A novel adoptive transfer model of chronic lymphocytic leukemia
suggests a key role for T lymphocytes in the disease
Davide Bagnara1, Matthew S. Kaufman1,2,3, Carlo Calissano1, Sonia Marsilio1,
Piers Patten1, Rita Simone1, Philip Chum1, Xiao Jie Yan1, Steven L. Allen1,3,5,
Jonathan E. Kolitz1,3,5, Sivasubramanian Baskar6, Christoph Rader6, Hakan Mellstedt7,
Hodjattallah Rabbani7, Annette Lee1,4, Peter K. Gregersen1,4,5, Kanti R. Rai1,2,3,
and Nicholas Chiorazzi1,3,5,8
1The Feinstein Institute for Medical Research, North Shore-LIJ Health System, Manhasset, NY
2Department of Medicine, Long Island Jewish Medical Center, North Shore-LIJ Health System,
New Hyde Park, NY
3Department of Medicine, Albert Einstein College of Medicine, Bronx, NY
4Department of Medicine, NYU School of Medicine, New York, NY
5Department of Medicine, North Shore University Hospital, North Shore-LIJ Health System,
6Experimental Transplantation and Immunology Branch, Center for Cancer Research, National
Cancer Institute, National Institutes of Health, Bethesda, MD
7Cancer Centre Karolinska, Karolinska Institute and Karolinska University Hospital Solna,
8Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY, USA
Address correspondence to:
The Feinstein Institute for Medical Research
350 Community Drive
Manhasset, NY 11030, USA.
Phone: (516) 562-1090; Fax: (516) 562-1011; E-mail: NChizzi@NSHS.edu
Nonstandard abbreviations used: CB, cord blood; CLL, chronic lymphocytic leukemia;
hMSC, human mesenchymal stem cell; ib, intrabone; NSG, NOD/SCID/γcnull
Blood First Edition Paper, prepublished online March 8, 2011; DOI 10.1182/blood-2010-12-324210
Copyright © 2011 American Society of Hematology
Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology.
Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has
been impeded by the lack of a reproducible adoptive transfer model.
We report a simple, reproducible system in which primary CLL cells proliferate in
NOD/SCID/γcnull mice under the influence of activated CLL-derived T lymphocytes. By co-
transferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth
of primary carboxyfluorescein succinimidyl ester-labeled CLL cells in vivo is achieved and
quantified. Using this approach, we have identified key roles for CD4+ T cells in CLL
expansion, a direct link between CD38 expression by leukemic B cells and their activation, and
support for CLL cells preferentially proliferating in secondary lymphoid tissues.
The model should simplify analyzing kinetics of CLL cells in vivo, deciphering
involvement of non-leukemic elements and non-genetic factors promoting CLL cell growth,
identifying and characterizing potential leukemic stem cells, and permitting pre-clinical studies
of novel therapeutics. Because autologous activated T lymphocytes are two-edged swords,
generating unwanted graph-versus-host and possibly autologous anti-tumor reactions, the model
may also facilitate analyses of T-cell populations involved in immune surveillance relevant to
hematopoietic transplantation and tumor cytoxicity.
The most common leukemia among Caucasian adults, B-cell chronic lymphocytic
leukemia (CLL) remains incurable and its pathogenesis poorly defined.1 Currently no system
permits differentiation and long-term growth of CLL cells in vitro; therefore an in vivo animal
model that reproducibly supports engraftment and growth of human CLL cells would help
elucidate key features of CLL cell biology and lead to better treatments.
