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Normal B Cells Express 51p1-Encoded Ig
Heavy Chains That Are Distinct From Those
Expressed by Chronic Lymphocytic
Leukemia B Cells
George F. Widhopf II and Thomas J. Kipps
J Immunol 2001; 166:95-102; ;
doi: 10.4049/jimmunol.166.1.95
http://www.jimmunol.org/content/166/1/95
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Copyright © 2001 by The American Association of
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References
Normal B Cells Express 51p1-Encoded Ig Heavy Chains That
Are Distinct From Those Expressed by Chronic Lymphocytic
Leukemia B Cells1
George F. Widhopf II, and Thomas J. Kipps2
B
cell chronic lymphocytic leukemia (CLL)3 is characterized by the accumulation of mature resting lymphocytes
in the blood, marrow, and lymphoid tissues (1). The leukemic cells of ⬎90% of CLL patients also express CD5, a cell
surface Ag involved in modulating lymphocyte cell signaling and
activation (2). Prior studies by many groups have examined the
nature of Ig expression in CLL (3, 4). Several previous studies
revealed that leukemic B cells of patients with CLL express a
restricted IgH V region (VH gene) repertoire.
Studies indicate that as many as 20% of CLL patients have
malignant cells that express 51p1 (5–7), a VH gene that also appears to be over-represented in the fetal and adult primary B cell
repertoire (8, 9). 51p1 is an Ig VH1 gene and is one of several
alleles that belong to the VH1-69 H chain locus. Restriction fragment length polymorphism analysis using oligonucleotide probes
identified at least 13 distinct variants (10). These variants represent
nine related sequences that can be divided into two groups based
on several single-base differences in the second complementaritydetermining region (CDR2). These nonconservative changes result
in the expression of different H chains that can be distinguished by
the murine anti-idiotypic mAb G6 (11, 12). G6 recognizes IgH
Division of Hematology/Oncology, Department of Medicine, University of California
San Diego, La Jolla, CA 92093.
Received for publication July 17, 2000. Accepted for publication September 28, 2000.
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 in part by National Institutes of Health Grant R37CA49870. G.F.W. was supported by National Institute of Allergy and Infectious
Diseases Training Grant AI07384 and National Institutes of Health Grant
T32-CA09290.
2
Address correspondence and reprint requests to Dr. Thomas J. Kipps, Division of
Hematology/Oncology, Department of Medicine, University of California San Diego,
La Jolla, CA 92093. E-mail address: [email protected]
3
Abbreviations used in this paper: CLL, chronic lymphocytic leukemia; VH gene, H
chain V region gene; CDR, complementarity-determining region; FW, framework
region.
Copyright © 2001 by The American Association of Immunologists
encoded by nonmutated VH1-69 alleles that are homologous to the
five 51p1-like variants, but does not react with any of the eight
VH1-69 variants that are more closely related to hv1263 (12).
Genetic analysis demonstrated that several variants likely arose
from VH1-69 gene duplications (13). As such, individuals may
possess zero to four copies of any one VH1-69 variant. Using the
G6 Id, prior studies determined that the expression of the 51p1-like
variants is proportional to its germline gene copy number (9).
However, this alone cannot account for the high frequency at
which 51p1 is used in CLL.
A prior study of CLL samples that express an Ig encoded by a
VH1 H chain found that use of the 51p1-like alleles of the IgH
VH1-69 locus is favored (5). Furthermore, leukemic B cells that
express the 51p1-like allele of VH1-69 have a distinctive use of
certain D and JH gene segments, in particular, D3-3/DXP4 and
JH6, respectively. This is not the case with CLL B cells that express H chains encoded by VH1 genes other than VH1-69 or with
nonneoplastic tonsillar B cells that express 51p1 (14). In addition,
the average length of the third complementarity-determining region (CDR3) of 51p1-expressing leukemia cells was found to be
significantly longer than that of normal tonsillar B cells that express Ig reactive with the G6 mAb that are encoded by 51p1.
It is unclear whether the distinctive molecular features of 51p1encoded IgH expressed by CLL B cells represent a disease-associated restriction or are features seen in the B cell repertoire of
normal adults. Contrary to our previous findings, a more recent
report using single-cell PCR to study VH expression in individual
blood B cells concluded that the characteristics of VH1-69-encoded Ig expressed by healthy adult donors were similar to those
described for VH1-69-encoded Ig expressed in CLL (15). These
characteristics are distinctive from those previously described for
VH1-69-encoded Ig expressed by normal tonsillar B cells (5, 14).
