1. No category

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1994 84: 2597-2603
Expression of CD28 and CD40 in human myeloma cells: a
comparative study with normal plasma cells
C Pellat-Deceunynck, R Bataille, N Robillard, JL Harousseau, MJ Rapp, N Juge- Morineau, J
Wijdenes and M Amiot
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Expression of CD28 and CD40 in Human Myeloma Cells:
A Comparative Study With Normal Plasma Cells
By Catherine Pellat-Deceunynck, Regis Bataille, Nelly Robillard, Jean-Luc Harousseau, Marie-Jose Rapp,
Nadine Juge-Morineau, JohnWijdenes, and Martine Amiot
CD28 and CD40 are important activation pathways for T and
B lymphocytes, respectively. The aim of this study was to
determine the phenotype of plasma cells (PCs) and the expression of these two molecules, CD28 and CD40. Therefore,
we have compared their expression on normal PCs from
bone marrows and tonsils with that of freshly explanted
malignant PCs from 31 patients with multiple myeloma
(MM) and those from 12 human myeloma cell lines. For this
purpose, we first described a new approach to identify
plasma cells in bone marrow using two-color immunofluorescence analysis with anti-CD38 and B-B4 antibodies. B-B,
specifically recognizes all PC; all B-B4cells are located within
the CD38 bright fraction and vice versa. CD19 and CD56 expression, which was previously shown to discriminate normal from malignant PCs, was also evaluated. In the current
report, we show that normal PCs express CD19, CD40, and
CD56 (weakly as a subset) and lack CD28. Regardless of
whether they express CD19, CD56is clearlyupregulated during the medullary chronic and accelerated phases of MM,
but is absent in patients with extramedullary involvement.
Although the level of CD40 expression is variable, only patients in accelerated phases expressed high CD40 levels. Finally, whereas CD28 was negative in chronic phase (as in
normal PCs), it was expressed in 63% of the patients in accelerated phases and 100% of cell lines. Our data strongly suggest that both disease activity and medullary homing (or
not) are correlated with theexpression of CD19, CD40, CD28,
and CD56 on human myeloma cells.
0 1994 by The American Societyof Hematology.
B
countereceptors B7 and B7.2 present on antigen-presenting
Although CD28 is one of the receptors best characterized on T cells, almost nothing is known about CD28
expression and function on plasma cells.
Therefore, in this study, we have used a new approach
using B-B, MoAb to identify the plasma cells, and compared
the phenotype of normaland myeloma plasma cells for
CD40 and CD28 expression in relationship withthat of
CD19 and CD56. Analyzing a large panel of myeloma cells
from patients with chronic to accelerated phases ofMM
disease, we have looked for a possible correlation between
the CD40 and CD28 expression and the disease activity.
The physiologic significance of these antigens on PCsis
discussed.
-CELL MATURATION is accompanied by the coordinated acquisition and loss of cell surface antigens.’.’
Plasma cells (PCs) are characterized by the expression of
cytoplasmic Ig and a recent report clearly indicates that PCs
in the bone marrow (BM) can be easily identified by their
very high level of CD38.3-5Traditionally, multiple myeloma
(MM) has been regarded as a B-cell malignancy located in
the BM and associated with the late stage of differentiation;
nevertheless, the myeloma cell phenotype and morphology
appear to be very heterogeneous from early to mature PCcx
The immature PC or plasmablasts are the proliferating cells
and represent a restricted compartment of the total malignant
population. Despite the phenotypic heterogeneity of PCs,
substantial progress has been made in establishing the phenotype of normal and malignant PCS.’.~These previous reports
suggest that CD19 and CD56 expression profile can be used
to distinguish myeloma from normal PCS.~.”.‘~
Indeed, it has
been reported that all normal PCs from various tissues are
CD19’ CD56-, whereas myeloma cells are often CD19CD56’ and never CD19+ CD56-.*
Recently, immunophenotypic studies on human myeloma
cell lines have found that CD40 can be expressed on few of
them.“.” CD40 isusually expressed on B cells, dendritic
cells, and carcinomas. The function of CD40 is of particular
interest, because it has been shown that, in the presence
of different cytokines (interleukin-4 [ G 4 1 and IL-lo), the
activation through CD40 by monoclonal antibodies (MoAbs)
or gp39 (the CD40 ligand) can initiate both B-cell growth
and differentiati~n.’~”~
A recent study indicates that stimulation through CD40 increases the proliferation of ANBL6, an
IL-6-dependent human myeloma cell line.16 In contrast to
the CD40 expression on fewcell lines, it has been shown that
all freshly isolated myeloma cells from 7 patients express
CD40.I6
In 1987, CD28, a T-cell-restricted antigen, was proved
tobe present on the cell surface of several myeloma cell
lines.I7 On T cells, two signals are required to stimulate
proliferation: one is mediated through the T-cell receptor
and the second one is delivered through CD28I8.I9 via
the two
Blood, Vol 84, No 8 (October 154, 1994 pp 2597-2603
MATERIALSANDMETHODS
Patients. Phenotypic analysis of myeloma cells was performed
in 31 patients with MM: 14 previously untreated patients, 1 in induction treatment with partial remission, and 16 with progressive disease
(ie, relapse or primary treatment failure, despite one line of treatment
or more). The diagnostic criteria were those of the American South-
From the Laboratoire d’OncoginnPse Immunohimatologique, Institut de Biologie, Nantes; the Dkpartement d’Himatologie, Hdteldieu, Nantes; the Laboratoire d’Himatologie, Institut de Biologie,
Nantes; and Innothirapie, Besaqon, France.
