Human chronic lymphocytic leukemia modeled in
mouse by targeted TCL1 expression
Roberta Bichi*, Susan A. Shinton†, Eric S. Martin*, Anatoliy Koval*, George A. Calin*, Rossano Cesari*,
Giandomenico Russo‡, Richard R. Hardy†, and Carlo M. Croce*§
*Kimmel Cancer Center, Jefferson Medical College, 233 South 10th Street, Philadelphia, PA 19107;†Institute for Cancer Research, Fox Chase Cancer Center,
7701 Burholme Avenue, Philadelphia, PA 19111; and‡Laboratory of Molecular Oncogenesis, Istituto Dermopatico dell’Immacolata, Via dei Monti
di Creta 104, 00167 Rome, Italy
Contributed by Carlo M. Croce, March 27, 2002
The TCL1 gene at 14q32.1 is involved in chromosomal transloca-
tions and inversions in mature T cell leukemias. These leukemias
are classified either as T prolymphocytic leukemias, which occur
very late in life, or as T chronic lymphocytic leukemias, which often
arise in patients with ataxia telangiectasia (AT) at a young age. In
transgenic animals, the deregulated expression of TCL1 leads to
mature T cell leukemia, demonstrating the role of TCL1 in the
initiation of malignant transformation in T cell neoplasia. Expres-
sion of high levels of Tcl1 have also been found in a variety of
human tumor-derived B cell lines ranging from pre-B cell to mature
established with TCL1 under the control of a VHpromoter-IgH-E?
Flow cytometric analysis reveals a markedly expanded CD5?pop-
ulation in the peritoneal cavity of E?-TCL1 mice starting at 2 mo of
age that becomes evident in the spleen by 3–5 mo and in the bone
marrow by 5–8 mo. Analysis of Ig gene rearrangements indicates
monoclonality or oligoclonality in these populations, suggesting a
preneoplastic expansion of CD5?B cell clones, with the elder mice
eventually developing a chronic lymphocytic leukemia (CLL)-like
disorder resembling human B-CLL. Our findings provide an animal
model for CLL, the most common human leukemia, and demon-
strate that deregulation of the Tcl1 pathway plays a crucial role in
10,000 new cases reported each year in the United States (1, 2).
Characteristically, B-CLL is a disease of elderly people resulting
from the progressive accumulation of a leukemic clone that may
be derived from a normal CD5?B lymphocyte (3). B-CLL has
a consistent association with CD5 expression (3), and, although
there is still a debate on the role and significance of CD5
expression on B cells, it remains reasonable to consider CD5?B
cells as the normal counterpart of B-CLL (4, 5).
Human hematopoietic malignancies are often caused by chro-
mosome translocations involving either T cell receptor (TCR) or
immunoglobulin loci (6). These chromosome breakpoints jux-
at chromosome 14q32.1 (9) that is commonly activated by
inversions or translocations that juxtapose it to a T cell receptor
in sporadic and ataxia telangiectasia-associated T prolympho-
cytic leukemia (T-PLL; refs. 10 and 11). We also provided
evidence that TCL1 is a bona fide oncogene, developing a
transgenic mouse model where ectopic expression driven by the
lck promoter in the T cell compartment results in the develop-
ment of mature T cell leukemias after a long latency period, in
a pattern closely resembling human mature T cell leukemia (12).
In normal T cells, TCL1 is expressed only at the very early
CD4??CD8?double negative stage, whereas more mature T
cells lack TCL1 expression (9). In the B cell lineage, the product
cell chronic lymphocytic leukemia (B-CLL) is the most
common leukemia in the Western world, with as many as
of the TCL1 gene, Tcl1, has been found in pre-B cells, surface
IgM-expressing virgin B cells, mantle cells and germinal center
B cells, whereas it is down-regulated at later stages of B cell
differentiation, i.e., plasma cells (9). Interestingly, high levels of
Tcl1 have been found in a broad variety of human tumor-derived
B cell lines and in many cases of B cell neoplasias (13, 14). To
elucidate the role of TCL1 in B cell development and in B cell
neoplasia, we generated transgenic mice under the control of a
promoter and enhancer whose activity specifically targets ex-
pression of the transgene to the B cell compartment (15). Here,
we show that E?-TCL1 transgenic mice develop a disease
resembling human CLL. The mice develop at first a preleukemic
state evident in blood, spleen, bone marrow, peritoneal cavity,
and peripheral lymphoid tissue, developing later a frank leuke-
mia with all characteristics of CLL. These findings strongly
indicate that TCL1 and?or other gene(s) in the TCL1 pathway
are responsible for the initiation of human CLL.
