*Thomas Enzler,1-3*Arnon P. Kater,1,4*Weizhou Zhang,2George F. Widhopf II,1Han-Yu Chuang,1Jason Lee,1EstherAvery,1
Carlo M. Croce,5Michael Karin,2and Thomas J. Kipps1
1Moores Cancer Center and2Laboratory of Gene Regulation and Signal Transduction, Departments of Medicine, Pharmacology and Pathology, School of
Medicine, University of California San Diego, La Jolla;3Department of Medicine, Stanford University School of Medicine, CA;4Department of Hematology,
Academic Medical Center,Amsterdam, The Netherlands; and5The Ohio State University Cancer Center, Columbus
Results of heavy-water labeling studies
have challenged the notion that chronic
lymphocytic leukemia (CLL) represents
an accumulation of noncycling B cells.
We examined leukemia cell turnover in
E?-TCL1 transgenic (TCL1-Tg) mice,
which develop a CLL-like disease at 8 to
12 months of age. We found that leukemia
cells in these mice not only had higher
proportions of proliferating cells but also
apoptotic cells than did nonleukemic lym-
phocytes. We crossed TCL1-Tg with
BAFF-Tg mice, which express high levels
of CD257. TCL1?BAFF-Tg mice devel-
oped CLL-like disease at a significantly
younger age and had more rapid disease
progression and shorter survival than
TCL1?BAFF-Tg mice had similar propor-
tions of proliferating cells, but fewer pro-
portions of dying cells, than did the CLL
cells of TCL1-Tg mice. Moreover, leuke-
mia cells from either TCL1?BAFF-Tg or
TCL1-Tg mice produced more aggressive
disease when transferred into BAFF-Tg
mice than into wild-type (WT) mice. Neu-
tralization of CD257 resulted in rapid re-
duction in circulating leukemia cells.
These results indicate that the leukemia
cells of TCL1-Tg mice undergo high lev-
els of spontaneous apoptosis that is off-
set by relatively high rates of leukemia
cell proliferation, which might allow for
acquisition of mutations that contribute
to disease evolution. (Blood. 2009;114:
Chronic lymphocytic leukemia (CLL), the most common adult
leukemia in Western countries, is a disease of neoplastic, monoclo-
nal CD5?B cells, which accumulate in the blood, marrow, and
secondary lymphoid tissues. For years this disease has been viewed
as a prime example of a tumor with very low levels of cell turnover
in which intrinsic resistance to apoptosis was responsible for a
gradual accumulation of neoplastic CD5?B cells.1Consistent with
this notion, CLL cells express high levels of antiapoptotic Bcl-2
family members as well as inhibitors of apoptosis proteins.2-5
However, recent studies have challenged this view. CLL cells
were found to undergo spontaneous apoptosis in vitro under
conditions that could support growth of established B-cell lines.6
Spontaneous apoptosis of CLL cells could be inhibited by acces-
derived marrow stromal cells,7follicular dendritic cells,8or nurse-
like cells (NLCs),6,9-11suggesting that CLL cells do not have an
intrinsic resistance to apoptosis. In particular, NLCs produce
factors that can enhance leukemia cell survival in vitro.6,12Among
these factors is a member of the tumor necrosis factor family, most
notably CD257 (B cell–activating factor of tumor necrosis factor
family [BAFF], also called tumor necrosis factor and apoptosis
lator).10CLL B cells express 3 receptors for CD257, namely
CD269 (formerly called B-cell maturation antigen), CD267 (for-
merly called transmembrane activator and CAML interactor), and
CD268 (formerly called BAFF receptor, or BR3).13,14Interaction of
CD257 with these receptors can enhance leukemia cell survival in
vitro, a mechanism that potentially could contribute to disease
progression in vivo.14The existence of cycling cells in CLL
patients is suggested by recent studies using heavy water to label
leukemia cells in vivo.15,16These studies demonstrated that some
patients with apparently indolent disease might generate up to 1%
of their entire leukemia cell population each day, implying that in
CLL there might be high rates of cell turnover that counterbalance
such high rates of leukemia cell proliferation. As such, we
hypothesize that B-cell survival factors such as CD257 could
enhance disease progression merely by inhibiting leukemia cell
turnover, thereby allowing for enhanced accumulation of nascent
leukemia cells in vivo.