Submitted February 28, 1994; accepted June 14, 1994.
Supported by the Ligue Rigionale de Lutte contre le Cancer (no.
411). C.P-D. isarecipient
of Conseil RigionaldesPaysdela
Loire fellowship and M.A. is supported by the Centre National de
la Recherche ScientiJque (CNRS).
Address reprint requests CO Martine Amiot, PhD,Laboratoire
d’Oncogkntse Immunohimatologique, Institut de Biologie, 9 Quai
Moncousu. 44035 Nantes cedex 01, France.
The publication costsof this article were defrayedin part by page
chargepayment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Sociev of Hematology.
0006-4971/94/8408-0016$3.00/0
2597
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PELLAT-DECEUNYNCK ET AL
2598
west Oncology G r o ~ p . ’Progressive
~
disease was defined as follows:
greater than 50% increase of monoclonal Ig above previous levels,
worsening anemia, and occurrence of new lytic bone lesions. Five
of the patients with progressive disease presented extramedullary
involvement. They were 18 IgG, 9 IgA, and 4 pure Bence-Jones
only. The ratio of K/X was 19:12.With 4 exceptions, previously
untreated patients have been described asin chronic phase, and
patients with progressive disease as in accelerated phasewith or
without extramedullary involvement. The 4 exceptions were the following: the unique patient (patient no. 5) in induction of treatment
(less than 6 months from diagnosis) has been considered to be in
chronic phase; 3 previously untreated patients (patients no. 17, 19,
and 27) have been considered to be in accelerated phase, because
of massive BM invasion (>50%), extensive new lytic bone lesions,
and extramedullary involvement, in case no. 27. Thus, three homogeneous populations of patients have been referred in thetext, as shown
in Table 2: 12 patients in chronic phase, mainly previously untreated
( l 1/12): 13 patients in accelerated phase without extramedullary
involvement, mainly heavily treated ( 1 1/13); and 6 patients in accelerated phasewith
extramedullary involvement, mainly heavily
treated (5/6).
Human myeloma cell lines. IL-6-dependent human myeloma
cell lines XGl to XG8 were established in the laboratory.12 LP-I”
and L363” were obtained from DSM (Braunschweig, Germany).
Antibodies. MoAbs against the following antigens were used:
CD19, CD28, CD38, CD40, and CD56; as well as the B-B,antibody.
Anti-CD19-PE (phycoerythrin), anti-CD28-FITC (fluorescein), antiCD38-FITC, control IgGI-FITC, and control IgGl-PE were obtained
from Immunotech (Marseilles, France); anti-CD56-PE from Becton
Dickinson (Heidelberg, Germany); MoAb89” anti-CD40 and B-B4
antibody were, respectively, a gift from Dr J. Banchereau (SheringPlough, Dardilly, France) and from Dr J. Wijdenes (InnothCrapie,
BesanGon, France).
Cell preparations. Onthe occasion of a diagnostic iliac crest
puncture, IO to 20 mL of BM aspirates was obtained and BM mononuclear cells were isolated by Ficoll-Hypaque centrifugation. Adherent cells were then removed by allowing total mononuclear cells to
adhere to plastic flasks in RPM1 1640 5% fetal bovine serum (FBS)
for 90 minutes at 37°C ina 5% COz humidified atmosphere. BM cells
from healthy donors were, in addition, depleted of T lymphocytes by
sheep erythrocyte rosetting. Tonsillar tissues were obtained from
subjects undergoing tonsillectomy for chronic tonsillitis and purification of PCs was performed as described for normal BM.
