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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3390-3396
Expression of the T-Cell-Specific Tyrosine Kinase Lck in Normal B-1
Cells and in Chronic Lymphocytic Leukemia B Cells
By
M. Bernardetta Majolini,
Mario M. D'Elios, Piero Galieni,
Marianna Boncristiano,
Francesco Lauria,
Gianfranco Del Prete,
John L. Telford, and
Cosima T. Baldari
From the Department of Evolutionary Biology, University of Siena,
Siena, Italy; Internal Medicine and Immunoallergology, University of
Florence, Florence, Italy; the Division of Hematology, Ospedale A. Sclavo, Siena, Italy; and Chiron Research Center, Siena, Italy.
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ABSTRACT |
Src family kinases play a key role in mitogenesis. The exquisitely
tissue-specific distribution of different Src family members suggests
that a fine tuning of their expression might be a key prerequisite for
cell homeostasis. We tested B cells from patients affected by B-cell
chronic lymphocytic leukemia (B-CLL) for expression of Src family
kinases. The T-cell-specific tyrosine kinase Lck was found to be
expressed at significant levels in CLL B-cells. This finding could be
accounted for either by ectopic expression of Lck in B-CLL or by
specific expression of this kinase in normal B-1 cells, which are
believed to be the normal counterpart of CLL B cells. To answer this
question B cells from different sources, characterized by a different
size of the B-1 subpopulation, were tested for Lck expression. The
results show that Lck expression is a feature of CD5+,
B-1 cells, suggesting a potential role for Lck in the self-renewal capacity of this B-cell subpopulation and supporting the notion that
B-1 cells are the subset undergoing oncogenic transformation in B-CLL.
Furthermore, we show that the CD5 , B-2 subpopulation,
while normally lacking Lck expression, acquires the capacity to express
Lck ectopically upon transformation by EBV.
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INTRODUCTION |
B-CELL CHRONIC lymphocytic leukemia
(B-CLL) is the most common form of leukemia in white adults in Western
countries, where it accounts for about 9% of all cancers and for about
30% of all leukemias.1 A characteristic feature of CLL B
cells is the expression of CD5, a 67-kD antigen formerly considered an
antigen restricted to T cells, which is also found on a limited subset of normal B cells.2,3 These normal CD5+ B
cells, designated B-1,4 represent a self-replenishing
population arising early in ontogeny in both mice and humans and are
normally present in fetal tissue, whereas their distribution in the
adult is limited essentially to the peritoneal cavity and to the mantle zone of lymph nodes.2-4 Although conventional B cells (B-2)
can be induced to express CD5 in response to activation,5
evidence accumulated primarily in mice strongly supports the notion
that B-1 and B-2 cells are derived from separate lineages of progenitor cells.6,7 In addition to surface marker expression, B-1
cells indeed display unique features as compared to B-2 cells,
including the capacity of self-renewal and a restricted antibody
repertoire, with a bias toward the production of polyreactive,
low-affinity autoantibodies.8 Furthermore, constitutive
nuclear expression of STAT3 was recently shown to be a distinctive
feature of B-1 cells.9
Because of their surface expression of CD5, CLL B cells are believed to
be the tumoral counterpart of normal B-1 cells. However, although some
unifying features can be found between normal B-1 cells and CLL B
cells, such as the production of polyreactive autoantibodies, the
ability to form rosettes with mouse erythrocytes and the expression of
myelomonocytic antigens,10 the differences are too
significant to unambiguously mark B-1 cells as the target of the
transforming events leading to B-CLL. For example, as opposed to normal
B-1 cells, CLL B cells accumulate in the G0 phase of the
cell cycle and express low to undetectable levels of surface immunoglobulins.10 Furthermore, they are characterized by a defective signal transduction in response to B-cell antigen receptor triggering, resulting in a reduced proliferative
response.10-13 Thus, the precise relationship of the CLL
B-cell to normal B-cell ontogeny is still incompletely understood.
