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1994 84: 2966-2970
Transfer of the tumor necrosis factor alpha gene into hematopoietic
progenitor cells as a model for site-specific cytokine delivery after
marrow transplantation
T Kuhr, GJ Dougherty and HG Klingemann
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Transfer of the Tumor Necrosis Factor (Y Gene Into Hematopoietic
Progenitor Cells as a Model for Site-Specific Cytokine Delivery
After Marrow Transplantation
By Thomas Kuhr, Graeme J. Dougherty, and Hans-G. Klingemann
Relapse of leukemiais the major
cause of failure after autologous stem cell transplantation due t o reinfusion of residual
clonogenic cells and the absence of an immune-mediated
graft-versus-leukemia effect. To provide an antileukemia effect, immune-activating cytokines have been given t o patients after transplantation. Systemicadministration of such
cytokines early after transplantation is often accompanied
suffiby substantial side effects, and it is unknown whether
cient concentrations reach the sites of residual disease in
the marrow. As a method of site-directed immunotherapy
provided earlyafter stem celltransplantation, we have
(1) marrow can be retrovitested in a murine model whether
rally transduced with the tumor necrosis factor a (TNFa)
gene, (2) local production of TNFa by marrow cells after
transplantation can be achieved, and (3) adverse effects of
TNFa occurred. Balb/c mice were treatedwith 5-fluorouracil
and bone marrow (BM) was obtained 4 days later. Whole
BM was transduced in the presence of interleukin-3 (IL-31,
IL-6, and stem cell factor by coculture with the packaging
cell line GP+E-86, producing thecDNA for TNFa. Irradiated
(1,300 cGy) syngeneic recipient mice were given I O 6 trans-
duced BM cells on day 0. Integration of theTNFa gene into
the host genome was documented by Southern blotting in
spleen and BMcells on days 7 and 12 and in BM onday 40
after marrow infusion, but was no longer found on day 90.
Messenger RNA for TNFa was present on day 12, but could
no longer be shown on day 40 or 90. Although no measurable (L929 bioassay) levels of TNFa were found in serum
of mice who had received TNFa transduced marrow, the
supernatant of 10’ unstimulated BM cells obtained 12 days
after marrow infusionwas found t o have 7 pg of TNFa compared with 1.8 p g in nontransduced marrow. Mice that had
received TNFa transduced marrow showed no side effects
suggestive of systemic TNFa release, and cellularity of the
TNFa-transduced marrow was not different from control
mice that had
received unmanipulated marrow or cells transduced with the neomycin resistance gene only. The studies
suggest that gene transfer of immune-activating cytokines
into hematopoietic cells could be
used as a means t o achieve
their temporary local productionearly after transplantation
by cells located in the BM.
0 1994 by The American Society of Hematology.
P
lated to inflammation and immunomodulation.’ In addition,
both in vitro and clinical studies have shown that TNFa has
antitumor/antileukemic activity and kills target cells both
by cytolytic mechanisms and by inducing fragmentation of
DNA”O-i2 It further induces various immunologic effector
mechanisms that are able to mediate leukemic cell kill.’3,’4
Despite its documented antitumor effects, TNFa has proven
to be unsuitable for systemic administration to patients because of the short half-life of the molecule in the circulation
(about 20 to 30 minutes after injection) and the severe side
effects it produces when given at therapeutic doses.I5 Conversely, TNFa has remarkable antitumor/antileukemic effects even at much lower doses when continuously released.
Such effects include the activation of phospholipase A, the
intracellular production of hydroxyradicals, or the stimulation of secondary antitumor/antileukemic ~ e l l s . ’ ~ Thus,
~’’
there i s a rationale for the application of low doses of TNFa
that, ideally, should be released locally in the marrow where
residual leukemia may reside. Furthermore, if immunotherapy is to have any adjunct antileukemia effect after BMT,
it has to be administered early, before the malignancy would
be likely to recur.