Previous attempts to engraft human CLL cells into mice have been hampered for two
reasons. First, xenogeneic recipients were not sufficiently immune deficient to prevent human
cell rejection.2-5 Although Dürig et al. successfully transferred CLL cells into NOD/SCID mice,5
the level of CLL cell growth was not sufficient to correlate kinetics with essential interactions
with different cell subpopulations. Second, optimal engraftment and growth may have been
impaired by inability of a murine microenvironment to support CLL cells in vivo. Indeed, in vitro
studies suggest three cell lineages are involved in CLL survival and growth: lymphoid (T
cells6,7), myeloid (monocytes and monocyte-derived nurse-like cells8), and mesenchymal
To provide a more physiologic microenvironment for CLL cells within highly immune
incompetent recipients, we introduced precursors of human hematopoietic and mesenchymal
lineages into NOD/Shi-scid,γcnull (NSG) mice, a NOD/SCID-derived strain that lacks the IL-2
family common cytokine receptor gamma chain gene (γc), rendering animals completely
deficient in lymphocytes, including NK cells. We found activated autologous T cells were
essential for leukemia cells to successfully engraft, survive, and proliferate in vivo, and to
recapitulate cardinal features of human CLL cells: kinetics, CD38 expression, and growth in
secondary lymphoid tissues. This adoptive transfer model may facilitate definition of leukemic
and non-leukemic elements involved in the interactions and kinetics of CLL cells in patients.
MATERIALS AND METHODS
Patients and samples. The Institutional Review Board and the Institutional Animal Care
and Utilization Committee of the North Shore – LIJ Health System sanctioned these studies.
After obtaining informed consent, in accordance with the Declaration of Helsinki, blood was
collected from 37 CLL patients for whom clinical information, laboratory data, and IGH and IGL
variable region gene DNA sequences11 were available. PBMCs were isolated by density gradient
centrifugation (Ficoll-Hypaque, Pharmacia LKB Biotechnology, Piscataway, NJ).
Carboxyfluorescein succinimidyl ester (CFSE) labeling. Cells (2x107/ml) were
incubated 10 minutes at 37ºC with CFSE (10μM; Invitrogen, Carlsbad, CA) and washed before
injection into irradiated mice.
Isolation of human cord blood (hCB) CD34+ cells. Anonymous, fresh samples were
collected at North Shore University Hospital by The New York Blood Center. CD34+ cells were
enriched using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Inc., Auburn, CA),
cryopreserved, and stored in liquid nitrogen until used.
Isolation of mature human antigen presenting cells (APCs). Leukocyte-enriched
blood from healthy volunteers (Long Island Blood Services, Melville, NY) were selected for
CD14+ and CD19+ cells using CD14 and CD19 MicroBeads (Miltenyi Biotec, Inc., Auburn,
CA); in some experiments, CD14+ cells were negatively selected with RosetteSep custom
cocktail (StemCell Technologies, Toronto, Canada) containing anti-CD19, -CD3, and -CD56
mAbs. Isolated cells were cryopreserved.
Human bone marrow (BM)-derived mesenchymal stromal cells (hMSCs). Human
MSCs were purchased (Lonza Walkersville, Inc., Walkersville, MD) and cultured with low
glucose DMEM supplemented with 20% FBS (Gemini Bio Products, West Sacramento, CA),
2mol/l L-glutamine, 1000U/ml penicillin, 100U/ml streptomycin (Invitrogen). Cells were used
prior to 6th passage.
Xenogeneic mouse transplantation. 4-8 week old NSG mice (Jackson Laboratory, Bar
Harbor, ME) were gamma-irradiated (220 cGy) within 24 hours of intrabone (ib) or intravenous
(iv) injection of hCB-derived hCD34+ cells (105), hMSCs (106), or mature APCs (CD14+ or
CD19+, 2x107). After anesthesia (tribromoethanol, 400μg/g body weight), mice received CFSE-
labeled PBMCs (50-100x106) either ib (20μl) into lateral condyles of both tibiae or iv (50-100μl)
into retro-orbital plexus. Some mice received ip twice weekly 20μg anti-CD3 mAb (clone
OKT3, Ortho Biotech, Raritan, NJ) or anti-CD4 mAb (OKT4), or anti-CD8 mAb (OKT8).