Src family protein tyrosine kinases (PTK) play a key role in cell
homeostasis. Although the mode of regulation is identical for different
family members, the tissue distribution is remarkably specific,14 suggesting that an altered balance in the
levels of Src family kinases, either qualitative or quantitative, might have profound effects on cell proliferation and survival. While the
outcome of ectopic expression of Src family PTKs has to date not been
investigated, dramatic effects of the overexpression of specific family
members have been reported. In this respect, the best-characterized Src
family PTK is Lck, which is normally specifically expressed in the
T-cell lineage.15 When overexpressed in thymocytes, Lck
induces thymic tumorigenesis.16
This report shows that Lck is expressed in both B-CLL and normal B-1
cells but not in conventional, B-2 cells. This result supports the
hypothesis of separate B-1 and B-2 cell lineages in humans and provides
evidence toward the concept of B-1 cells as the target of the
transforming event leading to B-CLL. Furthermore, it suggests a role
for Lck in B-1 cells. We also show that, although undetectable in
peripheral blood B cells from healthy donors, Lck is expressed in B-2
cells from the same individuals following transformation by
Epstein-Barr virus (EBV), suggesting that induction of ectopic Lck
expression in B-2 cells by EBV might play a role in transformation.
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MATERIALS AND METHODS |
Patients, purification of lymphocyte populations, and flow cytometry.
Blood samples were obtained from 33 patients who satisfied diagnostic
criteria for B-CLL,1 as well as from two patients diagnosed
for hairy cell leukemia (HCL) and two patients diagnosed as having
non-Hodgkin's lymphoma (NHL) in the leukemic phase. The patients had
not received any treatment for at least 6 months before blood sampling.
Two independent samples were taken from most patients. Peripheral blood
from healthy donors was used as a control. Cord blood and peripheral
blood mononuclear cells were isolated from whole blood by density
centrifugation on Ficoll-Paque (Pharmacia, Uppsala, Sweden). After
removal of macrophages by adherence to plastic, B cells were purified
by a double step of rosetting with sheep erythrocytes. Lymphocytes from
adenoid and spleen were recovered by disaggregation, followed by
rosetting as described above. The purity of B-cell and T-cell
populations was checked by flow cytometry using fluorochrome-conjugated
monoclonal antibodies (MoAbs) to CD19 and CD3, respectively (Becton
Dickinson, San Jose, CA). Purification of the
CD5 and CD5+ subpopulations from cord blood
B cells was carried out by fluorescence-activated cell sorter (FACS)
sorting using fluorochrome-conjugated anti-CD19 MoAbs (Becton
Dickinson) and the anti-CD5 MoAb B36.1,17 followed by a
fluorochrome-conjugated anti-mouse Ig. Alternatively, the CD5 and CD5+ subpopulations from cord blood
B cells were purified by immunomagnetic sorting using anti-CD5 MoAb and
magnetic beads conjugated with anti-mouse Ig (Dynal Corp., Oslo,
Norway). The purity of these populations was checked by flow cytometry,
using the same anti-CD5 MoAb, followed by FITC-conjugated anti-mouse Ig
(Life Technologies Italia srl, Milan, Italy). B-cell
activation was carried out by incubating purified B cells on plastic
wells precoated with anti-human µ chain antibodies (Cappel
Laboratories, Durham, NC) for 12, 24, 48, or 72 hours. The activation
was checked by a [3H]-TdR proliferation assay on a sample
of the same B-cell population activated in the same conditions for 48 hours. Flow cytometric analysis of CD5 expression in these activated B
cells showed that, beginning at 24 hours after activation, the
proportion of CD5+ B cells was increased on the average by
a factor of 1.5. The tumor cell lines, all obtained from American Type
Culture Collection (ATCC) (Rockville, MD), included the
EBNA+ B-cell lines Daudi, Raji, and RPMI-1788, the
epidermoid line A431, the neuronal line PC12 and the monocytic line
THP-1. EBV lymphoblastoid B-cell lines were obtained from peripheral
blood B cells of healthy donors by transformation with EBV, using
standard procedures.
Immunoblots and in vitro kinase assays.