Therefore, this study raised the possibility that the features of the
Ig expressed in CLL may be more reflective of those expressed by
blood B cells of normal adults. Moreover, the distribution of
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51p1 is an allele of VH1-69 that frequently is expressed by chronic lymphocytic leukemia (CLL) B cells with little or no somatic
mutation. The rearranged 51p1 genes expressed by CLL B cells have a distinctive use of D segments D3-3/DXP4 and D3-10/DXPⴕ1,
a favored use of JH6, and a longer third complementarity-determining region than the rearranged Ig genes used by CLL B cells
that express VH1 genes other than VH1-69. We examined the 51p1-encoded Ig expressed by blood B cells of healthy donors. In
contrast to the infrequent use of JH4 by 51p1-expressing CLL (e.g., 4%), 36% of the rearranged 51p1 sequences from normal blood
B cells used JH4. Furthermore, the D segment use of the rearranged 51p1 sequences from normal blood B cells was not restricted,
but reflected the D segment use of nonselected IgH of normal B cells. Finally, the mean length of the third complementaritydetermining region for the 51p1 genes of normal blood B cells was 14.6 ⴞ 4.3 (SD) codons. This is significantly shorter than that
noted for 51p1-expressing CLL B cells (18.8 ⴞ 3.2; p < 0.0001, n ⴝ 51). This study demonstrates that the 51p1-encoded IgH
expressed in CLL are not representative of the 51p1-encoded IgH expressed by normal blood B cells, indicating that CLL B cells
express IgH that are distinctive from those found in the normal adult blood B cell repertoire. The Journal of Immunology, 2001,
166: 95–102.
96
PROPERTIES OF 51p1-ENCODED Ig EXPRESSED BY NORMAL B CELLS
CDR3 lengths of blood B cells may be distinct from that of G6reactive tonsillar B cells.
To examine for this, we analyzed the molecular characteristics
of rearranged 51p1 genes expressed by blood B cells of normal
adults. For this, we used an allele-specific PCR to determine the
length distribution of the CDR3 of 51p1-encoded IgH rearrangements in the blood B cells of several normal adult subjects. In
addition, we examined the nucleotide sequences of IgM transcripts
encoded by 51p1 from a single adult donor. Our results indicate
that 51p1-encoded IgH expressed by blood B cells of normal
adults do not display the same distinctive molecular features as
those noted for 51p1-encoded Ig expressed in CLL.
Materials and Methods
Patient material
Genomic DNA isolation, RNA isolation, and cDNA synthesis
Genomic DNA was isolated from PBMC using QIAmp DNA blood reagents (Qiagen, Valencia, CA) according to the manufacturer’s instructions.
Total cellular RNA was isolated from PBMC using RNAeasy reagents
(Qiagen). First-strand cDNA was synthesized from one-third of the total
RNA using an oligo(dT) primer and Superscript II reverse transcriptase
(Life Technologies, Grand Island, NY). Reverse transcription was performed for 1 h at 42°C. Afterward, the mixture was heated to 70°C for 15
min, cooled on ice for 10 min, and then incubated with RNase H (Life
Technologies) at 37°C for 20 min. Four microliters of this reaction mixture
was used for a 100-␮l total volume PCR.
PCR assay
51p1 gene rearrangements were amplified by PCR of cDNA or genomic
DNA from normal PBMC using CDR2-specific oligonucleotide sense
primers corresponding to the sequence encoding amino acid positions
50 –54 of either the 51p1 (5⬘-AGGGATCATCCCTATCTT-3⬘) or hv1263
(5⬘-AAGGATCATCCCTATCCT-3⬘) allelic subsets and either a C␮ consensus primer (5⬘-TTGGGGCGGATGCACT-3⬘) or JH consensus oligonucleotide (5⬘-ACCTGAGGAGACRGTGACC-3⬘). Cycling parameters were
94°C for 30 s, 55°C for 30 s, 72°C for 1 min, using 1⫻ Pharmacia amplification buffer (Piscataway, NJ) and Taq polymerase in a 100-␮l total
reaction volume.
DNA cloning and sequencing
PCR products were size-selected by electrophoresis in 2% agarose containing 0.5 ␮g/ml of ethidium bromide (Life Technologies). The expected
products were excised and purified using Geneclean III (Bio 101, Carlsbad,
CA) per the manufacturer’s instructions and cloned into pBluescript (Stratagene, La Jolla, CA). Following transformation of XL-1 Blue competent
cells (Stratagene), plasmid DNA was isolated from overnight cultures of
randomly selected bacterial colonies using Wizard mini-preps (Promega,
Madison, WI). Sequencing was conducted using a fluorescent dideoxy
chain termination method and an automated nucleic acid sequence analyzer
(Applied Biosystems, Foster City, CA). Sequences were analyzed using
DNASTAR (DNAstar, Madison, WI) and by comparison with sequences
deposited in the V BASE and GenBank sequence databases.
