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*
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;
Departments of Pediatrics and Pathology, Children’s Hospital of Philadelphia, Philadelphia, PA 19104; and
Department of Laboratory Medicine/Pathology and University of Minnesota Cancer Center, University of Minnesota Medical School, Minneapolis, MN 55455
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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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In a very recent study, it was found that the human VH locus on chromosome 14 consists of 123 VH gene segments grouped into seven families (5). Although the total number of VH segments is higher than that reported in an earlier study (6), only 44 genes were found to be functional as compared with 51 found in the earlier study (6). This difference in the number of functional genes reported in the two studies can be attributed to the insertion/deletion polymorphic region that, when present, results in the gain of some functional genes (6). VH3 is the largest family, followed by VH4 and VH1, and these three families contain 44 of the functional genes. It has been found that 13–14 gene segments from these families account for a very large percentage of expressed heavy chains in the peripheral repertoire (7, 8, 9). In the VH3 and VH4 families, which together have 34 functional genes, only about 10–12 genes are frequently rearranged and account for 70%–80% of the peripheral repertoire (7, 8, 9, 10, 11). Overall these studies indicate that only about 25% of the functional VH repertoire is utilized in the formation of the diverse B cell repertoire. The reasons for this biased representation of VH genes are unclear. In a recent study (9), we have shown that the biased usage of VH3 and VH4 family gene segments starts at an early sIg-negative pre-B cell stage (CD19+CD20-IgM-) and persists until the sIg+ mature B cell stage. It has been postulated that during early B cell development, mechanistic processes leading to preferential Ig gene rearrangement and/or functional processes resulting in B cell selection could account for the nonrandom Ig repertoire in mature B cells.
The suggestion that some VH genes may be preferentially rearranged was derived from analysis of VH gene representation in pre-B and mature B cells (7, 8, 9, 12, 13, 14, 15). In all these studies, including our previous study, the contribution of preferential VH gene rearrangement could not be definitively assessed because more than 70% of VDJ rearrangements were productive even in the sIg- pre-B cells. The relative high frequency of individual gene segments among productive rearrangements in these B lineage cells may thus have reflected both rearrangement frequency at the earlier pro-B cell stage as well as the contribution of selection mechanisms occurring at later stages of development, e.g., efficiency of µ-chain/surrogate light chain (SLC) pairing at the pre-B stage and/or ligand selection at the immature B cell stage.
The goals of the current study were 1) to examine the influence of VDJ rearrangement on V gene repertoire generation independent of the influence of cellular selection processes, and 2) to evaluate the changes in the VH gene repertoire during the transition of B cells from a selection-independent pro-B cell stage to a VH-dependent selection at the pre-B cell stage. Our approach was to analyze Ig V gene rearrangement libraries from a sorted pro-B cell population in which VDJ rearrangement has occurred (16), but more than 90% of the cells have not yet expressed the µ protein (17), and from a population of pre-B cells, in which most of the cells express µ-heavy chain protein. Using a high resolution (HR) PAGE system, we classified individual rearrangements as in frame (IF) or out of frame (OF) based on the length of HCDR3. OF rearrangements were considered nonproductive, because there is no functional µ-heavy chain product or influence of VH-mediated selection. We determined the V gene usage among only OF rearrangements from pro-B cells to eliminate selection processes mediated by the product of the V gene, the variable portion of the µ-heavy chain. These studies demonstrate that certain VH genes (e.g., V4–34, V3–23, and V3–30) may be preferentially rearranged, in the absence of selection, at the pro-B cell stage. In addition, when compared with the pro-B cell population, the change in the IF/OF ratio for rearranged Ig V genes in the pre-B cell population provides evidence for positive and negative selection mechanisms operating during the transition of cells from pro-B to pre-B cell stage.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A pool consisting of CD19+/surface
µ- B cell
precursors and CD34+/CD19-
hemopoietic stem cells was initially isolated from the bone marrow of a
fetus of 19–21 wk of gestation, by mAb/magnetic bead depletion, as
previously described (18).