When required cells were activated by treatment with the anti-CD5 MoAb
B36.117 as described.18 Purified B cells and T
cells were lysed at about 106 cells/10 µL in 1% Nonidet
P-40 in 20 mmol/L Tris, pH 8, 150 mmol/L NaCl in the presence of 0.2 mg/mL Na orthovanadate, 1 µg/mL leupeptin, aprotinin, and pepstatin
and 10 mmol/L phenyl methyl sulfonyl fluoride (PMSF) (Boehringer,
Mannheim, Germany). The concentration of total proteins in postnuclear
supernatants was determined using a kit from Pierce (Rockford, IL) and
albumin as a standard. Equal amounts of proteins were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on
12% gels, transferred to nitrocellulose filters, and probed by
immunoblot using the following MoAbs: anti-Lck, anti-Lyn, and anti-Fyn
(Transduction Laboratories, Mamhead, UK), and anti- -tubulin
(Amersham, Buckinghamshire, UK). Peroxidase-labeled secondary
antibodies were revealed by chemiluminescence using reagents from
Pierce. To rule out a cross-reaction of the anti-Lck MoAb with other
known members of the Src family tyrosine kinases expressed in
lymphocytes, lysates of Jurkat T cells and of EBV transformed B cells
were tested by immunoblot with MoAbs to Fyn and Lyn. While an Lck
immunoreactive band could be detected in both types of cells, Fyn
expression was restricted to T cells, whereas Lyn expression was
restricted to B cells. Furthermore, no cross-reaction could be detected
in a Fyn-specific immunoprecipitate probed by Western blot with the
anti-Lck MoAb (Fig 1 and data not shown).
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| Fig 1.
Expression of Lck in B-CLL. (A) Immunoblot analysis using
an anti-Lck MoAb of peripheral blood B-cell lysates from healthy controls and from representative B-CLL patients. A lysate from normal T
cells is included as a positive control. (B) Immunoblot analysis using
an anti-Lyn MoAb of peripheral blood B-cell lysates from a healthy
control and from representative B-CLL patients.
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Lck was immunoprecipitated from postnuclear supernatants of
106 cells, using a polyclonal anti-Lck antiserum (Upstate
Biotechnology, Lake Placid, NY) and protein A-Sepharose (Pharmacia) as
described.18 Lck-specific immunoprecipitates were washed
three times in lysis buffer and resuspended in 1× kinase buffer
containing 20 mmol/L Tris pH 7.2, 10 mmol/L MgCl2, 10 mmol/L MnCl2, 5 µmol/L ATP, 10 µg acid-denatured
enolase, and 10 µCi [32P]- ATP per sample.
32P-labeled proteins were resolved by SDS-PAGE and
visualized and quantitated using a Phosphorimager (Molecular Dynamics,
Sunnyvale, CA). Titration of the levels of Lck recovered from
increasing amounts of a control Jurkat T-cell lysate by
immunoprecipitation with a fixed amount of the anti-Lck polyclonal
antiserum was carried out by Western blot, using the anti-Lck MoAb. The
results showed that Lck was quantitatively recovered in these
conditions (data not shown).
Reverse transcriptase-polymerase chain reaction.
Total RNA was extracted in guanidinium isothiocyanate from
107 CLL B cells, or from the same number of EBV-transformed
B cells. Jurkat T-cell RNA was used as a control. mRNA was
affinity-purified from 30 µg total RNA, using oligo-dT cellulose
(Boehringer), and an Lck specific first-strand cDNA was generated using
an oligonucleotide complementary to positions 1563 to 1578 of human Lck
mRNA (5 -AGGCTGAGGCTGGTA-3) and AMV reverse transcriptase
(Boehringer), according to the manufacturer's instructions. A 570-bp
fragment spanning positions 55 to 624 of human Lck mRNA was amplified
by polymerase chain reaction (PCR) in an Eppendorf Mastercycler 5330 temperature cycler using Taq polymerase (Boehringer) with the following
primers: 5-GGCTGTGGCTGCAGC-3 and 5-CCACCACCTCTCCCTG-3. Positive
controls included the product of a reverse transcriptase (RT)-PCR
carried out on mRNA from Jurkat T cells, as well as the product of a
PCR reaction carried out on Lck cDNA cloned in a plasmid vector. A
negative control reaction was set up using mRNA extracted from the
monocytic line THP-1. The amplification products were separated by
electrophoresis on 2% agarose gels and subsequently transferred to
nitrocellulose and hybridized with a 32P-labeled human Lck
probe prepared by random priming from a pGEX-Lck plasmid
construct (M. Boncristiano and C.T. Baldari, unpublished). The
1-kB-ladder molecular weight marker was purchased from Life Technologies Italia srl.
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RESULTS |
Expression of Lck in B cells from B-CLL patients.
Src family kinases play a key role in mitogenesis.14,15 As
opposed to the broad distribution of Src, other members of this family
of PTKs show a distinct cell type specificity. In lymphocytes, Lck and
Fyn are specifically expressed in T cells, whereas Lyn is found
predominantly in B cells.14 We hypothesized that ectopic expression of an Src family kinase could underlie the acquisition of an
altered proliferative potential in leukemia. To test this possibility,
we analyzed B cells from 33 B-CLL patients for the presence of Lck.
