1. No category

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1996 87: 717-724
B-chronic lymphocytic leukemias can undergo isotype switching in
vivo and can be induced to differentiate and switch in vitro [see
comments]
F Malisan, AC Fluckiger, S Ho, C Guret, J Banchereau and H Martinez-Valdez
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B-Chronic Lymphocytic Leukemias Can Undergo Isotype Switching In Vivo
and Can Be Induced to Differentiate and Switch In Vitro
By Florence Malisan, Anne-Catherine Fluckiger, Stephen Ho, Christiane Guret, Jacques Banchereau,
and Hector Martinez-Valdez
B-chronic
lymphocytic
leukemias
(B-CLL)
represent
expandedclones of resting B lymphocytes
that mostly express
surface IgM (slgM). The present study shows that B-CLL
cells, freshly isolatedfrom two patients, were slgM+, slgG-,
and slgA- but expressed IgG and IgA transcripts. cDNA cloning and sequencing showedthat the VDJ segments associto those
ated to y and Q heavy chain transcripts are identical
from p transcripts, thus showing that B lymphocytes giving
rise to CLL cells have undergone isotype switchingin vivo.
Stimulation of theseB-CLL cellsthrough surface CD40in the
presence of interleukin-l0 induced them to secrete IgG and
lgA, proving that they can alsodifferentiate into Ig-secreting
cells. Finally, CM-stimulated B-CLL cells were induced to
switch towards IgE in response to interleukin-4, as shown
by the presence of specific VDJ-Ce transcripts
and the secretion of IgE. Therefore, B-CLL cellstested herein can undergo
isotype switching in vivo and can be induced to undergo
further isotype switching anddifferentiation in vitro.
0 1996 by The American Societyof Hematology.
T
mined by standard isotype-specific enzyme-linked immunosorbent
assay (ELISA).~~
Factors. Purified recombinant interleukin-4 (IL-4; lo7 U/mg)
and highly purified recombinant I L - I O from Chinese hamster ovarytransfected cells (both from Schering-Plough Research Institute,
Kenilworth, NJ) were used at the final concentration of 500 U/mL
and 100 ng/mL, respectively.
B-cell cultures. Purified leukemic B cells were cultured in Iscove’s medium (Flow Laboratories, Irvine, CA) enriched with S0
pg/mL human transfenin, O S % bovine serum albumin (Sigma), and
S pg/mL bovine insulin (all from Sigma Chemical CO, St Louis,
MO); and S% selected heat-inactivated fetal calf serum, 1 0 0 U/mL
penicillin, and 1 0 0 pg/mL streptomycin (all from Flow Laboratories). For activation of B-CLL through the CD40 antigen, leukemic
B cells were cultured in the presence of the anti-CD40 MoAb89
presented by a mouse Ltk- cell line stably expressing CDw32
(CDw32 L cells). Briefly, IO5 B-CLL cells were cultured in roundbottom microtiter trays under a final volume of 1 0 0 pL, with S,OOO
irradiated (70 Gy) CDw32 L cells and 0.5 pg/mL of anti-CD40
MoAh89. Cytokines were added at the onset of the culture.”
RNA preparation and northern blotting. Isolation of total RNA
was performed by the method of Chomczynski.” Extracted unfractionated RNA (20 pg) was electrophoresed, under denaturing conditions, on 1% agarose gel (stained with ethidium bromide) using the
method of LerachI3 and was transferred onto nylon membranes as
described by Thomas.14The blot was hybridized to constant region
cDNA probe Cp, Cy, and Ca labeled by 32P using the random
priming kit according to manufacturer’s instructions (Boehringer,
Mannheim, Germany).
