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Division of Hematology/Oncology, Department of Medicine, University of California San Diego, La Jolla, CA 92093.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies indicate that as many as 20% of CLL patients have malignant cells that express 51p1 (5, 6, 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 complementarity-determining 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 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 51p1-encoded 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 CDR3 lengths of blood B cells may be distinct from that of G6-reactive 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x g.
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-µl 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 Cµ 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 1x Pharmacia amplification buffer (Piscataway, NJ) and Taq polymerase in a 100-µl total reaction volume.
DNA cloning and sequencing
PCR products were size-selected by electrophoresis in 2% agarose containing 0.5 µg/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 µl of deionized formamide, 0.5 µl of GS 500 TAMRA internal size standard (PE Biosystems, Foster City, CA), and 1 µl 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 multiple 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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). 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 51p1-encoded Ig with
a calculated mean CDR3 length of 14.6 ± 3.5 and 14.8 ± 3.8,
respectively. The calculated CDR3 lengths ranged from 6 to 27 codons. 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, 18, 19, 20, 21), as well as shorter
than these 36 sequences combined with 15 additional
51p1-expressing CLL sequences reported in the literature
(18.80 ± 3.2: p < 0.0001, n = 51
unpaired t test) (6, 7, 14, 17, 18, 19, 20, 21, 22, 23, 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).
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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 D6-13/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 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, 28, 29, 30, 31, 32) or other
VH genes (25, 26).
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). This pattern
is
similar to that of 42 previously characterized sequences of
51p1-encoded H chains expressed by tonsillar and other
nonmalignant B cells (8, 12, 25, 27, 28, 29, 30, 31, 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, 18, 19, 20, 21, 22, 23, 24).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We found that the mean length of the CDR3 for the
51p1-encoded IgH expressed by normal blood B cells is
significantly shorter than that noted for 51p1-expressing
CLL B cells. Allele-specific 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, 18, 19, 20, 21, 22, 23, 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
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, 28, 29, 30, 31, 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 IgM-expressing 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 51p1-encoded IgH expressed by CLL B cells use only 12 different D segments from just four families (6, 7, 14, 17, 18, 19, 20, 21, 22, 23, 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 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, 18, 19, 20, 21, 22, 23, 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, 18, 19, 20, 21, 22, 23, 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 sequences reported in the literature that are encoded by 51p1, 36 (71%) have a CDR3 of 18 codons or more (6, 7, 14, 17, 18, 19, 20, 21, 22, 23, 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. Twenty-five 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, 18, 19, 20, 21, 22, 23, 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, 18, 19, 20, 21, 22, 23, 24). Similarly, six of the eight (75%) 51p1-expressing 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 D segments and JH genes by CLL B cells that express 51p1 provides for certain conserved amino acid motifs within the CDR3 of IgH. The CDR3 is the most variable region of the H chain and is directly involved in Ag binding, suggesting that the B cell receptor may be involved in this process.
CLL B cells have been shown to express IgM Abs that display reactivity to self-proteins. In addition, several 51p1-encoded Ig expressed in CLL are polyreactive, having reactivity to IgG, cardiolipin, DNA, actin, and thyroglobulin (35, 36, 37, 38, 39, 40). Such polyreactivity is characteristic of some "natural" autoantibodies produced early in B cell development. A recent study predicted the tertiary structure of polyreactive IgM molecules expressed by CLL B cells selected for reactivity with mouse IgG (24). One expressed 51p1 and had a 19-aa CDR3 encoded by D3-9/DXP1 and JH6. Molecular modeling predicted a CDR3 that forms a flat binding surface covered by several aromatic side chains. The aromatic side chains predicted to be important for binding are contributed by both the D and JH and are most prevalent in the gene segments of the D2, D3, and JH6 gene families. However, polyreactivity alone cannot account for the noted restriction seen in the primary structure of 51p1-encoded IgH expressed in CLL, because not all 51p1-encoded polyreactive autoantibodies expressed by non-CLL B cells are encoded by the same D and JH genes.
A recent report has suggested that CLL may be classified into two groups based on the extent of somatic mutation in their expressed Ig V regions (22). The presence or absence of somatic mutation is thought to reflect B cells transformed at different stages of differentiation and/or activation. However, nearly all 51p1-encoded IgH expressed by CLL B cells are not somatically mutated (14). As such, CLL B cells that express 51p1 constitute a large proportion of the cases that lack somatic mutation.
The absence of mutation in 51p1-encoded H chains expressed
by CLL B cells does not necessarily indicate an immature phenotype, but
could reflect a selective pressure for maintaining germline
configuration. Greater than 90% of Ig expressed in CLL that are
encoded by VH1-69 use a 51p1-like
allele (14). Assuming an equal expression frequency, the
1263-like alleles would be expected to be present in 
30% of
VH1-69-encoded rearrangements. The 1263-like
variants primarily differ from 51p1 by only three amino acid
substitutions in CDR2 (10). These changes are enough 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.
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Acknowledgments |
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Footnotes |
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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.
3 Abbreviations used in this paper: CLL, chronic lymphocytic leukemia; VH gene, H chain V region gene; CDR, complementarity-determining region; FW, framework region.
Received for publication July 17, 2000. Accepted for publication September 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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antibody expressing typical features of a NPAB. Immunol. Lett. 41:261.[Medline]
heavy chain expression in synovial tissue of a patient with rheumatoid arthritis. Arthritis Rheum. 36:631.[Medline]
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, 39 sequences
of 51p1-encoded H chains from normal B cell sample A examined in this
study; B 
, 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 (
, 39 sequences reported in the GenBank or the
literature of CLL samples that express H chains encoded by
VH1 genes other than 51p1 (
, 51 sequences
reported in GenBank or the literature of CLL samples that express
51p1-encoded H chains (
, 146 random Ig
VH rearrangements reported in the literature (