[CANCER RESEARCH 59, 1029 –1035, March 1, 1999]
Antitumor and Immunotherapeutic Effects of Activated Invasive T Lymphoma
Cells That Display Short-Term Interleukin 1a Expression1
Elena Voronov, Yacob Weinstein, Daniel Benharroch, Emanuela Cagnano, Rivka Ofir, Maria Dobkin,
Rosalyn M. White, Margot Zoller, Vivian Barak, Shraga Segal, and Ron N. Apte2
Departments of Microbiology and Immunology [E. V., Y. W., R. O., M. D., R. M. W., S. S., R. N. A.] and Pathology [D. B., E. C.], Faculty of Health Sciences, and The Cancer
Center [E. V., Y. W., R. O., M. D., R. M. W., S. S., R. N. A.], Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; The German Cancer Research Center (Deutsches
Krebsforschungscentrum), Heidelberg D-69120, Germany [M. Z.]; and Department of Oncology, Hadassah University Hospital, Jerusalem, 91120, Israel [V. B.]
ABSTRACT
Expression of cytokines in malignant cells represents a novel approach
for therapeutic treatment of tumors. Previously, we demonstrated the
immunostimulatory effectiveness of interleukin 1a (IL-1a) gene transfer
in experimental fibrosarcoma tumors. Here, we report the antitumor and
immunotherapeutic effects of short-term expression of IL-1a by malig-
nant T lymphoma cells. Activation in culture of T lymphoma cells with
lipopolysaccharide-stimulated macrophages induces the expression of IL-
1a. The short-term expression of IL-1a persists in the malignant T cells
for a few days (;3– 6 days) after termination of the in vitro activation
procedure and, thus, has the potential to stimulate antitumor immune
responses in vivo. As an experimental tumor model, we used the RO1
invasive T lymphoma cell line. Upon i.v. inoculation, these cells invade the
vertebral column and compress the spinal cord, resulting in hind leg
paralysis and death of the mice. Activated RO1 cells, induced to express
IL-1a in a short-term manner, manifested reduced tumorigenicity: ;75%
of the mice injected with activated RO1 cells remained tumor free. IL-1
was shown to be essential for the eradication of activated T lymphoma
cells because injection of activated RO1 cells together with IL-1-specific
inhibitors, i.e., the IL-1 receptor antagonist or the M 20 IL-1 inhibitor,
reversed reduced tumorigenicity patterns and led to progressive tumor
growth and death of the mice. Furthermore, activated RO1 cells could
serve as a treatment by intervening in the growth of violent RO1 cells after
tumor take. Thus, when activated RO1 cells were injected 6 or 9 days after
the inoculation of violent cells, mortality was significantly reduced. IL-1a,
in its unique membrane-associated form, in addition to its cytosolic and
secreted forms, may represent a focused adjuvant for potentiating anti-
tumor immune responses at low levels of expression, below those that are
toxic to the host. Further assessment of the immunotherapeutic potential
of short-term expression of IL-1a in activated tumor cells may allow its
improved application in the treatment of malignancies.
INTRODUCTION
A significant percentage of lymphoid tumors in humans are non-
Hodgkin’s T-cell lymphomas, which are weakly or nonimmunogenic,
highly aggressive, and frequently resistant to conventional therapeutic
approaches. Until now, only limited attempts have been made to treat
T-cell malignancies using immunotherapeutic methods; the feasibility
of recombinant cytokines and techniques of gene transfer has opened
new and promising avenues for such approaches (1–9).
Received 7/13/98; accepted 12/31/98.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
E. V. was supported by the “Gileadi and Kamea Programs” of the Israel Ministry of
Immigrant Absorption, the Chief Scientist’s Office, the Israel Ministry of Health, the
Israel Cancer Research Fund, and the Israel Cancer Association. R. N. A. was supported
by the Israel Ministry of Science jointly with the Deutsches Krebsforschungscentrum
(Heidelberg, Germany), the Chief Scientist’s Office, the Israel Ministry of Health, the
Israel Cancer Research Fund, the Israel Cancer Association, and the Israel Science
Foundation (The George and Eva Klein Fund), which was founded by the Israel Academy
of Sciences and Humanities.
2
To whom requests for reprints should be addressed, at Department of Microbiology
and Immunology, Faculty of Health Sciences, Ben-Gurion University of The Negev,
Beer-Sheva 84105, Israel. Phone: 972-7-6477-270; Fax: 972-7-6477-626; E-mail:
rapte@bgumail.bgu.ac.il.
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IL3-1 is a pleiotropic cytokine with multiple antitumor activities
that are potentially more effective than those of other cytokines used
in immunotherapy (10 –14). Thus, IL-1 activates nonadaptive immune
surveillance cells (i.e., natural killer cells and macrophages) that have
the potential to limit tumor growth before specific immunity develops,
and it also facilitates the development of antitumor specific immune
responses, primarily by affecting IL-2 secretion or the expression of
high-affinity IL-2 receptors, thus serving as a costimulatory signal for
T-cell activation (reviewed in Refs. 15 and 16). In addition, IL-1 is an
inducer of several cytokines in diverse cells with the potential to
amplify and sustain antitumor immune responses. Antitumor effects
of IL-1 have been described in experimental tumor systems and in
Phase I and Phase II clinical trials (17–24). In those trials, treatment
with IL-1 has consisted of repeated bolus injections of the cytokine,
which result in unfavorable side effects for the patients (i.e., hypo-
tension, fever, and so on), and studies were terminated before bene-
ficial antitumor effects could be observed.
We have initiated studies to demonstrate the antitumor effects of
IL-1a expression by malignant cells (1, 10, 25–29). IL-1a-transduced
fibrosarcoma cells, which constitutively express the cytokine, lose
their tumorigenicity; they either do not grow at all in mice, or they
start to grow and thereafter regress. Regression of IL-1a-positive
fibrosarcomas mainly involves the activation of T cell-mediated im-
mune responses and the development of a long-term specific immune
memory, which protects against a challenge of violent parental cells
(IL-1a negative). On the basis of IL-1a expression by fibrosarcoma
cells, we developed immunotherapeutic approaches in which small
existing tumors of violent fibrosarcoma regressed following treatment
with syngeneic cells expressing IL-1a (1, 10, 25–29).
Gene transfer approaches, although widely used to intervene in the
growth of tumors in laboratory animals (1–9), have not yet been
adequately applied in clinical trials. Induction of short-term expres-
sion of cytokines in tumor cells at critical intervals for mounting
antitumor immune responses may serve as an additional immunother-
apeutic approach. Indeed, we have previously shown that fibrosar-
coma cells that express short-term activity of IL-1a following in vitro
activation with cytokines and bacterial LPS lose their tumorigenicity
and can serve as vaccine cells in treating violent tumors (1, 10, 25, 26,
28, 29). In fibrosarcomas, similar levels of IL-1a expression, regres-
sion kinetics and immunotherapeutic effects are manifested following
expression of IL-1a in constitutive or short-term manners. It was of
interest to assess whether induction of short-term activity of IL-1a in
malignant cells is also feasible in tumors of other lineages and
whether it may be effective in reducing metastasis. In this study, we
have evaluated the antitumor effects of short-term expression of IL-1a
by activated T lymphoma cells and its possible immunotherapeutic
potential in treating metastatic T-cell malignancies.
3
The abbreviations used are: IL, interleukin; LPS, lipopolysaccharide; IL-1Ra, inter-
leukin-1 receptor antagonist; PEC, peritoneal exudate cell; MTT, 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide.
 SHORT-TERM EXPRESSION OF IL-1a REDUCES TUMORIGENICITY

