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: stefano.indraccolo@unipd.it

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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