Allele-specific PCR for mean CDR3 length determination
One microliter from a reaction mixture of a 30-cycle PCR for rearranged
51p1 genes (described above) of each specimen was mixed with 12 ␮l of
deionized formamide, 0.5 ␮l of GS 500 TAMRA internal size standard (PE
Biosystems, Foster City, CA), and 1 ␮l of a PCR mixture of 51p1-encoded
fragments of known CDR3 length. All samples were heated to 90°C for 3
min and placed on ice before loading. One microliter of each sample was
size-separated on an ABI PRISM 310 capillary electrophoresis system (PE
Biosystems) using the POP-4 polymer (16).
Plasmid standards representing fragments of known CDR3 length were
used to define the lengths of specific fragments within the ladder of mul-
Results
We examined the blood B cells of normal adults for rearrangement
and expression of 51p1 using RT-PCR or genomic PCR. Rearranged 51p1 genes of circulating blood B cells were amplified
using fluorochrome-labeled sense-strand oligonucleotide primers
specific for the 51p1 CDR2 and anti-sense strand primers complementary to either the IgM C region or a consensus region of JH.
Specific fragments were amplified using cDNA synthesized from
the RNA of circulating B cells of four individuals, genomic DNA
of blood B cells from two others, or cDNA and genomic DNA of
the tonsil sample JES, from a donor that has at least one copy
of 51p1.
Amplified products were analyzed following size separation by
denaturing capillary electrophoresis. Both genomic DNA and
cDNA derived from tonsil samples from individuals who lacked
51p1 alleles did not produce any detectable PCR product, whereas
each of these derived from blood or tonsillar B cells from individuals who had 51p1 alleles generated PCR products of various
lengths (Fig. 1). Each peak seen in Fig. 1 represents fragments of
the same CDR3 length. Individual peaks differ from each other by
multiples of three nucleotide bases, with each multiple representing a single amino acid codon. PCR fragments generated from a
group of plasmids containing 51p1 rearrangements of known
CDR3 length were included as size markers and used to determine
the range of the length of the CDR3 for each sample (Table I). For
all samples, the deduced CDR3 lengths of rearranged 51p1 genes
ranged from a minimum of six to seven codons to a maximum of
24 –27 codons.
The mean length of the CDR3 of the 51p1 genes expressed in
each sample was determined by analysis of the relative contribution of each band to the total signal present in each ladder. For each
sample, we assumed that the distribution of amplified products was
normal and that each distinct 51p1 fragment contributed equally to
the signal detected in each band. After determination of the largest
integral unit value that could divide the signal detected for each
band, we deduced the “IU,” or the minimum number of “integral
units” of distinct 51p1 fragments that could generate the observed
distribution of fragments (Table I). This allowed us to calculate the
mean CDR3 length of the rearranged 51p1 genes for each sample.
The rearranged 51p1 genes amplified from the genomic DNA of
blood B cells from two unrelated adults were calculated to have
mean CDR3 lengths of 15.0 ⫾ 3.6 (mean ⫾ SD) and 14.8 ⫾ 4.1
codons, with a range of 6 –26 codons (Table I). Simultaneous analysis of the CDR3 lengths of the 51p1 genes expressed by tonsillar
B cells revealed a mean CDR3 length of 15.5 ⫾ 3.4 codons that
was not significantly different from that of 51p1 genes expressed
by blood B cells. Additional samples were analyzed using cDNA
to examine the CDR3 length distribution of expressed 51p1 IgH.
Tonsillar B lymphocytes expressed 51p1-encoded Ig that has a
calculated mean CDR3 length of 15.0 ⫾ 3.3 codons. B cell samples A and B expressed 51p1-encoded Ig that have a calculated
mean CDR3 length of 15.1 ⫾ 4.2 and 15.1 ⫾ 4.0 codons, respectively. Additional normal B cell samples C and D expressed 51p1encoded Ig with a calculated mean CDR3 length of 14.6 ⫾ 3.5 and
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PBMC were obtained with consent from normal volunteers or from the San
Diego Blood Bank. For this, the blood was collected into heparinized tubes,
and the PBMC were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma, St. Louis, MO). Some samples were depleted of
adherent cells by incubation at 37°C for 60 min. Nonadherent cells were
removed and combined with additional medium used to rinse residual cells
from the plate. Total RNA or genomic DNA was isolated from the nonadherent cells that were harvested by a 10-min centrifugation at 300 ⫻ g.
