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Use of a Lethally Irradiated Major Histocompatibility Complex
Nonrestricted Cytotoxic T-cell Line for Effective Purging of Marrows
Containing Lysis-Sensitive or -Resistant Leukemic Targets
By Alessandra Cesano, Giuliana Pierson, Sophie Visonneau, Anna Rita Migliaccio, and Daniela Santoli
Improved marrow purging protocols are needed in autologous bone marrow transplantation (BMT) t o achieve complete eradication of minimal residual disease. This study investigates the potential of a human major histocompatibility
complex (MHC) nonrestricted killer T-cell line (TALL-104) as
a new marrow purging agent in a clinical setting. TALL-I04
cells can be irradiated without losing cytotoxic activity
against tumor targets in vitro. In vivo, the irradiated killers
can be adoptively transferred into immunodeficient and immunocompetent leukemia-bearing mice, and reverse their
disease even in advanced stages. The present study shows
that y-irradiated TALL-I04 cells, cultured for 18 hours with
marrows from healthy donors, do not impair the viability
and long-term growth of committed and pluripotent hematopoietic progenitors. However, as determined by polymer-
ase chain reaction (PCR) and colony assays, TALL-104 cells
could completely purge marrows containing up t o 50% lysissusceptible myelomonocytic leukemia cells (U937). When
marrows were admixed with a pre-B leukemia cell line (ALL1). which is fairly resistant t o TALL-104 cell lysis in l o n g
term “Cr-release essays but can be totally growth inhibited
by TALL-I04 cells in proliferation assays, residual ALL-1 cells
were detectable by PCR after TALL-104 purging. However,
importantly, these PCR’ marrows were devoid of tumorigenic activity when transplanted into the human hematopoietic microenvironment of human severe combined immunodeficient (SCID) chimeras. These data indicate the strong
potential of the TALL-IO4 cell line in future marrow purging
strategies against lysis-susceptible and -resistant leukemias.
0 1996 by The American Society of Hematology.
successful BMT must be followed by effective consolidation
ONE MARROW transplantation (BMT) is commonly
strategies to completely eliminate MRD from the patient
used in the treatment of a variety of hematologic maligbody, after conditioning treatments.
nancies refractory to conventional therapy to ameliorate the
We have developed a new experimental approach to BM
bone marrow (BM) suppression that occurs as “side-effect’’
purging that uses an interleukin-2 (IL-2)-dependent major
of most intensive chemoradiotherapeutic protocols. Alhistocompatibility complex (MHC) nonrestricted cytotoxic
though transplanting marrows from healthy donors has obviT-cell line, TALL-104, established in this laboratory from a
ous advantages when treating patients with a primary hemachild with acute T-lymphoblastic leukemia (TALL- 104).16
topoietic disease or intramedullary metastases, only 25% to
TALL-104 cells viggered by IL-2 are able to lyse with high
40% of all patients have suitable donors, despite the increasefficiency a wide variety of tumor cells, including freshly
ing number of donors available through the National Marrow
explanted leukemic samples, without toxic effects on cells
Donor Program.’ This percentage is even lower in the case
from normal tissue^.'^.'^ The tumoricidal activity of TALLof minorities. Furthermore, graft-versus-host-disease, which
develops in 30% to 70% of allografted patients despite im104 cells can also be induced in vitro by IL-12 and the
munosuppressive therapy, is a major cause of morbidity and
combination of the two cytokines have additive effects.”
mortality in such patients.’
Because of their malignant origin and ability of inducing
The use of autologous BMT eliminates the problem of
leukemia in severe combined immunodeficient (SCID)
donor selection and graft-versus-host reactions. However, a
mice?’ we have used y-irradiation (4,000 rads) as a means
potential problem that arises with this kind of graft is that
to irreversibly arrest their in vitro growth as well as their
therapy-resistant residual malignant cells that remain in the
possible dissemination in animal tissues. Importantly, this
autologous marrow at the time of the harvest, will be reintreatment does not impair the tumoricidal activity of this cell
fused back to the patient without prior exposure to the “conline both in vitro and in vivo,2’.22thus suggesting that lethally
ditioning” therapy received by the patient. Although the
irradiated TALL-104 cells could be safely and effectively
exact number of tumor cells required to initiate tumor growth
in humans is unknown, substantial evidence in animal studies
indicates that one tumor cell might be sufficient to cause a
From The Wistar Institute, Philadelphia, PA; the Children’s Hosre~urrence.~.~
To eliminate residual malignant cells in autolopital of Philadelphia, Deparzment of Pediatrics, Philadelphia, PA;
gous BM grafts, several purging methods are being used,
and the Lindsley F. Kimball Research Institute, New York, NY.
such as antibody-mediated cytotoxicity in combination with
Submitted March 17, 1995; accepted August 16, 1995.
~omplement,~
magnetic beads depletion,6or immunot~xins,~ Supported by American Cancer Society Grant No. DHP-107A.
use of drugs (such as 4-hydroperoxycyclophospha1nide),~~~the Eleanor Maylor Dana Charitable Trust, and the Maxfield Founheat,” photosensitization,” long-term culture of BM
dation. A. C. is partially supported by the Jose‘ Carreras International
Leukemia Foundation.
cell^,''^'^ and combinations of drugs and antib~dies.’~
HowAddress reprint requests to Daniela Santoli, PhD, The Wistar
ever, despite the advances in purging procedures and detecInstitute,
3601 Spruce St, Philadelphia, PA 19104.
tion of minimal residual disease (MRD), the relapse rate in
The publication costs of this article were defrayed in part by page
patients with leukemia undergoing autologous BMT is still
charge payment. This article must therefore be hereby marked
higher than the one seen in patients receiving allografts in
“advertisement” in accordance with I8 U.S.C. section 1734 solely to
similar conditioning regimens.” In the latter group, the only
indicate this fact.
factor leading to relapses is the inadequacy of the applied
0 1996 by The American Sociery of Hematology.
conditioning regimens in eliminating MRD. Furthermore, a
0006-4971/96/8701-0I40$3.00/0
B
Blood, Vol 87, No 1 (January I), 1996 pp 393-403
393
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394
CESANO ET AL
used to cleanse remission marrows and might even be used
in consolidative therapy after autologous BMT.