Reverse-transcription polymerase chain reaction (RT-PCR)
assays. Total RNA (1 @g) was converted into single-stranded
cDNA by a standard RT reaction using oligodT,,,.,,, (Pharmacia,
LKB, Uppsala, Sweden) and Superscript (RNase H- Moloney murine leukemia virus reverse transcriptase; BRL, Gaithersburg, MD)
kit, according to manufacturer’s instructions. The PCRwas per-
HE GENERATION OF functional antibodies is the result of multiple genetic changes at the Ig loci. During
B lymphopoiesis, gene segments at the Ig heavy (H) and
light (L) chain loci assemble in a defined, ordered manner.’.*
This developmental pathway, allows the commitment and
the maturation of hemopoietic progenitors cells into B lymphocytes, with functional heavy and light chains, expressing
IgM at their ~ u r f a c e . ~
The
. ~ non-self-reactive low-affinity
cells develop further to express sIgD, giving rise to mature
B cells, also known as naive or virgin B cells. At a later
stage, which is driven by antigen and is T-cell-dependent
(referred herein as immunopoiesis), B lymphocytes undergo
affinity maturation through somatic mutations and isotype
switching. During these distinct stages, B lymphocytes become the target of abnormal development, resulting in diverse forms of leukemia^.^,^ Thus, B-chronic lymphocytic
leukemias (B-CLL) could phenotypically be identified in a
transition stage between immature sIgM+, sIgD- and mature
sIgM+, sIgD+ naive B cells. In this context, B-CLL cells
are characterized by a marked increase of resting immature/
mature B cells expressing low quantities of surface Ig, in
which the isotype most frequently found is IgM or the combination of IgM and IgD.7 It has been previously reported
that B-CLL cells can proliferate and differentiate into Igsecreting cells after in vitro stimulation.’ However, it has
not been formally shown whether such Ig secretion indeed
results from in vivo and/or in vitro B-CLL isotype switching
or from the contribution of contaminating normal B lymphocytes within the CLL population. In the present report, by
analysis of the cDNA sequences of Ig genes expressed by
B-CLL cells from two patients, we show that B lymphocytes
that give rise to CLL cells have the capacity to undergo
isotype switching and differentiation.
MATERIALS AND METHODS
Patients. Clinical features of patients FLA and PAS and purification of B lymphocytes from peripheral blood of patient FLA and
from spleen of patient PAS have been previously described? Briefly,
mononuclear cells were separated by standard FicolVHypaque gradient method and were next submitted to E-rosetting with sheep red
blood cells. Nonrosetting cells were labeled with anti-T-cell and
antimonocyte monoclonal antibodies (MoAbs) and were subsequently incubated with magnetic beads coated with anti-IgG antibodies (Dynal, Oslo, Norway).
Ig secretion. The secretion of IgM, IgG, IgA, and IgE was deterBlood, Vol 87, No 2 (January 15). 1996 pp 717-724
Fromthe
Laboratory for Immunological Research, Schering
Plough, Dardilly, France.
Submitted March 7, 1995; accepted September I , 1995.
F.M. is a recipient of a fellowship fromFondation Mkrieux.
Address correspondence to Hector Martinez-Valdez, PhD, Schering-Plough, Laboratory for Immunological Research, 27 chemin des
Peupliers. BP 11, 69571, Dardilly, France.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
oooS-4971/96/8702-01$3.00/0
717
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MALISAN ETAL
718
W
a
F M
PAS
Y
[r
Z K h K h
+ 650 bp
ATACAGCTlTACC 3’; and VH6, S‘ TGCCATCTCCGGGGACAGTGTCTCT 3‘.
Cloning u ~ t d.sc.yrrenring. TheproductsfromtheRT-PCRwere
directly cloned, without further processing. by the TA cloning kit
as described by themanufacturer (Invitrogen, SanDiego, CA).”
Sequencing reactions were performed using the Taq DyeDeoxyTermination Cycle sequencingkit (catalog no. 401 150; Applied Biosystems Inc. Roissy. France) and were analyzed on an Applied Biosystems automatic DNA sequencer.
RESULTS
Freshly isolated sIgM’, slgG-, slgA- R-CLL cells express
y and a herny-chain trclnscripts. As previously described,”
F M PAS
1 2 3 4
- 4.7
Fig 1. Homogeneity of the B-CLLcells. Monoclonality of the BCLL populations was evaluated through RT-PCR on the expression
of K or A light chains. Amplifying sense primers were either VK (lanes
1 and 3)or V h (lanes2and 4) consensus primers, usedin combination
with corresponding CK or CA antisense primers, respectively.
formed as described by Saiki et al.’5 with minor modifications. A
total of S pL of theRT reactionwasamplifiedusing
1 0 0 ng
of each sense and antisenseprimers, 2.5 U ofTaq Polymerase
(Cetus. Norwalk, CT). and S% dimethyl sulfoxide. Primers used
were as followsforIgL
chain: VK, S‘ GTGTTGACCCAGTCTCCAGCCTCC 3’; CK. S’ GCGCCGTCTAGAACTAACACTCTCCCCTGTTGAAGCTCITTGTGACGGGCAAG 3’; VA. 5‘
GCTGACTCAGCCGCCCTCTGTGTC 3‘; andCA. S’ GCCTCGAGCTATGAACATTCTGTAGGGGCCAC 3’.