Lysates of purified B cells from peripheral blood of B-CLL patients, as
well as of control peripheral blood B cells from healthy donors, were
subjected to immunoblot analysis using an anti-Lck MoAb. The results of
13 representative patients are presented in Fig 1. While no Lck could
be detected in normal peripheral blood B cells, significant amounts of
Lck were found in B cells from all B-CLL patients (Fig 1A and data not
shown). The levels of Lck were variable; however, they were always
lower as compared to control T cells (Fig 1A). These differences were
confirmed by reprobing the stripped filters with an anti-tubulin MoAb
(data not shown). In some patients, an Lck immunoreactive band
characterized by a slower electrophoretic mobility could be detected
(Fig 1A). In T cells this difference has been correlated with
phosphorylation of Lck on serine residues following exposure to
activating signals.19 CLL B cells and control B cells
expressed similar levels of the B-cell-specific Src family PTK Lyn
(Fig 1B). As opposed to B cells, no difference in the levels of Lck
could be detected in peripheral blood T cells from the patients as
compared to control T cells from healthy donors (data not shown).
Although the anti-Lck MoAb did not cross-react with other known
lymphocyte-specific Src family PTKs (see Materials and Methods), a
potential cross-reaction with an as yet unidentified new family member
could not be ruled out. mRNA purified from CLL B cells was therefore
tested by RT-PCR for the presence of an Lck specific transcript. A
primer complementary to the 3 end of the coding region of Lck was used
to generate the first strand by retrotranscription. A fragment spanning
570 bp at the 5 end of the Lck coding region was then amplified by
PCR. This region is the least conserved among the sequences encoding
Src family kinases. The results from two representative B-CLL patients
are shown in Fig 2A. A fragment of the
expected size was obtained, which comigrated with the fragment obtained
from the positive controls. The specificity of the fragment for Lck
sequences was confirmed by Southern blot analysis of the same samples,
using a 32P-labeled human Lck cDNA probe (Fig 2B).
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| Fig 2.
RT-PCR analysis of Lck expression in B-CLL. (A) Ethidium
bromide staining of the amplification products of an Lck-specific first-strand cDNA retrotranscribed from the mRNA from either CLL B
cells (lanes 3 and 4) or control T cells (lane 2) or THP-1 cells (lane
6). PCR amplification of the same region of the Lck coding sequence
from a pGEX-Lck construct is shown (lane 1). Lane 2, molecular-weight
marker. (B) Phosphorimager print of a Southern blot hybridization of
the same samples with 32P-labeled Lck cDNA.
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While the data show that Lck is expressed in B cells from B-CLL
patients, they do not provide any information about Lck activity in
these cells. To understand whether Lck from CLL B cells is functional,
Lck-specific immunoprecipitates were subjected to in vitro kinase
assays using enolase as exogenous substrate. As shown in Fig
3, a significant phosphorylation of enolase
could be detected in in vitro kinase assays of Lck-specific
immunoprecipitates from CLL B cells, showing that Lck expressed in B
cells from B-CLL patients is functional. The levels of Lck activity
correlated with the levels of protein (Fig 3), suggesting that Lck is
not hyperactive in CLL B cells.
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| Fig 3.
Lck is functional in CLL B cells. Quantitation of
32P labeling of enolase by Lck in in vitro kinase assays of
lysates of either normal peripheral blood B cells or B cells from three
representative B-CLL patients (B-CLL), a hairy cell leukemia (HCL)
patient, a patient with non-Hodgkin's lymphoma (NHL) in the leukemic
phase, or normal T cells. Equal amounts of proteins for each lysate
were used for Lck immunoprecipitation. An anti-Lck immunoblot of the same samples is shown below the graph. Each kinase assay was repeated at least twice.
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To understand whether Lck expression is restricted to B-CLL, we tested
different leukemic B cells for expression of Lck. They included B cells
isolated from two patients with HCL and two patients with NHL in the
leukemic phase. As expected, B cells from both types of leukemias
lacked CD5 expression (data not shown). In addition, three tumor lines
derived from different cell types (ie, the monocytic line THP-1, the
neuronal line PC12, and the epidermoid line A431) were analyzed.