tiple bands that was generated from each sample. Using the area integration
tools of GeneScan 3.1 software (PE Biosystems), we defined the relative
contribution of individual CDR3 length fragments to the total signal
present in each ladder. The data then were transferred to Microsoft Excel
(Redmond, WA) spreadsheets for further analyses. The signal from the
band with the lowest intensity in each ladder was defined as one fragment
unit, and the total number of fragments contributing to the ladder was
determined by dividing the total ladder intensity by this number. The mean
CDR3 length of the 51p1 genes expressed in each sample was determined
by analysis of the relative contribution of each band to the total signal
present in each ladder.
The Journal of Immunology
97
FIGURE 1. CDR3 length distribution of 51p1-encoded H chains of normal blood B cells as determined by allele-specific PCR and size separation by denaturing
capillary electrophoresis. The lengths of the PCR fragments are presented on the abscissa as the deduced codon size of the CDR3. The codon size represented by
each fragment is determined by subtracting the size of the non-CDR3 region (200 bp) from the size of the detected PCR fragment and then dividing the remainder
by three. Depicted on the ordinate are the relative fluorescence intensities of the various PCR fragments as detected using the ABI PRISM 310 genetic analyzer.
The height of each peak is proportional to the relative number of distinct PCR fragments of the given size.
Sequence analysis of 51p1 expressed in normal blood B cells
We also examined the nucleotide sequence of 51p1-encoded IgH
expressed by the blood B cells of a normal adult. For this we
isolated 40 distinct cDNA encoded by rearranged 51p1 genes from
B cell sample A. Of the 39 IgH analyzed, the size of the CDR3
ranged from eight to 25 codons, with a mean length of 14.6 ⫾ 4.3
codons (Fig. 2). This mean value is significantly shorter than that
previously reported for 51p1-expressing CLL B cells (19.08 ⫾ 3.5:
p ⬍ 0.0001, n ⫽ 36 unpaired t test) (6, 7, 14, 17–21), as well as
shorter than these 36 sequences combined with 15 additional 51p1expressing CLL sequences reported in the literature (18.80 ⫾ 3.2:
p ⬍ 0.0001, n ⫽ 51 unpaired t test) (6, 7, 14, 17–24). The mean
CDR3 length of the 39 sequences is also not significantly different
from that calculated for the same and similar blood B cell samples
using allele-specific PCR (Fig. 3).
Comparison of the nucleotide sequences of each clone with one
another and with the germline VH1-69 revealed that 39 of 40 sequences are unique and that each is derived from a productive
rearrangement of a 51p1-like allele of VH1-69. The oligonucleotide primer used for allele-specific amplification of 51p1 corresponds to the nucleotide sequence encoding amino acids 50 –54 of
CDR2 and therefore prevents conclusive determination of somatic
mutation in this sample. However, compared with the nucleotide
sequence encoding the last 43 aa of 51p1, 27 sequences are identical to the most homologous 51p1-like allele. Of the twelve samples that display differences, nine vary by only a single nucleotide,
two have two nucleotide differences, and one differs by four from
the 51p1 sequence reported in the literature. Seven of the 12 sequences vary from germline by a single nucleotide substitution at
the end of the third framework region (FW3). This is the only
difference in five of the sequences that display only a single difference, and is likely attributed to junctional processing during
rearrangement. The other nucleotide disparities are scattered randomly throughout FW3.
The CDR3 of each of the functional H chains was first analyzed
by identifying the D and JH segments with the highest nucleotide
sequence homology. Identification of the D gene segments was
possible for 36 of 39 sequences (Fig. 2). Eighteen different D segments were identified in all three reading frames and represent six
of the seven D segment families. Only one sample appears to use
more than one D segment, possibly secondary to D-D fusion.
Of the 39 sequences, we determined that five (13% of the total)
used D3-22/D21-9, five (13%) used D1-26, four (10%) used D613/DN1, and three (7.5%) each used D3-9/DXP1 or D6-6/DN4.
