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Blood, 1 November 2002, Vol. 100, No. 9, pp. 3068-3076
PLENARY PAPER
The alternative transcript of CD79b is overexpressed in
B-CLL and inhibits signaling for apoptosis
Mark S. Cragg,
H. T. Claude Chan,
Mathew D. Fox,
Alison Tutt,
Aimée Smith,
David G. Oscier,
Terry J. Hamblin, and
Martin J. Glennie
From the Tenovus Research Laboratory, Cancer Sciences
Division, School of Medicine, University of Southampton, United
Kingdom; and the Department of Haematology, Royal Bournemouth Hospital,
Bournemouth, United Kingdom.
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Abstract |
The B-cell receptor (BCR) for antigen is composed of surface
immunoglobulin (sIg), which provides antigen specificity, and a
noncovalently associated signaling unit, the CD79a/b heterodimer. Defects in CD79 can influence both BCR expression and signaling and may
explain why cells from certain malignancies, such as
B-chronic lymphocytic leukemia (B-CLL), often express
diminished and inactive BCR. Recently, an alternative transcript of
CD79b ( CD79b) has been reported that is up-regulated in B-CLL and
may explain this diminished BCR expression. Here we assess the
expression of CD79b in B-CLL and other lymphoid malignancies and
investigate its function. High relative expression of CD79b was
confirmed in most cases of B-CLL and found in 6 of 6 cases of splenic
lymphomas with villous lymphocytes (SLVLs) and hairy cell leukemia. In
a range of Burkitt lymphoma cell lines, expression of CD79b was
relatively low but correlated inversely with the ability of the BCR to
signal apoptosis when cross-linked by antibody (Ab).
Interestingly, when Ramos-EHRB cells, which express low CD79b, were
transfected with this transcript, they were transformed from being
sensitive to anti-Fcµ-induced apoptosis to being highly resistant.
Although CD79b was expressed as protein, its overexpression did not
reduce the level of cell surface BCR. Finally, we showed that the
inhibitory activity of CD79b depended on an intact leader sequence
to ensure endoplasmic reticulum (ER) trafficking and a
functional signaling immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic tail. These results point to
CD79b being a powerful modulator of BCR signaling that may play an
important role in normal and malignant B cells.
(Blood. 2002;100:3068-3076)
© 2002 by The American Society of Hematology.
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Introduction |
The CD79a/b heterodimer is critical to the
structure and function of the B-cell receptor (BCR), being necessary
for both its signaling capacity1-5 to regulate processes
such as allelic exclusion, proliferation, differentiation, anergy, and
apoptosis6-8 and for the transport of the complete
receptor to the cell surface.9 The individual a and b
chains of CD79 are coded by the mb-110 and
B2911 genes, respectively, and are
linked to each other by a disulfide bridge in the extracellular portion
of the complex, just adjacent to its immunoglobulin (Ig)-like
domains.1 Abnormalities in the BCR have often been
associated with cells in certain neoplastic diseases, particularly
B-chronic lymphocytic leukemia (B-CLL),12 which is
characterized by the progressive accumulation of circulating monoclonal
B cells, which tend to be CD5+ and to express low levels of
surface BCR.13 Furthermore, recently it has been found
that these tumors can be subdivided into 2 distinct groups depending on
whether the Ig V regions are mutated or not, with the unmutated group
being associated with a more aggressive type of disease.14
The low level of surface Ig may explain the reduced ability of cells
from many cases of B-CLL to capture, present, and respond to
antigen.15 Defective BCR signaling has also been
associated with low levels of CD38 on subsets of B-CLL cells.16 Interestingly, defects in the BCR of B-CLL have
now been attributed to functional deficiency in the CD79 heterodimer, especially the CD79b, which is expressed at low levels on these tumors.17 Among the mechanisms proposed to explain these
observations are reduced expression of CD79b mRNA,18
somatic mutation of the B29 gene18 or,
most recently, through alternative splicing of CD79b to yield
CD79b.19
Alternative gene splicing is emerging as an increasingly important
mechanism for regulating gene expression, whereby a single pre-mRNA
gives rise to different mature mRNA species by altering which exons are
spliced together and in what order. Alternatively spliced forms of both
CD79 a and b genes have been described in both normal and malignant B
cells.20,21 The mb-1 gene encodes for a
variant that is truncated by 110 extracellular base pairs as a result
of splicing at cryptic sites in the gene, while the alternative
transcript of the B29 gene is spliced at
conventional sites, which removes the entire exon 3 and results in a
 chain lacking the extracellular, Ig-like, domain.20 In
both alternative transcripts, the Cys residues necessary to form the
disulfide bridge between CD79a and CD79b are lost. As such, these
variants, if translated, would not be expected to form stable
heterodimers at the cell surface.
Although described some years ago, the importance or possible function
of CD79a and CD79b has not been fully addressed. However,
recently, Alfarano et al19 demonstrated that B-CLL cells
consistently show elevated levels of the mRNA for CD79b, indicating
a possible physiological role. For this reason we set out to explore
the role of CD79b more fully and to determine if CD79b influences
the activity of the BCR in neoplastic B cells.
