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The Journal of Immunology, 1998, 160: 2915-2922.
Copyright © 1998 by The American Association of Immunologists

Resistance to Melanoma in Mice Immunized with Semiallogeneic Fibroblasts Transfected with DNA from Mouse Melanoma Cells1

Edwin F. de Zoeten, Victoria Carr-Brendel and Edward P. Cohen2

Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor-associated Ags (TAA) that characterize a population of malignant cells are recognized by CTLs in the context of determinants specified by the MHC class I locus. Nevertheless, most progressively growing neoplasms do not induce antitumor immune responses that can control tumor cell growth. The TAA may be insufficiently antigenic. We found previously that immunization of mice with a cellular immunogen prepared by transfecting tumor DNA into allogeneic mouse fibroblasts resulted in strong antitumor immune responses that were specific for the type of tumor from which the DNA was obtained. Since the fibroblasts differed at the MHC from the immunized mice, we postulated that the immunogenic properties of the allogeneic transfected cells might be enhanced if the cells were modified to express syngeneic class I determinants. In a mouse melanoma model system, the H-2Kb gene was introduced into LM mouse fibroblasts (H-2k). Afterward, the cells were transfected with DNA from B16 melanoma cells (H-2b). The transfected cells were tested for their immunotherapeutic properties in C57BL/6J mice (H-2b) with melanoma. Mice with melanoma treated solely by immunization with the semiallogeneic transfected cells developed strong, long-term resistance to the growth of the tumor. In some instances, the mice survived indefinitely. Intact rather than disrupted transfected cells were required to induce the antimelanoma response, consistent with direct presentation of TAA by the transfected cells. The augmented resistance to melanoma in mice treated with the semiallogeneic transfected cells points toward an analogous form of therapy for cancer patients.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Accumulating evidence from multiple sources (reviewed in 1 indicates that neoplastic cells form weakly immunogenic, tumor-associated Ags (TAA).3 Under appropriate circumstances, the Ags can stimulate tumor-specific, cellular immune responses (2, 3, 4) that may be useful in the treatment of patients with malignant disease.

To augment their immunogenic properties, neoplastic cells have been genetically modified to secrete various cytokines, such as IL-2 (5, 6, 7), IL-4 (8), IL-6 (9), IL-7 (10), IL-12 (11), TNF-{alpha} (12, 13), IFN-{alpha} and {gamma} (14, 15), and granulocyte-macrophage CSF (16, 17), among others (18). The weakly antigenic cells may become immunogenic as a result. Similar effects are found if genes specifying allogeneic MHC determinants are introduced into neoplastic cells (19, 20, 21).

Immune rejection of tumor cells genetically modified to increase their antigenic properties resulted in generalized, antitumor cellular immune responses that were reactive with both the modified as well as unmodified cells. Since, in most instances, normal, nonmalignant cells of the immunized, tumor-bearing host were unaffected, it was likely that the antitumor immune responses were directed toward unique antigenic determinants expressed by the malignant cells.

The origin of genes that specify TAA is not established. It may be the consequence of the expression of dysregulated, or mutated genes in cancer cells that ordinarily code for various normal cellular constituents (22, 23). The products of genes that specify TAA, such as BAGE (24), GAGE-1 (25), MAGE (26, 27, 28), and tyrosinase (29), among others (30, 31, 32), are expressed by human neoplastic cells. These may be only several representations of an undefined, and possibly large number of analogous genetic events in a malignant cell population that result in the expression of an array of different TAA. Genetic instability is a characteristic phenotype of malignant cells (33, 34, 35, 36, 37).

We found previously that immunization of C57BL/6J mice (H-2b) with a mouse fibroblast cell line (LM; H-2k) modified for IL-2 secretion that was transfected with DNA from malignant cells (of C57BL/6 mouse origin) resulted in potent T cell-mediated cellular immune responses that were specific for the type of tumor from which the DNA was obtained (38, 39, 40, 41). Mice with melanoma treated solely by immunizations with IL-2-secreting LM fibroblasts transfected with melanoma DNA survived significantly longer than mice with melanoma treated with LM cells transfected with DNA from other types of mouse neoplasms, or with DNA from non-neoplastic cells. The finding was consistent with the expression of unique TAA characteristic of the neoplasm by a subpopulation of the highly immunogenic, transfected cells.

The fibroblasts used as recipients of DNA from the neoplastic cells were allogeneic in C57BL/6 mice. Hence, presentation of the TAA may have followed Ag uptake by APCs of the host. Conceivably, the immunogenic properties of the allogeneic cells could be enhanced if the fibroblasts were modified to form syngeneic H-2Kb class I determinants before they were transfected with DNA from the neoplasm. The self MHC class I determinants might provide a mechanism for the direct presentation of antigenic peptides to CTLs of the tumor-bearing host, further enhancing the cells’ immunogenic properties.