In an attempt to deliver TNFa to BM cells without doselimiting toxicityearly after grafting, we have transduced
murine hematopoietic progenitor cells with a retroviral vector containing the cDNA for TNFa. When transplanted into
irradiated syngeneic recipients, temporary integration and
expression of the TNFa gene in repopulating cells could be
achieved. Because only local production of TNFa occurred,
no systemic side effects were observed in these mice and
marrow engraftment in the presence of TNFa production
was not delayed.
ATIENTS RECEIVING a bone
marrow
transplant
(BMT) from an allogeneic donor for the treatment of
acute or chronic leukemia have a significantly lower relapse
rate than those receiving an autologous or syngeneic transplant for the same disease.’.’ This finding appears to be
caused by (1) the infusion of residual clonogenic cells with
the marrow3 and (2) the lack of a graft-versus-leukemia
(GVL) effect that can eliminate residual malignant cells remaining
in the
There is evidence that
the alloantigen-driven immune response after allogeneic BMT involves
the clonal expansion of T lymphocytes that release, among
other cytokines, interleukin-2 (IL-2).’ This cytokine can
stimulate the production of tumor necrosis factor a (TNFa)
by monocytes, T lymphocytes and natural killer cells.” Although TNFa is suspected to be involved in the pathology
of the tissue damage seen in graft-versus-host disease, it is
also believed to contribute to the GVL effect.’.’
TNFa has a number of noncytotoxic actions that are reFrom the LeukemidBone Marrow Transplant Program of British
Columbia, Terry Fox Laboratory, Divisionof Hematology, BC Cancer Agency, Vancouver, Canada
Submitted May 2, 1994; accepted July 7, 1994.
Supported by a grant from the Leukemia Research Fund of Canada (to H.-G.K.)and Grant No. J W 5 - M E D from the Schroedinger
Foundation (to T.K.). G.J.D. is a Research Scientist of the National
Cancer Institute of Canada.
Address reprint requests to Hans-G. Klingemann, MD, The L a k e midBone Marrow Transplant Program of British Columbia, 910 W
10th Ave. Vancouver, BC, .Canada VSZ 4E3.
The publication costs of this article weredefrayed in part by page
charge payment. Thisarticle must therefore hr hereby murked
“advertisement” in accordclnce with 18 U.S.C. section 1734 solelyto
indicate this fact.
0 1994 by The American Sociery of Hematology.
0006-4971/94/8409-00I9$3.00/0
2966
MATERIALS AND METHODS
Vector construction and characterizution. A full-lengthmurine
TNFa cDNA flankedby Xba I restriction enzyme sites was generated
Blood, Val 84, No 9 (November 1). 1994: pp 2966-2970
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TNF GENE TRANSFERFOR
MARROW TRANSPLANTATION
Fig 1. JmTNFa retroviral vector. (0)
indicates long teminal repeat that contains the retrovirus
promotor and
enhancer.
(B)indicate the coding region of the
TNFa gene. I.)
Indicates the
coding region for the TK-neo
gene. For details see Materials
and Methods.
2967
JZEN .l
m
D
D
D
D
X
X
by reverse-transcription polymerase chain reaction (RT-PCR) using
RNA isolated from the murine fibrosarcoma Fsa-N (Dougherty ST,
et al; manuscript in preparation). To generate the retroviral vector
JmTNFa , the cDNA for TNFa was first inserted into the Xhu I site
of plasmid pTZI9Rtk-neo.IX A cassette containing both the TNFa
gene and the Tk-promoted neomycin resistance gene was then isolatedby Sma IiHindIlldigestionand
ligated into H p IfHindIII
cut Jzen.1 (Fig l ) . " The plasmid obtained was introduced into the
ecotropic packaging line GP+E-86 by calcium-phosphate precipitation and transfected cells selected using the neomycin analog G4 I8
(0.5 mg/mL,activeweight)(GIBCO-BRL,Grand
Island, NY).2"
Supernatants conditioned by the retroviral packaging line had active
X IO5 and 3 X 10" colony-forming
viraltitersrangingbetween2
units (CFUs)/mL when assayed for their ability to generate G418resistant NIH3T3 cells. A control virus designated Jzen-neo was also
constructed by inserting a 954-bp Mlu IIHincII fragment containing
the coding region of the neoK gene from pMCIneo into the H p 1
site of Jzen. l ." Supernatants of this construct had active viral titers
of greater than 1 X 10' CFU/mL.
