Heterogeneous intracellular expression of B-cell receptorcomponents in B-cell chronic lymphocytic leukaemia (B-CLL) cellsand effects of CD79b gene transfer on surface immunoglobulinlevels in a B-CLL-derived cell line
The B cell antigen receptor (BCR) is a multi-chain complex
that consists of the membrane-bound immunoglobulin (Ig)
molecule in non-covalent association with a disulphide-linked
CD79a/CD79b heterodimer (Hombach et al, 1990; Reth,
1992). The CD79a/CD79b components serve as a signalling
subunit (Sanchez et al, 1993), are strictly required for Ig
transport to the cell surface (DeFranco, 1993), and contribute
to antigen presentation in B cells (Tarlinton, 1997). In addition
to its functions in mature B cells, the CD79a/CD79b complex
regulates the early stages of B cell development, as shown in
transgenic mice (Gong & Nussenzweig, 1996; Torres et al,
1996). Abnormalities in the BCR have often been associated
with certain haematopoietic malignancies, including B-chronic
lymphocytic leukaemia (B-CLL), which is usually characterised
by the progressive accumulation of monoclonal CD5+ B cells,
expressing low amounts of surface Ig (sIg) (Hamblin & Oscier,
1997; Matutes & Polliack, 2000; Caligaris-Cappio & Ghia,
2004). This phenotype may explain the reduced ability of
B-CLL cells to capture, present, and respond to antigens
(Lankester et al, 1995), as well as their defective tyrosine
phosphorylation when stimulated through the BCR pathway
(Semichon et al, 1997).
Defects in the BCR of B-CLL cells have recently been
attributed to functional deficiency of CD79b, which is
expressed at low levels in most patients (Zomas et al,
1996). Among the mechanisms proposed to explain these
Sonia Minuzzo,1 Stefano
Indraccolo,1,2Valeria Tosello,1 Erich
Piovan,1 Anna Cabrelle,3 Livio Trentin,3
Giampietro Semenzato3 and Alberto
Amadori1
1Department of Oncology and Surgical Sciences,
University of Padova, 2Istituto Oncologico Veneto
& Azienda Ospedaliera, and 3VIMM &
Department of Clinical and Experimental
Medicine, Clinical Immunology Branch,
University of Padova, Padova, Italy
Received 18 March 2005; accepted for
publication 23 June 2005
Correspondence: Stefano Indraccolo, MD,
Department of Oncology and Surgical Sciences,
University of Padova, Via Gattamelata,
64–35128 Padova, Italy.
E-mail: [email protected]
Summary
B-cell chronic lymphocytic leukaemia (B-CLL) cells display low amounts of
surface immunoglobulins (sIg). To investigate the mechanisms underlying
this phenomenon, we performed a thorough study of surface and
intracellular expression of the B-cell receptor (BCR) components in B-CLL
cells using flow cytometry. There was an heterogeneous pattern of expression.
Overall, 20 of 22 samples showed reduced sIgM levels, compared with normal
B cells. Among them, three (15%) had very low to undetectable intracellular
IgM levels and variable amounts of CD79a and CD79b; nine (45%) had low
intracellular CD79b levels but appreciable levels of IgM and CD79a; and eight
(40%) had relatively normal intracellular levels of all BCR components. To
investigate whether surface BCR levels could be controlled by the rate of
CD79b synthesis, adenoviral vectors encoding CD79b were generated and
used for gene transfer experiments. Delivery of CD79b to non-B cells
transfected with IgM and CD79a lead to high-level expression of a functional
BCR. Moreover, CD79b gene transfer in a B cell line derived from a B-CLL
patient and characterised by low intracellular levels of endogenous CD79b
consistently increased sIgM levels. These findings indicate that the phenotype
of B-CLL cells in a subset of patients may depend primarily on poor CD79b
expression, and suggest that upregulation of CD79b expression may correct
the phenotype of these cells.
Keywords: chronic lymphocytic leukaemia, B-cell receptor, CD79b, adeno-
viral vector, gene transfer.
research paper
doi:10.1111/j.1365-2141.2005.05699.x ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889
observations are reduced expression of CD79b mRNA
(Thompson et al, 1997), somatic mutations of the B29 gene
(Thompson et al, 1997), over-expression of a product of
alternative splicing of CD79b, termed DCD79b (Alfarano
et al, 1999), and abnormal assembly of the BCR chains,
leading to their accumulation in the intracellular compart-
ments (Payelle-Brogard et al, 2002). Possible alterations of
CD79a in B-CLL have been studied in less detail, although
this molecule shares many functional features with CD79b
(Minegishi et al, 1999). Recently, however, Vuillier et al
(2005) reported glycosylation and folding defects of the
CD79a chains in B-CLL patients.
Although the reduced surface expression of the Ig and
CD79b components of the BCR in B-CLL cells is well
established, most studies have measured surface levels of these
components. We have investigated the expression of IgM,
CD79a, and CD79b on the surface of B-CLL cells and
compared it to intracellular levels by flow cytometric analysis.
We found that B-CLL cases were heterogeneous in this respect
and that different reasons possibly explain the low sIgM levels
found in the great majority of the patients. Notably, however,
lack of intracellular CD79b represented the most common
finding and it was observed in a high proportion of B-CLL
samples (45%). Since CD79b is strictly required for efficient
transport of the BCR to the cell surface (Costa et al, 1992;
Grupp et al, 1995), we hypothesised that its rate of synthesis
could regulate membrane Ig levels in B-CLL cells. To
investigate this, we exploited a gene transfer approach and
observed that adenoviral vector-mediated CD79b gene transfer
consistently increased sIg levels in a B-CLL-derived cell line
selectively lacking CD79b. These findings underline that
reduced intracellular availability of CD79b protein is found
in a considerable subset of patients and suggest that upregu-
lation of CD79b synthesis could represent a valuable approach
to restore BCR expression in B-CLL patients.
