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Blood, Vol. 91 No. 10 (May 15), 1998:
pp. 3850-3861
Stable Transduction of the Interleukin-2 Gene Into Human Natural
Killer Cell Lines and Their Phenotypic and Functional Characterization
In Vitro and In Vivo
By
Shigeki Nagashima,
Robbie Mailliard,
Yoshiro Kashii,
Torsten E. Reichert,
Ronald B. Herberman,
Paul Robbins, and
Theresa L. Whiteside
From the Departments of Pathology, Otolaryngology, Medicine, and
Biochemistry/Molecular Biology, University of Pittsburgh School of
Medicine, Pittsburgh, PA; and University of Pittsburgh Cancer
Institute, Pittsburgh, PA.
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ABSTRACT |
A variety of strategies have been attempted in the past to stably
transduce natural killer (NK) cells with cytokine or other cellular
genes. Here, we demonstrate the successful delivery of the
interleukin-2 (IL-2) gene into two human NK cell lines, IL-2-dependent NK-92 and IL-2-independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 and
neor markers from a polycistronic message was
constructed and transduced into a CRIP packaging cell line. By
coincubation of NK cells with monolayers of CRIP cells or by using
retrovirus-containing supernatants in a flow-through method, 10% to
20% of NK cells were stably transduced. Upon selection in the presence
of increasing G418 concentrations, transduced NK cells were able to
proliferate independently of IL-2 for more than 5 months and to secrete
up to 5.5 ng/106 cells/24 h of IL-2. IL-2 gene-transduced
NK-92 cells had an in vitro cytotoxicity against tumor targets that was
significantly higher than that of parental cells and secreted
interferon gamma (IFN ) and tumor necrosis factor alpha (TNF ) in
addition to IL-2. Moreover, the in vivo antitumor activity of IL-2
gene-transduced NK-92 cells against established 3-day liver metastases
in mice was greater than that of parental nontransduced NK cells.
Stable expression of the IL-2 transgene in NK cells improved their
therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells
secreted sufficient quantities of bioactive IL-2 to proliferate in
vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer
therapy in the future.
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INTRODUCTION |
NATURAL KILLER (NK) cells are immune
effector cells that are able to lyse virally infected or transformed
targets without previous sensitization. Freshly purified NK cells from
human peripheral blood have been recently shown to use both
perforin-mediated secretory and nonsecretory apoptotic pathways of
killing.1 In addition, NK cells activated by interleukin-2
(IL-2), especially a subset designated as A-NK cells, were found to be
able to kill a variety of tumor cell targets in vitro and in
vivo.2 Previously, we showed that a single adoptive
transfer of human A-NK cells and IL-2 induced regression of established
liver metastases in nude mice.3,4 Similarly, transfers of
murine A-NK cells into immunocompetent mice bearing pulmonary
metastases of B16 melanoma were shown to be therapeutically
effective.5 Antitumor activity of A-NK cells in vitro and
in vivo was strictly dependent on the presence of exogenous IL-2 at
doses sufficient to support functions of these effector
cells.3-5 At high concentrations, exogenous IL-2
administered in vivo to tumor-bearing hosts induces toxicity, which has
been a limiting factor in the wider utilization of adoptive
immunotherapies with IL-2-dependent effector cells for the treatment
of animals or patients with cancer.6 To bypass the need for
exogenous IL-2, a strategy of genetically modifying the effector cells
to express IL-2 can be considered. Such genetically modified effector cells secreting IL-2 are likely to be independent of exogenous IL-2 but
retain their functions, particularly in the ability to kill tumor cell
targets.
A variety of tumor cell targets, normal tissue cells, or progenitor
cells have been successfully transduced with cytokine genes.7,8 However, NK cells appear to be resistant to
retroviral transduction, as well as to infection by other viral
vectors. For reasons that are not understood, NK cells do not survive
the infection and selection process. On the other hand, NK cell lines appear to be more permissive to gene transfer. Two recent reports described instances of stable gene transfer into NK cell lines, one of
the CD18 gene into mutant YT-1 cells by electroporation,9 and the other, of the chimeric  -chain gene into NK 3.3 cells by a
retroviral vector.10
In this report, we demonstrate the successful retroviral transduction
of the human IL-2 gene into two NK cell lines: IL-2-dependent NK-92
and IL-2-independent YT2C2. The transduced and selected NK cell lines
produced and secreted bioactive IL-2 in quantities sufficient to
support various functions, including the growth of the NK-92 line.
Furthermore, upon adoptive transfer, NK-92 cells transduced with the
IL-2 gene (TR-IL-2-NK-92) significantly prolonged the survival of nude
mice with established liver metastases. This report describes the
process of NK cell transduction, characteristics of the stable NK cell
transfectants generated, and their use for immunotherapy in
tumor-bearing hosts.
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MATERIALS AND METHODS |
Cell lines.
The NK-92 cell line was established from a patient with rapidly
progressive non-Hodgkin's lymphoma11 and was kindly
provided by Dr H.G. Klingemann. This line was maintained in myeloid
long term culture medium (MLTC; Terry Fox Laboratory, Vancouver, BC, Canada) containing 12.5% fetal calf serum (FCS), 12.5% horse serum, and 10 4 mol/L 6-mercaptoethanol and supplemented with
106 mol/L hydrocortisone (Sigma, St Louis, MO) and 1,200 IU/mL IL-2 (Chiron, Emeryville, CA). The surface marker expression and
cytolytic activity of this line against K562 and Daudi are similar to
those of A-NK cells.11 The YT2C2 cell line was established
from a patient with thymic lymphoma12 and was kindly
provided by Dr Kendal Smith. This line was maintained in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% (vol/vol) FCS
(GIBCO). K562, a chronic myelogenous leukemia cell line, and Daudi, a
Burkitt lymphoma cell line, were maintained in RPMI 1640 medium
supplemented with 10% FCS. CTLL-2, an IL-2-dependent murine cytotoxic
T-cell line, was maintained in RPMI 1640 medium supplemented with 10% FCS and 300 IU/mL IL-2. HR, a human gastric carcinoma cell line, was
established in our laboratory from a liver metastasis, as previously
described, and was maintained in DMEM (GIBCO) supplemented with 10%
FCS.13 These lines were tested monthly for mycoplasma using
Gen Probe reagents (Gen Probe Inc, San Diego, CA).