Flow cytometric analyses. PB samples were analyzed biweekly for CFSE+ cells co-
expressing markers using anti-human -CD45AmCyn, -CD3APCcy7, -CD5PerCPcy5.5, -
CD38PEcy7 (Becton Dickinson) and -CD19Pacific Blue (eBioscience, San Diego, CA). In some
instances, CFSE+ cells were also analyzed for ROR1 expression using the murine 2A2 mAb12
plus goat anti-mouse IgG-FITC. CountBright beads (Invitrogen) were used for absolute cell
counts. When mice succumbed or were sacrificed, BM, spleen and PB were collected for
Histology and immunohistochemistry. Antigen retrieval was performed on formalin-
fixed, paraffin-embedded spleen and liver slices (5μm) in 10mM citrate buffer with a high-
pressure decloaking chamber. After incubation with primary antibodies (anti-human -CD20, -
IgG, -IgM, -kappa and -lambda antibodies [Dako, Carpinteria, CA]), binding was detected using
the DAKO EnVision system. For detection of ROR1 (mAb clone 1D813), antigen retrieval was
not used. Immunofluorescent confocal microscopy was performed as described.7
SNP analysis. Genomic DNA (~200ng) was genotyped using Illumina Human Linkage-
12 beadchip containing 6,090 SNPs or a custom iSelect Infinium beadchip of 12,108 SNPs.
Samples were processed according to manufacturer’s instruction and imaged on Illumina Bead
Array Reader. Normalized bead intensities were converted into SNP genotypes by Illumina
Single cell IGH variable region gene sequences. CD19+CD5+CFSE+ or CD19+CD5-
CFSE- lymphocytes were sorted (FACS Aria; BD Bioscience) as single cells as reported14. After
an initial amplification with CH-specific and VH family–specific FR1 primers,11 IGH cDNA was
reamplified by semi-nested PCR using the same VH family–specific primers and a JH consensus
primer (5’ctga(ag)gagac(ag)gtgacc3’). Resulting products were sequenced.
EBV detection. Presence of EBV episomal DNA or RNA was identified in mouse PB
and spleen as reported.15
Statistical analyses. Differences in CD38 expression and CLL cell numbers in mouse
blood were found using Wilcoxon’s one-sample sign rank test. Significance of infiltration of
CLL cells in BM and spleen was calculated using the Mann-Whitney test.
Human allogeneic hematopoietic elements support survival and proliferation of CLL cells.
Previous attempts to adoptively transfer CLL cells into mice were possibly suboptimal because
recipient animals were not sufficiently immune deficient to prevent rejection and the murine
microenvironment was not adequate to support survival and growth of leukemic cells. Therefore,
we created a human microenvironment in NSG mice that lack all lymphocytes, including NK
cells, by transferring 105 normal hCB-derived hCD34+ cells and 106 normal hBM-derived
hMSCs, either by iv or by ib injection.
Following transfer, human hematopoietic engraftment was considered adequate if 1-10%
hCD45-expressing cells were detected in the PB by flow cytometry. At this point, we injected
108 CLL PBMCs, either into the same tibiae for mice pre-conditioned by ib injection, or into the
systemic circulation for mice pre-conditioned intravenously. Animals were bled every 2 weeks
after injection of leukemic cells to document CLL cell engraftment and division. PBMCs were
transferred to retain non-leukemic elements that might facilitate CLL cell engraftment.
CD5+ is not a reliable marker for CLL cells in this model because normal human B cells
developing from hCD34+ cells in NSG mice are mainly CD5+.16 To discriminate normal from
leukemic cells and quantify cell divisions, patient PBMCs were labeled before injection with
CFSE. We defined CLL cells as hCD45+hCD3-hCD19+hCD5+CFSE+ cells.