We examined for this in an animal model for CLL, namely mice
made transgenic (Tg) for the human T-cell leukemia 1 (TCL1) gene
controlled by the immunoglobulin ? heavy-chain promoter/
enhancer, known as E?-TCL1-Tg mice (here noted as TCL1-Tg
mice). Such animals have constitutive high-level expression of
Tcl1 in mature B cells. At 8 to 12 months of age, these mice
develop a CLL-like disease that shares many features with human
CLL, such as excessive accumulation of monoclonal CD5?B cells
in blood and lymphoid tissues.17Originally identified in T-cell
leukemia, Tcl1 enhances Akt kinase activity, mediates its nuclear
translocation, and influences cell activation and survival.18More
recently it was shown that Tcl1 is expressed by human CLL cells
and also interacts with c-Jun, JunB, and c-Fos to inhibit activator
Submitted June 29, 2009; accepted August 17, 2009. Prepublished online as
Blood First Edition paper, September 15, 2009; DOI 10.1182/blood-2009-06-
*T.E.,A.P.K., and W.Z. shared first authorship.
The online version of this article contains a data supplement.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2009 by TheAmerican Society of Hematology
4469BLOOD, 12 NOVEMBER 2009?VOLUME 114, NUMBER 20
protein 1 transcriptional activity. Furthermore, Tcl1 was found to
play a role in the activation of nuclear factor ?B (NF-?B) by
interacting with p300/cyclic adenosine monophosphate response
element binding protein.19Because the leukemia cells that develop
in TCL1-Tg mice express high levels of Tcl1, it was assumed that
these cells had low rates of spontaneous apoptosis, in contrast to
what now is speculated to occur in at least some patients with CLL.
In our study, we examined whether the leukemia cells of TCL1-Tg
mice instead undergo high rates of cell turnover and whether
CD257 could suppress spontaneous apoptosis of leukemia cells,
thereby offsetting cell turnover in favor of disease development and
progression in this animal model of CLL.
E?-TCL1 mice (TCL1-Tg mice) were backcrossed for at least 9 generations
onto the C57Bl/6 background. BAFF-Tg mice were from L. Gorelik
(Biogen Idec). All mice were housed under conventional barrier protection
in accordance with University of California San Diego and National
Institutes of Health guidelines, and mouse protocols were approved by the
University of California San Diego Institutional Animal Care Committee.
Survival data were obtained by observing cohorts of 12 mice (6 females and
6 males) of each genotype.
Adoptive cell transfer
For adoptive transfer, splenic cells from TCL1XBAFF-Tg mice were
labeled with CD5-allophycocyanin and CD3–fluorescein isothiocyanate
(FITC) antibodies and separated using a FACSAria cell sorter (Becton
Dickinson). CD3?CD5?sorted cells (106) in 100 ?Lof phosphate-buffered
saline (PBS) were injected retro-orbitally into either wild-type (WT) or
BAFF-Tg mice that were gamma-irradiated with 6 Gy rad and anesthetized
using isoflurane (Abbott Labs).
netetraacetic acid–coated blood collection tubes (Becton Dickinson).
Single-cell suspensions were obtained from spleen, lymph nodes (axillary,
brachial, and inguinal or mesenteric), and thymus by grinding through
nylon sieves (BD Falcon). For some experiments, B cells were positively
enriched from WT mice splenocytes with B220 magnetic-activated cell
sorting (MACS) beads and leukemic cells were negatively enriched with
CD3 MACS beads, followed by positive selection with CD5 MACS beads
(Miltenyi Biotec) according to manufacturer’s protocol.
Single-cell suspensions were stained for surface expression with phyco-
erythrin (PE)–labeled anti-CD5, allophycocyanin-labeled anti-B220
(CD45R), and FITC-labeled anti-CD3 antibodies (Pharmingen). For intra-
cellular staining, cells were stained for surface markers before fixation and
permeabilization using the Fix & Perm kit (Caltag) and staining with a
PE-labeled monoclonal human TCL1 antibody (Pharmingen). Propidium
iodide was used to exclude dead cells. Flow cytometry analyses were
performed with either BD FACSCalibur or Accuri C6 flow cytometers.
Plots were done with FlowJo software (TreeStar).
In vitro apoptosis measurements
Purified B cells from 12-month-old WT control mice and CD5?leukemic
cells from TCL-Tg mice were treated with 200 ng/mL recombinant human
CD257 (rhCD257; a kind gift from Dr G. Zhang, National Jewish Medical
and Research Center) for the indicated time periods and terminal deoxynu-
cleotidyl transferase deoxyuridine-triphosphatase nick-end labeling
(TUNEL) assays were performed with In Situ Cell Death Detection Kit
TMR Red (Roche) and analyzed with an Accuri C6 flow cytometer. Cell
viability was also examined by trypan blue exclusion.