Neither THP-1 nor PC12 nor A431 cells expressed Lck (data not shown).
Furthermore, no Lck could be detected in B cells from either HCL or NHL
in the leukemic phase (Fig 3 and data not shown). This result was
confirmed in in vitro kinase assays of Lck-specific immunoprecipitates
(Fig 3). Thus Lck expression is a distinctive feature of CLL B cells.
Expression of Lck in normal CD5+ B-1 cells.
Two alternative explanations could account for the expression of Lck in
B-CLL. One possibility is that Lck is ectopically expressed in B cells
from this type of leukemia but not in normal B cells. In this case Lck
might potentially be involved in the development of B-CLL, as
alterations in the levels of Lck expression, even in the absence of an
activating mutation, have been shown to result in thymic tumorigenesis
in genetically engineered mouse models.16 The other
possibility is that Lck expression is a feature of normal B-1 cells and
that uncontrolled expansion of this B-cell population would account for
the relatively high levels of Lck found in B cells from B-CLL patients.
To address this question we analyzed lysates of normal B cells from
different lymphoid organs characterized by different ratios of
CD5/CD5+ B cells.
B cells were purified from peripheral blood, spleen, adenoid, and cord
blood and the relative numbers of CD5 and
CD5+ B cells were determined by flow cytometry. The mean
proportions of CD5+ B cells in these organs were 11.8%,
17.3%, 28.8%, and 57.2% of total B cells, respectively. Lysates of
unfractionated B cells from these sources were analyzed by immunoblot
for the presence of Lck. The results are presented in Fig
4. No Lck was detectable in B cells
purified either from adult peripheral blood or from spleen, which were
mostly CD5 negative. No induction of Lck expression was observed, even
following activation of peripheral blood B cells by surface IgM
cross-linking (Fig 4). By contrast, B cells from both adenoid and cord
blood expressed relatively high levels of Lck, albeit not comparable to
the levels found in T cells (Fig 4). Thus Lck can be found in B-cell
preparations that include a significant CD5+ B-cell
subpopulation, suggesting that Lck is expressed specifically by
CD5+, B-1 cells. To confirm this point, CD5
and CD5+ cells were separated from purified cord blood B
cells by FACS sorting and tested by immunoblot for the presence of Lck.
As shown in Fig 4, Lck could only be detected in CD5+ B
cells. Thus, Lck is expressed in normal B-1 cells, and its presence in
B cells from B-CLL patients is likely due to the selective expansion of
the B-1 cell subpopulation.
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| Fig 4.
Expression of Lck in normal CD5+ B cells.
Immunoblot analysis using an anti-Lck MoAb of lysates from normal B
cells from different lymphoid organs. On the right is shown a similar
immunoblot of lysates of total cord blood B cells (CB), as well as of
the purified CD5 and CD5+ B-cell
populations from the same cord blood sample. A lysate from normal T
cells is included as a positive control. PB, resting peripheral blood B
cells; PB act, peripheral blood B cells activated by surface IgM
cross-linking; spleen, B cells purified from spleen; adenoid, B-cells
purified from adenoid, CB, cord blood B cells.
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CD5 B-2 cells acquire Lck expression after
transformation by EBV.
Our data show that CD5 B-2 cells, the major subset of
normal B cells, fail to express Lck, even following in vitro activation by surface IgM cross-linking. An interesting question is whether the
gene encoding Lck in B-2 cells is irreversibly switched off or,
conversely, B-2 cells could acquire the capacity to express Lck
ectopically in anomalous settings, ie, following transformation. As
shown in Fig 3, CD5 B cells freshly isolated from two
types of B-cell malignancy, HCL and NHL, lack Lck expression,
suggesting that Lck is not a general marker of transformed B cells. We
then tested chronically activated CD5, EBV-transformed B
cells for Lck expression. Fifteen lymphoblastoid B-cell lines were
derived from B cells of healthy donors by transformation with EBV. Two
B-cell lines derived from Burkitt's lymphoma (Daudi and Raji) and the
EBV+ B-cell line RPMI-1788 were also assessed for Lck
expression. With the exception of two of the EBV-transformed lines, all
other lines were CD5 (data not shown). The results of a
representative immunoblot experiment with anti-Lck MoAb are shown in
Fig 5A. Significant levels of Lck
expression were consistently found in all EBV transformed B-cell lines
derived from healthy donors, as well as in Daudi, Raji, and RPMI-1788
cells, independently of CD5 expression. In all these cell lines, two
Lck immunoreactive species could be detected, with a high ratio of the
species migrating with a slower electrophoretic mobility (Fig 5A). The
identity of Lck was confirmed by RT-PCR on mRNA purified from
EBV-transformed B-cells (data not shown). Thus, while B-1 cells
normally express Lck, B-2 cells ectopically express Lck following EBV
transformation.