IL-2 expression vector.
The DFG-hIL-2-Neo vector was constructed in our laboratory as
previously described.14 Briefly a 490-bp DNA fragment
encoding human IL-2 cDNA was obtained from Dr T. Taniguchi (Osaka
University, Osaka, Japan) and digested with the restriction enzymes
BamHI and NcoI. The neomycin resistance (neor)
gene was generated by PCR using a pMC1 Neo Poly A plasmid (Stratagene, La Jolla, CA) as a selectable marker and placed under the regulation of
the internal ribosome entry site (IRES) of encephalomyocarditis virus.
The DFG-hIL-2-Neo vector contains a full-length human IL-2 cDNA under
the regulation of the retroviral LTR.
The DFG-LacZ-Neo vector was similarly constructed and used as a control
in some experiments.
IL-2 gene-producing packaging cell line.
The packaging cell line, CRIP, was derived from NIH-3T3 mouse
fibroblasts.15 The DFG-IL-2-Neo vector was transduced into this line by a calcium phosphate method.16 Transfectants
were selected by culture in the presence of 400 µg/mL G418 (GIBCO), and G418-resistant colonies were tested by enzyme-linked immunosorbent assay (ELISA) for the ability to produce IL-2. The best colony, which
produced 103 ng IL-2/mL/5 ×105 cells in 48 hours, was
expanded and IL-2 gene-producing cells were used as a source of
infectious virus. Supernatants of these cells contained a high titer of
the virus (105 to 106 colony-forming units
[cfu]/mL). The DFG-LacZ-Neo vector was also transduced into CRIP
cells by the same procedure.
IL-2 gene transduction into NK-92 cell lines.
NK-92 cells cultured at a concentration of 4 × 105 to 1 × 106 cells/mL were supplemented with fresh MLTC medium
24 hours prior to transduction by the flow-through method described by
Chuck and Palsson.17 NK-92 cells were pelleted and
resuspended in an aliquot of the CRIP supernatant
(1 × 106 cells/mL plus 8 µg/mL polybrene). Aliquots
(2 mL) of this suspension were added to each well of a six-well plate
containing Transwell-Col tissue culture membranes (pore size, 0.4 µm;
Costar, Cambridge, MA). The supernatant was allowed to filter through
by gravity (30 to 40 minutes at 37°C). It was then harvested from the
lower chamber and reapplied to the cells remaining on the surface of the membrane. This process was repeated six or seven times. After the
last cycle of flow-through, the cells were harvested from the
membranes, pooled, and resuspended in the conditioned culture medium
(4 × 105 cells/mL) containing 1,200 IU/mL IL-2. The
flow-through transduction process was repeated after 2 to 3 days of
culture.
YT2C2 cells were transduced by cocultivation with the
retrovirus-producing packaging cell line at an E:T cell ratio of 5:1 to
10:1 for 48 hours in the presence of 2 µg/mL polybrene (Sigma). This
cocultivation was repeated twice and was followed by selection in the
presence of 200 µg/mL G418 for 4 weeks. After selection, the cells
were maintained in RPMI 1640 medium containing G418 without exogenous
IL-2.
As a control, NK-92 cells and YT2C2 cells were also transduced with the
LacZ gene, using the LacZ gene-producing packaging cell line or its
supernatant and the procedure already described.
Selection of IL-2/Neo-transduced NK cells.
Selection was accomplished by gradually increasing the concentration of
G418 in the medium while decreasing IL-2. Forty-eight hours after
transduction, the cells were suspended in medium containing 50 µg/mL
G418 and 1,200 IU/mL IL-2 at a concentration of 4 × 105
cells/mL and cultured for 14 days. Control cultures established in
parallel contained parental (nontransduced) cells plated in the same
medium and transduced NK cells cultured in the absence of G418. The
cultures were centrifuged on Ficoll-Hypaque gradients to remove dead
cells, washed, and replated at 4 × 105 cells/mL in medium
containing 0.1 mg/mL G418 and 1,200 IU/mL IL-2. After 1 week of
culture, the cells were transferred to the medium containing 300 IU/mL
IL-2 and 0.2 mg/mL G418. Following 1 additional week of selection, the
cells were cultured in medium containing only IL-2 (300 IU/mL) and then
were transferred to IL-2-free medium. During the selection process,
cell viability was determined by the trypan blue exclusion method.
Parental NK cells are referred to as P-NK-92 or P-YT cells. Transferred
and selected NK cells are referred to as TR-IL2-NK-92 or TR-IL2-YT cells throughout.
 -Galactosidase staining.
Forty-eight hours after transduction and 6 weeks after selection in the
presence of G418 medium, parental or LacZ gene-transduced NK cells
were stained using the fluorescent LacZ substrate, fluorescein di--galactopyranoside (FDG), as previously described.4
Briefly, 3 × 105 cells were washed twice in PBS and
resuspended in a 100-µL aliquot of 1 mmol/L FDG (Sigma) in medium
prewarmed to 37°C. The cell suspension was incubated in the 37°C
water bath for 1 minute and then placed on ice, and 0.5 mL ice-cold PBS
was added to each tube. After 15 minutes on ice, the cells were
analyzed by flow cytometry.
Proliferation assay.