CD34+/CD19+ pro-B cells and
CD34-/CD19+ pre-B cells
were then purified by FACS using biotinylated anti-CD19, detected
with streptavidin-PE (Caltag, San Francisco, CA), and
anti-CD34- FITC (Becton Dickinson Immunocytometry Systems,
San Jose, CA). The cells were sorted on a FACSVantage (Becton Dickinson
Immunocytometry Systems). Fig. 1
shows
the light scatter profile of the B cell precursor/hemopoietic stem cell
pool (A), the CD19/CD34 staining profile of the pool
(B), the postsort purities of the
CD34+/CD19+ pro-B cells
(C), and the
CD34-/CD19+ pre-B cells
(D).
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For the construction of libraries, amplification of the
VH gene rearrangements from genomic DNA of pro-B
and pre-B cells was performed in two steps using nested primers. In the
first step, a 30-cycle amplification of VH4 or
VH3 family genes was performed using
family-specific leader primers VH4-L,
5'-CCGAATTCATGAAACACCTGTGGTTCTT-3' and VH3-L,
5'-CTGAATTCCATGGAGTTTTGGCTGAG-3' (8) paired with a
consensus 3' JH primer,
5'-GCGAGCTCTAGACTTACCTGAGGAGACGGTGACC-3', as described previously
(9). A total of 10 µl of the PCR product was used to
perform a second round of amplification for 20 cycles using
family-specific 5' internal primers: VH4-LF,
5'-CAGAATTCCTGTCCCAGGTGCAGC-3' (9) and
VH3-LF, 5'-CAGAATTCCAGTGT(C/G)AGGTGCAGC-3', which
is 100% homologous to all of the VH3 genes
studied, and a 3' JH primer,
5'-GACTCTAGACT(T/C)ACCTGAGGAGACGGTGA-3'. The 5' internal primers were
complementary from the -4 codon corresponding to the 3' regions of the
leader sequence to the +4 codon in the FR1 region of their respective
families. Construction of phage libraries and screening for individual
genes using gene-specific probes were conducted as described previously
(8, 9, 19, 20, 21). A family-specific gene probe was used to
find the total number of positive clones. Gene-specific oligonucleotide
hybridization (8, 9) was used to determine the frequency
of each gene in the libraries. The validity of any data pertaining to
the frequency of VH genes is based on the ability
of the sense primers to amplify the individual VH
genes with equal efficiency. The VH4 and
VH3 5' primers used in the present study have
been shown previously to amplify with equal efficiency all of the
VH4 genes and 
25 VH3
genes, which possibly represent the entire VH3
component (8, 9, 21, 22, 23, 24). Using the same primers, it was
also determined that the frequency with which individual
VH genes are recovered correlates with germline
copy number (8). In control experiments, to reconfirm that
the primers amplify all of the VH genes with
identical efficiency, unrearranged VH4 and
VH3 genes from genomic DNA were amplified using
the same 5' primers used to amplify rearrangements and paired with a
consensus family-specific FR3 3' primer (8). The
individual genes were then identified with gene-specific
oligonucleotides using the library screening approach. Furthermore,
aliquots of PCR products were blotted on nylon membranes, and phosphor
imaging analysis was conducted after gene-specific oligo hybridization.
In both of the experiments, we found that all of the genes under study
were amplified equally even after the second round of amplification
(data not shown).
Screening for IF and OF rearrangements
We have developed a novel, HR acrylamide gel method to identify rearrangements as IF or OF. A size-standard DNA ladder was constructed consisting of 16 bands representing 16 different HCDR3 sizes ranging from 24–69 bp. A smaller ladder consisting of five different lengths ranging from 12–21 bp was also constructed to analyze sequences smaller than 24 bp. These bands represent IF sequences with a 3 bp difference between any two HCDR3 sizes. The size standards were prepared by amplifying individual VDJ rearrangements that were cloned into a vector and for which HCDR3 length had been determined by sequencing. Each plasmid containing a size-standard VDJ was grown overnight in culture, and the plasmid DNA was purified using a Qiagen Plasmid Maxi Prep kit (Qiagen, Chatsworth, CA). The concentration of DNA was determined by spectrophotometry, and appropriate dilutions were made to normalize the concentrations. A total of 5 µl of a 1/10,000 dilution was used in amplification reactions.