This report investigates at the cellular and molecular levels
the marrow purging efficacy of lethally irradiated TALL104 cells against susceptible and resistant leukemic targets,
and the effects of this approach on the growth of normal
BM precursors, which are necessary for the patient's hematopoietic reconstitution,
MATERIALS AND METHODS
Cell lines. The TALL-104 cell line was maintained at 37°C in
10% CO2in Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal bovine serum (FBS) (complete medium) and
100 U/mL of recombinant human (rh) IL-2 (a gift from Dr M.
Gately, Hoffman LaRoche, Nutley, NJ). The human tumor cell lines
U937 (myelomonocytic leukemia), Raji (B-cell lymphoma), ALL- 1
(pre-B-ALL), U-87 MG (glioblastoma), A549 (lung carcinoma),
CHP- 100 (neuroblastoma), and WM 1985 (melanoma) were cultured
in complete medium. All cell lines were free of mycoplasma contamination on repeated testing. Human melanocytes were obtained from
Dr M. Herlyn (The Wistar Institute) immediately after their separation as described.2'
Tagging of U937 cells with U neomycin-resistance (neo-R) gene.
The N2A double copy retroviral vector, containing the neo-R gene
and the human IL-2 cDNA driven by the thymidine kinase prom ~ t e r ; was
~ obtained from Drs E. Gilboa and B. Gansbacher (Memorial Sloan-Kettering Cancer Center, New York, NY) and used to
transfect U937 cells by lipofection, according to published methods." G-4 18 (geneticin)-resistant U937 clones were subcloned by
limiting dilution. Some of the clones were found to produce high
levels of IL-2 and others were nonproducers as measured by enzymelinked immunoabsorbent assay (ELBA). In this study, we selected
to use a nonproducer U937 clone. G-418 ( I mg/mL) was added
biweekly to the culture medium of this clone throughout the study.
As reported previously;' the susceptibility of the transfected U937
clone to TALL-IO4 cell lysis, measured in a 4-hour "Cr release
assay, was identical to that of the parental U937 cell line.
Normal and leukemic BM cells. BM aspirated from the iliac
crest of healthy donors was centrifuged on a Ficoll-Hypaque gradient
(Westbury, NY). Mononuclear cells were procured, washed twice
in phosphate-buffered saline, and used in cytotoxicity and clonogenic
assays. Leukemic BM samples from pediatric and adult cases of
lymphoid and myeloid leukemias (of various French-American-British [FAB] classifications) were provided by Drs B. Lange (Children's
Hospital of Philadelphia, Philadelphia, PA), J. Hoxie, and D. Biggs
(Hospital of the University of Pennsylvania, Philadelphia). Use of
excess diagnostic material for research was approved by the Institutional Review Boards of the two hospitals and The Wistar Institute
in accord with an assurance filed with and approved by the Department of Health and Human Sciences. Mononuclear cells were separated on a Ficoll-Hypaque gradient and immediately used as targets
in cytotoxicity assays. Leukemic marrows contained greater than
90% blasts.
Collection and separation of cord blood cells. Human umbilical
cord blood samples were collected at Mount Sinai Hospital (New
York, NY) under protocols approved by the Institutional Review
Boards of both Mount Sinai Hospital and the New York Blood
Center. Light-density cord blood cells were separated by centrifugation over a density gradient (1.077 g/mL; Ficoll). The numbers of
progenitor cells (mean 2 SD from nine experiments) in lo5 highdensity cord blood cells were 44 t 57 BFU-E (primitive erythroid
progenitor colonies), 270 2 172 CFU-GM (granulocyte and macrophage colonies), and 97 2 82 CFU-GEMM (mixed lineage colonies).
CD34' cells were separated directly from the light-density cell fraction by affinity chromatography with the Ceprate device as described
by the manufacturer (Cellpro, Bothell, WA)." The frequency of
CD34+ cells in the samples varied from 30% to 60%. Twenty-five
percent of the CD34-enriched populations were CD38-. The number
of progenitor cells in lo3 CD34' cord blood cells was 163 5 30
(mean 2 SD from four experiments). In clonogenic assays, these
cells give rise to 1% to 2% BFU-E, about 90% CFU-GM, and
approximately 10%CFU-GEMM.26The latter colonies are very large
in size and similar to the recently described early cell population with
very high proliferative and replating potential, which are abundant in
CD34' human umbilical cord blood samples."
Cytotoxic assays. Direct TALL-104 cell killing of "Cr-labeled
targets was measured in an 18-hour assay, as previously described."
The effectors were stimulated overnight with rhIL-2 (100 U per mL)
and IL-12 (10 ng/mL) (a gift of Dr S. Clark, The Genetics Institute.