To assess proliferation, NK cells were resuspended in RPMI 1640 supplemented with 10% FCS but without IL-2 at a concentration of 1 × 104 cells/200 µL in wells of a U-bottom microliter plate
(Costar) and incubated for 4 days. For the final 16 hours of
incubation, 1 µCi 3H-thymidine (New England Nuclear,
Boston, MA; 147.9 GBq/mmol) was added to each well. Cells were
harvested onto glass-fiber filters using a semiautomatic cell harvester
(Scatron, Sterling, VA). 3H-thymidine uptake was determined
using an LKB Betaplate counter (Pharmacia, Gaithersburg, MD).
Flow cytometry.
Expression of IL-2 and the IL-2R , , or chains or other
surface markers on parental and transduced NK-92 or YT cells was measured by flow cytometry as previously described.18 The
following antibodies were used for staining: anti-IL-2 mAb (BioSource
International, Camarillo, CA), anti-CD25 mAb for IL-2R chain (Becton
Dickinson, San Jose, CA), anti-IL-2R chain mAb (Endogen, Boston,
MA), and TUGh4 mAb for IL-2R chain (kindly provided by Dr K. Sugamura). Rat IgM kappa (BioSource, International, Camarillo,
CA) was used as an isotype control for anti-IL-2 mAb. Rat
IgG (TAGO, Burlingame, CA) was used as an isotype control of TUGh4 mAb.
Labeled anti-CD3, -CD56, -CD28, and -CD11a mAbs and isotype control
antibodies were purchased from Becton Dickinson (San Jose,
CA). Anti-CD54 (ICAM-1) was purchased from Immunotech (Marseille,
France). For staining, cell suspensions were adjusted to the
concentration of 1 × 106 cells/mL in 0.1% (wt/vol)
sodium azide-PBS and incubated with pretitered mAb for 30 minutes at
4°C. Cells were then washed with 0.1% sodium azide-PBS, fixed with
1% (wt/vol) paraformaldehyde (PFDH)-PBS, and analyzed in a
FACScan flow cytometer (Becton Dickinson). Isotype
controls were routinely included. To detect intracellular IL-2 or IL-2R
chains, cells were first prefixed with 0.5% (wt/vol) PFDH for 20 minutes at 4°C and then permeabilized with cold acetone for 3 minutes, washed, and stained with Abs to IL-2 or IL-2R chains. Indirect
immunostaining was used for detection of the -chain protein, in
which pretitered TUGh4 mAb (1:100 dilution) was the primary reagent,
and FITC-labeled goat anti-rat IgG was used to develop the staining
reaction.
ELISA.
Parental or transduced NK cells were washed, resuspended in enriched
medium at the concentration of 1 × 106 cell/mL, and
incubated for 48 hours. Supernatants were collected, filtered, and
tested for the presence of IL-2, TNF, or IFN by ELISA (Endogen,
Cambridge, MA). Supernatants of NK-92 cells transiently transduced with
the IRAP gene were also tested for IRAP by ELISA (R & D Systems,
Minneapolis, MN). The kits were calibrated against World Health
Organization International Cytokine Standards, and the assays were
performed under quality-control conditions described previously by
us.19
Immunohistochemistry for IL-2.
Cell suspensions were washed in PBS and cytocentrifuged onto glass
slides using a Shandon centrifuge (Sewickley, PA). The cells were
prefixed for 5 minutes in 2% PFDH, permeabilized, and fixed with 0.1%
Triton X/PFDH 2% for 2 minutes. Cytospins were stained for the
presence of IL-2 using anti-IL-2 polyclonal Ab (Becton Dickinson) at an
optimal working dilution of 20 µg/mL, which was determined in
preliminary titration experiments with phytohemagglutinin
(PHA)-stimulated Jurkat cells. Immunostaining for IRAP was performed
using anti-human Ab (Amgen, Boulder, CO) followed by goat
anti-mouse Ig. A standard streptavidin-biotin complex immunoperoxidase
(sABC-HRP) technique was used for staining, and the color was developed
with 3-amino-9-ethyl-carbazole ([AEC] Biomeda, Foster City,
CA).20 The slides were counterstained with hematoxylin
(Biomeda), mounted in a glycerol-based mounting medium,
and evaluated in an Olympus BH-2 light microscope
(Olympus, Tokyo, Japan).
In some experiments, Cy3 staining was used. Slides were fixed in 4%
PFDH for 10 minutes, permeabilized in 0.1% saponin in PBS for 5 minutes, and incubated with polyclonal anti-IL-2 Ab (Becton Dickinson)
at a working dilution of 1:700. Goat anti-rabbit IgG conjugated with
Cy3 (Jackson Immuno Research, West Grove, PA) was used as a second
antibody (1:1,000). The slides were counterstained with 2 µg/mL
Hoechst dye 33342 for 2 minutes and then examined in a fluorescent
microscope.
In all experiments, isotype Abs purchased from Dako and Sigma were used
as controls. Prior to staining, polyclonal anti-IL-2 Ab was
preincubated with an excess of IL-2 (Chiron, Emeryville, CA) to verify its specificity. This absorption was shown
to completely eliminate cytokine-specific staining.
IL-2 bioassay with CTLL-2 cells.
Supernatants of NK cells transduced with the IL-2 gene were tested for
the presence of bioactive IL-2 in 4-day 3H-thymidine (TdR)
incorporation assays using the IL-2-dependent CTLL-2 cell line as
described by us previously.14
RT-PCR for IL-2 and IL-2R mRNA.
For amplification of the IL-2 gene, the following oligonucleotide
primers were used: IL-2 sense, 5 -GTCACAAACAGTGCACCTAC-3; and IL-2
antisense, 5-CCCTGGGTCTTAAGTGAAAG-3. To analyze expression levels of
IL-2 mRNA from parental or transduced NK cell lines, quantitative
competitive (QC) RT-PCR established in our laboratory was performed as
previously described.21
Cytotoxicity assays.
Untreated NK cells or PFDH-fixed NK cells were used as effectors.