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| Fig 5.
Expression of Lck in EBV-transformed B cells. (A)
Immunoblot analysis using an anti-Lck MoAb of lysates from
EBV-transformed lymphoblastoid B-cell lines, or Daudi, Raji, and
RPMI-1788 B-cell lines. A lysate from normal T cells is included as a
positive control, as well as a lysate from normal peripheral blood B
cells as a negative control (PB). (B) Immunoblot analysis using an
anti-Lck MoAb of lysates from either freshly isolated peripheral B
cells from healthy controls (PB1, PB2) or EBV-transformed
lymphoblastoid B-cell lines from the same controls (B-EBV1, B-EBV2). A
lysate from normal T cells is included as a positive control.
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The finding that Lck is present not only in EBV-transformed
lymphoblastoid B-cell lines, but also in Daudi, Raji, and RPMI-1788 cells, which are all positive for EBV nuclear antigens, suggests the
possibility that Lck expression might be induced by B-cell transformation by EBV. To test this hypothesis we probed peripheral blood B cells from healthy donors from which EBV-transformed
lymphoblastoid, Lck positive, B-cell lines were derived. As shown in
Fig 5B, Lck could be detected in B cells from healthy donors only after
EBV transformation, strongly supporting the notion that Lck expression in these B-cell lines is a consequence of EBV transformation.
Lck is functionally coupled to CD5 in CLL B cells.
In T cells, Lck has been shown to bind to the cytoplasmic domain of
CD5.20 Although this interaction is significantly weaker than the one involving CD4, it is sufficient to functionally couple CD5
to Lck, as CD5 triggering results in Lck activation.20 We asked whether CD5 engagement on either CLL B cells or EBV-transformed B-cells could initiate a signaling pathway involving Lck activation. Purified CLL B cells, as well as purified T cells, from three different
patients were triggered with anti-CD5 MoAb. Lck-specific immunoprecipitates from these cells were then analyzed by immunoblot using an anti-phosphotyrosine MoAb. As a positive control, we used the
leukemic T-cell line Jurkat, shown by flow cytometry to express high
levels of CD5 (data not shown). The results obtained with a
representative patient are presented in Fig 6. A
significant induction of tyrosine phosphorylation of Lck in response to
CD5 triggering could be detected in CLL B cells, at levels comparable to those detected in T cells from the same patients. Thus, CD5 is
functionally coupled to Lck in CLL B cells.
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| Fig 6.
Lck is functionally coupled to CD5 in CLL B cells.
Immunoblot analysis using anti-phosphotyrosine MoAb of Lck-specific
immunoprecipitates from lysates of either purified B and T cells from a
representative B-CLL patient or from control Jurkat T cells. The
migration of Lck, as revealed by immunoblot with an anti-Lck MoAb of a
control Jurkat T-cell lysate on the same gel, is indicated.
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DISCUSSION |
Our data shed light on two controversial questions regarding B-cell
biology, on the one hand mature B-cell lineage, and on the other the
identity of the B-cell population, which is the target for the
transforming events underlying B-CLL. The CD5+ B-cell
subset, designated B-1, can be distinguished from conventional B cells
by a number of features such as anatomic localization, functional
characteristics, and gene expression.1-3 In contrast to
conventional B cells, B-1 cells maintain their number by
self-replenishment and exert a feedback regulation on de novo
production of B-1 cells from progenitors. Furthermore, their antibody
repertoire is built on a restricted set of V genes and is highly
enriched in autoreactive antibodies,1-3 suggesting the
potential implication of this B-cell subset in autoimmune diseases.
Strong evidence of the independent origin of B-1 and conventional B
cells from different lineages of progenitor cells has been obtained in
mouse.6,7 In humans, the origin of B-1 cells from a
distinct lineage, rather than from a modification of phenotypical and
functional features of B-2 cells, is still under debate. Taken together
with the recent demonstration of a constitutive STAT3 activity
restricted to B-1 cells,9 our finding of a differential
expression of Lck in B-1 cells, but not conventional B cells, supports
the existence of separate B-cell lineages of progenitor cells in man.