Biotinylution of purijied untibodies. PurifiedB-B,and
antiCD40 antibodies were biotinylated using the following method. Antibodies (0.2 to 1 mg) were preliminilarily dialyzed in 0.1 moln
NaHCO? pH 8.3 at 4°C andthe biotinylation reactions wereperformed for 2 hours at roomtemperature with 50 pg of biotinamidocaproate N-hydroxysuccinimide ester (Sigma B2643; Sigma, St Louis,
MO) per milligram of antibody, freshly prepared in dimethyl sulfoxide (DMSO) at the concentration of 1 mg/mL.
Phenotype unalysis of PCs. For immunofluorescence staining, 2
to 5 X IO5 cells were incubated with different FITC- or PE- or
biotinylated MoAbs for 30 minutes at 4°C in the presence of 20%
humanAB serum followed by incubation (30 minutes at 4°C) of
streptavidin coupled to PE (Immunotech). Cells were fixedin 4%
formaldehyde and analyzed on a FACScan flow cytometer (Becton
Dickinson).
PCa were identified by specific B-B4’++and CD38”- immunofluorescence. For phenotype analysis, each MoAbwas associated
with PE-streptavidin-biotinylated B-B4or anti-CD38-FITC. MoAbs
against CD28-, CD38-FITC-conjugated were associated with biotinylated-B-B,, and MoAbs against CD19- and CD56-PE-conjugated and also biotinylated anti-CD40 antibodies were used in combination with anti-CD38-FITC. For Fig2E,B-B,-FITCwasused
in combination with anti-CD56-PE.
To determine the level of positivity for each marker, we divided
the mean fluorescence intensity of each marker by the background
staining. By this procedure, we distinguished three levels of positivity: weakly positive (+), 1.4 < fluorescence ratio c 10; positive
(+ +), I O < fluorescence ratio 5 50; and brightly positive (+++),
fluorescence ratio >50.
RESULTS
B-B4 specijicallyrecognizesall
known thatnormal andmyeloma
Pes. It i s nowwell
PCs arefound only in
A
I
l
t
+.
m
x
a
U)
h
0
S0 l 0 0 150 288 256
FSC\Forward S c a t t e r --
I
CD30
F
a
m
0
58
1 B B 150 288 250
FSC‘ Forward S c a t i e r
--
I
CD30
I
Fig 1. Identificationof PCs in
normal and myeloma BM. Human BM cells were freshly iroleted from BM aspirates from
healthy donor MW IA, B, and C)
and MM patient no. 12 (D.E, and
F) as described under Materials
and Methods. Scatter cytograms
show the gate of the phenotype
study (Aand D). Two-color analysis of BM cells with anti-CD38FlfC
[x-axis,
Log
scale)
and
B-B,-biotin-streptavidin-PE lyaxis, Log scale) are presented (C
and F); the PCs are locatedin the
upper-rightquadrantregion. The
corresponding controls with an
irrelevant antibody are shown in
16) and (E).
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2599
EXPRESSION OF CD28 AND CD40 INHUMAN MYELOMA CELLS
E
B
A
d
8
P
0
l
CD38
I
I
CD38
I
I
BB4-FlTC
1
D
C
Y
CD38
CD28
Fig 2. Immunofluorescence profile of normal PCs. Two-color analysis of normal BM cells with anti-CDlS-PE and anti-CD38-FITC (A), with
anti-CD56-PE and anti-CD38-FITC (B), with anti-CD40-biotin-streptavidin-PEand anti-CD38-RTC (C), and with anti-CD28-RTC and B-B,-biotinstreptavidin-PE (D) are presented.When the two-color staining was performedwith anti-CD38, PCs were found in theright part of the cytogram,
whereas PCs were found in the upper part of the cytogram when B-B4-biotin-streptavidin-PE was used for the staining. (E) represents atwocolor analysis of normal BM cells stained with anti-CD56-PE and B-B4-FITC; plasma cells were located in the right part of the cytogram. (A)
and (C) were from MN3 and (B), (D), and (E) were from MN8.
the CD38-bright fraction when analyzed by flow cytometry.