TUNEL and CD5 staining on frozen splenic sections
Frozen spleen sections were fixed and permeabilized with Perm/Fix
solution (BD Biosciences) according to instruction. TUNEL staining was
performed as described above and washed 3 times with PBS, followed by
CD5-biotin and streptavidin-FITC staining after blocking with 2% goat
serum, 1% fetal calf serum, and 1% bovine serum albumin in PBS. Slides
were counterstained with 4,6 diamidino-2-phenylindole before mounting
with antifade solution.
Cell fractionation and immunoblots
Cytosolic and nuclear fractions were prepared as follows: 106B cells or
leukemic cells were treated with 200 ng/mL CD257 or control vehicle for
the indicated periods. Cells were collected and suspended in buffer A
(10mM N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid, pH 7.8;
1.5mM MgCl2; 10mM KCl; 0.5mM dithiothreitol, 0.05% Nonidet P-40,
and protease inhibitor cocktails from Roche) for 30 minutes on ice; nuclei
and cell debris were subjected to 500g for 10 minutes at 4°C and
supernatants (cytosol) were collected. Nuclear pellets were suspended in
buffer B (5mM N-2-hydroxyethylpiperazine-N?-2-ethanesulfonic acid,
pH 7.8; 1.5mM MgCl2; 0.2mM ethylenediaminetetraacetic acid, 0.5mM
dithiothreitol, 26% glycerol, and protease inhibitors cocktail). Cytosolic
and nuclear fractions were gel separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis for immunoblot analyses with antibod-
ies specific for: Bcl-2, Bcl-XL, RelB, p65, histone deacetylase 1 (HDAC1;
Santa Cruz Biotechnology), A1/Bfl1, Mcl-1 (Cell Signaling Technology),
?-tubulin (Sigma-Aldrich), or p52 (Upstate Biotechnology).
Cell proliferation assays
For in vivo 5-bromo-2-deoxyuridine (BrdU) labeling, 10 mg/mLBrdU (BD
Biosciences) in PBS was injected intraperitoneally and 12 hours later
splenocytes were collected and stained with anti–BrdU-FITC antibody
(Pharmingen) after fixation with 2% paraformaldehyde, permeabilization
using 3M HCl plus 2%Tween 20 solution, and neutralization with 1M boric
acid. For in vitro Ki-67 staining, 106cells were surface labeled with
CD5-PE antibody. Labeled cells were permeabilized with HCI plus Tween
and labeled with Ki-67 antibody (Pharmingen).
Decoy receptors CD268-Fc and CD269-Fc
Decoy receptors CD268-Fc and CD269-Fc were kindly provided by
Genentech and Biogen Idec, respectively.
Southern blot analysis
Splenic leukemia cells were processed to single cells and cryopreserved.
DNA was isolated and digested with HindIII and analyzed by Southern
blotting using a JH-probe as described.20Detection limit of was around 20%
of clonal expansion, as determined by serial dilutions of hybridoma-derived
DNAwith genomic DNAof NIH3T3 cells.
Student t tests assuming equal variance were performed for most of our
studies. For comparison of TUNEL-positive cells in splenic sections of the
4 different mice strains, a 1-way, repeated measure using analysis of
variance with Bonferroni correction was performed. For survival curve
shown by Kaplan-Meier plot, log-rank (Mantel-Cox) test and log-rank test
for trend were performed with Prism V.5 software (GraphPad). P values are
indicated in the figures.