Materials and methods
Patient population
A group of 22 unselected B-CLL patients who met the
diagnostic criteria of the National Cancer Institute-Working
group was studied; 12 were men and 10 women, with a mean
age of 70 years (range 44–84). The patients were staged
according to Rai criteria and studied for the expression of
CD5, CD19, CD38, and IgVH gene mutational status (Table I).
Subsequently, these patients underwent detailed analysis of the
expression of BCR components.
Table I. Characteristics of B-CLL patients.
Patient number Stage* CD19 CD38� IgVH somatic hypermutation� Sex Age (years) Therapy
1 IV 85 POS ) M 78 Chlorambucil
2 IV 98 BIMOD ) F 76 Mitoxantrone
3 IV 89 NEG ) M 78 Prednisone, chlorambucil
4 IV 73 BIMOD n.d. M 63 No
5 I 89 NEG non amp. F 69 Prednisone, chlorambucil
6 0 95 NEG + F 61 No
7 IV 95 NEG + F 78 Chlorambucil
8 II 90 BIMOD ) M 44 No
9 III 87 NEG + M 81 No
10 III 98 BIMOD ) F 71 Chlorambucil
11 III 98 BIMOD ) M 75 No
12 II 80 NEG + F 75 No
13 III 91 BIMOD ) F 72 Chlorambucil
14 III 78 BIMOD ) F 65 No
15 II 96 BIMOD ) M 57 Cyclophosphamide
16 I 87 NEG n.d. M 77 Prednisone, azathioprine
17 II 97 POS ) M 76 Prednisone, chlorambucil
18 IV 78 NEG ) M 59 Prednisone
19 II 84 POS ) M 73 Prednisone
20 IV 97 BIMOD ) M 77 Prednisone, chlorambucil
21 IV 79 NEG ) F 84 No
22 III 65 NEG ) F 62 Chlorambucil
*Rai stage at diagnosis.
�CD38 expression profiles as defined by Ghia et al (2003). POS, samples homogeneously positive for CD38; NEG, samples homogeneously negative
for CD38 or with <2% positivity; BIMOD, samples with a bimodal expression profile.
�Hypermutated samples were defined as those bearing <98% sequence homology with the nearest germ line gene; n.d., not done; non amp, sample
not amplificable.
Intracellular expression of BCR components in B-CLL cells
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889 879
Primary cell isolation and cell lines
B-CLL cells were obtained by Ficoll-Hypaque (Pharmacia
Biotech, Uppsala, Sweden) gradient centrifugation from per-
ipheral blood samples (Coppola et al, 1998). Circulating B cells
from normal donors were also obtained by Ficoll–Hypaque
centrifugation of peripheral blood samples followed by positive
purification, as detailed elsewhere (Piovan et al, 2003), with
anti-CD19 monoclonal antibody (mAb)-coated microbeads
(Miltenyi Biotec GmbH, Bergish Gladbach, Germany); their
purity was 82–97%, as assessed by flow cytometry with anti-
CD19 mAb. MEC2 is an Epstein–Barr virus (EBV)-positive
lymphoblastoid B cell line derived from a B-CLL patient,
described in detail elsewhere (Stacchini et al, 1999). The
human Burkitt lymphoma cell line Daudi served as a positive
control for BCR expression. Both cell lines, as well B-CLL cells
and normal B cells, were maintained in Roswell Park Memorial
Institute (RPMI)-1640 medium (Cambrex Bio-Science, Milan,
Italy) supplemented with 10% fetal calf serum (FCS), 1%
l-glutamine (both from Gibco-BRL, Grand Island, NY, USA),
1% Na pyruvate (Cambrex). The human embryonal kidney-
derived 293T cell line was obtained from the American Type
Culture Collection (Manassas, VA, USA) and grown in high-
glucose Dulbecco’s modified Eagles medium (Sigma-Aldrich,
Milan, Italy) supplemented with 10% FCS and 1%
l-glutamine.
Immunophenotypic analysis
The following mAb were used: anti-CD5 phycoerythrin (PE)-
labelled (Immunotech, Marseille, France); fluorescein isothio-
cyanate (FITC)-labelled anti-CD19 (Becton Dickinson, San
Jose, CA, USA); anti-CD38-PE (Immunotech); anti-CD54-PE
(Caltag, Burlingame, CA, USA). To measure surface expression
of BCR components we used a rabbit anti-IgM-PE (Dako,
Glostrup, Denmark), an anti CD79a-FITC mouse mAb (clone
ZL7Æ4; Caltag), and an anti-CD79b-PE mouse mAb (clone
CB3Æ1; Immunotech). Staining for intracellular molecules
(IgM, CD79a, CD79b, HAtag) was performed on permeabi-
lised cells, using Cytofix/Cytoperm Plus kit (Becton Dickin-
son). To measure intracellular expression we used a mouse
anti-IgM antibody (Southern Biotechnologies, Birmingham,
AL, USA), an anti-CD79a mAb (clone HM57; Dako) and an
anti-CD79b-FITC mAb (clone SN8; Dako). A mouse mAb
against the haemagglutinin (HA) tag (BabCO/Covance,
Princeton, NJ, USA) was used in the experiments involving
detection of the HA-tagged CD79b molecule. Fluorescence
intensities were assessed in comparison with that given by an
isotype-matched control antibody. Indirect immunofluore-
scence was performed with species-specific PE- or FITC-
conjugated anti-Ig antibodies (Dako) as the second reagent.