A totalof 35 cycles of amplification were performed ( I minute
at 94°C. 2 minutes at 60°C. and 3 minutes at 72°C). A second round
10 pL of the
was performed using the same primers. starting with
first round.
as described
For IgH chain. cDNAandPCRwereperformed
above. Two rounds of28 cycles wereperformed (30 seconds at
94°C. 30 seconds at 60°C. and I minute at 72°C). The primers used
in thefirstroundwere
as follows: S’VH consensus S’ TCTGAGGTGCAGCTGGTGGAGTCTG3‘ and Cp, S’ GAATTCTCACAGGAGACGAGGGG 3’; Ch. S’ TTCTTCCTCTAGAAGGCGACCGGT 3‘; Cy. S’ AAGTAGTCCTTGACCAGGCAG 3’: Ca. 5’
GGGTCAGCTGGGTGCTGCTGG3‘; CC.S’ TGTAAGGGAGGTACGGTGGAGGCA 3’; or JH, 5‘ TGAGGAGACGGTGACCAGGGTCCC 3‘.
The second round was performed using 10 pL of the first round,
usingthesameprimersexceptfor
Cy , Cc,and Ca forwhich
more 3’ internal primers were used in a seminested fashion: Cy, 5’
TTCCCGGGTAGCCAGAAG3‘; Ca. S’ CCCGGAGGCATCCTGGCTGGG 3’: andCc. S’ AGCGAGTGGCATTGGAGGGAATGT
3’.
Sourhent blotrirtg m d /tyhrir/i:ofinns. PCR products were transferred to nylon membranes, as previously reported,“’ and hybridized
with VH probeslabeledusingthedigoxygenin-deoxyuridinetriphosphate (dUTP) kit (Boehringer, Mannheim, Germany). The
probes usedwere consensus oligonucleotide sequences to thesix
VH families: VHI. S’ CAAGGCTTCTGGATACACCTTCACC 3‘;
VH2. S’ CACCTTCTCTGGGTTGTCAGTCACC 3’; VH3. S‘
TGCAGCCTCTGGATTCACCTTCAGT 3’; VH4, 5’ CGCTGTCTCTGGTGGCTCCATCAGC 3‘: VHS, S’ TAAGGGTI’CTGG-
1.9
”8- “ ‘
- 4.7
- 1.9
l
- 4.7
Ca
- 1.9
Fig 2. Comparative Northern blot analysis of IgM, IgG, and IgA
mRNA levels expressed by the B-CLL cells FLA and PAS.Total unfractionated RNA (20 pg) from each B-CLL was electrophoresed on 1%
agarose and transferred onto nylon membranes.The three blots prepared were hybridizedto constant region Cp. Cy, or Ca probes. Size
markers (in kilobases) are indicated at right. Molecular sizes correspond to those reported e l s e ~ h e r e ? ~Autoradiography
-~’
exposure
times are as follows: Cp. 30 minutes; Cy, 1 hour; Ca. 16 hours.
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SWITCH
B-CLL
ISOTYPE
CELLS CAN
IN VIVO AND IN VITRO
719
5Y
FM
A
450
550 bp
z
f- 660 bp
1 2 3 4 5
FLA
VH1
6 7 8 9 1 0
B-CLL cells
B
Fig 3. Expression of IgH
genes by freshly isolated B-CLL
cells FLA and PAS. (AI Presence
of IgG and IgA transcriptsin addition t o IgM andIgD was evaluated by isotype-specific RT-PCR.
This experiment was performed
using a 5' consensus VH primer
and a 3' primer Cp (lanes 1 and
6). Cy (lanes 2 and 7). C(I (lanes
3 and 8). C& (lanes 4 and 9). or
CE (lanes 5 and 10). The upper
bands correspond to the expected PCR products. The lower
bands seem t o be RT artifact,
because sequencing ofthose
showed that theycorrespond t o
truncated
forms
of
the
Igs
(Songsvilai et at3' and our own
unpublished observations). (B)
To determine the origin
of y and
(I transcripts, the PCR products
VDJCp (lanes 1 and 6). V D J G
(lanes 2 and 7). VDJCy (lanes 3
and 8). VDJCa (lanes 4 and
9) and VDJCE (lanes 5 and 10)
werehybridized t o VH-specific
probes. Allthetranscripts
expressed by FLA and PAS used
VH3 and VH1 family, respectively. Lane 11 represents a control performed on normal B cells
after consensus VH-JH amplification andVH-specific hybridization.
