surface expression of
CD19 and CD5 by CLL B cells. The T
cell-depleted circulating lymphocytes from one healthy adult subject
(1-0) and from three patients diagnosed with CLL (1.19, 1.32, and
1.69) were reacted with phycoerythrin-labeled mAb to CD5 and
FITC-labeled mAb to CD19, and
then applied to the FACS for analysis
of their fluorescence intensities. B
cells were identified by their high
levels of expression of CD19.
Greater than 99% of the B lymphocytes were surface CD5 positive in
the three CLLs, compared with a proportion of 10 to 25% in the healthy
subject.
family primers, VH6, VH4, or VH1, yielded a product of appropriate size (approximately 500 bp) when used in conjunction
with the antisense Cm primer to amplify cDNA reverse transcribed from mRNA isolated from each CLL sample, 1.19, 1.32,
and 1.69, respectively (Fig. 3). Accordingly, only one of the
five L chain primers, Vk1,2 or Vl2– 6, yielded an amplification
product of appropriate size (approximately 350 bp) in each CLL
sample when used in conjunction with the antisense Ck or Cl
primer (data not shown).
Sequences of the CLL B cell VHDJH genes
The PCR-amplified Ig VHDJH gene DNAs were cloned and sequenced. Each sequence was derived from the analysis of four to
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Patient
The Journal of Immunology
823
FIGURE 2. Southern blot analysis of genomic
CLL B cell DNA digested with BamHI, HindIII, and
EcoRI restriction enzymes and hybridized with a
radiolabeled human JH genomic probe. HL60 denotes the germline position in each of the digests. In
both CLLs 1.19 and 1.69, one germline and one Ig
gene rearrangement were detected (monoclonal).
In CLL 1.32, a double Ig gene rearrangement was
detected (monoclonal).
VH gene segment was found to be unique to the CLL B cell clone;
thus, the likelihood that these differences were due to allelic polymorphism is highly improbable. Finally, the contention that the
differences detected in the VH gene segment sequences expressed
by 1.19, 1.32, and 1.69 CLL cells represented somatic mutations,
as compared with their respective germline VH gene sequences,
was supported further by the presence of point mutations in the
sequences of the JH gene segments of CLLs 1.19 and 1.32, and in
the sequences of the Jk and Jl gene segments of each CLL B cell
clones.
The nucleotide and deduced amino acid sequences of the expressed junctional VHDJH gene segments from the CLL B cell
clones were compared with those of the reported germline D, DIR,
and JH genes (Fig. 4) (45– 49). The best-fit D and/or DIR genes
were identified according to the two following criteria: first, priority was given to the VH or JH gene sequence when VH and D, or
D and JH gene sequences overlapped; and second, if more than one
candidate D and/or DIR gene was identified, the germline D gene
sequence, displaying the longest stretch of identity (with a minimum match of six nucleotides in a stretch of 7 bp, or with a minimum match of five nucleotides in row) to that of the expressed D
gene segment, was assigned. One of the three clones, 1.69, expressed a D segment that resulted from the utilization of a single
germline D gene; the second, 1.19, resulted from the utilization of
two different germline D genes, possibly as result of a D-D fusion,
one of which was inverted; and the third, 1.32, from the utilization
of three different D genes (Fig. 4 and Table II). Thus, the D gene
segments expressed by the CLL B cells represented the rearrangement of a heterogeneous assortment of germline D genes in conventional, fused, or inverted configurations. Two of the three CLLexpressed D genes (1.19 and 1.32) were 59 flanked by
nontemplated residues (N additions); and all but that encoding
CLL 1.32 were also 39 flanked by nontemplated nucleotides. CLL
1.19 and 1.32 utilized JH6b and JH4b genes, respectively, that were
both truncated and mutated (Fig. 4 and Table II). CLL 1.69 utilized
a JH6b gene that was both intact and unmutated. The deduced
amino acid sequences of the DJH segments of the three CLLs were
divided into CDR3 and FR4 stretches (Fig. 4), according to Kabat
et al. (50). The CDR3 sequences were highly divergent in composition and ranged in length from 15 to 22 amino acids; the FR4
sequences were invariable in length and displayed little diversity.