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Materials and methods |
Tumor samples and cell lines
Tumors were classified according to the standard World Health
Organization (WHO) clinical criteria and included 16 B-CLL, 3 follicle center cell lymphomas (FCLs), 8 diffuse large cell lymphomas
(DLCLs), 3 splenic lymphomas with villous lymphocyte (SLVL) samples, 3 hairy cell leukemias (HCLs), and 5 myelomas. Disease status was
not used as a criterion in selecting patients. Peripheral blood
lymphocyte (PBL) samples were isolated from the venous blood of
patients or healthy volunteers on a Hypaque density gradient
(Lymphoprep; Nicomed, Invitrogen Life Technologies, Paisley, United
Kingdom) before washing in phosphate-buffered saline (PBS). DLCLs were taken from lymph node sections. Nodes were excised and RNA
isolated and converted to cDNA as detailed below. Normal cell
contamination, assessed by V region analysis, did not usually exceed
20% in these DLCL samples.
Daudi, Ramos, Ramos-EHRB, Raji, and Namalwa cells were obtained from
the European Collection of Cell Cultures (ECACC, Salisbury, United
Kingdom). K-562 and COS-7 cells were kind gifts from Dr A. Al-Shamkani
(Cancer Sciences Division, University of Southampton, United
Kingdom). All cell lines were maintained in supplemented RPMI
1640 (RPMI 1640 medium containing glutamine [2 mM], pyruvate [1
mM], penicillin and streptomycin [100 IU/mL], amphotericin B [2 µg/mL], and 10% fetal calf serum [FCS;
Myoclone]; all supplied by Gibco, Paisley, United
Kingdom) at 37°C in a 5% CO2 humidified incubator. Cells
used for apoptosis studies were maintained in log phase of growth for
24 hours prior to experiments.
Antibody preparation
All hybridoma lines secreting monoclonal antibody (mAb)
were expanded in tissue culture and purified from culture supernatant using a protein A column.22 Monoclonal Ab used in the
study included CP1/17 (irrelevant, IgG1), M15/8 (anti-µ chain), ZL7/4 (anti-CD79a), ZL9/1 and ZL9/2 (anti-CD79b), and AT80 (anti-CD20), which
were all raised in this laboratory and have been reported previously.23,24 AT105/1 is a murine mAb recently raised
in this laboratory to a CD79b peptide-KLH construct. CB3-1 and SN8 (anti-CD79b) were purchased from BD Pharmingen (Cowley, United Kingdom)
and Ancell (Bayport, MN), respectively. Binding of anti-CD79 mAb to
CD79 Fc fusion protein was tested by a standard "capture" enzyme-linked immunosorbent assay (ELISA) on plates (Maxisorb; Nunclon, Invitrogen Life Technologies) coated with rabbit antihuman Fc
(2.5 µg/mL) as first layer followed by the fusion proteins as a
second layer.
Isolation of RNA and conversion to cDNA
Total RNA and mRNA were isolated using the Puregene (Gentra
Systems, Minneapolis, MN) total RNA and Microquickprep mRNA
(Amersham Pharmacia Biotech UK, Little Chalfont, United Kingdom) kits,
respectively. RNA was converted to cDNA using the first-strand cDNA
synthesis kit (Amersham Pharmacia Biotech United Kingdom) according to
the manufacturer's instructions.
PCR
Polymerase chain reaction (PCR) was performed in
thin-walled PCR tubes with approximately 100 ng cDNA, 100 ng 5' and 3'
primers, 1 unit DNA polymerase, in the presence of deoxyribonucleoside triphosphates (dNTPs), and 1 × reaction buffer (all from
Promega, Southampton, United Kingdom). PCR reactions carried out to
generate chimeric CD79b constructs detailed below or to
sequence CD79b genes from tumor samples were performed with Pfu
polymerase. Reverse transcriptase (RT)-PCR was performed
using Taq polymerase. DNA was first denatured at 95°C for 5 minutes, followed by 25 to 30 amplification cycles. Annealing
temperatures for each set of primers used are shown in Table
1. PCR products were analyzed by
electrophoresis on 1.5% to 2% agarose gels and visualized under UV
light after staining with ethidium bromide.
Construction of CD79 chimeric molecules
Construction of the CD79b-Fc fusion protein, displaying the
extracellular domain of CD79b, was reported previously.23
Essentially the same technique was applied to yield an Fc fusion
protein with the extracellular region of CD79b. Briefly, the
extracellular domain of CD79 or CD79b was PCR amplified from
Ramos-EHRB cells using primers c) and d), which incorporate a
splice donor site (ACAGGTAAGT) at the 3' end. The products were cloned
into pGEM-T vector (Promega), sequenced, and subcloned into the pIG1
vector, which contains the genomic Fc region (hinge, CH2, and CH3) of human IgG1. These, and all other constructs, with the exception of the
yellow fluorescent protein (YFP) chimeras, were further subcloned into
pcDNA3 (Invitrogen Life Technologies) for expression.
The full-length CD79b molecule retaining the CD79b leader sequence
was amplified from Ramos-EHRB cDNA using primers g) and h).