To investigate this question, a plasmid (pBR327H-2Kb) was used to introduce expression-competent genes for H-2Kb determinants into the fibroblasts. Afterward, the semiallogeneic cells were cotransfected with DNA from B16 melanoma cells, along with a second plasmid (pHyg) conferring resistance to hygromycin, used for selection. Pooled colonies of the hygromycin-resistant, transfected fibroblasts were then tested for their immunotherapeutic properties in C57BL/6J mice with melanoma. The results indicated that tumor-bearing mice treated solely by immunizations with the genetically modified cells developed generalized antimelanoma immunity and survived for prolonged periods, in some instances, indefinitely. The data suggest that transfection of semiallogeneic fibroblasts with DNA from autologous neoplasms could be a convenient means of preparing cellular immunogens useful in the overall management of cancer patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and experimental animals

Eight- to ten-week-old specific pathogen-free C57BL/6J mice (H-2b), obtained from The Jackson Laboratory (Bar Harbor, ME), were maintained in the animal care facilities of University of Illinois (Chicago, IL), according to National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The mice were 8 to 12 wk old when used in the experiments.

B16 cells, a highly malignant melanoma cell line derived from a spontaneous neoplasm occurring in a C57BL/6 mouse, were obtained from I. Fidler (M. D. Anderson Cancer Center, Houston, TX). EO771 cells, a mammary adenocarcinoma cell line derived from a C57BL/6 mouse, were from Tumor Repository of Division of Cancer Treatment, Diagnosis and Centers of National Cancer Institute (Frederick, MD). Both cell types were maintained by serial passage in histocompatible C57BL/6J mice, or at 37°C in a humidified 7% CO2/air atmosphere in DMEM (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Sigma Chemical Co., St. Louis, MO) and antibiotics (Life Technologies) (growth medium). LM cells, a fibroblast cell line of C3H mouse origin, were from American Type Culture Collection (ATCC), Rockville, MD. Like B16 cells, they were maintained at 37°C in a humidified 7% CO2/air atmosphere in growth medium. BLK-SV (BLK), a fibroblast cell line of C57BL/6 mouse origin, was from ATCC. Like B16 and LM cells, the cells were maintained at 37°C in a humidified 7% CO2/air atmosphere in growth medium.

Modification of LM and BLK fibroblasts for IL-2 secretion

LM and BLK fibroblasts were modified for IL-2 secretion by transduction with the retroviral vector pZipNeoSVIL-2 (from M. K. L. Collins, University College, London, U.K.) (LM-IL-2 cells). The vector, packaged in GP+env AM12 cells (from A. Bank, Columbia University, New York, NY), included a human IL-2 cDNA and a neor gene, both under control of the Moloney leukemia virus long-terminal repeat. The neor gene conferred resistance to the aminoglycoside antibiotic G418 (Life Technologies). For use as a control, LM fibroblasts were transduced with the retroviral vector pZipNeoSV(X) (from M. K. L. Collins), also packaged in GP+envii AM12 cells (LM-ZipNeo cells). pZipNeoSV(X) specified the neor gene, but lacked the gene for IL-2. Virus-containing supernatants of GP+envii AM12 cells transfected with pZipNeoSVIL-2, or pZipNeoSV(X), were added to LM or BLK fibroblasts, followed by overnight incubation at 37°C in growth medium to which polybrene (Sigma Chemical Co.; 5 µg/ml, final concentration) had been added. The cells were maintained for 14 days in growth medium containing 400 µg/ml G418 (Life Technologies). One hundred percent of nontransduced LM or BLK cells died in the medium supplemented with G418 during this period. Colonies of cells proliferating in the G418-containing growth medium were pooled for later use in the experiments.

IL-2 secretion by LM-IL-2 or BLK-IL-2 cells was detected by the capacity of supernatants from the transduced cells to sustain the growth of CTLL-2 cells, an IL-2-dependent cell line (from A. Finneagan, Rush Medical College, Chicago, IL) (42). Varying dilutions of the filtered culture supernatants (0.2 um nitrocellulose; Gelman, Ann Arbor, MI) were transferred to 96-well plates containing 5 x 103 CTLL-2 cells in a final volume of 200 µl of growth medium per well. After incubation for 16 h, 0.5 µCi [3H]thymidine (Amersham, Arlington Heights, IL) was added to each well for additional 6 h of incubation. A standard curve was generated by adding varying amounts of human rIL-2 (Life Technologies) to an equivalent number of CTLL-2 cells. Afterward, the cells were collected onto glass fiber filters (Whittaker M.A. Products, Walkerville, MD) using a PhD multiple harvester (Microbiologic Associates, Bethesda, MD). After washing with ethanol (95%), radioactivity in the insoluble fraction was measured in a liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, IL). One unit of IL-2 resulted in half-maximal proliferation of CTLL-2 cells under these conditions. Every third passsage, the transduced cells were cultured in growth medium containing 400 µg/ml G418.