PCR amplification of HCDR3
All of the VDJ sequences used as size standards were members of the VH4 family in germline configuration. Therefore, a consensus 5' VH4-FR3 primer, 5'-GCGAGCTCTAGAGTGTATTACTGTGCGA-3', and a downstream consensus JH oligo, 5'-GCGAGCTCTAGACTTACCTGAGGAGACGGTGACC-3', were used to amplify the HCDR3 regions of the VDJ rearrangements used as standards, as well as VH4 gene rearrangements from the B lineage cell libraries. For amplifying HCDR3 regions of VH3 members, a consensus VH3FR3 5' primer, 5'-GCGAGCTCTAGAGT(A/G)TAT(C/T)ACTGT-3', was used. The highlighted regions in the primers are XbaI restriction sites. PCR amplifications were performed in 100-µl reaction volumes containing 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 0.2 mM dNTPs, 0.4 µM primer, 2.5 U of Taq polymerase, and 5 µl of the 1/10,000 dilution of the standard template. The template was initially denatured at 95°C for 3 min in Perkin-Elmer DNA Thermal Cycler (model 480; Norwalk, CT), and PCR was performed with 1-min denaturation at 95°C, 30-s annealing at 60°C, and 1-min extension at 72°C for 30 cycles, with a 5-min extension at 72°C in the last cycle. The products were ethanol precipitated, washed, vacuum dried, and resuspended in 35 µl of sterile distilled water. The DNA from all tubes were pooled and digested with 300 U of XbaI for 4 h. A total of 100 µl of 6x loading buffer type III (25) was added to the digested DNA.
To identify the frame of the rearrangements, 1.75 µl phage supernatant of individual VH4 or VH3 clones from the pro-B or pre-B cell libraries was amplified using the same conditions as described for the size standards, except that the reactions were scaled down to 50 µl. A total of 25 µl of the PCR was digested for 1 h with 10 U of XbaI, followed by the addition of 4 µl of 6x loading buffer type III to each digested product.
HR PAGE for determining HCDR3 frame
We modified the standard polyacrylamide sequencing gel to detect single base differences between HCDR3 sizes of individual VH gene rearrangements. A 14% nondenaturing, vertical polyacrylamide gel in 1x TBE buffer (0.13 M Tris base, 0.04 M boric acid, and 0.05 M EDTA) was prepared using a Model S2 sequencing gel electrophoresis apparatus (Life Technologies, Gaithersburg, MD). A mixture consisting of 3 µl of the standard ladder and 1.5 µl of the unknown was subjected to electrophoresis in 1x TBE buffer at a constant voltage of 750 V for a total of 18,000 V hours, typically about 21 h. As a control, an amplification product from VH rearrangements, for which the HCDR3 length and the frame had been determined by sequence analysis, was included with every run. The gel was stained in 2 µg/ml of ethidium bromide and photographed on a UV transilluminator (Fotodyne, Hartland, WI) after destaining in distilled water. Digital images of the gels were captured and saved as TIFF files using a video camera (COHU, San Diego, CA).
Because codons consist of three bases, the length of segments of DNA
that contain IF rearrangements will occur at an interval of three
bases, and those that contain OF rearrangements will fall within those
three base intervals (Fig. 2). The
unknown DNA is loaded in greater quantity than the ladder controls, so
they stand out visually as more intense bands superimposed on the less
intense standards (Fig. 2). In a few samples, we found more than one
band, possibly due to a PCR artifact or the presence of two
rearrangements in a clone (9). In such samples, if both
the bands in the unknown were IF, the sample was scored as one IF
rearrangement. A similar strategy was followed for OF rearrangements,
that is, if both the bands in the sample were OF, it was considered as
one (Fig. 2B). Samples were not considered in the analysis
for the VH gene assigned to that clone if IF and
OF complementarity-determining region 3 bands were present.