4470ENZLER et al BLOOD, 12 NOVEMBER 2009?VOLUME 114, NUMBER 20
CD5?B220lowB cells in TCL1-Tg mice do have a high
In comparison with normal B cells, leukemic cells of TCL1-Tg
mice express less B220 but are positive for the T-cell marker CD5,
which is not expressed normally by mature B cells. These features
are similar to the expression patterns of CD5 and CD20 in human
CLL.17,21To test whether the proliferation rate of leukemic
B220lowCD5?cells of TCL1-Tg mice is different from normal
B cells we injected 5-bromo-2-deoxyuridine (BrdU) into 4- to
9-month-old TCL1-Tg mice and into WT littermates. We mea-
sured BrdU incorporation of CD5?B220?, CD5?B220low, and
CD5?B220?cells by flow cytometry. We found that BrdU
incorporation into leukemic cells that represented cells entering
S phase was significantly higher than BrdU incorporation into
normal B or T cells (Figure 1A-B). Surprisingly, BrdU incorpora-
tion into normal B or T cells did not differ from TCL1-Tg mice and
WT mice. This may explain why young TCL1-Tg mice have
normal B- and T-cell counts. BrdU incorporation correlated well
with expression of the proliferation marker Ki-67 that was en-
hanced in leukemic cells of TCL1-Tg mice (Figure 1B-C; supple-
mental Figure 1A, available on the Blood website; see the Supple-
mental Materials link at the top of the online article).Assuming that
the leukemic cells proliferate constantly at this high rate cell counts
are expected to double every 2 to 3 weeks. However, this was by
far not the case, and disease development observed in TCL1-Tg
mice was much slower. Consequently, a substantial amount of
leukemic cells must undergo cell death. Indeed, there is a much
higher death rate of CD5?B220?cells in spleens TCL1-Tg mice
than of normal CD5?B220?B cells, as measured by terminal
deoxynucleotidyl transferase deoxyuridine-triphosphatase nick end
labeling (TUNEL) and costained with either CD5-FITC or CD45R-
FITC (Figure 1D-E; supplemental Figure 1B-C). Thus, although
cell proliferation of leukemic cells in TCL1-Tg mice is high, a
substantial part of leukemic cells underwent apoptosis, slowing
down disease progression. Taken together, we found that CLL cell
turnover in TCL1-Tg mice is unexpectedly high, closely resem-
bling in vivo kinetics of human CLL,15which is a new and
important feature of the TCL1-Tg mouse model that is shared with
CD257 increases survival of leukemic cells from TCL1-Tg mice
As we demonstrated earlier, CD257 is a potent survival factor for
human CLL cells in vitro.10We examined the effect of CD257 on
CD3?CD5?leukemic B cells from TCL1-Tg mice. We monitored
for apoptosis of CD3?CD5?B cells from TCL1-Tg mice (n ? 3)
that were cultured with or without recombinant human (rh) CD257,
using TUNEL assay (Figure 2A-B; supplemental Figure 2). Cul-
ture with rhCD257 significantly reduced the proportion of cells
Figure 1. CD5?B220lowB CLL cells from TCL1-Tg mice have a high
cell turnover. (A) In vivo cell proliferation of different cell populations of
WT (top panel) or TCL1-Tg (bottom panels) mice was studied by injecting
10 mg/mL BrdU intraperitoneally into the mice strains indicated after
analysis of splenic cells by flow 12 hours later using the antibodies
indicated (n ? 6 mice each strain). We gated on CD5?B220?T cells (P1),
CD5?B220?B cells (P2), or CD5?B220lowB cells (P3), as indicated in the
far left panels. We determined the proliferation rates for each of these
subpopulations by evaluating for the proportion of cells that incorporated
BrdU (rectangles), as indicated in the panels to the right. Each of these
panels depicts the BrdU fluorescence of gated cells in P1, P2, or P3,
respectively. (B) Statistical analysis of the results obtained in panel A.
(C) CD5?B220lowB CLLcells from TCL1-Tg were also tested for levels for
Ki-67 using intracellular flow cytometry and those results were compared
with B220?splenic B cells from WT mice (n ? 3 mice each strain).
(D-E) Frozen splenic sections of mouse strains indicated were costained
for CD5-FITC and TUNEL-TMR-Red. Data shown were average of
TUNEL-positive cells/field from splenic sections (n ? 6 mice). Magnifica-
tion of the objective lense: 40?/1.3 Oil DIC. Camera: ZeissAxioCam HR.
CLLCELLTURNOVER IN TCL1-Tg MICEAND CD257 IMPACT 4471BLOOD, 12 NOVEMBER 2009?VOLUME 114, NUMBER 20
undergoing apoptosis. As we showed previously, rhCD257 acti-
vated both classical and alternative NF-?B signaling in normal
B cells and human CLL cells.14,22Similarly, stimulation of normal
or neoplastic B cells from WT or TCL1-Tg mice with rhCD257
resulted in their having increased amounts of nuclear p65/RelA,
RelB, and p52 (Figure 2C). Correspondingly, stimulation of such
B cells with rhCD257 induced increased expression of several
NF-?B target genes, including those encoding the antiapoptotic
proteins Bcl-2, Bcl-XL, Mcl-1, and A1/Bfl-1 (Figure 2D). Thus,
rhCD257 acted in a very similar way in leukemic B cells from
TCL1-Tg mice as it did in human CLLcells.
