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Originally published online as doi:10.1189/jlb.0905509 on May 17, 2006

Published online before print May 17, 2006
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(Journal of Leukocyte Biology. 2006;80:96-106.)
© 2006 by Society for Leukocyte Biology

Immune phenomena involved in the in vivo regression of fibrosarcoma cells expressing cell-associated IL-1{alpha}

Tatyana Dvorkin*, Xiaoping Song*,1, Shmuel Argov{dagger}, Rosalyn M. White*, Margot Zoller{ddagger}, Shraga Segal*,2, Charles A. Dinarello§, Elena Voronov* and Ron N. Apte*,3

* Departments of Microbiology and Immunology and
{dagger} Pathology, Faculty of Health Sciences and the Cancer Research Center, Ben-Gurion University of the Negev, Beer Sheva, Israel;
{ddagger} Department of Tumor Progression and Tumor Defense, German Cancer Research Center, Heidelberg; and
§ University of Colorado Health Sciences Center, Denver

3 Correspondence: Department of Microbiology and Immunology, Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. E-mail: rapte{at}bgumail.bgu.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Constitutive expression of cell-associated, but not secreted, interleukin-1{alpha} (IL-1{alpha}) by oncogene-transformed fibrosarcoma cells induced regressing tumors in mice, a phenomenon that was abrogated by the IL-1 inhibitor, the IL-1 receptor antagonist (IL-1Ra). On the contrary, non-IL-1{alpha}-expressing tumor cells induce progressive tumors in mice. In vivo and ex vivo experiments have shown that regression of IL-1{alpha}-positive fibrosarcoma cells depends on CD8+ T cells, which can also be activated in CD4+ T cell-depleted mice, with some contribution of natural killer cells. In spleens of mice bearing the non-IL-1{alpha}-expressing fibrosarcoma cells, some early and transient manifestations of antitumor-specific immunity, such as activation of specific proliferating T cells, are evident; however, no development of cytolytic T lymphocytes or other antitumor protective cells could be detected. In spleens of mice bearing the non-IL-1{alpha}-expressing fibrosarcoma cells, the development of early tumor-mediated suppression was observed, and in spleens of mice injected with IL-1{alpha}-positive fibrosarcoma cells, protective immunity developed in parallel to tumor regression. Treatment of mice bearing violent fibrosarcoma tumors with syngeneic-inactivated, IL-1{alpha}-positive fibrosarcoma cells, at a critical interval after injection of the malignant cells (Days 5–12), induced tumor regression, possibly by potentiating and amplifying transient antitumor cell immune responses or by ablation of tumor-mediated suppression. Membrane-associated IL-1{alpha} may thus serve as an adhesion molecule, which allows efficient cell-to-cell interactions between the malignant and immune effector cells that bear IL-1Rs and function as a focused cytokine with adjuvant activities at nontoxic, low levels of expression. Our results also point to the potential of using antitumor immunotherapeutic approaches using cell-associated IL-1{alpha}.

Key Words: interleukin-1 • protective immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin-1 (IL-1) represents one of the most pleiotropic, proinflammatory, and immunoregulatory cytokines acting by itself, but it is more important that it induces the generation of a wide variety of cytokines and the expression of adhesion molecules on diverse cells, which amplify and sustain its responses (reviewed in refs. [1 2 3 4 5 6 7 ]). The most studied members of the IL-1 family include two agonistic proteins, namely, IL-1{alpha} and IL-1ß, and one antagonistic protein, the IL-1 receptor antagonist (IL-1Ra), which binds to IL-1Rs without transmitting an activation signal and represents a physiological inhibitor of preformed IL-1. In their recombinant form, IL-1{alpha} and IL-1ß bind to the same receptors and exert the same biological activities. However, in their in vivo native milieu, within the producing cell or its microenvironment, IL-1{alpha} and IL-1ß differ dramatically in the subcellular compartments in which they are active [1 2 3 4 5 6 7 ]. IL-1ß is active only in its secreted form (17.5 kD), whereas its cytosolic precursor is inactive. IL-1{alpha} is mainly active as an intracellular precursor (31 kD) or as a membrane-associated form (23 kD) but is only marginally active in the mature form (17.5 kD), as it is secreted mainly by activated macrophages and other cells express cell-associated IL-1{alpha}. Most of the data about the in vivo and in vitro effects of the IL-1 molecules have been deduced from the use of recombinant IL-1 molecules; however, the differential effects of the IL-1 molecules in their native milieu have not been studied thoroughly.

IL-1 is present at tumor sites; it is produced by stromal/immune cells or by the malignant cells in response to local inflammatory stimuli. Aberrant secretion of IL-1 by tumor cells has been described in experimental and human tumors; this has usually been associated with increased invasiveness and a bad prognosis (reviewed in refs. [1 , 7 ]). Secreted IL-1{alpha} and IL-1ß diffuse into the microenvironment and activate a cascade of proinflammatory cytokines and mediators that potentiate tumor invasiveness and angiogenesis, tumor cell dissemination, and metastasis (reviewed in refs. [1 , 7 , 8 ]), similar to recombinant IL-1{alpha} and IL-1ß. As indicated above, both IL-1 molecules bind to the same receptors and exert similar effects on inflammtory responses. However, many cells, except macrophages/monocytes, do not secrete IL-1{alpha} but rather express the cell-associated cytokine. For example, we have shown the biological functions of IL-1{alpha} in normal and transformed fibroblasts, which express cell-associated (membrane and cytosolic-associated cytokine) and not secreted IL-1{alpha} [9 , 10 ]. The role of nonsecretable tumor cell-associated IL-1{alpha} in tumor-host interactions has not been studied thoroughly.