MATERIALS AND METHODS olated from a standard curve consisting of recombinant IL-1a, ranging from 15
to 405 pg/ml.
Animals. BALB/c female mice were purchased from Harlan Laboratories Assessment of Tumorigenicity and Immunotherapeutic Treatments.
(Jerusalem, Israel) and were housed at the Animal Facilities of the Faculty of BALB/c mice were injected i.v., into the tail vein, with violent or activated
Health Sciences, Ben-Gurion University of the Negev (Beer-Sheva, Israel). RO1 cells (106 cells/mouse), and tumorigenicity was scored by development of
Six- to 8-week-old mice were used for experimental studies. hind legs paralysis and mortality rate. Immunotherapeutic treatment consisted
Tissue Culture Reagents. Cells were grown in RPMI 1640 containing 100 of single i.v. injections of activated cells (106 cells/mouse) at different intervals
IU/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 2-mercaptoeth- after the inoculation of violent RO1 cells. Vaccinating cells were treated with
anol (5 3 1025 M), and 5% FCS, hereafter referred to as complete medium. All mitomycin C; 5 3 106 cells/ml were exposed to the drug (100 mg/ml) for 1 h
media and supplements were purchased from Biological Industries (Beth- at 37°C and then thoroughly washed four times before use.
Haemek, Israel). Disposable tissue culture flasks and plates were purchased Inhibition of Antitumor Effects of Activated T Lymphoma Cells with
from Greiner Labortechnik (Frickenhausen, Germany). the IL-1Ra and the M 20 IL-1 Inhibitor. Mice were treated with the IL-1Ra
The IL-1Ra was kindly provided by Dr. Daniel Tracey (Upjohn, Kalama- or M 20 IL-1 inhibitor before and after the inoculation of activated T lym-
zoo, MI). The M 20 IL-1 inhibitor was purified from the M20 cell line as phoma cells. The protocols of treatment with the IL-1Ra or the M 20 IL-1
described (30, 31). Briefly, crude supernatants of serum-free cultures were inhibitor consisted of multiple inoculations of the inhibitors, and their exact
concentrated by vacuum ultrafiltration using dialysis tubing and purified on a schedules are mentioned in “Results.”
high-performance liquid chromatography-DEAE column. The conditioned me- Proliferation Assay. Violent and activated T lymphoma cells were plated
dium was also purified by molecular sieving on a Sephacryl S-300 (Pharmacia in 96-well plates at a concentration of 5 3 104 cells/ml (0.1 ml/well) in
Biotech Inc., Uppsala, Sweden) column, followed by isoelectric focusing in medium containing 1% FCS for different intervals, and thereafter, proliferation
free solution, using the Rotofor cell (Bio-Rad Laboratories, Richmond, VA). was measured by the colorimetric MTT assay (34).
Fractions were collected, dialyzed, and bioassayed. The active fractions cor- Histological Staining. Formalin-fixed and paraffin-embedded samples,
responding to a Mr ;52,000 (6 4000) and pI 4.1– 4.2 were pooled and stored from the vertebral region of mice injected with violent and activated RO1 cells,
at 270°C before use. One unit of M 20 is characterized as the amount of were cut into 4-mm-thick sections and stained with H&E.
high-performance liquid chromatography-DEAE partially purified material Statistical Analyses. Each experiment was repeated at least three to five
that causes 50% inhibition of mouse thymocyte proliferation stimulated by 1 times, unless otherwise indicated, with similar patterns of results. In vivo
unit of recombinant IL-1b, as described previously (30 –32). LPS from Esch- experiments consisted of groups of 6 –10 mice. The results shown are from
erichia coli 055:B5 and mitomycin C were purchased from Sigma Chemical individual experiments. ELISAs were performed in triplicates, and the results
Co. (St. Louis, MO). Fluid thioglycollate medium was purchased from Difco represent the means. Triplicate values did not differ from the mean by .20%.
Laboratories (Detroit, MI).
T Lymphoma Cell Lines. The RO1 and RO2J T lymphoma cell lines were
RESULTS
derived from tumors recovered from the thymus of adult mice that were Expression of IL-1a in Activated T Lymphoma Cells. None of
neonatally infected with the murine Moloney leukemia virus. The establish-
the T lymphoma cell lines screened expressed constitutive activity of
ment and characteristics of these cell lines were described previously (33). The
IL-1a. Direct activation of T lymphoma cells with T-cell mitogens,