PFDH-fixed NK-92 cells were prepared as previously
described1 to prevent secretion of cytoplasmic granules and
cytokines while preserving cell surface-bound ligands. Briefly, cells
were fixed with 1% (wt/vol) PFDH in PBS for 10 minutes at room
temperature (RT), washed twice with PBS, and incubated overnight in
RPMI 1640 medium at 37°C. Supernatants of NK-92 cells were used in
some assays to assess their effects on the viability or growth of tumor cell targets.
51Cr-release cytotoxicity assays were performed as
previously described,1,4 using target cells labeled with
100 µCi 51Cr (NEN; 5 µCi/mmol/L) and incubated with
effector cells at E:T ratios of 8:1 to 0.5:1 in U-bottom 96-well plates
(Costar) for 4 hours. Spontaneous release and maximal release were
determined by incubating target cells in medium alone or in 5% Triton
X-100, respectively. The assay was always performed in triplicate. The percent specific lysis was calculated according to the formula, percent
specific lysis = (mean cpm experimental release  mean cpm
spontaneous release/mean cpm maximal release mean cpm spontaneous release) × 100.
3H-thymidine release assay was a modification of the JAM
test described by Matzinger.22 Target cells were labeled
with 5 µCi/mL methyl 3H-thymidine (NEN; 147.9 GBq/mmol)
and incubated with effector cells at E:T ratios of 16:1 to 2:1 for 1 hour at 37°C. Cells were disrupted by repeated (three times) freezing
(at 20°C) and thawing (at RT) and harvested onto fiberglass
filters, and their radioactivity was counted in an LKB Betaplate
counter (Pharmacia). The percentage of specific
3H-thymidine release was determined using the formula,
percent cytotoxicity = 100 × (C E)/C, in which E (experimental)
is the cpm of target cells in the presence of effector cells, and C
(control) is the cpm of target cells alone.
MTT assay was also performed as previously described.23
Briefly, adherent target cells were plated at the density of 5,000/well in 96-well flat-bottom plates (Costar) and cultured for 24 hours at
37°C in 5% CO2 in air to prepare the monolayers for
assay. Effector cells were added to the monolayer cultures at E:T
ratios of 16:1 to 2:1, and following centrifugation, the plates were incubated for 24 hours. Effector cells and detached target cells were
then removed by washing. Next, a 200-µL aliquot MTT (Sigma) solution
(0.5 mg/mL in DMEM supplemented with 10% FCS) was added to each well
and the plates were incubated for additional 4 hours at 37°C. MTT
solution was then removed, and a 150-µL aliquot of dimethyl sulfoxide
(Sigma) was added to each well to solubilize the formazan crystals that
formed in viable target cells. The plates were rotated on a plate
shaker for 10 minutes at RT. Absorbance was read immediately at a
wavelength of 540 nm on a scanning multiwell spectrophotometer. The
percentage of cell death was calculated using the formula, percent cell
death = 100 × (C E)/(C B), where C is the optical density
(OD) reading in wells containing target cells alone (control), B is the
OD reading of wells containing medium (background), and E is the OD
reading of adherent targets remaining in the wells after incubation
with effector cells (experimental).
Adoptive transfer of NK cells.
P-NK-92 or TR-IL-2-NK-92 cells or human A-NK cells generated as
previously described1 were used for immunotherapy of
established 3-day liver metastases in BALB/c nude mice.4
Liver metastases were induced by a single intrasplenic injection of
human HR cells (5 × 106/0.2 mL) into nude mice
immunosuppressed by treatment with cyclophosphamide (200 mg/kg; Sigma)
and anti-asialo GMI (AS-GMI) Ab (0.2 mg/mouse; Waco, Dallas, TX). Three
days after intrasplenic injection of HR cells, 5 × 106
effector cells/mouse (P-NK-92 and exogenous IL-2, TR-IL-2-NK-92 alone,
or A-NK cells and exogenous IL-2) were delivered intrasplenically to
mice in groups of 10 animals per treatment. As a negative control, HBSS
(GIBCO) was injected. Within 10 minutes of these injections, the
spleens were removed. Exogenous IL-2 (60,000 IU in 0.5 mL HBSS) was
delivered IP to mice treated with P-NK-92 and A-NK cells twice daily
for 5 days. During this period of IL-2 therapy, all mice were treated
with anti-AS-GMI Ab by IP injection twice to eliminate endogenous NK
activity. Mice were evaluated for 60 days to assess survival.
Statistical analysis.
Statistical analysis of the results was performed using the
Mann-Whitney U test. Differences were considered significant
for P values less than .05.
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RESULTS |
Transient transduction of NK cells.
To determine the ability of an MuLV-based retroviral vector to stably
infect NK cells, expression of IL-2 and Neo markers was evaluated. The
efficiency of a flow-through method was determined by immunostaining
for expression of the transgene. As a control, MFG-IRAP-infected NK-92
cells were used. Figure 1 shows staining for IL-2 of cultured P-NK-92 cells, which were negative for
intracytoplasmic IL-2, and of transiently transduced NK-92 cells, which
contained 10% to 20% stained cells. The percentage range of positive
cells was determined by counting a total of 200 cells on each
immunostained cytosmear prepared from three different cultures of
transiently transduced NK-92 cells. These cultures were not tested by
ELISA for the level of IL-2 in the supernatants because of the presence of exogenous IL-2. Instead, a culture of NK-92 cells transduced with
the IRAP gene and containing 2% to 3% IRAP-positive cells by
immunostaining (not shown) was tested in ELISA for the level of IRAP in
the supernatant. At 72 hours posttransduction, the supernatant
contained 8.4 ng IRAP/106 cells/48 h. P-NK-92 cells did not
produce IRAP.
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| Fig 1.