The same primers were used to amplify the full-length CD79b molecule. A
CD79b molecule lacking the CD79b leader sequence was constructed
using primers i) and h) and subcloned into the pHA-CMV vector
(BD Clontech United Kingdom, Basingstoke, United Kingdom). This
construct, hereafter referred to as leaderless CD79b, possesses an
additional 30-bp sequence encoding for a 10 amino acid-hemagglutinin
(HA) tag at the end of the extracellular domain, which facilitates
detection of the transfected construct above endogenous levels of
CD79b. Rat CD4 leader sequence (CD4L) was added to the leaderless
CD79b molecule using a 2-step recombinant PCR strategy. In the first
step, leaderless CD79b and rat CD4L constructs with overlapping
regions were amplified using primer pairs i) and h) and j) and
k); 100 ng of each amplified DNA was then used in the second
step. This second reaction was initially carried out for 15 cycles
without end primers i) and k), which were then added, and a
further 15 cycles were performed. This construct was ligated directly
into the PCR-Blunt II-TOPO vector (Invitrogen Life
Technologies) before subcloning into pcDNA3. A CD4L-CD79b molecule with
Tyr207 of the CD79b immunoreceptor tyrosine-based activation motif
(ITAM) mutated to Ala (Tyr207-Ala207) was created in a similar
fashion using primers j) and l) and m) and h) in the first step and
primers j) and h) in the second. To construct yellow
fluorescent protein (YFP) CD79 chimeric molecules, full-length and
truncated CD79 transcripts were amplified from Ramos-EHRB cDNA using
primers h) and n), prior to subcloning into the pEYFP-N1
vector (BD Clontech United Kingdom).
Transfection
Transfection of Ramos-EHRB and K-562 cell lines was achieved via
electroporation using a Gene Pulser (Bio-Rad, Hemel Hempstead, United
Kingdom), with voltage and capacitance settings of 0.3 to 0.32 mV and
960 microfarads (µF), respectively. Transfection of COS-7 cells was
performed in chamber slides using a standard diethylaminoethyl
(DEAE) dextran method.25 For stable expression, cells were
seeded onto 96-well plates and subjected to selection with geneticin
(1-2 mg/mL) 24 to 48 hours later. Expression of the relevant CD79b
transcript was determined by RT-PCR. YFP transfectants were screened by
fluorescence-activated cell sorter (FACS) analysis and
positive clones sorted using a FACS Vantage cell sorter (BD Pharmingen).
SDS-PAGE and Western blotting
Whole cell lysates were prepared from 5 × 106 to
10 × 106 cells in lysis solution containing 1% Nonidet
P-40 (NP-40), 150 mM NaCl, 10 mM Tris (tris(hydroxymethyl)aminomethane)
HCl, 2.5 mM EDTA (ethylenediaminetetraacetic acid), 1 mM
phenylmethylsulfonyl fluoride (PMSF), 2.5 mM iodoacetic acid, and 1 mg/mL aprotinin. Insoluble material was removed by
centrifugation at 15 000 rpm in a Kendro microcentrifuge for 15 minutes at 4°C. Samples were then diluted 1:3 in sample buffer and
heated at 100°C for 3 minutes prior to loading. For Western blotting,
proteins were transferred immediately onto nitrocellulose paper
(Hybond; Amersham Pharmacia Biotech United Kingdom) using a semidry
transfer system (TE 22 system; Hoeffer, Amersham Pharmacia Biotech
United Kingdom). The blot was blocked overnight with PBS/10% bovine
serum albumin (BSA) buffer and then incubated with the desired
primary Ab (1-5 µg/mL) in PBS/10% BSA containing 0.1% Tween 20 at
room temperature for 1 to 2 hours. Bound mAb was detected using
F(ab')2 rabbit antimouse horseradish peroxidase (HRP) for
60 to 90 minutes and enhanced chemiluminescence (ECL) reagents
(Amersham Pharmacia Biotech United Kingdom) before exposure to
light-sensitive film (Hyperfilm ECL, Amersham Pharmacia Biotech
United Kingdom).
In vitro translation
In vitro translation was performed using the coupled
transcription translation TnT rabbit reticulocyte system
(Promega). Briefly, the alternative transcripts of CD79 were cloned
into pcDNA3 as detailed above and then retranscribed into mRNA by
reverse transcriptase and translated into protein using rabbit
reticulocytes in the presence of [3H]Leu.
[3H]Leu was incorporated as the labeled amino acid due to
low numbers of Met and Lys in CD79b. Transcription/translation of
luciferase cDNA was used as a positive control. Following translation,
the proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Translated protein was
detected by fluorography using the Amplify (Amersham Pharmacia Biotech United Kingdom) reagent and exposing the gel to light-sensitive film
(Hyperfilm; Amersham Pharmacia Biotech United Kingdom).
Flow cytometry
Flow cytometry was performed on a FACScan cytometer (BD
Pharmingen) equipped with a 488-nm argon ion laser. Data were collected using CellQuest or Consort 30 software and analyzed by Lysis II or
CellQuest (BD Pharmingen). Cell debris was excluded using the forward
scatter (FSC) threshold, and at least 7500 events were collected
per sample.
Measurement of surface antigens by flow cytometry has been reported
previously.23 Briefly, cells were incubated with the fluorescein isothiocyanate (FITC)-conjugated (direct) or
unlabeled (indirect) mAb of choice (50 µg/mL final concentration)
and, in the case of indirect immunofluorescence, detected with an
appropriate FITC-conjugated secondary Ab before washing and analyzing.
Fluorescence intensities were assessed in comparison to that given by
an isotype-matched control Ab and expressed as histograms of
fluorescence intensity versus cell number.
Detection of apoptosis
Apoptosis was detected and quantified using 3 different methods.