Materials and Methods
E?-TCL1 Transgenic Mice. A 350-bp fragment possessing the entire
human TCL1 coding region was generated by PCR and cloned
into the EcoRV and SalI sites of the pBSVE6BK (pE?) plasmid
containing a mouse VH promoter (V186.2) and the IgH-?
enhancer along with the 3? untranslated region and the poly(A)
site of the human ?-globin gene. The construct containing TCL1
free from vector sequences was injected into fertilized oocytes
from B6C3 animals. Mice were screened for the presence of the
transgene by Southern blot analysis on tail DNAs digested with
XhoI. Blots were hybridized with the same BssHII DNA frag-
ment used to inject the oocytes. Two founders were obtained (F3
and F10) and bred. Transgenic heterozygote mice issued from
these founders were studied and compared with nontransgenic
siblings raised in identical conditions. Genotyping was per-
formed on tail DNAs by PCR.
Western Blot Analysis. Cell proteins were extracted with Nonidet
P-40 lysis buffer, quantified by using the BCA kit (Pierce), size
fractionated on 15% Tris-glycine SDS?PAGE gels, and electro-
transferred onto nitrocellulose (Immobilon-P, Millipore). The
membrane was blocked overnight in 10% nonfat dried milk in
PBST (7.6 g/liter NaCl?0.7 g/liter Na2PO4?0.2 g/liter KPO4?
0.1% Tween 20). Expression was detected with the MoAb
(Amersham Pharmacia). Ponceau-S staining was used to verify
equivalent protein loading.
Cell Preparations. Bone marrow cells were isolated by flushing the
cavities of the femur and tibia with ice cold staining medium
Abbreviations: TCL-1, T cell leukemia 1; B-CLL, B chronic lymphocytic leukemia; PI, pro-
pidium iodide; LDI-PCR, long distance inverse PCR; MZ, marginal zone.
§To whom reprint requests should be addressed. E-mail: email@example.com.
The publication costs of this article were defrayed in part by page charge payment. This
article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.
§1734 solely to indicate this fact.
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(Deficient RPMI, Irvine Scientific) containing 10 mM Hepes,
0.1% NaN3, and 3% FCS. Spleens were dissociated in staining
medium between two frosted slides. Peritoneal cells were re-
moved by injection of 10 ml of staining medium into the
peritoneal cavity followed by withdrawal of the peritoneal
exudates. Erythrocytes were lysed by brief treatment with 0.165
M ammonium chloride, and the cells then washed in staining
WBC Preparation. Blood was collected from the cavernous sinus
with a capillary tube in a tube coated with EDTA (Becton
Dickinson). Smears were immediately prepared and stained with
May-Gru ¨nwald-Giemsa. Full counts were made on a cell counter
(Beckman). For immunofluorescence staining, cells were
treated with 0.165 M ammonium chloride to eliminate red cells
and washed in staining medium.
Immunofluorescence Analysis and Cell Sorting. Single cell suspen-
sions of the indicated cell type were prepared and stained for
surface expression as described previously (16). Cells were
stained for surface expression of IgM and CD5, then fixed and
permeabilized by using the Fix&Perm kit (Caltag, South San
Francisco, CA) and stained for expression of Tcl1 by using a
phycoerythrin-labeled anti-Tcl1 monoclonal antibody 27D6?20
(14). IgM?CD5 distributions were gated as indicated, and his-
tograms of the Tcl1 staining were determined. Plots were done
with FLOWJO software (Tree Star, San Carlos, CA). Exclusion of
propidium iodide (PI) was used to eliminate dead cells, and
samples shown were also gated by forward and right angle scatter
to exclude non-lymphoid cells and debris. Flow cytometry and
sorting were done on a dual-laser dye-laser FACStarPlus
equipped with detectors for five color immunofluorescence.