Cambridge, MA), and y-irradiated (4,000 rads) before the assay. A
fixed number (lo4per well) of "Cr-labeled target cells were tested
against four effector cell concentrations. Percent-specific "Cr-release was calculated from the mean of three replicates and the results
were expressed as 30% LU/lOx effectors, as described." In cold
target inhibition assays, unlabeled normal BM cells were tested for
ability to compete against TALL-104 cell binding to "Cr-labeled
leukemic U937 or Raji targets. In these experiments, cells were
seeded in round-bottomed 96-well plates at the effector-to-target
(E:T) ratio of 50:1, and cold BM cells were added to the mixture
at four ratios ranging from 1OO:l to 12:l relative to the labeled
targets. Cold U937 or Raji cells were used at the same ratios as
positive controls for inhibition.
E T conjugate formation assay. E:T conjugates were counted
using the dual-color flow cytometric method as described." Briefly,
the effector cells (1 x IO6), labeled with Calcein-AM (Molecular
Probes, Eugene, OR), and the targets, labeled with hydroethidine,
were mixed at a 1:l ratio, and incubated for I O minutes at 37°C.
Conjugates were analyzed in an Epics Elite machine (Coulter Corp,
Hialeah, FL) and a total of 10,000 events was counted. The percentage of conjugated cells was determined by dividing the number of
dual-labeled particles by the total number of effectors and multiplying this number by 100.
Proliferation assays. ALL-I cells were seeded in wells of microplates (104/100 yL per well). Irradiated TALL-104 cells were
added at the same four E:T ratios used in "Cr release assays ( I 0 0
pL per well). After a 72-hour incubation at 37"C, 'H-TdR was added
( 1 yci/50 yL per well) and its incorporation measured 6 hours later,
as d e ~ c r i b e d . ' ~ ~ ' ' . ' ~
Hematopoietic progenitor cell assay. To assess the growth of
hematopoietic progenitors, I x 105 total BM mononuclear cells or
1 x IO' CD34-enriched cord blood samples were cultured in Iscove's
methylcellulose medium containing erythropoietin- and phytohemaglutinin-stimulated leukocyte conditioned medium (CM) (Methocult HCC-4432; Stem Cell Technologies, Vancouver, BC, Canada)
using standard procedures.28 Briefly, cells were plated in 35-mm
culture dishes (Nunc Inc, Naperville, IL) in duplicates and incubated
at 37°C in a humidified atmosphere of 5% CO2.Cultures were scored
on day 14 under an inverted microscope to identify and count BFUE, more mature erythroid progenitor colonies (CFU-E), CFU-GM,
and CFU-GEMM. The latter are identified by the presence of erythroid and myeloid elements in the same colony; BFU-E are red,
hemoglobinized colonies composed of two or more large clusters;
CF'U-E contain red blood cells (RBCs) in a single cluster; CFUGM colonies are composed of non-RBCs and have a translucent
appearance. Colonies are defined as aggregates of SO cells or more.
Cyrokine assa.ys. The presence of tumor necrosis factor-a (TNFa ) and interferon-y (IFN-y) in the CM of TALL-104 cells cultured
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395
A CYTOTOXIC T-CELL LINE FOR MARROW PURGING
for 18 hours either alone or with BM/tumor cell mixtures was tested
by radioimmunoassays, as de~cribed?~
using monoclonal antibodies
supplied by Dr G. Trinchieri (The Wistar Institute). The presence
of granulocyte-macrophage colony-stimulating factor (GM-CSF) in
the same CM was quantitated by ELISA using a commercially avalable kit (R&D, Minneapolis, MN). The sensitivity of the ELISA test
was 16 pg/mL. In some experiments, TALL-104 cells were pretreated for 2 hours with Actinomycin D (Calbiochem, Novabiochem
Corp, San Diego, CA; 10 pglmL) and Puromycin (Sigma, St Louis,
MO; 50 pglmL) to block protein synthesis, and washed three times
in saline before incubation for 18 hours with untreated BM cells.
The same ActinomycinDPuromycin treatment was applied to BM
cells before incubation with untreated TALL-104 cells. CM from
all of these cultures were then collected and tested for the presence
of cytokines.
RNA blot analysis. Total RNA was extracted from the above
ActinomycinDPuromycin-treatedTALL- 104/BM cultures using the
RNAzol method16 (CinnalSiotech Laboratories International Corp.
Friendswood, TX). RNA samples were fractionated in a 1% agarose
formaldehyde gel, transferred to nylon membrane filters (Zetabind
AMF; Cuno, Meridian, CT), and covalently bound to the membranes
by UV irradiation. Filter-bound RNA was hybridized” at 42°C for 16
hours with ’*P-labeled probes at 1 to 2 X IO6 cpm/mL hybridization
solution. Filters were washed and autoradiographed to X-OMAT AR
films (Eastman Kodak, Rochester, NY) between double intensifying
screens (DuPont CO,Wilmington. DE) at -70°C for 1 to 2 days.
cDNA probes (whole plasmid preparations, provided by Dr G. Trinchieri and the Genetics Institute) specific for GM-CSF, IFN-y, and
TNF-a were used for RNA analysis.
Engrafrment of human tissue in immunodejkient mice. Human
fetal bone tissue was obtained with informed consent from the International Institute for the Advancement of Medicine (Exton, PA) in
compliance with regulations issued by each state and the Federal
government. The tissues, derived from curettage operations, were
individually placed in sterile 50-mL tubes containing complete medium and antibiotics, shipped on wet ice, received within 16 to 20
hours, and transplanted immediately into SCID mice. The construction of SCID-human chimeras with fetal long bone implants was
obtained by implanting subcutaneously small fragments (approximately 5 X 5 X 10 mm) from femurous and tibias of 19- to 23gestational-week-old fetuses, as described by Namikawa et aL3’This
system allows for the closed expansion of leukemic cells within the
human bone grafts precluding dissemination in the peripheral blood
(PB), marrow, and other murine tissues.