Immunostaining for IL-2 in transduced but nonselected
NK-92 cells. (A) P-NK-92 cells incubated for 48 hours in the absence of
exogenous IL-2 prior to staining. (B) Transduced nonselected (48 hours
posttransduction) IL-2/Neo/NK-92 cells incubated for 48 hours in the
absence of exogenous IL-2 prior to staining. Insert shows an
IL-2-expressing NK cell (original magnification ×1,000). The
staining reaction was developed with AEC, and the cells were counterstained with hematoxylin. For A and B, original magnification is
×500.
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The transduced YT cells were assessed for the ability to secrete IL-2
by ELISA performed 48 hours after transduction. The supernatants
consistently contained low levels of IL-2 (7 to 10 pg/106
cells/48 h), while those of parental YT cells contained none.
Selection of transduced NK cells.
The process of selection was modified to include a series of stepwise
increases in the concentration of G418 separated by periods of culture
in the absence of G418 to give the surviving cells an opportunity to
recover. Throughout selection, the growth medium was supplemented with
exogenous IL-2 (1,200 IU/mL) to avoid massive cell death due to IL-2
withdrawal. The selection process was usually accomplished in 4 to 5 weeks, at which time the surviving cells were transferred to the
IL-2-free medium and thereafter cultured without exogenous IL-2.
YT cells, which are IL-2-independent, were selected as already
described, but the medium used for selection was not supplemented with
IL-2.
Growth of NK cell lines transduced with the IL-2 gene.
TR-IL-2-NK-92 cells were initially cultured in the presence or absence
of exogenous IL-2, and their proliferation was compared with that of
parental cells. Figure 2A shows that
P-NK-92 cells did not continue proliferating in the absence of
exogenous IL-2. In contrast, TR-IL-2-NK-92 cells proliferated
exceedingly well, continuing to double in number every 24 hours. These
cells have been maintained in a continuous culture for over 6 months.
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| Fig 2.
(A) Proliferation of P-NK-92 and selected TR-IL-2-NK-92
in cultures not supplemented with exogenous IL-2. For culture of
P-NK-92 cells, exogenous IL-2 was removed just before day 0. One of 2 experiments performed is shown. (B) Proliferation of TR-IL-2-NK-92 cells and NK-92 cells transduced with the retroviral vector containing the IRAP gene as control. The latter were IL-2-dependent and were cultured in the presence of exogenous IL-2. Note that the growth rates
of transduced experimental and control NK-92 cells were similar.
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It was possible that transfer of a retroviral vector into NK-92 cells
enhanced their growth irrespective of the IL-2 gene. To test this
possibility, we also transduced NK-92 cells with the retroviral IRAP
gene and cultured them in the presence of exogenous IL-2 (1,200 IU/mL).
Proliferation of TR-IRAP-NK-92 + IL-2 was similar to that of P-NK-92 + IL-2 and of TR-IL-2-NK-92 cultured in the absence of IL-2 (Fig 2B). In
contrast, control TR-IRAP-NK-92 cells cultured in the absence of IL-2
did not proliferate beyond day 25 (data not shown). These control
experiments demonstrated that transfer of a retroviral vector into
NK-92 cells was not itself responsible for spontaneous proliferation of
NK cells.
Growth of transduced and parental NK-92 cells was also compared in
short-term 3H-TdR incorporation assays (Fig
3). In the absence of exogenous IL-2 or in
the presence of low IL-2 concentrations, TR-IL-2-NK-92 cells
proliferated significantly more rapidly than parental cells. Growth of
P-NK-92 cells was strictly IL-2-dependent, and at the concentration of
60 IU/mL, it reached a plateau. Addition of exogenous IL-2 at
concentrations greater than 60 IU/mL had little effect on growth of the
parental cell line. On the other hand, growth of TR-IL-2-NK-92 cells
was independent of the presence of exogenous IL-2.
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| Fig 3.
Proliferation of parental or selected and transduced NK
cell lines in short-term cultures. Cells were adjusted to the
concentration of 1 × 104 cells/200 µL in wells of a
U-bottom microtiter plate and incubated ± IL-2 at various
concentrations for 4 days. 3H-TdR incorporation was
measured during the last 16 hours of culture. *P < .05 for
differences in growth between parental and transduced NK cells.
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Only two cycles of coincubation with CRIP cells were necessary for
transduction of the IL-2 gene into YT cells, which then became
resistant to G418. Irrespective of the presence or absence of exogenous
IL-2, transduced and selected YT cells grew significantly more rapidly
than parental cells (175% ± 2% v 100% with parental cells in the absence of IL-2). Since P-YT cells are IL-2-independent and addition of exogenous IL-2 at levels of 6 to 6,000 IU/mL had no
effect on their rate of growth, this rapid growth of TR-IL-2-YT cells
may be attributable to activation of the autocrine IL-2 pathway in the
transfectants. Control YT cells transduced with the LacZ gene did not
proliferate better than P-YT cells (data not shown).
Expression of IL-2 protein and secretion of IL-2 by transduced NK
cells.
To determine the ability of TR-IL-2-NK-92 or TR-IL-2-YT cell lines to
secrete IL-2, supernatants of transduced and parental cell lines were
tested by ELISA. IL-2 was secreted by transduced NK-92 cells
consistently in the range of several ng/106 cells/24 h
(Table 1). Fluctuations in the level of secreted IL-2 over time may be
attributed to its utilization by IL-2-dependent NK-92 cells.
TR-IL-2-YT cells secreted considerably lower quantities of IL-2 than
TR-IL-2-NK-92 cells. Both transduced NK-92 and YT cells have continued
to secrete IL-2 for up to 3 months in culture (Table
1). In contrast, the parental cell lines
produced no detectable IL-2.
The bioactivity of IL-2 secreted by TR-IL-2-NK-92 cells was next
assessed in a CTLL bioassay. A standard curve based on the rate of
CTLL-2 proliferation in the presence of various concentrations of
exogenous IL-2 was prepared to calibrate the level of IL-2 in
supernatants of NK-92 cells (data not shown). TR-IL-2-NK-92 cells
secreted bioactive IL-2 in quantities comparable to those measured in
immunoassays (850 pg/mL). In contrast, P-NK-92 cells contained minimal
levels of bioactive IL-2 (16 pg/mL), probably representing residual
exogenous IL-2 (data not shown).
