1. Art and Diseño

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Differential Usage of an Ig Heavy Chain Variable Region Gene
by Human B-Cell Tumors
By Freda K. Stevenson, Myfanwy B. Spellerberg, Joanna Treasure, Caroline J. Chapman, Leslie E. Silberstein,
Terry J. Hamblin, and David B. Jones
A monoclonal anti-idiotypic antibody has been raised that
recognizes Igs with heavy chains encoded by a member of
the V,4 family, the V,4-21 gene segment. The idiotope
(Id) is detectable on a high percentage of early B cells in
fetal spleen, and is expressed by certain autoantibodies,
particularly cold-reactive anti-red blood cell antibodies.
Therefore, it was of interest to investigate usage of this V,
gene by neoplastic B cells; 81 chronic lymphocytic leukemias (CLLs) involving CD5+ B cells and 6 2 B-cell lymphomas of varying histologic type have been analyzed. The Id
was expressed by only 3 of 81 (3.7%)of the CLLs, indicating a relatively low usage by these tumors. In contrast, the
Id was expressed by 9 of 62 (14.5%) of the lymphomas
across a range of histologic types, indicating a differential
use of the V,4-21 gene among B-cell neoplasms. For three
of the Id-positive lymphomas, each of a different histologic
class, the nucleotide sequence of the tumor-derived V,
gene was determined; the V,4-21 gene was identified, as
expected. The sequence from the CLL was identical to the
germline sequence, and the marginal zone lymphoma
showed only 3 nucleotide changes, 2 of which gave rise to
amino acid substitutions. In contrast, the sequence from
the follicular lymphoma showed 29 nucleotide changes
giving rise to 1 4 amino acid substitutions, which were
scattered among the CDR and FW regions.
0 1993 by The American Society of Hematology.
D
quency of V, family use was similar to that reported for
normal blood lymphocytes, indicating no selective bias at
this level, although there was a suggestion of nonrandom
use of individual members of the V,4 family.'
During our studies of the idiotypic determinants expressed by cells of B-cell lymphomas, we raised a monoclonal anti-idiotype (anti-Id), 9G4, that recognizes a conformation-dependent Id encoded by the heavy chain of a member
of the V,4 family, the V,4-2 1 gene.'-'' This deduction was
made after profiling the reactivity of a large panel of sequenced Igs with the anti-Id"'," and has been confirmed by
others.I2 An assessment of the involvement of this gene in a
panel of human monoclonal Igs showed a highly restricted
usage, with 0 of 55 IgGs and 1 of 19 IgM paraproteins being
Id-positive; interestingly, the positive IgM was an autoantibody directed against the Ii carbohydrate antigen on the
surface of red blood cells (RBCs), a so-called cold agglutinin,'and we have found subsequently that 48 of48 autoantibodies of this specificity express the Id and, in 8 cases studied by sequence analysis, use the V,4-21
This
degree of restriction of a V, gene for an antibody specificity
is highly unusual, and implies some structural requirement
for the framework sequence encoded by the gene for antigen
recognition. However, although the V,4-2 I gene appears
mandatory for cold agglutinins, it can also be used by other
antibodies, and has been reported to encode an IgG autoanti-DNA antibody."