Transduction ofmurine BM with the TNFa gene. BM cells were
isolated from normal male Balbic mice (8 to I 2 weeks old, Charles
River, Montreal, Quebec, Canada) that had received 5-Fluorouracil
(150 mg/kg body weight, interperitoneally) (Sigma Chemical CO,St
Louis, MO) 4 days previously. Mice were keptin a standard animal
facility and fed ad libitum in a nonprotective environment. BM was
harvested by flushing the marrow cavities of the femurs and tibiae
with a-minimalessentialmedium
(aMEM) supplementedwith2
mmol/L glutamine, penicillin/streptomycin and 2% fetal calf serum
(FCS; catalog no. SLM-4100; StemCell Technologies Inc, Vancow
4 X IO") withoutfurther
ver,BC,Canada).Marrowcells(2to
separation were cocultured with the viral producer cell line (GP +
E-86 cells) at 70% confluence in
100-mm tissue-culture dishes (catalog no.3025;Falcon,Cockeysville,MD)
in Dulbecco'smoditied
mediumsupplementedwith
10% newborn calf serum, 2 mmol/L
glutamine
and
penicillin/streptomycin
(catalog
no. SLM-2000;
StemCellTechnologies) and then irradiatedwith 1,500 cGy. To
increase transfectionefficiency, recombinant murinesteel factor ( I 00
ng/mL)(provided by DrWidmer[Immunex Corp, Seattle,WA]),
L 3 (20 ng/mL), IL-6 (15 ng/mL) (both cytokines provided by the
Terry Fox Laboratory, Vancouver, BC), and 8 g/mL of polybrene
36 hours of coculture, nonadherent
were added to the cultures. After
cellswereremoved, washed twice with phosphate-bufferedsaline
(PBS), and resuspended at I d cells 10.5 mL for transplantation into
irradiated recipients.
Transduction efficiency in the nonadherent cells was determined
after plating IO4 Jzen-neo or JmTNFa-infected viable cells
in culture
dishes(GreinerGmbH,Frickenhausen,Germany)containing
Iscove's methylcellulose (catalog no. HCC-4100; StemCell Technol30% FCS,
M2-mercaptoethanol
and
ogies)
supplemented
with
l mmol/L L-glutamine supplemented either with or without G418
(neomycin sulfate) (Geneticin, GIBCO-BRL) at 1.2 mg/mL (active
weight). This concentration of G418 completely suppressed growth
of nontransfectedhematopoieticcells.Cultureswereincubatedat
37°C in 5% CO, for 12 to 14 days and colonies (>S0 cells) were
I
l.lkb
I
1.2kb
l
counted under an inverted microscope (Nikon Canada Instrurnenls
Inc, Mississauga, Ontario, Canada). Transduction efticiencywas calculated according to the formula:
% Efficiency
-
No. of Colonies Growing in the Presence of G418
No. of Colonies Growing in the Absence of G418
Transplantation protocol and docwnentutionof enCqrafiment. Recipient Balb/c mice were irradiated with 1,300 cGyof total body
radiation given by a Cesium source overnight ( I .45 cGy/min). This
dose of irradiation has proven to be fatal to 100% of mice by day
15 if no marrow support is given. Syngeneic marrow cells(It)'' cells/
mouse) were injected into the tail vein of recipient mice. On days
12 and 40 after transplant, recipient mice were
killed by cervical
dislocationand BM cellswereharvestedfrom
all fourlimbs by
flushing with PBS. Spleen cellson day 12 were obtainedby stripping
cells free from individual mouse spleens
in aMEM supplemented
with 10%FCS. Before counting nucleated cells,red blood cells were
lysed with 3% acetic acid and remaining cells stained
with trypan
blue.