chemically induced EL4 cell line was obtained from the American Tissue
Culture Collection (Manassas, VA). Cells were incubated at 37°C in an
such as phytohemagglutinin, concanavalin A, and phorbol 12-myris-
atmosphere of 5% CO2-95% air. tate 13-acetate, also did not result in the expression of IL-1a (results
Activation of T Lymphoma Cells. The optimal conditions for inducing not shown).
IL-1a activity in T lymphoma cells consist of activation of malignant cells in Significant levels of IL-1a were induced following activation of the
culture with macrophages (adherent PECs) and LPS. PECs were harvested on malignant T cells in culture with macrophages and LPS. Optimal
day 3 following thioglycollate injection, thoroughly washed, suspended in levels of IL-1a were observed in mixed cultures of T lymphoma cells
complete medium, and cultured (at 104 cells/ml) for 2 h to allow cell adherence (106 cells/ml) and adherent PECs (104 cells/ml) that were cultured for
to the tissue culture Petri dishes. Thereafter, nonadherent cells were removed 24 h in the presence of LPS (1 mg/ml). IL-1a activity was assessed in
by vigorous washings and T lymphoma cells (at 106 cells/ml) and LPS (1 nonadherent lymphoma cells that were separated from macrophages
mg/ml) were added to the adherent cells. At indicated intervals, nonadherent and recultured in plain medium for an additional 24 h, as described in
cells were collected and separated from adherent cells by two cycles of the “Materials and Methods.” In Fig. 1, the patterns of IL-1a induc-
adherence on tissue culture Petri dishes at 37°C (2 h each), followed by
tion are shown in the RO1 T lymphoma cell line. In activated RO1
collection of nonadherent T lymphoma cells.
cells, IL-1a is expressed at similar levels in cell lysates (Fig. 1A) and
Cytokine activity was assessed in supernatants and cell lysates. Superna-
tants from activated T lymphoma cells were obtained following reculture of
supernatants (Fig. 1B). Untreated T lymphoma cells and lymphoma
nonadherent T lymphoma cells. Thus, T lymphoma cells were recultured (at cells stimulated with either LPS or macrophages alone generated only
10 cells/ml) in complete medium for various intervals; the medium was low background levels of IL-1a. Fig. 1A also shows IL-1a levels in
6
4
collected and centrifuged; and supernatants were collected, filtered through macrophage controls, 10 cells/ml fresh LPS-stimulated macrophages,
0.45-mm syringe filters (Corning Glass Works, Corning, NY), and kept at and the adherent fraction obtained after separation of nonadherent
220°C before assay. For the preparation of lysates, cells were collected, RO1 cells in the activation cultures. Thus, macrophages, at the same
thoroughly washed three times in PBS, counted, and resuspended (at 106 concentration as that added to T cells, generate only small amounts of
cells/ml) and thereafter lysed by three freeze-thaw cycles at 270°C. Lysates IL-1a (;30 –50 pg/ml). From these results, it can be concluded that
were cleared from cell debris by centrifugation, and supernatants were col- the IL-1a activity detected in our experiments is of T cell lymphoma
lected, filtered as described, and kept at 220°C before assay. origin. The presence of contaminating macrophages in the nonadher-
IL-1a ELISA. Murine IL-1a ELISAs were performed using commercial ent fraction of activated cells was excluded by negative staining with
pairs of antibodies and recombinant cytokine (Genzyme Immunodiagnostics,
anti-Mac-1 antibodies (results not shown).
Cambridge, MA). The following antibodies were used: a primary anti-IL-1a
Induction of IL-1a expression could also be demonstrated in other
capture monoclonal antibody (1 mg/ml; code 1837-01), which recognizes the
IL-1a precursor and secreted and membrane-associated forms of natural malignant T cell lines using the same activation protocol. Table 1
mouse IL-1a; biotinylated anti-IL-1a polyclonal antibodies (1:1000 dilution; demonstrates the IL-1a content in lysates of two additional T cell
code 1P-110); and for detection, peroxidase-conjugated affinity pure donkey lymphoma lines, EL4 and RO2j, which were generated and assayed as
antirabbit IgG polyclonal antibodies and their substrate, o-phenylenediamine described above (Fig. 1). In general, activated T lymphoma cells
dihydrochloride (Sigma). The ELISA procedure was performed according to generate IL-1a in amounts ranging from 150 to 500 pg/ml by 10
6