Modification of LM-IL-2 fibroblasts for the expression of H-2Kb class I determinants

pBR327H-2Kb (Biogen Research Corp., Cambridge, MA), a plasmid encoding MHC H-2Kb determinants (43), was used to modify LM-IL-2 fibroblasts to express H-2Kb determinants (LM-IL-2Kb cells). Ten micrograms of pBR327H-2Kb and one microgram of pBabePuro (from M. K. L. Collins), a plasmid conferring resistance to puromycin (44), were mixed with lipofectin (Life Technologies), according to the supplier’s instructions, and then added to 1 x 106 LM-IL-2 cells in 10 ml of DMEM without FBS. For use as a control, an equivalent number of LM-IL-2 cells was transfected with 1 µg of pBabePuro alone. The cells were incubated for 18 h at 37°C in a CO2/air atmosphere, washed with DMEM, followed by the addition of growth medium. After incubation for 48 h, the cell cultures were divided and replated in growth medium supplemented with 3 µg/ml puromycin (Sigma Chemical Co.), followed by incubation at 37°C for 7 additional days. The surviving colonies were pooled and tested by staining with specific FITC-conjugated Abs for the expression of H-2Kb determinants. One hundred percent of nontransfected LM-IL-2 cells maintained in growth medium containing puromycin died during the 7-day period of incubation.

Immunofluorescent staining and cytofluorometric measurements

Quantitative immunofluorescence measurements were used to detect the expression of H-2Kb determinants by LM-IL-2 cells transfected with pBR327H-2Kb. The measurements were performed in an Epic V flow cytofluorograph (Coulter Electronics, Hialeah, FL) equipped with a multiparameter data acquisition and display system (MDADS). For the analysis, a single cell suspension was prepared from the monolayer cultures with 0.1 mM EDTA in PBS. The cells were washed with PBS containing 0.2% sodium azide and 0.5% FBS. Afterward, FITC-conjugated H-2Kb, H-2Db, H-2Kk mAbs (PharMingen, San Diego, CA) or FITC-conjugated IgG2a isotype serum (Dako Corp., Carpenteria, CA) was added to the cells, followed by incubation at 4°C for 1 h. The cells were then washed with PBS containing 0.5% FBS and 0.2% sodium azide. One-parameter fluorescence histograms were generated by analyzing at least 1 x 104 cells. Background staining was determined by substituting cells stained with FITC-conjugated mouse IgG2a alone for cells stained with the specific Abs. The 15% of cells that stained with the highest intensity was separated into 15-ml conical tubes (Falcon, Franklin Lakes, NJ) containing DMEM supplemented with 50% FBS. Immediately afterward, the cells were centrifuged at low speed and resuspended in growth medium in plastic tissue culture plates (Falcon), followed by incubation at 37° in a humidified 7% CO2/air atmosphere.

Transfection of LM-IL-2Kb cells with DNA from B16 melanoma cells

DNA from B16 cells was used for the transfection of LM-IL-2Kb cells, or BLK-IL-2 cells, using the method described by Wigler et al. (45), as modified (40, 41). Briefly, high m.w. DNA was sheared by three passages through a 25-gauge needle. Afterward, 100 µg of the sheared DNA was mixed with 10 µg of pHyg (from L. Lau, University of Illinois), a plasmid that encodes the Escherichia coli enzyme hygromycin B phosphotransferase (46), conferring resistance to hygromycin B. The sheared DNA and pHyg were then mixed with lipofectin, according to the manufacturer’s instructions (Life Technologies). The DNA/lipofectin mixture was added to a population of 1 x 107 LM-IL-2Kb cells, or BLK-IL-2 cells, that had been divided into ten 100-mm plastic cell culture plates 24 h previously. Eighteen hours after addition of the DNA/lipofectin mixture to the cells, the growth medium was replaced with fresh growth medium. As a control, DNA from B16 cells was omitted, and 1 µg of pHyg alone mixed with lipofectin was added to an equivalent number of LM-IL-2Kb cells, or BLK-IL-2 cells. In both instances, the cells were maintained for 14 days in growth medium containing 500 µg/ml hygromycin B (Boehringer Mannheim Corp., Indianapolis, IN). One hundred percent of nontransfected LM-IL-2Kb cells, or BLK-IL-2 cells, maintained in the hygromycin growth medium died within this period. The surviving colonies (at least 5 x 104) of LM-IL-2Kb cells, or BLK-IL-2 cells, transfected with pHyg and DNA from the melanoma cells (LM-IL-2Kb/B16 cells, or BLK-IL-2/B16 cells, respectively) or with pHyg alone (LM-IL-2Kb cells or BLK-IL-2, respectively), were pooled and used in the experiments.

In some instances, the cells were disrupted by homogenization and sonication before injection into the mice. A quantity amounting to 4 x 106 LM-IL-2Kb/B16 cells suspended in 400 µl of growth medium was homogenized in a Takmar Tissue Mizer (Cincinnati, OH) for 1 min at 4°C, followed by sonication for 1 min at 4° in a Sonifier Cell Disrupter (VWR Scientific, Philadelphia, PA).