Further investigation of Id expression showed that a significant proportion (-6%) of B cells in the fetal spleen were
positive,16 and that the Id was present on CD5' B cells in
cord blood." In fact, 24% of Epstein-Barr virus (EBV)transformed B-cell clones established from CD5+ cord
blood lymphocytes expressed the Id.I7 However, this figure
does not necessarily reflect the frequency of Id-positive cells
in the untransformed population, because there could be
differences in the responses of B cells to EBV. Sequence
analysis of 2 of 2 of these Id-positive EBV clones has confirmed usage of the V,4-2 1 gene (Dean et al, submitted for
publication). Taken together, the evidence suggested that
the V,4-2 1 gene was active early in normal B-cell maturation, was associated with CD5 positivity, and was used by
neoplastic B cells involved in secreting autoanti-RBC antibodies. It was therefore of interest to investigate the various
URING MATURATION of a normal B cell, selection
and recombination ofa particular V, gene with other
genetic elements occurs, giving rise to a functional V,-D-J,
unit. This selection is from among the six known V, families in the germline (V, I to V,6), with family sizes varying
from I (v,6) to more than 30 (v,3) of an estimated total of
100 to 200 V, gene segments.lS2In fact, the frequency of use
of the individual V, families by normal adult blood lymphocytes appears to correlate roughly with estimates of family size based on Northern blotting or in situ hybridizati~n.~.~
With regard to neoplastic B cells, there is evidence for
deviation from this random use of V, families by certain
tumors; the most studied group of these is chronic lymphocytic leukemia (CLL), in which biased expression of genes
from the V,5 and V,6 families, accompanied by an underrepresentation of V,1 and V,2, has been reported.s.6
The neoplastic cell typical of CLL is usually CD5+ and
normal or neoplastic B cells expressing this marker apparently derive from a separate lineage that, in both human and
mouse, appears to be associated with synthesis of autoantibodies.' Considerably less is known of V, use by other Bcell tumors, although V, restriction could give rise to crossreactive idiotypes that might have a diagnostic or
monitoring potential.' However, in a recent survey of 36
cases of follicular lymphoma, it was found that the fre-
From the Molecular Immunology Group, Tenovus Laboratory,
and the Department of Pathology, Southampton University Hospitals, Southampton. UK;and the Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA.
Submitted November 17, 1992; accepted February 15, 1993.
Supported by Tenovus, the Cancer Research Campaign, the Wessex Medical Trust, UK, and by a collaborative grant from NATO.
Address reprint requests to Freda K. Stevenson, DPhil, Molecular
Immunology Group, Southampton University Hospitals, Tenovus
Laboratory, Tremona Rd, Southampton SO9 4XY. UK.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1993 by The American Society of Hematology.
0006-49 7l/93/820I-0008$3.00/0
224
Blood, VOI 82, NO 1 (July 1). 1993: PP 224-230
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VH GENE USAGE BY HUMAN B-CELL TUMORS
categories of B-cell tumors for Id expression, anticipating
that it would be found to be preferentially expressed by
those tumor cells that are thought t o arise from the early
CD5 lineage, ie, CLL.
+
MATERIALS AND METHODS
Cell suspension analysis. Blood was obtained from patients
with CLL and from two patients with lymphoma; lymphocytes
were separated using Ficoll-Hypaque. Cells were then suspended in
warm medium (RPMI 1640, HEPES buffered) containing fetal calf
serum (FCS; lo%), streptomycin (0.1 mg/mL), and penicillin (60
pg/mL) and washed twice. Cell surface antigens were analyzed in
the FACS-SCAN (Becton Dickinson Lab Systems, Mountain View,
CA) by indirect immunofluorescence using monoclonal anti-Ig reagents as described" and anti-CD5 (OKTl; ATCC, Rockville,
MD), with fluorescein isothiocyanate (FIX)-labeled sheep antimouse IgG (Sigma Chemical Co, Poole, Dorset, UK) for detection.
In one case of lymphoma (JJ), frozen cells were cytocentrifugedand
analyzed by the sensitive single-stage alkaline phosphatase-antialkaline phosphatase (APAAP) technique, with sheep antimouse Ig as
second bridging antibody followed by complexes of AP with its
mouse antibody.16The rat anti-idiotypic antibody, 9G4, which detects the VH4-21-associated Id, has been
It is of the
IgG2a subclass, and was used with a control subclass-matched rat
antibody; detecting antibody was FITC-sheep antimouse IgG selected for cross-reactivitywith rat IgG.