CD257 accelerates leukemogenesis in TCL1-Tg mice
We crossed TCL1-Tg mice17with BAFF-Tg mice23and monitored
cohorts of WT, BAFF-Tg, TCL1-Tg, or TCL1?BAFF-Tg mice for
development of CLL-like disease (6 males and 6 females each
cohort).Whereas TCL1-Tg mice develop a leukemic CD5?B220low
population at approximately 7 months of age, TCL1?BAFF-Tg
mice develop such a population before 4.5 months of age
(Figure 3A). We did not observe a sex bias in disease development.
WT or BAFF-Tg mice did not develop a leukemic cell population
throughout their life spans. The CD5?B cells expressed human
TCL1 (hTCL1), as assessed by intracellular flow cytometry
(Figure 3B). At 9 months of age the mean blood count of CD5?
B cells in TCL1?BAFF-Tg mice (45 ? 106cells/mL) was signifi-
cantly greater than that of TCL1-Tg mice (10 ? 106cells/mL;
Figure 3C). In accordance with this, blood smears of 9-month-old
mice revealed that both TCL1-Tg and TCL1?BAFF-Tg mice had
mononuclear cells that predominately were mature lymphocytes
with numerous cells appearing similar to smudge cells typically
identified on blood smears of CLL patients (supplemental Fig-
ure 3).24Southern blot analyses of splenocytes harvested at various
ages detected oligoclonal, or monoclonal, splenic CLLcell popula-
tions already by 4 months of age in all TCL1?BAFF-Tg mice,
whereas such cells could not be uniformly detected until 12 months
of age in TCL1-Tg mice (Figure 3D). Collectively, these data
indicated that TCL1?BAFF-Tg mice developed oligoclonal, or
monoclonal, CD5?B-cell lymphoproliferative disease resembling
CLLmuch earlier than TCL1-Tg mice.
We found the life span of TCL1?BAFF-Tg mice was signifi-
cantly shorter than that of TCL1-Tg or BAFF-Tg mice (Figure 3E).
Whereas both TCL1?BAFF-Tg mice and TCL1-Tg mice primarily
died of leukemia, most BAFF-Tg mice died of renal failure due to
autoimmune disease, as previously noted.23None of the
TCL1?BAFF-Tg mice examined on necropsy had developed overt
renal pathology, apparently because they had died too young for
kidney disease manifestation. In summary, presence of extrinsic
CD257 in TCL1-Tg mice led to a more aggressive disease course
and shorter survival.
CD257 enhances survival rather than proliferation of leukemia
Because double-transgenic mice developed leukemia at a signifi-
cantly younger age than TCL1-Tg mice, we investigated whether
constant CD257 exposure increased the rate of leukemia cell
proliferation by labeling cells in vivo with BrdU. Importantly, we
did not observe a significant difference in BrdU incorporation by
leukemia cells from TCL1-Tg compared with cells from
TCL1?BAFF-Tg mice (Figure 4A, supplemental Figure 4A). Fur-
thermore, the proportions of cells labeling with the proliferation
marker Ki-67 were not different between leukemia cells from
TCL1-Tg mice versus those obtained from TCL1?BAFF-Tg mice
(Figure 4B, supplemental Figure 4B). TUNEL staining of spleen
sections revealed that TCL1-Tg mice had 3 times more apoptotic
Figure 2. CD257 enhances survival of CD3?CD5?CLL cells from
TCL1-Tg mice and activates NF-?B. (A) CD3?CD5?CLL cells from
TCL1-Tg mice were cultured in absence (top line) or presence (bottom
line) of 200 ng/mL rhCD257 and apoptosis was determined by TUNEL
assay at different time points as indicated (n ? 3 independent experi-
ments).One representativeassayisshownforeachexperiment.(B) Statis-
tical analysis of data obtained in panel A. Error bars represent SD of
3 independent experiments. (C) Immunoblot from nuclear extracts of cells
treated for different times with 200 ng/mL rhCD257, or left untreated,
using antibodies to detect activation of the classical (p65) or alternative
a representative blot (n ? 3 independent experiments). (D) Immunoblot of
cytosolic extracts of the same cells used in panel C immunoblotted for the
antibodies indicated. Tubulin was used as a loading control. Shown is a
representative blot (n ? 3 independent experiments).
4472 ENZLER et alBLOOD, 12 NOVEMBER 2009?VOLUME 114, NUMBER 20
cells in their spleen than TCL1?BAFF-Tg mice (Figure 4C-D),
suggesting that CD257 promotes B-cell expansion mainly by
inhibiting B-cell apoptosis in vivo.
