Proliferation Response of Leukemic Cells to CD70-Ligation Oscillates with
Recurrent Remission and Relapse in a Low Grade Lymphoma
Yael Kaufmann2*‡, Ninette Amariglio*†, Esther Rosenthal*, Jasmine Jacob Hirsch†,
Amira Many*, and Gideon Rechavi*†‡
*Institute of Hematology, and †Pediatric Hematology-Oncology, Safra Children's Hospital,
The Chaim Sheba Medical Center, Tel-Hashomer, and ‡ Sackler School of Medicine
Tel-Aviv University, Tel-Aviv, 69978, Israel.
Running title: Recurrent responsiveness in CD70-signaling
Key words: Human, Tumor immunity, B cells, Surface molecules, Co-stimulation
1
This work was supported by the Carol and Leonora Fingerhut fund for Cancer Research,
Tel-Aviv University.
2
Address correspondence to Dr. Yael Kaufmann, Institute of Hematology, The Chaim Sheba
Medical Center, Tel-Hashomer 52621, Israel. Phone: (972) 3-649-6273; FAX: (972) 3-530-3506;
email: yaelka@sheba.health.gov.il
Abbreviations used in this paper: CLL, B cell chronic lymphocytic leukemia; FCS, fetal calf
serum; mAb, monoclonal antibody; PBMC, peripheral blood mononuclear cells; PMA, phorbol
myristate acetate; sCD27, soluble CD27; TNF-R, TNF receptor.
Abstract
Interactions of the tumor necrosis factor-related cell surface ligand CD70 with its receptor CD27
provide a co-stimulatory signal in B- and T- cell activation. Functional CD70-CD27
interactions could contribute to lymphoma and leukemia progression. This possibility was
studied using DNA microarrays on a unique case of low-grade lymphoma/leukemia
characterized by recurrent cycles of acute leukemic phase alternating with spontaneous
remission. Upon induction of the acute phase expression of CD70 and CD27 in the leukemic
cells increased 38 and 25 fold, respectively. Co-expression of membrane CD70 and CD27 on
the leukemic (CD5+CD19+) cells was maximal 2-3 days following initiation of the attack.
Soluble CD27 in the patient's serum was elevated during remission and further increased in the
attack. Functional tests showed that neither anti-CD70 nor anti-CD27 antibodies affect the rate
of apoptosis. However, the anti-CD70 antibody specifically enhanced proliferation of the
remission phase leukemic cells whereas proliferation of the acute-phase counterparts that
express higher level of membrane CD70 was unaffected. Hence, in this lymphoma/leukemia,
membrane CD70 is presented on the leukemic cells in a responsive state during the remission
and a non-responsive state during the attack. Presumably, CD70 in its responsive state provides
a co-stimulatory receptor for initiating the next acute phase while its non-responsive state
enables the remission.
2
Introduction
CD27 and its ligand CD70 are members of the tumor necrosis factor-receptor (TNF-R)
family and TNF superfamily, respectively. Interactions between TNF-R family members and
their ligands control lymphocyte death, survival, proliferation and differentiation (1, 2). Tightly
regulated expression of CD70 and CD27 on normal lymphocytes ensures the transient
availability of these co-stimulatory signals. CD70 is transiently induced upon activation of B
cells (3) and T cells (4). CD27 is absent from immature and mature B cells but is found on
memory B cells (5, 6) T cells (7) and NK cells (8). Differentiation to effector T cells is
associated to loss of CD27 (9). Cell surface CD27 can be proteolytically cleaved to produce a
32-kDa soluble CD27 (sCD27) molecule, which can be detected in the serum and urine (10).
Elevated serum sCD27 levels are seen in a number of infectious and autoimmune disease states
and are believed to be a marker of T cell activation. Also, various types of B cell malignancies
express the CD27 molecule and elevated serum levels of sCD27. There appeared to be a
correlation between tumor load and sCD27 levels (11). Studies on the biological role of this
receptor-ligand pair have mainly addressed the ability of CD27 to regulate the immune response.
CD27 signaling appears to play a co-stimulatory role promoting T cell expansion and
differentiation (7, 12-15) as well as enhancement of NK cell activity (16). CD70 has been
generally envisaged as a passive element merely required for specific CD27 engagement. Yet,
reverse signaling of CD70 was reported. Thus, anti-CD70 monoclonal antibodies (mAb) were
observed to induce proliferation in a subset of B cell chronic lymphocytic leukemia (CLL) (17)
and stimulate NK and  T cell-mediated redirected killing (18), suggesting that CD70 might be
a signaling molecule. Recently Arens et al showed that CD70 has reverse signaling properties in
murine B cells, initiating a signaling cascade that regulates expansion and differentiation (19).
In contrast to the tightly regulated expression of CD70 on normal cells, constitutive expression
of CD70 is found on several human leukemias and lymphomas (17, 20, 21). The finding that
cross-linking of CD70 on malignant B cells augments proliferation (17) raised the possibility
that CD70 might contribute to progression of these B-cell malignancies.