Detection of residual leukemia. BM samples were mixed with
various concentrations of neo-R’ U937 cells or ALL-1 cells, as
indicated. IL-2/IL-l2-stimulated, y-irradiated TALL-I04 cells were
added for 18 hours. The cell mixture was washed and treated with
DNAse I (10 &mL) to rid cell-free DNA. Cellular DNA was extracted by lysing the cells in 100 p L lysis buffer (50 mmol/L KCI,
IO mmoVL Tris-HC1, pH 8.3, 2.5 mmoVL MgCl, 0.45% NP-40, and
0.45% Tween 20) with an added 0.6 p L proteinase K (10 mg/mL).
Lysates were left for 1 hour at SYC, and 10 more minutes at 95°C.
DNA (1 pgll00 pL) was subjected to polymerase chain reaction
(PCR) amplification using either neo-R gene-specific primers, to
detect U937 cells, or VJ primers specific to the CDR3 region of
ALL-1 cells. The sequences amplified by these primers are of 501
and 189 base pairs, respectively. Oligonucleotide probes recognizing
20 nucleotides in the middle of the amplified sequences were used
to show the specificity of the PCR products.
In some experiments, indicated numbers of ALL-I cells were
resuspended in 25 pL IMDM containing 10% FBS, and injected
with a microtiter syringe (Hamilton CO,Reno, NV) into human bone
grafts implanted into SCID mice 8 weeks earlier. After 10 more
weeks, the mice were killed, the human bone grafts removed, and
single-cell suspensions prepared by mincing tissues with scissors in
cold complete medium. Ammonium chloride treatment was then
applied to lyse erythrocytes. Cellular DNA was extracted and subjected to PCR amplification, as previously described.
RESULTS
Irradiated TALL-I04 cells lyse tumor targets, sparing normal cells. As measured in extended (18-hour) 5’Cr-release
assays, IL-2AL-12-stimulated, y-irradiated TALL-104 cells
display high levels of cytotoxicity against a broad spectrum
of tumor targets, including established hematopoietic (U937
and Raji), and nonhematopoietic (U-87 MG, A549, CHP100, and WM1985) tumor cell lines (Fig lA), and primary
lymphoid (B- and T-ALL) and myeloid leukemic samples
(Fig 1B and C). A great variability was observed in the lytic
efficiency of irradiated TALL- 104 cells against leukemic
samples from different patients; however, all of the B-ALL
or myeloid leukemias that were poorly sensitive to TALL104 cell killing in a % release assay were growth arrested
by 100% on a 3-day incubation with TALL-104 cells, as
measured in 3H-TdRincorporation assays (not shown). Thus,
these effectors can exert either cytotoxic or cytostatic effects
on different leukemias. Growth inhibition was always accompanied by apoptotic cell death (not shown).
Importantly, normal cells, such as healthy melanocytes or
cells derived from normal marrows and PB are completely
resistant to TALL-104 cell killing (Fig 1D). Healthy donors’
BM cells cultured in rh growth factors, such as IL-3 or GMCSF, for 3 days and displaying high levels of proliferative
activity are also resistant to TALL-104 cell lysis (Fig 1D).
None of PB lymphocyte (PBL) and BM samples from the
healthy donors tested were sensitive to the cytostatic effects
of TALL-104 cells in 3H-TdR incorporation assays (not
shown). To determine whether the failure of TALL-104 cells
to lyse normal marrow cells was caused by binding defects,
E:T conjugate assays were performed comparing the ability
of TALL-104 cells to bind normal versus leukemic targets.
As shown in Fig 2, TALL-104 cells can efficiently bind to
U937 cells but are totally unable to form conjugates with
normal BM cells. Competition experiments further confirmed that normal marrow cells do not compete for TALL104 cell binding and killing of the leukemic targets U937
and Raji (Fig 3).
Cytokine production during TALL-104/BM incubation.
Based on the ability of both nonirradiated and y-irradiated
TALL-104 cells to produce high levels of lymphokines when
triggered by IL-2 or IL-12 or exposed to tumor target^,'^
experiments were performed to measure possible cytokine
release resulting from incubation of cytokine-TALL-104
cells with normal marrows. High levels of GM-CSF (10 ng/
mL), IF”-y (1,500 U/mL), and TNF-a (250 U/mL) were
detected in the CM of IL-2/IL- 12-stimulated, y-irradiated
TALL-104 cells admixed with healthy marrows for 18 hours
(Fig 4). To determine the respective contributions of the two
cell populations to cytokine production, TALL-104 cells, or
BM cells, were pretreated for 4 hours with the protein synthesis inhibitors Actinomycin D and Puromycin, mixed with
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CESANOETAL
396
BJLL
I4.L
I-ALL
Bob11
14LL
0
i o 0 0 2000 loo0 l o o 0 1000 6000 7000
b?L
90% LU/lO( crll*
30%
LUnd
the untreated BM,
or TALL-l04 cells, respectively, and incubatedfor18hoursinthepresenceof
rhL-2 and L-12.
TALL-l04 cells were the main producers of
IFN-y and GMCSF whereas the two cell populations contributed equally
totheproductionof
TNF-a (Fig 4). Results of cytokine
expression were in accordance with data of mRNA expression for IFN-y,
TNF-a,and GM-CSF by the
two cell populations, shown in Fig 5 . Specifically, the basal levels of all
three cytokine mRNAs expressed by TALL-l04 cells (lane
1) increased upon incubation with Actinomycin DPuromycin-pretreated BM cells (lane 2); by contrast, BM cells did
notexpressmRNAforanycytokine
(lane 3) but TNF-a
mRNAwas detectable after a 4-hour incubation of these
cells withActinomycin
DPuromycin-treated TALL-l04
cells.