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A and lanes 4 and 7 in Fig. 2B, the unknown band and the underlying marker band shift to
a lower position on the gel. To confirm that this shift was due to the
amount of DNA loaded, increasing quantities of known IF rearrangements
were loaded into adjacent lanes on a HR PAGE gel (not shown). The shift
in band position was measured in pixels using the linear distance tool
of the National Institute of Health Image software package. The degree
of shift in band position was found to be proportional to the amount of
DNA loaded, with the heaviest loading resulting in as much as 35%
shortening of the distance between the overloaded band and the next
lower marker band. Furthermore, the marker DNA comigrated with the
unknown DNA, so that a single bright band, shifted to a lower position
on the gel, was observed, rather than two bands. The presence of a
single band was confirmed when gel images were examined with
magnification and with variation of image contrast. Accordingly,
unknown bands were read as IF when a single heavy band shifted down on
the gel was observed. In a few cases, heavily overloaded unknowns were
repeated with reduced amounts loaded to confirm that only a single band
was present. As an independent confirmation of the accuracy of the IF
and OF assignments, a sample of 71 clones was sequenced to determine
reading frame. The assignments from the gel were found to be in 100%
agreement with the sequence data (data not shown). Although some error
could be made in scoring a rearrangement to be IF or OF due to reasons
discussed above, our sequencing data strongly suggest that such errors
are minimal. Statistical methods
The 
2 tests were used to compare the distribution of
the observed VH gene frequencies with the
frequencies of individual genes, as expected by random usage based on
the genomic complexity of the VH4 and
VH3 genes. Test of significance was also
performed to compare the average HCDR3 length of rearrangements from
different repertoires, with the assumption that
n1 is not equal to n2.
Fischer’s Exact test was used to compare the IF/OF ratios of
individual genes between the pro-B and the pre-B cell stages with the
null hypothesis that the frequency of IF rearrangements of a particular
gene is similar at the pro- and pre-B cell stages. The p
values equal to or less than 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pro-B lineage (CD34+/CD19+) cells were isolated from a fetal bone marrow to assess 1) the frequency of individual VH4 and VH3 family gene segments among all rearrangements; 2) the proportion of IF vs OF rearrangements for individual gene segments; and 3) the distribution of individual genes among only the OF rearrangements. We chose to study Ig gene usage among only OF rearrangements from pro-B cells because we predicted, based on murine studies, that this B cell fraction would contain a large number of OF rearrangements and would exclude bias due to µ/SLC pairing or ligand selection.
Frequency of VH4 and VH3 gene rearrangements in pro-B cells
The VH4 and VH3 gene
usage in CD34+/CD19+ pro-B
cell population is presented in Table I.
The frequency of individual VH4 genes was
assessed by screening more than 600 independent rearrangements in two
libraries (PV42a and PV42b) generated from independent PCRs (Table I).
Because we used probes specific to all the known functional genes,
including the pseudogene of the VH4 family, a
definitive assignment to a VH4 gene segment was
possible for 98% of the clones (641 of 657). V4–34 was the most
frequent gene accounting for 32% of all the VH4
rearrangements. Other genes such as V4–39, V4–59, and V4–31 were
also rearranged at frequencies higher than expected. These four genes
accounted for 86% of the VH4 repertoire. The
gene segments V4–34, V4–59, and V4–39 are also overexpressed in the
mature B cells (9) (D. F. Friedman, M. Scully, and L. E.
Silberstein, unpublished data). V4–61 was found at the expected
frequency, while other genes such as V4–4b and V4–28 were rare or
absent. Interestingly, the pseudogene V4–55, which has never been
reported in rearrangements from pre-B or mature B cells from earlier
studies (7, 8, 9), was found in 4% of all rearrangements.
Although some of the rearrangements of V4–55 were classified as being
IF (see Table III), a functional protein
cannot be made due to the presence of a stop codon in the V4–55
germline gene segment (5, 6).