Recently, the B-B4 MoAb was successfully used to purify
PCs from BM cells of myeloma patients.28329
Using twocolor staining with anti-CD38 and B-B, MoAbs, we showed
that allB-B,-positive
cells (B-B:++) are located in the
CD38+ bright fraction (CD38+++)and vice versa. Representative cytograms of a healthy donor and a multiple myeloma
patient are presented in Fig 1C and F. For healthy donors
and MM patients, we next confirmed that the percentage of
PCs determined byflow cytometry is identical to the one
evaluated by morphology in May-Grunwald Giemsastained cytospin (data not shown). We took advantage of
the B-B4 specificity in all the following phenotype analyses
to first identify the PCs and then to study the expression of
other cell surface markers that were usedin combination
with B-B4 or anti-CD38 for two-color staining analysis by
flow cytometry.
CD19 and CD56 are expressed on normal PCs. In this
study, we were interested in defining the CD28 and CD40
expression in relation with CD19 and CD56, which were
known to distinguish normal from malignant PCs. For this
purpose, we first established the expression of CD19 and
CD56 antigens on normal PCs (Fig 2A). As expected from
previous reports, normal PCs from BM (6/6 cases) and tonsil
(212 cases) were positive for CD19 (Table 1). We can see
that the CD19' fraction ranged from 63% to 90%. Although
it was initially reported that normal PCs do not express
CD56, we show that 5 of 6 donors possess a subpopulation
of PCs that express a low level of CD56, as shown in Fig
2B. To confirm this result, we performed two-color staining
using B-B,-FITC antibody that is restricted to the PCs. As
expected, a subpopulation of B-B,-positive cells express
CD56 (Fig 2E).
Normal PCs are CD40+ but CD28-. The CD40 and
CD28 molecules have been detected on myeloma cells, but
their expression profile on normal PCs remains unknown.
Also, we analyzed the CD28 and CD40 expression on normal
plasma cells from tonsil and BM. We observed a single and
homogeneous population CD40+ and CD28- (Table 1 and
Fig 2C and D), but the CD40 level expression is weak on
PCs from BM and higher on PCs from tonsils (data not
shown).
CD19 and CD56 expression on myeloma cells. The
phenotype of 3 1 MM patients was analyzed and the results
D40
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2600
PELLAT-DECEUNYNCK ET AL
Table 1. Phenotypic Analysis of Normal PCs
Phenotype of PCs
CD56
Healthy
Donor
CD19
% of
PCS
BM
MNI
MN2
MN3
MN4
MN5
MN6
MN7
MN8
Tonsil
TI
T2
2.5
1
1.5
0.4
1.6
2
1
1.33
++ (90)
++ (66)
++ (66)
++ (85)
++ (80)
ND
ND
(63)
++
-
++ (36)
+ (60)
++ (36)
+ (90)
ND
ND
+ (35)
is variable, we, however, can notice that only patients
with progressive myeloma present a high level o f CD40
expression, as shown in Fig 3E. Concerning CD28 expression, interestingly, none of the 8 patients with MM
i n chronicphaseexpressedCD28,whereasCD28was
detected in 63% of thepatients in acceleratedphase.
Positive and negative representative CD40 and CD28 expression profiles are presented in Fig 3E and F and Fig
3G and H, respectively.
DISCUSSION
In this study, with the two-color immunofluorescence
B-B4 antibodies, we demethodusinganti-CD38and
+++
+ (40)
+++
0.6
scribed a new and efficient approach to identify the PCs
++
+ (331
2
++
in the BM. Using this approach, we were able to examine
Intensity of fluorescence is defined: +, weakly positive; ++, posithe expression of cell surface markers of PCs in BM contive; +++, highly positive, asdescribed under Materials and Methods. taining as little as 0.5% of PCs among the total mononuWhen both positive and negative cells were found, the percentage of clear cells.
positive cells is indicated.
The results presented here first establish that normal PCs
Abbreviation: ND. not done.
are CD19' and that CD56 is expressed on a subpopulation.