PAS
550 bp
450 bp
-
P 6Y a&
Normal
B cells
PAS
p 6 y a & JH
3
f-400
bp
VH2
VH3
660 bp
550 bp
450 bp
3
+400 bp
VH4
4
+
VH5
400 bp
VH6
freshly purified B cells from the patients PAS and FLA are
CD5'IgMK' and CD5' IgMA', respectively. Both surface
and intracytoplasmic IgG as well as surface IgA were negative (data not shown). By using RT-PCR amplification of
light chain transcripts (Fig I), it was shown that the CLL
1 2 3 4 5
6 7 8 9 1 0
11
sample FLA exclusively expressed A light chain, whereas
sample PAS only expressed K. These results stress the clonality of the B-CLL cells.
In spite of the absence of both intracytoplasmic and surface IgG as well as surface IgA expression, y and (Y heavy-
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720
MALISAN ET AL
Fig 4. B-CLL cells FLA and PAS undergo isotype switching in vivo. The VDJ regions associatedto Cp. Cy, and Cm of the freshly isolated BCLL cells FLA (A) and PAS (B) were cloned, sequenced, and compared with the corresponding germline sequence.".40
chain W A S could be detected by Northern analysis (Fig
2). The majority of the transcripts were p, but significant
levels of y and a transcripts could also be detected. Such
level of expression of p, 7 , and a , reflecting corresponding
mRNA frequency, was indicated by the sensitivity of detection, ie, p > y > a.
Freshly isolated B-CLL cells show p, y, and a transcripts
with a unique VDJ region. The expression of IgH genes
was further analyzed using an isotype-specific RT-PCR (Fig
3A). y and a transcripts could be detected in addition to p
and 6 transcripts, confirming the results obtained by Northern
analysis. To establish the B-CLL origin of the y and a
transcripts, analysis of their VH repertoire was performed.
As shown inFig 3B, after amplification and VH-specific
hybridization, the p (450 bp), y (450 bp), and a (550 bp)
transcripts expressed by B-CLL FLA or PAS used the same
VH family genes. Therefore, whereas the B-CLL cells FLA
exclusively expressed a VH3 family gene, PASonly expressed a VH1 family gene, indicating that p, y , and a
transcripts originated from the same leukemic clone. Furthermore, we investigated whether the freshly isolated B-CLL
cells expressed transcripts showing identical VDJ segments
associated to different constant region genes. To this end,
the cDNAs corresponding to the p, y , and a transcripts, from
B-CLL cells FLA and PAS, were cloned and sequenced.
Consistent with the VH repertoire analysis, sequencing data
shown in Fig 4A and B showed that p, y , and a heavychain transcripts shared the same VDJ segments, proving
that the y and a transcripts expressed by B-CLL cells FLA
and PAS is the result of in vivo isotype switching. Comparison and alignment of these sequences to corresponding
germline genes, showed little or no somatic mutation, suggesting that the cells did not undergo affinity maturation and
selection.
IL-10 induces B-CLL cells to differentiate into IgG- and
IgA-secreting cells. B-CLL cells were cultured in the
CD40 system in the presence of IL-10, conditions that result
in efficient differentiation of normal B lymphocytes."." Ig
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B-CLL CELLSCAN ISOTYPE SWITCH IN VIVO AND IN VITRO
FLA
lo00
800
600
400
200
r
FLA
1200
lo00
800
600
400
200
0
PAS
PAS
PAS
L
FLA
Fig 5. IL-10 induces anti-CD40-activated B-CLL cells FLA and PAS
to secrete IgM, IgG, and IgA. B cells were cultured for 14 days in the
lo6 Bcells were cultured with 5,000 irradiated
CD40system;ie,
C M 2 L cells and 0.5 pg/mL of anti-CD40 MoAb8S in the presence
or absence of 100 ng/mL IL-10. Their Ig content was measured in
standard ELISA, and results were expressedas mean of triplicate
determination. Standard deviation was less than 10%. (m),no cytokine: (01, IL-10.