Tissue section analysis. Frozen tissue sections obtained from
reactive lymph node, or from lymph node, spleen, and lung from
patients with lymphoma were used to assess expression of Id. Cases
of B-cell lymphoma were obtained from the frozen tissue files ofthe
Department of Pathology, University ofsouthampton, and the original histologic diagnosis, made according to the Keil classification," was confirmed by independent review. Cryostat sections ( 5
pm) were air-dried at room temperature and stored at -70°C until
required. Immediately before staining, the sections were fixed in
acetone for 10 minutes and then stained by an indirect immunoperoxidase technique using HRP-rabbit antimouse IgG or HRP-rabbit
antirat IgG, followed by development of reaction product by diaminobenzidine tetrahydr~chloride.'~
A diagnostic monoclonal antibody (MoAb) panel, containing antibodies against CD37, CD19, CD22, CD5, and CDlO, in conjunction with antibodies against Ig heavy and light chains, was used to
confirm the presence of a B-cell tumor and allowed identification of
areas of tumor involvement. Morphologicallyidentified tumor cells
were considered positive if a clear membrane immunoperoxidase
reaction product could be seen in the frozen section.
Patients. Patients with a clinical diagnosis of CLL were identified from the routine immunology laboratory; those with a white
blood cell count (WBC) ofgreater than 10 X 109/Land with tumor
cells expressing the CD5 antigen were included in the study.
Three patients with Id-positive tumor cells were selected for nucleotide sequence analysis; each of these represented a distinct category of B-cell neoplasia. The first patient, KR (a 69-year-old
woman) was a typical long-standing case of CLL, having a lymphocytosis (8 X 109/L)comprising small K-positivemonomorphic lymphocytes. The second patient was a 63-year-old man, JJ, who presented in 1986 with a lymphocytosis and an enlarged spleen that
was removed surgically.The histologic diagnosis was of a lymphocytic lymphoma with greater than 90% tumor cells in the involved
spleen and bone marrow. However, a more recent assessment ofthe
histologic appearance of the spleen has led to an assignment to the
category of marginal zone lymphoma. The blood lymphocyte
count was 7.5 X 109/L,of which the majorityi>95%) were X-positive tumor cells. These cells were stored frozen in liquid nitrogen for
further analysis. The third patient, PS (a 43-year-old woman), pre-
225
sented in 1987 and had a lymph node removed surgically;the histologic diagnosis was of follicular lymphoma involving cells of centroblastic and centrocytic morphology. The patient also had a
lymphocytosis (3.7 X 109/L)consisting of greater than 90% K-positive tumor cells; treatment with several courses of chemotherapy
failed to control disease and tumor cells in the blood were at 4.5 X
109/Lwhen collected for the study.
Establishment of cell line from JJ. Active EBV was harvested
from the marmoset cell line B95/8 and filtered (0.45 pm) before
use. Blood lymphocytes from patient JJ were suspended in B95/8
culture fluid at 2 X IO6 cells/mL. After incubation for 1 hour, cells
were centrifuged, resuspended in medium, and plated at 5 X IO5
cells/well in 200 pL medium in a 96-well plate. After a minimum of
4 days, cells received fresh medium and were monitored for growth
before transfer. When in flasks, supernatant was taken for assay of
IgM and Id by enzyme-linked immunosorbent assay (ELISA) as
described,16and cells were examined for clonality by staining for Id
using the single-stage APAAP technique.
Cloning and sequencing of the productively rearranged VH
gene. For the V, of the EBV line established from JJ, the method
of cloning and sequencing has been described in detail." For patients KR and PS, a more rapid method of direct sequencing from
cDNA was used. Total RNA was isolated from -5 X IO6 tumor
cells (>95% of the blood lymphocyte population) using RNAzol B
(Cinna Biotecxlaboratories Inc, Houston, TX) and was reverse
transcribed with Moloney murine leukemia virus reverse transcriptase and a Not-l-d(T),, primer (Pharmacia LKB, Uppsala, Sweden).
One-twentieth of the cDNA was amplified by polymerase chain
reaction (PCR) using an oligonucleotide primer specific for the VH4
heavy chain leader (5'-ATGAAACACCTGTGGTTCTT)and a C p
primer (5'-CGAGGGGGAAAAGGGTTGG).12Amplified products were electrophoresed through a 1.5% agarose gel and purified
using Geneclean I1 (Bio 101 Inc, La Jolla, CA). Purified DNA (100
ng) was sequenced directly by the dideoxy chain termination
method with T7 DNA polymerase (Pharmacia) and 100 ng of the
relevant PCR primer. PCR amplification and sequencing was performed three times.