In addition, the proportion of cells in culture expressing surface
and/or intracellular IL-2 was determined by both
immunoperoxidase staining and flow cytometry (Fig
4). Both NK-92 and YT cells expressed a
minimal amount of surface-associated IL-2 detectable by flow cytometry
(data for YT cells are shown in Fig 4A), and there were no differences
between transduced cells and parental cells. Intracellular IL-2 protein
was detectable in all transduced NK-92 (Fig 4C) and all YT cells (Fig
4B), as indicated by the shift of the IL-2 peak to the right. P-NK-92
cells also contained low levels of intracellular IL-2 (NK-92 cells are
IL-2-dependent), but YT cells did not. To more precisely determine the
cellular localization of IL-2, immunoperoxidase or Cy5 staining was
performed in the presence of brefeldin, and all TR-IL-2-NK-92 cells
were found to express IL-2 protein in the Golgi zone, an indication
that the protein was synthesized in the transduced cells (data not
shown). In contrast, weak and diffuse expression of IL-2 protein was
observed in the cytoplasm of the parental cells (not shown), perhaps
due to uptake of exogenous IL-2 from the medium.
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| Fig 4.
IL-2 expression in parental or IL-2 gene-transduced and
selected NK cell lines. (A) and (B) Nonpermeabilized or permeabilized cells, respectively, were stained with the FITC-labeled antibody for
IL-2 and examined for surface or intracytoplasmic expression of IL-2 by
flow cytometry. One representative experiment of 3 performed is shown
for YT cells. Similar data were obtained in 3 experiments performed on
NK-92 cells. (C) Immunostaining for IL-2 in stably transduced and
selected NK-92 cells (original magnification ×1,000).
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RT-PCR for IL-2 and IL-2R mRNA.
Both transduced and selected NK-92 and YT cells expressed a high number
of IL-2 mRNA copies as determined by QC-RT-PCR (Fig 5; TR-IL-2-NK-92 cells, 1,400 copies/ng
total RNA; TR-IL-2-YT cells, 2,000 copies/ng). In contrast, parental
cells expressed a marginally small number of IL-2 mRNA copies (P-NK-92
cells, 0.304 copies/ng total RNA; P-YT cells, 0.117 copies/ng).
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| Fig 5.
Expression of IL-2 mRNA in the parental or IL-2
gene-transduced and selected NK cell lines by QC-RT-PCR. Total RNA
extracted from NK cell lines (100 ng from parental cells, 1 ng from
IL-2 gene-transduced cells) was reverse-transcribed and amplified
using primers specific for IL-2. Southern hybridization was performed with radiolabeled cDNA for IL-2 to confirm the identify of the PCR
product. The ratio of cpm in the internal control to cpm in cellular
RNA was plotted to calculate the number of copies of IL-2 mRNA/ng total
cellular RNA (not shown). (A) NK-92 cells, (B) YT cells.
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Expression of IL-2Rs and other markers in parental and transduced NK
cell lines.
The ability to produce and secrete IL-2 could lead to changes in IL-2R
expression on transduced NK cells. Therefore, surface expression of
IL-2R , , and was measured by flow cytometry in the parental
and IL-2 gene-transduced and selected NK cell lines. With P-NK-92
cells, flow cytometry was performed after 48 hours' incubation in the
absence of exogenous IL-2. The flow cytometric data shown in Fig
6 indicate that TR-IL-2-NK-92 cells have
lower surface expression of IL-2R and than P-NK-92 cells. Expression of the chain was also decreased in transduced NK-92 cells relative to P-NK-92 cells (data not shown). Similarly, TR-IL-2-YT cells have lower cell surface expression of IL-2R and chains than P-YT cells. YT cells do not express IL-2R . These patterns of
surface expression of IL-2Rs are expected, because transduced NK cells
produce IL-2 and thus IL-2Rs are likely to be occupied by the
endogenously produced ligand and internalized or shed from the cell
surface.
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| Fig 6.
IL-2R expression on the parental and IL-2
gene-transduced NK cell lines as determined by flow cytometry.
Nonpermeabilized or permeabilized cells were stained with the
appropriate antibodies for IL-2 , , and chains and examined
by flow cytometry. One representative experiment of 3 performed is
shown. (A) NK-92 cells, (B) YT cells.
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Intracytoplasmic expression of IL-2R in the IL-2 gene-transduced and
selected NK cell lines, as detected by flow cytometry, was also lower
than in parental cells (Fig 6). This observation is consistent with a
rapid turnover of IL-2R chains in transduced NK cells, which secrete
IL-2.
No changes in expression of other surface markers commonly seen on
lymphocytes were observed on parental or transduced NK cell lines by
flow cytometry. Thus, all transduced and selected NK-92 cells were
CD3CD56+CD11a+CD54+CD45+,
as were YT cells, except for CD56 expression. In addition to determining the proportion of cells positive for the various markers, we also looked for possible quantitative differences in their level of
expression. However, no changes in the MFI of these markers were seen
on transduced cells, with the exception of CD11a (LFA-1), which showed
mildly increased MFI in transduced NK-92 but not YT cells (data not
shown).
Production of TNF and IFN by
transduced NK cell lines.
It was also of interest to examine the production of TNF and IFN,
since these cytokines are known to be produced by human NK
cells.24 ELISA was used to determine the levels of these two cytokines in supernatants of TR-IL-2-NK cells. P-NK-92 cells produced little or no TNF and considerable levels of IFN in the
presence of exogenous IL-2 (Table 2). In
contrast, TR-IL-2-NK-92 cells spontaneously produced IFN, but at
levels substantially lower than in P-NK + IL-2. TR-IL-2-YT cells
secreted no TNF and less IFN than P-YT cells.