Sequences of the CLL B cell VkJk and VlJl genes
The nucleotide and deduced amino acid sequences of the VLJL
gene segments expressed by the three CLL B cells are depicted in
Figure 5, A and B, respectively, and summarized in Table II. Like
the VH gene locus, the human Ig k- and l-chain loci have been
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six independent bacterial isolates, that is, discrete Ig VHDJH gene
cDNAs. The nucleotide and deduced amino acid sequences of the
VHDJH gene segments are depicted in Figures 3 and 4, and summarized in Table II. The sequence of the Ig VH gene expressed by
CLL 1.19 contained 12 nucleotide differences when compared
with that of the germline V6-1 gene, the single member of the VH6
family; the sequence of the VH gene expressed by CLL 1.69 displayed one nucleotide difference when compared with that of the
closest reported germline gene DP-88 (an allelic variant of the
V1-69 gene) (33); and the sequence of the Ig VH gene expressed
by CLL 1.32 displayed seven nucleotide differences when compared with the closest reported germline gene V4-59 (formerly
referred to as VH4-15). These VH gene nucleotide differences were
assumed to result from somatic point mutations because of the
following considerations. First, the whole human Ig H chain locus
has now been fully characterized, and all VH germline gene segments have been sequenced (34 –37). Like many other human VH
genes, including V3-23 (formerly referred to as VH26) and V4-34
(formerly referred to as VH4.21), the V6-1 gene displays no allelic
polymorphism, and recurs in an absolutely conserved form within
each individual ethnic population (38 – 42). To further confirm the
conserved nature of V6-1 in CLL 1.19, we utilized DNA from the
patients’ autologous PMNs and a pair of primers encompassing
sequences identical in the expressed 1.19 and the reported V6-1
gene to amplify an approximately 400-bp DNA segment for cloning. Sequences from multiple independent isolates were identical
to each other and throughout the overlapping area to that of V6-1
(Fig. 3), therefore pointing at this gene segment as the template of
the expressed and mutated 1.19. The remaining two germline
genes, DP-88 and V4-59, putatively utilized by the CLLs analyzed
in this study, have been reported to occur in polymorphic variants.
DP-88 is one of 13 related variants (variant 7) of the V1-69 gene.
Each of these variants has been sequenced, thereby producing a
complete profile of this gene’s polymorphism (41, 42). The sequences of the V1-69 allelic set differ by a limited number of
nonconservative substitutions that may have functional significance. Sasso and coworkers concluded that 6 of these 13 elements,
variants 1, 5, 7, 10, 12, and 13, comprise the alleles present in most
people (33, 41). When compared with the sequences of all V1-69
variants, the transition (C to T) at position 20 was found to be
unique to the expressed CLL 1.69 VH gene segment, suggesting
that this substitution that yields an amino acid replacement is the
result of a somatic point mutation. The polymorphic variants of the
V4-59 gene, although not yet fully characterized, are believed to
consist of only few nucleotide differences. When compared with
the sequences of all known V4-59 variants (38, 43, 44), each of the
seven nucleotide differences detected in the expressed CLL 1.32
824
CD51IgM1CLL B CELL Ig V(D)J GENES
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FIGURE 3. Nucleotide and deduced amino acid sequences of the VH gene segments expressed by the three CLL B cell clones (1.19, 1.32, and
1.69). Dashes indicate identities. The top sequence in each cluster is that of the germline gene to which the remaining sequences of the cluster
are compared. Solid lines on top of each cluster depict CDRs. Lower case letters denote untranslated sequences. Sequences encompassed by the
VH leader and VH6 23-bp spacer primers are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers
U31936, U31937, and U31962.
characterized recently in detail, and virtually all germline Vk and
Vl gene segments have been sequenced (50 –53), thus providing a
representative assortment of unmutated germline templates to
which the expressed Vk and Vl genes can be compared.