levels were measured in day-l4 supernatants in standard
ELISAs. The addition of IL-IO resulted in a significant production of IgM, IgG, and IgA (Fig 5) without evidence of
IgE secretion (data not shown), suggesting that IL-10 can
induce CD40-activated B-CLL cells to differentiate into IgGand IgA-secreting cells. Note that the response of B-CLL to
IL-l0 was not as efficient as that of normal B cells; ie, 100
to 1,100 ng/mL of IgG and IgA were detected for B-CLL
721
FLA and PAS as compared with 1,000 to 5,000 ng/mL for
normal B-cell samples. Therefore, it is conceivable that the
defect behind the B-CLL cells FLA and PAS studied herein
lies on their incapacity to differentiate in vivo into Ig-secreting cells, rather than their inability to undergo isotype
switching. Although such defect can be partly restored by
physiological differentiation signals known to operate in normal B lymphocytes, no quantitative correlation is expected
between the Ig transcripts observed in vivo and the levels
of Ig secreted after in vitro stimulation.
To rule out the possibility that secretion of Igs derived
from contaminating normal B lymphocytes, which actively
proliferate in the CD40
RT-PCR-amplified
cDNAs, corresponding to the y and a transcripts from the
CD40-activated B-CLL FLA in the presence of IL-10, were
cloned and sequenced. We chose to examine FLA VDJ sequence because of its higher Ig secretion levels, in which
the risk of contribution by contaminating normal B cells
required to be ruled out. Sequence analysis of the y and a
transcripts of stimulated B-CLL FLA cells and p transcripts
of freshly isolated B-CLL FLA cells, as shown in Fig 6,
showed that VDJ segments were identical, confirming that
the secretion of IgG and IgA was of CLL origin.
Expression of IgE by B-CLL cells is the result of isotype
switching in vitro. As shown in Figs 3A and B, freshly
isolated B-CLL cells FLA and PAS did not express E transcripts, indicating that they did not undergo isotype switching towards IgE in vivo. Therefore, the induction of in vitro
isotype switching towards IgE was analyzed after the culture
of the B-CLL cells FLA and PAS in the CD40 system in
the presence of IL-4. As determined by ELISA experiments
shown inFig 7, after in vitro stimulation, IgM and IgE
secretion could be detected, but there was no induction of
IgA and IgG secretion (data not shown). To establish, at the
molecular level, that the secretion of IgE resulted from in
vitro isotype switching, RT-PCR-amplified cDNAs, corresponding to E transcripts from B-CLL cells stimulated in
the CD40 system in the presence of IL-4, were cloned and
sequenced. As shown in Fig 8, the E heavy-chain transcripts
show the same VDJ domain as p, thus showing that B-CLL
cells can be induced to switch in vitro.
DISCUSSION
The present report shows at the molecular level that BCLL cells express isotypes other than IgM, as shown by the
presence of p, y , and a heavy chain transcripts showing the
same VDJ segments. However, the detection of IgG and IgA
transcripts in these B-CLL cells does not discriminate from
isotype switching taking place either before or after the malignant transformation event.
On the other hand, although these cells express significant
levels of mature y and a heavy-chain transcripts, they do
not show cytoplasmic or cell surface expression of mature
IgG and surface IgA (data not shown). Because the light
chain is not a limiting factor for the assembly of the mature
Ig, inasmuch as sIgM can be detected on the cell surface,
these data suggest that transcription and translation of these
B-CLL cells for IgG and IgA expression may be uncoupled.
This apparent impaired translation can be released on cultur-
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MALISAN ET AL
FR1
CDRl
TCTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTTCCTATA
CH
GAGAAGCACTATGGACGTCTGGGGCCAAGGGGACCACGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAGCCCTTTTC
GCCTCCACCAAGGGCCCATCGGTCTTC
"""_""""""-"""""""""""""""GCATCCCCGACCAGCCCCAAGGTCTTC
..........................
Fig 6. Sequence analysis of
the VDJ regions of y, a,and p transcripts confirmsthat IgG and IgA secretion isof CLL origin. RT-PCR-amplified
cDNAs corresponding to y and a transcripts from the stimulated B-CLL FLA cellswere cloned and sequenced, and VDJ regions were compared
with those of p transcript from the freshly isolated B-CLL cells.
ing B-CLL cells in the CD40 system in the presence of IL10, resulting in a significant secretion of IgG and IgA. It is
worth noting that it is necessary to apply this strong differentiation stimulus for these B-CLL to express IgG and IgA,
because polyclonal activation with Staphylococcus aureus
Cowan (SAC; data not shown) or CD40 activation without
'8007
IgM
a
b
r
400
r
0
FLA
PAS
n
FLA
PAS
Fig 7. lL-4 induces anti-CW-activated B-CLL cells to switch towards IgE. A total of 10' B cells was cultured for 21 days with 5 x
10' irradiated CDw32 L cells and 0.5 p g h L of anti-CD40 MoAb89
with or without 500 UlmL IL-4, and their Ig content was measured
by ELISA. Results were expressed asthe mean of triplicate determination. Standard deviation was less than 10%. (MI,no cytokine; (01.