RESULTS
Phenotypic analysis of blood cells from patients with
CLL. Samples of blood lymphocytes from patients with a
diagnosis of CLL and WBC ofgreater than 10 X 109/L were
analyzed in the FACS-SCAN. All expressed CD5, and those
that were also clearly positive for surface Ig were selected for
further study, giving a total of 8 1. The surface Ig was identified as IgMK (43) or IgMh (38), and ofthese, 2 IgMK and 1
IgMX also expressed the V,4-2 1-associated Id. The overall
percentage of positivity ofthe Ig-expressingtumor cell populations in CLL was therefore 3.7%(Table 1).
Expression of Id in normal lymphoid tissue. The fact
that the Id is expressed by 3% of B cells in normal lymph
nodes has been described,16 and the detailed distribution of
those cells in a typical follicle of a normal lymph node is
shown in Fig I . Staining with anti-CD22, a pan-B-cell antigen (Fig lA), shows a high density of B cells in the mantle
zone, with a mixed population containing some B cells in
the follicle center. Figure 1B then shows a similar distribution for the Id-positive cells; there is a clear subpopulation
of Id-positive B cells in the mantle zone, comprising a small
percentage of the total B-cell population. However, Id-positive cells are not confined to the mantle zone, because there
are also some scattered cells visible within the folliclecenter.
-
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STEVENSON ET AL
226
Table 1. Expression of Id by 6-Cell Tumors
No. of
No. of
Cases
Positive Cases
CLL
Lymphocytic lymphoma
Marginal zone lymphoma
81
9
1
3
Follicle center cell lymphoma:
Follicular, low grade
Intermediateand high grade
18
28
2
6
1'
Tumor Category
Lymphoma of mucosa-associated
lymphoid tissue
0
1
5
This single positive case showed morphologic transformation to a
high-grade tumor.
E.ypre.ssion qf Id in R-cell ljwplioma. The incidence of
Id positivity among lymphomas of various histologic type is
also shown in Table I. In each case, the Id was expressed by
the morphologically identifiable tumor cell population. and
there were 9 of 62 clear positives (14.5%). which is significantly higher than that for CLL (. 1 > P > .05 by xzanalysis).
Although numbers in each histologic category are too small
to detect any significant differences, there is an indication of
a low incidence in lymphocytic lymphoma (0 of 9 positive).
consistent with the similarity of this neoplasm in morphology and behavior to CLL." Expression of CD5 was heterogeneous among the lymphocytic lymphomas, with 6 of 8 being positive ( 1 case was not evaluable).
An example of the pattern of staining in the follicle center
of a lymph node infiltrated with Id-positive tumor is shown
in Fig 2. Again, the pan-B-cell marker CD22 has been used
to stain the total B-cell population (Fig 2A). and the positive
cells (marked with arrows). identifiable by darkly stained
surface membranes. are clearly visible among a background
of T cells, with the morphology of the stained population
being strongly suggestive of tumor cells. This feature is also
seen in Fig 2B using the anti-Id antibody that stains the large
pleiomorphic tumor cells (marked with arrows) in a pattern
similar to that with anti-CD22, suggesting that most ofthe B
cells are neoplastic.
The phenotypic analysis of the Id-positive cases, including those from the CLL group (Table 2), indicates that there
was a mixture of K and X light chain types and that all identifiable heavy chains were p. In one case of A-expressing highgrade lymphoma. technical reasons prevented allocation of
the heavy chain class. All the cases of Id-positive CLL were
CD5'. but the Id-positive lymphomas were all C D Y (Table
2).