Previously, we have demonstrated that expression of cell-associated IL-1{alpha} (cytosolic and membrane-associated but not secreted) by a malignant cell correlates with reduced invasiveness, as evidenced by the inability to develop tumors in mice or by the in vivo regression of tumors, which had developed initially [7 , 8 , 10 11 12 13 14 15 ]. This was demonstrated in tumor cells, which express IL-1{alpha} "spontaneously" following in vitro transformation with oncogenes [10 11 12 13 14 ], tumor cells with constitutive expression of IL-1{alpha} following transfection with the precursor of IL-1{alpha} [8 , 11 ], and also in tumor cells that have been activated in vitro with cytokines/immunomodulators to express IL-1{alpha} in a transient manner [14 , 15 ]. Furthermore, mice that had rejected IL-1{alpha}-positive tumors or mice that had been immunized with such cells become resistant to challenge with the wild-type parental cells, as both types of cells share epitopes. Innate and specific effector cells were shown to be activated in response to live or inactivated IL-1{alpha}-expressing cells [8 , 10 11 12 13 14 15 ]; however, the nature of IL-1{alpha}-activated signals, which lead to their development, has not yet been characterized.

Here, we describe the nature of the cellular responses that develop in mice following the injection of IL-1{alpha}-positive fibrosarcoma cells, as compared with those developed following injection of the corresponding violent cells. We also demonstrate the immunotherapeutic potential of tumor cell-associated IL-1{alpha} in intervention in the growth of existing, violent fibrosarcomas and characterize the therapeutic window of its effectiveness.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
NFS/N mice, for which NIH/3T3 cells are syngeneic [16 ], were kindly obtained from Dr. I. Fossar Larsen (Animal Section, The Fibiger Institute, Copenhagen, Denmark) and were subsequently bred at our animal facilities. Two- to 3-month-old mice were used for experiments. The mouse experiments were approved and performed according to the guidelines of the Ben-Gurion University of the Negev, Faculty of Health Sciences, Animal Safety Committee (Beer-Sheva, Israel).

Cell lines
The 277a fibroblastoid cell line was derived from the NIH/3T3 line (Clone 490NT3) following transfection with TPR-MET cDNA as described previously [10 ]. 277a cells were cloned in soft agar, as described [17 ], and separate colonies were picked and propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin G (100 U/ml), and streptomycin (100 µg/ml; each from Biological Industries, Kibbutz Beit Haemek, Israel). Cell lysates were obtained by three cycles of freezing/thawing of 106 cells per 1 ml DMEM and tested for IL-1{alpha} and IL-1ß content. Representative clones differing in IL-1{alpha} production were used in the present experiments.

In vivo assessment of tumor growth
Cells were harvested by trypsin-EDTA, washed in saline, and injected intrafootpad (i.f.p.) or subcutaneously (s.c.) at the indicated cell concentrations in a final volume of 50 µl (i.f.p. injection) or 100 µl (s.c. injection) using an insulin syringe. Tumor development was assessed every 3–4 days using a caliper. Tumor size is expressed as its diameter.

Histological and immunohistochemical analysis
Fibrosarcoma sections were performed 3 weeks after i.f.p. inoculation of the malignant cells (2x105 cells/mouse). For histological analysis, formalin-fixed, paraffin-embedded slides of tumor fragments were stained with hematoxylin and eosin (H&E). Immunohistochemical stainings of tumor fragments were performed on frozen tissue sections applying the avidin-biotin peroxidase complex, using the commercial HistomouseTM SP kit (Zymed Laboratories Inc., San Francisco, CA), according to the manufacturer’s instructions. The following monoclonal antibodies (mAb) were used: rat anti-mouse anti-CD4 (GK1.5 hybridoma, L3T4), rat anti-mouse anti-CD8 (53.6.72 hybridoma, Lyt-2), murine anti-mouse NK1.1 (PK-136 hybridoma; kindly provided by Professor Lea Eisenbach, The Weizmann Institute of Science, Rehovot, Israel), and rat anti-mouse membrane-activated complex-1 (Mac-1; M1/70.15.11.5.HL hybridoma; kindly provided by Professor Jacob Gopas, Ben-Gurion University). Preliminary calibration experiments have confirmed NK1.1 expression in lymphoid cell populations from NFS/N mice, similar to the patterns detected in C57BL/6 mice but distinct from BALB/c mice, which do not express NK1.1.

In vivo depletion of lymphoid cell subpopulations
In vivo depletion of CD4+, CD8+, and NK1.1+ cells was achieved by multiple intraperitoneal (i.p.) injections of ascitic fluids containing the relevant mAb starting 1 day before i.f.p. inoculation of tumor cells (2x105 cells/mouse) and then twice a week for 4 weeks. Nontreated mice injected with tumor cells or mice treated with an irrelevant antibody served as controls. Lymphoid cell depletion was verified 1 day after termination of the depletion by fluorescein-activated cell sorter (FACS) analysis performed on spleen cells of experimental mice by incubation with the appropriate antibodies followed by staining with mouse anti-rat phycoerythrin-conjugated immunoglobulin (PharMingen, San Diego, CA).

Assessment of antitumor immune responses
Antitumor immune responses were assessed in comparative studies using spleen cells from mice injected with an IL-1{alpha}-positive (Clone 2) cell line or a non-IL-1{alpha}-expressing (Clone 5) fibrosarcoma cell line.