Routine assessment of apoptotic cell death was performed by flow
cytometry using a method modified from Dive et al26 based
on the observation that apoptotic cells have lower FSC and higher side
scatter (SSC) properties compared with viable cells. Alternatively,
cells were stained with annexin V-FITC (BD Pharmingen) and 10 µg/mL
propidium iodide (PI) and assessed by flow cytometry, as
detailed by Vermes et al.27 The percentage of annexin
V-positive cells were scored as apoptotic. In addition, cell samples
were assessed for apoptosis on a basis of DNA fragmentation,
essentially according to the method of Nicoletti et al28
as detailed previously.29
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Results |
Expression of alternative transcripts of CD79 in B-CLL and other
B-cell malignancies
In our initial work we wished to confirm whether the alternative
transcripts of CD79a and CD79b (CD79a and CD79b) were present in
B-CLL. Using a semiquantitative RT-PCR, the CD79b but not the
CD79a was present at high levels in these cells (Figure
1A). Full-length CD79a and CD79b
transcripts were also present in all samples, although in some cases
for CD79b only at low levels (Figure 1A, lanes 3, 10, 13, 14). Although
PCR techniques are not quantitative, simultaneous amplification of 2 different transcripts using the same primers, in the same reaction, as
was done here, does provide a good estimate of the relative frequency
of each species. In most cases of CLL assessed, the level of CD79b
was high relative to the full-length CD79b transcript (0.89 ± 0.36)
and elevated in comparison with the relative level seen in normal
peripheral blood samples (0.20 ± 0.17) as suggested
previously.19
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| Figure 1.
Messenger RNA expression of alternative transcripts of
CD79, CD79a and CD79b.
Shown are DNA fragments resulting from semiquantitative RT-PCR of RNA
isolated from malignant B cells with primers specific for CD79a, CD79b,
or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 1).
Messenger RNA was first isolated from samples and then reverse
transcribed to yield cDNA for PCR. Primers for GAPDH were used to
verify integrity and quantity of cDNA. (A) RT-PCR from 16 B-CLL
samples. (B) RT-PCR from a selection of other B-cell malignancies:
follicle center cell lymphoma (FCL) (lanes 1-3); diffuse large B-cell
lymphoma (DLCL) (lanes 4-11); myeloma (lanes 12-16); splenic lymphoma
with villous lymphocytes (SLVL) (lanes 17-19); and hairy cell leukemia
(HCL) (lanes 20-22). The ratios in each panel show the relative level
of CD79b/CD79b.
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The level of CD79b in other B-cell malignances was generally lower
than that seen in the B-CLL cases (0.19 ± 0.11 for FCL and
0.33 ± 0.3 for DLCL), and in several individuals no alternative transcript could be detected (Figure 1B). For example, lanes 1, 4, 7, and 11 in the FCL and DLCL cases showed no evidence of the CD79b
transcript, and in 4 of 5 myeloma samples tested, neither the full nor
the truncated transcript of CD79b could be detected. However, in 3 of 3 cases of SLVL and 3 of 3 cases of hairy cell lymphoma (Figure 1B),
CD79b was also highly expressed (0.6 ± 0.15 for SLVL and
0.92 ± 0.27 for HCL), demonstrating that up-regulation of the
variant transcript is not restricted to B-CLL.
Correlation of CD79b expression and decreased sensitivity
to BCR-induced apoptosis
Having confirmed that the CD79b was present in B-CLL and having
found high levels in certain other B-cell tumors, we next considered
what function this product might have in malignant B cells. In our
previous work, we described a range of Burkitt lymphoma cell lines that
differed in their susceptibility to growth inhibition and apoptosis
after BCR ligation with mAb.29 Therefore, we decided to
assess whether the level of the CD79b transcript differed in these
lines using the RT-PCR approach (Figure
2A). The results showed a spectrum of
expression, from very low levels of CD79b in Ramos-EHRB cells to
relatively high levels, compared with the full-length transcript, in
Raji and Namalwa. The relative levels of the CD79b transcript in
these latter 2 lines was analogous to that seen in B-CLL and in all 3 samples from SLVL patients. Figure 2B illustrates the surface
expression levels of CD79a, CD79b, and suface Ig (sIg) in EHRB, Daudi,
Raji and Namalwa cells, indicating that levels of CD79b message do not
directly correlate with surface expression of CD79a, CD79b, and sIg.
Intriguingly, those Burkitt cell lines that expressed high levels of
the alternative transcript were also the same lines that were most
refractive to the apoptosis induced through ligation of their BCR with
anti-Fcµ mAb (Figure 2C). Conversely, cell lines that were sensitive
to apoptosis displayed low levels of the alternative transcript. Figure
2D shows the clear inverse relationship between CD79b expression and
insensitivity to anti-µ-induced apoptosis. Such data suggested that
CD79b transcript might protect cells against apoptosis signaled via
the BCR.
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| Figure 2.
Correlation of expression of CD79b and sensitivity to
anti-Fcµ-induced apoptosis of Burkitt lymphoma cell lines.
(A) Level of CD79b transcript in Burkitt cell lines. To assess the
level of CD79b in a range of Burkitt lymphoma cell lines, RT-PCR
analysis was performed using the specific primers for CD79b as detailed
in Figure 1. Samples assessed were Ramos-EHRB (E), Ramos (R), Daudi
(D), Raji (Rj), and Namalwa (N). For comparison, the PCR products from
an example of a B-CLL (C) and an SLVL (S) are also shown. The ratios in
each panel show the relative level of CD79b/CD79b. (B) The levels of
surface BCR on EHRB (tightly grouped dotted outline [...]),
Namalwa (spaced dotted outline [. . .]), Raji (solid histogram), and
Daudi (light gray solid outline) cells as measured by flow cytometry.