Long-term effects of TALL104 cells on normal hematopoietic progenitors in BM and cord blood samples. Twoweek clonogenic assays were performed to exclude the
possibility that the release of toxic mediators, such
as TNF-a
and IFN-.)I (see Fig 4). would impair the growth of normal
hematopoietic precursors in BM samples. Figure 6 shows
that the clonogenic growth
of committed (CFU-GM, BFU-E,
and CFU-GEMM) progenitorsin marrows from two donors
admixed for 4or 18 hours withTALL-l04 cells at0.1: 1 and
1:l ratios, was very similar to that of untreated marrows.
Experiments were then performed to evaluate the effects of
TALL-l04 cells on pluripotent CD34+ stem cells that are
indispensableforsuccessfulhematopoieticreconstitution.
The capability of cord blood-derived CD34+ cells to form
colonies of various types (CFU-C, BFU-E, CFU-GM, and
CFU-GEMM)in a 2-week clonogenic assay was not impaired after an 18-hour pre-incubation withTALL-l04 cells
COth
Fig 1. Selective killing of tumor targets byTAUl 0 4 cells. “Cr-labeled tumor cell lines (A), primary
ALL (B), and myeloid (C) leukemic samples, normal
B M samples from five donors uncultured or cultured
for 3 days in rhlL-3 or GM-CSF (D), PBL from three
donors, and freshly explanted normal melanocytes
(Dlwere tested for susceptibility to lysis by TALL104 cells at four E:T ratios. Results of the 18-hour
cytotoxic assay were expressed as 30% LU/108 effectors. In (Cl, APL is acute promyelocytic leukemia,
CML/BC is a chronic myeloid leukemia in blastic crisis, and samples MO, M1, M2, M4, and M5 are AML
of various FAB classifications.
(Table 1). Both before and after purging, the CFU-GEMM
could be replated at least 4 times, thus being similar to the
early progenitor with high proliferative and replating capacity described byLu et al” in human umbilical cord blood
samples. Altogether, these results indicate that TALL-l04
cells do notaffecttheviabilityandlong-termgrowthof
pluripotent stem cells, norof lineage-committed precursors.
EfJiciency of lethally irradiatedTALL-IO4 cells in purging
sensitive andresistantleukemias.
To testtheefficacyof
lethally irradiatedTALL-l04 cells in marrow purging different approaches were taken. In a first series of experiments,
we testedpurgingefficacyagainstatargetthatishighly
susceptibleto TALL-l04 cellkilling:neO-R+U937
cells
were admixed at various concentrations (0% to 50%) with
cells fromnormalBM.IL-2DL-12-activatedy-irradiated
TALL-l04 cells were then added at the ratio of 1: 1 relative
to BM cells; the cell mixtures were incubated for 18 hours
inthepresenceofDNAse
I, todigestDNAreleasedby
dying cells and, therefore, reduce false-positive results.
PCR
amplification of the neo-R gene 18 hours after addition of
TALL-l04 cells to BMN937 samples showed the total disappearance of U937 cells from such samples at all
E T ratios
used (Fig 7). Furthermore, no U937 cell colonies could be
detected microscopically after a 2-week culture period in
methylcellulose (not shown).
In a second set of experiments, we proceeded to determine
whether TALL-l04 cells would efficiently purge marrows
infiltratedwithlysis-resistanttargets,suchastheALL-l
(pre-B) cell line. The susceptibility
of this target to the cytotoxickytostatic effects of TALL-l04 cells is shown in Fig
8A. Specifically, the killing efficiency of
TALL-l04 cells
against ALL-l cells is very low, even at high E:T ratios and
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A CYTOTOXICT-CELLLINE
FOR MARROW PURGING
397
Fig 2. E:T conjugateformation by TALL-l04 cells with U937 cells and normal BM cells. The abscissa representsthe fluorescence of calceinlabeled effectors, and the ordinate represents the fluorescence of hydroethidine-labeledtarget cells. The number of double-labeled particles
is in quadrant 2, the totalnumber of effectors is the sum of quadrants2 and 4. (A and D) Fluorescence of TALL-l04 cells alone.(B) Fluorescence
of U937 cellsalone. (C) Double fluorescenceof TALL-l04/U937 conjugates. (E) Fluorescence ofBM cells alone.IF) Double fluorescence of TALL104/BM conjugates.
in a long-term (18-hour) cytotoxic assay (no killing at all is
observed in a 4-hour assay, data not shown). By contrast, in
a 72-hour proliferation assay, irradiated TALL-IO4 cells can
inhibit almost completely the growth of ALL-l cells (Fig
SA). When ALL-I cells were admixed at various concentrations (0% to 40%) with cells from normal marrows, and IL2/IL- 12-activated, y-irradiated TALL- 104 cells were added
at the 1:1 ratio relative to BM cells for 18 hours, PCR amplification of the CDR3 specific region of ALL-l cells always
showed the presence of residual ALL-l cells even when
their initial concentration was 0.5% (Fig 8B). To determine
whether the residual ALL-l cells detected byPCR were
tumorigenic on transfer into a human BM microenvironment,
aliquots of unpurged and TALL- 104-purged ALL- l/marrow
samples were injected into human fetal bone grafts previously implanted in SCID mice, and were allowed to expand
for 2 months. Before injection in the fetal bones, both unpurged and purged samples were PCR+, although to a different extent (Fig 8C). Interestingly, PCR analysis of the cells
recovered from the fetal bones 2 months later showed the
local expansion of ALL- 1 cells only from the unpurged mar-
row (Fig 8D, lane 6 ) .A known number of ALL-l cells (from
2 X IO' to IO5) was injected inhuman bone grafts of a
separate group of mice to measure the sensitivity of the
system. The minimal number of ALL-l cells that resulted
in a PCR detectable growth in these grafts after 2 months
was 2 X IO4 (Fig 8D, lane 2).