Spleen cell-mediated cytotoxicity determinations

A standard 51Cr release assay was used to detect the presence of cells with cytotoxic activity toward B16 cells. A spleen cell suspension was prepared by forcing the spleens though a number 40-gauge stainless steel screen in approximately 5 ml of ice-cold growth medium. Cells were collected and overlaid onto a Histopaque 1077 gradient (Sigma Chemical Co.) and then centrifuged (400 x g) for 30 min at room temperature. The viability of the mononuclear cells collected from the gradients at this point was greater than 98%, as determined by trypan blue dye exclusion (0.4%). Cells of the same type used to immunize the mice were treated with mitomycin C (Sigma Chemical Co.) (50 µg/ml for 45 min at 37°C). After washing, they were then coincubated in growth medium at 37°C for 5 days with aliquots of the spleen cell suspensions. The ratio of spleen cells to mitomycin C-treated cells was 30:1. (The incubation medium consisted of RPMI 1640 medium (Life Technologies) supplemented with 100 U/ml human IL-2, 10% FBS, 5 x 10-2 mmol 2-ß-mercaptoethanol, 15 mmol HEPES, 0.5 mmol sodium pyruvate, and penicillin/streptomycin (Life Technologies).) At the end of the 5-day incubation, the population that failed to adhere to the plastic cell culture flasks was collected and used as the source of effector cells for the cytotoxicity determinations.

For the assay, 5 x 106 target cells were labeled with 51Cr during a 1-h incubation at 37°C in growth medium containing 100 µCi 51Cr (Amersham). After three washes with DMEM, 1 x 104 of the 51Cr-labeled cells were incubated for 4 h at 37°C with the nonplastic-adherent population of spleen cells from the immunized mice, at varying E:T ratios. Afterward, the percentage of specific cytolysis was calculated as:

The spontaneous release ranged from 10 to 15% of the maximal 51Cr release.

Statistical analyses

The Student t test was used to determine the statistical differences between the survival and cytotoxic activities in mice in various experimental and control groups. A p value of less than 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Modification of LM mouse fibroblasts for IL-2 secretion

A replication-defective retroviral vector, pZipNeoSVIL-2, was used to modify LM cells (H-2k) for IL-2 secretion. The vector specified the gene for human IL-2, along with a gene (neor) conferring resistance to the neomycin analogue, G418. After selection in growth medium containing sufficient quantities of G418 to kill 100% of nontransfected cells, the surviving colonies were pooled and a biologic assay for IL-2 was performed. The results (Table IGo) indicated that 1 x 106 retrovirally transduced LM cells (LM-IL-2), or LM-IL-2 cells transfected with DNA from B16 cells (LM-IL-2/B16) formed approximately 100 U IL-2 in 48 h, as determined by the capacity of the culture supernatants to sustain the growth of IL-2-dependent CTLL-2 cells. LM-IL-2 cells modified for H-2Kb determinants (LM-IL-2Kb), LM-IL-2Kb cells transfected with DNA from B16 cells, or BLK cells modified for IL-2 secretion formed equivalent amounts of IL-2. The culture supernatants of LM cells, or BLK cells, transduced with the IL-2-negative vector, pZipNeoSV(X) (LM-ZipNeo cells) failed to sustain the growth of CTLL-2 cells. Every third passage, the IL-2-secreting cells were placed in medium containing 400 µg/ml G418. Under these conditions, equivalent quantities of IL-2 were detected in the culture supernatants of each of the cell types for more than 6 mo of continuous culture (these data are not presented).


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  		Table I. IL-2 secretion by genetically modified LM mouse fibroblasts transfected with pZipNeoSV-IL-21

 
Modification of LM-IL-2 cells for the expression of H-2Kb class I determinants

LM-IL-2 cells were modified to express H-2Kb determinants, by transfection with the plasmid pBR327H-2Kb (43). A second plasmid, pBabePuro (44), conferring resistance to puromycin, was included for selection. (The ratio of pBR327H-2Kb to pBabePuro used for transfection was 10:1.) The transfected, puromycin-resistant cells were selected in growth medium containing sufficient quantities of puromycin (3 µg/ml) to kill 100% of nontransfected LM-IL-2 cells. The surviving colonies were pooled, and the cell number was expanded in vitro.

The expression of H-2Kb determinants by the cells was measured by quantitative immunofluorescence, using FITC-labeled mAbs for mouse H-2Kb. As controls, aliquots of the puromycin-resistant cell suspension were incubated with FITC-labeled IgG2a isotype serum, or with FITC-labeled mAbs for H-2Kk, or H-2Db determinants. The results (Fig. 1Go) indicated that puromycin-resistant LM-IL-2 cells cotransfected with pBR327H-2Kb and pBabePuro stained positively with H-2Kb and H-2Kk mAbs, but not with IgG2a isotype serum or H-2Db mAbs. (LM cells are of C3H mouse origin.) The expression of H-2Kb determinants appeared to be a stable property of the cells. They stained with equivalent intensity with H-2Kb mAbs after 3 mo of continuous culture (these data are not presented).



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  		FIGURE 1. The expression of H-2Kb determinants by LM-IL-2 cells transduced with pBR327H-2Kb. A quantity amounting to 1 x 104 LM-IL-2 cells transduced with the plasmid pBR327H-2Kb (LM-IL-2Kb cells) was incubated for 1 h at 4° with FITC-conjugated anti-H-2Kb, anti-H-2Kk, or anti-H-2Db mAbs, as described in Materials and Methods. The cells were then analyzed for fluorescent staining by flow cytofluorography. Light line, cells incubated with IgG2a isotype serum. Bold line, cells incubated with anti-H-2Kb, anti-H-2Kk, or anti-H-2Db mAbs.