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We generated two (PV43a and PV43b) VH3
rearrangement libraries (Table I) from independent PCRs and screened
750 independent clones for eight VH3 genes. Table I shows that these eight genes accounted for 74.6% (567 clones) of the
VH3 repertoire. V3–30 (24%) and V3–23 (20%)
were the most frequently rearranged genes. The gene V3–30 has two
alleles, V3–30b and V3–30.3. However, we did not detect V3–30.3
sequence in any of the rearrangements (see discussion of deletion
polymorphism, above); therefore, the data for V3–30 are entirely
accounted for by V3–30 and its other allele, V3–30b. The
contributions of V3–74, V3–15, and V3–20 to the rearranged
repertoire were low and ranged from 1%–3%, while the frequencies of
V3–33, V3–11, and V3–53 were between 6% and 9%.
The ratio of IF/OF rearrangements in pro-B cells suggests the absence of cellular selection
We hypothesized that the ratio of IF and OF rearrangements found
in pro-B cells would be unaffected by selection mechanisms because
VH-DJH rearrangement has occurred in this
population of cells, but the µ protein is not yet produced. In a
random rearrangement process without selection, two-thirds (66.7%) of
all rearrangement events for any gene segment will be OF, and one-third
(33.3%) will be IF, resulting in an IF/OF ratio of 0.5
(27, 28, 29). As shown in Table II, of 997
VH4 and VH3 clones
analyzed, 665 (66.7%) were OF and 332 (33.3%) clones were IF,
resulting in an IF/OF ratio of 0.5, in agreement with the predicted
value. Furthermore, the IF/OF ratio determined for most of the
VH4 and VH3 genes
individually (Tables III and IV) also
approximated the expected ratio, consistent with an unselected
population of progenitor B lineage cells. The sorted
CD34+/CD19+ pro-B cell
population that we have analyzed contains a small number (3%) of
CD34- pre-B cells (Fig. 1C).
Cells that have synthesized cytoplasmic µ may be subject to a µ
protein-dependent selection mechanism. It has been found from previous
studies that 5–10% of the
CD34+/CD19+ human pro-B
cells are positive for cytoplasmic µ (17). However, the
overall IF/OF ratio in the pro-B cell libraries indicates that the
presence of a small number of cytoplasmic µ-positive B cells did not
significantly alter the IF/OF ratios expected based on the random
rearrangement process.
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Next, we determined the rearrangement frequency of individual genes among only the OF rearrangements to focus on preferential rearrangement. We reasoned that, because the OF rearrangements are not translated, their frequency in a library of VDJ rearrangements from CD34+/CD19+ pro-B cells should not be biased by µ-SLC pairing or ligand selection, and the proportion of any particular V gene among OF rearrangements should reflect only the rearrangement process. Because there are 12 VH4 and 22 VH3 functional genes (6, 8, 26), the frequency of each gene in a random distribution would be 1/12 or 8% for VH4 and 1/22 or 4.5% for VH3. Our results (Tables I and II) indicate that V4–34, V4–59, and V4–39 genes within the VH4 family and V3–30 and V3–23 genes within the VH3 family are found at a significantly higher frequency than expected among OF rearrangements from VH4 and VH3 families. Others genes such as V4–4b, V4–61, and V3–20 are found at lower frequency than expected (see Tables I and II). In the previous study on sIg- pre-B cells (9) as well as in the current study of fetal pro-B cells, the gene V4–28 was not found in any of the rearrangements. Thus, this gene is not rearranged at all or is rearranged at such low frequency that it was not identified in the sample of 641 clones from the VH4 library.
Comparison of Ig gene repertoires during ontogeny
The frequency of most individual V gene segments determined in the
fetal bone marrow in the present study is similar to that observed in
fetal liver (12, 13, 14, 30, 31) and adult bone marrow
repertoires (9, 15). However, V4–34 and V4–39
rearrangements are either rare or absent in fetal liver B cells,
although they predominate in fetal and adult bone marrow B cells. An
additional difference between fetal bone marrow and fetal liver was
observed when we determined the average HCDR3 size of the
VH rearrangements in fetal bone marrow and
compared our findings with the previously published data on HCDR3
lengths in fetal liver and in adult repertoires (Fig. 3). We found that the average lengths of
the HCDR3 in VH4 and VH3
rearrangements from fetal bone marrow are significantly longer than
from the fetal liver (p < 0.001) and closer to
the average adult HCDR3 size, although still significantly different
from adult (p < 0.05). Taken together, these
observations suggest that the fetal bone marrow
VH repertoire shares more features with the adult
B cell repertoire than with the fetal liver repertoire.