We have previously reported a case of CD5-positive B cell lymphoma, diagnosed as
atypical CLL/SLL, which is characterized by recurrent acute waves of active disease (22, 23).
The patient experienced within 8 years 27 cycles of leukemic waves followed by spontaneous
3
partial remissions. The first week of the wave characterized by fever, generalized
lymphadenopathy and increasing number of clonal IgM-positive leukemic cells usually above
60.000 cells/mm3. After a few days the symptoms of the acute illness disappeared. Following 2-3
weeks most of the transformed leukemic cells disappeared and the white blood cell count returned
to almost a normal level. Nonetheless, 10-60% of the peripheral lymphocytes were monoclonal B
cells. The remission periods lasted 2-7 months (22). Transient bouts of serum TNF and IL-10
preceded the acute phases, which were characterized by co-existence of CD40-ligand+
T lymphocytes and lymphoma cells in the bone marrow (23). During remissions, residual IgM+
leukemic cells exhibited resting phenotype, low proliferative response to CD40-ligand and
delayed apoptosis in culture. In contrast, the acute phase counterparts were phenotypically
activated, underwent rapid apoptosis and proliferated extensively in response to membraneanchored CD40-ligand (23, 24).
The existence of a clonal leukemic cell population throughout the course of the disease
(22) provides a unique opportunity to characterize molecular changes in the neoplastic cells
associated with recurrent activation and regression of the disease. Microarray analysis of the
leukemic cells done in this study showed consistent pattern of gene expression characterizing
different stages of the disease. Because interactions between CD70 and CD27 provide costimulatory signal in B cell activation we examined their involvement in progression of this
"cyclic" lymphoma/leukemia. Indeed, CD70 and CD27 were among the genes whose expression
transiently increased simultaneously with initiation of the acute phase. The leukemic cells from
remission periods, but not those from acute phases, responded to CD70-ligation by increased
proliferation response to PMA. The results are consistent with transition of cell surface CD70
from responsive to non-responsive state that occurs concomitantly with initiation of the attack and
is coupled to activation and blast transformation of the leukemic cells.
4
Materials and Methods
Isolation of leukemic cells and determination of sCD27 in the serum.
Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood by Ficol
(Nycomed Pharma AS) density gradient centrifugation and stored in liquid nitrogen. Where
indicated the patient's leukemic cells were purified from PBMC on CD19 magnetic beads
(DynalR, Oslo, Norway) according to the manufacturer's instructions. The purified cells
contained 94-98% CD19+CD5+ double positive leukemic cells. Viability of the leukemic cells,
examined by trypan blue exclusion, was always above 92%. Sera were analyzed for sCD27 by
sandwich ELISA (Sanquin Reagents, CLB, Amsterdam, Netherlands) according to the
manufacturer's instructions. This study was approved by the Institutional Review Board, and
informed consent was obtained.
Microarray cDNA analysis.
CDNA analyses were performed using Affymetrix Hu95A oligonucleotide arrays, as described at
http://www.affymetrix.com/support/technical/datasheets/human_datasheet.pdf.(URL1). Total
RNA was isolated from 1X107 CD19-purified leukemic cells by TrizolTM reagent (Life
Technologies). Quality and amount of RNA was confirmed using an agarose gel. Biotinylated
target RNA was prepared, with minor modifications from the manufacturer's recommendations.
(http://www.affymetrix.com/support/technical/manual/expression_manual.affx)(URL2). When
scaled to a target intensity of 100 (using Affymetrix MAS 5.0 array analysis software), scaling
factors for all arrays were within acceptable limits. Details of quality control measures can be
found at (http://eng.sheba.co.il/genomics)(URL3).
Real-time quantitative polymerase chain reaction (RTQ-PCR) assay.
RTQ-PCR assays were performed to determine the expression level of CD70 and CD27.
Forward and reverse Primers (MWG-Biotech AG), were designed in different exons;
CD70 forward primer 5'- TGGGTGGGACGTAGCTGAG-3'
and reverse, 5'- TCCTGCTGAGGTCCTGTGTG- 3'.
CD27 forward primer 5’-GCCAGGAACATTCCTCGTGA-3’
5
and reverse, 5’- TTACAGTGCCGACAGCTCTCA-3’. Thymosinbeta was used as an internal
standard. The RTQ-PCR reactions were done using Applied Biosystems 7900HT prism realtime PCR instrument, in a total volume of 20l containing 500nM of each forward and reverse
primer, QuanTitect SYBR green PCR kit (Quiagen) for CD70 and CD27 assays, with cDNA
equivalent to 25ng RNA of each sample. Appropriate negative controls were designed for each
run. Ratio was calculated by dividing each gene expression with that of an internal standard.
Flow cytometry.