The presence of CD19 on these cells is in agreement with
a previous observation.' The finding that normal PCs express
are summarized in Table2. In this study, we distinguished CD56 was contrary to previous reports, which may be extwo major groupsof patients according to the stageof the
plained by the differences in the methods of identification
disease: 12 were in chronic phase and 19 in accelerated
of PCS.',~ With regards to the CD19 and CD56 expression
on myeloma cells, our resultsconfirmthatmostPCsare
phase. For CD19 and CD56 expression, irrespective
of the
CD19- CD56' or CD19+CD56+.' Comparing the level of
different stages of the disease, two frequent phenotypes
CD56 expression on myeloma andnormal PCs, we can conwere observed: CD19- CD56'(13 of 31 cases) and CD19'
clude that CD56 is often upregulated on myeloma PCs. The
CD56+ (1 1 of 31 cases). Moreover, as it had been indibiologic role of CD56 on PCs remains unknown, but it is
cated in a previous report, we confirmed that the CD19'
CD56- phenotype was never observed inMM patients. In
reasonable to think that CD56 could be involved in homomost of the cases, the level
of CD56 is higher than on
typic adhesion with some cellular components of the BM
environment or in heterotypic adhesion with some extracelnormal PCs, indicating that CD56 expression is upregulular matrix components, as has been described in other cellated on myeloma cells (Table 2). Analysis of the results
lular models.20"2Analysis of the data obtained from patients
5 patients were CD19shows that myeloma cells from
with extramedullary involvement supportsthenotion
that
and CD56-; the cytograms of 1 of these patients is shown
in Fig 3B and D. It is of particular interest to note that 4
the loss of CD56 could berelated to thecapacity of myeloma
cellstodisseminateout
of theTheideathatCD56
of 5 of these patients in accelerated phase presented not
upregulation and downregulation is underthe control of very
only a BM infiltration butalso an extramedullary involveprecise mechanisms is consistent with earlier reports showment. This result strongly suggests a correlation between
the subsequent lack of CD56 expression and the spreading ingthat CD56 can be downregulated during embryogenic
migratory events and then reexpressed when target organs
of malignant PCs.
are rea~hed.".'~In relation to this observation, we can notice
These patients who did
not express CD56 on PCs still
that the myeloma cells from the patient no. 31 were excluexpressed CD56 on the CD38+population corresponding to
sively located in the peritoneal cavity. Because thecells had
natural killer (NK) cells. This NK cell population CD56'
all left the BM and were sited then in the peritoneal cavity,
can be observed in theBM from myeloma patients(Fig 3D)
where they highly proliferated, it is reasonable to think that
as well as in normal BM (Fig 2B).
CD56 would have been reexpressed once thenew organ was
CD28 and CD40 expression on myelomacells.
On
all myeloma cell lines examinedso far, we observed that reached, ie, the peritoneal cavity. Finally, regardless of the
ascitis, all BM or peripheral (case no.30) myeloma cells
CD28 is always expressed and, furthermore, with a high
from patients
with
extramedullary
involvement
are
level of intensity (Table 3). On the other hand,7 of these
C D 1 9 - C D K . Concerning CD19 and CD56 expression on
12 cell lines expressed different levels of CD40, and 5
cell lines were totally negative (Table 3). We next exam- human myeloma cell lines, the most frequent phenotype is
CD19-CD56-. This result reflects our findings, because at
ined the CD40 and CD28 expression on the 31 patients.
least all XG celllineswere obtainedfromtheperipheral
Theresultsanalyzedindependently
of thedifferent
blood of MM patients with extramedullary progression.
groups of patients indicated that CD40 and CD28 were
In the present report, whichfirst confirms the recent obserexpressed somewhat in two-thirds and 40% of MM patients, respectively (Table 2). Although the level of CD40 vation that CD40 is present on some human myeloma cell
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EXPRESSION OF CD28 AND CD40 IN HUMAN MYELOMA
2601
CELLS
Table 2. Phenotypic Analysis of Myeloma Cells
Phenotype of Myeloma Cells
MM
Patient
No.
-
Disease Status
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Chronic
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (M)
Accelerated (EM)
Accelerated (EM)
Accelerated (EM)
Accelerated (EM)
Accelerated (EM)
Accelerated (EM)
mlg
D
D
D
D
T
D
D
D
D
D
D
D
T
T
T
T
D
T
D
T
T
T
T
T
T
T
D
T
T
T
T
% of
PCS
5
10
4.5
2
1.3
31
6
33
7
3
3
28
5
4
3
2
85
22
50
20
8
8
2.5
20
19
40
65
26
40
90
85
CD19
CD56
CD40
++C
-
+++
++
+++
+++
+++
-
CD28
+
+
+
-
+ (42)
++
-
+ (60)
++
+
-
++
+++
+++
+
+
-
++
++
++ (80)
-
++
ND
+++
+++
++
++
++
+
++
++
+
++
+ (25)
+ (25)
++
++
-
+
++
+++
-
-
+ (22)
-
-
-
+++
-
-
+
+
In all cases, cells were purified from BM except for patients no. 30 and 31. The cells were obtained from peripheral blood lymphocytes for
highly positive, as described
no. 30 and from ascitis for no. 31. Intensity of fluorescence is defined: +, weakly positive; ++, positive;
under Materials and Methods. When both positive and negative cells were found, the percentage of positive cellsis indicated.