D2-2/D4, D2-15/D2, D3-10/DXP⬘1, and D6-19 were each identified twice (5%), whereas D1-7/DM1, D2-21/D3, D3-16/D21-10,
D4-11/DA1, D4-17, D4-23, D5-12/DK1, D5-24, and DIR were
each found only once (2.5%) (Fig. 4). D segments belonging to the
Table I. Length distribution of 51p1 gene rearrangements in normal blood and tonsillar cellsa
Sample
Source
Material
CDR3 Length
⫾SD
Min
Max
IU
A
B
C
D
JES
E
F
JES
Blood
Blood
Blood
Blood
Tonsil
Blood
Blood
Tonsil
RNA
RNA
RNA
RNA
RNA
DNA
DNA
DNA
15.1ⴱ
15.1ⴱ
14.6ⴱ
14.8ⴱ
15.0ⴱ
15.0ⴱ
14.8ⴱ
15.5ⴱ
4.2
4.0
3.5
3.8
3.3
3.6
4.1
3.4
6
6
7
6
7
7
6
7
27
26
24
25
24
24
26
25
195
215
178
174
246
120
116
257
a
Length distribution analyses for 51p1 gene rearrangements in normal human blood and tonsillar B cells as determined by allele-specific PCR and denaturing capillary
electrophoresis. Data from analysis of eight different samples is presented. Column 2 lists the origin of the B cells of each specimen. Column 3 designates the genetic material
analyzed for that B cell sample. The columns marked “CDR3 length” provide the mean number of codons found within the CDR3, whereas the one marked “⫾SD” provides
the standard deviation about the mean. The column labeled “Min” provides the number of codons found in the smallest CDR3 segments of the population, whereas the one marked
“Max” lists the CDR3 length of the longest CDR3 detected within the population. The column marked “IU” lists the minimum number of integral units of disparate PCR fragments
that can define the spectratype. ⴱ, Significantly different ( p ⬍ 0.0001) from the mean CDR3 length of 51p1 rearrangements found in CLL (18.8 ⫾ 3.2, n ⫽ 51).
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14.8 ⫾ 3.8, respectively. The calculated CDR3 lengths ranged
from 6 to 27 codons.
98
PROPERTIES OF 51p1-ENCODED Ig EXPRESSED BY NORMAL B CELLS
D3 family were found most frequently, being represented in 11
sequences (28%), followed by those of D6 (23%). D segments
D3-22/D21-9 and D6-13/DN1 are members of these two families,
respectively, and are two of the more frequent D gene segments
used by blood B cells of normal adults (25, 26). Moreover, the
distribution of D gene segment use by blood B cells that use 51p1
is similar to that of other non-neoplastic B cells that express 51p1
(8, 12, 25, 27–32) or other VH genes (25, 26).
The CDR3 and FR4 were further analyzed by comparison to the
germline JH segment with the greatest nucleotide sequence homology. Of the 39 sequences, we found that one (3%) used JH1, three
(8%) used JH2, four (10%) used JH3, 14 (36%) used JH4, four
(10%) used JH5, and 13 (33%) used JH6 (Fig. 5). This pattern is
similar to that of 42 previously characterized sequences of 51p1encoded H chains expressed by tonsillar and other nonmalignant B
cells (8, 12, 25, 27–32). Of this group, none used JH1, one (2%)
used JH2, four (10%) used JH3, 15 (36%) used JH4, seven (17%)
used JH5, and 15 (36%) used JH6 in 51p1 rearrangements that
encode Abs of unknown specificity. In contrast, 51p1 H chains
expressed by CLL B cells predominantly use JH6 (30 of 51, 59%)
and infrequently use JH4 (4 of 51, 8%) (6, 7, 14, 17–24).
The frequent use of JH6 in these samples contributes to the
unique properties of 51p1-encoded H chains expressed in CLL.
Although JH6 is used frequently in the sequences presented in this
study that display longer CDR3s, it is also found in several IgH
genes that have a CDR3 length less than the calculated mean of
14.6 ⫾ 4.3 codons. Finally, the D segments D3-22/D21-9 and
D6-13/DN1 that are present in multiple H chains expressed by
these blood B cells are each rearranged to JH genes that belong to
different JH gene families. D3-22/D21-9 is present in five clones
and rearranged twice to JH4 or once each to either JH1, JH5, or JH6.
D6-13/DN1 is present in four sequences associated once each with
JH2, JH4, JH5, or JH6, consistent with the random association of D
and JH segments by normal blood B cells.
Discussion
This study demonstrates that the 51p1-encoded IgH expressed by
blood B cells of normal adults do not display the same distinctive
molecular features as 51p1-encoded Ig expressed by CLL B cells.