DISCUSSION
Our previous studies have shown the ability of lethally
irradiated TALL-IO4 cells to kill a broad range of malignant
targets, including leukemias, with MHC nonrestricted mechanisms~l".17.19.21We have also documented the antitumor efficacy of these potent effectors in SCID mice engrafted with
human leukemia (U937 cell line) or glioblastoma (U-87 MG
These observations have prompted us to investigate the ability of irradiated TALL-IO4 cells to purge leukemic marrows ex vivo, with the idea that this might provide
an alternate and highly effective marrow purging approach
for potential clinical use in an autologous BMT setting.
Considerable controversy exists about the role of natural
killer (NK) cells in the regulation of hematopoiesis.3' It has
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398
CESANO ET AL
direct cytolytic activity against marrow cells from healthy
donors, the mean lysis being always below IO%, even at
high E:T ratios (100: 1) in long-term (18 hour) "Cr-release
A
assays. This observation confirms studies done with LAK
cells in rats" and humans.40Importantly, E:T conjugate formation assays and competitive inhibition experiments both
showed that the failure of TALL-104 cells to lyse normal
marrow cells was due to binding defects, ie, to the inability
of these effectors to recognize and physically interact with
this type of target. These results are in accordance with
another study on LAK cells.4'
The lack of direct cytotoxicity of TALL-104 cells against
normal hematopoietic elements is a necessary condition for
the possible applicability of these killer cells in a clinical
setting of BM purging. The ability of TALL-104 cells to
actually distinguish leukemic progenitor cells from normal
progenitors in the same individual, thus killing only the malignant blasts and sparing healthy hematopoietic elements,
0
1
is very difficult to show at the molecular level, because
7
1OO:l
50:l
25:l
12:l
6:l
v)
of both the scarcity of PCR reagents specifically detecting
different leukemia subsets, and the intrinsic limitations of
the PCR technology. In fact, the sensitivity of PCR detection
is always below one out of lo6cells and, as discussed below,
a PCR-positive result may very well reflect a state of irreversible quiescence induced by TALL- 104 cells in certain
ap
types of leukemias. To address the crucial issue of whether
TALL-I04 cells spare normal progenitors, we used two approaches. One approach was to examine the clonogenic potential of BM cells from a relatively large number of healthy
60
donors on incubation with the irradiated effectors. We found
that in long-term (2-week) clonogenic assays, BM cells from
10 normal individuals preincubated with IL-2AL-12-acti40
vated, y-irradiated TALL-I04 cells for 4 or 18 hours at E:T
ratios of 0.1:1 and 1:1 were able to form colonies (CFU and
BFU) at the same extent as control marrows. The second
20
approach was to examine the effects of TALL-104 cells on
CD34-positively selected cells from human umbilical cord
blood samples, which compared with adult human marrow,
are a rich source of hematopoietic stem and progenitor
cells.27
An early CD34+ cell population with very high prolif1OO:l
5O:l
26:l
12:l
6:l
erative and replating potential was recently isolated from
Blocker to target ratio
human umbilical cord blood by Lu et al.27These colonyforming cells are present in about eightfold higher frequency
Fig 3. Cytotoxicity of TALL-I04 cells against "Cr-labeled U937 (0) in cord blood than in adult BM and appear to be associated
(A) or Raji ( 0 )(E) targets using E M cells from three healthy donors
with the high hematopoietic repopulating capacity of umbilias cold targets (blockers). The percentage of killing of 5'Cr-labeled
cal cord blood cells.27Our data with CD34-enriched cord
targets in the absence of blockers is indicated by the continuous
straight line in both panels. Cold U937 and Raji cells were used as
blood cells show the failure of TALL-104 cells to reduce
positive controls for blocking. ( 0 1, BMI; (O),BM 2; (A), E M 3.
the number of any type of colony forming cells detectable
in a 14-day clonogenic assay, including the CFU-GEMM.
This type of CFU morphologically resembled the human
been reported, in fact, that NK cells can either inhibit,33,'4 early progenitors with high-proliferative potential found in
cord blood samples" and we were able to replate them at
~ t i m u l a t e ? ~or. ~not
~ affect at all3' the growth of various
least 4 times. These data confirm that TALL-104 cells do not
hematopoietic progenitors. BM precursors appear to be more
affect the self-renewal potential of very early hematopoietic
susceptible than PB progenitors to the inhibitory action of
precursors.