the manufacturer’s instructions. The amount of IL-1a in samples was extrap- malignant cells in 24 h.
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 SHORT-TERM EXPRESSION OF IL-1a REDUCES TUMORIGENICITY

of IL-1a in activated T lymphoma cells were retained in cell lysates

after 48 and 96 h, respectively. In supernatants, high levels of IL-1a
are detected for longer periods, up to 120 h following activation. The
kinetics of IL-1a expression in supernatants and cell lysates of acti-
vated T cells, compared to cell viability, indicate that, at early inter-
vals (; up to 48 h), IL-1a levels in supernatants reflect active
secretion, whereas at later intervals they may also represent IL-1a
released from disintegrating tumor cells. Mitomycin C-treated acti-
vated RO1 cells retain their ability to express IL-1a expression for a
few days, similarly to untreated activated cells (Fig. 2B). This is of
importance because only inactivated cells can be used in tumor
vaccines.
Effects of IL-1a Expression by Malignant T Cells on Prolifer-
ation. The in vitro proliferation patterns of activated RO1 cells were
compared to those of violent cells. RO1 were activated in culture with
macrophages and LPS, according to the protocol demonstrated in Fig.
1, thoroughly washed, and thereafter recultured in plain medium, and
their proliferative capacity was assessed by the MTT colorimetric
assay. The proliferation of RO1 cells (5 3 104 cells/ml) was assessed
in medium containing 1% FCS, to assess whether IL-1a expression
provides some “proliferative advantage” to cells cultured at limiting
conditions. As shown in Fig. 3, violent and activated RO1 cells
manifested similar proliferative rates.
Effects of IL-1a Expression by Malignant T Cells on Tumori-
genicity Patterns. The effects of short-term IL-1a expression on
tumorigenicity of the malignant cells were studied in the RO1 exper-