Ana1jsi.s of liimor cells . f h n patients KR, JJ, and
PS. The phenotypic profiles of the tumor cells from the
three patients available for nucleotide sequence analysis,
KR, JJ, and PS, are included in Table 2 and are indicated by
an asterisk. For patient KR (CLL), the blood lymphocytes
included 73% of IgMr-positive cells, all of which were positive for both Id and CD5: the majority of the remaining
lymphocytes (22%)were T cells. For JJ (marginal zone lymphoma), the blood lymphocytes that were used for genetic
analysis had a profile similar to that of the splenic tumor
cells, with the majority (>95%) of cells expressing Id-positive IgMX. but no CD5, and few detectable T cells. After
transformation with EBV. the same IgMld profile was
maintained in greater than 95% of the cells in the line, and
transformed cells secreted IgMX that was all idiotypic by
ELISA.I6 consistent with a clonal population. For patient
PS (follicular lymphoma), the Ig phenotype of tumor cells in
the lymph node sections was IgMK and C D Y (Table 2);
again, the neoplastic blood lymphocytes used for genetic
analysis were of the same phenotype being greater than 90%
positive for Id-positive IgMK.
Moleciilar analwis qf e.ypre.s.sed l/, genes qf KR, PS, and
JJ. The nucleotide sequence of the V, region of the IgM
from patient KR indicates that it is identical to the germline
V,4-2 1 gene sequence (Fig 3). The V, sequence of JJ is also
derived from the V,4-2 1 germline gene, and there are only
three mutations. However, as shown in Fig 4, two of the
Fig 1. Expression of 964 Id by normal 6 cells in a reactive lymph node. (A) 6-cell population stained with anti-CD22. (6) Id-positive cell
population stained with anti-Id (964). Positively stained cells are indicated by arrows. Original magnification X 450.
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227
V, GENE USAGE BY HUMAN E-CELL TUMORS
three mutations givc rise to amino acid substitutions. ic. a
glycine to aspartic acid in C D R I and a lysine to arginine in
IW3. Interestingly. the same two substitutionsarc found in
the PS V, (Fig 4). and the gly to asp substitution via the
same nucleotide change has been observed in four other
Table 2. Phenotypic Analysis of Id-Positive Cases
lg
Tumor Category
CLL
Marginal zone lymphoma
Follicle center cell lymphoma:
Follicular, low grade
Intermediate and high grade
Lymphoma of mucosa-associated
lymphoid tissue
Tissue
Expression
CD5
Blood
Blood
Blood'
Blood,' spleen
lgMX
lgMx
lgMh
IgMDh
+
Lymph node,'
blood
Lymphnode
Lymph node
Lymph node
Lymph node
Lymphnode
Lymphnode
1gMx
-
lgMX
lgMX
lgMx
lgMx
lgMX
-
Lung
+
+
-
At
-
lgMX
-
Nucleotide sequence analysis of V. genes was performed on the
tissues indicated.
t The heavy chain was not identified in this case.
lgMs derived from V,4-2 I . all of which had cold agglutinin
activity: however. it is not mandatory for such sp~cificity.'~
The V, sequence of PS is also similar to the V,4-2 I germline gene. but in this case thew is only 90.0a homology with
29 nucleotidc ditrerenccs. giving rise to 14 amino acid substitutions. Among the recently updated analysis of the VH4
family.'" the sequence of PS lies within the VS8 group.
which also includcs V,4-2 I . However, homology with the
other members of the group is less than 90%and the VH4-2 I
gene is the closest match. The D segments of KR and JJ are
both long (Fig 4) and JH6is used in each case. In fact. K R
has a strctch of 26 nucleotides homologous to the D X P 4
germline gene and JJ hasa stretch of 23 nucleotides homologous to the D L R 3 germline gene.'' with flanking sequences
showing shorter sequence homologies to two other D segments. In contrast. the D segment of PS is a short sequence
with a stretch of 7 nucleotides homologous to the D M ?
germline gene." In all thrcc cases. thcre are probable N-termind additions.
DISCUSSION
Anti-idiotypic antibodies have been used for more than
20 years to idcntify common structures in the variable regions of Ig moleculcs.'' Thc advcnt of monoclonal anti-Ids
Fig 2. Expressionof 964 Id by neoplastic B cells in a lymph node
from a patient with follicular lymphoma. Note that follicle mantle
zones seen in normal lymph node have been ablated. (A) E-cell population stained with anti-CD22 MoAb (original magnification
X 250). (B) Id-positive cell population stained with anti-Id (9G4);
arrows indicate positive membrane staining of clusters of neoplastic cells (original magnification X 250). For comparison, see the
negative lymphocytes in the follicle center of Fig 1E. (C) Id-positive
cell population stained with anti-Id (9G4) at higher magnification
(original magnification X 400). with small reactive lymphocytes
appearing to be negative.