For comparison of cell-surface marker expression, cells that had been cryopreserved during
different disease phases were thawed, processed, and analyzed simultaneously. 4105 PBMC
were incubated in 0.4ml RPMI containing 10% fetal calf serum (FCS) (GIBCO) for 20 min. at
200C with CD19 PerCP, CD5 APC, CD27 R-PE and CD70 FITC, washed in PBS and analyzed
on dual laser-FACScalibur-flow cytometer from Becton Dickinson, San Jose, CA.
Discrimination of leukemic cells from the normal hematopoitic cells was done by gating on cells
expressing CD19 and CD5. CD70 and CD27 expression on the leukemic cells was scored
within the gated CD19+ CD5+ cells. The following monoclonal antibodies (mAbs) were used for
4 color FACS analysis; (R-PE)-labeled CD27 (clone M-T271) and (FITC)-labeled CD70 (clone
Ki-24) Becton Dickenson (BD) Biosciences Pharmingen, (APC)-labeled CD5 (clone L17F12)
and (perCP)-labeled CD19, BD Biosciences, San Jose, CA.
Tests of apoptosis and cell proliferation.
The patient's PBMC or CD19+ purified leukemic cells were cultured at 2106/ml in RPMI-1640
supplemented by L-glutamine, 10% FCS and gentamycin sulphate 50g/ml (CM medium), at
37oC in 5% CO2. Spontaneous apoptosis of the leukemic cells in culture was examined by
Anexin-V-FITC staining. 4105 leukemic cells were cultured in 96-well flat-bottom plates in
CM medium in presence or absence of 1-9g/ml azide-free preparation of anti-CD70 mAb.
(clone Ki-24, BD Biosciences Pharmingen) or anti-CD27 mAb. (clone 1A4, Immunotech,
Marseille, France). Apoptosis of the cultured leukemic cells was determined following 7 or 24 h
incubation in CM medium at 37oC in 5% CO2. The cells were washed and resuspended in a
buffer containing 10 mM HEPES (pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2. Annexin-V6
FITC 1:50 (Roche, Mannheim, Germany) and Propidium Iodide 50g/ml (Sigma), were added
and the cells were stained for 20 min. at 200C and immediately assessed on a Beckman Coulter
Epics MCL flow cytometer. Proliferation of the leukemic cells was tested by 3H-thymidine
uptake. 2105 cells were cultured in 96-well plates in CM medium in tetraplicates. To examine
the effect of the antibodies on proliferation the leukemic cells were first allowed to bind the
antibodies by preincubtion at 200C for 30 min. with 1-9g /ml azide-free CD70 mAb.(clone Ki24), or 1-9g/ml CD27 mAb (clone 1A4), followed by addition of 1ng/ml phorbol myristate
acetate (PMA, Sigma, St.Louis). To examine the proliferative response of the leukemic cells to
CD40L, purified leukemic cells were cultured with -irradiated CD40L-presenting L cell, as
detailed previously (23). The cells were incubated at 37oC in 5% CO2 for 4 days. During the last
18 h of the culture 1Ci 3H-thymidine (20 Ci/mmol, PerkinElmer, Boston, MA) was added to
measure proliferation. All apoptosis and proliferation assays were repeated three times.
Online supplemental material.
The results of the microarray analysis representing all probe sets (pivot for supp) and those
representing the 216 probe sets which were upregulated upon initiation of the acute phase (216
subcluster gene list) will be accessible at http://eng.sheba.co.il/genomics.
7
Results
Transcriptional profiling of the leukemic cells during remission and the acute phase.
The recurrent leukemic attacks enabled us to search for changes occurring consistently in the
leukemic cells during the spontaneous induction and regression of the acute phase. Thus,
initiation of the attacks was indicated by high fever followed by a burst of blast-like
lymphocytes in the blood peaking on the third day and decaying spontaneously within 2-3
weeks. During remission periods 10-60% of the mononuclear cells were the clonal IgM-k, CD5+
CD19+ leukemic cells (22). cDNA microarrays, representative of ~12,000 genes, were
employed to compare transcriptional profiles of the leukemic cells during remission periods and
on day 3, 5, 7 and 11 of the attack. The leukemic cells were purified by magnetic cell separation
of CD19+ cells. Expression of hundreds of genes correlated with the phase of the leukemic
cycle. Furthermore, each stage of the attack was characterized by a different expression profile
(fig. 1A). Figure 1B depicts a cluster of 216 genes including CD70 and CD27, which were upregulated during transition from remission to the third day of the leukemic attack. This cluster of
stimulated genes includes among others genes related to immune response (16), cell adhesion
(14), response to external stimulus (21), signal transduction (30), cell-cell signaling (5),
regulation of cell proliferation (5) and cell death (15). Table 1 indicates some selected genes
whose expression was elevated during the leukemic attack. The first and second groups include
genes implicated in cell activation, signal transduction and proliferation. Specific examples of
these gene classes are (i) the CD80 antigen involved in the co-stimulatory signal essential for T
cell activation and PBEF1 secreted by activated lymphocytes, (ii) galectin 1, affecting the MEK
and PI3K signaling pathways, and MKP2 capable of controlling the MEK pathway, (iii) CD70
and CD137 ligand reported to promote reverse signaling in activated B cells and (iv) cyclin B1
and M-phase phosphprotein 1 required for control of the cell cycle. The third and fourth groups
of genes whose expression increased during the attack include the growth inhibiting genes
TGFRIII, CDK2AP1, and calgizzarin and the pro-apoptotic genes BIK, BAX, MCG10, PIG3,
DNASE1L3 and caspase 9. It is interesting that elevated expression of both cell growth
enhancers and inhibitors was evident at an early stage of the acute phase. Here we chose to
focus on CD70 and CD27 and investigate their possible involvement in the induction and
regression of the recurrent leukemic attacks.