For each sample at least 20 000 events were acquired on an
EPICS XL cytofluorimeter equipped with a 488 argon ion laser
(Coulter, Hialeah, FL, USA), and analysed with EXP032
Software (Coulter). Results were expressed as percentage of
positive cells and show the mean fluorescence intensity (MFI),
which was calculated according to the following formula:
MFI ¼ log10 (mean · 10) · (1024/4).
CD79bHA cloning and adenoviral vector generation
Total RNA isolated from Daudi cells was used for the
synthesis of first strand cDNA using reverse transcriptase
and an oligod(T) primer as described elsewhere (Indraccolo
et al, 2002). Aliquots of the cDNA samples were then
amplified with CD79b-specific primers, whose sequences are
listed below:
CD79b-for: 5¢-TGAAGATCT-GTGACCATGGCCAGGCT-GGCGTTGT-3¢CD79bHA-rev: 5¢-AAGCTT-TCAAGCATAATCTGGAACA-
TCATATGGATA-CTCCTGGCCTGGGTGCTC-3¢The CD79bHA-rev primer carried an HA tag, which was
inserted to enable the exogenous CD79b molecule to be
discriminated from the endogenous one in B cells. A BglII and
a HindIII site were also included in the forward and reverse
primers respectively to allow subsequent subcloning of the
insert. PCR analysis was performed in a 50-ll volume
containing 0Æ2 lmol/l of each primer and 0Æ7 U of Taq
polymerase (Applied Biosystems, Niauwekerk, The Nether-
lands), under the following conditions: 94�C denaturation for
1 min, 60�C annealing for 30 s, 72�C extension for 1 min, for
25 cycles. The amplified products were separated on 1Æ5%agarose gels, purified by GFX gel purification kit (Pharmacia
Biotech), and first cloned in pcDNA3Æ1 (Invitrogen, San
Giuliano Milanese, Italy). After enzymatic digestion with BglII
and HindIII, the CD79bHA fragment was cloned by standard
protocols into the corresponding sites of the shuttle vector
pAdTrack-CMV, which was used for production of green
fluorescent protein (GFP)-trackable adenoviruses containing
both CD79b and GFP under the control of two serial CMV
promoters and SV40 polyadenylation sites. All constructs were
verified by molecular analysis and sequencing before perform-
ing functional assays.
The adenovirus (Ad) vectors were produced by the Gene
Vector Production Network (Nantes, France) using the
method described by He et al (1998). As a control vector we
used the parental Ad-GFP, which encodes only the GFP
marker. The titres were 2Æ4 · 1011 infectious particles/ml for
Ad-GFP and 2Æ5 · 1010 infectious particles/ml for Ad-GFP-
CD79bHA.
Transfection and transduction protocols
In experiments aimed at characterising the Ad-GFP-CD79b
vector, 293T cells were infected with Ad-GFP or Ad-GFP-
CD79b [both multiplicity of infection (MOI) ¼ 10], or
transfected by calcium–phosphate co-precipitation with the
construct pCD79b, as previously described (Indraccolo et al,
2002). Twenty-four-hour later CD79b expression was analysed
by immunofluorescence or immunoblotting as detailed above.
S. Minuzzo et al
880 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889
In experiments aimed at restoring BCR expression, 293T cells
were first co-transfected with IgM- and CD79a-coding plas-
mids (6 and 3 lg respectively) and 24 h later infected with
Ad-GFP or Ad-GFP-CD79b, both at a MOI ¼ 10. One day
later, the cells were analysed for sIgM expression; as a positive
control for BCR reconstitution, 293T cells were co-transfected
with plasmids coding for IgM, CD79a and CD79b (6, 3 and
3 lg respectively), as reported (Indraccolo et al, 2002). Simi-
larly, the analysis of CD79b phosphorylation was performed on
293T cell lysates transfected with IgM- and CD79a-coding
plasmids (6 and 3 lg respectively), and then infected with
Ad-GFP or Ad-GFP-CD79b at a MOI ¼ 10. In gene transfer
experiments in B cells, 0Æ5–1 · 106 MEC2 cells were infected
with adenoviral vectors at a MOI ¼ 100 or ¼ 1000 respec-
tively; 36 h after infection, the cells were analysed for sIgM and
GFP expression, and for intracellular expression of the HA tag.
Western blot analysis
To identify CD79b in 293T cells infected by adenoviral vectors,
a goat anti-CD79b antibody was used, followed by incubation
with horseradish peroxidase-conjugated anti-goat-IgG anti-
body (both from Santa Cruz Biotechnology, Santa Cruz, CA,
USA).
In experiments aimed at detecting phosphorylated
CD79bHA, cell lysates were immunoprecipitated overnight at
4�C with an anti-pTyr (PY99) antibody (Santa Cruz Biotech-
nology), coupled to protein A-Sepharose; bound proteins were
then released by boiling, separated on 12Æ5% SDS-polyacryl-
amide gels, and incubated with an anti-HA antibody (BabCO/
Covance). To verify that homogeneous amounts of proteins
were loaded in the different lanes, the same lysates were
separated by sodium dodecyl sulphate polyacrylamide gel
elctrophoresis (SDS-PAGE) and analysed by Western blotting
with an anti-Syk antibody (N-19) (Santa Cruz Biotechnology).