Comparison of genome-wide gene expression of CD3?CD5?
splenic B cells from 12-month-old TCL1-Tg mice (n ? 3) versus
age-matchedTCL1?BAFF-Tgmice(n ? 3)showedsimilarexpres-
sion levels of genes encoding proteins involved in cell proliferation
(P ? .39;supplementalTable 1).Takentogether,thesedatasuggest
that CD257 does not promote leukemogenesis in TCL1-Tg mice by
enhancing the rate of leukemia cell proliferation but rather by
elongating their life span.
Transfer of CD3?CD5?leukemic cells to BAFF-Tg mice results
in rapid disease progression
To exclude the possibility that TCL1?BAFF-Tg mice have intrin-
sic factor produced governing the disease course and to fully test
our hypothesis that factors extrinsic to the leukemia cells play a
prominent role in CLL development, we performed adoptive cell
transfers of leukemic cells from TCL1?BAFF-Tg and from
TCL1-Tg mice (data from TCL1-Tg mice not shown). For this, we
isolated CD3?CD5?CLL cells from spleens of 9-month-old
TCL1?BAFF-Tg or TCL1-Tg mice, infused 106such cells into
Figure 3. Constitutive CD257 production enhances leu-
kemia development. (A) Flow cytometric analysis of blood
cells from different mouse strains (TCL1?BAFF-Tg mice
labeled as BAFF/TCL1; TCL1-Tg, as -/TCL1; and BAFF-Tg,
as BAFF/-) collected at different ages as indicated and
stained with CD5 and B220 antibodies. Each analysis was
done in triplicate. One representative blot for each analysis
is shown. (B) Representative contour plots of blood cells
from 9-month-old mice (n ? 3 in each cohort) stained with
CD5 and hTCL1 antibodies. (C) Blood-derived CD3?CD5?
cells in the indicated mouse strains at different ages were
measured by flow cytometry using CD3 and CD5 antibodies
(n ? 12 mice per cohort; error bars indicate SD). (D) Clonal
expansion of TCL1-Tg and TCL1?BAFF-Tg mice spleno-
cytes (n ? 2 per cohort per indicated age) as determined by
Southern blot analysis of genomic DNA using a JH-probe.
(E) Survival (Kaplan-Meier) plots of cohorts of 12 age- and
sex-matched mice of the indicated strains. Mean survival
times were TCL1?BAFF-Tg mice, 11.2 ? 3.4; TCL1-Tg,
20.7 ? 3.9; and BAFF-Tg, 21.0 ? 3.8 months. More than
50% of WT mice were still alive at the end of the 25-month
Figure 4. CD257 does not affect cell proliferation but enhances
survival of leukemia cells. (A) Proliferation of CD3?CD5?lymphocytes
obtained from TCL1-Tg or TCL1?BAFF-Tg mice was measured by BrdU
incorporation. For this, mice were pulsed with BrdU and 12 hours later
splenocytes were collected and stained with anti-BrdU. Mouse immuno-
globulin G was used as an isotype control for anti-BrdU. Splenocytes from
TCL1-Tg and TCL1?BAFF-Tg mice were gated for CD3?CD5?and
analyzed by flow using anti-BrdU. Results are from triplicate experiments
(n ? 3 mice each strain). (B) Ki-67 antibody stainings. Cells were obtained
and gated as in panel A and stained for intracellular Ki-67 expression.
Mouse immunoglobulin G was used as isotype control for anti–Ki-67.
Results are from triplicate experiments (n ? 3 mice each strain).
(C-D) Frozen splenic sections of mouse strains indicated were stained for
TUNEL-TMR-Red. Data shown were average of relative TUNEL-positive
cells from splenic sections (n ? 5 mice). Magnification of the objective
lense: 20?/0.50 Camera: Zeiss AxioCam HR. Software: AxioVs40AC
CLLCELLTURNOVER IN TCL1-Tg MICEAND CD257 IMPACT 4473 BLOOD, 12 NOVEMBER 2009?VOLUME 114, NUMBER 20
either BAFF-Tg or WT mice, and monitored the growth of CD5?
hTCL1?leukemia cells over time (Figure 5A). Whereas CD5?
hTCL1?cells were undetectable inWTmice 30 days after adoptive
transfer, such cells were readily identified in the blood of BAFF-Tg
recipient mice at this time. To trace the leukemic cells after
adoptive transfer, we transduced the CD3?CD5?leukemic B cells
cence-based imaging 10 and 20 days after adoptive transfer re-
vealed accumulation of leukemia cells primarily in BAFF-Tg
recipients, but not in WT recipients (Figure 5B). Necropsy at
6 months after adoptive transfer revealed that BAFF-Tg mice had
massively enlarged spleens that were extensively infiltrated with
leukemia cells, accounting for more than 90% of total splenocytes
(Figure 5C). These mice also had enlarged lymph nodes that were
heavily infiltrated with leukemic B cells as well (Figure 5D), but a
normal-appearing liver and thymus. WT recipients, on the other
hand, had normal-sized spleens with leukemic cells accounting for
only 10% (10.4% ? 1%; n ? 3) of all splenocytes (not shown).