resting and &%-activated lym~hocytes.~'
Although TALLBased on the negative effects of IFN- y and TNF-a on the
104 cells kill tumors more potently than IL-2-activated lymclonogenic growth of hematopoietic cells,3z-34and on the
phocytes (LAK cells), the present study shows that IL-ZLLability of TALL-104 cells to produce high levels of these
12-activated, y-irradiated TALL-104 cells have little or no
lo0l
e
I
""1
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
A CYTOTOXIC T-CELL LINE FOR MARROW PURGING
cytokines upon exposure to IL-2, IL-12, or tumor
we also investigated, in this study, whether in the absence
of a direct lytic activity, TALL-IO4 cells could still exert
inhibitory effects on the growth of hematopoietic precursors
through the release of toxic mediators. Interestingly, we
found that normal BM cells are able to produce TNF-a
during an 18-hour incubation with TALL-I04 cells, thus
GM-CSF
100000
1
TNF-a
IFN-y
GM-CSF
28 S
TNF-a
Fig 5. mRNA levels of IFN-y, TNF-a, and GM-CSF in TALL-l04/BM
mlxture. TALL-104 and BM cells from the same donor as in Fig 4,
were pretreated for 4 hours with 10 pg/mL of ActinomycinD/Puromycin. and incubated for 4 hours with untreated BM and TALL-104 cells,
respectively. Total RNA was extracted for Northern blot hybridization
with specific cytokine probes. Ethidium bromide gel is shown for
RNA normalization. Lanes 1 through 4 show results of cytokine RNA
expression by TALL-104 cells alone (lane 11, TALL-104 cells incubated
with ActinomycinD/Puromycin-treatedBM cells (lane 21, BM cells
stone (lane 3). and BM cells incubated with ActinomycinDIPuromycin-treated TALL-104 cells (lane 4). Similar data were obtained when
BM cells from two other donors were tested.
'0°1
250
200
7
3
150
100
50
0
IFNy
1000
indicating that there is a subpopulation in nonleukemic,
healthy marrows that recognizes TALL-I04 cells as foreign
stimulus and responds to it by producing cytokines.
The absence of inhibition of CFU growth, even in the
presence of high levels of endogenous TNF-a and IFN-y
(as indicated in Fig 4), could be attributed to the antagonizing
activity in the same milieu of GM-CSF, another cytokine
produced by activated TALL-104 cell^^^ which has potent
stimulatory effects on marrow progenitors. Altogether, these
results indicate that TALL-104 cells do not affect the viability and long-term growth of pluripotent stem cells and lineage-committed precursors, and are in accordance with other
data in the literature showing the lack of inhibitory effects
by other MHC nonrestricted effectors (both CD3/TCR-y6'
and -cup' T-cell clones) against the same cells.42These data
1so0
a
3
1000
100
0
CI
YI
0
.-+
Y
w
0
i
2
c
Fig 4. Levels of cytokine production in TALL-l04/BM cell mixtures.
TALL-104 and BM cells, pretreated for 4 hours with 10 pg/mL of Actinomycin D (ActD) and 50 p g l m L Puromycin, were incubated for 18
hours with untreated BM and TALL-104 cells, respectively. Cell-free
CM were obtained and the levels of IFN-y, TNFa, and GM-CSF measured by radioimmunoassays or EUSA. Results from one donor are
shown; similar data were obtained with BM cells from two more BM
donors. Control TALL-104 cells cultured alone produced <1 U/mL of
TNFa, 3 UlmL IFN-y, and 58 pg/mL GM-CSF. After ActD/Puromycin
treatment TALL-104 cells produced 4 6 pg/mL GM-CSF, this being the
sensitivity limit of the ELISA assay used (see Materials and Methods).
Control BM cells cultured alone produced <1 UImL TNFa, 30 UImL
IFN-y (15 UlmL if ActDIPuromycin-~eated),and 34 pglmL GMGSF
(116 pglmL if ActD/Puromycin-treated). Control K562 cells cultured
alone produced <1 UlmL IFN-y, and TNF-a, and c 1 6 pglmL GM-CSF.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
CESANO ET AL
400
Donor A
4 hr
Donor B
TALL-104IBM
too,
ratlo:
18 hr
"ol'8
0°1
-
hr
100
60
C
-e
0
s
eo
40
60
P
E
=
40
20
20
0
0
CFU-GM
BFU-E
CFU-GEMM
CFU-GM
BFU-E
are further supported by findings in rats, in which marrow
grafts pretreated with LAK cells retained the ability to reconstitute lethally irradiated recipients?' Our recent findings in
the B6D2Fl murine model of syngeneic 7 0 2 pre-B cell
leukemia provide strong evidence that heavily infiltrated leukemic marrows from these animals, cultured for 18 hours
with irradiated TALL-104 cells retain the ability to fully
reconstitute lethally irradiated recipients without inducing
leukemia (Cesano et al., manuscript in preparation). These
data in an animal model strongly support the ability of
TALL-IO4 cells to effectively purge leukemic marrows without affecting the repopulating potential of early marrow pre-
Table 1. Clonogenic Potential of Cord Blood-Derived CD34' Cells
After Incubation With TALL-104 Cells
CFU-GEMM
Fig 6. Long-term effects of TALL-104 cells on normal BM progenitors. TALL-104 and BM cells were
mixed at the indicated ratios for 4 or 18 hours, and
subjected t o a 14-day colony assay.
cursors. Experiments are underway to extend these findings
to human CD34' marrow and cord blood progenitors by
examining their ability to reconstitute SCID mice after
TALL- 104 purging.
% Neo+ U937 cells
'0 5 10 25 50
a
'
BM alone
-501bp
BM +
TA LL-104
- 501 bp
cells
No. of Colonies
TALL-104/
CD34 Ratio
CFU-E
BFU-E
CFU-GM
CFU-GEMM
01
1:l
43 -c 1
56 2 22
538 ? 63
732 2 183
1,418 ? 115
1,617 2 61
32 2 21
44 2 0
lo5 CD34 positively selected cells from a cord blood sample were
incubated alone or with irradiated TALL-104 cells for 18 hours, and
then subjected to a 14-day colony assay. Both purged and unpurged
CFU-GM and CFU-GEMM could be replated 2 and 4 more times, respectively. No toxic effects on colony formation were seen with CD34
cells positively selected from two more cord blood samples.