Winn assay
The Winn assay was performed to demonstrate antitumor protective cells in the spleen of tumor-bearing mice. Thus, mice were injected s.c. with mixtures of violent tumor cells (Clone 5; 2x105 cells/mouse) and splenocytes derived from tumor-bearing mice (inoculated 5 days previously with 2x105 cells of Clone 2 or 5) or from control mice (4x106 cells/mouse; effector:tumor cell ratio 20:1) [18 ]. Mice injected with violent fibrosarcoma cells alone served as a control. Tumor development was assessed as described above.

Mixed lymphocyte tumor-cell reaction (MLTR)
In the MLTR, spleen cells derived from mice injected i.f.p. with 2 x 105 cells of Clone 2 or 5 were cultured in 24-well plates (5x106/ml). Mitomycin C (Sigma Chemical Co., St. Louis, MO)-treated fibrosarcoma cells (100 µg/107 cells for 1 h at 37°C) served as stimulators and were added to cultures at a concentration 2.5 x 106 cells/ml (effector:stimulator cell ratio 2:1) [13 ]. Cytokines in culture supernatants or cell lysates were assayed by commercial enzyme-linked immunosorbent assay (ELISA) kits, according to the manufacturer’s instructions. Lymphocyte proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method in parallel experiments of 5-day cultures performed in 96-well plates (2x105 effector cells/ml, effector:stimulator cell ratio 2:1). The stimulation index (SI) was calculated by the following formula: SI = optical density (OD; effector cells+stimulator cells)/OD (effector cells).

Cytotoxicity assays
Natural killer (NK) cell activity, in fresh spleen cell suspensions from tumor-bearing or control mice, was assayed in a 51Cr-release assay using YAC-1 cells, which are immunospecific for murine NK cells as targets [12 ].

For cytolytic T lymphocyte (CTL) activation, splenocytes were suspended in complete RPMI 1640 (Biological Industries; 5x106 cells/ml) and cultured for 7 days in the presence of Mitomycin C-inactivated fibrosarcoma cells (2.5x106 cells/ml). Blasts were harvested, and cytotoxic activity was determined in a standard 4-h 51Cr-release assay, as described previously [13 ]. Briefly, effector spleen cells were added at different ratios to target cells labeled with 100 mCi 51Cr, and 51Cr-release was determined in culture supernatants using a {gamma}-counter (Packard, Downers Grove, IL). Data are expressed as percent of specific lysis according to the formula: specific lysis (%) = [experimental counts per minute (cpm)–spontaneous cpm)/(total cpm–spontaneous cpm) x 100%. Spontaneous release was 8–12% in all experiments. The results are presented as the mean percent of specific 51Cr-release of triplicates of one representative experiment out of four performed.

Statistical analysis of the results
Each experiment was repeated three to five times or as indicated otherwise, with similar patterns of results. Individual in vivo experiments consisted of groups of five to 10 mice. In vitro assays were performed in triplicates, and triplicate values did not differ by more than 20% from the mean. Most results shown are from single assays and represent the mean of triplicates ± 1 SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fibrosarcoma cells expressing IL-1{alpha} fail to induce tumors in mice
We have assessed patterns of IL-1{alpha} expression and tumorigenicity of six arbitrarily chosen fibrosarcoma clones derived from NIH/3T3 cells, which were freshly transfected in vitro with the oncogene TPR-MET. Three of the clones (Numbers 1, 2, and 10) constitutively expressed IL-1{alpha} at levels of 100–200 pg/ml/106 cells cultured for 24 h, as detected by ELISA (Table 1 ). IL-1{alpha} was detected in cell lysates of fibrosarcomas, representing the cytosolic and membrane-associated compartments, but not in supernatants. IL-1{alpha}-positive clones also manifested bioactivity of IL-1 in cell lysates and paraformaldehyde-fixed cells, the latter representing the membrane-associated cytokine, as detected by the costimulatory effects of IL-1{alpha} on T cell proliferation (results not shown). These effects were neutralized by anti-IL-1{alpha} antibodies or the IL-1Ra. No IL-1ß expression was detected in supernatants or cell lysates of the fibrosarcoma cell lines (results not shown).


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  		Table 1. IL-1{alpha} Production by Fibrosarcoma Clones

 
All three IL-1{alpha}-positive fibrosarcoma clones induced regressing tumors when injected i.f.p. into NFS/N mice; initially, cells started to grow for 10–15 days and subsequently regressed within 20–40 days (Fig. 1 ). On the contrary, cells of non-IL-1{alpha}-expressing clones grew progressively and resulted in the death of tumor-bearing mice. Both types of cell lines (IL-1{alpha}-positive and -negative) show similar growth patterns in vitro and in immune-compromised mice (nude and sublethally irradiated mice), which may rule out nonimmune-related mechanisms that affect the observed tumorigenicity patterns [11 12 13 ].


Figure 1
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  		Figure 1. In vivo growth of fibrosarcoma clones expressing IL-1{alpha}. Cells were injected i.f.p. (2x105/mouse), and tumor development was scored. Shown are means of tumor diameters (five mice/group) of one representative experiment.

 
In subsequent experiments, two clones—the IL-1{alpha}-positive Clone 2 and the non-IL-1{alpha}-expressing Clone 5—were assessed in a comparative manner in studies on the immune mechanisms, which are activated by tumor cell-associated IL-1{alpha}, which subsequently leads to tumor regression.