Abbreviations: M, medullary involvement only: EM, extramedullary involvement; D, diagnosis; T, under treatment; ND, not done.
+++,
lines,",'* we describe the CD40 expression on PCs. Indeed,
we show that all normal plasma cells express CD40, whereas
two thirds of myeloma patients express CD40. Our percentage of myeloma cells expressing CD40 contrasts with the
recent finding that all myeloma cells are CD40+.I6This discrepancy likely reflects differences in the number of patients
studied, which was (l) larger in our study and (2) representative of the different disease stages. Moreover, our results on
MM are in agreement with the variability of CD40 expression observed on cell lines. IL-6 has been shown to be the
major myeloma cell growth factor, most probably functioning both as an autocrine and a paracrine f a ~ t o r . ~A' ,recent
~~
study indicated that signaling through CD40 induced an autocrine IL-6 production on one myeloma cell line, ANBL6,
which has been described to proliferate only in presence of
IL-6.I6 Because 3 of 8 myeloma cell lines that proliferated
in response to IL-6 are CD40- in our hands, this important
functional observation regarding CD40 does not seem to be
a common feature of myeloma cells. In addition, this finding
is also supported by the fact that one third of MM patients
have been reported negative for CD40 and we can raise
the hypothesis that IL-6 autocrine responsiveness triggered
through CD40 cannot be the unique pathway for in vivo
myeloma cell proliferation.
Finally, the results presented in this report provide the
first documentation of CD28 expression on normal and myeloma PCs. We determine that all myeloma cell lines established to date express CD28 and the results on myeloma
patients clearly indicate that CD28 is only detected on MM
cells from aggressive stages of disease. It is of importance
to note that at least all myeloma cell lines established successfully in the laboratory'* were exclusively obtained from
patients with aggressive stages of the disease and even
with terminal disease, which is in good agreement with our
results on CD28 expression depicted in this report. These
findings could support the hypothesis that CD28 appears
at one precise stage of B-cell differentiation. Therefore, a
possible explanation for the absence of CD28 on normal
PCs might be that this stage exists for a short period of
time during normal development and is only accessible
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PELLAT-DECEUNYNCK ET AL
2602
E
C
A
3>
cwel
B
F
D
H
I
"
Fig 3. Heterogeneous immunofluorescence profile of malignant PCs of BM. Representative positive (A, C, E, and G) and negative (B, D, F,
and H) profiles of PCs for CD19 (A and B), CD56 (C and D), CD40 (E and F), and CD28 (G and H). (A and C) Patient no. 18; (B and D) patient no.
27; (E and G) patient no. 29; (F and HI patient no. 8.
ACKNOWLEDGMENT
when this compartment has been immortalized on cell lines
or considerably enhanced in aggressive MM. It may repreWe thank the Department of Hematology (Prof J.-L. Harousseau)
sent the plasmablastic compartment, overlapping the prolif- forproviding us withbone marrow samples;DrBanchereaufor
erative cells. To address this hypothesis, we have started
providing CD40 antibody: M. Etrillard and D. Puthier for technical
looking at the labeling indexof myeloma cells. Despite the assistance; Drs R. Garand and F. Accard for their cytologic expertise;
Dr L. Bataille-Zagury for editing the English text; and N. Raimbault
small number of cases examined so far, we have observed
for secretarial assistance.
a high labeling index in 5 of 8 patients CD28' (62%) and
only in 3 of 12 patients CD28- (27%). These preliminary
REFERENCES
results seem to strongly indicate that
a correlation exists
1. Banchereau J, Rousset F: Human B lymphocytes: phenotype.
between the CD28 expression and
a high labeling index.
proliferation and differentiation. Adv Immunol 52: 12.5, 1992
A possible close relation between these two events is now
2. Uckun FM:Regulation
of human B-cell ontogeny: Blood
under investigation.