Fig 7. Purging efficacy of TALL-104 cells on incubation with normal marrows containing neo-R-tagged U937 cells. A EM sample was
admixed with different numbers of neoR' U937 cells, as indicated.
ylrradiated TALL-104 cells were added for 18 hours in the presence
of 10 UlmL DNAse I. DNAse was also added t o the BM/U937 cell
mixtures incubated in the absence of TALL-104 cells. Cellular DNA
was extracted and subjected t o PCR amplification using primers s p e
cific t o the neo-R gene. An oligonucleotide probe specific for the
amplified sequences was used t o show the specificity of the PCR
products.
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A CYTOTOXIC T-CELL LINE FOR MARROW PURGING
40 1
A
Fig 8. Purging efficacy of TALL-104 cells upon incubation with
normal marrows containing lysis-resistant ALL-1 cells. (A) The percentage of specific ALL-1 cell lysis by TALL-104 cells in an 18 hour
"Cr-release assay and the percentage of inhibition of 'H-TdR uptake
in a 72-hour assay. (B) PCR amplification of ALL-1-specific CDRB in
normal BM admixed with various numbers of ALL-1 cells. DNAse I
was added t o both control (untreated) and TALL-104 cell-treated BMI
ALL-1 cell mixtures. (C) PCR amplification of ALL-1-CDR3 in TALL-104
unpurged (-1 and purged (+I marrows before injection in hu-SCID
chimeric mice. Before purging the normal marrow/ALL-1 cell mixture
was composed of 1 x 10' BM cells and lo5 ALL-1 cells. (D) PCR
amplification of ALL-1-specific CDRB in cells recovered from human
fetal bone grafts that had been injected 2 months earlier with different numbers of ALL-1 cells (los, 2 x 10'. lo', 2 x lo3 on lanes 1, 2,
3,and 4, respectively) or with purged (lane 5) and unpurged (lane 6)
marrows.
40
50:l
25:l
6:l
12:l
VT ratios
?Ao ALL-I cells
B
'4O20l052.5l.50'
~~
- 199 bp
BM alone
BM +
- 199 bp
TALL-104
cells
D
C TALL-104
5& cells
rI-+I
-
54-
ALL-1
I 1 2 3 4 5 6
- 199 bp
After establishing the lack of direct and cytokine-mediated
toxicity of TALL- 104 cells on normal hematopoietic elements, experiments were designed to test the purging efficacy of these effectors against lysis-susceptible and -resistant
leukemic targets admixed with healthy marrows. In the case
of leukemic targets displaying high susceptibility to MHC
nonrestricted lysis (such as U937 cells), TALL-104 cells
were able to completely eliminate all the leukemic cells,
even when present at 50% concentration in the marrows, as
measured both by PCR amplification of the neo-R marker
gene, and by long-term clonogenic assays. By contrast, when
a more resistant leukemic target was used, such as the ALL1 cell line, TALL-I04 cells were not able to eliminate them,
as shown by both "Cr-release assays and PCR, even when
marrow contamination was limited to 1.5%. Because, however, TALL-I04 cells can inhibit almost completely the
growth of most resistant leukemias, including ALL-1 cells,
as measured in 'H-TdR incorporation assays, we wanted
to determine whether the PCR technique was giving falsepositive results by simply identifying growth arrested ALL1 cells that would eventually become apoptotic. To prove
this point, in vivo experiments were performed to test
whether PCR detected ALL-1 cells had maintained their
tumorigenicity in vivo. SCID mice engrafted with human
fetal bones were chosen as a model system because it allows
the growth of most human leukemic cells in a human marrow
microenvironment and the expansion of very low numbers
of freshly separated human leukemia^.^'
Results showing the failure of TALL-104-purged marrows, which were PCR" for ALL-l cells, to induce leukemia
in this immunodeficient host demonstrated that the residual
ALL-1 cells were, at the moment of the injection, either
dying or permanently unable to further proliferate. These
results are conceptually very important as they introduce a
word of caution in interpreting PCR data. Namely, the PCR
technique, although excluding necrotic cells, does detect
cells that are either growth arrested or in a pre-apoptotic
phase. Therefore, a PCR-positive result after purging with
any biologic agent does not necessarily indicate a failure of
the purging procedure, but may indicate that the applied
treatment has impaired the growth or tumorigenic potential
of the target. Likely, under certain conditions, PCR detectable residual malignant cells may even be eliminated by the
host's immune system.
In conclusion, the data presented in this report indicate
the ability of irradiated TALL- 104 cells to eliminate malignant blasts either by cytotoxic or by cytostatic mechanisms,
yet sparing normal hematopoietic precursors. This creates an
advantageous therapeutic index for this human killer clone in
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
402
CESANO ET AL
potential marrow purging strategies and even in consolidative immunotherapy in combination with autologous BMT.
Importantly, in addition to leukemias and lymphomas, this
approach might be feasible for eradicating residual disease in
other forms of cancer displaying BM involvement, including
neuroblastoma in children and breast carcinoma in adult patients, whenever treatment with high-dose chemotherapy or
radiotherapy followed by rescue BMT is indicated.
ACKNOWLEDGMENT
We thank Jeffrey Faust for assistance at the cell sorter, Mike
Sidelsky for animal care and maintenance, and the Editorial Department of The Wistar Institute for preparation of this manuscript.
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1996 87: 393-403
Use of a lethally irradiated major histocompatibility complex
nonrestricted cytotoxic T-cell line for effective purging of marrows
containing lysis-sensitive or -resistant leukemic targets [see
comments]
A Cesano, G Pierson, S Visonneau, AR Migliaccio and D Santoli
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