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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3531-3540
By
From the Departments of Adult Oncology and Pediatric Oncology,
Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; and
the "Abel Salazar" Institute for Biomedical Sciences, Oporto
University, Oporto, Portugal.
We have previously shown that leukemia-specific cytotoxic T cells
(CTL) can be generated from the bone marrow of most patients with
B-cell precursor acute leukemias. If these antileukemia CTL are to be
used for adoptive immunotherapy, they must have the capability to
circulate, migrate through endothelium, home to the bone marrow, and,
most importantly, lyse the leukemic cells in a leukemia-permissive bone
marrow microenvironment. We demonstrate here that such antileukemia
T-cell lines are overwhelmingly CD8+ and exhibit an
activated phenotype. Using a transendothelial chemotaxis assay with
human endothelial cells, we observed that these T cells can be
recruited and transmigrate through vascular and bone marrow endothelium
and that these transmigrated cells preserve their capacity to lyse
leukemic cells. Additionally, these antileukemia T-cell lines are
capable of adhering to autologous stromal cell layers. Finally,
autologous antileukemia CTL specifically lyse leukemic cells even in
the presence of autologous marrow stroma. Importantly, these
antileukemia T-cell lines do not lyse autologous stromal cells. Thus,
the capacity to generate anti-leukemia-specific T-cell lines coupled
with the present findings that such cells can migrate, adhere, and
function in the presence of the marrow microenvironment enable the
development of clinical studies of adoptive transfer of antileukemia
CTL for the treatment of ALL.
DESPITE THE SUBSTANTIAL successes
observed in the treatment of childhood acute lymphoblastic leukemia
(ALL), treatment of adults with ALL and treatment of children who have
either failed to enter remission or who have relapsed presents a
significant obstacle to the cure for this disease. Moreover, curative
chemotherapeutic strategies are accompanied by severe long-term adverse
complications that significantly compromise the quality of life of
long-term survivors. Because presently available treatment modalities
for these patients have approached their therapeutic limit, the
challenge is to develop new therapeutic strategies with nonoverlapping
toxicities. Novel strategies could be used either at the attainment of
minimal residual disease, at the quantitative increase in minimal
residual disease, or at the time of clinical relapse. One such novel
therapeutic approach is the induction of anti-ALL-specific immunity.
We and others have failed to detect clinically significant
anti-leukemia-specific immunity in patients with B-cell ALL. Moreover, a defective cytolytic activity has been observed in leukemia
patients.1-4 The generation of cytolytic cells capable of
lysing leukemia cells has been attempted by several groups. For
example, lymphokine-activated killer (LAK) cells have been generated
from the peripheral blood and bone marrow of ALL
patients.4-10 However, this strategy is based on the
overall expansion of cytolytic cells and can also result in significant
cytotoxicity of nonmalignant cells.5 In another approach,
TCR Therefore, a system that facilitates the generation of
leukemia-specific cytotoxic T cells (CTL) offers obvious advantages for
the development of an immunotherapeutic strategy that can be translated
into clinical practice. We have previously shown that human B-cell
precursor leukemia cells are either inefficient or ineffective
allogeneic antigen-presenting cells (APC) but that they can be modified
to become efficient APC by cross-linking the CD40 molecule expressed on
their cell surface.13 In fact, cross-linking of CD40
results in the induction and/or upregulation of adhesion, major
histocompatibility complex (MHC), and, more importantly,
B7-family molecules.13 By using the CD40-stimulation strategy, we have developed a methodology that allows for the generation and/or expansion of anti-leukemia-specific autologous T-cell lines from the bone marrow of patients with B-cell
leukemias.14 Generation of such CTL requires both
improvement of the antigen-presenting capacity of the leukemia cells as
well as culture conditions favorable for the stimulation and expansion
of anti-leukemia-specific T cells.14
Although autologous anti-leukemia-specific CTL have been generated
from more than half of the B-cell precursor ALL patients tested, it is
not known whether these cells can function when adoptively transferred.
To be successful in adoptive immunotherapy, antileukemia T cells must
be able to circulate, migrate through endothelium, home to the bone
marrow microenvironment, and, most importantly, lyse the leukemia cells
in a microenvironment that favors tumor cell survival and
proliferation. In the present study, we demonstrate that antileukemia T
cells exhibit an activated phenotype and are capable of
transendothelial migration through both venous and bone marrow
endothelium and that these migrated cells preserve their cytolytic
capacity. Moreover, these antileukemia T cells are capable of adhering
to autologous bone marrow stroma. Finally, and most importantly, these
antileukemia T cells are capable of lysing the leukemia cells in the
presence of autologous bone marrow stroma without significant damage to
the stromal cells.
B-cell leukemia samples.