Southern blot analysis. Genomic DNA was isolated from spleen
coloniesondays7and
12 andBMondays
7, 12, 40, and90
after marrow injection by enzymatic digestion followed by phenol/
chloroformextractionandethanol
precipitation." Resuspended
DNAwasdigestedtocompletion
with Xbu 1 to excise the entire
0.75-kb TNFa cDNA. The digested DNA was separated in a 1 %
agarose gel, and the fragments were transferred to a nylon membrane
andhybridizedtoa
"P-labeled TNFacDNAprobe.Filterswere
imaged by autoradiogl-aphy at -70°C usingKodak XAR-S film
(Eastman-Kodak, Rochester, NY).
Norrhern /dol anul.y.sis. For documentationofgeneexpression,
total RNA was isolated from spleen and marrow cells using the acid
guanidinium-isothiocyanate phenol-chloroformsingle-stepextraction method as describedpreviously." Samples prepared from spleen
cells on day I2 and marrow cells on days 12,40, and 90 were probed
with the murine TNFa cDNA. A full-length murine &actin cDNA
probe was used asacontrol.Probeswereoligolaheledwith
[12P]
deoxycytidine triphosphate (dCTP), hybridized, and imaged by autoradiography as described above.
Determinalion of TNFaactivily by LY2Y assuy. Bioactivityof
TNFa in serum and supernatants of cultured marrow cells was dctermined based on the lysis of the transformed mouse fibroblast cell
line L929 (American Type Culture Collection, Rockville, MD)
as
described p r e v i ~ u s l ySerum
. ~ ~ ( I mL) from mice that had received
Jzen-neo or JmTNFa-transduced marrow was concentrated
threefold
usingaCentricon I O Microconcentrator(Amicon,Beverly, MA).
BM cells (IO") from the same mice were culturedin aMEM medium
for 24 hours and the supernatant
was also concentrated through a
Centricon filter. For the L929 assay, target cells were incubated with
the test samples in flat-bottom microtiter wells (Costar, Cambridge,
MA). After 18 hours of incubation, L929 cells remaining intact were
stained with crystal violet and dye uptake was measured using an
ewyme-linked immunosorbent assay microplate reader (Microplate
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
2968
KUHR,DOUGHERTY, AND KLINGEMANN
E1309; BIO-TEKInstruments, Winooski, VT). A dose-response
curve using a recombinant murine TNFa standard (Genentech Inc.
South San Francisco, CA) was included in each assay. TNFa bioreactivity was inversely related to the amountof staining, which represented viable nonlysed cells.
cy
c
Q)
Q,
= 5
v)
RESULTS
Transduction efJiciency of transplanted marrow. Equivalent numbers of nontransfected, Jzen-neo or JmTNFa-infected marrow cells were assayed for hematopoietic progenitor cell content in semisolid media in the presence of G418.
Transduction efficiency was 30% for Jzen-neo and 10% for
JmTNFa. This difference in transduction efficiency was
likely caused by the consistently lower viral titer for the
JmTNFa construct.
Integration and expression of the transduced TNFa gene
in spleen and BM cells. Several groups of recipient mice
were injected with BM transduced with either the Jzen-neo
or JmTNFa retroviral vectors. On day 7, 12,40, and 90 after
transplantation, spleen and/or BM cells were isolated and
the DNA analysed by Southern blotting. Both spleen and
marrow cells obtained on day 12 after BMT showed the
0.75-kb DNA fragment that hybridized with a TNFa specific
DNA probe (Fig 2). Data from day 7 were identical, but are
not presented. The gene was also documented in BM cells
on day 40 after transplantation, but was not found on day
90 (Fig 2).
These findings were correlated with the expression of the
TNFa gene as documented by Northern blotting. TNFa
mRNAwaspresent
in spleen cells onday 7 (result not
shown) and day 12 (Fig 3). Despite the presence of the gene
on day 40 (documented by Southern blotting), no expression
of the gene in spleen or marrow cells could be documented
at that timepoint.
Bioactivity of TNFa in serum and marrow supernatant.