The Ig Vk gene expressed by CLL 1.19 B cells was of the Vk1
subgroup, and its sequence contained 19 somatic point mutations
as compared with that of the germline gene 02/012. The CLL 1.69
Ig Vk gene sequence contained two somatic point mutations as
0
p 5 0.348
Jl2/
Jl3
Jk2
1
p 5 0.409
Jk5
2
99.5
98.3
JH6b
0
p 5 0.244
Dxp1
JH4b
DIR1/D21-9
3
p 5 0.296
0
NA
1
NA
99.7
97.6
1.69
1.32
R, number of detected and (expected) replacement mutations; S, number of detected silent mutations; p, probability that the R mutations occurred by chance; NA, not applicable.
a
D gene in inverted form.
b
Statistically significant.
1
NA
0
p 5 0.071
5
(3.2)
1
(1.1)
1
(0.5)
3
p 5 0.065
6
(3.4)
3
(1.1)
0
(0.5)
93.3
02/012
(Vk1)
1v318
(Vl3)
A23
(Vk2)
JH6b
DK1/D2a
7
p 5 0.042b
3
(6.1)
3
(3.4)
1
(0.6)
2
(2.4)
0
(1.2)
0
(0.2)
96.0
V6-1
(VH6)
V4-59
(VH4)
DP-88
(VH1)
1.19
CLL
Closest
Germline
Gene
(family)
Nucleotide
% Identity
R
CDR
p 5 0.286
S
0
FR
R
S
CDR
R
FR
S
D
Gene
JH
Gene
Closest
Germline
Gene
(family)
Nucleotide
% Identity
R
VL Gene
VH Gene
compared with that of the Vk2 germline gene A23. Finally, the
CLL 1.32 Ig Vl gene sequence contained five somatic point mutations as compared with that of the Vl3 germline lv318 gene.
Comparison of the expressed Ig JL gene nucleotide and deduced
amino acid sequences with those of the germline Jk and Jl genes
(Fig. 5B) showed that CLL 1.19 utilized a mutated Jk5 gene; CLL
1.69 utilized a mutated Jk2 gene; and CLL 1.32 utilized a mutated
Jl2/Jl3 gene that was 59 flanked by a CCT triplet coding for a Pro
residue. This additional CCT triplet could have resulted from the
direct juxtaposition of the two deoxcytosine nucleotides (CC) 59 of
the heptamer/nonamer sequences of the donor Vl gene segment
and the deoxythymidine residue (T) 39 of the heptamer/nonamer
sequences of the donor Jl gene segment (53).
Analysis of the somatic point mutations in the CLL B cell Ig
V(D)J genes
In the absence of negative or positive selective pressure on a gene
product, R and S mutations distribute randomly throughout the
coding sequence. If a DNA segment displays a number of R mutations higher than that expected by chance alone, it is likely that
a positive pressure was exerted on the gene product to select for
those mutations, as it occurs in the V segment CDRs of affinity
mature Abs in which nucleotide changes yield a high R:S mutation
ratio. Conversely, if a DNA segment displays a number of R mutations lower than that expected by chance, it is likely that a negative pressure was exerted on the gene product to select against
mutations such that the protein structure is preserved, as it is in the
FRs of a functional Ab, in which nucleotide changes yield a low
R:S mutation ratio. We calculated the number of expected R mutations in the CLL VH and Vk or Vl gene segments, as proposed
by Chang and Casali (26). In our calculations, the general replacement frequency (Rf) value of 0.75, originally calculated by Jukes
and King (54) for random hypermutation of a gene product that
need not be conserved in structure, was substituted with the Rf
values derived from the analysis of each individual germline VH,
Vk, or Vl gene sequence, as listed in Materials and Methods. The
introduced correction yields a more accurate estimate of the frequency of R mutations expected by chance only, given the demonstration by Chang and Casali (26) that a significant number of
human Ig V gene segments display a CDR codon composition that
is inherently more prone to R mutations.