Cytolytic functions of transduced NK cells.
Because exogenous IL-2 is known to upregulate the cytotoxicity of NK
cells, we wished to study the effect of endogenous secreted IL-2 on
antitumor functions of transduced NK cells. Cytolytic activities of
parental or transduced NK cells were measured in short- or long-term
51Cr-release assays against NK cell-sensitive targets
(K562) and NK cell-resistant targets (Daudi) (Fig
7). While K562 targets were lysed by
P-NK-92 cells in 4-hour 51Cr-release assays, the cytolytic
activity of TR-IL-2-NK-92 cells against K562 was significantly higher
than that of parental cells (Fig 7A). In contrast, lysis of Daudi
targets by these TR-IL-2-NK-92 cells was comparable to that of parental
cells (Fig 7A). Neither P-YT nor TR-IL-2-YT cells lysed K562 or Daudi
targets in 4-hour 51Cr-release assays (data not shown).
However, both types of cells lysed these targets in 24-hour
51Cr-release assays, and TR-IL-2-YT cells had significantly
higher cytotoxicity than P-YT cells (Fig 7B).
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| Fig 7.
Cytotoxicity of parental or IL-2 gene-transduced NK
cells against NK-sensitive K562 targets or NK-resistant Daudi targets. Cytotoxicity was tested in the absence of exogenous IL-2 in 4- or
24-hour 51Cr-release assays. One representative experiment
of 3 performed is shown. *P < .05. (A) NK-92 cells, (B) YT
cells.
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Figure 8 shows the cytotoxicity of parental or transduced NK cell lines
against HR human gastric carcinoma targets, which are considered "NK
cell-resistant." HR targets were found to be only slightly
sensitive to perforin-mediated killing in 4-hour 51Cr-release assays by both transduced or parental NK-92
targets or YT cells (Fig 8). However, these
targets were sensitive to nonsecretory apoptotic killing
(3H-TdR-release assays) by both NK-92 and YT cells.
Furthermore, HR targets were killed significantly better by transduced
versus parental NK cells in 1-hour 3H-TdR-release assays.
TR-IL-2-NK-92 cells were also significantly more growth-inhibitory to
HR targets than parental cells in 24-hour MTT assays. In contrast,
TR-IL-2-YT were not, perhaps because they secreted considerably less
IL-2 than transduced NK-92 cells.
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| Fig 8.
Cytotoxicity of parental or IL-2 gene-transduced NK
cells against human gastric carcinoma targets in 4-hour
51Cr-release, 1-hour 3H-TdR-release, and
24-hour MTT assays was tested. One representative experiment of 3 performed is shown. *P < .05. (A) NK-92 cells, (B) YT
cells.
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We have shown previously that PFDH-fixed NK cells are unable to secrete
granules and mediate cytolysis via the classic perforin-mediated pathway.1 On the other hand, PFDH-fixed NK cells can induce apoptosis and cytostasis in tumor cell targets.1 In
agreement with these prior data, PFDH-fixed NK-92 cells did not lyse HR targets in 4-hour 51Cr-release assays. However, they did
mediate HR cell apoptosis in 1-hour 3H-TdR-release assays
and caused growth arrest of HR targets in 24-hour MTT assays.
TR-IL-2-NK-92 cells were significantly more effective than parental
cells in these in vitro assays (Fig 9A).
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| Fig 9.
Cytotoxicity of PFDH-fixed parental or IL-2
gene-transduced NK cells or their supernatants against HR targets was
tested in various cytotoxicity assays. One representative experiment of 3 performed is shown. *P < .05.
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Supernatants of NK-92 cells, which contain spontaneously released
cytokines, had little effect on HR targets in 51Cr-release
assays (Fig 9B). However, HR targets were readily killed by
supernatants in 3H-TdR-release and 24-hour MTT assays, and
supernatants produced by TR-IL-2-NK-92 were significantly more
effective than those of P-NK-92 cells. Taken together, these data
indicate that NK cells transduced with the IL-2 gene and secreting IL-2
mediate cytotoxicity and/or growth inhibition of solid tumor
targets in vitro significantly better than nontransduced NK-92 cells.
In vivo antitumor effects of TR-IL-2-NK-92 cells.
A subset of IL-2-activated human NK cells, A-NK cells, have been
previously shown by us to rapidly eliminate established liver metastases of HR, a human gastric carcinoma, and to prolong survival of
nude mice treated by adoptive transfer of A-NK cells and
IL-2.4,24 In view of our results indicating that
TR-IL-2-NK-92 cells have significantly enhanced in vitro antitumor
activity against HR targets, we next performed a series of in vivo
adoptive immunotherapy experiments in the HR model of liver metastasis,
comparing TR-IL-2-NK-92 and P-NK-92 cells. Single adoptive transfer of
TR-IL-2-NK-92 cells resulted in significantly prolonged survival of
nude mice with 3-day established liver metastases as compared with
3-day therapy with P-NK-92 cells administered together with IP IL-2
(Fig 10). Nevertheless, the therapeutic effect of TR-IL-2-NK-92 cells
was not as impressive as with A-NK cells transferred together with exogenous IL-2. P-NK-92 cells plus IL-2 had no therapeutic effect against established liver metastases as compared with tumor-bearing control mice not treated with AIT (Fig
10).
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| Fig 10.
Survival curves of nude mice with 3-day established
liver metastases following immunotherapy with HBSS control, P-NK-92
cells plus exogenous IL-2, TR-IL-2-NK-92 cells, or A-NK cells plus
IL-2. Statistical differences in survival between groups of 10 animals each are indicated.