The cell lines were stained with FITC-conjugated mAb to CD79a (ZL7-4),
CD79b (ZL9-1), and sIg (M15/8). (C) Induction of apoptosis with
anti-Fcµ mAb. Namalwa, Daudi, and EHRB Burkitt cell lines were
assessed for their sensitivity to anti-µ-induced apoptosis by
exposure to 10 µg/mL anti-µ mAb for 24 hours. Apoptosis was
assessed by flow cytometry using annexin V-FITC and PI. (D)
Correlation of CD79b expression and sensitivity to anti-Fcµ mAb.
The ratio of CD79b/CD79b is taken from panel A, and the sensitivity
to apoptosis is taken from the same experiments as those shown in
panel C.
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To test this hypothesis, we next cloned and overexpressed CD79b in
Ramos-EHRB, the cell line that was most sensitive to
anti-Fcµ-induced apoptosis. As can be seen in Figure
3A, overexpression of the CD79b gene
rendered certain clones for example, 4 and 12resistant to this form
of apoptosis. When the mRNA was assessed in these clones, inhibition of
apoptosis correlated closely with the level of CD79b transcript
(Figure 3B). For example, clones 4 and 12, which express relatively
high levels of CD79b, were resistant to apoptosis, while clones 9 and 23, which were transfected in the same way but did not show
overexpression of the gene, were still sensitive to anti-µ mAb.
Resistance to apoptosis induced by anti-µ mAb was not simply due to
down-regulation of the BCR, because the levels of surface Ig, CD79a,
and CD79b on all the transfected clones remained unchanged (Figure 3C).
Similarly, the rate and extent of internalization in these clones when
these molecules were cross-linked by the appropriate mAb were
indistinguishable from that seen in the wild-type EHRB cells (data not
shown). These data are important because they demonstrate that the
overexpression of the CD79b transcript relative to the full-length
CD79b molecule per se did not cause changes in the surface level of the
BCR or alter the way in which it responded to being cross-linked by
mAb.
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| Figure 3.
Sensitivity of Ramos-EHRB clones to anti-Fcµ mAb following
transfection with CD79b.
(A) Untreated cells (NT) and 4 CD79b-transfected clones of
Ramos-EHRB were cultured (5 × 105/mL) in the presence of
anti-Fcµ mAb for 24 hours before assessing apoptosis via flow
cytometry using the annexin V-FITC and PI as detailed in Figure 2.
Apoptosis was also verified using the hypo-PI method to detect
fragmented DNA (data not shown). The histogram shows average results,
± SD, for 3 experiments on each clone. (B) The expression of CD79b
and CD79b in the nontransfected (NT) and transfected clones, 4, 9, 12, 23, was confirmed by RT-PCR as detailed in Figure 1. Ratios show
the relative level of CD79b/CD79b. GAPDH message was also determined
as a control to ensure that equal amounts of DNA were present in each
sample. (C) The levels of surface BCR on wild-type and
CD79b-transfected Ramos-EHRB clones as measured by flow cytometry.
The cells were stained with FITC-conjugated mAb to CD79a (ZL7-4), CD79b
(ZL9-1), sIg (M15/8), and an irrelevant control mAb (Irr, CP1/17). No
difference was seen in the various clones compared with untransfected
cells.
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Protein expression of the alternative transcripts of CD79
Next, we wished to ascertain whether protein could be translated
from the alternative transcripts of CD79. Initially, we performed in
vitro coupled transcription-translation assays on both CD79a and
CD79b expressed in pcDNA3. The alternative transcripts were both
well translated and produced proteins of the correct molecular weight
(Figure 4A). A fusion protein approach
was also undertaken using YFP as a reporter partner, wherein YFP was
joined to the ends of the cytoplasmic domains of both the full-length
and the alternative transcripts of CD79b (see "Materials and
methods"). The YFP fusion constructs were transfected into various
cell lines and expression assessed either by fluorescence microscopy or
flow cytometry. Expression of both products was observed in COS-7, J558L, and K562 cells, and also in Ramos-EHRB B cells (data not shown).
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| Figure 4.
Protein expression of alternative transcripts of CD79.
(A) The results obtained when the alternative transcripts of CD79 were
cloned into pcDNA3 and translated into protein using a coupled
transcription translation system (see "Materials and methods").
Proteins were resolved on a 15% SDS-PAGE gel and detected by
fluorography. The positive control used was a vector coding for the
luciferase protein. (B) An ELISA using a panel of anti-CD79b mAbs was
used to assess binding to the full-length (full-CD79b-Fc ) or
alternately spliced (CD79-Fc) extracellular domain of
CD79b. (C) COS-7 cells were transiently transfected with
full-length or truncated CD79b and then harvested 24 to 48 hours later
for blotting with different anti-CD79b mAbs and detection with ECL
reagents. All mAbs show specific reactivity with both the full and
truncated forms of CD79b, shown as bands of 33 and 17.5 kDa,
respectively. "Vector" indicates cells transfected with
empty pcDNA3 plasmid, and "M" indicates the molecular weight
markers. (D) Expression of CD79b in Raji, Daudi, and EHRB B-cell
lines and the EHRB transfectant clones 9 and 12 detailed in Figure 2.
Expression was determined following Western blotting with anti-CD79b
mAb AT105/1.
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In an attempt to address directly whether the native CD79b
transcript is expressed as a protein, we considered a Western blot
approach. First we analyzed a panel of 4 anti-CD79b mAbs for their
ability to bind to fusion proteins of Fc linked to the extracellular
regions of either the full-length or the truncated CD79b. A
"capture" ELISA was used for this investigation (Figure 4B). First
a polyclonal anti-Fc Ab was used to capture the Fc domain of the
fusion proteins and then the various anti-CD79b mAb used to detect
either of the products in a standard format. As expected, all
anti-CD79b mAbs reacted with the Fc fusion protein displaying the
extracellular domain of the full-length CD79b molecule (Figure 4b).