Fig. 1. Induction of IL-1a expression in T lymphoma cells. RO1 cells were incubated
alone (LC), with LPS (LC1LPS), with macrophages (LC1MF), or with macrophages and
LPS (LC1MF1LPS) for 24 h, as described. IL-1a expression in lysates (A), prepared by
freeze-thawing cells, and in supernatants (B) of purified nonadherent lymphoma cells was
assessed by ELISA. Also shown in A are IL-1a levels generated by fresh LPS-stimulated
peritoneal macrophages (MF1LPS) and in lysates of residual adherent cells recovered
from the activation culture, after removal of nonadherent RO1 cells (Adherent cells).
Columns, IL-1a levels obtained from one activation experiment, expressed as means of
ELISA triplicates.

Table 1 IL-1a expression upon activation in various T lymphoma cell lines

Cells IL-1a (pg/ml)
RO1 lymphoma 0
Activated RO1 lymphoma 380 6 40
EL4 lymphoma 0
Activated EL4 lymphoma 230 6 34
RO2J lymphoma 0
Activated RO2J lymphoma 140 6 7

T lymphoma cells stimulated in culture with macrophages and LPS

to express IL-1a in a short-term manner will be hereafter referred to
as activated T lymphoma cells. All subsequent experiments described
herein were performed with the RO1 T cell lymphoma line.
Duration of IL-1a Expression in Activated T Lymphoma Cells.
To use activated T lymphoma cells for in vivo immunotherapeutic
purposes, we found that it was, thus, essential to assess whether IL-1a
activity in malignant T cells persists for some time after removal of
the activators from the culture. Thus, RO1 cells were stimulated in
culture with macrophages and LPS for 24 h, as indicated above. Then
nonadherent activated RO1 cells were separated from macrophages, Fig. 2. Duration of expression of IL-1a in activated T lymphoma cells. RO1 cells were
extensively washed to remove residual activators, and recultured in activated with macrophages and LPS for 24 h (act LC), as described in the Fig. 1 legend.
Purified nonadherent RO1 cells were separated and recultured in plain medium (without
plain tissue-culture medium (without the activators). At various inter- the inducers), and at different intervals, supernatants and lysates were prepared for IL-1a
vals, supernatants were collected and lysates from samples of 106 assay. Data points (A), IL-1a levels obtained from one activation experiment, expressed
cells/ml were prepared and assayed for IL-1a content. As can be seen as means of ELISA triplicates. IL-1a levels were compared to the viability of the cells,
which was assessed by trypan blue vital staining. In B, in a single activation experiment,
in Fig. 2A, IL-1a expression in activated RO1 cells persists for a few the duration of IL-1a expression in activated RO1 cells was compared to that of activated
days in supernatants and lysates; 75% and 50% of the original levels and mitomycin C-treated RO1 cells.
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 SHORT-TERM EXPRESSION OF IL-1a REDUCES TUMORIGENICITY
Fig. 3. Proliferation rate of activated and violent T lymphoma cells. Untreated (violent)
RO1 T lymphoma cells (LC) and 24-h activated cells (act LC), according to the protocol
described in the Fig. 1 legend, were recultured in plain medium containing 1% FCS.
Proliferation was assessed at different intervals by the MTT colorimetric assay. Data
points, means of triplicates obtained from one representative proliferation experiment.
imental tumor model. RO1 cells manifest exceptionally high patterns
of malignancy upon i.v. inoculation. The tumor cells disseminate into
the vertebral column, resulting in compression of the spinal cord and
in paralysis of the hind legs, followed by death of 100% of the injected
mice (within 4 –7 weeks). Lymphoid organs and the liver are also
affected. The invasion of violent lymphoma cells into the vertebral
column is demonstrated in Fig. 4. Sections from the vertebral column
of mice injected with violent cells are shown in Fig. 4, A and B, 30
days after tumor cell inoculation and illustrate invasiveness of lym-
phoma cells to the spinal cord (Fig. 4A) and peripheral nerves (Fig.
4B). The cervical region of mice injected with activated T lymphoma
cells, 60 days after injection of the malignant cells, demonstrated
normal histological features (Fig. 4C).
The tumorigenicity of activated RO1 cells injected i.v. is depicted
in Fig. 5. Survival rates of mice injected with activated and violent
RO1 cells in a single experiment are shown. One hundred % of the
mice injected with violent cells died within 25 days, whereas 75% of
the mice injected with activated cells remained alive after 3 months.
Similar patterns of tumorigenicity were observed in 10 additional
experiments with groups of 6 –10 mice. In each experiment, 50 –100%
of the mice injected with activated RO1 cells survived 3– 6 months,
until the termination of the experiment, whereas each mouse that was
injected with cells not expressing IL-1a died.
Similar retardation of tumor growth and increase in survival rate
were observed with activated cells of other T lymphoma cell lines
(results not shown).
Recombinant IL-1a at the range of concentrations expressed by