IL-4.
IL-l0 does not allow IgG and IgA secretion. Furthermore,
IL-10 alone does not allow production of IgGandIgA."
Because isotype switching of normal B cells occurs within
secondary lymphoid organs and, more specifically, within
germinal centers of secondary follicle^,*^ it may be that
translation of switched isotype transcripts necessitates signals that
are given within these microenvironments.24.25
As a matter
of fact, T cells bearing CD40 ligand and producing IL-l0
and IL-4 have been detected within germinal centers.*'.*'
Thus, it is possible that the translation blockade of some
leukemic cells may be because of the lack of germinal center
microenvironment components related to the altered secondary lymphoid organ structure that is observed in CLL." It
may be that the defect in FLA and PAS B-CLL cells lies in
their incapacity to differentiate in vivo, rather than in their
inability to undergo isotype switching. However, other reports described isotype-switched variants of CLLZyand BCLL cells expressing surface IgG."" It is likely that these
variants may represent B-CLL cells that have differentiated
after an encounter withthe relevant signals (eg, an auto
antigen, as described in some CLL cases)."
Although IL-10 has been suggested to act as a switching
factor for CD40-activated B cell^,^'.^^ we cannot conclude
whether the IgG and IgA production of E A and PAS BCLL cells obtained in response to the combination of antiCD40 and IL- 10 is the result of further isotype switching in
vitro and/or the induction of IgG and IgA synthesis and
secretion. In this context, the secretion of IgE, whose corresponding transcripts show the original VDJ gene, in response
to CD40- and IL-4 signaling shows that CLL cells can be
induced to undergo isotype switching in vitro. Such a result
confirms and further extends the results of a previous study
that showed the secretion of IgE by B-CLL cells cultured
with hydrocortisone and IL,-4.34
In conclusion, the present study has shown that B cells
giving rise to CLL cells have the capacity to undergo isotype
switching and differentiate into Ig-secreting cells. Whether
the presence of IgG and IgA transcripts in B-CLL represents
a stochastic event or, indeed, is the consequence of a specific
signal remains to be determined.
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E-CLLCELLSCAN
723
ISOTYPE SWITCH IN VIVO AND IN VITRO
F LA
CH
GACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTTTTTC
GCCTCCACACAGAGCCCATCCGTCTTCC
"""""""""""""""""""~
PAS
FR1
CDRl
TCTGAGGTGCAGCTGGTGGAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTG~GGTCTCCTGCAAGGCTTCTGGATACACCTTCACC~CTACTATAT
""~"""_"""__""""~"""""~""""""~"""""~""""""""~""""""""-"""
FRZ
FR3
CDRZ
GCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG~ATGGATC~CCCTAACAGT~TGGCAC~CTATGCACAGAAGT~CAGGGCTGGGTCA
"""""_""__"""""~"~"""~"""""""""""""""""""~"""""""""""""-
D
CCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGCGTTGGT~TAGG
""--""~"""""~"""""""""""""""""""""""""""~"""""""""""--"-
CB
TACTACTATGATAGTAGTGGTCCTGATGCTTTTGATATCTGGGGCCAAGGGACAAT~TCACCGTCTCTTCAGGGAGTGCATCCGCCCCAACCCTTTTTC
--T---------------------------------------------------------------------GCCTCCACACAGAGCCCATCCGTCTTCC
Fig 8. Sequence analysis of p and c transcripts VDJ regions shows that IgE secretion is the result of an in vitro isotype switching. RTPCR-amplified cDNAs correspondingto B transcripts from B-CLL cells FLA and PAS cultured in theCD40 system in the presence of IL-4 were
cloned and sequenced, and VDJ regions were compared with those of p transcript from freshly isolated B-CLL cells.
ACKNOWLEDGMENT
We are grateful to Dr J. Chiller for support, to Drs F. Briltre
and S. Saeland (Schering-Plough Laboratories, Dardilly, France) for
critical review of the manuscript, and to MS M. Vatan and N. Courbiltre for editorial assistance.