B-cell precursor ALL cells were obtained from the bone marrow and/or
peripheral blood of patients with high leukemia involvement (>90%;
except ALL-83: 72%). The ALL patients' characteristics are shown in
Table 1. Appropriate informed consent and
Institutional Review Board approval was obtained for all sample
collections. Samples were enriched by density centrifugation over
Ficoll-Hypaque and then washed twice in RPMI-1640 supplemented with
10% (vol/vol) fetal bovine serum (FBS) and 2 mmol/L L-glutamine
(further referred to as RPMI-10 media).
Generation of anti-leukemia-specific T-cell lines.
Autologous anti-leukemia-specific T-cell lines were generated and
expanded as previously described.14 Briefly, high-density bulk cultures of bone marrow were initiated in the presence of 25% to
40% (vol/vol) soluble CD40L (sCD40L) in RPMI supplemented with 2%
human AB serum (further referred to as RPMI-HS2; human serum was
obtained from NABI, Miami, FL) and incubated at 37°C, 5%
CO2. After 4 days, cells were harvested and dead cells were eliminated by density centrifugation over Ficoll-Hypaque. Live cells
were then cultured at 2 × 106/mL in Iscove's
modified Dulbecco's medium (IMDM; Mediatech, Herndon, VA)
supplemented with 4% human AB serum (further referred to IMDM-HS4) and
recombinant human interleukin-2 (rhu-IL-2; 20 U/mL) and were incubated
at 37°C, 5% CO2. On day 7, fresh IMDM-HS4 media and IL-2 were added. On day 10, cells were harvested, dead cells were removed, and the live cells were restimulated with syngeneic
CD40-stimulated leukemia cells. The same sequence of expansion and
restimulation was repeated twice.14 For the T-cell
restimulation, B-cell precursor ALL cells were stimulated by
CD40-cross-linking using NIH3T3 cells stably transfected with the CD40L
coding region (t-CD40L), as previously described.13 sCD40L
was then used during the initial step (priming) of the generation of
antileukemia T-cell lines, whereas tCD40L was used to obtain the
CD40-stimulated leukemia cells (CD40-ALL) necessary for the
restimulation of the antileukemia T-cell lines.
Endothelial cells and bone marrow stroma.
The bone marrow endothelial cell line BMEC15
was used as human bone marrow endothelium. This cell line
was kindly provided by Dr J. Ascensão (University of Nevada,
Reno, NV) and was cultured in IMDM supplemented with 5% (vol/vol) FBS.
Pooled human umbilical vein endothelial cells (HUVEC) in early passages
were used as vascular endothelium. These HUVEC were
obtained from Clonetics (San Diego, CA) and were cultured in EGM-2-MV
media (Clonetics).
Monoclonal antibodies (MoAbs) and fusion proteins.
MoAbs were used as purified Ig. The anti-CD11a (clone TS1/22) and
anti-CD18 (clone TS1/18) antibodies were purified in our laboratory and
produced from hybridomas obtained from ATCC (Manassas, VA). The anti-CD54 MoAb (clone R6.5) was kindly provided
by Dr T. Springer (Boston, MA). The blocking antibodies CD29 (clone 4B4), anti-CD49d/VLA-4 (clone HP2/1), and anti-CD106/VCAM-1 (clone 1G11) were obtained from Immunotech/Coulter (Marseille, France). Fluorochrome-conjugated MoAbs anti-CD3 (clone UCHT1), anti-CD4 (clone
13B8.2), anti-CD8 (clone B9.11), and anti-CD29 (clone 4B4) were kindly
provided by Coulter (Miami, FL). Fluorochrome-conjugated MoAbs
anti-CD95 (clone UB2), anti-CD45RO (clone UCHL1), anti-CD26 (clone
BA5), and anti-TCR Chemokines and supernatants with chemoattractant activity.
RANTES and MIP-1 Phenotypic analysis.
Expression of cell surface molecules was determined by direct labeling
using standard methodology. Fc receptors were blocked by incubation
with mouse Ig before the addition of the specific MoAbs. The MoAbs used
were fluorescein isothiocyanate (FITC)-conjugates anti-CD3, anti-CD95,
anti-CD100, and anti-4-1BB and phycoerythrin (PE)-conjugates anti-CD4,
anti-CD8, anti-TCR Transendothelial chemotaxis assay.
The transendothelial chemotaxis assay was performed as previously
described.19,20 The migration through endothelium was performed using HUVEC or BMEC. Briefly, 1 × 105
endothelial cells (HUVEC or the BMEC cells) were seeded on 6.5- or
24-mm diameter microporous collagen-I-coated Transwell inserts (Costar, Cambridge, MA) and cultured at 37°C to establish the endothelial layers (4 to 7 days or until monolayer confluence was
reached). To assess endothelial cell confluence and the establishment of monolayers, sample inserts were stained with May-Grunwald-Giemsa and
visualized by microscopy. For the chemotaxis assay, the assay media
consisted of 1:1 mixture of AIM V media and IMDM. Chemokines (RANTES,
MIP-1 Cytotoxic assay.