To show that tumor cell-associated IL-1{alpha} is indeed the active moiety that mediates regression of tumors, systemic blockage of IL-1Rs by multiple injections of the IL-1Ra was performed in mice injected with cells of Clone 2, as described in Materials and Methods. This treatment abrogated regressions and resulted in a shift to progressive growth (Fig. 2 ). The results point to a role of tumor cell-associated IL-1{alpha} in tumor regression, possibly by direct immune cell activation, through ligation of membrane-associated IL-1{alpha} to IL-1Rs, which are distributed abundantly on immune cells. However, one cannot exclude the possibility that tumor cell-associated IL-1{alpha} can also act indirectly by activating IL-1 expression in stromal and immune cells in the microenvironment of the developing tumor, and this IL-1 is neutralized by the IL-1Ra.


Figure 2
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  		Figure 2. In vivo neutralization of IL-1 by the IL-1Ra abrogated regression of tumors of IL-1{alpha}-expressing cells. Mice were injected with IL-1{alpha}-positive cells (Cl.2, 2x105 cells/mouse, i.f.p.) and treated with the IL-1Ra (0.125 mg/mouse) by i.p. injections 1 day before tumor inoculation and then twice a week during 5 weeks. Tumor development was scored. Shown are mean tumor diameters ± SD (five mice/group) of one representative experiment.

 
The nature of infiltrating cells, which are associated with the regression of IL-1{alpha}-positive fibrosarcomas
Histological sections from tumors were analyzed at the peak of regression of tumors induced by cells of Clone 2 (Day 21, tumor size 6–7 mm); violent tumors induced by cells of Clone 5 reached at that time the size of ~22 mm. As can be seen in Figure 3 , the site of injection of IL-1{alpha}-positive fibrosarcoma cells of Clone 2 was infiltrated heavily with mononuclear leukocytes, and the tumor’s mass was replaced by granulation tissue, characterized by fibroplasia, extracellular matrix deposition, and neoangiogenesis. IL-1{alpha}-positive tumors are invaded early by leukocytes, approximately 1 week after the inoculation of the malignant cells; the tumor’s mass was replaced gradually by fibrotic scar tissue, possibly after destruction of the malignant cells by immune-surveillance mechanisms. In sections from tumors induced by cells of the non-IL-1{alpha}-expressing Clone 5, actively proliferating tumor cells were observed with only a sparse infiltrate of leukocytes, mostly polymorphonuclear cells that localize to necrotic areas.


Figure 3
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  		Figure 3. Histological analysis of tumor sections from IL-1{alpha}-positive or violent fibrosarcomas. Tumor fragments of IL-1{alpha}-positive (Clone 2) and violent (Clone 5) fibrosarcomas were obtained 3 weeks after tumor cell inoculation and stained with H&E (original magnification, x160). In addition, frozen sections were fixed in acetone and immunostained with anti-CD4, anti-CD8, anti-NK1.1, and anti-Mac-1 mAb using the commercial Histomouse SP kit (original magnification, x250).

 
In immunohistochemical stainings, to characterize the nature of the infiltrating cells at injection sites of cells of Clone 2, we have observed the accumulation of CD8+ T cells, NK cells, and macrophages, and only few CD4+ T cells were observed. Infiltrating immune cells were not evident in sections from violent tumors induced by cells of Clone 5. Preliminary calibration experiments were performed to minimize the background staining patterns of the secondary antibodies (results not shown). The pattern of immunohistochemical stainings observed in sections of tumors of Clone 5, which do not contain significant numbers of inflammatory cells, may serve as an internal, negative control to staining patterns in sections from mice injected with IL-1{alpha}-positive cells that are invaded by infiltrating leukocytes.

CD8+ T cells, but not CD4+ T cells, are essential for the regression of IL-1{alpha}-positive fibrosarcomas
We next wanted to verify whether the infiltrating cells that are found in sections of regressing tumors of IL-1{alpha}-positive fibrosarcoma cells are indeed essential for eradication of the malignant cells. For this purpose, in vivo depletion experiments, using anti-CD4, anti-CD8, and anti-NK1.1 mAb, were performed in tumor cell-injected mice to characterize the nature of effector cells that mediate the regression of IL-1{alpha}-positive fibrosarcomas. Treatment consisted of multiple i.p. injections of the antibodies twice a week in a protocol, which is specified in Materials and Methods. Depletion was verified by FACS analyses. At the end of the depletion procedure, spleens contained 1% of CD4+ T cells, as compared with 63% CD4+ T cells in nondepleted, tumor-bearing mice, and 2% of CD8+ T cells, as compared with 17%. NK1.1 depletion, reduced by 90–95% of the initial number of positive cells in the spleen (2.5%) and peripheral blood mononuclear cells (7.5%).

Depletion of CD4+ T cells did not impair the regression of IL-1{alpha}-positive fibrosarcoma cells of Clone 2, and growth patterns were similar to those in nontreated mice (Fig. 4A ). CD8+ T cell depletion resulted in a shift in growth patterns of the tumors, resulting in the generation of progressive tumors. Growth patterns of fibrosarcoma cells of Clone 2 in NK1.1+-depleted mice were heterogeneous. In some mice, progressive growth was observed, and in most mice, tumor regressions occurred later, after the primary tumor had reached an initial, larger size, in comparison with tumors that developed in nontreated mice or in mice depleted with anti-CD4 antibodies. Thus, CD8+ T cells are essential for the regression of cells of Clone 2, whereas CD4+ T cells are not obligatory for this process. NK cells were also shown to control tumor growth, possibly at early stages of tumor regression, before specific immunity develops.