The observed distribution of the R and S mutations in the VH and
VL gene segment CDRs and FRs is summarized in Table II, which
also reports the expected number of R mutations in the CDRs and
FRs. In the CLL 1.19 B cells, the expressed VH gene contained five
R mutations, two of which were located in the CDR2, and seven S
mutations, all of which were located in the FRs, yielding an R:S ratio
of 2:0 in the CDRs and 3:7 in the FRs; the Vk gene contained 11 R
mutations, six of which were located in the CDRs, and five S mutations, two of which were located in the FRs, yielding an R:S mutation
ratio of 6:3 in the CDRs and 5:2 in the FRs. In CLL 1.32 B cells, the
expressed VH gene contained three R mutations, all of which were
located in the FRs, and four S mutations, three of which were located
in the FRs, yielding an R:S mutation ratio of 0:1 in the CDRs and 3:3
in the FRs; the Vl gene contained four R mutations, three of which
were located in the CDRs, and one S mutation located in the FR1,
yielding an R:S mutation ratio of 3:0 in the CDRs and 0:1 in the FRs.
In the CLL 1.69 B cells, the expressed VH gene contained only one R
mutation located in the FR1, yielding an R:S mutation ratio of 0 in the
CDRs and 1:0 in the FRs; the Vk gene contained one R mutation,
located in the FR1, and one S mutation, located in the CDR3, yielding
an R:S mutation ratio of 0:1 in the CDRs and 1:0 in the FRs. In all
three VH genes, the number of R mutations in the CDRs was lower
than expected (Table II). In one VL gene segment (1.69), it also was
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Table II. Structure of the expressed CLL B cell Ig V(D)J genes
825
p 5 0.118
S
JL
Gene
The Journal of Immunology
826
CD51IgM1CLL B CELL Ig V(D)J GENES
FIGURE 4. Nucleotide and deduced amino acid sequences of the D and JH gene segments expressed by the three CLL B cell clones (1.19, 1.32,
and 1.69). Each expressed sequence represents a composite sequence of the multiple time point and multiple transcript analysis. Dashes indicate
identities. The nucleotide sequences of relevant portions of the germline D and JH genes are given for comparison, and appear above or below
the expressed Ig D and JH gene segments as underlined strings. Unencoded nucleotides (N) are segregated 59 and/or 39 of the D gene segment.
The deduced amino acid sequences are divided in CDR3 and FR4.
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FIGURE 5. Nucleotide and deduced amino acid sequences of the VLJL gene segments expressed by the three CLL B cell clones (1.19, 1.32, and
1.69). Dashes indicate identities. A, V gene segments; the top sequence in each cluster is that of the germline gene to which the remaining
sequences of the cluster were compared. Solid lines on top of each cluster depict CDRs. Sequences encompassed by the VL leader primers are
underlined. B, J gene segments; the nucleotide sequences of relevant portions of the germline J genes are given for comparison, and appear above
the expressed J gene segments as underlined strings. The deduced amino acid sequences are divided in CDR3 and FR4. These sequence data are
available from EMBL/GenBank/DDBJ under accession numbers U31963, U31964, and U31965.
The Journal of Immunology
827
lower then expected; in the two others (1.19 and 1.32), it was higher
than expected (Table II); however, this excess can be due to chance
only. Thus, these results demonstrate that in each of the CLLs analyzed, both the mutated Ig VH and VL gene segments did not show
any preferential enrichment for R mutations in the CDRs. In addition,
in all CLL VH and VL gene segments but one (1.19 VH), the number
of R in the FRs was not different from that expected on the basis of
chance only. In the 1.19 VH gene segment, the scarcity of R mutations
in the FRs reached a level of significance, demonstrating that a negative pressure was probably exerted on the gene product so that the
protein structure is preserved.