Samples were held on ice during sorting. Preparation of labeled
reagents has been described previously (17).
Analysis of VH11 Sequences. Cells were stained for IgM?CD5
expression, and 1 ? 105IgM?CD5?cells were sorted directly
into lysis?denaturation buffer. RNA and cDNA were prepared
as described previously (18), and a VH11-C? fragment was
amplified by using a VH11 leader and C? primer using Pfu
polymerase and PCR. The amplified material was cloned by
using the TOPO TA cloning kit (Invitrogen) following the
manufacturer’s instruction. Colonies with insert were expanded,
and plasmid DNA was isolated and sequenced by using an
Applied Biosystems ABI 377 automated sequencer as described
Cell Cycle. For PI staining, 105cells were sorted directly into ice
cold 95% ethanol, and nuclei were pelleted and then resus-
pended in PI-labeling solution (1 mg/ml RNase A?20 ?g/ml PI
in PBS containing 0.01% Nonidet P-40). After 30 min, cells were
analyzed for PI fluorescence on a FACScan (Becton Dickinson)
using doublet discrimination gating.
Cell Proliferation. Mice were injected i.p. with BrdUrd in PBS at
a dose of 50 ?g per gram of body weight daily for 4 days. Mice
were killed, and cells were stained for expression of IgM and
CD5, then sorted to obtain the indicated populations. Cells (5 ?
105) were fixed and permeabilized, then treated with DNase in
acid buffer and stained with an anti-BrdUrd monoclonal anti-
body labeled with FITC. Samples were analyzed on a FACScan
for BrdUrd staining.
IgH Gene Rearrangement. Southern blots of DNA digested with
EcoRI and StuI were prepared following conventional methods
and hybridized with a32P-labeled DNA probe PJ3 representing
the JH4 region of the IgH locus. The probe was synthesized
by PCR amplification from mouse DNA with primers F2
(5?-TGTGGTGACATTAGAACTGAAGTA-3?) and R1
Long Distance Inverse PCR (LDI-PCR). High molecular weight DNA
was digested with StuI. LDI-PCR was performed as described
(20). Primers designed within the mouse JH and IGH enhancer
regions were used to amplify the purified DNA, and the gel-
purified products were ligated into pCR 2.1-TOPO vector
(Invitrogen). Plasmids containing the correct size insert were
sequenced by using an ABI 377 automated sequencer and
compared with the GenBank database by using the BLAST
program (http:??www.ncbi.nlm.nih.gov?BLAST?). The VH, DH
Histopathology and Immunohistochemistry. Animals were autop-
sied, and tissues were fixed in 10% buffered formalin and
embedded in paraffin. Sections were stained with hematoxylin
and eosin according to standard protocols and analyzed by
mouse pathologists (University of Missouri, Research Animal
Diagnostic Laboratory). Immunohistochemistry was performed
on representative sections. For the dewaxing step, the sections
were heated for 1 h at 55°C, followed by rehydration steps
through a graded ethanol series and distilled water, immersed in
PBS, and then treated with 0.1% trypsin solution in Tris buffer
for 30 min at 37°C. Endogenous peroxidase was blocked with
10% normal serum. The 27D6?20 MoAb specific for recombi-
nant human Tcl1 protein (14) was used as a primary antibody,
and the immunohistochemical staining was performed by using
streptavidin-biotin peroxidase labeling method according to the
manufacturer’s instructions (Histomouse-SP kit, Zymed).
Results and Discussion
Production and Characterization of E?-TCL1 Transgenic Mice. We
generated transgenic mice in which the expression of TCL1 was
under the control of a VH promoter-IgH-E? enhancer whose
tion of the construct used to generate the mice. Restriction sites: X, XhoI; S, SalI;
first TG progeny for both founders and non-TG control. (c) Immunoblot analysis
(d) TCL1 expression on gate subsets of splenic B cells. (Upper) Refers to the F3
progeny. (Lower) F10 progeny (Blue ? TG, Red ? non-TG).