REFERENCES
l . Tonegawa S: Somatic generation of antibody diversity. Nature
302575, 1983
2. Alt F W , Oltz EM, Young F, Groman J, Taccioli G, Chen J:
VDJ recombination. Immunol Today 13:306, 1992
3. Rolink A, Kudo A, Karasuyama H, Kikuchi Y, Melchers F
Long-term proliferating early pre B cell lines and clones with the
potential to develop to surface Ig-positive, mitogen reactive B cells
in vitro and in vivo. EMBO J 10:327, 1991
4. Banchereau J, Rousset F Human B lymphocytes: Phenotype,
proliferation and differentiation. Adv Immunol 52:125, 1992
5. Sawyers CL, Denny CT, Witte ON: Leukemia and the disruption of normal hematopoiesis. Cell 64:337, 1991
6. O'Brien S, del Giglio A, Keating M: Advances in the biology
and treatment of B-cell chronic lymphocytic leukemia. Blood
85307, 1995
7. Dighiero G, Travade P, Chevret S, Fenaux P, Chastang C,
Binet J-L, The French Cooperative Group on CLL: B-cell chronic
lymphocytic leukemia: Present status and future directions. Blood
78:1901, 1991
8. Totterman TH, Nilsson K, Sundstrom C: Phorbol ester-induced
differentiation of chronic lymphocytic leukaemia cells. Nature
288:176, 1980
9. Fluckiger AC, Rossi JF, Bussel A,BryonP, Banchereau J,
Defrance T: Responsiveness of chronic lymphocytic leukemia B
cells activated via surface Igs or CD40 to B-celltropic factors. Blood
803173, 1992
10. Chrktien I, Pltne J, Briltre F, De Waal Malefyt R, Rousset F,
De Vries JE: Regulation ofhuman IgE synthesis: I. Human IgE
synthesis in vitro is determined by the reciprocal antagonistic effects
of IL-4 and IFN-7. Eur J Immunol 20:243, 1990
1 1. Fluckiger A-C, Gamone P, Durand I, Galizzi JP, Banchereau
J: IL-l0 upregulates functional high affinity IL-2 receptors on normal
and leukemic B lymphocytes. J Exp Med 178:1473, 1993
12. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162156, 1987
13. Lerach J, Diamond D, Wozney J, Baaltken H: RNA molecular
weight determinations by gel electrophoresis under denaturating conditions, a critical re-examination. Biochesmistry 16:4743, 1977
14. Thomas P: Hybridization of denaturated RNA and small DNA
fragments transferred to nitrocellulose. Proc NatlAcad Sci USA
775201, 1980
15. Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn
GT, Mullis KB, Erlich HA: Primer-directed enzymatic amplification
of DNAwith a thermostable DNA polymerase. Science 239:487,
1988
From www.bloodjournal.org by guest on October 21, 2014. For personal use only.
724
16. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A
Laboratory Manual (ed 2). Cold Spring Harbor, NY, Cold Spring
Harbor Laboratory, 1989
17. Holton TA, Graham MW: A simple and efficient method for
direct cloning of PCR products using ddT-tailed vectors. Nucleic
Acids Res 19:1156, 1990
18. Banchereau J, de Paoli P, Vallt A, Garcia E, Rousset F Long
term human B cell lines dependent on interleukin 4 and antibody to
CD40. Science 251:70, 1991
19. Rousset F, Garcia E, Defrance T, Ptronne C, Vezzio N, Hsu
DH, Kastelein R, Moore KW, Banchereau J: Interleukin 10 is a
potent growth and differentiation factor for activated human B lymphocytes. Proc Natl Acad Sci USA 89:1890, 1992
20. Rousset F, Garcia E, Banchereau J: Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes
triggered through their CD40 antigen. J Exp Med 173:705, 1991
2 I . Banchereau J, Rousset F: Growing human B lymphocytes in
the CD40 system. Nature 353:678, 1991
22. Fluckiger A-C, Durand I, Banchereau J: Interleukin 10 induces apoptotic cell death of B-chronic lymphocytic leukemia cells.