Cell-mediated toxicity was determined using either a standard
51Cr-release assay or a fluorescence cytotoxicity assay.
Adhesion assays.
For the adhesion assays, human bone marrow stroma layers were
established in 96-well flat-bottom plates using stromal cells expanded
as described above. The cell adhesion assay was performed as previously
described.24,25 Antileukemia T cells (day 30 of CTL
generation system) were labeled by incubation with the fluorescent dye
BCECF-AM (Molecular Probes) for 30 minutes at 37°C. After 2 washes
in phosphate-buffered saline (PBS), cells were resuspended in RPMI
supplemented with 0.1% heat-inactivated BSA. Labeled cells (5 × 104 cells/well) were then added to the wells
covered with the BM stroma layer and incubated for 30 minutes at
37°C, 5% CO2. Wells coated with BSA (0.1% in PBS)
were used as a control. All of the conditions were tested in
triplicate. After incubation, plates were gently washed 3 times with
RPMI containing 10 mmol/L HEPES to remove unbound cells. The plates
were then analyzed in a fluorescence analyzer (Cytofluor 2300;
Millipore Corp, Bedford, MA). After subtraction of background cell
binding to BSA-coated wells, the number of cells bound per square
millimeter was calculated as described.26
Statistical analysis.
The statistical significance between the treatment groups was
determined using the Wilcoxon test for matched pairs.
Autologous antileukemia T cells are mostly CD8+ T
cells and exhibit an activated phenotype.
We have previously developed a culture system that allows for the
generation and/or expansion of autologous antileukemia
CTL.14 This system takes advantage of the expression of
CD40 by the ALL cells (Table 2) and is
based on our previous demonstration that cross-linking of this molecule
results in significant increase in the APC capability of ALL
cells.13,14 Using this culture system, T-cell lines were
generated from the bone marrow of 14 of 19 B-cell precursor leukemia
patients (Table 2). Note that significant CD40 expression is also
observed in patients in which we failed to generate antileukemia T-cell
lines using this system. As shown in Table 2, the median expansion of T
cells observed was 29-± 14-fold (range, 6- to 50-fold), and these T
cells are capable of killing autologous leukemia cells with specific
lysis ranging from 28% to 63%.
Antileukemia CTL are capable of transendothelial migration in
response to chemokines.
The first requirement for the potential use of these antileukemia
T-cell lines for adoptive immunotherapy is that these cells are capable
of migrating through endothelium. To determine whether the antileukemia
CTL were capable of transendothelial migration, we used a chemotaxis
assay using human endothelial cell layers established on microporous
membranes. The endothelial cell layers were established using a human
bone marrow endothelial cell line (BMEC) or HUVEC cells. Antileukemia
T-cell lines were placed in the Transwell insert and their capacity to
migrate through the endothelium in response to chemokines added to the
lower compartment was assessed. As shown in
Fig 2, T cells are capable of
transendothelial migration in response to both MIP-1
Antileukemia CTL are capable of adhering to human bone marrow stroma.
In light of the capacity of the antileukemia T cells to migrate through
endothelium, we next sought to determine whether these T-cell lines
were capable of adhering to the bone marrow microenvironment. Although
it does not prove that the T cells will home to the bone marrow in
vivo, adherence to autologous bone marrow stroma in vitro is an
indication of the functionality of the antileukemia T-cell lines
generated in our system. To assess their adhesive properties,
antileukemia T cells were labeled with the dye BCECF-AM and plated on a
layer of autologous bone marrow stroma. As shown in
Fig 5, antileukemia T cells are capable of
adhering to autologous bone marrow stroma, with this adhesion ranging
from 172 to 885 cells bound/mm2. This assay shows that 19%
to 67% of the plated T cells could adhere to the autologous stroma
(data not shown). This adhesion to the autologous bone marrow stroma
could be significantly inhibited (P < .01) by the addition of
a cocktail of anti-integrins antibodies anti-CD29 (
Antileukemia CTL lyse leukemia cells in the presence of bone marrow
microenvironment but do not damage the stromal cells.
A final requirement necessary before undertaking a clinical adoptive
immunotherapy trial is to demonstrate that antileukemia T cells are
capable of lysing the leukemia cells in their bone marrow
tumor-permissive microenvironment. Cytolytic assays were thus performed
to assess the capacity of autologous antileukemia T-cell lines to lyse
leukemia cells in the presence of stroma derived from the patient's
bone marrow (autologous stroma). As shown in
Fig 6, T-cell lines (day 30) are capable of
lysing both unstimulated and CD40-stimulated autologous leukemia cells
in the presence of the autologous bone marrow stroma, although the tumor cell lysis was consistently inferior to that observed in the
absence of stroma. Stromal cells were also labeled and used as targets
to assess whether the T-cell lines are capable of lysing the stroma.