Production of E?-TCL1 transgenic (TG) mice. (a) Schematic representa-
www.pnas.org?cgi?doi?10.1073?pnas.102181599Bichi et al.
activity specifically targets expression of the transgene to im-
mature and mature B cells (ref. 15; Fig. 1a). Two transgenic
founders on a B6C3 background, designated F3 and F10, were
generated and bred to establish two transgenic lines (Fig. 1b).
The expression of the transgene in each was evaluated by
Western blot of total protein extracted from spleen, bone
marrow, and liver of 3-mo-old mice, by using a monoclonal
antibody specific for human Tcl1 protein (14). The two trans-
genic lines expressed Tcl1 in spleen and bone marrow whereas
no expression was detected in liver or in nontransgenic siblings
(Fig. 1c). We also used fluorescence-activated cell sorting
(FACS) to investigate the distribution of TCL1 expression on
of 3-mo-old mice, in both transgenic lines. The combination of
cell surface markers with intracellular detection of Tcl1 revealed
a high level of TCL1 expression in normal resting B cells, with
a 2.5-fold higher level in the CD5?cells (Fig. 1d).
The Immunophenotyping of E?-TCL1 Transgenic Mice Reveals an
Expanded CD5??IgM?Population. We used flow cytometry to
monitor the immunophenotypic profile of peripheral blood
lymphocytes from mice of these two lines between 1 and 9 mo
of age. The results revealed the presence of a B220low?IgM?
population that was detected starting at 6 mo of age in 100% of
the transgenic mice, but in the absence of any sign of disease. A
normal distribution of B cell populations was found in the
nontransgenic controls. T cell subsets were normal and identical
between transgenic animals and their littermate controls. E?-
TCL1 transgenic mice were further characterized to identify the
B cell subsets affected. The expanded B220low?IgM?popula-
tion was found to coexpress CD5 and Mac-1?CD11b. This result
suggested that the E?-TCL1 transgenic mice had an expanded
population of CD5??B1 cells in peripheral blood, where such
cells are normally infrequent (21). In mice, CD5 is a pan-T cell
surface marker that is also present on a subset of B lymphocytes
cavity in TG animals and a non-TG littermate. (b) Hematoxylin and eosin-stained spleen of mouse showing an expanded MZ in ??-TCL1 animals. (c)
Immunodetection of Tcl1 protein in lymphoid cells of the MZ. (d) Cell cycle analysis on IgM and CD5 subsets of cells by PI-labeling. (e) Cell proliferation analysis
by BrdUrd incorporation.
Table 1. Analysis of VH11 sequences in CD5?splenic B cells
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that appear during fetal?neonatal time, and whose development
appears quite distinct from the majority of B cells (19, 22). CD5
is also frequently expressed on murine B cell lymphomas and
leukemias (23, 24). We analyzed a group of animals at 2, 4,
and 8 mo of age to assess the expansion of the CD5??IgM?
population in bone marrow, spleen, and peritoneal cavity. FACS
analysis revealed a phenotypically homogeneous population
markedly expanded in the peritoneal cavity of the transgenic
mice starting at 2 mo of age (44%) that became evident in spleen
(8.6%) by 4 mo and bone marrow by 8 mo (43%; Fig. 2a).
Eight-month-old transgenic mice presented a slightly enlarged
spleen, 1.5-fold compared with littermate controls and moreover
a very high cellularity in the peritoneal cavity, ranging between
50- to 100-fold increased. Histopathology of enlarged spleens of
E?-TCL1 mice demonstrated a consistent increase in the size of
the marginal zone (MZ; Fig. 2b). Immunostaining of lymphoid
cells in the white pulp of the spleen showed Tcl1 staining more
intensely in the MZ. As expected, no immunostaining was
observed in the spleen of littermate controls (Fig. 2c). Interest-
ingly, the anatomical localization of the expanded CD5?cells
was in the MZ whereas they did not have the precise phenotype
of typical MZ B cells, i.e., not CD21-high but rather CD21-low,
like a normal CD5?B cell (25). The histological analysis of other
tissues from the same animals—including thymus, liver, kidney,
and intestine—did not reveal any pathologic alteration (not
Analysis of VH11 Sequences in the Expanded CD5?Population. The
increased frequency of CD5?B cells in these transgenic mice
could represent either the induction of CD5 expression on cells
normally not CD5?or else the expansion of normally gener-
ated CD5?B cells. To distinguish between alternatives, we
investigated V gene usage in the expanded cell population.