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228
STEVENSON ET AL
CDRl
1
I
VH4.21 (GL) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGPAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCC TTCAGTGGTTACTACTGGAGC TGWTCCGCCAGCCCCCAGGGAAGGGGCTG
w(
(1gH)
JJ
(IgM)
-------A------------PS
(1gH) --------A-----A-----------------A-----------------T--A----------------------------------C-A-----------A------------T-------A---------
..............................
.................................................................................... .....................
....................................................................................
..............................
CDR2
I
I
VH4.21 (GL) GAGTGGATTGGG GAAATCAATCATAGTGGPAGCACCPACTACAACCCGTCCCTCPAGAGT CGAGTCACCATATCAGTAWCACGTCCAAGAACCAGTTCTCCCT~GCTGAGCTCTGTGACCGCCGCGGACACG
KR
(IgH)
---------------____-____________________-------JJ
(Ign)
----------_-____________________________-----------------------------------------------------G------------------A--------pS
(IgH) A&-------------------C------C-G-------------------------- ------G----C---A-T-------------CG------------CG-T---C----C----G------------
...........................................................................
_----------_
-_-----_--__
D SEGMENT
VH4.21 (GL)
GCTGTGTATTACTGTGCGAGA GG
.....................
JJ
(IgH)
(IgH)
pS
(IgH) ---C-----------------
w(
1
I
GTAGGCTGGTATTACGATTTTTGGAGTGGTTATTCGGTCTCCGAC
TACTACTACATGGACGTCTGGGGCMAGGCACCACGGTCACCGTCTCCTCA JH6
AATGGAACCTCTGGCGAT
TTTGACTACTGGGGCCAGGWCCTGGTCACCGTCTCCTCA
_--_---_--___________
AGCTCCGCCCCACCTATTGTGGTGGTGACTGCTATTCCATTGACCGCT~CCGGGC
TACTACGGTATGGACGTCTGGGGCCAACCCAeCACGCTCACCGTCTCCTCA JH6
JH4
Fig 3. Nucleotide sequence comparison of the V,,4-21 germline gene segment and the VMgene segments expressed in three patients
with B-cell tumors. Patient KR, CLL; patient JJ, marginal zone lymphoma; patient PS,follicular lymphoma.
allowed more precise localization of the sequences involved
in Id expression, and such reagents have proved useful for
investigations of the clonal Igs characteristic of B-cell lymphomas,' and of the oligoclonal autoantibodies synthesized
in autoimmune diseases such as systemic lupus erythematosus (SLE).23The monoclonal anti-idiotypic antibody 9G4
that we raised in 1986' has proved particularly useful because it recognizes an Id that is expressed by Igs with heavy
chains encoded by a defined VHgene, V,4-2 1, a member of
the V,4 family. The Id has been reported to be expressed by
13 of 13 Igs that use this gene,l33I4and we have since found it
to be present on a further 29 of 30 sequenced Igs with heavy
chains encoded by the V,4-2 I gene (unpublished observations). In contrast, it is not expressed by other sequenced Igs
encoded by different members of the vH4 family, or by Igs
from other V, families.",12
Another interesting point is that the Id is expressed by
autoantibodies that are specific for the I/i carbohydrate antigen on the RBC surface, the so-called cold agglutinins, and
it appears that these autoantibodies are all encoded by the
VH4-21 gene.Lo.'2,'4This degree of restriction of a V, gene
for a particular antibody is highly unusual; however, the
V,4-2 I gene is not confined to anti-RBC antibodies, be-
CDRl
n
cause it has been found also to encode autoanti-DNA antibodies."