 
The survival of C57BL/6J mice injected with B16 melanoma cells and LM-IL-2Kb cells transfected with DNA from B16 cells

C57BL/6J mice exhibit no apparent resistance to the growth of B16 melanoma cells. One hundred percent of naive mice injected s.c. with 5 x 103 B16 cells die from progressive tumor growth.

The possible immunotherapeutic properties of LM-IL-2Kb/B16 cells were determined by comparing tumor growth in C57BL/6J mice injected s.c. with B16 cells alone with C57BL/6J mice injected with a mixture of B16 cells and LM-IL-2Kb/B16 cells and injected i.p. with LM-IL-2Kb/B16 alone. The mice were injected s.c. and i.p. twice more, at weekly intervals, with the same number of modified cells as in the initial injections, but without additional B16 cells. The results (Fig. 2Go) indicated that 100% of mice injected with the mixture of B16 cells and LM-IL-2Kb/B16 cells remained tumor free. The animals survived more than 120 days (Fig. 3Go). Under similar conditions, progressively growing neoplasms developed in mice injected with B16 cells alone (Fig. 2Go); the animals died from progressive tumor growth in approximately 38 days (Fig. 3Go; p < 0.001 for mice injected with B16 cells and LM-IL-2Kb/B16 cells and mice injected with B16 cells alone). Mice injected with a mixture of equivalent numbers of B16 cells and non-IL-2-secreting, non-DNA-transfected LM-ZipNeo cells developed progressively growing neoplasms and died in approximately 40 days (Figs. 2Go and 3Go). Other naive C57BL/6J mice were injected with a mixture of B16 cells and non-DNA-transfected, IL-2-secreting LM-IL-2 cells. The difference in the survival of mice injected with B16 cells alone, and with the mixture of B16 cells and LM-IL-2 cells was not significant (p > 0.1) (Fig. 3Go).



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  		FIGURE 2. Tumor growth in C57BL/6J injected with a mixture of B16 melanoma cells and LM-IL-2Kb/B16 cells. C57BL/6J mice (five per group) were injected s.c. with a mixture of 5 x 103B16 cells and 2 x 106 LM-IL-2Kb/B16 cells. At the same time, the mice received a second injection i.p. of 2 x 106 LM-IL-2 Kb/B16 cells alone. As controls, the mice were injected according to the same protocol with equivalent numbers of B16 cells alone, with B16 cells and LM-ZipNeo cells, with B16 cells and LM-IL-2 cells, or with B16 cells and LM-IL-2/B16 cells. The mice were injected s.c. and i.p. twice more, at weekly intervals, with the same number of modified cells as in the initial injections, but without additional B16 cells. Mean tumor volume was derived from two-dimensional measurements obtained with a dial caliper. —{blacksquare}—, Injected with B16 cells alone; —{diamond}—, injected with B16 cells and LM-ZipNeo cells; —{circ}—, injected with B16 cells and LM-IL-2 cells; —{blacktriangleup}—, injected with B16 cells and LM-IL-2/B16 cells; —•—, injected with B16 cells and LM-IL-2Kb/B16 cell.

 


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  		FIGURE 3. Survival of C57BL/6J mice injected with a mixture of B16 melanoma cells and LM-IL-2Kb/B16 cells. C57BL/6J mice (five per group) were injected s.c. with a mixture of 5 x 103B16 cells and 2 x 106 LM-IL-2 Kb/B16 cells. At the same time, the mice received a second injection i.p. of 2 x 106 LM-IL-2 Kb/B16 cells alone. As controls, other naive C57BL/6J mice were injected according to the same protocol with equivalent numbers of B16 cells and LM-ZipNeo cells, with B16 cells and LM-IL-2 cells, with B16 cells and LM-IL-2/B16 cells, or with B16 cells alone. The mice in each treatment group were injected twice more, at weekly intervals, with the same number of LM-IL-2Kb/B16, LM-ZipNeo cells, LM-IL-2, or LM-IL-2/B16 cells, but without additional B16 cells. Mean survival times: mice injected with viable B16 cells alone, 38.4 ± 2.8 days; mice injected with viable B16 cells and LM-ZipNeo cells, 39.4 ± 7.1 days; mice injected with viable B16 cells and LM-IL-2 cells, 47.7 ± 9.6 days; mice injected with viable B16 cells and LM-IL-2/B16 cells, 62.2 ± 12.2 days; and mice injected with viable B16 cells and LM-IL-2Kb/B16 cells, >120 days. Survival of mice injected with viable B16 cells and LM-IL-2Kb/B16 cells, relative to survival of mice in each of the other groups, p < 0.001. —•—, Injected with B16 cells alone; —{square}—, injected with B16 cells and LM-ZipNeo cells; —{triangleup}—, injected with B16 cells and LM-IL-2 cells; —{circ}—, injected with B16 cells and LM-IL-2/B16 cells; —{blacktriangleup}—, injected with B16 cells and LM-IL-2Kb/B16 cells.