Importantly, the autologous T cells generated in our system did not
lyse autologous bone marrow stroma (Fig 6). These studies demonstrate
that the antileukemia T cells are capable of lysing the leukemia cells
in the presence of autologous bone marrow stroma without significant
cytolysis of the stroma.
Our previous studies demonstrating that leukemia-specific CTL could be
consistently generated from the bone marrow of patients with B-cell
precursor leukemias have suggested a potential use of these cells for
adoptive T-cell immunotherapy. However, to be used in such strategies,
the antileukemia CTL must have the capability to circulate, migrate
through endothelium, home to the bone marrow, and, most importantly,
lyse the leukemia cells in a leukemia-permissive bone marrow
microenvironment. In the present study, we analyzed the functional
properties of autologous antileukemia T-cell lines to assess whether
these cells are appropriate for adoptive immunotherapy. Our findings
show that these cells are capable of migrating through the endothelium
and that this migration does not affect their capacity to lyse tumor
cells. Moreover, we observed that these autologous antileukemia T cells can adhere to an autologous bone marrow stroma and are capable of
lysing the leukemic cells in the presence of that stroma.
Submitted October 4, 1998; accepted July 12, 1999.
Supported by National Institutes of Health Grants No. P01-CA68484-02
and P01-CA66996-01 to L.M.N. J.P.V. is a recipient of a fellowship from
Fundação para a Ciência e para a Tecnologia (Portugal). P.G. received support from the American-Italian Cancer Foundation; Toby S. Meyerson, Esq. of Paul, Weiss, Rifkind, Wharton & Garrison; and Jerry I. Speyer, President of Tishman Speyer Properties Inc. H.M.A. is supported by a joint program of Protech (Private Industry Council, Boston, MA) and the Dana-Farber Cancer Institute.
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. section
1734 solely to indicate this fact.
Address reprint requests to Angelo A. Cardoso, MD, PhD, Dana-Farber
Cancer Institute, D-538, 44 Binney St, Boston, MA 02115; e-mail:
cardoso{at}mbcrr.harvard.edu.
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TOP
ABSTRACT
INTRODUCTION
T-cell clones capable of lysing leukemia cells were
generated.
4 mol/L 2-mercaptoethanol, 0.2 mmol/L i-inositol,
20 µmol/L folic acid, and 1 µmol/L hydrocortisone sodium succinate
(further referred to as BMS media). Bone marrow stroma was expanded and
maintained as described
(clone BMA031) were kindly provided by
Immunotech-Coulter. Fluorochrome-conjugated MoAb anti-4-1BB/CD137 (clone 4B4-1) was obtained from Ancell (Bayport, MN).
Fluorochrome-conjugated MoAbs anti-CD100 (clone A8) and
anti-CD40L/CD154 (clone TRAP1). The soluble CD40L (sCD40L), a fusion
protein of CD40L and CD8
spontaneous 51Cr release]/[maximum
51Cr release 
were used at 100 ng/mL. The supernatant is from the culture of autologous T-cell lines
(day 30) restimulated for 48 hours by irradiated CD40-stimulated
leukemia cells (2:1 ratio). The combination of anti-LFA-1 (5 µg/mL),
anti-ICAM-1 (5 µg/mL), and anti-CD18 (5 µg/mL) was used as
blocking antibodies. Peripheral blood and/or bone marrow T cells
purified by negative selection were used as negative controls; no
significant transendothelial migration was observed through either
HUVEC (<3%) or BMEC (<2%). The results shown are from 1 experiment (2 patients) and are representative of 3 different patients
studied using the HUVEC and the BMEC layers.
) were capable of lysing the
autologous leukemia cells, and their cytolytic efficiency was not
significantly different from that of the input T cells (
). Because
of limitations of the cell numbers available to perform the experiments
with migrated T cells, no other cell targets could be used to confirm specificity. Nevertheless, cytolytic assays performed with the input
T-cell populations clearly confirmed the anti-leukemia-specificity of
these autologous T-cell lines (Fig 
), autologous CD40-stimulated leukemia cells (
),
autologous PHA blasts (
), and K562 cells (
). Target cells were labeled with
DiOC18(3) and used at the effector/target ratios displayed.
(B) Migrated T cells were recovered after the 6-hour chemotaxis assay
and used for the cytotoxicity assay. Primary leukemia cells were
labeled with DiOC18(3) and used as targets at the
effector/target ratios displayed. Input cells (