Figure 4
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  		Figure 4. In vivo growth patterns of IL-1{alpha}-positive fibrosarcoma cells in CD4, CD8, and NK cell-depleted mice. Cells of the IL-1{alpha}-positive clone 2 (A) or the violent clone 5 (B) were injected i.f.p. (2x105 cells/mouse) into mice depleted of various effector cell populations. Treatments with anti-CD4+, anti-CD8+, and anti-NK1.1+ were performed as indicated in Materials and Methods. Shown are tumor diameters of individual mice of one representative experiment (five mice/group).

 
Depletion of CD4+ T cells in mice injected with cells of Clone 5 resulted in a reduced growth of the tumors, as compared with growth in nontreated mice (Fig. 4B) , and depletion of CD8+ or NK1.1+ cells did not alter tumorigenicity patterns (data not shown). These data indicate that the cells that infiltrate the sites of injection of tumor cells of Clone 2 are indeed the cells that participate in their eradication.

Characterization of antitumor effector mechanisms in the spleens of mice bearing IL-1{alpha}-positive and violent fibrosarcomas
In a time-course kinetics experiment, we further compared antitumor cell immune responses in spleens of mice injected with cells of Clone 2 or 5 at different intervals after inoculation of the malignant cells. Parameters of specific antitumor cell immunity were assessed in mixed cultures consisting of spleen cells from tumor-bearing mice, which were rechallenged in vitro with the relevant, inactivated (Mitomycin C-treated) tumor cells (MLTR cultures).

Antigen-specific T cell proliferation with the concomitant secretion of T helper cell type 1 (TH1) cytokines, especially IL-2, is essential for the expansion and differentiation of CTL precursors. Cell proliferation was assessed by the MTT method, as described in Materials and Methods. As shown in Figure 5A , injection of violent cells of Clone 5 induced a significant but transient T cell-proliferative response, peaking on Day 5 and returning to baseline levels on Day 10, and IL-1{alpha}-positive cells of Clone 2 induced a potent, proliferative response that increased continuously as the tumors regressed (Fig. 5B) . Similar patterns of response were observed for IL-2 and interferon-{gamma} (IFN-{gamma} secretion; results not shown). T cell proliferation was observed only upon in vitro challenge of spleen cells from tumor-bearing mice with the sensitizing tumor cells, indicating that these responses are antigen-specific. No response was observed in response to an irrelevant methycholanthrene-induced fibrosarcoma cell line originated in NFS/N mice (results not shown).


Figure 5
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  		Figure 5. Induction of antitumor cell-proliferative responses in spleens of mice bearing IL-1{alpha}-positive or violent fibrosarcomas. Mice were injected i.f.p. with IL-1{alpha}-positive cells (Clone 2) or violent tumor cells (Clone 5; 2x105 cells/mouse). At different intervals thereafter, spleens were removed, and MLTR cultures were established (effector:stimulator cell ratio 2:1). Proliferation of spleen cells was assessed by the MTT method after 5 days of culture. Shown are proliferative responses in MLTR cultures (SI) versus in vivo growth of fibrosarcoma clones (five mice/group) of one representative experiment. Values of Day 0 represent the control of naïve spleen cells.

 
A significant CTL response, peaking on Days 10–14, was observed in spleens of mice injected with IL-1{alpha}-positive fibrosarcoma cells of Clone 2 (Fig. 6A ). CTL activity remained high throughout tumor regression. In contrast, in spleens of mice bearing progressive, non-IL-1{alpha}-expressing tumors induced by cells of Clone 5, no CTL activity was observed at any of the intervals tested.


Figure 6
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  		Figure 6. CTL and NK cell lytic activity in spleen cells from mice injected with IL-1{alpha}-positive or violent fibrosarcoma cells. Spleens of mice bearing IL-1{alpha}-positive (Clone 2) or violent (Clone 5) fibrosarcomas were removed at different intervals after tumor cell inoculation (2x105 cells/mouse). (A) For CTL activation, splenocytes were cultured for 7 days in the presence of Mitomycin C-treated fibrosarcoma cells (effector:stimulator cell ratio 2:1). CTL-specific activity against fibrosarcoma cells was assessed in a 4-h 51Cr-release assay (effector:target cell ratio 100:1). (B) NK cell lytic activity in fresh spleen cell suspensions was assessed in a 4-h 51Cr-release assay using YAC-1 cells as targets (effector:target cell ratio 100:1). Each data point represents the mean values of lysis ± SD, which were obtained from cultures of five individual mice per time-point in a single experiment. Statistical significance was tested by Student’s t-test.

 
A transient peak in NK cell activity was detected in the spleens of tumor-bearing mice, 1–2 days after tumor cell injection. NK activity was prominent in spleens injected with both types of tumor cells; however, it was significantly higher in the spleens of mice injected with cells of Clone 2 (P=0.001, Fig. 6B ).