76: 1908, 1990
Table 3. Phenotypic Analysis of Human Myeloma Cell Lines
~
~~
CD56 CD19
XG 1
XG2
XG3
XG4
XG5
XG6
XG7
XG8
U266
RPMI-8226
L363
LP1
-
(%)
++ (80)
~
-
+
-
-
~
~
-
~
-
++
~
CD28 CD40
+++
~
+++
+
+
++
+
++
~
++
-
+ (25)
+
-
+ (70)
-
-
+
+
-
-
~
++
++
+
++
++
++
++
++
++
Intensity of fluorescence is defined: +, weakly positive; ++, positive; +++,highly positive, asdescribed under Materialsand Methods.
When both positive and negative cells were found, the percentageof
positive cells is indicated.
3. Van Camp B, Durie BGM, Spier C, De Waele M, Van Riet I,
Vela E, Frutiger Y, Richter L, Grogan TM: Plasma cells in multiple
myelomaexpressa
natural killercell-associatedantigen:CD56
(NKH-I; LW-19). Blood 761377, 1990
4. Leo R, BoekerM,PeestD,
Hein R, Bart1 R, GessnerJE,
Selbach J, WackerG, Deicher H: Multiparameter analyses ofnormal
and malignant human plasma cells: CD38'+, CD56+, CD54'. clg'
is the common phenotype of myeloma cells. Ann Hematol 6 4 : 132,
1992
S . Harada H, Kawano MM, Huang N, Harada Y, lwato K, Tanabe
0, Tanaka H, Sakai A, Asaoku H, Kuramoto A: Phenotypic difference of normalplasmacellsfrommaturemyelomacells.
Blood
81:2658, 1993
6. Hata H, Xiao H, Petrucci MT, Woodliff J, Chang R, Epstein
J: Interleukin-6 gene expressionin multiple myeloma: A characteristic of immature tumor cells. Blood 81:3357, 1993
7. Omedt P, Boccadoro M, Fusaro A, Gallone G, Pileri A: Multiplemyeloma: "Early" plasmacellphenotype
identifies patients
with aggressive biologicaland clinical characteristics. BrJ Haematol
85:504, 1993
8. Kawano M, Huang N, Harada H. Harada Y, Sakai A, Tanaka
From www.bloodjournal.org by guest on November 20, 2014. For personal use only.
EXPRESSION OF CD28 AND CD40 IN HUMAN MYELOMA CELLS
H, Iwato K, Kuramoto A: Identification of immature and mature
myeloma cells in the bone marrow of human myelomas. Blood
82:564, 1993
9. Terstappen LWMM, Johnsen S, Segers-Nolten IMJ, Loken
MR: Identification and characterization of plasma cells in normal
human bone marrow by high-resolution flow cytometry. Blood 76:9,
1990
10. Barker HF, Hamilton MS, Ball J, Drew M, Franklin IM:
Expression of adhesion molecules LFA-3 and N-CAM on normal
and malignant human plasma cells. Br J Haematol 81:331, 1992
11. Jelinek DF, Ahmann GJ, Greipp PR, Jalal SM, Westendorf
JJ, Katzmann JA, Kyle RA, Lust JA: Coexistence of aneuploid
subclones within a myeloma cell line that exhibits clonal immunoglobulin gene rearrangement: Clinical implications. Cancer Res
53:5320, 1993
12. Zhang XG, Gaillard JP, Robillard N. Lu ZY, Gu ZJ, Jourdan
M, Boiron JM, Bataille R, Klein B: Reproducible obtaining of human
myeloma cell lines as a model for tumor stem cell study in multiple
myeloma. Blood 83:3654, 1994
13. Banchereau J, De Paoli P, Val16 A, Garcia E, Rousset F:
Long-term human B cell lines dependent on interleukin-4 and antibody to CD40. Science 251:70, 1991
14. Rousset F, Garcia E, Banchereau J: Cytokine-induced proliferation and immunoglobulin production of human B lymphocytes
triggered trough their CD40 antigen. J Exp Med 73:705, 1991
15. Rousset F, Garcia E, Defrance T, PBronne 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
16. Westendorf JJ, Ahmann GJ, Armitage RJ, Sprigs MK, Lust
JA, Greipp PR, Katzmann JA, Jelinek DF: CD40 expression in
malignant plasma cells. Role in stimulation of autocrine IL-6 secretion by a human myeloma cell line. J Immunol 152:117, 1994
17. Kozbor D, Moretta A, Messner HA, Moretta L, Croce CM:
Tp44 molecules involved in antigen-independent T cell activation