Surprisingly, however, these mAbs also bound to the Fc-CD79b
molecule. Thus, despite having only 25 amino acids remaining as an
extracellular domain, CD79b is recognized by all the mAbs tested. We
next assessed these anti-CD79b mAbs in Western blots with cell lysates
from COS-7 cells transfected with either full-length CD79b, CD79b,
or empty vector. The data show (Figure 4C) that all mAbs tested reacted
with both the full-length and truncated forms of CD79b and confirm a
protein product from the CD79b mRNA transcript. Next, we wished to
address whether the alternative transcript was present in our various
cell lines and EHRB transfectants. Figure 4D demonstrates that the
expression of the CD79b protein corresponds well with the expression
of the CD79b mRNA seen in Figure 2A-B, such that Raji cells
expressed high levels of CD79b protein and EHRB cells very little,
with Daudi cells intermediate. EHRB transfectant clone 12 showed
elevated levels of CD79b protein compared with mock transfectant
clone 9, as expected. Interestingly, additional bands were seen on the Western blots in these B-cell lines, which may correspond to other, posttranslationally modified, forms of CD79b not produced in COS-7 cells.
Functional importance of the leader sequence and the ITAM
of CD79b
We postulated that, because the apoptosis induced via the BCR
requires the signaling activity of the CD79 heterodimer,7 the CD79b transcript might inhibit this process by interfering with
a signaling pathway from the BCR. Furthermore, we reasoned that the
CD79b molecule might perform this function by competing for vital
signaling adaptor molecules at the plasma membrane. Therefore, we
undertook a mutation strategy to probe the CD79b, first to determine
if a leader sequence was required to traffic the inhibitory CD79b
into the ER and, second, to see if, like the full-length transcript, it
utilizes the ITAM.
The results in Figure 5 show again that
the alternative transcript inhibits anti-Fcµ-induced apoptosis
(compare the first and second pair of bars) and that this inhibitory
activity is completely lost once the leader sequence had been removed.
Thus, clones transfected with an empty vector (Figure 5; first pair of
bars) and cells transfected with a leaderless CD79b (Figure 5; third
pair of bars) are equally sensitive to anti-Fcµ mAb despite the
transfected gene being readily detectable by RT-PCR (data not shown).
Interestingly, when we used a construct in which the leader sequence of
rat CD4 had been added back to the leaderless CD79b, the inhibitory
activity was fully restored (Figure 5; fourth pair of bars). To assess
whether the ITAM of CD79b might be involved in the inhibition, a
mutation of the second Tyr in the ITAM (Tyr207-Ala207), known to ablate
signaling function,5 was performed. As shown in Figure 5
(last pair of bars), loss of ITAM function rendered the construct
unable to inhibit apoptosis triggered by anti-Fcµ mAb.
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| Figure 5.
Functional analysis of CD79b.
Constructs encoding the wild-type CD79b, a leaderless CD79b, a
CD79b construct in which the endogenous leader was replaced with
that from rat CD4 (CD4L), and the CD4LCD79b possessing a mutated
ITAM (CD4LCD79b [Y207A]) were transfected into Ramos-EHRB
cells and then positive clones assessed for sensitivity to
anti-µ-induced apoptosis after 48 hours by annexin V-FITC/PI
analysis. Two clones from each construct are shown. The average of 3 experiments, ± SD, are shown for each clone. Underneath, expression
levels of CD79a, CD79b, and sIg are shown for each clone, determined as
indicated in Figure 2, illustrating that all clones expressed similar
levels of all BCR components at the cell surface.
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Specificity of apoptotic inhibition
Lastly, we wished to address whether the inhibitory properties of
CD79b were specific for the BCR apoptosis pathway. Therefore, we
stimulated our various cell lines and transfectant EHRB cells with
anti-CD20 mAb to investigate whether this pathway was also blocked by
overexpression of CD79b. As shown in Figure
6, Raji, Daudi, and EHRB cells seemed
similarly sensitive to anti-CD20 apoptosis, although they are
differently susceptible to BCR apoptosis and express different levels
of CD79b. CD79b overexpressing EHRB clones 4 and 12 were slightly
less sensitive to anti-CD20 apoptosis compared with the mock
transfectant clones 9 and 23. However, when the level of surface
expression of CD20 was assessed, it was apparent that the resistant
clones expressed less CD20 on their surface, possibly accounting for
this difference in sensitivity.
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| Figure 6.
Sensitivity of Ramos-EHRB clones to anti-CD20 mAb
following transfection with CD79b.
Four CD79b-transfected clones of Ramos-EHRB were cultured
(5 × 105/mL) in the presence of anti-CD20 mAb for 48 hours before assessing apoptosis via flow cytometry using the annexin
V-FITC and PI assay as detailed in Figure 2. Underneath, the lower
panel shows the levels of surface CD20 on wild-type and
CD79b-transfected Ramos-EHRB cells as measured by flow cytometry.
The 4 clones, 4, 9, 12, and 23, were stained with FITC-conjugated mAb
to CD20 (AT80), washed, and then assessed by flow cytometry. The solid
histogram represents cells stained with an irrelevant control mAb
(CP1/17). The 2 least intense FACS profiles represent clones 4 and
12.