Our results indicated that extrinsic CD257 was able to greatly
accelerate CLLdisease course.
Inhibition of CD257-CD257 receptor interactions in vivo results
in rapid decline of leukemic cell count
Incubation of B cells with soluble CD268-Fc decoy receptor
completely abrogated the prosurvival effect of rhCD257 in vitro
(Figure 6A). We next examined whether such decoy receptors
could antagonize the effect of CD257 on leukemia B cells in vivo.
To test this, we injected CD268-Fc or CD269-Fc decoy recep-
tors,25,26or control protein, into the peritoneum of BAFF-Tg mice
that were previously adoptively transferred with CD3?CD5?
leukemic B cells from TCL1?BAFF-Tg mice and we monitored
leukemia blood counts over time. Injection of 200 ?g of CD268-Fc
reduced the number of circulating CD3?CD5?cells relative to that
found in control-treated mice by almost 20% (18.2% ? 5.3%;
n ? 3)within5 days(Figure 6B).Injectionof200 ?gofCD269-Fc
was even more effective in reducing the number of circulating
CD3?CD5?leukemia cells (37.4%?4.7%; n?3). These findings
indicate that strategies that target CD257 and/or CD257-expressing
A long-held view was that CLL represented an accumulation of
slowly dividing, incompetent pathologic B cells that had an
intrinsic resistance to apoptosis.1However, recent studies have
challenged this assumption, suggesting that CLL is a much more
dynamic process in which relatively high rates of leukemia cell
proliferation are offset by high rates of leukemia cell turnover.15
This different view of CLLmay explain in part the activity of drugs
Figure 5. CLL cells expand faster in vivo in the
presence of constantly elevated levels of CD257.
(A) Splenocytes from TCL1?BAFF-Tg mice were labeled
with CD3 and CD5 antibodies and CD3?CD5?were
enriched by cell sorting. Sorted leukemic cells (106) were
injected into either BAFF-Tg or WTrecipients (n ? 6 each
flow cytometry. (B) Expansion of transferred CD3?CD5?
cells transduced with a luciferase-expressing lentivirus
before injection was measured by bioluminescence at the
indicated time points. (C) Representative autopsy and
flow cytometry of splenocytes from a BAFF-Tg mouse
5 months after adoptive transfer (n ? 6). Camera: Nikon
COOLPIX 995. (D) Representative flow cytometry of cells
obtained from inguinal lymph nodes from WT or BAFF-Tg
mice 5 months after adoptive transfer (n ? 6 mice each
Figure 6. Blockade of CD257 induced the regression of leukemic cells from
TCL1?BAFF-Tg mice grown in vitro and in BAFF-Tg mice. (A) In vitro viability of
CD3?CD5?cells from TCL1-Tg mice was examined by trypan blue exclusion in
absence or presence of 200 ng/mLrhCD257 and absence or presence of 200 ?g/mL
decoy receptor CD268-Fc (n ? 3). (B) BAFF-Tg mice inoculated with leukemic cells
were injected with 200 ?g of decoy receptors CD269-Fc or CD268-Fc. Peripheral
CD3?CD5?cell counts were determined 5 days after treatment with decoy receptors.
Values represent relative mean ? SD CD3?CD5?cell counts of treated mice
compared with normalized actual CD3?CD5?cell counts of untreated mice (n ? 3
mice per each treatment group; each experiment was done in triplicate).
4474ENZLER et al BLOOD, 12 NOVEMBER 2009?VOLUME 114, NUMBER 20
in this disease that target cells undergoing replication.27It also
provides a model for the clonal evolution theory in which
subclones of daughter cells are thought to establish predominance
and possibly overtake the cell population if the subclones incur
chance somatic mutations that provide them with a competitive
advantage.28Validation of this in future clinical studies would
provide incentive for re-evaluating the current watch-and-wait
strategies that withhold treatment for patients until they develop
overtly progressive and/or symptomatic disease.29
Here we demonstrate that an animal model for CLL, the
TCL1-Tg mouse, does indeed experience high rates of leukemia
cell turnover in vivo and thus mimics human CLLin this important
feature. This allows us to use this animal model to investigate the
effect of factors that can offset or inhibit cell turnover on the
pathogenesis and progression of this disease. It also allows for
studies on the influence of factors that potentially could offset the
balance between cell proliferation and survival that apparently
governs the overall rate of disease progression.