First we compared survival and growth of CLL cells in mice reconstituted ib solely with
hMSCs to those receiving hMSCs plus hCD34+. With one exception, proliferation occurred only
in animals receiving hCD34+ cells (9 CLL samples; Table S2). The number of dividing cells and
their divisions varied between samples, with cells from some patients dividing at least 6 times
after which CFSE fluorescence was too dilute to distinguish positive cells (Figure 1A).
Although proliferative responses were consistent for each CLL sample, the interval between
CLL cell injection and the start of proliferation varied, perhaps due to differences in the
microenvironment. In addition, mice receiving both hCD34+ cells and hMSCs had more
leukemic cells at first bleeding (2.4±1.8 fold, P<0.0001). As most cells in the samples had not
yet divided, proliferation was not responsible for differences in leukemic cell numbers. These
studies indicated hMSCs were not necessary for CLL proliferation (Figure 1B), suggesting the
murine mesenchymal environment could support human CLL cell growth as well as transferred
Finally, we tested if ib injection was optimal for CD34+ cell and CLL cell engraftment.
Despite variability, there was no evidence for superiority of ib over an iv route of injection
(Figure S2). However, there were differences in the degree of autologous T-cell expansion and
subsequent CLL cell proliferation occurring in the absence of allogeneic cells; with ib injection
only 1 of 10 cases exhibited T-cell expansion, while T-cell expansion occurred in 7 of 10
experiments after iv injection (not shown). With either route, CLL proliferation was ~2-3 fold
less in mice not receiving allogeneic cells. Notably, there was no obvious difference in CLL cell
numbers or proliferation when using fresh or frozen PBMCs.
Proliferating human B lymphocytes are leukemic in origin. Cells proliferating in vivo
expressed hCD45+hCD3-hCD19+hCD5+CFSE+ cells, indicating they derived from the CLL
PBMC inoculum. In some experiments, expression of ROR1 further supported a CLL origin
(Figure S2). Finally, sequence identity of the IGHV/D/J rearrangements from single
CD19+CD5+CFSE+ cells with the CLL clone assured that the dividing human B cells were
leukemic (Figure S3).
Autologous T lymphocytes are necessary for CLL cell survival and proliferation. We
sought to identify the hematopoietic element(s) that enabled CLL cells to efficiently engraft and
grow in NSG mice. There was a direct correlation between T-cell levels in mouse blood and
leukemic cell proliferation (Figure 2A). Animals with circulating T cells exceeding a threshold
exhibited more robust CLL cell proliferation than animals with low T-cell numbers. In animals
without T-cell expansion, CLL cell proliferation was not observed (CLL 515 and 598; Figure
2A); in animals in which T-cell numbers were initially low but subsequently increased, CLL cell
division was not evident until T-cell expansion occurred (CLL 439, 505, and 639 - Figure 2A;
CLL753 - Figure 2A and B).
To directly document T-cell proliferation in vivo and to relate this to CLL cell expansion,
we simultaneously analyzed CFSE dilution among CD3+ cells and CD5+CD19+ cells from mice
receiving CLL cells from distinct donors, Figure 3A). This could be accomplished because
CFSE labeling was carried out with CLL PBMCs that contained both CLL cells and autologous
T cells. CLL cell proliferation was consistently preceded and accompanied by T-cell division
(Figure 3A), indicating a direct correlation between the two and suggesting that the relevant T
cells were derived from the leukemic inoculum. SNP marker panel analyses confirmed that
these T cells were indeed of patient origin and had not differentiated from hCD34+ cells. This
assay, which detected T-cell populations if they exceeded 5% of the total, indicated that the vast
majority of T lymphocytes sorted from recipient mice were patient-derived (5 different CLLs;
In vivo elimination of CD3+ or CD4+ cells abrogates CLL cell survival and proliferation. To
prove T cells were crucial in the model, animals were treated with anti-CD3 mAbs at the time of
CLL PBMC injection. For 15 samples (Table S2), leukemic cell proliferation did not occur in
animals receiving anti-CD3 mAbs (Figure 3B). In three samples (CLL 321, 505, and 1281),
proliferation occurred after T-cell depletion, although cell outgrowth required more time than
samples with adequate numbers of T lymphocytes, and these samples came from patients with
more aggressive disease (Table S1). Administration of anti-CD4 mAbs also completely aborted
CLL cell growth, whereas anti-CD8 mAbs had little effect (Figure 3C).