8
Expression of CD70 and CD27 oscillates in correlation with the leukemic phase.
The expression level of CD70 and CD27 in the purified leukemic cells was verified by RTQPCR analysis. In an attempt to obtain expression ratios that more reliably reflect the transition
from remission to acute phase, leukemic cells from three different remission periods were used
as reference for comparison of gene expression during the attack. Three days following the
burst of the attack the level of CD70 and CD27 increased 38 and 25 fold, respectively. During
the next 8 days, in parallel with the spontaneous decay of the attack, expression of CD70 and
CD27 gradually decreased approaching the remission phase level (Fig. 2). The higher folds of
expression compared to those indicated in Table 1 (7 and 2.6, respectively) probably reflect the
use of a different remission cell reference. Higher folds of expression were also found in this
assay for TGFRIII and BIK (240 and 150 vs. 42.2 and 32.0, respectively). The time of
maximal expression of TGFRIII and BIK remained the 5th day of the attack (not shown).
Cell surface expression of CD70 and CD27 was determined on CD19+ CD5+ leukemic
cells by 4 colors FACS analysis. Flow cytometric dot plots show that expression of CD70 and
CD27 on the majority of the leukemic cells increased upon induction of the attack (Fig. 3A).
The cells were examined during 4 different remission periods and 5 out of 9 leukemic attacks
occurred during the first 3 years of the disease. CD70 was expressed on the majority of
CD19+CD5+ leukemic cells during the attack but on less than 10% of them during remission
periods while CD27 was consistently expressed on all the leukemic cells. The level of both
CD70 and CD27 expression was maximal on the second-third day of the attack (Fig. 3B). Thus,
during the peak of the acute phase 80% of the leukemic cells co-expressed high levels of
membrane CD70 and CD27 while during remission periods 90% of the leukemic cells expressed
CD27 but no detectable CD70 and 5-10% of them co-expressed CD70 and CD27. The intensity
of CD70 staining during remission indicates that the small fraction of leukemic cells included in
the CD70-positive subpopulation express high levels of CD70. This oscillation pattern of
membrane CD70 and CD27 expression was consistent during the first 3 years of the disease
while the patient did not receive any specific medication. Later on, following the 9th leukemic
attack, prednisone at 30 mg/day was instituted at the onset of the attack and the dose was
subsequently tapered off over a period of 30 days. The stimulation of membrane CD70
9
expression during the attack was inhibited by the prednisone treatment while elevated
expression of membrane CD27 was stabilized (Fig. 4).
Soluble CD27 in the patient's serum.
Soluble CD27 (sCD27), which is capable of binding cell surface CD70, may impede the
cellular response of membrane CD27 to CD70 presenting cells (25). Hence, examination of the
involvement of CD70-CD27 interaction in progression of the disease requires determination of
sCD27 levels in the patient's serum during different phases of the disease. The concentration of
sCD27 was determined by ELISA and was expressed as units per milliliter in reference to sera
of 11 healthy blood donors. The concentration of sCD27 in the sera of normal donors was
185±80 (U/ml). The patient's sera were examined while prednisone treatment was given during
the attack, as no sera were available prior to this period. The patient's sera were taken on the 6th
or 7th day of 4 different attacks, during a regression period (on day 20 of the attack), and during
3 remission periods (30, 90 and 150 days from initiation of the recent attack). During the 6th and
7th day of the attack the levels of sCD27 were 1600-3500 U/ml. These levels decreased to 600
(U/ml) during regression of the attack in the next 2 weeks and remained at the same level during
remission periods (Fig. 5). Thus, the level of sCD27 in the patient's serum oscillates with the
leukemic cycle being 3-5 folds of normal level during remission periods and further increased 35 folds during the attacks.
Functional analysis of CD70 and CD27 ligation on the leukemic cells.
Ligation of CD70 or CD27 on the leukemic cells membrane does not affect the rate of apoptosis.