Lack of intraclonal diversification in the CLL B cell clones
To determine whether selective forces were influencing the clonal
expansion of the three CLL B cells in vivo at times different from that
initially considered, we analyzed the intraclonal diversity of each case
by selecting multiple time points during a 11⁄2- or 2-yr time period.
The expressed Ig VHDJH gene from CLL 1.19 was analyzed over a
1–1/2-yr period at four separate time points (Figs. 3 and 4, and Table
I). Each of the VH gene segments as well as the sequences of the
CDR3 and FR4 were absolutely identical at each of the four time
points, and no additional point mutations were present. The expressed
Ig VHDJH genes from CLLs 1.32 and 1.69 were both analyzed over
a 2-yr period by selecting three separate time points (Figs. 3 and 4,
and Tables I and II). Like CLL 1.19, all of the somatic point mutations
in the VH gene segment (1.32 and 1.69, respectively), as well as the
sequences encoding the CDR3 and FR4, were absolutely identical at
each of the three time points, and no additional point mutations were
observed.
bcl-1 and bcl-2 proto-oncogene configuration
Translocation of the bcl-1 and bcl-2 proto-oncogenes has been associated with other B cell neoplasia that display significant traces of
intraclonal diversification and Ag-dependent selection of somatic
point mutations. bcl-1 is translocated in mantle zone lymphoma and
B-prolymphocytic leukemia (2), and bcl-2 is translocated in low grade
follicular non-Hodgkin lymphoma (55). In both cases, translocation of
these proto-oncogenes has been associated with the high degree of
clonal expansion and cellular division characteristic of these B cell
disorders. To analyze whether these proto-oncogenes were translocated in the three CLLs, B cell genomic DNA from each of the CLL
B cells was subjected to Southern blot analysis using four different
probes for either the bcl-1 or bcl-2 loci, as detailed in Materials and
Methods. The hybridization pattern was consistent with that of both
proto-oncogenes being in germline configuration (Fig. 6).
Discussion
These studies show that IgM1CLL B cells can express Ig V(D)J
gene segments containing variable numbers of somatic point mutations. They also show that these mutations do not display any
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FIGURE 6. Bcl-1 and bcl-2 proto-oncogene gene rearrangement analysis. Southern blot analysis of genomic CLL B cell DNA (1.19, 1.32, and
1.69) digested with BamHI, HindIII, and EcoRI restriction enzymes and hybridized with specific probes. A, MTC on the HindIII digest for the major
translocation cluster of bcl-1 and p94PS on both EcoRI and BamHI digests for the second break point of bcl-1. B, pFL-1 and pFL-2 on both HindIII
and BamHI digest for the major and minor break points of bcl-2, respectively. HL60 cells (promyelocytic leukemia cell line) were used as controls
displaying unrearranged bcl-1 and bcl-2 proto-oncogenes.
828
expressed H or L chain V gene and in each CLL patient, the number of expected CDR and FR R mutations was calculated, and used
to determine, on the basis of a binomial distribution model, the
probability that any excess and scarcity of R mutations in the
CDRs and FRs were due to chance only. The results of these calculations clearly indicated that in none of the three CLL B cell H
and L chain V segments was there evidence for positive selection
of R mutations in the CDRs, a result, in general, of a process of
Ag-driven clonal selection. In some cases, it could be hypothesized
that a negative, rather than a positive, selective pressure applies to
R mutations, that is, the unmutated product rather than a somatically mutated form of the germline gene is selected by Ag. This is
a distinct possibility (58), but has never been experimentally substantiated in vivo. Even in such a case, however, negative pressure
on R mutations in CDRs should be accompanied by a similarly
negative pressure in the FRs to preserve a structurally sound Ab
molecule. Thus, a significant scarcity of FR R mutations should be
an important feature of Ag-selected Ab-producing cell clones. The
present findings show, with the exception of the CLL 1.19 VH gene
segment, no significant negative selection of R mutations in the
FRs of the Ig H and L chain V segments expressed by the three
CLL B cell clones.