To determine whether the expression of TNFa mRNA on
day 12 resulted in production of bioactive TNFa production,
10' marrow cells obtained from the femur and tibia of the
mice were cultured for 24 hours in medium and the bioactivity of TNFa in the supernatant measured using the L929
assay. Results were compared with those obtained with marrow cells obtained from mice that had received marrow transduced with the neoRgene (Table l ) . In addition, serum from
recipients of transduced (neoKor TNFa) or nontransduced
marrow wasconcentrated and TNFa measured. No bioactive
TNFa could be detected in the serum of mice thatwere given
the TNFa transduced marrow. In contrast, their marrow cells
produced an amount of TNFa that was near that measured
in the supernatant of the packaging cells and significantly
higher thanthat found in control micethathad
received
Jzen-neo transduced marrow (Table I ) .
Engrqjiment of Jzen-neo- and JmTNFa-transduced
marrow. Because TNFa is believed to be myelosuppressive, the cellularity of spleen (obtained on day 12 after BMT)
and BM (obtained on day 12 and 40 after BMT) was determined by counting the number of nucleated cells from the
spleen andor from all four limbs. Results suggest that the
number of nucleated cells from the TNFa-transduced BM
early after BMT (day 12) was slightly lower than that from
D
P
m
E
Q
E
m
6.1 kb
0.75 kb
Fig 2. Southern hybridizationof cells from recipients' spleenand/
or BM obtained onday 12, 40, and 90 after transplantation of
JmTNFa-transduced BM. Southern hybridizationwas performed using a '*P-labeledTNFa cDNA probe. Beside the endogenous band for
TNFo (6.1 kb), the 0.75-kb transducedTNFa is detectable on days 12
and 40 in spleen and marrow cells, respectively.
the Jzen-neo-transduced or control mice (Table 2). However, the difference was not statistically significant. By day
40 after BMT, marrow cellularity had increased in all mice
compared with day 12, and no differences were found between the different treatment groups.
DISCUSSION
Immunotherapy is increasingly being considered as a noncross-reactive treatment modality to prevent relapse, especially after autologous marrow transplantation, where no allogeneic GVL effect occurs. Because
the
function
of
cytotoxic cells recovers early after BMT, cytokines such as
IL-2 and interferon a have been given to marrow recipients
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TNFGENETRANSFERFORMARROW
TRANSPLANTATION
2969
Table 2. Effect of TNFa Transduction on BM
and Spleen Cellularity Post-BMT
CI
a
BM Cellularity
l
Fig 3. Northern blot analysis
of total cellular RNA obtained
frommurine
spleen cells on
day 12 after transplantation of
JmTNFa- or Jzen-neo (spleencontroll-infected marrow. RNA
was extracted from spleen cells
using the acid guanidinium-isothiocyanate
phenol-chloroform
single-step extraction methodas
described in Materials and Methods. Probes were oligolabeled
with f3'Pl dCTP, hybridized, and
analyzed as described. The3.8kb JmTNFa transcript has the
expected
position of
sizethe relation
in t o 18s
28s
rRNA. A cross-hybridizing 2.4-kb
ribosomal band is seen in both
TNFa-transduced and
control
spleen cells.
( ~ 1 0 %femurshibiasl
I
5
P
I
P
5
I
Day 12
Day 40
Day 12
15.5 2 4.3
14.8 2 6
9.2 2 2.4
32 -+ 2.7
31.9 2 2.7
27 2 8.2
14.2 -c 2.4
13.1 2 3.2
21.1 2 5
"
% E ?
$-. - ~ .
.
"~
3.8 kb
2the
4 kb
and
pactin
BM control"
BM Jzen-neo transduced
BM JmTNFo transduced
.
l+-
in an attempt to stimulate those cells toward antileukemic
The rationale for the experiments presented here
is based on the observation that the systemic administration
of such cytokines can cause a number of side effects; it
is also uncertain whether high enough concentrations are
obtained locally in the marrow where residual leukemia is
located.