activated RO1 cells and even at much higher concentrations (in the
range of nanograms), when applied as a single treatment or in multiple
injections did not impair the tumorigenicity patterns of violent RO1
cells (results not shown). This points to the immunotherapeutic sig-
nificance of tumor cell-associated IL-1a in activated RO1 cells.
Systemic Inhibition of IL-1 Activity Reverses the Reduced Tu-
morigenicity Patterns of Activated T Lymphoma Cells. We next
assessed whether IL-1, expressed in a short-term manner in activated
RO1 cells, is the moiety responsible for reduced tumorigenicity. IL-1
activity was reduced by using either the IL-1Ra or the M 20 IL-1
inhibitor. The IL-1Ra was injected on days 25, 23, and 21; before
tumor cell inoculation; on the day of tumor cell inoculation; and
subsequently, on days 1, 4, 10, and 19 (i.p. injections; 125 mg/0.1 ml
per injection). The protocol of treatment with the M 20 IL-1 inhibitor
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consisted of one i.v. injection, one day after tumor cell inoculation,
followed by biweekly i.p. inoculations of 2 units of M 20 per mouse.
As seen in Fig. 6, in the group of mice injected with activated cells
alone, a 75% rate of survival was recorded after 105 days (termination
of the experiment), whereas 100% of the mice injected with untreated
(violent) RO1 cells died within 25 days. Treatment of mice with M 20
IL-1-inhibitor completely abrogated the reduced tumorigenicity pat-
terns of activated RO1 cells and 100% of the mice died within 30
days, whereas only 25% of the mice injected with activated RO1 cells
together with the IL-1Ra survived after 105 days (Fig. 6). The use of
IL-1-specific inhibitors did not alter the tumorigenicity patterns of
violent cells, possibly ruling out the involvement of nonspecific
metabolic effects induced by the inhibitors in the malignant cells
(results not shown). It is noteworthy that treatments with the IL-1Ra
were performed only until day 19 after the inoculation of the malig-
nant cells, whereas the M 20 IL-1 inhibitor was applied throughout the
experiment. This may explain why the M 20 IL-1 inhibitor exerted
more pronounced effects on tumorigenicity. Our results on the degree
of in vivo neutralization of IL-1-mediated effects with the IL-1Ra or
the M 20 inhibitor are comparable to those obtained with these agents
in other biological systems (13, 30 –32). These results suggest a
dominant role of IL-1 in determining reduced tumorigenicity of acti-
vated RO1 cells.
Effects of Activated T Lymphoma Cells Given to Mice with
Established Tumors. The ability of activated T lymphoma cells to
intervene in the growth of violent RO1 cells after tumor establishment
was assessed. Mice were inoculated i.v. with violent RO1 cells, and at
different intervals thereafter, activated cells (treated with mitomycin
C) were inoculated i.v. All mice injected with violent RO1 cells died
within 60 days (Fig. 7A). As can be seen in Fig. 7B, inoculation of
activated cells at critical intervals (day 6 or 9) improved the survival
rate of the mice. Thus, in this experiment, all mice injected only with
violent cells died within 30 days, whereas 30% and 66% of the mice
treated with activated cells on days 6 and 9, respectively, survived for
3 months (termination of the experiment). In contrast, mice treated on
day 14 did not manifest reduced tumorigenicity patterns; they dis-
played the patterns of violent cells (Fig. 7B). When using IL-1a-
expressing cells in various experimental tumor systems, we always
characterize a specific “therapeutic window” in which treatment is
efficient (usually between days 5 and 12). However, the peak day for
achieving maximal immunotherapeutic effects may slightly vary
among experiments.
DISCUSSION
We have shown here that short-term expression of IL-1a by acti-
vated malignant T cells endows reduced tumorigenicity patterns. In
addition, we have demonstrated that tumor cells expressing IL-1a can
serve as a treatment when they are used to intervene in the growth of
violent metastatic T-cell lymphomas. IL-1a activity was optimally
induced by activating malignant T cells in culture with macrophages
in the presence of LPS, in agreement to other reports on induction of
IL-1a in normal and malignant T cells following stimulation with
mitogens or antigens in the context of antigen-presenting cells (35–
37). IL-1a generated by T lymphoma cells was detected in cell
lysates, on the membrane of activated T lymphoma cells as well as in
its secreted form. The mechanism of induction of short-term activity
of IL-1a in T lymphoma cells is still under investigation. Activated T
lymphoma cells manifest similar proliferative patterns to those of the
violent cells, even in “limiting conditions,” i.e., in low concentrations
of serum in which IL-1a or cytokines induced by IL-1a may poten-
tiate the growth of the cells, thus excluding a possible autocrine role
of endogenous IL-1a in the proliferation of RO1 malignant T cells.
 SHORT-TERM EXPRESSION OF IL-1a REDUCES TUMORIGENICITY