As shown by the study of a variety of specific experimental Ab
responses, Ag-driven selection and expansion of a B cell clone not
only result in positive selection of R mutations in the Ig V segment
CDRs, but also lead to a significantly high degree of intraclonal
diversification, revealed at the DNA transcription level by the appearance of colinear Ig V(D)J DNA sequences sharing and differing in various numbers of somatic point mutations. Intraclonal
diversification has been shown in neoplastic equivalent of GC B
cells, such as follicular lymphoma (57, 59, 60), and one patient
with CD52CLL (61). In these cells, as in normal cells undergoing
specific expansion and selection by Ag, somatic diversification has
been found to be associated with positive selection of CDR R
mutations and negative selection of FR R mutations. To better
verify whether selective forces were influencing the biologic behavior of the CLL clones in vivo, we addressed the issue of intraclonal diversification by not only analyzing multiple independent
bacterial isolates from a given time point, but by also examining
multiple time points of interest during a 11⁄2- to a 2-yr period. By
following these clones individually through their natural history in
vivo, we failed to detect any nucleotide variation throughout our
time point analysis study, therefore concluding that these CLL B
cells lacked traces of intraclonal diversification. Lack of intraclonal diversification in CLL B cells is supported further by Fais et
al. (62, 63), who demonstrated that clonally related IgG- and IgAswitched progeny of IgM1CLL B cells fail to accumulate appreciable numbers of new somatic mutations in their Ig V genes.
However, IgM1 progenitors of IgG1CLL B cells retain the ability
to accumulate somatic mutations (64), presumably because they
have yet to receive the final “hit” in the transformation process.
The occurrence of switching without the accumulation of V gene
mutations suggests that the processes of differentiation and diversification are not necessarily linked, and that in CLL B cells, clonal
differentiation can occur in the absence of V gene mutation.
Taken together, our findings suggest that antigenic stimulation is
unlikely involved in the clonal diversification of our panel of
CD51IgM1CLL B cells. They differ from those by Hashimoto and
coworkers (65), who detected a high number of somatic mutations
in the expressed Ig H chain genes in two (CLLs 055 and 030) of
seven IgG1CD51CLLs, as compared with their respective germline genes (V4-34 and H11, respectively). The distribution of R
mutations in both of these CLL IgG1 clones was consistent with
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preferential enrichment of R changes within the CDRs, and, in
each CLL patient, they are absolutely conserved not only in multiple Ig cDNAs from the same time point, but also throughout
multiple time points over a 2-yr period. The scarcity of R mutations in the CDRs, and absolute conservation of the sequences of
the Ig V(D)J gene cDNA transcripts among different B cells of the
same clonotype, are consistent with an absence of intraclonal diversification. These findings were derived from the analysis of
three patients diagnosed with CD51IgM1CLL, each of whom fulfilled the established clinical and morphologic diagnostic criteria
for the disease, including B cell surface expression of both CD5
and CD23 at high density. The CLL nature and the Ag-independent emergence of these neoplastic clones were strengthened further by the germline configuration of the bcl-1 and bcl-2 protooncogenes, which translocate (to chromosome 14) in neoplastic B
cells undergoing a high degree of intraclonal somatic diversification and selection by Ag, such as in mantle cell lymphoma and
follicular lymphoma (56, 57).
The first issue addressed by these studies was whether (IgM1)
CLL B cells can express somatically mutated Ig V genes. In each
of the three B cell clones, the expressed VH gene segment sequences displayed a number of differences as compared with those
of the closest reported germline genes V6-1, V4-59, and DP-88.