Membranes were blocked overnight in phosphate buffered
saline (PBS; Sigma) containing 0Æ1% Tween-20 (PBST) and
1% non-fat dried milk (Sigma) and incubated with the
appropriate antibody, in PBST-3% BSA. Blots were then
washed in PBST and incubated with horseradish peroxidase-
conjugated sheep anti-mouse, donkey anti-rabbit IgG (both
from Amersham, Paris, France) or donkey anti-goat IgG
(Santa Cruz Biotechnology). After several washes, probed blots
were developed using an enhanced chemiluminescence West-
ern blotting detection system (Pierce, Rockford, IL, USA),
according to the manufacturer’s instructions.
Protein tyrosine phosphorylation assay
One day after infection with Ad-GFP-CD79b or the control
adenoviral vector, 293T cells (5Æ5 · 106/ml) were incubated
with 10 lg/ml anti-IgM antibody for different times (0, 0Æ5, 5,20 min). The cells were then washed twice in cold PBS,
pelleted by centrifugation and lysed in ice-cold lysis buffer
(150 mmol/l NaCl, 50 mmol/l Tris pH 7Æ5, 1% NP40, 2 mmol/
l EDTA, 50 mmol/l NaF, 1 mmol/l sodium orthovanadate,
protease inhibitor cocktail (Sigma)]. An aliquot of the lysates –
corresponding to 0Æ5 · 106 cells/lane – was then separated by
SDS-PAGE and analysed by immunoblotting, as described
above. The remaining lysate – corresponding to 5 · 106 cells –
was immunoprecipitated with an anti-pTyr antibody and then
analysed to identify phosphorylated CD79b.
Endoglycosidase H (Endo-H) digestion
MEC2 or Daudi cells were lysed on ice by incubation for
30 min with MBS buffer (25 mmol/l morpholinoethanesul-
phonic acid, 150 mmol/l MaCl pH 6Æ6, 0Æ5% Triton X-100,
1 mmol/l EDTA, 10 mmol/l NaF, 1 mmol/l sodium ortho-
vanadate, protease inhibitors). Cell lysates were pelleted by
centrifugation at 10 000 g for 10 min, and supernatants were
collected. Proteins were treated with Endo-H (Roche Molecu-
lar Biochemicals, Mannheim, Germany) in 50 mmol/l potas-
sium acetate pH 5Æ5, 0Æ2% SDS, 0Æ1 mol/l 2-mercaptoethanol,
protease inhibitors for 18 h at 37�C and then analysed by
Western blotting, as described above.
Statistical analysis
Data were managed using the Statgraphics software. The
mean ± 2 SD were used where appropriate to compare the
percentage of IgM expression or the MFI in B cells transduced
by the Ad-GFP or the Ad-GFP-CD79b vectors.
Results
B-CLL samples show different patterns of surface andintracellular expression of IgM, CD79a, and CD79b
We analysed 22 B-CLL samples from patients in different
stages of disease by flow cytometry for the expression of the
BCR components. CD19 expression was high in all samples
and ranged between 65% and 98% (Table I); CD19+ cells were
also invariably CD5+ (not shown). The surface marker CD38
was homogeneously expressed on three of 22 CLL samples,
whereas 10 of 22 samples did not express it; interestingly, a
bimodal profile was observed in further nine of 22 samples, as
recently reported by Ghia et al (2003). Ig somatic hypermu-
tation was detected in four samples, and these were all CD38)
B-CLL (Table I).
A large majority of the samples (20/22) presented reduced
sIgM expression, compared with normal B cells (Table II),
particularly in terms of MFI. This agrees with previously
reported findings in different CLL study groups (Alfarano
et al, 1999; Payelle-Brogard et al, 2002). When we compared
the surface and intracellular distribution of IgM, CD79a, and
CD79b, we found heterogeneous patterns of expression of the
BCR components; representative cases are shown in Fig 1, and
the analysis is summarised in Table II. Two B-CLL samples
presented with a phenotype similar to normal B cells and
Intracellular expression of BCR components in B-CLL cells
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889 881
Daudi cells, which were used as controls. These samples
showed high and similar percentages and MFI values of
intracellular versus surface IgM, CD79a, and CD79b reactivity
(Fig 1 and Table II).
The remaining B-CLL samples (20/22) presented variably
reduced surface IgM, CD79a, and CD79b levels compared with
normal B cells (Table II). As isotype switching may have
accounted for lack of IgM expression, we analysed these
samples for IgG expression, and found them invariably
negative (not shown). These B-CLL samples could be tenta-
tively classified into three groups, according to the expression
of BCR components. Three samples (group low I; 15%) had
very low to absent surface IgM, CD79a, and CD79b reactivity,
which correlated with undetectable intracellular IgM; intracel-
lular CD79a and CD79b, however, were readily detected in
these cells (Fig 1, panel CLL no. 3). Nine of twenty samples
(group low II; 43%) had reduced but appreciable surface and
intracellular IgM and CD79a levels, while presenting a
dramatic reduction in the expression of intracellular CD79b
(Fig 1, panel CLL no. 6). Finally, eight of 20 samples (group
low III; 42%) had reduced sIgM expression levels in spite of
normal or close-to-normal intracellular levels of the other BCR
components (Fig 1, panel CLL no. 19).
Immunoprecipitation analysis of B-CLL cell lysates con-
firmed the lack of CD79b in group low II samples and
demonstrated variable amounts of the full-length CD79b
protein in other B-CLL samples analysed (Fig 2). Notably, the
truncated CD79b protein encoded by the alternatively spliced
CD79b transcript, which is readily expressed at the mRNA
level in these samples (data not shown), was apparently not
detected (Fig 2).