 
To determine whether modification of the cells to form H-2Kb determinants augmented the cells’ immunotherapeutic properties, the survival of mice injected with B16 cells and LM-IL-2Kb/B16 cells was compared with the survival of mice injected with equivalent numbers of B16 cells and LM-IL-2/B16 cells. As indicated (Fig. 3Go), the survival of mice injected with the mixture of B16 cells and LM-IL-2Kb/B16 cells was significantly longer than that of mice injected with the mixture of B16 cells and LM-IL-2/B16 cells (p < 0.001).

Thus, the greatest immunotherapeutic benefit was in the group of mice treated with IL-2-secreting, DNA-transfected cells that expressed both syngeneic and allogeneic MHC determinants.

The possible long-term immunotherapeutic properties of LM-IL-2Kb/B16 cells were investigated by reinjecting mice that survived as a result of the initial treatment with a second injection of B16 cells. The second injection of B16 cells took place 150 days after the first. As indicated (Fig. 4Go), the first appearance of tumor was significantly (p < 0.001) delayed in the previously treated mice. The mice that received prior injections of B16 cells and LM-IL-2Kb/B16 cells survived significantly (p < 0.01) longer than naive mice injected with B16 cells alone (53 ± 7.1 days vs 34.3 ± 2.2 days, respectively).



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  		FIGURE 4. The time to the first appearance of tumor in mice surviving a prior injection of B16 cells and LM-IL-2Kb/B16 cells injected a second time with B16 cells alone. C57BL/6J mice surviving 150 days after the prior injection of B16 cells and LM-IL-2Kb/B16 cells were injected s.c. a second time with 5 x 103 B16 cells alone. As a control, naive C57BL/6J mice were injected s.c. with an equivalent number of B16 cells. There were five mice in each group. —•—, Surviving mice injected with B16 cells; —{circ}—, naive mice injected with B16 cells.

 
Treatment with LM-IL-2Kb/B16 cells prolonged the survival of mice with pre-existent melanoma

As described, mice injected with a mixture of B16 cells and LM-IL-2Kb/B16 fibroblasts survived significantly longer than mice injected with a mixture of B16 cells and LM fibroblasts that lacked one or more immunogenic properties. The treated animals exhibited long-term resistance to the growth of the melanoma. To determine whether LM-IL-2Kb/B16 cells had a similar immunotherapeutic effect on mice with pre-existent melanoma, C57BL/6J mice were injected s.c. with B16 melanoma cells, followed at varying times afterward by injections of LM-IL-2Kb/B16 fibroblasts. As indicated (Fig. 5Go), mice injected with B16 cells 5 or 10 days before the first injection of LM-IL-2Kb/B16 fibroblasts survived significantly longer than mice injected with B16 cells alone (p < 0.003 and p < 0.04, respectively). Mice injected with B16 cells 20 days before the first injection of LM-IL-2Kb/B16 fibroblasts failed to survive significantly longer than mice injected with B16 cells alone (median survival time = 34.4 ± 4 and 31.8 ± 6 days, respectively; p = 0.1).



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  		FIGURE 5. Survival of C57BL/6J mice with melanoma treated with LM-IL-2Kb/B16 cells. C57BL/6J mice were injected s.c. with 5 x 103 B16 cells. At varying times afterward, the mice were injected s.c. and i.p. with 2 x 106LM-IL-2Kb/B16 cells at each injection site. As controls, other naive C57BL/6J mice were injected s.c. with a mixture of 5 x 103 B16 cells and 2 x 106LM-IL-2Kb/B16 cells, and i.p. with 2 x 106 LM-IL-2Kb/B16 cells, or s.c. with an equivalent number of B16 cells alone. Mean survival time: mice injected with B16 cells alone, 31.8 ± 6.1 days; mice injected with a mixture of B16 cells and LM-IL-2Kb/B16 cells, 52.8 ± 9.9 days; mice injected with LM-IL-2Kb/B16 cells 5 days after the injection of B16 cells, 44.2 ± 5.8 days; mice injected with LM-IL-2Kb/B16 cells 10 days after the injection of B16 cells, 39.3 ± 3.6 days; and mice injected with LM-IL-2Kb/B16 cells 20 days after the injection of B16 cells, 34.4 ± 4 days. p for survival of mice injected with the mixture of B16 cells and LM-IL-2Kb/B16 cells vs mice injected with B16 cells 5 days before LM-IL-2Kb/B16 cells <0.005; for mice injected with B16 cells 10 days before the injection of LM-IL-2Kb/B16 cells, <0.04; and for mice injected with B16 cells 20 days before the injection of LM-IL-2Kb/B16 cells, <0.1. —•—, Injected with B16 cells alone; —{square}—, injected with B16 cells 20 days before LM-IL-2Kb/B16 cells; —{triangleup}—, injected with B16 cells 10 days before LM-IL-2Kb/B16 cells; —{circ}—, injected with B16 cells 5 days before LM-IL-2Kb/B16 cells; —{blacktriangleup}—, injected with a mix of B16 cells and LM-IL-2Kb/B16 cells.

 
Immunization of C57BL/6J mice with LM-IL-2Kb/B16 cells stimulated CD8+ T cell-mediated antimelanoma responses

Antimelanoma responses were generated in C57BL/6J mice immunized with LM-IL-2Kb/B16 cells. The involvement of CD8+ T cells and/or NK/lymphokine-activated killer cells in the antimelanoma response was determined in vitro by treating spleen cell suspensions from the immunized mice with CD8+ or asialo-GM1 mAbs before the cytotoxicity determinations toward B16 cells were performed.