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The IF/OF ratios of members of VH4 and
VH3 families are summarized in Tables III and IV.
The ratio of IF/OF at the pro-B cell stage in both of the families is
0.5 (Table II), which is similar to the expected ratio based on the
random rearrangement process. This is also characteristic for
VH segments at the pro-B cell stage in mouse bone
marrow populations (28). In general, the IF/OF ratio
of individual members of VH3 and
VH4 families was low, indicating the predominance
of OF rearrangements at the pro-B cell stage. At the pre-B cell stage,
however, a significant increase (p < 0.01) in
the IF/OF ratio was observed. VH3 genes and some
members of the VH4 family such as V4–34, V4–59,
and V4–39 show an increase in the IF/OF ratio, indicating the
selection of cells bearing productively rearranged genes. In contrast,
among V4–31 rearrangements, the predominance of OF rearrangements
persists in the pre-B library with an IF/OF ratio of 0.3, which is
lower than the expected value of 2.3 (32, 33, 34). The V4–61
gene was infrequently rearranged at the pro-B cell stage relative to
other VH4 genes, which possibly explains its
relative low frequency at the later, more mature B cell stages
(9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IF and OF rearrangements have been analyzed in mature B cells (7, 24), in which OF rearrangements occur in mature B cells solely by virtue of their coexistence in cells with productive rearrangements of the second VH allele. However, in these studies it was difficult to draw definitive conclusions regarding the preferential rearrangement of VH gene elements due to the small number of observed OF rearrangements. In addition, a large proportion of rearrangements may not be observed in the mature B cell repertoire because both positive and negative selection may have occurred during B cell maturation (38). In the present study, however, more than 90% of the cells are unaffected by µ-chain-mediated selection mechanisms, permitting an unbiased sample of all rearrangement events. This analysis of OF rearrangements suggests that, at the pro-B cell stage, the V4–34, V4–39, and V4–59 gene segments are the most frequently rearranged VH4 family genes, and the V3–23 and V3–30 gene segments are the most frequently rearranged VH3 family genes during the recombination process.
The molecular mechanisms that result in the preferential rearrangement of particular VH elements during Ig gene assembly are not known. However, inherent differences in accessibility to recombinase machinery (39), a more favorable spacer in the RSS (13), a more efficient cleavage in the RSS (12, 40, 41, 42), and proximity between elements (43, 44, 45, 46) have all been suggested as possible explanations. More recently, the complete nucleotide sequence of the VH locus, including the heptamer/nonamer and the 5' regulatory sequences for all VH genes, has been reported (5). For the genes analyzed in the present study, including the pseudogene (V4–55) and the unrearranged V4–28 gene segment, the 5' regulatory regions, spacers, splice sites, and heptamer/nonamer are all intact; no structural defects have been detected (5). The differences in the frequency of rearrangement of the VH genes may be due to the influence of yet unknown factors outside the VH locus, as previously suggested (23, 47). The position of genes within the VH locus has also been suggested to contribute to the preferential rearrangement of some genes. In mice, the most D-proximal VH segment, VH81X, a member of VH7183, predominates early in ontogeny (44, 48). In humans, however, proximity to JH does not appear to influence the rearrangement frequency of VH genes in early or later stages of ontogeny (14, 31, 49, 50).
While the current data demonstrate the preferential rearrangement of
certain VH genes in early lineage B cells of
fetal bone marrow, we have not shown that this process also occurs in
early B lineage cells of adults. However, based on the comparison of
HCDR3 size (Fig. 3) and VH gene usage, it appears
that the Ig repertoire of fetal bone marrow more closely resembles that
of adult bone marrow (9, 15) than that of fetal liver
(13, 14, 30).