We found that the mean length of the CDR3 for the 51p1-encoded IgH expressed by normal blood B cells is significantly
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FIGURE 2. Sequence of the CDR3 and FW4 of 51p1 cDNA isolated from blood B cells. On top of the figure are descriptors indicating the region that
encodes the CDR3 and FW4. Indicated on the left margin are the names of the heavy chain and the known D segments that have the highest homology
with each cDNA. Listed on the right margin are the names of the deduced lengths of the CDR3 for each PCR fragment, the names of each heavy chain,
and the JH segments that have the highest homology with that of each cDNA. The nucleotide sequence of each clone is presented between the names of
the cDNA. Below each sequence is the comparison with the D or JH segment of the highest nucleotide base homology. A dot indicates homology with the
cDNA sequence. Above each nucleotide sequence is the deduced amino acid sequence designated by the single-letter amino acid code.
The Journal of Immunology
99
FIGURE 3. CDR3 length distribution in
normal and CLL B cells. Proportionate distribution of CDR3 lengths of 51p1 rearrangements from both normal and neoplastic B
cells. A , 39 sequences of 51p1-encoded H
chains from normal B cell sample A examined in this study; B f, Distribution of CDR3
length in normal blood B cells of sample A
as determined by allele-specific PCR and
size separation in this study; C , 51 sequences reported in GenBank or the literature of CLL samples that express 51p1-encoded H chains (6, 7, 14, 17–24).
,
51p1-encoded G6-reactive IgH (13.5 ⫾5.5) isolated from human
tonsillar B cells (12). Of note, there is no significant difference
between the calculated mean CDR3 length of this group of sequences derived from blood B cells compared with the mean
CDR3 length of 42 rearranged 51p1 genes expressed by nonmalignant B cells reported in the literature (13.6 ⫾ 4.0) (8, 12, 25,
27–32), or with 146 Ig expressed by random blood B cells of two
normal adults (13.9 ⫾ 4.2) (25, 26).
The use of longer CDR3 regions is not a property of CLL B cells
in general, because CLL B cells that express Ig encoded by VH
genes other than 51p1 also display mean CDR3 lengths similar to
those of normal blood B cells. A recent study of 64 random IgMexpressing CLL samples reported an average CDR3 length of 15.1
codons (22). This group includes samples expressing IgH encoded
by VH genes belonging to all seven H chain families, including five
encoded by 51p1. If one considers only the IgH encoded by VH
genes other than 51p1, the mean length of these sequences is only
14.9 codons. Additionally, the 39 CLL B cell samples that express
H chains encoded by VH1 genes other than 51p1 have an average
CDR3 length of 14.5 ⫾ 3.5 codons (7, 14, 20, 22, 23), indicating
that the tendency toward longer-length mean CDR3 regions is not
a property of CLL B cells in general.
We note that the distribution of D segments used by 51p1-encoded H chains expressed in normal blood B cells is distinct from
that of 51p1-expressing CLL B cells. Blood B cells are not restricted in their use of D genes, because 39 51p1-encoded H chain
sequences derived from a normal adult use 17 of the 26 D genes
represented by the six major D segment families (33). This distribution is not significantly different from that observed for 146 H
chain sequences derived from random Ig rearrangements of two
normal adults (25, 26). In contrast, the 51 characterized 51p1encoded IgH expressed by CLL B cells use only 12 different D
segments from just four families (6, 7, 14, 17–24). Moreover, 74%
(50 of 68) of the D genes identified in these CLL-derived sequences use D2 or D3. Three D segments, D3-3/DXP4, D3-10/
DXP⬘1, and D2-2/D4, accounted for 63% (32 of 51) of the rearranged IgH. However, these same D segments are conspicuously
under-represented in the set of D segments that we found expressed by normal blood B cells. D3-3/DXP4 was not present in
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shorter than that noted for 51p1-expressing CLL B cells. Allelespecific PCR of rearranged 51p1 genes from eight normal blood
and tonsillar B cell samples revealed a mean CDR3 length that
ranges from 14.6 to 15.5 codons. Both individually and collectively, the mean CDR3 lengths determined using either genomic
DNA or cDNA from normal blood or tonsillar B cells were significantly different from the 18.8-codon (⫾3.2, n ⫽ 51) mean
CDR3 length noted for 51p1-encoded Ig expressed by CLL B cells
( p ⬍ 0.0001; see Table I) (6, 7, 14, 17–24). However, the calculated mean CDR3 lengths of 51p1-encoded Ig of normal blood B
cells were not significantly different from the mean CDR3 lengths
of 146 Ig encoded by random VH genes expressed by normal adult
B cells (13.9 ⫾ 4.2) (25, 26) or the mean CDR3 lengths of CLL B
cells that express IgH encoded by VH1 genes other than 51p1 (7,
14, 20, 22, 23). The mean CDR3 length of these 39 samples is 14.5
⫾3.5 (n ⫽ 39) codons and ranges in size from 10 to 26 codons.