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Discussion |
B-CLL is a malignancy characterized by a low expression of sIg,
diminished response to antigen, poor antigen capture and presentation, and defective apoptosis (reviewed by Rozman and
Montserrat,12 Hamblin and Oscier,13 and
Alfarano et al19). This intriguing series of related
aberrations has led workers to speculate that B-CLL possesses
deficiencies in BCR function and recently that these may be due to
defects in CD79b gene expression. Alfarano et al19 have
suggested that increased alternative splicing of the CD79b mRNA is the
cause of the diminished surface expression and function of the BCR,
while in contrast Thompson et al18 have attributed it to
reduced levels of CD79b mRNA or to mutations in CD79b. In contrast to
other reports,19-21 the latter authors found no evidence
for CD79b in B-CLL or in normal PBLs, but this may reflect the
forward primer positioning used in this study.
Here we found relatively high levels of the alternatively spliced
variant of CD79b, CD79b, in most B-CLL cells. Only 4 cases (25%)
showed somewhat lower levels, which were more typical of the pattern
found in PBLs. These results are consistent with those reported by
Alfarano et al19 showing that most cases of B-CLL overexpress CD79b. Interestingly, in addition to CD79b, all cases
of B-CLL were found to express the full-length CD79b transcripts. This
agrees with the results of Alfarano et al,19 Verschuren et
al,30 and Rassenti and Kipps31 but disagrees
with Thompson et al,18 who found B29 message undetectable
in a number of B-CLL samples using Northern blotting or RNase
protection assays. We have no clear explanation for such discrepancies,
but they may reflect either the different detection techniques used or
the fact that in our study mRNA was isolated from fresh cells and immediately converted to cDNA, whereas samples assessed by Thompson were from total RNA isolated from cells stored at  70°C, as we have
observed degradation of CD79b message levels in CLL cells stored at
80°C (data not shown). We did not assess the CD79b base
sequences of any of the samples to check for mutations. Others have
found that, in B-CLL samples with normal or at least detectable surface
expression of CD79b, mutations are present within the B29 gene in one
or both alleles18 and have demonstrated that some of these
mutations could severely affect BCR expression and/or function.32
Three other B-cell malignancies, FCL, DLCL, and myeloma, did not
express high levels of CD79b. Four of 5 myeloma samples did not
express readily detectable levels of any CD79b transcripts, in
agreement with the notion that CD79b gene expression becomes down-regulated in plasma cells.33 Conversely, all SLVL and
HCL samples expressed high levels of the CD79b transcript, as did other non-Hodgkin lymphoma samples (data not shown). Clearly, therefore, overexpression is not confined to B-CLL. Because
up-regulation of CD79b is known to occur when normal B cells are
activated with stimuli such as interleukin-4 (IL-4), lipopolysaccharide (LPS), and anti-IgM,21 it is perhaps not
surprising to also find it in a range of malignant B cells. One
interpretation of such results is that overexpression of CD79b
relates more to the activation status of a given neoplasm than to its causation.
Our most unexpected observation was that the level of CD79b in a
range of Burkitt lymphoma lines correlated inversely with the
sensitivity of these cells to anti-Fcµ-induced apoptosis. These
cells all express the BCR, albeit at variable levels, and are, with the
exception of the Ramos cell line, Epstein-Barr virus (EBV)
positive29 (data not shown). By way of confirming
the importance of CD79b to the apoptosis triggered via the BCR, we transfected a highly sensitive cell line, Ramos-EHRB, with a vector carrying CD79b. A range of clones (2 examples shown in Figure 3)
confirmed a clear relationship between expression of this transcript and readiness to undergo apoptosis when engaged by anti-BCR mAb but not
anti-CD20 mAb. These results gave a strong indication that the product
of the CD79b mRNA interferes specifically with signaling from the
BCR and inhibits the readiness of the cells to undergo programmed cell death.
Various assays were undertaken to show that the CD79b transcript was
translated into protein. In vitro transcription-translation assays
revealed that the alternative transcripts of both CD79a and CD79b
produced proteins of about 25 and 17.5 kDa, respectively, in close
agreement with that predicted from the gene sequences.20 Furthermore, transfection of the YFP-CD79b fusion constructs into
COS-7, K-562, and Ramos-EHRB cells demonstrated that protein product is
expressed in whole cells (data not shown). Finally, we also used
Western blotting experiments with a panel of anti-CD79b mAbs directed
to the extracellular domain of CD79b to confirm the existence of
truncated CD79b in transfected COS-7 cells, B-cell lines, and
transfected EHRB cells. These experiments revealed that protein species
are generated from the alternative transcripts of CD79 and confirm the
work of Benlagha et al,34 which has recently demonstrated
the truncated form of CD79a in normal B cells by Western blotting.
Intriguingly, all of the anti-CD79 mAbs available bound to both
full-length and truncated forms of the CD79b molecule, despite the fact
that the truncated molecule has lost the complete extracellular Ig-like
domain and is left with only 25 amino acids to provide the Ab epitope.
Presumably, all of these mAbs recognize the same short extracellular
peptide region distal to the transmembrane domain, which is present in both the full-length and truncated CD79b molecule, because the proximal
peptide would probably be inaccessible in the full-length molecule.
This indicates either that there is only one immunodominant epitope in
the CD79b protein or that other sites are obscured by the binding
orientation of CD79b in the BCR. Importantly, these data also
demonstrate that current anti-CD79b mAbs are incapable of
differentiating between the full-length and alternative transcripts of CD79b.