are expressed on human plasma cells. J Immunol 138:4128, 1987
18. June CH, Ledbetter JA, Linsley PS, Thompson CB: Role of
the CD28 receptor in T-cell activation. Immunol Today 11:211, 1990
19. Thompson CB, Lindsten T, Ledbetter JA, Kunkel SL, Young
HA, Emerson SG, Leiden JM, June CH: CD28 activation pathway
regulates the production of multiple T-cell-derived lymphokines/
cytokines. Proc Natl Acad Sci USA 86:1333, 1989
20. Schwartz RH: Costimulation of T lymphocytes: The role of
CD28, CTLA-4, and B7/BBl in interleukin-2 production and immunotherapy. Cell 71:1065, 1992
21. Linsley PS, Clark EA, Ledbetter JA: T-cell antigen CD28
mediates adhesion with B cells by interacting with activation antigen
B7BB-I. Proc Natl Acad Sci USA 87:5031, 1990
22. Azuma M, Ito D, Yagita H, Okumura K, Phillips JH, Lanier
2603
LL, Somoza C: B70 antigen is a second ligand for CTLA-4 and
CD28. Nature 366:76, 1993
23. Hathcock KS, Laszlo G, Dickler HB, Bradshaw J, Linsley P,
Hodes RJ: Identification of an alternative CTLA-4 ligand costimulatory for T cell activation. Science 262:905, 1993
24. Dune BGM: Staging and kinetics of multiple myeloma.
Semin Oncol 13:300, 1986
25. Diehl V, Schaadt M, Kirchner H, Hellriegel KP, Gudat F,
Fonatasch C, Laskewitz E, Guggenheim R: Long-term cultivation
of plasma cell leukemia cells and autologous lymphoblasts (LCL)
in vitro: A comparative study. Blut 36:331, 1978
26. Pegoraro L, Malavasi F, Bellone G, Massaia M, Boccadoro
M, Saglio G , Guerrasio A, Benetton G, Lombardi L, Coda R, Avanzi
GC: The human myeloma cell line LP-l : A versatile model in which
to study early plasma-cell differentiation and c-myc activation.
Blood 73:1020, 1989
27. Val16 A, Zuber CE, Defrance T, Djossou 0, De Rie M, Banchereau J: Activation of human B lymphocytes through CD40 and
interleukin 4. Eur J Immunol 19:1463, 1989
28. Borset M, Helseth E, Naume B, Waage A: Lack of IL-1
secretion from human myeloma cells highly purified by immunomagnetic separation. Br J Haematol 85:446, 1993
29. Portier M, Rajzbaum G , Zhang X-G, Attal M, Rusalen C,
Wijdenes J, Mannoni P, Maraninchi D, Piechaczyk M, Bataille R,
Klein B: In vivo interleukin 6 gene expression in the tumoral environment in multiple myeloma. Eur J Immunol 21: 1759, 1991
30. Goridis C, Brunet JF: NCAM: Structural diversity, function
and regulation of expression. Semin Cell Biol 3:189, 1992
31. Sadoul R, Him M, Deagostini-Bazin H, Rougon G, Goridis
C: Adult and embryonic mouse neural cell adhesion molecules have
different binding properties. Nature 304:347, 1983
32. Acheson A, Sunshine JL, Rutishauser U: NCAM polysialic
acid can regulate both cell-cell and cell-substrate interactions. J Cell
Biol 114:143, 1991
33. Edvardsen K, Chen W, Rucklidge G, Walsh FS, Obrink B,
Bock E: Transmembrane neural cell-adhesion molecule (NCAM),
NCAM, down-regubut not glycosyl-phospbatidylinositol-anchored
lates secretion of matrix metalloproteinases. Proc Natl Acad Sci
USA 90:11463, 1993
34. Edelman GM, Crossin KL: Cell adhesion molecules: Implications for a molecular histology. Annu Rev Biochem 60:155, 1991.
35. Kawano M, Hirano T, Matsuda T, Taga T, Horii Y, Iwato K,
Asaoku H, Tang B, Tanabe 0, Tanaka H, Kuramoto A, Kishimoto
T: Autocrine generation and essential requirement of BSF-2aL-6
for human multiple myeloma. Nature 322:83, 1988
36. Klein B, Zhang XG, Jourdan M, Content J, Houssiau F,
Aarden LA, Piechaezyck M, Bataille R: Paracrine rather than autocrine regulation of myeloma-cell growth and differentiation by interleukin-6. Blood 73:517, 1989