In summary, this analysis showed heterogeneous patterns of
intracellular staining for BCR components in B-CLL samples,
suggesting that their common feature, i.e. reduced sIg levels,
may be accounted for by different mechanisms.
Adenoviral vector-mediated gene transfer of CD79b in293T cells restores surface expression of a functional BCR
In view of our observation of a selective lack of CD79b in a
subset of B-CLL samples and of previous literature suggesting
that abnormal CD79b expression could be responsible for the
low sIg levels in these cells (Zomas et al, 1996; Thompson et al,
1997; Alfarano et al, 1999), we set out to explore whether gene
transfer of CD79b may suffice to restore normal levels of
surface IgM expression. To investigate this, we generated an
Table II. Expression of IgM, CD79a, and CD79b on the surface or in the intracellular (intra) compartment of B-CLL cells by FACS analysis.
Patient number Group
IgM CD79a CD79b
Surface Intra Surface Intra Surface Intra
% MFI % MFI % MFI % MFI % MFI % MFI
1 High 85 668 99 613 70 457 99 533 79 451 95 404
2 High 95 704 99 622 95 498 98 437 91 526 99 512
3 Low I 12 333 14 315 25 348 89 423 4 561 83 362
4 Low I 0 n.d. 8 353 7 495 80 465 4 447 65 358
5 Low I 0 n.d. 0 n.d. 20 362 0 n.d. 11 428 100 423
6 Low II 23 321 91 477 40 343 98 439 9 301 17 285
7 Low II 37 314 94 433 16 389 96 423 6 321 0 n.d.
8 Low II 10 491 94 446 3 352 97 454 0 – 0 –
9 Low II 0 n.d. 42 453 15 465 38 423 5 453 0 n.d.
10 Low II 58 311 20 291 13 295 98 450 5 307 0 n.d.
11 Low II 54 305 78 317 0 n.d. 89 330 0 n.d. 0 n.d.
12 Low II 58 319 69 346 15 380 96 393 0 n.d. 0 n.d.
13 Low II 64 395 98 404 12 313 99 500 0 n.d. 0 n.d.
14 Low II 26 466 80 566 20 348 98 n.d. 9 347 0 n.d.
15 Low III 74 338 98 525 54 353 99 491 8 321 45 358
16 Low III 71 395 98 487 n.d. n.d. 99 467 n.d. n.d. 100 384
17 Low III 82 445 97 480 61 348 97 487 53 321 94 392
18 Low III 55 378 88 597 35 385 96 485 4 636 90 358
19 Low III 88 509 94 478 57 381 92 441 25 353 90 362
20 Low III 89 430 99 567 54 362 98 439 33 407 84 327
21 Low III 53 445 98 471 25 348 99 487 21 389 82 343
22 Low III 76 443 85 453 77 401 97 492 58 362 87 370
Normal controls
1 79 593 68 418 88 474 91 462 76 428 88 373
2 81 560 75 414 89 431 74 330 82 473 74 355
MFI, mean fluorescence intensity; n.d.: not determined.
S. Minuzzo et al
882 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889
Fig 1. Patterns of surface and intracellular IgM, CD79a, and CD79b expression in normal B, primary B-CLL, and Daudi cells. Cells were analysed for
surface or intracellular expression of the BCR components after incubation with anti-IgM, anti-CD79a, and anti-CD79b conjugated with different
fluorochromes, as detailed in the Materials and methods. Intracellular staining was performed on cells permeabilised with Cytofix/Cytoperm Plus kit.
The histograms show the expression profiles of normal B cells, different B-CLL samples representative of the groups described in the text (high, low I,
low II, low III) and Daudi cells, with the intensity and the number of events shown on the x-axis and the y-axis, respectively. The percentage and the
mean fluorescence intensity (MFI) are shown in the upper right corner of each histogram. At least 20 000 cells were analysed in each histogram; the
shaded areas refer to negative controls.
Intracellular expression of BCR components in B-CLL cells
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889 883
adenoviral vector encoding human CD79b tagged at the 3¢-endwith an HA tag to enable discrimination between the product
of the transgene and endogenous CD79b. Furthermore, the Ad
also encoded GFP from an independent expression cassette, to
allow easy detection of the transduced cells, as reported for the
prototype of the vector (He et al, 1998). As a control vector,
we used the parental Ad encoding only GFP from the same
promoter.
In preliminary experiments, these vectors were used to
transduce 293T cells (a non-B cell line highly sensitive to Ad
infection) using nominally identical amounts of vector parti-
cles. Flow cytometry analysis of GFP expression indicated high
efficiency of gene transfer in these cells (Fig 3A); CD79b was
expressed at the cell surface, however, only by Ad-GFP-
CD79b-infected cells (Fig 3A). The production of Ad-encoded
CD79b in transduced 293T cells was confirmed by immuno-
blotting with an anti-CD79b antibody, which indicated a
major band of about 40 kDa, slightly higher than the size of
the native protein encoded by a CD79b expression plasmid
previously described (Indraccolo et al, 2002), probably because
of the presence of the HA tag (Fig 3B).
To test whether gene transfer of CD79b could suffice to
rescue BCR expression in cells selectively lacking this compo-
nent, we transfected 293T cells with plasmids encoding IgM
and CD79a and subsequently infected them with the Ad
encoding CD79b or the control vector. 293T cells transfected
with IgM/CD79a expressed sIgM in a small percentage of cells
and infection of these cells with the control Ad encoding GFP
did not change these figures (Fig 3C). Transduction of these
cells by the Ad encoding CD79b resulted in a clear-cut increase
in sIg expression, and IgM expression was detected in 55% of
the cells at very high levels (Fig 3C); these figures were
comparable with those observed following co-transfection of
these cells with plasmids encoding all BCR components, which
was used as a positive control for the molecular reconstitution
experiment (Indraccolo et al, 2002), and led to sIgM expres-
sion in 52% of the cells (Fig 3C).