It has been suggested by one group,21 based upon L-cell
reconstitution studies, that overexpression of CD79b might account for the low levels of surface Ig that are usually found on B-CLL cells.
Three lines of evidence presented here indicate that this is not a
dominant effect of CD79b: First, forced overexpression of CD79b
in transfectant cell lines was shown not to modulate the surface levels
of CD79a, CD79b, or Ig; second, Namalwa cells express high levels of
endogenous CD79b but still display high levels of surface Ig; and
third, SLVL cells express high levels of CD79b but are characterized
by their high level of surface Ig expression.35 Therefore,
we feel that these data argue that CD79b expression per se does not
account for a decrease of Ig at the cell surface. Although decreased
expression of full-length CD79b, occurring as a result of elevated
splicing to the CD79b form, may account for decreased CD79b protein
expression, this was not indicated by our RT-PCR analysis. Rather, it
suggests that B-CLL cells may possess a defect in posttranslational
pathways of intracellular synthesis and transport or enhanced
proteolysis, such as discussed by Payelle-Brogard et al.36
Furthermore, these properties may well be a product of an anergic state
similar to that detailed by Bell and Goodnow37 for
tolerant mouse B cells exposed to endogenous hen egg lysozyme
(HEL) self-antigen in the HEL-transgenic mouse system.
It is not clear by what mechanism CD79b could control apoptosis
induced via the BCR. Alternative splicing is common in signaling receptors of the immune system. Examples include the coreceptors CD22
and CD45, T-cell receptor (TCR) components, cytokine
receptors, Fc receptors of the  and all 3 types, as well as Ig
itself (reviewed by Hashimoto et al20). The purpose of
alternative splicing is not always apparent, but the literature tends
to support the notion that alternatively spliced truncated receptors
are negative regulators of their full-length counterparts, acting either as inert decoy molecules or as proteins with directly opposing functions.38,39 A good illustration is seen with the Bcl-2 family member Bcl-x, wherein the full-length form of this molecule (Bcl-xL) is a potent inhibitor of apoptosis, while the truncated form
(Bcl-xS) is a proapoptotic protein. Furthermore, altering the ratio of
splice forms can drastically alter cellular fate (ie, life or
death).40
We would speculate that CD79b is a decoy molecule that acts as a
negative regulator of the BCR by sequestering critical signaling adaptor molecules away from the functional CD79a:CD79b heterodimer. First, our data clearly show that overexpression of this molecule inhibits apoptosis induced via the BCR. Although overexpression studies
are not always reliable exponents of real biologic systems, we would
argue that the levels of CD79b induced in the transfectants are
within the physiological spectrum of expression seen in normal and
malignant B cells. Second, we have found that a leaderless version of
the short transcript does not function. This is consistent with the
CD79b protein needing to traffic through the ER perhaps for export
to the cell surface to deliver its inhibitory activity. Koyama et
al21 would argue against this suggestion, because they
showed, using reconstitution experiments in murine fibroblast L cells,
that transfection of CD79b (along with CD79a and Ig) does not evoke
transport of the BCR to the cell surface, indicating that CD79b
cannot facilitate exit from the ER. However, this work was done in
mouse fibroblasts, which may lack important trafficking molecules
expressed in B cells. Third, we have found that the CD79b molecule
needs a functional ITAM in order to provide inhibitory function.
Mutation of Y207 in the ITAM has previously been shown to ablate the
signaling potential of CD79b,7 and in our system this
mutation was sufficient to remove the inhibitory capacity of CD79b.
A similar dependence on the ITAM motif was recently demonstrated for
the inhibitory effect of the Ig R.41 In this instance,
the binding and sequestering of important signaling molecules was
responsible for the inhibition of productive signaling. We are
currently exploring the immediate downstream signaling events that are
induced in the absence and presence of CD79b, although preliminary
data indicate that global tyrosine phosphorylation and Ca++
release are similar in cells irrespective of their CD79b
status (data not shown).
Another possible mechanism for how CD79b might function is through a
continual signaling or desensitization model. Recent studies by Vilen
et al42 have shown that the BCR can become desensitized
and dissociated from the CD79 heterodimer when ligated by low- to
moderate-affinity antigens and that this destabilization requires only
a small proportion of the available BCR to be ligated. In this way, it
is conceivable that CD79b might only interact or interfere with a
small number of receptors, desensitizing the cell by interacting with,
for example, Syk, but not causing mass down-regulation of surface
expression. Furthermore, it is a phenomenon not observed with
high-affinity antigens but with low- to moderate-affinity antigens such
as those feasibly encountered by self-specific BCR of B-CLL. To survive
constant antigen stimulation, the cell would require not only
down-regulation of surface Ig but also dampening of intracellular
signaling pathways. Perhaps CD79b is capable of this function.
Whatever the mechanism, we would postulate that up-regulation of
CD79b is a physiological response of B cells that allows control of
signaling via the BCR. As such, we would predict it to be present at
times of B-cell activation, perhaps preventing cells from being
overstimulated and killed via apoptosis, such as might occur in the
germinal center. We are currently pursuing this proposal and examining
the expression levels of CD79b in apoptosis-sensitive B-CLL cells
bearing CD38.
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Acknowledgments |
The authors thank Dr Ruth French for help with cell sorting and
preparation of the manuscript and Dr Will Howatt for help with
fluorescence microscopy. We are indebted to Dr Mike Neuberger and
Theresa O'Keefe for reagents and helpful discussion. Thanks |
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Abstract
Introduction