Studies on fetal spleen showed that the Id was expressed
early in development, being identifiable on -6% of B cells
at 20 weeks of gestation.I6 The id was also detected on cord
blood lymphocytes, in which it appeared to be preferentially
expressed by CD5+ B cells.'7 This finding ofexpression ofan
Id, known to be associated with both cold agglutinins and
anti-DNA autoantibodies, by immature CD5+ B cells led us
to anticipate that the Id would be present on CD5' neoplastic cells found in CLL, thereby supporting the proposed link
between this normal cell population and the cells found in
CLL.24In fact, it is known that a significant proportion of
such tumors synthesize autoantibodies, particularly rheumatoid factor.25The proposal that CLL could represent the
neoplastic counterpart of immature B cells found in fetal
spleen has been supported by the finding of common highfrequency cross-reactive Ids,26some of which have been located to usage of certain V-genes, particularly V,IIIb.27 Assymetric usage of V, genes has been also reported for CLL,
with increased involvement of v H 4 , vH5, and VH6.' However, the finding in this report is that expression of the Id
associated with a member of the vH4 family, V,4-2 1, which
cDR3
CDRP
I
I
I
VH4-21 (GL) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWS MIRQPPGKGLEWIG EINHSGSTNYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR
KR
(IgH) ____________________________
VGWYYDFWSGYSVSD
YYYHDV JH6
JJ
(IgH) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _--D---__
---------------R---------------SWPIVWTAIPLTAQPG YYGHDV JH6
PS
(IgM) _________________-_-________
--D---N ----S-----T------R-TA---------A--I----T----R-T-L------L----NGTU;D
FDY JH4
_______ ______________ ________________ ________________________________
______________ ________________
Fig 4. Deduced amino acid sequence comparison of the VM4-21germline gene segment and the V. gene segments expressed in three
patients with B-cell tumors. Patient KR, CLL; patient JJ, marginal zone lymphoma; patient PS, follicular lymphoma.
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VH
GENE USAGE BY HUMAN B-CELL TUMORS
is expressed by -6% of fetal B cells, is found on only 3 of 8 1
(3.7%) cases of CD5' CLL. This indicates a consistency between the use of the V,4-21 gene by early B cells and its
incidence in CLL. However, usage in CLL is low in comparison with other V, genes such as the 5 l p l V, 1 gene, which is
associated with the G6 idiotope.26For G6, although expression in the fetal spleen (6.9%) is comparable with our results
for the VH4-21-associated Id, expression by cases of CLL is
high (20%).26Loss of Id by mutation is an unlikely explanation for this relatively low level of expression because Vgenes in CLL tend to be unmutated"; also, the Id appears to
tolerate quite extensive mutations without loss (Pascual et
all0 and unpublished observations).
In contrast to CLL, the V,4-2 1 gene is used by neoplastic
B cells that secrete IgM with cold agglutinin activity,I0,l2and
these tumors represent a significant proportion (13 of 105,
ie, 12%) of the IgM-secreting neoplasms known as Waldenstrom's macr~globulinaemia.~~
Because the tumor cells
tend not to be in the blood, there is less information on their
CD5 status; however, in our preliminary studies using double staining for Id and CD5, we have found that the majority
of cases (7 of 8) do express CD5 (unpublished observations).
The relatively high incidence of use of the VH4-21gene in
this group of tumors could indicate that they develop from a
normal, possibly CD5+, B cell that is distinct from that
which gives rise to CLL. Our findings that the gene appears
to be switched on after certain common infections such as
EBV29asuggest that those postinfective IgMId-secreting cells
could represent the population that undergoes neoplastic
transformation. The frequency of Ig class switching events
among this normal B-cell population is not yet clear; certainly the V,4-2 I gene can be found in IgG,'o,'5but a search
of our myeloma IgG protein panel found 0 of 55 positive for
indicating a low use of the gene in plasma cell tumors.
With regard to expression of the 9G4-associated Id in
normal lymphoid tissue, we have shown already that it has a
wide distribution.16 Within the lymph node there is a clear
population of Id-positive B cells in the mantle zone, but
there is also expression by some of the cells in the germinal
center. This distribution is consistent with the specificity of
the antibody for an Id that is effectively a V-region subgroup
determi~~ant,~'
and it appears to hold for the 9G4 Id that has
a restricted specificity within the V,4 family.