TNFa has not been givenafter BMT because of its known
short half-life and significant toxicities when given systemically.'' On the other hand, even low concentrations of this
cytokine have a potent antitumor effect. To overcome the
drawbacks of systemic application, we exploited the possibility of using TNFa gene transfer into hematopoietic progenitor cells as a means to deliver this antileukemic cytokine
locally to the marrow.'6 Hematopoietic progenitor cells were
Table 1. TNFa Bioactivity in Serum and Supernatants of Cedis
From the Marrow and the
Packaging Line
TNFu*
GP+E-86
(TNFa
transduced)
Serum
Control (neoRtransduced)
TNFa transduced
Supernatant-marrow
Control (neoRtransduced)
TNFa transduced
Spleen Cellularity
(xlO*/spleen)
8.9 2 1.00
ND
ND
1.78 t 1.43
7.08 2 2.18 ( P i.05)
Abbreviation: ND, not detectable.
*The bioactive concentration of TNFa was measured by L929
assay." The amount ofTNFo (in pg) was
determined in thesupernatant of lo6 marrow cells cultured for 24 hours in a 10-cm2flask in 10
mLaMEM medium. The supernatant was then concentrated threefold
using a Centricon filter. TNFa was also measured in 1 mL mouse
serum or supernatant of the packaging cell line, both of which were
also concentrated through a Centricon filter. The mean t SE is presented for results obtained from three different mice.
Numbers present the mean 2 SE of nucleated cells obtained from
three animals.
Balblc recipients were given syngeneic unmanipulated marrow
after whole body radiation(1,300 cGy).
chosen as target cells as they home to the marrow cavity
after myeloablative treatment. The entire marrow harvest
was cocultured with the retroviral vector-producing packaging cell line and then injected into irradiated syngeneic recipients. Stable integration and expression of the TNFa gene
in BM and spleen cells for at least 2 weeks after marrow
infusion was documented. At 6 weeks posttransplant, marrow cells still had the TNFa gene present, but no expression
of the gene could be documented by Northern blotting at
that time. The gene was below detectable level on day 90
after BMT. Using a bioassay, we could not detect any TNFa
in the serum of mice thathad received TNFa-transduced
marrow cells. Conversely, significant TNFa activity was
found in the supernatant of TNFa-transduced BM cells compared with cells transduced with the neoR gene.
Althoughunproven at this point, it is hopedthat these
locally produced TNFa concentrations could be sufficient
to have an antileukemic effect, either directly or through
activation of secondary effector cells. The temporary expression of the TNFa gene confined to the time early after BMT
may be desirable for the clinical situation, because immunotherapy should be delivered early after stem cell transplantation when only minimal disease is believed to be present.
Hence, no attempt was made to achieve gene expression in
early stem cells or devise strategies to maintain its expression. It is conceivable that gene integration and expression
occurs only temporarily because of the lack of any selective
pressure for maintaining this gene.
Before proceeding to any clinical studies, it was important
to document in a murine model that marrow recovery after
BMT is not delayed, and TNFa ,produced locally, does not
induce unwanted effects (such as wasting with weight loss
and fever) in recipients." These side effects seem to occur
only at higher serum concentrations of TNFa that are not
achieved with this approach. A marrow-suppressive effect
has been reported in some~x.29
but not all studies." We have
previously observed that TNFa has no negative effect on
human long-term-culture initiating cells, which represent a
cell population at the early stage of hematopoiesis (Gong
and Klingemann, unpublished observation, February 1994).
Hence, it was not unexpected to see maintained hematopoietic function in murine recipients of TNFa-transduced marrow.
Autologous hematopoietic progenitor cells could be useful
vehicles for cytokine genes whose products can mediate anti-
From www.bloodjournal.org by guest on October 15, 2014. For personal use only.
KUHR, DOUGHERTY, ANDKLINGEMANN
2970
leukemic effects, but which would cause sideeffects if given
systemically.” Inadditiontobeinganadjuncttreatment
modalitytoarrestor
eliminateresidualdiseaseafterstem
cell grafting for leukemia, this approach might also be useful
for other malignant diseases in which minimal residual disease may persist after stem cell transplantation.
ACKNOWLEDGMENT
We thank T. Jewall for typing the manuscript and L. Williams
for editing.
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