Co-transfer of mature allogeneic APCs supports CLL proliferation. We reasoned that
autologous T lymphocytes were activated in vivo to provide “help” for B-cell proliferation and
that this activation resulted from stimulation by allogeneic APCs arising from hCD34+ cells that
matured down myelomonocytic or B-cell lineage pathways.16,17 To test this and to define
necessary normal allogeneic hematopoietic element(s), we transferred normal mature allogeneic
APCs (CD14+ or CD19+ cells) simultaneously with patient PBMCs. Co-administration of
normal mature monocytes or B cells with CLL PBMCs supported robust leukemic cell
proliferation (Figure 4A).
We next compared mice reconstituted with immature hCD34+ cells to those receiving
mature monocytes at the time of CLL cell injection. Both hCD34+ cells and mature CD14+ cells
were isolated from the same CB sample, with the hCD34+ fraction transferred immediately into
NSG animals and the CD14+ fraction used ~4 weeks later. In addition, the same CLL PBMC
sample was inoculated into the two sets of mice. Since injection of CLL cells into each set of
animals was performed at the same time, a temporal comparison of CLL cell engraftment and
growth between the conditions was made. CLL engraftment and growth were almost
indistinguishable between the two sets of animals (Figure 4B).
To ensure that CLL cells were not dividing because of in vivo growth of patient B cells
latently infected with EBV,3 we searched for EBV DNA in spleen cells from 35 mice (26
receiving hCD34+ cells and 9 allo-APCs) reconstituted with 14 different patient samples. EBV
DNA was found in only 5 mice, and two of these were CD5- B-cell expansions (not shown).
Finally to define which cell fractions contained EBV, we searched for EBV RNA in
CD5+ and CD5- splenic B-cell populations from 27 mice (6 receiving hCD34+ cells and 21 allo-
APCs) given 14 different patients’ PBMCs (not those above). EBV RNA was not found in any
CD5+ B cells; EBV transcripts were identified in CD5- B cells from 1 mouse that had received
hCD34+ cells (not shown).
Thus, the key to CLL cell survival and expansion in this model was activation of
autologous T lymphocytes, resulting from recognition of allogeneic APCs that either developed
in vivo after pre-conditioning animals with hCD34+ cells or were transferred as mature cells.
CD38 expression on CLL cells in the murine circulation reflects that in patient’s blood.
CD38 expression by CLL clones has prognostic and physiologic importance; patients with more
circulating CD38+ cells follow a more aggressive clinical course,18 possibly due to signaling
through CD38.19 Consistent with this, the CD38+ fraction of CLL clones is enriched in cells
expressing proliferation markers.20,21
We therefore studied CD38 expression on CLL cells in mouse blood. The highest levels
of CD38+ cells were found in animals reconstituted with conditions promoting CLL proliferation
in vivo (Figure 5), while the lowest levels were in mice where division never occurred or prior to
the onset of CLL cell division (Figure 5A). Remarkably, the percentage of circulating CD38+
CLL cells in mice closely approximated that in patient’s blood (Figure 5B). Both of these
findings were similar in mice pre-conditioned with hCD34+ cells or given mature APCs at the
time of transfer (Table 1).