Extreme changes in the rate of apoptosis of the leukemic cells during the leukemic cycle (22,
23) and oscillation in expression of some BCL-2 family genes (24) prompted us to examine the
possible involvement of membrane CD70 and CD27 in apoptosis. Leukemic cells from acute
phases and remission periods were incubated with 1-9g/ml mAb directed to CD70 or CD27,
(KI-24 or 1A4, respectively). The effect of the antibodies on spontaneous apoptosis of the
leukemic cells in culture was examined by Annexin-PI analysis. The indicated mAbs were
previously reported to affect some lymphocyte activities. Binding of KI-24 anti CD70 mAb to
10
CD70-positive glioblastoma cells increased allo-proliferation and decreased apoptosis of T
lymphocytes in a mixed tumor-T lymphocyte reaction (25), while 1A4 anti CD27 mAb inhibited
lympohcytes activation and proliferation (26, 27). Figure 6 shows that the bound CD70 and
CD27 mAbs did not affect the rate of apoptosis, either that of the acute phase leukemic cells
exhibiting rapid apoptosis or that of the remission phase leukemic cells exhibiting slower
apoptosis. This result suggests that cell surface CD70 and CD27 are not directly involved in
apoptosis of the leukemic cells.
Ligation of CD70 enhances proliferation of the leukemic cells from remission but not those from
the acute phase.
The leukemic cells of this patient do not proliferate in culture spontaneously. CD70 ligation
with anti-CD70 mAb strongly enhanced proliferation of the remission phase leukemic cells
submitogenically stimulated with PMA. In contrast, leukemic cells from the acute phase did not
exhibit such CD70-mediated proliferation. Ligation of CD27 on the leukemic cells from either
remission or the acute phase had no significant effect on proliferation (Fig. 7). The
enhancement of proliferation by CD70-ligation resulted from response of the leukemic cells
rather than that of normal lymphocytes. Thus, leukemic cells from 3 different remission periods
were examined following selection of CD19+ cells on magnetic beads. The purified cells,
containing 94-98% CD19+CD5+ leukemic cells, exhibited elevated proliferation as a result of
CD70 ligation (Fig. 8). CD70 mAb did not induce proliferation in absence of PMA (data not
shown). Ligation of CD70 on leukemic cells from 4 different acute phases at different stages of
the attack did not enhance proliferation. In contrast, ligation of CD40 on the leukemic cells
from acute phases, but not remission periods, enhanced proliferation (Fig. 8). Thus, during
remission the leukemic cells proliferate in response to CD70 ligation; with induction of the acute
phase they lose this property and acquire the ability to proliferate in response to CD40 ligaton.
11
Discussion
In this study we have investigated a possible contribution of CD70 and CD27 to progression of
an unusual B cell lymphoma/leukemia characterized by cycles of leukemic phase alternating
with spontaneous remission. We focused here on CD70 and CD27 because the interaction
between them may affect lymphocyte activation, proliferation, and death (1, 2) raising the
possibility that they are involved in the induction and regression of the recurrent acute attacks.
The results presented herein were mostly obtained during the first 3 years of the disease during
which the patient experienced 9 leukemic cycles without any specific therapy. However, Cell
surface expression of CD70 and CD27 and sCD27 in the serum, were also examined during a
later stage of the disease while the patient received prednisone during the attack.
Ttranscriptional profiling of the leukemic cells by microarray analysis showed massive changes
in gene expression between different stages of the disease. Increased expression of cell growth
enhancers and inhibitors as well as apoptosis enhancers was observed at an early stage of the
leukemic attack. For example, initiation of the attack was followed by highly increased
expression of galectin 1, implicated in enhancements of the Ras signal transduced to ERK (28)
via the MEK pathway and PKB activity (29) via the PI3K pathway. Relevant to this may be the
observation of Arens et al (19) who have reported that CD70 signaling in murine B cells
depends on initiation of MEK and PI3K pathways. On the other hand, termination of the MEK
signal could be affected by elevated expression of MKP2 that dephosphorylates MAP kinases
ERK1 and ERK2. This in turn could downregulate the MEK pathway during decay of the
attack. As for the suppressors of cell growth, the elevated expression of TGFRIII (and
CDK2AP1, which is induced by TGF could be relevant to the regression of the attack. This
possibility is suggested by two observations. First, TGF functions in CLL as an auto-reactive
growth inhibitor accounting for reduced proliferative response of the leukemic cells to CD40L
(30). Second, increased expression of TGFRIII is sufficient to enhance TGF signaling (31).