We demonstrate that during the transition from pro-B to pre-B cell stage, there is reversal of the IF/OF ratio, reflecting a predominance of IF rearrangements of frequently rearranged genes such as V4–34. In contrast, the IF/OF ratio of V4–31 is not reversed during the pro- to pre-B cell transition. The differential ability of VH- µ-chains to pair with surrogate light chain (51, 52, 53) may explain this difference. For example, µ-chains that contain VH gene products such as V4–34 and V3–23, which are preferentially rearranged at the pro-B cell stage, may form stable complexes with surrogate light chain at the pre-B cell stage fostering clonal expansion and maturation of B cells expressing these VH genes. In contrast, VH genes such as V4–31, which is also frequently rearranged at the pro-B cell stage, may not pair efficiently with surrogate light chain. Consequently, they would not have a survival advantage and would occur less frequently in mature B cell populations, similar to the VH81X gene in the mouse (51, 54).
In summary, the current experiments provide evidence that certain VH gene segments are preferentially rearranged during VDJ recombination at the pro-B cell stage. Further studies are needed to determine whether selection mechanisms at later B cell developmental stages also contribute to the biased VH gene usage of mature peripheral B cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Leslie E. Silberstein, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 284 John Morgan Building 36th and Hamilton Walk, Philadelphia, PA 19104. E-mail address:
3 Abbreviations used in this paper: sIg, surface Ig; FR, framework; HCDR3, third complimentarity-determining region of the heavy chain; HR, high resolution; IF, in-frame; OF, out-of-frame.
Received for publication February 25, 1999. Accepted for publication June 28, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chain (CD25,TAC) expression defines a crucial stage in pre-B cell development. Int. Immunol. 6:1257.This article has been cited by other articles:
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  J.-H. Weitkamp, N. L. Kallewaard, A. L. Bowen, B. J. LaFleur, H. B. Greenberg, and J. E. Crowe Jr VH1-46 Is the Dominant Immunoglobulin Heavy Chain Gene Segment in Rotavirus-Specific Memory B Cells Expressing the Intestinal Homing Receptor {alpha}4{beta}7 J. Immunol., March 15, 2005; 174(6): 3454 - 3460. [Abstract] [Full Text] [PDF]
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 A. Li, M. Rue, J. Zhou, H. Wang, M. A. Goldwasser, D. Neuberg, V. Dalton, D. Zuckerman, C. Lyons, L. B. Silverman, et al. Utilization of Ig heavy chain variable, diversity, and joining gene segments in children with B-lineage acute lymphoblastic leukemia: implications for the mechanisms of VDJ recombination and for pathogenesis Blood, June 15, 2004; 103(12): 4602 - 4609. [Abstract] [Full Text] [PDF]
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 R J Looney, J Anolik, and I Sanz B lymphocytes in systemic lupus erythematosus: lessons from therapy targeting B cells Lupus, May 1, 2004; 13(5): 381 - 390. [Abstract] [PDF]
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F. Y. Mortuza, I. M. Moreira, M. Papaioannou, P. Gameiro, L. A. Coyle, C. S. Gricks, P. Amlot, H. G. Prentice, A. Madrigal, A. V. Hoffbrand, et al. Immunoglobulin heavy-chain gene rearrangement in adult acute lymphoblastic leukemia reveals preferential usage of JH-proximal variable gene segments Blood, May 1, 2001; 97(9): 2716 - 2726. [Abstract] [Full Text] [PDF]
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A. Wilson, C. Marechal, and H. R. MacDonald Biased V{beta} Usage in Immature Thymocytes Is Independent of DJ{beta} Proximity and pT{{alpha}} Pairing J. Immunol., January 1, 2001; 166(1): 51 - 57. [Abstract] [Full Text] [PDF]
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T. W. LeBien Fates of human B-cell precursors Blood, July 1, 2000; 96(1): 9 - 23. [Abstract] [Full Text] [PDF]
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