These results contrast with those of a previous study that found
the CDR3 lengths of 51p1 genes expressed by blood B cells to be
similar to those noted for CLL B cells (15). However, this previous
study averaged the CDR3 length of only six different 51p1 gene
sequences that were isolated via single-cell PCR from three different individuals. Instead, the current analyses used allele-specific
PCR spectratyping to examine the average CDR3 lengths of hundreds of disparate PCR fragments isolated from the blood B cells
of several different normal donors (Table I). Conceivably, the limited sample size of 51p1 genes examined in the previous study
accounted for the apparent overestimation of the CDR3 lengths of
51p1 genes expressed by the blood B cells of normal adults.
Consistent with this, the nucleotide sequence analyses of 39
51p1-encoded IgM H chains expressed by the blood B cells of one
adult confirm the estimation of average CDR3 length for expressed
51p1 genes that was obtained via allele-specific PCR. The mean
CDR3 length of 39 51p1-encoded H chain transcripts isolated from
the blood B cells of one healthy adult is 14.6 ⫾ 4.3 codons, with
a range of 8 –25 aa. This mean CDR3 length and size distribution
is comparable to those of any one of the B cell samples analyzed
by allele-specific PCR, including sample A, from which the cDNA
were isolated for nucleic acid sequence analyses (see Table I). It is
also similar to the previously reported mean CDR3 length of 13
100
PROPERTIES OF 51p1-ENCODED Ig EXPRESSED BY NORMAL B CELLS
FIGURE 4. D segment use in normal and
CLL B cells. Values are expressed as a percentage of samples found to use each of the
different D gene segments. A f, 39 sequences of 51p1-encoded H chains from normal B cell sample A examined in this study;
B , 51 sequences reported in GenBank or
the literature of CLL samples that express
51p1-encoded H chains (6, 7, 14, 17–24); C
_, 39 sequences reported in the GenBank or
the literature of CLL samples that express H
chains encoded by VH1 genes other than
51p1 (7, 14, 20, 22, 23).
,
quences reported in the literature that are encoded by 51p1, 36
(71%) have a CDR3 of 18 codons or more (6, 7, 14, 17–24). Of
these, 29 of 36 (81%) express an Ig that uses JH6. The remaining
15 samples range in length from 13 to 17 codons, and only two
(13%) use a JH6 gene segment.
Many of the longer CDR3 regions of 51p1-encoded IgH that use
JH6 also use a D segment belonging to the D2 or D3 family. The
genes of these two families typically encode three or four more
codons than the D segments from any of the other families. Twentyfive of the 30 51p1-expressing CLL samples that encode an IgH
using JH6 also use a D segment from these two families (6, 7, 14,
17–24). Twenty-one of those 25 (84%) are D3-3/DXP4, D3-10/
DXP⬘1, and D2-2/D4. Most of the 51p1-encoded IgH from blood
B cells with longer CDR3 also are encoded by JH6 genes (72%),
but most (63%) use D segments D6-6/DN4, D6-13/DN1, D5-24,
D3-9/DXP1, and D3-22/D21-9, which are not frequently expressed by CLL B cells that use 51p1.
Analyses of the primary amino acid sequences of 51p1-encoded
IgH of CLL B cells revealed common motifs in the CDR3 that
reflected frequent use of a particular reading frame for each commonly used D segment (14). For example, eight of 14 (57%) CLL
B cells that had 51p1 rearrangements with D3-3/DXP4 had the
CDR3 motif of (Tyr)-Asp-Phe-Trp-Ser-Gly-Tyr-(Tyr)-(Pro) encoded by the second reading frame of the D3-3/DXP4 gene segment (6, 7, 14, 17–24). Similarly, six of the eight (75%) 51p1expressing CLL that used D2-2/D4 had a common CDR3 motif of
Ile-Val-Val-Val-Pro-Ala-Ala encoded by the third reading frame
of D2-2/D4. However, these common motifs were not observed in
any of the 39 51p1-encoded H chains of normal blood B cells
except in B9, which used D2-2/D4 and had a truncated motif of
Val-Pro-Ala-Ala. Otherwise, even the 51p1-encoded IgH of normal B cells with longer CDR3 that used D3-10/DXP⬘1 or D2-2/D4
did not display the conserved amino acid motifs that commonly
were detected in the CDR3 of 51p1-encoded IgH of CLL B cells.
As such, even the longer CDR3 of rearranged 51p1-encoded H
chains of normal blood B cells do not appear representative of the
CDR3 for rearranged 51p1 genes expressed in CLL.