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To explain the relatively weak but significant survival advantage seen
in mice treated with TR-IL-2-NK-92 cells, we determined the level of
IL-2 in serum and in homogenized liver tissues of animals treated as a
control with P-NK-92 (no IL-2) or with TR-IL-2-NK-92 cells. No IL-2,
IFN, TNF, or IL-12 was detectable in the sera of these animals 24 hours after injection of effector cells (Table 3). Only liver homogenates of mice treated
with transduced cells contained a detectable but low level of IL-2,
which probably explains why the survival benefits of AIT were not as
pronounced as those with A-NK cells supplemented with a relatively high
dose of exogenous IL-2. On the other hand, levels of TNF and IFN
were higher in liver homogenates of mice treated with TR-IL-2-NK-92
versus P-NK-92 cells. In this experiment, IL-12 levels in liver
homogenates were also assayed to show that secretion of IL-2 by
TR-IL-2-NK-92 cells did not induce IL-12 production in the liver.
To ascertain that mice do not develop a lymphoproliferative disease
following adoptive transfer of TR-IL-2-NK-92 cells, we injected two
normal animals with these cells and observed them for a period of 5 months. No evidence of sickness, splenomegaly, or wasting has been
observed in these animals so far.
| |
DISCUSSION |
NK cells are thought to play an important role in protection against
various infectious agents and to mediate immune surveillance against
metastases.25-28 Antitumor functions of NK cells in vitro and in vivo are known to be dependent on the presence of
IL-2.3-5 In fact, human NK cells, which constitutively
express IL-2R, are the first lymphoid cells to respond to exogenous
IL-2 by upregulation of IL-2R chain expression29,30 and
by adherence to solid substrates.6 The dependence of NK
cells on exogenous IL-2 necessitates that this cytokine be present in
concentrations sufficient to support NK activities, when the cells are
adoptively transferred for therapy of established tumor or tumor
metastases in experimental animals4,24 or in patients with
advanced tumors.31 However, toxicities associated with
high-dose IL-2 therapy have limited its use alone or together with
effector cells in patients with cancer.32 Therefore, we wished to test the hypothesis that human NK cells transduced with the
IL-2 gene and producing IL-2 might be able to sustain and mediate
antitumor activities without the addition of exogenous IL-2.33
To test this hypothesis, we initiated attempts to introduce the IL-2
gene into human NK cells. However, these effector cells proved to be
resistant to gene transfer under a variety of experimental conditions
tried. Techniques that have been successful with tumor or tissue
cells14,34-36 failed to work with NK cells, as reported by
us and others.33 Transfection with viral supernatants has been the most widely used strategy for genetic modification of a
variety of cell types.14,36,37 Since NK cells have been previously reported to be susceptible to viral
infections,38,39 we decided to deliver the IL-2 gene by
transduction of NK cell lines with a retroviral vector. The NK-92 cell
line established from a patient with lymphoma11 is
IL-2-dependent and cytotoxic against K562 and Daudi targets. The YT
cell line, on the other hand, is IL-2-independent and unable to lyse
NK-sensitive targets in 4-hour 51Cr-release assays. The use
of these NK cell lines for gene transfer gave us an opportunity to
establish and test a model system for transduction and selection of
fresh NK cells. By cocultivation of NK cells with the CRIP cell line,
producing high titers of the viral particles (105 to
106 cfu/mL) containing IL-2 and NeoR genes, we readily
obtained transfectants of YT cells. In contrast, cocultures of NK-92
cells with CRIP resulted in a complete destruction of CRIP cell
monolayers mediated by cytolytic NK-92 cells. This observation
indicated that NK-92 cells were capable of killing virally infected
targets rapidly, and that retroviral-mediated transduction of the IL-2
gene into these effectors might be much more difficult than such
transduction into YT cells that are unable to kill virally infected
targets.
Based on this initial experience, a transduction method facilitating a
high rate of gene transfer (ie, rapid binding and internalization of
the virus) was sought. A recently described technique of flow-through gene transfer17 appeared to offer this advantage, and we
applied it to transduction of NK-92 cells with the viral supernatants produced by CRIP cells. Transduction of 10% to 20% NK-92 cells, which
was achieved in this system, allowed for subsequent selection in
G418-containing medium, leading to the establishment of stably transduced NK-92 cells expressing the IL-2 gene.
The TR-IL-2-NK-92 cells we have consistently been able to generate are
resistant to G418, proliferate well in the absence of exogenous IL-2,
express abundant mRNA for IL-2 and IL-2 protein localized to the Golgi
zone, and secrete biologically active IL-2 into culture supernatants.
In addition, these TR-IL-2-NK-92 cells have an increased ability to
kill tumor cell targets in vitro compared with P-NK-92 cells.
Furthermore, TR-IL-2-NK-92 cells produce supernatants with antitumor
activity. These supernatants were shown to contain IFN in addition
to secreted IL-2, but little measurable TNF. The near absence of
TNF in death-inducing supernatants of transduced cells indicates
that other members of the TNF family of proteins, such as FasL, might
be responsible for tumor cell apoptosis.40,41
IL-2-activated NK cells and the transduced NK cell lines express FasL
and their supernatants contain sFasL, while HR targets express Fas
(N.L. Vujanovic and T.L. Whiteside, unpublished data, October
1997). Thus, sFasL could be responsible for apoptosis of
HR cells observed in vitro. The most important observation in these in
vitro studies is that TR-IL-2-NK-92 cells kill NK-resistant solid tumor
cell targets more efficiently than parental cells. This could
translate into a more efficient antitumor effect in vivo, because the
transduced cells are likely to survive and function in the absence of
exogenous IL-2, while the parental cells depend on the presence of
exogenous IL-2. Furthermore, the presence of considerable levels of
TNF in liver homogenates indicates that in vivo TNF might
contribute to elimination of established liver metastases.
In vivo experiments with TR-IL-2-NK-92 cells showed that adoptive
transfer of these effector cells in the absence of exogenous IL-2 was
therapeutic in an experimental metastasis model. Survival of nude mice
with 3-day established liver metastases from HR, |
Abstract
Introduction
Abstract