Comparison of circulating and tissue bound leukemic cell activation and proliferation. In
patients, clonal proliferation occurs primarily in solid lymphoid tissues within structures known
as proliferation centers (PCs);22 these are aggregates of larger CLL cells with denser CD38
expression, admixed with activated T lymphocytes.7 We compared levels of proliferation and
CD38 expression of cells in mouse BM, spleen, and peritoneal cavity with those in mouse blood
(Figure 5C). Proliferation (measured by CFSE dilution) and activation (measured by CD38
expression) of circulating CLL cells were often significantly less than in solid tissues (Figure
5C). There was a hierarchy in proliferation and CD38 expression in the various sites
(spleen>BM>peritoneum and PB; Figure 5C), although occasionally more CD38+ cells were
found in BM than spleen.
Localization and characterization of CLL cells in solid tissues. Using flow cytometry, we
found many more CD19+CD5+CFSE+ cells among the total nucleated cells in spleen than BM
(23.6±19.3 fold; P<0.0001; Figure 5D). To correlate CFSE+ cell numbers by flow cytometry
with immunohistochemical findings, we divided tissues into two fragments and performed flow
cytometry on dissociated cells from one fragment and immunohistochemistry on the non-
dissociated fragment. In this way, we identified follicular structures, comprised of CD20+ cells,
in mouse spleens. These cellular accumulations, which were usually in close proximity to blood
vessels and contained monotypic L chain aggregates expressing the same IgH chain, were
confirmed as CLL-derived by IHC analyses that documented ROR1 expression (Figure 6), a
marker expressed almost exclusively on human CLL cells.23-25 CD20+ lymphocytes were also
interspersed in the tissue and outside of the follicular structures, but these cells usually contained
more Ig than those in the follicular structures and expressed IgM or IgG as well as kappa and
lambda L chains; these were presumably residual normal B and plasma cells. Finally, T
lymphocytes were found in and around follicular structures (Figure 7), further suggesting a role
for T cells in CLL growth in these animals.
T-cell expansion is eventually associated with GVHD and B-cell elimination. Although T-
cell expansion was virtually required for CLL cell survival and proliferation, eventually all
human B cells disappeared from recipient mice. This loss was seen initially among normal
CFSE- cells (Figure 1C) and subsequently among CFSE+ CLL cells. Animals succumbed within
~12 weeks of CLL cell injection (not shown). Before death, mice exhibited lethargy, weight
loss, hunched posture, ruffled fur, and hair loss. Post-mortem examination revealed marked liver
infiltration by human T cells (not shown), consistent with a graft versus host reaction.26
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In this study, we set out to develop a reproducible adoptive transfer model for
engraftment and growth of human CLL cells in alymphoid NSG mice, immunologically inert
recipients that allow growth of human cells.27 By a series of exclusionary experiments, we
identified minimal conditions from those we originally considered optimal for CLL cell growth,
i.e., a human microenvironment of mesenchymal and hematopoietic elements.
We determined that the murine mesenchymal microenvironment was adequate, as CLL
cells grew well in mice with an endogenous murine BM microenvironment (Figure 1B). This is
consistent with in vitro findings that murine stroma and cell lines support CLL cell survival28 and
suggest that trophic signals between CLL cells and biochemical and cellular elements within the
BM are evolutionarily conserved between mice and humans.
We also found that two distinct human hematopoietic cell types facilitated growth of
CLL cells in vivo. One of these was an autologous element - T lymphocytes from the CLL
patient (Figures 2 and 3). Even though they represented a minor component of the transferred
PBMCs (Table S1), autologous T lymphocytes were key mediators of leukemic cell growth.
Derivation of these cells from the patient inoculum was documented by SNP analyses and by
transferring only mature allogeneic CD14+ cells with CLL PBMCs. There was a direct
correlation between T-cell levels (Figure 2) and their level of proliferation (Figure 3A) and CLL
cell division in vivo and selective depletion of CD3+CD4+ cells aborted CLL proliferation
(Figure 3B and C).
These findings appear at variance with those of Shimoni et al.4 and Durig et al.5 who
found that transfer of CLL PBMCs from early stage (Rai 04 or Binet A5) stable patients did not