Fig. 4. Dissemination of RO1 cells into the ver-

tebral column. Violent and activated RO1 cells
were injected i.v. (106 cells/mice), and after 30 and
60 days, respectively, mice were sacrificed and
sections from the cervical vertebral region were
microscopically analyzed. A, invasion of violent
RO1 cells into the vertebral column. Shown is the
cervical vertebral bone. Neoplastic cells have in-
vaded the spinal cord. B, compression of the spinal
nerves and adjacent muscles by invading violent
RO1 cells. C, unaltered morphology of the vertebral
region of mice injected with activated RO1 cells.
Shown is a peripheral nerve and striated muscle.
Unaltered morphology of the spinal cord is demon-
strated (top left).

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 SHORT-TERM EXPRESSION OF IL-1a REDUCES TUMORIGENICITY

The antitumor effects observed in this study possibly stem from

IL-1a, generated by activated T lymphoma cells, because systemic
neutralization by IL-1-specific inhibitors reversed the tumorigenicity
patterns of activated T lymphoma cells. The M 20 IL-1 inhibitor was
shown to specifically abrogate IL-1-dependent effects in various in
vitro and in vivo biological systems, i.e., T-cell proliferation, inflam-
mation in rheumatoid arthritis, and the proliferation of chronic my-
elogenous leukemia, in an as yet unknown mechanism (30 –32),
whereas the IL-1Ra binds to IL-1 receptors, without transmitting
activation signals, and thus serves as a blocking agent that protects
cells from external IL-1 (reviewed in Ref. 13).
Tumor cell-derived IL-1a possibly serves as an adjuvant to increase
the immunogenicity of malignant T cells and to potentiate specific
antitumor immune responses. Cell-mediated immune responses, i.e.,
development of cytotoxic cells and the secretion of TH1 cytokines, are
involved in reduced tumorigenicity patterns of activated RO1 cells
(results not shown). In addition, an immune memory is established
following the rejection of activated cells expressing IL-1a. Thus, 75%

Fig. 7. Immunotherapeutic effects of activated T lymphoma cells on the development

Fig. 5. Mortality rate of mice injected with activated T lymphoma cells. Violent (LC)
and activated (act LC) RO1 cells, according to the protocol described in the Fig. 1 legend,
were injected i.v. into mice, as described in Legend to Fig. 4, and mortality was scored.
The results represent mortality rates obtained in one experiment; each group consisted of
10 mice. The differences in survival of mice injected with activated RO1 cells compared
to violent cells were statistically significant (P , 0.01, ANOVA test; the value given for
survived mice was 125, the day of termination of the experiment).
Fig. 6. Effects of the IL-1Ra and M 20 IL-1 inhibitor on the tumorigenicity of activated
T lymphoma cells. Mice were injected i.v. with activated RO1 cells (act LC), as described
in the Fig. 1 and 4 legends, and concomitantly treated with the IL-1Ra or the M 20 IL-1
inhibitor, by multiple injections in protocols described in the text. Mortality was scored in
the various experimental groups. The differences in survival of mice injected with
activated RO1 cells compared to those treated with the M 20 IL-1 or the IL-1Ra violent
cells were statistically significant (P , 0.01, ANOVA test; the value given for survived
mice was 125, the day of termination of the experiment).
1034
of violent tumors. Mice were injected with violent RO1 cells (LC), as described in the Fig.
4 legend. On days 6, 9, and 14, mice were treated with autologous activated RO1 cells
(treated with mitomycin C), as described in the Fig. 1 legend. Mortality was scored in the
various experimental groups. The results represent mortality rates obtained in one exper-
iment; each group consisted of eight mice. The differences in survival of mice injected
with violent RO1 cells versus mice also treated with activated cells on days 6 and 9 were
statistically significant (P , 0.01, ANOVA test; the value given for survived mice was 75,
the day of termination of the experiment).
of the mice preimmunized with activated RO1 cells (treated with
mitomycin C) are protected from a challenge with violent cells,
whereas no protection was observed with violent cells (results not
shown). Similarly, a significant proportion of mice that had survived
following injection with activated RO1 cells develop an immune
memory, which protects them from a challenge with violent RO1
cells. The nature of immune mechanisms that control reduced tumor-
igenicity patterns of activated RO1 cells is currently under investiga-
tion and will be described elsewhere.
The effects of IL-1a may be direct, due to its pleiotropic
antitumor effects, or indirect, through its ability to induce the
generation of a cytokine cascade by the malignant cells in their
microenvironment or in the draining lymph nodes. It is noteworthy
that, in supernatants and cell lysates of activated T lymphoma
cells, in which IL-1a is abundant, we could not detect “classical”
T cell cytokines, such as IL-2, IL-4, IL-10, or IFN-g. Thus, we
assume that tumor cell-derived IL-1a possibly initiates antitumor
T cell-mediated immunity in the host. As indicated, IL-1 is a
unique cytokine with significant potential as an antitumor immu-
notherapeutic agent (10 –15). The unique subcellular compartmen-
talization of IL-1a in tumor cells is important for exerting its
efficient antitumor effects and differs from IL-1b and most cyto-
kines, which are active only in their secreted form. Thus, the
membrane-associated form of IL-1a (Mr 23,000) may serve as an
adhesion molecule, allowing efficient cell-to-cell interactions be-