As some transient, antitumor immune responses were observed in the spleen of mice injected with cells of Clone 5 early after the injection of the malignant cells, we wanted to assess whether they are able to confer the mice with antitumor cell-protective capacity. To assess this, we used the modified Winn assay, in which spleen cells from tumor-bearing mice, serving as effector cells, were admixed in vitro with violent tumor cells (Clone 5) at a ratio of 1:20, respectively, and subsequently injected s.c. into naïve, recipient mice, and tumor development was assessed (Fig. 7 ). We took spleens 7 days after tumor cell inoculation, as early antitumor immune responses are crucial for TH cell polarization and subsequently, for the development of specific antitumor cell CTLs and other effector cells. Growth patterns of violent cells of Clone 5 injected alone, without any supplemental spleen cells, are also shown in Figure 7A . Spleen cells from control mice had no effect on tumor growth (results not shown). Spleen cells from mice injected with cells of Clone 2 completely inhibited the growth of the violent cells (Fig. 7B) , and spleen cells from mice injected with violent cells of Clone 5 did not have any protective effect on the progressive growth of cells of Clone 5 (Fig. 7C) . Spleen cells from CD4+ T cell-depleted mice injected with cells of Clone 2 retained their antitumor, protective capacity (Fig. 7D) , although depletion of CD8+ T cells abrogates the protective effects (Fig. 7E) , further substantiating the results demonstrated in Figure 4A .


Figure 7
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  		Figure 7. Induction of antitumor-protective cells in spleens of mice injected with IL-1{alpha}-positive fibrosarcomas. Mice were injected i.f.p. with 2 x 105 of IL-1{alpha}-positive cells (cl.2) or violent tumor cells (cl.5). On Day 7, spleen cells (SPC; 4x106) from various experimental groups were mixed with 2 x 105 cells of the violent fibrosarcoma cells (cl.5; effector:tumor cell ratio 20:1). The cell mixure was injected s.c., and tumor development of individual mice was assessed. Shown are growth patterns of tumors in one representative experiment (five mice/group).

 
As initial and transient antitumor cell responses were generated after the inoculation of cells of Clone 5 (Fig. 5A) , it was of interest to assess whether tumor-mediated suppression accounts for the decay in antitumor cell-immune responses, which ultimately leads to progressive growth. To assess the latter, violent, non-IL-1{alpha}-expressing fibrosarcoma cells of Clone 5 were coinjected with mixtures of spleen cells from tumor-bearing mice, previously inoculated with cells of Clone 2 or cells of Clone 5 (Fig. 7) . In this experiment, 2 x 105 cells of Clone 5 were injected with a total number of 4 x 106 spleen cells (ratio 1:20, respectively); however, the ratios between the two types of spleen cells varied. As the number of spleen cells from mice bearing tumors induced by Clone 5 increased (Fig. 7 F-H) , the protective effects were diminished. Thus, mixtures of spleen cells from mice bearing tumors of Clone 2 and mice bearing tumors of Clone 5 at a 1:1 ratio, respectively, induced complete protection, and no tumor development of the violent cells of Clone 5 was observed (Fig. 7F) . At a 1:2 ratio, regression occurred only in part of the mice (Fig. 7G) , and at a 1:3 cell ratio, all mice developed progressive tumors (Fig. 7H) . This indicates that spleen cells from mice bearing progressive tumors of Clone 5 have imposed dominant suppression on the function of antitumor effector cells that reside in the spleen of mice injected with cells of Clone 2, which by themselves, manifest protective capacity at these cell concentrations.

Intervention in the growth of progressive tumors by IL-1{alpha}-positive fibrosarcoma cells
Violent cells of Clone 5 induce early and transient antitumor T cell-mediated responses, which are insufficient to induce tumor regression. It was thus of interest to test if we can intervene in the growth of violent tumor cells of Clone 5 using IL-1{alpha}-positive tumor cells, which will potentiate and sustain the weak antitumor immune responses or alternatively, intervene in tumor-mediated suppression. For this purpose, mice were injected i.f.p. with cells of Clone 5, and concomitantly or at different intervals thereafter, Mitomycin C-treated cells of Clone 2 were injected into the same footpad (a single injection), and tumor growth was assessed. In this immunotherapeutic intervention protocol, IL-1{alpha}-positive tumor cells induced positive antitumor effects at critical intervals (Days 5–12) after the injection of the violent tumor cells (Fig. 8 ). When the IL-1{alpha}-positive-treating cells (Clone 2) were injected together with the violent cells, at early intervals thereafter (up to Days 3–5) or at late intervals (from Day 14 onwards), after injection of violent cells of Clone 5, no antitumor effects were observed. Regressions were observed only after treatment with IL-1{alpha}-positive cells; treatment of mice bearing violent tumors with Mitomycin C-treated tumor cells of Clone 5 did not alter the progressive growth of tumors (results not shown). In mice with regressing tumors after treatment, elevated IL-2 levels were detected in MLTR cultures, indicating the activation of antitumor cell immune responses in the treated mice (results not shown).