Finally, we addressed whether the reconstructed BCR was
also functional. To this end, we cross-linked the BCR on the
surface of transduced 293T cells with an anti-IgM antibody
and analysed phosphorylation of the vector-encoded CD79b-
HA. As shown in Fig 3D, CD79b-HA was readily immuno-
precipitated in transduced 293T cells, and it showed a basal
level of phosphorylation that increased as a function of time
following BCR stimulation. Endogenous Syk, a component of
the signal transduction machinery associated to the BCR in B
cells, was also detected in 293T cells. Thus, we conclude that
the CD79b-HA molecule encoded by the adenoviral vector is
functional both in terms of Ig transport and as a signalling
molecule.
Effects of Ad-mediated CD79b gene transfer on BCRexpression in B cells
Having established that Ad-GFP-CD79b transduction into a
non-B cell line was able to mediate surface expression of a
fully functional BCR complex, we sought to investigate
whether this could also occur in a B cell line, termed MEC2,
derived from a B-CLL patient in prolymphocytic transfor-
mation (Stacchini et al, 1999). Interestingly, these cells had a
phenotype very similar to that of group II primary B-CLL
samples, with low intracellular levels of CD79b. In fact,
MEC2 cells expressed low-levels of sIgM and sustained IgM
and CD79a levels in the intracellular compartment, but
showed a clear lack of the CD79b component at both the
surface and intracellular level (Fig 4A). As found in 293T
cells, MEC2 cells transduced by the Ad-GFP-CD79b
(MOI ¼ 100), but not control cells which received Ad-GFP,
showed a significant increase in sIg levels; results obtained in
three consecutive experiments are shown in Table III.
Intriguingly, however, CD79b gene transfer did not restore
BCR expression on the totality of MEC2 cells. To investigate
whether higher levels of BCR expression could be achieved,
we repeated transduction of MEC2 cells by using a higher
input of vector particles (MOI ¼ 1000). Moreover, as not all
GFP+ cells co-express the HA-tagged CD79b, as evidenced by
our experiments in 293T cells (Fig 3A), we restricted analysis
of sIg levels selectively on cells expressing the exogenous
CD79b. Intracellular staining with the anti-HA antibody,
followed by fluorescence-activated cell sorting analysis con-
firmed expression of the HA-tagged vector-encoded CD79b
in a sizable fraction of MEC2 cells infected by the corres-
ponding Ad vector (Fig 4B). Moreover, when IgM expression
was measured separately on the surface of HA+ and HA)
MEC2 cell subsets, a very marked increase in sIgM levels was
observed (Fig 4B) and this was confirmed in five independent
experiments (Fig 4C).
Fig 2. Immunoprecipitation analysis of CD79b expression in B-CLL
cell lysates. B-CLL samples (107 cells/lane) were lysed and immuno-
precipitated with a rabbit anti-CD79b serum directed against the
cytoplasmic tail of human CD79b (Indraccolo et al, 2002); SDS-PAGE
was performed under reducing conditions. 293T cells transiently
transfected with CD79b or DCD79b expression plasmids were used as
positive controls (Indraccolo et al, 2002). Following transfer, the filter
was probed with a goat anti-CD79b antibody. CD79b was absent in
group low II samples, highly expressed in group ‘high’ samples, and
showed variable expression levels in the other B-CLL samples analysed.
Truncated CD79b protein was not detected in these B-CLL samples.
The arrows indicate different isoforms of full-length CD79b protein.
S. Minuzzo et al
884 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889
Immature l chains are detected in MEC2 cells
To investigate whether other factors may also limit BCR
expression in MEC2 cells, we analysed the glycosylation status
of the CD79a and IgM chains, which could affect the BCR
transport, as recently observed in B-CLL patients (Vuillier
et al, 2005). Figure 5 displays the differences in IgM and
CD79a glycosylation status in MEC2 and Daudi cells, which
were also included in the experiment as a prototype of B cell
line with very high levels of BCR expression. To determine the
glycosylation status of these cells we used Endo-H treatments,
as previously suggested (Vuillier et al, 2005). Endo-H cleaves
high mannose oligosaccharides associated with glycoproteins
present in the endoplasmic reticulum (ER), and proteins
become resistant to digestion after transport to the medial
Golgi apparatus. Thus, Endo-H digestion can be used to
distinguish ER-resident (immature) from Golgi-processed
(mature) glycoforms. In MEC2 cells, the majority of the lchains present in the cell lysates shifted to a 70 kDa band
following Endo-H treatment, thus indicating that these chains
were largely immature. On the other hand, Daudi cell lysates
contained an apparently larger fraction of Endo-H-resistant lchains, which migrated with a size of 82 kDa and indicated
mature glycoforms (Fig 5). Moreover, most of CD79a corres-
ponded to mature glycoforms in MEC2 cells, whereas more
immature forms were found in Daudi cells (Fig 5). Thus,
defects in glycosylation of l chains could possibly explain why
CD79b gene delivery did not result in sIgM upregulation in the
totality of MEC2 cells.