Recurrent expression of certain VHVL combinations is a
characteristic feature of normal and neoplastic CD5?B cells
(19, 26). Using antibodies specific for variable regions, we
found that one of these combinations, VH11Vk9, was repeat-
edly represented at 5–10% in the expanded CD5?B cell
population in all mice analyzed, similar to the frequency seen
in normal CD5?B cells (data not shown). Furthermore,
analysis of VH11 sequences from sorted IgM??CD5?cells
from the spleen of a 3-mo-old transgenic mouse (Table 1)
showed normal VH11 rearrangements, with low levels of
N-region addition, typical of CD5?B cells that are pre-
dominantly generated fetally?neonatally when levels of TdT
are low (27).
were analyzed by Southern blot on EcoRI and StuI-digested splenocyte
DNAs. Transgenic mice (?) of 7, 8, and 9 mo show rearranged bands
(asterisks). No predominant rearrangement is observed in the youngest
mice. Controls (?) are non-TG mice with the genomic 6.5-kb EcoRI and
4.7-kb StuI fragments. (b) Southern blots on DNA isolated from bone
marrow, spleen, and peritoneal cavity of TG mice (nos. 40 and 41) with the
CD5??IgM?expanded population. IgH gene predominant rearrangements
were detected in spleen and peritoneal cavity (asterisks). DNA from spleen
of non-TG mouse was used as control.
Analysis of IgH gene configuration. (a) IgH gene rearrangements
Table 2. Results of VDJ rearrangements in selected cases of E?-TCL1 transgenic mice
No. 41 PerC
No. 41 Spleen
Variations from the germ-line sequence are underlined. N regions are in lowercase.
www.pnas.org?cgi?doi?10.1073?pnas.102181599Bichi et al.
IgM??CD5?Populations Are Arrested in the G0?G1Phase of the Cell
Cycle and Do Not Actively Divide. Chronic lymphocytic leukemia
cells are characterized by a low proliferative activity and by the
progressive accumulation of clonal B lymphocytes blocked in the
early phases (G0?G1) of the cell cycle (28, 29). We investigated
the cell cycle distribution and the rate of cell proliferation in
spleen and peritoneal cavity of four transgenic mice and four
littermate controls at 7 mo of age. Detection of DNA content in
replicating cells by PI labeling and analysis of cell proliferation
from the distribution of BrdUrd incorporation in IgM?CD5?
sorted populations revealed that most of these cells are not
actively cycling in the transgenic mice (Fig. 2 d and e).
IgH Gene Configuration in Transgenic Mice with the Expanded CD5?
Population. Analysis of Ig gene rearrangement revealed the
presence of preleukemic or leukemic clones consistently in
E?-TCL1 mice over 7 mo of age. No clonality was observed in
the youngest transgenics or in nontransgenic mice (Fig. 3a). The
detection of clonal JHrearrangements indicated that there could
be a clonal expansion without evidence of disease. Further
analysis of Ig gene rearrangement in bone marrow, spleen, and
peritoneal cavity from 8-mo-old mice with a markedly expanded
CD5??IgM?population showed an identical size JHband de-
tected in spleen and peritoneal cavity, but not bone marrow (Fig.