We examined the effect of CD257 on this presumed balance.
CD257 is a known survival factor for normal and leukemia B cells
that is expressed at high levels by NLCs, which conceivably
operate in the leukemia microenvironment to enhance leukemia
cell survival in vivo.10For this purpose, we used BAFF-Tg mice,
which express high levels of CD257 in the liver, but also have
elevated circulating CD257 that can cause systemic effects.23
Despite high-level expression of CD257, BAFF-Tg mice do not
develop overt lymphoma per se, but rather systemic autoimmune
disease.22,23TCL1?BAFF-Tg mice, on the other hand, have
continuous, high-level expression of Tcl1, and systemically el-
evated levels of CD257, which can promote leukemia cell survival.
Consistent with the model that proposes this could offset the
balance between leukemia cell proliferation and cell turnover.
TCL1?BAFF-Tg mice more rapidly developed leukemia and had
more aggresive disease than TCL1-Tg mice. The studies involving
adoptive transfer of leukemia CD5?B cells into WT versus
BAFF-Tg mice provided further evidence that extrinsic CD257
progressive leukemia, WT recipients remained healthy for many
months, experiencing only slow disease progression. This effect of
CD257 could be inhibited by soluble CD268-Fc or CD269-Fc
decoy receptors, which resulted in an immediate and substantial
drop in leukemia cell blood counts of such mice.
The capacity of CD257 to enhance leukemia survival was
associated with elevated expression of antiapoptotic proteins,
whose genes are regulated in part by NF-?B. Whereas the TCL1
transgene had a relatively weak effect on NF-?B activation in CLL
cells, we observed a synergistic effect in presence of CD257 on
NF-?B activation in such cells. Similarly, although freshly isolated
circulating human CLL cells exhibited constitutively activated
NF-?B, which apparently could enhance survival and contribute to
disease progression,30,31microarray analyses demonstrated in-
creased levels of expression of NF-?B target genes in CLL cells
that resided within the leukemic cell microenvironment.32These
findings point to microenvironmental factors as key survival
signals, closely resembling the role of cytokines produced by
myeloid cells in solid malignancies.33-36Our results fit well with the
ute to leukemic cell survival and hence disease progression. It seems
leukemia microenvironment of lymphoid tissues,10is one such factor
Mouse models of human cancers can be used to investigate the
effectiveness of therapeutic approaches before human clinical
trials. We found relatively rapid leukemia cell turnover in TCL1-Tg
mice, making this mouse model one with which to study the
influence of factors or agents that alter either cell proliferation or
cell death. Moreover, the adoptive transfer of leukemia cells into
BAFF-Tg mice provides an excellent model with which to test
agents that can interfere with signaling pathways that promote
leukemia cell survival. Collectively, our findings reported here
suggest that agents that target proliferating cells might work
synergistically with agents that inhibit the survival factors in
eliminating the disease. Thus, future treatment strategies should
envelop consideration of the potentially dynamic nature of CLL so
as to provide the most effective therapy for this disease.
We thank Dr G. Zhang (National Jewish Medical and Research
Center) for the kind gift of rhCD257. We thank Genentech and
Biogen-Idec for the kind gifts of CD268-Fc and CD269-Fc,
respectively, and Dr Laura Z. Rassenti for technical support, Dr
Catriona Jamieson for providing the lentiviral construct, and Dr
Sonja Jain for doing the statistics.
This project was funded by a Leukemia & Lymphoma Society
Specialized Center of Research (SCOR) grant.A.P.K. is personally
supported by a “Veni” grant fromThe Netherlands Organization for
Health Research and Development. W.Z. is personally supported
by a postdoctoral fellowship from Susan G. Komen for the Cure.
Contribution: T.E. and A.P.K. designed the research, performed
most of the experiments, analyzed the data, and wrote the
manuscript; W.Z. designed the research, performed significant
experiments, and analyzed the data; G.F.W. contributed to the
design of the study and performed some in vitro experiments;
H.Y.C. and J.L. performed some in vitro experiments; E.A.
performed some in vitro experiments and provided help with
mouse work; C.M.C. significantly contributed to the design of the
and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Thomas J. Kipps, 3855 Health Sciences Dr,
Rm 4307, San Diego, CA92093-0820; e-mail: email@example.com.
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