Figure 8
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  		Figure 8. Immunotherapeutic effects of IL-1{alpha}-positive fibrosarcoma cells on the progressive growth of violent fibrosarcoma cells. Mice were inoculated i.f.p. with 105 cells of violent fibrosarcoma cells (Clone 5). At various intervals thereafter, 2 x 105 cells of IL-1{alpha}-producing Clone 2 (Mitomycin C-inactivated) were injected into the same footpad, and tumor development was assessed. Shown are growth characteristics of tumors in individual mice (five mice/group) in one representative experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study emphasizes the role of tumor cell-associated IL-1{alpha} in the induction of specific immune responses against epitopes on the malignant cells, ultimately leading to tumor regression and the development of an immune memory, which protects the mice from a challenge with the violent tumor cells (non-IL-1-expressing). Tumor cell-associated IL-1{alpha} activates TH1 cells and their derived cytokines, CTLs, as well as nonadaptive effector cells, such as NK cells and activated macrophages [7 , 8 , 10 11 12 13 14 15 ]. In mice injected with cells of Clone 5, which do not express IL-1, no effective antitumor immune responses could be detected. We hypothesize that the antitumor effects of fibrosarcoma cell-derived IL-1{alpha} are a result of its unique cell-associated compartmentalization in malignant fibroblastoid cells. Membrane-associated IL-1{alpha} (23 kD) possibly serves as an adhesion molecule, allowing efficient cell-to-cell interactions between the malignant and immune effector cells, which abundantly express IL-1Rs. As such, membrane-associated IL-1{alpha} represents a focused cytokine and an adjuvant that activates immune responses at low levels of expression (i.e., 100–250 pg/106 cells/24 h), below those that are toxic to the host. On the contrary, secretable IL-1, IL-1ß, and IL-1{alpha} have been shown to induce potent inflammatory responses at tumor sites that result in increased invasiveness of the malignant cells and also in tumor-mediated suppression (reviewed in refs. [1 , 7 , 8 ]. However, we cannot exclude the possibility that cell-associated IL-1{alpha} also acts indirectly by stimulating the production of small doses of IL-1 by cellular elements of the microenvironment, which mediate efficient antitumor cell immune responses.

A mononuclear cell infiltrate, consisting of CD8+ T cells (and not CD4+ T cells), NK cells, and macrophages, was shown to accumulate at injection sites of IL-1{alpha}-positive fibrosarcoma cells, and the tumor’s mass was ultimately replaced by a fibrotic scar tissue. These results were substantiated by in vivo studies showing that the regression of IL-1{alpha}-positive fibrosarcomas also occurs in CD4+ T cell-depleted mice, whereas depletion of CD8+ T cells results in a shift to progressive tumor growth. This may indicate that auxiliary TH1 cytokines, mainly IL-2, which are essential for CTL expansion/maturation, may be derived from some CD8+ T cells in an autocrine manner or from other cells, such as NK or NKT cells [19 20 21 22 23 ]. However, we cannot exclude that in intact mice, CD4+ T cells participate in the activation of tumor cell-specific CD8+ CTLs. For optimal activation of antitumor CTLs, especially during priming, CD4+ and CD8+ T cells have to be activated [24 25 26 ]. However, in some instances, CD8+ T cells can promote their own expansion/differentiation, provided that the target cells that present the tumor peptides to CTL precursors also express the second costimulatory signal [27 , 28 ]. Indeed, B7-transfected tumor cells were shown to activate CD8+ T cells directly, without the need for CD4+ T cells [29 , 30 ]. Membrane-associated IL-1{alpha}, by ligating to IL-1Rs on CTL precursors, may supply the second costimulatory signal for activation of the cells. In addition, endogenous IL-1{alpha} expression by fibrosarcoma cells concomitantly induces surface B7-1, but not B7-2, expression, which is an essential costimulatory molecule for the development of efficient antitumor immunity (T. Dvorkin et. al., in preparation). It was also demonstrated that ligation of B7 to CD28 or of soluble IL-1 to its receptors on T cells activates common signaling pathways that result in enhanced transcription and secretion of IL-2 via activation of the pathway of nuclear factor-{kappa}B [31 ]. Taken together, these results indicate that cell-associated or even soluble IL-1 can serve as a costimulatory signal for T cell activation.

The present study demonstrates the role of early antitumor immune responses on the fate of the malignant process and emphasizes the role of tumor cell-associated IL-1{alpha} in the induction of these responses. When violent fibrosarcoma cells of Clone 5 were injected into mice, we observed only initial and transient TH1 responses (antitumor cell-proliferative responses and IL-2 secretion), which did not lead to CTL maturation or the development of antitumor protective immunity. This may result from insufficient signals for the induction/maintenance of antitumor cell immunity or from tumor-mediated suppression. North and Bursuker [32 , 33 ] have shown that immunogenic fibrosarcomas grow progressively despite the induction of antitumor immunity, as a result of the development of tumor-mediated immune suppression induced by CD4+ T cells. In our experiments, the growth of violent fibrosarcoma cells (Clone 5) was slower in CD4+ T cell-depleted mice, as compared with intact mice (Fig. 4b) . The suppressive role of some CD4+ T cells on tumor growth was demonstrated in several experimental tumors; depletion of CD4+ T cells improves the development and function of antitumor effector cells (increased IFN-{gamma} secretion by CTLs) and also enables more CD8+ T cells to infiltrate into tumor sites [34 35 36 37 38 ]. Suppressor mechanisms known to impair the development/function of antitumor immunity may include CD25+CD4+ regulatory T cells, TH3 cells, and their derived suppressor cytokines (transforming growth factor-ß and IL-10) [34 35 36 37 38 39 40 41 ], as well as suppressive tumor-associated macrophages [42 ] or early granulocyte-macrophage (GM) precursors [43 , 44 ]. The mechanisms whereby cell-associated IL-1{alpha} attenuates tumor-mediated suppression are still to be elucidated.