3b). The clonal JHband was not always shared between spleen
and peritoneal cavity, as for example mouse no. 40 (Fig. 3b) with
two independent clonal populations, suggesting multiple inde-
pendent events in some cases. Clonal rearrangements were
subsequently confirmed in some samples by cloning and se-
approach, the transgenic mice exhibited additional clonal rear-
rangements, compared with the littermate controls. Table 2
shows sequence data referring to the (?) samples marked as 9m
and 8m in Fig. 3a, StuI digested. Some sequences had a low level
of N addition, whereas others had a higher level (Table 2), as has
been noted in sequence analyses of normal CD5?B cells (30).
The clonal population suggested by Southern blot for the
transgenic no. 41 (Fig. 3b) in spleen and peritoneal cavity was
also confirmed by LDI-PCR (Table 2).
E?-TCL1 Mice Developed Lymphocytic Leukemia on Aging. The onset
of the establishment of a murine model for B-CLL. All mice
around the age of 13–18 mo became visibly ill and presented with
enlarged spleens and livers associated with high WBC counts.
The weight of the transgenic spleens was between 1.5 g and 2.3 g
(normal splenic weight was 0.07 ? 0.01 g), and the mean of the
WBC was 180.0 ? 106cells?ml (the mean WBC?ml blood for
normal adult mice was 2.8 ? 106cells?ml). In addition, the mice
also developed advanced lymphoadenopathy, a hallmark of
human CLL. Cytological examination of blood smears showed
an increase in circulating lymphocytes, with many of them
displaying a clumped nuclear chromatin (Fig. 4 A and B). The
predominant cell type was represented by large lymphoid cells,
and smudged cells were also present. Histopathological exami-
nation demonstrated consistent infiltration of spleen, liver, and
lymph nodes by small and large lymphocytes (Fig. 4 C, E, and G).
Positive staining for Tcl1 protein was observed primarily in
lymphocytes found in these tissues (Fig. 4 D, F, and H), and flow
cytometric analysis confirmed the expansion of the CD5??IgM?
population in all tissues (data not shown). Clonality was shown
by Southern blot analysis of DNA isolated from leukemic
splenocytes by using the PJ3 probe (Fig. 5). DNA from spleens
of littermate controls showed the IgH gene in its germ-line
configuration, whereas DNA from leukemic splenocytes pre-
sented extra-rearranged bands, indicating the presence of clonal
B cell populations.
(B) High magnification of the blood smear. (C) Histology of spleen, liver (E), and cervical lymph node (G) after hematoxylin-eosin staining. (D) Immunodetection
of Tcl1 protein in spleen, liver (F), and cervical lymph node (H). (Insets) Negative control in which the primary antibody has been omitted.
Histopathological analysis of the E?-TCL1 mice. (A) Blood smear stained with Wright Giemsa showing an increased number of circulating lymphocytes.
TG mice. DNAs from leukemic mice and a littermate control were digested
with StuI. The strong 4.7-kb bands represent the gene in its germ-line config-
uration. Clonal rearrangements are indicated by asterisks. Lanes 1 and 2,
Leukemic mice from TG line F3; lanes 3 and 4, leukemic mice from TG line F10;
lane 5, non-TG mouse.
Bichi et al.PNAS ?
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Conclusions Download full-text
B-CLL is the most common leukemia in humans, and its
pathogenesis is still unknown. Transgenic mice in which we
targeted the expression of TCL1 in B cells develop a lympho-
proliferative disease closely resembling human CLL. Our data
strongly indicate that TCL1 and?or other gene(s) in the TCL1
pathway are responsible for the initiation of human B-CLL.
The present findings provide an adequate animal model to
investigate the mechanisms underlying the initiation and pro-
gression of human B-CLL, and we hope that ultimately this
model may aid in the development and test of novel anti CLL
We thank A. Shaw for the gift of the expression vector, T. Manser and
J. Rothestein for valuable suggestions, D. Remotti for reviewing some
spleen sections, J. Letofsky and S. Rattan for expert technical assistance,
J. Faust and A. Acosta for the preliminary flow cytometric analysis, and
the transgenic mouse facility and the laboratory animal services at the
Kimmel Cancer Center. This study was supported by National Institutes
of Health grants to R.R.H., by Associazione Italiana Ricerca sul Cancro
grants to G.R., and by National Cancer Institute grants to C.M.C.
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