In the experiment, naive C57BL/6J mice were injected three times at weekly intervals with LM-IL-2Kb/B16 cells. One week after the last injection, a suspension of spleen cells from the immunized mice was coincubated for 5 additional days with (mitomycin C-treated) LM-IL-2Kb/B16 cells. At the end of the incubation, 51Cr-labeled B16 cells were added and a standard cytotoxicity determination was performed in the presence or absence of excess quantities (3 times the amount required to saturate the relevant binding sites, as determined by flow-cytofluorometric analyses) of CD8+ or asialo-GM1 mAbs. As indicated (Table IIGo), the anti-B16 cytotoxic response was inhibited by CD8+ mAbs, and to a lesser extent by asialo-GM1 mAbs. Cytotoxicity responses toward B16 cells that were inhibitable by CD8+ mAbs were also present in spleen cell suspensions from mice immunized with (non-Kb-expressing) LM-IL-2 cells/B16 cells. The magnitude of these responses was less than that of cells from mice immunized with LM-IL-2Kb/B16 cells. Cytotoxic reactions toward B16 cells failed to develop in mice immunized with LM-ZipNeo, LM-IL-2, or non-DNA-transfected LM-IL-2Kb cells. As an additional control, C57BL/6J mice were immunized according to the same schedule with BLK fibroblasts (H-2b) modified to express IL-2 (BLK-IL-2), or with BLK-IL-2 cells transfected with DNA from B16 melanoma cells (BLK-IL-2/B16). The results indicated that unlike the responses following immunization with LM-IL-2Kb/B16 cells, significant antimelanoma responses failed to be detected in mice immunized with syngeneic BLK-IL-2 or BLK-IL-2/B16 cells (specific isotope release at a 100:1 E:T ratio for mice immunized with BLK-IL-2 or BLK-IL-2/B16 cells was 4.9 ± 1.6 and 5.8 ± 1.2, respectively). Reactions toward EO771 cells, a mammary carcinoma cell line of C57BL/6 origin, were not detected in mice immunized with LM-IL-2Kb/B16 cells or any of the other cell types.


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  		Table II. Cytotoxic responses toward B16 melanoma in C57BL/6J mice immunized with LM-IL-2Kb/B16 cells1

 
Intact LM-IL-2Kb/B16 cells were required for the generation of antimelanoma cytotoxic responses in C57BL/6J mice immunized with LM-IL-2Kb/B16 cells

The experiments described above were performed in mice immunized with viable LM-IL-2Kb/B16 cells. Injections with homogenized/sonicated LM-IL-2Kb/B16 cells were conducted to determine whether antimelanoma cytotoxic responses were generated in mice immunized with disrupted LM-IL-2Kb/B16 cells.

Naive C57BL/6J mice were immunized by i.p. and s.c. injections of viable, intact LM-IL-2Kb/B16 cells, or with an equivalent number of homogenized/sonicated, killed LM-IL-2Kb/B16 cells. The mice received two subsequent injections at weekly intervals of equivalent numbers of the viable or disrupted cells. One week after the last injection, a standard 51Cr release assay toward B16 cells was performed. As indicated (Fig. 6Go), cytotoxic reactions were detected in the group of mice injected with the viable, but not with the homogenized/sonicated cells. Spleen cells from naive C57BL/6J mice or mice injected with viable non-DNA-transfected LM-IL-2Kb cells, or viable LM-IL-2 cells failed to exhibit cytotoxic reactions toward B16 cells.



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  		FIGURE 6. Cytotoxicity toward B16 cells in C57BL/6J mice injected with viable or disrupted LM-IL-2Kb/B16 cells. C57BL/6J mice were injected s.c. and i.p. with 5 x 106 viable or disrupted (by homogenization/sonication) LM-IL-2Kb/B16 cells at each injection site. The mice received two subsequent injections at weekly intervals of the viable or disrupted cells. Other naive C57BL/6J mice were injected according to the same protocol with viable LM-IL-2Kb or LM-IL-2 cells, or with media. One week after the last injection, the mice were killed and pooled spleen cell suspensions from each group were tested for cytotoxic responses toward B16 cells in a standard 51Cr release assay. The experiment was performed three times with equivalent results in each instance.

 
H-2Kb mAbs reduced, but did not eliminate, the immunogenic properties of LM-IL-2Kb/B16 cells

The prior data indicated that the coexpression of H-2Kb determinants by LM-IL-2Kb/B16 cells augmented the cells’ immunogenic properties in C57BL/6J mice, and that intact cells were required. The data suggested that direct MHC-restricted presentation of TAA had occurred in the H-2b mice. To investigate this point in further detail, we determined the effect of H-2Kb mAbs on the induction of cellular anti-B16 melanoma responses in spleen cells from naive C57BL/6J mice coincubated with LM-IL-2Kb/B16 cells. As indicated (Table IIIGo), the presence of anti-Kb Abs during the induction phase (the Abs were added at the beginning of the incubation of the mixed cell culture) significantly reduced, but did not eliminate, the antimelanoma response. Under similar circumstances, H-2Kd mAbs failed to inhibit induction of the cellular immune response toward B16 cells. The experiment was conducted three times, with equivalent results (Table IIIGo).