The probability that these differences stemmed from the utilization
of heretofore unreported germline gene was estimated to be negligible in each of the three CLL VH gene segments due to the
following considerations. V6-1 has been shown to be among the
most conserved Ig VH genes throughout the human population.
V4-59 has been reported to be polymorphic, but the allelic variants
in this gene have been shown to consist of only few nucleotide
differences (38, 39). DP-88 is one of the 13 related variants of the
V1-69 gene. Each of these variants has been sequenced, thereby
producing a complete profile of this gene’s polymorphism (41, 42).
The contention that the expressed Ig VH genes constitute somatically mutated forms of V6-1, V4-59, and DP-88 templates is supported further by the presence of somatic mutations in the juxtaposed D and JH genes, and by the isolation of a VH gene template
identical to the germline V6-1 gene from the genomic PMN DNA
of patient 1.19. In each B cell clone, the load of somatic mutations
in the expressed Ig VHDJH gene segment was accompanied by a
comparable load of putative somatic mutations in the paired L
chain variable segments. For instance, the CLL 1.19 B cell Vk
gene sequence displayed 19 nucleotide differences when compared
with that of the closest reported k-chain germline gene, 02/012; the
CLL 1.32 B cell Vl gene sequence displayed five nucleotide differences when compared with that of the closest reported l-chain
germline gene, lv318; and the CLL 1.69 B cell Vk gene sequence
displayed two nucleotide differences when compared with that of
the closest reported k-chain germline gene, A23. Like their H
chain counterparts, the Jk and Jl genes were also mutated.
The next question asked by our experiments was whether the
nature and the distribution of the R mutations in the CLLs Ig V
segment were consistent with a selection by Ag. Ag-selected Abs
have been shown to include a higher frequency of R mutations in
the Ig V gene CDRs than in the FRs, in which the proportion of S
mutations may be greater. The recent findings by Chang and Casali
(26) suggest that when assessing the Ag-selected nature of somatic
point mutations in an expressed Ig V gene, the inherent susceptibility to amino acid replacement or replacement frequency, Rf,
needs to be calculated for the progenitor germline gene sequence.
The Rf is then used to calculate the theoretically expected number
of R mutations in the CDRs or FRs of that particular gene given a
random distribution of R mutations. This principle was applied to
the analysis of the Ig V genes of the three CLL subjects. For each
CD51IgM1CLL B CELL Ig V(D)J GENES
The Journal of Immunology
Acknowledgments
We are grateful to Drs. E. Newcomb and M. Potmesil for providing the
CLL samples. We thank Dr. A. Matolscy for his help with the protooncogene analysis, and Ms. N. Pacheco for providing her skillful technical
assistance.
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selection by Ag. Thus, different selective forces are present in IgGproducing CLL cell, as discussed by Hashimoto et al. (65), as
compared with IgM-producing CLL cells, as shown by this study.
The different selective pressures that are applied to IgM1 and
IgG1 CLLs need to be further investigated. The majority of studies
on CLL suggest that the B cells are clonally expanded, and use a
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for low affinity, polyreactive autoantibodies (8). These findings are
consistent with the general unmutated nature of the putative nonneoplastic CLL progenitors, B-1a cells. Recent findings by us and
others (16 –20), however, have demonstrated that normal human
B-1a lymphocytes can produce somatically mutated and Ag-selected Abs, indicating that these cells do indeed possess the machinery for somatic hypermutation, and can undergo an affinity
maturation process, and therefore suggesting that CLL B-1a cells
may also be able to mutate the expressed Ig V(D)J genes. By
indicating that mutations can occur in the V genes of both H and
L chains, and that the numbers and locations of these mutations are
closely paralleled in individual patients, our findings in
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show evidence of a pattern of Ag selection of R mutations, nor did
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somatic mutations are inherent to the clonal evolution of CLL, but
rather represent a pre-existing feature of the normal B-1a cell
clones “hit” by the transforming event(s).
829
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CD51IgM1CLL B CELL Ig V(D)J GENES