Given the key role of Lck in thymocyte mitogenesis,16,21
this differential expression of Lck might underlie the unique capacity
of self-renewal of B-1 cells.
The second question focuses on the cellular origin of B-CLL. Since this
type of leukemia is characterized by the accumulation of
CD5+ B cells, B-1 cells have been proposed as the normal
counterpart of CLL B cells. A number of similarities between normal
CD5+ B cells and CLL B cells have been highlighted,
particularly concerning their production of polyreactive, low-affinity
autoantibodies in the absence of extensive somatic
hypermutation.10 However, normal and malignant
CD5+ B cells differ significantly in a number of features.
In contrast to normal CD5+ B cells, CLL CD5+ B
cells express very low levels of surface immunoglobulins11 and show a reduced proliferative response to B-cell antigen receptor cross-linking,22 which correlates with a reduced induction
of protein tyrosine kinase activity and a defective mobilization of
intracellular Ca2+.11,13 Furthermore, they
accumulate in the G0 phase of the cell cycle, as documented
both by kinetic studies and by the lack of c-myc
expression.10 The latter property, combined with Bcl-2 overexpression,23,24 is likely to play a major role in the accumulation of CLL B cells. Our finding of Lck expression as a common
feature of both normal CD5+ B cells and CLL B cells
provides strong evidence in favor of B-1 cells being the target of the
transforming events leading to B-CLL. It should be noted that, in
addition to mitogenesis, Lck has been implicated in cell
survival.25 Thus the outcome of Lck expression might be
proliferation or survival, dependent on the different cellular context
of the normal and malignant B-1 cell, respectively. Since CD5 is
functionally coupled to Lck, an important factor in normal and
malignant cell fate determination might be the encounter with B cells
expressing the CD5 counterreceptor CD72.26
Our data suggest that the expression of Lck in CLL B cells and in
EBV-transformed B cells could have two different meanings. Lck
expression in B-CLL could be accounted for by the selective expansion
of malignant B-1 cells and would not be as such directly involved in
neoplastic transformation. By contrast, Lck expression appears to be an
anomalous feature of CD5 negative, B-2 cells, acquired following
transformation by EBV. Although our data do not rule out the
possibility that Lck might be expressed in other types of B-cell
malignancies, the lack of Lck expression in HCL and NHL B cells shows
that Lck is not a common feature of transformed CD5 B
cells. The presence of Lck in EBV-transformed B cells, but not in
normal peripheral B cells of the same donor, suggests that EBV nuclear
antigens, which have been shown to interfere with normal gene
expression,27-29 might contribute to the activation of the
Lck gene promoter, resulting in ectopic Lck expression in
CD5 B cells. As opposed to B-CLL, this event might play
a role in B-cell transformation by EBV. The crucial role of Lck both in thymocyte development and in T-cell activation has been clearly demonstrated.21,30 The levels of Lck expression in T cells must be strictly controlled, as shown by the dramatic alterations associated with Lck overexpression. Even in the absence of an oncogenic
mutation, Lck overexpression in the thymus results in thymic
tumorigenesis.16 Furthermore, the gene encoding Lck was first cloned from the mouse thymoma line LSTRA, which is characterized by abnormally high levels of wild-type Lck.31 Thus, an
alteration in the protein tyrosine kinase balance produced by the
ectopic expression of Lck is likely to have profound effects on the
proliferative potential of the B cell.
| |
FOOTNOTES |
Submitted June 20, 1997;
accepted December 22, 1997.
Supported by the Italian Association for Cancer Research (AIRC). The
contribution of the Regione Toscana (Ricerca Sanitaria Finalizzata) and
the MURST (quota 60%) is also acknowledged. M.B.M. is the recipient of
an FIRC fellowship.
Address reprint requests to Cosima T. Baldari, PhD,
Department of Evolutionary Biology, University of Siena, Via Mattioli 4, 53100 Siena, Italy.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Sonia Grassini for skilled technical assistance and
Leonardo Gamberucci for photographic assistance. They also thank B. Perussia for the generous gift of the B36.1 hybridoma, M. Ferrarini and
P. Ghiara for their kind gift of cell lines, and S. Plyte for critical
reading of the manuscript.
| |
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Abstract
Introduction
Abstract