Discussion
Many studies have reported the surface immunophenotypic
analysis of B-CLL cells, identifying reduced BCR expression as
(A) (B)
(D)(C)
Fig 3. Functional characterisation of an adenoviral vector encoding human CD79b. (A) Detection of high level GFP and surface CD79b expression
following transduction of 293T cells with Ad-GFP-CD79b. A MOI ¼ 10 was used; transgene expression was analysed 24 h after gene transfer using an
anti-CD79b-PE mouse mAb. The shaded histogram represents the control. (B) Immunoblotting of cell lysates obtained from 293T cells transduced by
Ad-GFP-CD79b (as shown in A) with a goat anti-CD79b antibody shows the presence of a band of about 40 kDa corresponding to CD79b, slightly
higher than the size of the native protein encoded by the pCD79b expression plasmid previously described (Indraccolo et al, 2002), probably due to
the presence of the HA tag. (C) Reconstitution of surface IgM expression in 293T cells transfected with IgM and CD79a expression plasmids and
subsequently infected with Ad-GFP-CD79b. A MOI ¼ 10 was used and cells were analysed 24 h after gene transfer. A clear-cut increase in sIgM
expression was observed following infection with Ad-GFP-CD79b, but not the control Ad-GFP vector; these figures were comparable with those
observed following co-transfection of these cells with the previously described pCD79b plasmid (Indraccolo et al, 2002), which was used as a positive
control for the molecular reconstitution experiment. The percentage and the MFI are shown in the upper right corner of each histogram. At least
20 000 cells were analysed in each histogram. This experiment was repeated twice with similar results. (D) The Ad-encoded CD79b-HA is phos-
phorylated following BCR activation. 293T cells were first transfected with expression plasmids for IgM, CD79a and then infected with the Ad-GFP-
CD79b vector or the Ad-GFP control vector. 293T cells were subsequently stimulated with anti-IgM (10 lg/ml) and cell lysates were analysed at
different time points by immunoprecipitation with an antibody to phosphotyrosine and immunoblotting with an anti-HA antibody (top panel).
CD79b-HA showed a basal level of phosphorylation in 293T cells, which increased as a function of time following BCR stimulation. Immunoblotting
of the same lysates with an antibody against Syk (bottom panel) was performed to estimate the amount of proteins loaded in each lane.
Intracellular expression of BCR components in B-CLL cells
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889 885
an hallmark of this disease (reviewed in: Caligaris-Cappio &
Ghia, 2004); yet, the intracellular expression of Ig, CD79a, and
CD79b has been less well evaluated. Because different mecha-
nistic explanations have been proposed for this phenomenon,
including lack of expression of selective components of the
complex (Thompson et al, 1997), or a defect in the transport
of normally synthesised BCR components to the cell surface
(Payelle-Brogard et al, 2002), and surface analysis cannot
discern between these possibilities, we performed a thorough
analysis of surface and intracellular expression of all BCR
components in B-CLL cells. We found an heterogeneous
pattern of expression of the BCR components in 22 B-CLL
samples, ranging from those whose phenotype was similar to
normal B cells (2/22), to others which almost lacked intracel-
lular IgM expression (3/22). The relative majority of the B-CLL
samples (17/22), however, presented with a sort of dichotomy
in the surface and intracellular expression levels of the BCR
molecules; intracellular staining was considerably stronger
than membrane-associated reactivity, and this was also
confirmed by confocal microscopy analysis of some samples
(not shown). Interestingly, about one-third of these samples
(9/22) showed low level intracellular reactivity principally for
CD79b. On the other hand, lack of cytoplasmic CD79a was
found only in one of 22 samples, indicating that molecular
defects of this component of the BCR may occur more rarely
compared to CD79b. Thus, our study provides a further
demonstration of B-CLL heterogeneity and one of its major
conclusions is that a similar surface phenotype of B-CLL cells
may be explained by different molecular mechanisms. For
instance, three samples had very low to absent surface IgM,
CD79a, and CD79b reactivity, which correlated with unde-
(A)
(B)
(C)
Fig 4. Ad-mediated CD79b gene transfer modulates sIgM expression in MEC2 cells. (A) Patterns of surface and intracellular IgM, CD79a, and CD79b
expression in MEC2 cells. Cells were analysed after incubation with anti-IgM, anti-CD79a, and anti-CD79b Abs conjugated with different fluoro-
chromes, as detailed in Fig 1 and in the Materials and methods. The percentage and the MFI are shown in the upper right corner of each histogram.
At least 20 000 cells were analysed in each histogram. The shaded areas represents the controls. (B) Infection of MEC2 B cells by Ad-GFP-CD79b leads
to detection of intracellular CD79b-HA and an increase in sIgM levels by flow cytometric analysis. A MOI ¼ 1000 was used and cells were analysed
36 h after gene transfer using a mouse mAb against the haemagglutinin (HA) tag and PE–conjugated anti-mouse Ig Ab as the second reagent, or a
rabbit anti-IgM-PE Ab. CD79b-HA and IgM expression were measured in the total and selectively GFP+ cell population respectively; at least 20 000
cells were analysed in each panel. (C) Surface IgM expression in MEC2 infected cells in five experiments performed as described in (B).
Table III. Expression of sIgM in MEC2 cells following CD79b gene
transfer.
Group Ad-GFP Ad-GFP-CD79b
GFP IgM GFP IgM
MEC2 % %� MFI % %� MFI
Experiment 1 40 18 437 20 39 454
Experiment 2 45 33 420 23 66 513
Experiment 3 13 28 337 3Æ6 46 395
MFI, mean fluorescence intensity; GFP, green fluorescent protein.
�P < 0Æ05. IgM expression was measured selectively in the GFP+ cell
population.