Several studies have postulated that CLL may arise from a
clonal outgrowth of B cells (14, 34). The restricted use of certain
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any of the 39 sequences, yet was identified in 27% (14/51) of the
51p1-expressing CLL samples reported in the literature (6, 7, 14,
17–24). D3-10/DXP⬘1 is used in only two (5%) of the sequences
presented in this study, whereas it accounts for 20% (10/51) of the
characterized 51p1-encoded Ig used in CLL. D2-2/D4 is present in
eight (16%) of 51p1-encoded CLL H chains, but in only two (5%)
of the IgH used by normal blood B cells. Furthermore, D3-3/DXP4
was found in only two (5%) of 39 sequences reported in the literature that are derived from CLL B cells that express an Ig VH
encoded by a VH1 gene other than 51p1 (7, 14, 20, 22, 23). In the
same group of samples, D2-2/D4 was also present in 2 (5%) samples, and D3-10/DXP⬘1 was only identified once (3%). Conversely, normal blood B cells express several D segments that are
not seen used in 51p1-expressing CLL. These include D1-7/DM1,
D1-26, D5-24, D6-6/DN4, and D6-19, as well as all members of
the D4 family. Because so many different D segments are used, it
is not possible to discern a bias toward use of a particular reading
frame for any one of the D segments used by the rearranged 51p1
genes of normal blood B cells.
In addition, we found that the distribution of JH segments used
by 51p1-encoded IgH expressed by normal blood B cells is different from that seen in CLL B cells. The 51p1-encoded IgH used
by normal blood B cells most commonly use JH4 (14 of 19, 36%)
and JH6 (13 of 39, 33%). In contrast, 51p1-encoded H chains expressed in CLL B cells predominantly use JH6 (30 of 51, 59%) and
infrequently use JH4 (4 of 51, 8%) (6, 7, 14, 17–24). However,
frequent use of JH6 is not a property of all Ig expressed by CLL B
cells. Only 26% (10 of 39) of CLL B cell samples that express H
chains encoded by VH1 genes other than 51p1 use JH6. Likewise,
two recent studies showed that JH6 is present in only 31% (20 of
64) and 37% (27 of 84) of CLL B cells that express IgM H chains
encoded by random VH genes from all seven H chain families (22,
23). JH4 genes were the JH segments most frequently used by the
CLL B cells in each of these three analyses.
Although the frequent use of JH6 may contribute to longer
CDR3s, this is not invariable. In this sample of 39 sequences from
blood B cells, 10 (26%) have a CDR3 of 18 codons or greater, and
seven of these (70%) use JH6. However, six of the 29 (21%) remaining sequences also contain a JH6 gene. Of the 51 Ig VH se-
The Journal of Immunology
101
FIGURE 5. JH segment use in normal and
CLL B cells. Values are expressed as the percentage of samples found to use genes from each
of the JH families. A f, 39 sequences of 51p1encoded H chains from normal B cell sample A
examined in this study; B , 51 sequences reported in GenBank or the literature of CLL samples that express 51p1-encoded H chains (6, 7,
14, 17–24); C , 39 sequences reported in GenBank or the literature of CLL samples that express H chains encoded by VH1 genes other than
51p1 (7, 14, 20, 22, 23); D 1, 146 random Ig VH
rearrangements reported in the literature (25,
26); E _, 42 rearranged Ig 51p1 genes expressed
by nonneoplastic B cells (8, 12, 25, 27–32).
,
,,
,,
to eliminate binding of the G6 mAb used to define the G6 Id. The
conservation of germline 51p1 indicates that structural portions of
the 51p1 H chain also may be important.
In summary, the 51p1-encoded IgH expressed in CLL are not
representative of the 51p1-encoded IgH expressed by normal
blood B cells. Instead, only a relatively small number of normal B
cells in the blood or lymphoid tissue appear to have rearranged
51p1 genes with CDR3 segments that are similar to those of the
IgH expressed by CLL B cells that also use this VH1-69 allele.
Nevertheless, it should be noted that each of the normal donors
examined in this study did have a small proportion of blood B cells
that expressed 51p1-encoded IgH with CDR3 lengths typical of
those observed for the CDR3 of 51p1-encoded IgH of CLL B cells.
Conceivably, a subset of such normal B cells may be selected to
undergo transformation by virtue of their expression of surface Ig
with some distinctive binding activity for an as-yet-unidentified
foreign or self-Ag. As such, these B cells may be predisposed to
leukemogenesis and may represent the true precursors of the CLL
B cell.
Acknowledgments
We thank Todd Johnson and Dr. Dieter Deforce for helpful discussions and
suggestions.
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