J Exp Med 179:91, 1994
23. Martinez-Valdez H, Malisan F, de Bouteiller 0, Guret C,
Banchereau J, LiuYJ: Molecular evidence that isotype switching
occurs within the germinal centers. Ann NY Acad Sci 764: 151, 1995
24. DiSanto JP, Bonnefoy JY, Gauchat JF, Fischer A, de Saint
B a d e G: CD40 ligand mutations in X-linked immunodeficiency
with hyper-IgM. Nature 361541, 1993
25. Korthauer U, Graf D, Mages HW, Brihre F, Padayachee M,
Malcolm S, Ugazio AG, Notarangelo LD, Levinsky RJ, Kroczek
RA: Defective expression of T-cell CD40 ligand causes X-linked
immunodeficiency with hyper-IgM. Nature 361539, 1993
26. Lederman S, Yellin MJ, Inghirami G, Lee JJ, Knowles DM,
Chess L: Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B cell-activating
molecule (5c8 antigen) and CD40 in contact-dependent help. J Immunol 149:3817, 1992
27. Toellner K-M, Scheel-Toellner D, Sprenger R, Duchrow M,
Trumper LH, Ernst M, Flad HD, Gerdes J: The human germinal
centre cells, follicular dendritic cells and germinal centre T cells
produce B cell-stimulating cytokines. Cytokine 7:344, 1995
28. Hams NL, Jaffe ES, Stein H, Banks PM, Chan JKC, Cleary
ML, Delsol G, De Wolf-Peeters C, Falini B, Gatter KC, Grogan
TM, Isaacson PG, Knowles DM, Mason DY, Muller-Hermelink
H-K, Pileri SA, Pins MA, Ralfkiaer E, Warnke RA: A revised
MALISAN ET AL
European-American classification of lymphoid neoplasms: A proposal from the International Lymphoma Study Group. Blood
84:1361, 1994
29. Friedman DF, Moore JS, Erikson J, Manz J, Goldman J,
Nowell PC, Silberstein LE: Variable region gene analysis of an
isotype-switched (IgA) variant of chronic lymphocytic leukemia.
Blood 80:2287, 1992
30. Ikematsu W, Ikematsu H, Okamura S, Otsuka T. Harada M,
Niho Y: Surface phenotype and Ig heavy-chain gene usage in chronic
B-cell leukemias: expression of myelomonocytic surface markers in
CDS-chronic B-cell leukemia. Blood 83:2602, 1994
31. Schroeder HW Jr. Dighiero G: The pathogenesis of chronic
lymphocytic leukemia: Analysis of the antibody repertoire. Immunol
Today 15:288, 1994
32. Defrance T, Vanbervliet B, Briere F, Durand I, Rousset F.
Banchereau J: Interleukin 10 and transforming growth factor p cooperate to induce anti-CD40-activated naive human B cells to secrete
immunoglobulin A. J Exp Med 175:671, 1992
33. Briere F, Servet-Delprat C, Bridon J-M, Saint-Remy J-M,
Banchereau J: Human interleukin 10 induces naive sIgD' B cells to
secrete IgG, and IgG,. J Exp Med 179:757, 1994
34. Sarfati M, Luo H, Delespesse G: IgE synthesis by chronic
lymphocytic leukemia cells. J Exp Med 170:1775. 1989
35. Islam KB, Nilsson L, Sideras P, Hammarstrom L, Smith CIE:
TGF-01 induces germ-line transcripts ofbothIgA
subclasses in
human B lymphocytes. Int Immunol 3: 1099, 1991
36. Kuziel WA, Word CJ, YuanD, White MB, Mushinski JF,
Blattner FR, Tucker PW: The human immunoglobulin Cp-Cb locus:
Regulation of p and 6 RNA expression during B cell development.
Int Immunol 1 :310, 1989
37. Kondo N, Inoue R, Kasahara K, Kaneko H, Kameyama T,
Orii T: Failure of IgG production due to a defect in the opening of
the chromatin structure of Iyl region in a patient with IgG and IgA
deficiency. Clin Exp Immunol 99:2 I. 1995
38. Songsvilai S, Bye JM, Marks JD, Hughes-Jones NC: Cloning
and sequencing of human X imunoglobulin genes by the polymerase
chain reaction. Eur J Immunol 20:2661. 1990
39. Tomlinson IM, Walter G, Marks JD, Llewelyn MB. Winter
G: The repertoire of human germline VH sequences reveals about
fifty groups ofVH segments with different hypervariable loops. J
MolBiol 227:776, 1992
40. Kuppers R, Fischer U, Rajewsky K, Gause A: Immunoglobulin heavy and light chain gene sequences of a human CD5 positive
imrnunocytoma and sequences of four novel VHIII germline genes.
Immunol Lett 3457. 1992