CD70 and CD27 were among the 216 genes whose expression consistently increased
upon induction of the acute phase concurrently with activation and blast transformation of the
leukemic cells. A detailed 4 colors FACS analysis showed that the level of membrane CD70
and CD27 on the CD19+CD5+ leukemic cells was maximal 2-3 days following initiation of the
leukemic attack. CD27 was consistently expressed on all the leukemic cells while CD70 was
12
expressed on 80% of the cells at the peak of the acute phase and on less than 10% of them
during remission. The fluctuations in cell surface expression of CD70 and CD27 characterized
the 9 leukemic cycles that occurred without specific therapy. A different situation was observed
during the second stage of the disease, in which prednisone treatment was given throughout the
acute phase. The prednisone treatment did not prevent the appearance of 18 additional attacks
but they were shorter and the lymphadenopathy less prominent (23). Cell surface CD70 levels
in presence of prednisone were not elevated during the attack while increased levels of CD27
were attenuated. The stimulation in CD70 expression occurring in this atypical CLL patient
concomitantly with cell activation and blastoid transformation is reminiscence of mantle cell
lymphoma in which up-regulation of CD70 coincides with blastoid transformation (32).
The consistent up and down regulation of cell surface CD70 and CD27 along the recurrent
leukemic attacks and remission periods, is compatible with a functional role of these proteins in
the cyclic nature of the disease. CD70 and CD27 mAbs were applied to examine the possible
involvement of cell surface CD70 and CD27 in activation and regression of the leukemic
attacks. We have found that the rate of apoptosis of the leukemic cells was not affected by
either CD70 or CD27 mAb, suggesting that cell surface CD70 and CD27 are not involved in the
accelerated apoptosis of the acute phase cells or the slow apoptosis of the remission phase cells
(22). However, PMA-mediated proliferation of the leukemic cells from remission phases, but
not that of the acute phase cells, was considerably enhanced by CD70 mAb. This augmented
proliferation of the remission phase cells was specific for CD70-ligation as mAbs directed to
several other membrane molecules, including CD27 mAb, had no effect on proliferation. The
finding that the remission phase cells respond to CD70 while the acute phase cells expressing
higher level of cell-surface CD70 are none-responsive, raises the possibility that other factors
determine whether ligation of CD70 may result in proliferation. A similar phenomenon has been
observed in CLL where, in addition to expression of surface Ig, the integrity of the BCRcoupled signaling complex determines the functional outcome of BCR-ligation (33). Our
finding is in agreement with that of Lens et al (17) who reported that leukemic cells of 4/18 CLL
patients proliferated in response to CD70 mAb. However, CD70 expression per se did not
predict whether CLL cells would proliferate in response to CD70 Ab. Expression of CD70
together with CD27 on the leukemic cells raises the possibility that this interaction may
13
constitute an autocrine circuit regulating the malignant cells proliferation. Alternatively, the T
cells infiltrating the lymphoid stroma in this patient (23) could as well provide the CD27 as
ligand for CD70 on the tumor cells.
We have previously found that the patient’s cells exhibited resting state during remission
periods and activated state at the initiation of the leukemic attack. Cell activation became
apparent by blast-transformation and elevated expression of CD23, CD25, ICAM-1, and LFA-3
(22, 23). The cell activation, the bouts of TNF and IL-10 detected in the serum during early
acute phases and the production of TNF and IL-10 by activated leukemic cells in culture (23)
suggested that the neoplastic cells responded to an activation signal at the onset of each
leukemic attack. The leukemic cells from the attack but not those from remission exhibited high
proliferative response to CD40L. The potent responsiveness of the acute phase leukemic cells to
CD40L is compatible with the finding that during acute phase the patient’s bone marrow was
heavily infiltrated with both CD40L-positive T cells and the neoplastic cells (23). These results
suggested that CD40L+, reactive T lymphocytes residing in the patient’s bone marrow support
proliferation of the neoplastic cells during the acute leukemic phases. Notably, however, the
proliferation response of the resting, remission phase cells to CD40L was significantly lower
than that of activated, acute phase cells (23). This observation implied that initiation of extensive
proliferation of the resting neoplastic cells at onset of acute phases requires an additional
activation signal. The finding herein that the remission phase leukemic cells proliferate
effectively in response to CD70-ligation and less effectively in response to CD40L while the
acute phase cells do not respond to CD70-ligation but proliferate in response to CD40L,
suggests that signaling via CD70 precedes that of CD40 in promoting the leukemic attack.
The recurrent change in CD70 responsiveness of the leukemic cells could be explained by
several ways: 1. The CD70 unresponsiveness of the acute phase cells could result from binding
of sCD27 to cell surface CD70 making it unavailable to the stimulating CD70 Ab. This
possibility seems however unlikely since the ratio of sCD27 to membrane CD70 is 3-5 times
higher during remission periods while the cells respond effectively to CD70 signaling. 2. The
second interpretation is based on a previous observation (22) that two B-cell clones with
differently rearranged heavy-chain genes consistently appeared in the blood of this patient
during recurrent acute phases. During the spontaneous remissions, one clone (termed the "acute
14
clone") regressed while the second clone (the "remission clone"), remained relatively stable.