S. Minuzzo et al
886 ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889
tectable intracellular IgM; CD79a and CD79b, however, were
readily detected in these cells. In these samples, the low level
BCR expression may be conceivably accounted for by defective
Ig synthesis. Nine of 20 samples had reduced but appreciable
surface and intracellular IgM and CD79a levels and presented
with a marked reduction in the expression of intracellular
CD79b; in this case, impaired assembly of the BCR complex
may be reasonably suspected. Finally, eight of 20 samples had
only slightly reduced sIgM levels, mainly in terms of MFI
values, in spite of normal or close-to-normal intracellular
levels of the BCR components; in this case as well, complex
abnormalities in BCR assembly and transport to the cell
surface – as recently hypothesised by Payelle-Brogard et al.
(Payelle-Brogard et al, 2002) – rather than impaired synthesis
of its components, might account for the phenotype of the
cells.
To test the idea that gene transfer of BCR accessory chains
into B-CLL cells could, at least in some cases, overcome the
BCR transport defect(s) thus increasing sIg expression, we
generated an Ad encoding CD79b fused to the HA tag. Ad is
a type of vector that has been used to deliver genes to B-CLL
cells (Cantwell et al, 1996; Huang et al, 1997; Takahashi et al,
2001). Molecular reconstitution experiments in 293T cells
clearly indicated that, in a controlled situation where lack of
sIg expression was merely because of absence of CD79b, the
introduction of the full-length molecule by Ad gene transfer
sufficed to correct the cell phenotype. The ability of our
construct to vicariate functional BCR assembly was also
confirmed in a B cell line, termed MEC2, derived from a
B-CLL patient (Stacchini et al, 1999). MEC2 cells were
characterised by low level sIgM expression associated with
very low intracellular levels of CD79b, which contrasted with
abundant cytoplasmic CD79a and IgM levels (Fig 4A). Thus
the phenotype of these cells resembled, in terms of BCR
expression, that of B-CLL samples belonging to the low II
group (Table II). In MEC2 cells, the amount of CD79b was
clearly one rate-limiting step of BCR export to the cell
membrane, as an increase of intracellular CD79b levels by
gene transfer resulted in marked and rapid changes in the
levels of sIg. However, the finding that surface IgM was not
detected on the totality of MEC2 cells, even following
infection with very high amounts of adenoviral vector
encoding CD79b, drew our attention to the possibility that
other molecular defects may also limit BCR export in these
cells. Intriguingly, very recently Vuillier et al (2005) reported
glycosylation and folding defects of the IgM and CD79a
chains in B-CLL patients, which contrasted with unimpaired
folding and structure of CD79b, in spite of its low levels of
surface expression. We investigated this in MEC2 cells and
found that a large proportion of the l chains, but not
CD79a, was immature in these cells, thus indicating that
glycosylation defects of some components of the BCR could
possibly contribute to the phenotypic abnormalities of these
cells.
We also attempted transduction of primary B-CLL cells. In
our hands, however, the efficiency of gene delivery in B-CLL
cells freshly isolated from nine patients was extremely low
(range 0Æ1–0Æ5% transduced cells), and we failed to demon-
strate any significant increase in sIg levels following gene
transfer of CD79b, both in terms of percentage of IgM+ cells
and MFI (data not shown). This was not because of
insufficient input of vector particles, because we have used
very high vector to target cell ratios (MOI ¼ 1 · 103),
comparable with those used in other studies (Cantwell et al,
1996). The observation that monocytes, which were occasion-
ally present in the samples, were efficiently transduced (data
not shown) may indicate an intrinsic resistance of non-
activated B-CLL cells to infection by the Ad. Previous reports
on the ability of Ad to transduce B-CLL cells are contradictory.
Some investigators have concluded that such cells are refrac-
tory to Ad infection (Wattel et al, 1996), whereas others report
successful transduction, generally following some sort of
cellular activation (Huang et al, 1997; Takahashi et al, 2001),
or employing very high MOI (>500) (Cantwell et al, 1996). As
inefficient gene transfer may preclude the possibility of
achieving high-level exogenous CD79b expression in resting
B-CLL cells, our study should prompt the investigation of
other pathways to upregulate endogenous CD79b synthesis in
B-CLL cells.
Fig 5. Glycosylation analysis of IgM and CD79a in MEC2 cells. MEC2
and Daudi cell lysates were incubated at 37�C in the presence (+) or
absence of Endo-H ()), separated by 10% SDS-PAGE, and then the
proteins were transferred to nitrocellulose. Filters were probed with a
goat anti-l heavy chain (panel l) or a mouse anti-CD79a Ab (panel
CD79a) and immunoreactive bands were detected with an appropriate
horseradish peroxidase-linked secondary Ab. Mature glycosylated (*)
proteins and forms deglycosylated (�) by Endo-H treatment are
indicated for each stain.
Intracellular expression of BCR components in B-CLL cells
ª 2005 Blackwell Publishing Ltd, British Journal of Haematology, 130, 878–889 887
Acknowledgements
Supported by AIRC (Associazione Italiana per la Ricerca sul
Cancro), FIRC (Fondazione Italiana per la Ricerca sul Cancro),
Ministero dell‘Universita e Ricerca Scientifica (MIUR 60%,
PRIN and FIRB), ISS-AIDS Project. V.T. is a recipient of an
AIRC fellowship. We thank the vector core of the University
Hospital of Nantes (France) supported by the Association
Francaise contre les Myopathies (AFM) for providing the
adenovirus vectors used in this study. The invaluable help of
P. Gallo and A. Azzalini in artwork preparation is also
gratefully acknowledged.
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