Positive immunotherapeutic effects of tumor cell-associated IL-1{alpha}, as indicated by interference in the growth of violent fibrosarcomas, have been shown in this study in a defined, therapeutic window. Thus, when the IL-1{alpha}-positive "treating" cells are applied at defined, early intervals (6–14 days) after injection of the violent cells when transient activation of antitumor T cells has been observed, the IL-1{alpha}-positive cells may potentiate and sustain antitumor immune responses or alternatively, act to abrogate tumor-mediated suppression. This ultimately leads to complete tumor regression in all tumor-bearing mice treated on Days 7–12. At earlier times, when no or subthreshold antitumor immune responses develop, or suppression has not yet developed, no effects of tumor cell-associated IL-1{alpha} were observed. Indeed, when the violent and inactivated IL-1{alpha}-positive tumor cells are injected together or when the treating cells are injected on Days 1 or 3 afterward, no antitumor effects are observed. In this protocol, the IL-1{alpha}-expressing, inactivated tumor cells possibly disintegrate quickly, and tumor cell-derived IL-1 disappears and thus cannot potentiate antitumor cell immune responses or alternatively, alleviate tumor-mediated suppression as they develop later. At late intervals, i.e., Day 14 and onward, there are no immunotherapeutic responses to treatment with IL-1{alpha}-positive tumor cells, because of the inability of the immune system to cope with established tumors or as a result of domination of tumor-mediated suppression. Thus, tumor cell-associated IL-1{alpha} efficiently induces antitumor cell immunity and/or ablates tumor-mediated immune suppression when applied at critical intervals. This is in contrast to the inconsistent reports about the therapeutic efficiency of recombinant IL-1; in most studies, application of high, systemic levels of the cytokine resulted in enhanced invasiveness as a result of the proinflammatory features of the cytokine, or treatments had to be terminated as a result of adverse side-effects induced by IL-1 (reviewed in refs. [1 , 7 ]). More stringent and multiple treatments with tumor cell vaccines expressing cell-associated IL-1{alpha} may broaden this therapeutic window. Membrane-associated IL-1{alpha} is among the few cytokines that has a "natural" membrane-associated form, which stems from myristoylation and cleavage of the IL-1{alpha} precursor (reviewed in ref. [1 ]). Membrane IL-1{alpha} is biologically active; its activity is neutralized by anti-IL-1{alpha} antibodies or the IL-1Ra. It is anchored to the membrane via a lectin interaction involving mannose residues. In experiments of prolonged fixation of cells expressing IL-1{alpha}, it was shown that leakage of the precursor of IL-1{alpha} from cells did not account for the activity of membrane IL-1{alpha}. In most cells, except macrophages, active IL-1{alpha} is cell-associated (located in the cytosol and on the membrane), and IL-1ß is only active in the microenvironment into which it is secreted. The antitumor effects of IL-1{alpha} have been shown by us in multiple experimental tumor systems, as specified in Introduction [7 , 8 , 10 11 12 13 14 15 ]. In vivo expression of IL-1{alpha} by malignant cells possibly occur during the process of tumorigenesis; however, IL-1{alpha}-positive cells are immunogenic and are eradicated by immune effector mechanisms. This occurs in the process of immune editing of the repertoire of the malignant cells during tumor progression, which culminates in the appearance of overt tumors consisting of weakly or nonimmunogenic malignant cells [45 ]. Therefore, we could not detect IL-1{alpha}-positive cells in tumors of transplantable cell lines recovered after in vivo passage (T. Dvorkin, R. N. Apte, unpublished results). However, by using our experimental tumor systems, we have highlighted the role of cell-associated IL-1{alpha} in potentiating the immunogenicity of malignant cells, findings that can be translated into immunotherapeutic protocols.

Other studies have also emphasized the antitumor effects of membrane-associated cytokines [IFN-{gamma}, GM-colony stimulating factor (CSF), M-CSF, and tumor necrosis factor {alpha} (TNF-{alpha})], acting via cell-to-cell contact to enhance the immunogenicity of the tumor cells and also to eliminate the potential toxicity attendant with cytokines released to the bloodstream via classic secretory pathways [46 47 48 ]. In the case of M-CSF, the superiority of the membrane-associated isoform, as compared with the secreted form, was demonstrated clearly in a model of rat glioma [49 ]. It was hypothesized that membrane M-CSF provides a molecular bridge between the macrophages and tumor cells and induces the tumoricidal effects of macrophages; this is followed by the induction of T cell-mediated antitumor immunity, including the establishment of an immune memory. Furthermore, adenovirus vectors engineered to express the membrane or the secreted forms of TNF-{alpha} showed similar antitumor effects in a murine transgenic breast cancer model; however, the membrane form of this cytokine showed much lower systemic toxicity, which is the most limiting factor regarding the use of TNF-{alpha} in tumor therapy [46 ]. The immunotherapeutic potential of membrane-associated IL-1 will be investigated further by using tumor cell vaccines or DNA vaccination protocols aimed at the expression of cell-associated IL-1{alpha}.


   ACKNOWLEDGEMENTS
 
R. N. A. was supported by the Israel Ministry of Sciences (MOS) jointly with the Deutsches Krebsforschungscentrum (DKFZ), the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities, the Israel Ministry of Health Chief Scientist’s Office, the Association for International Cancer Research (AICR), the United States-Israel Binational Science Foundation (BSF), and the German-Israeli DIP (Deutsch-Israelische Projektkooperation) collaborative program.

This paper is dedicated to the memory of Prof. Shraga Segal, friend, mentor and collaborator.


   FOOTNOTES
 
1 Current address: Diabetes Research Labs, Massachusetts General Hospital and Harvard Medical School, 65 Landsdown St., Cambridge, MA 02138. Back

2 Deceased. Back

Received September 11, 2005; revised February 5, 2006; accepted February 22, 2006.


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