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Research.
 SHORT-TERM EXPRESSION OF IL-1a REDUCES TUMORIGENICITY
tween the malignant and immune effector cells, and as a focused
cytokine with strong adjuvant activity. In addition, the active
cytosolic form of IL-1a (Mr 31,000) may be released from disin-
tegrating tumor cells and may be present in the tumor’s microen-
vironment during tumor eradication by the immune system. The
expression of active IL-1a in multiple compartments may allow
efficient activation of the immune system at levels below those that
are toxic to the host. Indeed, relatively moderate amounts of IL-1a
generated by activated malignant T cells, i.e., ;150 –500 pg per
106 cells over 24 h, significantly reduces their tumorigenicity, as
we showed previously in fibrosarcoma experimental tumors (1, 10,
25–29). Short-term expression of IL-1a persists in culture (Fig. 2)
and possibly also in vivo for a few days; this is sufficient for
stimulation of antitumor immune responses, as was also shown in
our studies on fibrosarcoma experimental tumor systems, in which
malignant cells expressing IL-1a in a short-term manner regress
due to the development of T cell-mediated immune responses
against the malignant cells (1, 10, 25–29).
We showed previously that IL-1a-positive fibrosarcoma cells can
serve as a treatment by intervening in the growth of existing violent
tumors. Successful immunotherapeutical intervention in the growth of
violent tumors, with IL-1a-positive fibrosarcoma cells, occurs at
certain critical intervals after tumor cell inoculation, usually 6 –12
days after the injection of the malignant cells. At that critical time, an
inefficient antitumor immune response develops, which is potentiated
by IL-1a, resulting in tumor regression. At later intervals, the tumor’s
mass is possibly too large for intervention. A similar immunothera-
peutic window was characterized here for the treatment of mice
previously inoculated with violent RO1 cells (Fig. 7). The immuno-
therapeutic manipulation described here is minimal (a single treat-
ment) and its efficiency (66% rate of survival for treatment on day 9)
can possibly be improved by multiple vaccinations or in combination
with other debulking treatments. The effects of tumor cell-associated
IL-1a, that are described here, are remarkable, especially considering
that T lymphomas represent disseminated metastatic tumors. The
antimetastatic effectiveness of short-term expression of IL-1a by T
lymphoma cells, is comparable and, in some cases, even better than
effects observed with constitutive expression of IL-1a or other cyto-
kines by tumor cells (gene transfer; Refs. 1–9 and 25–29). Short-term
expression of cytokines in tumor cell vaccines may represent a novel
immunotherapeutic modality of treatment, which may supplement the
current cytokine gene transfer approaches and may facilitate the better
use of cell-associated IL-1a in tumor cell vaccines.
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Antitumor and Immunotherapeutic Effects of Activated Invasive
T Lymphoma Cells That Display Short-Term Interleukin 1 α
Expression
Elena Voronov, Yacob Weinstein, Daniel Benharroch, et al.

Cancer Res 1999;59:1029-1035.

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