Thus, the 2 clones of the leukemic cells may differ in their CD70 responsiveness as follows; the
remission clone which comprise the majority of leukemic cells during remission respond to
CD70-ligation by enhanced proliferation while the second clone which dominates the acute
phase do not respond to CD70. However, preferential proliferation of the remission clone cells,
contradicts the previous results which demonstrated that proliferation and death of the acute
clone cells and not that of the remission clone cells, determine the cyclic nature of the disease
(22). 3. The overall results suggest that the leukemic cells of the acute clone and not those of
the remission clone, consistently express cell surface CD70 and respond to its ligation during
remission but not during the attack. We do not know what may cause this switch between
responsive and none-responsive state of cell surface CD70. A possible explanation will be a
change in the signaling cascade. An alternative explanation could be a conformational change of
membrane CD70 affecting its responsiveness. A structural rearrangement of cell surface
integrins resulting in change of their ligand binding activity was previously described (34).
In conclusion, we have found that in this lymphoma/leukemia case a switch from
responsiveness to unresponsiveness of CD70 occurs on the leukemic cells during initiation of
the acute phase. Prior to this transition, cell surface CD70 could supply a co-stimulatory
receptor for cell proliferation and thus provide a growth advantage to the responder leukemic
clone. Termination of this CD70 response concurrently with cell activation and blast
transformation of the leukemic cells could contribute to regression of the acute phase in this
'cyclic' disease. The finding that spontaneous remission follows the acute phase in spite of
elevated proliferation response to CD40 ligation, support the notion that continuous growth of
the leukemic cells requires CD70 response in this case. In human leukemias and lymphomas
that constitutively express cell surface CD70 it may provide a co-stimulator receptor for tumor
cells proliferation. Whether this responsiveness is required for tumor cell growth and could be
regulated by a switch in CD70 reactivity should be examined in a future study.
Acknowledgments
We thank R. Pinkas-Kramarsky, R. Dardik and A. Simon for valuable advices and G.
Kaufmann for critical reading of the manuscript.
15
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19
Figure Legends
Figure 1.
Comparative gene expression profiles of purified leukemic cells obtained from the
atypical B-CLL patient during remission and different stages of the acute phase.
The leukemic cells
were taken on day 3, 5, 7 and 11 of the acute phase and during a remission phase and purified on CD19
beads. Transcriptional profiles of purified leukemic cells containing 94-98% CD5+CD19+ cells was
obtained by microarray analysis. Figure 1A shows an unsupervised hierarchal clustering of 5963 valid
probe sets out of the total 12625 probe sets. A distinction was made between the samples taken close to
the onset of the attack (day 3 and day 5) as compared to the samples taken later (day 7 and day 11)
which exhibited expression profile closer to that of the remission period. This is emphasized in the
enlarged subcluster (figure 1B) representing 216 probe sets which are up regulated in the samples taken
on the third and fifth day of the attack and down regulated in the samples taken later in the attack (day 7
and day 11) and in the remission sample. Included in the 216 genes subcluster are: (a) CD70, TNFligand (TNFSF7) (AffID 34054-at Acc L08096) and (b) CD27, TNF-receptor (TNFRSF7) (AffID
38578-at Acc M63928). Yellow indicates high relative expression and blue low expression. Each
column represents a sample and each row a gene.
Figure 2.
RTQ-PCR analysis of CD70 and CD27 gene expression in the leukemic cells during
remission and the leukemic attack.
Comparative ratio analysis by RTQ-PCR of CD70 (TNFSF7) and
CD27 (TNFRSF7) gene expression in purified leukemic cells during the leukemic cycle. The Y axis
shows relative gene expression. The average expression level of CD70 and CD27 during 3 different
remission periods (R) was taken as 1. Expression level of CD70 and CD27 increased 37 and 25 folds,
respectively, during transition from remission to the third day of the attack followed by gradual
decrease to the remission phase level.
Figure 3.
Cell surface expression of CD70 and CD27 on the leukemic cells is increased
concomitantly with transition from remission to the acute phase. Four colors FACS analysis
shows cell surface expression of CD70 and CD27 gated on CD19+CD5+ double positive
leukemic cells. A. Flow cytometric dot plots illustrate labeling of CD19+CD5+ cells with
20
antibodies to CD70 and CD27 during remission and on the third day of the leukemic attack. The
percentage of cells in the upper right quadrant (CD70+CD27+) is indicated.
B. Accumulated data represent staining intensity and percentage of CD70+CD27+ and CD70CD27+ leukemic cells during early (R1) and late (R2 and R3) remission periods and on different
days (2nd-11th) following the initiation of the leukemic attack. The percentage of CD70+CD27+
cells increased from 5-10% during remission periods to 80-90% on the second-third day of the
attack. Maximal density of cell surface CD70 and CD27 was observed at an early stage of the
attack.
Figure 4.
Prednisone treatment during the attack prevents elevation of cell surface CD70
expression on the leukemic cells.