The finding of 9 of 62 Id-positive cases among the lymphomas indicates that the V,4-2 1 gene is used at about the
same frequency as in Waldenstrom's macroglobulinemia,
and more commonly than in CLL (. 1 > P > .05 by x2 analysis). Previously, we found the Id-positive population of normal lymph node to represent 3.2% 2.4% of the B cellsI6;
this would suggest that the incidence of 14.5%in B-cell lymphomas reflects a preferential use of the V,4-2 1 gene. It will
be ofinterest to expand the investigation ofthe various histologic types of lymphoma to see if there is any heterogeneity
of expression among the groups. In a previous study of lowgrade follicular lymphoma, usage of V, families by tumor
cells was comparable to that by normal B cells; however, use
of individual members of the V,4 family appeared asymetric, with 3 of 8 of the tumors of the 1 1 to 14 member V,4
family being assigned to V,4-2 1.'
To verify the association between Id expression and usage
-
*
229
of the V,4-21 gene, we investigated the nucleotide sequences of randomly chosen B-cell tumors from the panel.
Tumor-derived material was available from three patients,
one a typical CLL and two having lymphomas of different
histologic categories. For this particular V, gene, we have
the advantage first, that it has a sequence that is quite distinct from other members of the V,4 family3' and second,
that it has a very low degree of polymorphism, which allows
recognition of mutations from the germline gene.32In fact,
in a recent study of a random series of I 1 Id-positive clones
established from six patients with infectious mononucleosis, we have found the V,4-2 1 to be in germline configuration and to be identical in nucleotide
The sequences obtained from the three tumors were
closely related to the V,4-21 germline gene, which has a
characteristic sequence particularly in FWl . In fact, that
from patient KR was typical of the pattern found in C D Y
CLL in being unmutated from germline." Little is known
concerning V,-gene usage in marginal zone lymphoma, but
it was interesting to note a very low level of mutations in this
CD5- tumor. In contrast, the sequence of the V, gene from
the follicular lymphoma PS showed 29 mutations, ofwhich
18 were replacements. This result is consistent with previous findings in follicular lymphoma, and may reflect the
fact that normal B cells undergo hypermutation in the follicle center.33In fact, in a recent report of a case of follicular
lymphoma involving the V,4-2 1 gene, a similar number of
mutations from germline (23) was found.34However, if an
analysis of the pattern of mutations in the two cases is made,
there are differences, in that the changes in patient PS do
not show a high rep1acement:silent ratio in the complementarity determining regions (CDRs) that may result from antigen selection. For patient PS, the analysis of mutations
was performed in the same way. To assess the incidence of
replacement and silent mutations, each change was assumed to have occurred independently and was designated
as R or S and located in the CDRs or in framework regions
( F W R S ) .The
~ ~ majority ofthe mutations (23) are located in
FWregions, particularly FWR3, and the numbers of replacement and silent mutations in the FWRs ( 1 3R and 10s) and
in the CDRs (5R and 1S) are not sufficiently different from
those expected from a random distribution and composition (FWRs: 14R and 8s; CDRs: 4R and 3s) to indicate a
role for antigen selection. This finding contrasts with the
published case in which there were 23 mutations, with a
concentration of replacement mutations ( 1 1) in the CDRs
compared with 4 expected.34Clearly, more sequences of the
V-genes of tumor cells are needed and the relatively nonpolymorphic V,4-2 1 gene is ideal for investigating this crucial point. At present, it is difficult to assess the role of chemotherapy in inducing mutations, because the patient in
the previously reported
and in the current study had
both received chemotherapy. It will be of interest in the
future to analyze V-gene sequences from patients at presentation. Even if chemotherapy is not a perturbing factor, it is
possible that the situation in the lymph node is complicated
in that the hypermutation mechanism may be activated in a
B cell, but, after a neoplastic event, the tumor cell may or
may not be influenced by antigen.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
STEVENSON ET AL
230
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1993 82: 224-230
Differential usage of an Ig heavy chain variable region gene by human
B- cell tumors
FK Stevenson, MB Spellerberg, J Treasure, CJ Chapman, LE Silberstein, TJ Hamblin and DB
Jones
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