Comparative expression of cell surface CD70 and CD27 on
the leukemic cells in absence and presence of prednisone. Four colors FACS analysis of
CD19+CD5+ leukemic cells shows that prednisone treatment inhibited stimulation of cell surface
CD70 during the attack. In contrast, elevation of cell surface CD27 at the initiation of the attack
was unaffected by prednisone treatment and its reduction during regression was attenuated.
Figure 5.
Elevated levels of sCD27 in the patient's Serum.
Serum samples were examined
for sCD27 on the day indicated following the initiation of the attack. Sera representing 2
different "leukemic cycles" were analyzed. Sera of 11 normal controls contained 185±80 U/ml
sCD27. The levels of sCD27 in the patient's serum were elevated during remission and were
further increased during the attack.
Figure 6.
cells.
Antibodies to CD70 and CD27 do not affect the rate of apoptosis of the leukemic
Spontaneous apoptosis of the leukemic cells in culture was examined. The results
expressed by annexin positive cells show the percent of apoptotic cells following 7 h or 24 h
incubation. Leukemic cells from the peak of the acute phase exhibiting rapid spontaneous
apoptosis (39% in 7h and 94% in 24h ) or leukemic cells from remission period exhibiting
slower apoptosis (23% in 7h and 36% in 24h ), were incubated in absence or presence of 6
g/ml of either anti CD70 mAb (clone ki-24) or CD27 mAb (clone 1A4). Data are expressed as
21
average values of 4 different experiments with cells from 2 different "leukemic cycles". The
rate of spontaneous apoptosis of the leukemic cells was not affected by either mAb.
Figure 7.
The effect of CD70 and CD27 mAbs on proliferation of leukemic cells obtained
during remission and acute phase.
The patient's leukemic cells obtained during remission
period or on the fifth day of the acute phase were preincubated for 30 min at 200C in the absence
or presence of 1-
g/ml of either anti CD70 mAb (clone ki-24) or CD27 mAb (clone 1A4).
PMA (1ng/ml) was added and cell proliferation was tested after 4 days of culturing. The data of
one representative experiment of three are displayed as average cpm (SE values ranged between
1-5%). Cells from 3 different "leukemic cycles" were analyzed. Proliferation of leukemic cells
from the remission phase but not that of the acute phase cells was enhanced by CD70 mAb.
CD27 mAb did not significantly affect proliferation of the leukemic cells.
Figure 8.
CD70 mAb enhances proliferation of purified leukemic cells obtained during
remission whereas CD40L enhances proliferation of those obtained during the attack. The
leukemic cells were taken on the third and fifth day of the acute phase and during two different
remission periods and purified on CD19 beads. The purified leukemic cells were cultured in
g/ml of either anti CD70 mAb (clone
ki-24) or CD27 mAb (clone 1A4). To examine the effect of CD40 triggering, the leukemic cells
were cultured with -irradiated CD40L-presenting cells. Shown is one representative
experiment out of three. CD70 mAb enhanced proliferation of the leukemic cells obtained
during remission and did not affect the acute phase cells. CD40L induced proliferation of the
leukemic cells obtained during the attack and did not affect the remission phase cells.
22
Table I. Differentially expressed genes during the leukemic attack
Functional category/Gene name
GenBank
Acc No.a
Fold Change relative
to Remission
Day in the attack
Cell activation and signal transduction
CD80 antigen
pre-B-cell colony-enhancing factor, PBEF1
galectin 1
dual specificity phosphatase 4, MKP2
Proliferation
CD70, TNFSF7
CD137 ligand, TNFSF9
cyclin B1
M-phase phosphoprotein 1
Growth inhibition
transforming growth factor receptor III
CDK2-associated protein 1, CDK2AP1
S100 Ca-binding protein A11, calgizzarin
Apoptosis
BCL2 interacting killer, BIK
poly(rc) binding protein 4, MCG10
quinone oxidoreductase homolog, PIG3
deoxyribonuclease I-like 3, DNASE1L3
caspase 9
BCL2 associated protein, BAX
CD27, TNFRSF7
M27533
U02020
AI535946
U48807
3
5
7
11
___________________
3.3 4.0 3.5
2.8
4.0 3.0 1.4
2.5
48.5 27.9 19.7 8.6
8.6 3.2 2.6
5.7
L08096
U03398
M25753
L16782
7.5
4.3
4.3
1.6
3.0
1.6
1.6
3.0
3.5
3.2
1.4
3.5
L07594
AF006484
D38583
22.6 42.2 18.4
3.5 5.7 3.7
5.7 7.5 2.8
21.1
2.3
1.9
U34584
W22541
AF010309
U75744
U60521
U19599
M63928
13.9 32.0
6.5 4.6
4.6 3.5
4.0 4.0
3.0 3.7
1.6 3.5
2.6 2.6
-1.1
4.9
-1.6
2.3
1.3
1.7
1.3
a
GenBank accession number.
23
4.6
2.6
7.5
4.0
5.7
3.7
1.1
1.1
1.2
2.1
1.5