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  		Table III. The effect of H-2Kb mAbs on the cytotoxic response toward B16 melanoma in mixed cell cultures of spleen cells from C57BL/6J mice and LM-IL-2Kb/B16 cells1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Under ordinary circumstances, progressively growing neoplasms do not induce antitumor immune responses that can control the growth of neoplastic cells. TAA expressed by the cells are insufficiently immunogenic. A variety of strategies have been used to increase the antigenic properties of tumor cells in the expectation that the induced antitumor immune responses may be useful in the treatment of cancer patients. In some instances, objective evidence of tumor regression has been observed in patients receiving only immunizations with tumor cell immunogens, as evidence of the potential of this form of therapy in the management of patients with malignant disease.

In prior studies (40, 47), we found that antitumor immunity was generated in mice immunized with IL-2-secreting allogeneic fibroblasts transfected with DNA from different mouse neoplasms. Immunizations with cells transfected with DNA from non-neoplastic cells failed to result in tumor immunity. The antitumor immune responses were directed toward the type of tumor from which the DNA was obtained, consistent with the expression of an array of undefined TAA that characterize the tumor cell population in a highly immunogenic form by the transfected cells.

In this work, we report that the immunotherapeutic properties of IL-2-secreting allogeneic LM mouse fibroblasts transfected with DNA from B16 melanoma cells were enhanced if the fibroblasts were modified to express MHC class I determinants that were syngeneic in the tumor-bearing host. Mice with melanoma treated solely by immunizations with the genetically modified semiallogeneic transfected fibroblasts survived significantly longer than mice in various control groups, including mice with melanoma treated with genetically modified fibroblasts that formed allogeneic MHC determinants alone. In some instances, the animals appeared to have rejected the melanoma cells and survived indefinitely.

Genetic modification of a highly immunogenic, non-tumor-specific fibroblast cell line expressing defined allogeneic MHC determinants, rather than modification of the melanoma cells themselves, was chosen for multiple, nonexclusive reasons. In prior studies (38), we found that melanomas developed in C57BL/6J mice injected solely with (nonirradiated) B16 cells modified for IL-2 secretion. The melanomas grew progressively and led eventually to the animals’ death. In repeated studies, tumors have not been observed in immunocompetent mice injected with nonirradiated, viable IL-2-secreting or nonsecreting cells that expressed foreign (allogeneic) MHC determinants. Like other allografts, the cells were rejected; they were no longer detectable approximately 14 days after injection. In addition, allogeneic Ags expressed by the cells chosen as recipients of DNA from the neoplastic cells had the properties of an immune adjuvant. In prior investigations, antimelanoma immunity failed to develop in C3H mice (H-2k) immunized with syngeneic fibroblasts transfected with melanoma DNA, under circumstances in which antimelanoma immunity developed in mice with b, s, or d haplotypes (39). The coexpression of allogeneic Ags by the cells protected against proliferation of the genetically modified cellular immunogen as it aided in the development of the antimelanoma response.

Genetic modification of a cell line, rather than modification of cells from the primary neoplasm, had an additional, important advantage. The establishment of a tumor cell line, a technically challenging prerequisite for its successful modification, was not required. The fibroblast cell line, modified for IL-2 secretion to increase its overall immunogenic properties (48), was readily transfected and proliferated under standard cell culture conditions. The genetic modifications were persistent, and the cell number could be expanded as desired.

We considered at least two possible, nonexclusive mechanisms to explain the resistance to melanoma in C57BL/6J mice-immunized LM-IL-2Kb/B16 cells. Large numbers of CTLs with specificity toward TAA expressed by a subpopulation of the transfected cells may have been generated in the microenvironment of allograft recognition and rejection. The immunogenic properties of tumor cells transfected with genes specifying allogeneic determinants are supportive of this interpretation (19, 20). In addition, MHC class I genes that share identity with the tumor-bearing host may present tumor-associated T cell epitopes directly to CTLs. Immunization with disrupted LM-IL-2Kb/B16 cells failed to result in antimelanoma cellular immune responses, and the presence of mAbs to H-2Kb, but not H-2Kd determinants inhibited the immunogenic properties of LM-IL-2Kb/B16 cells. As an added advantage, LM fibroblasts, including the cells used in these experiments, formed B7.1, a costimulatory molecule required for Ag-specific T cell activation (49), increasing the likelihood that T cells were directly stimulated by syngeneic class I determinants. Fibroblasts are reported to act as efficient APC (50).

The finding that antimelanoma immune responses were generated in mice immunized with the semiallogeneic fibroblasts transfected with DNA from the tumor was incompletely understood. The proportion of the transfected cells expected to incorporate and express the products of genes specifying TAA was expected to be small. The fact that specific antitumor immune responses followed immunizations with cells transfected with tumor DNA may be an indication that multiple, and possibly large numbers of immunologically distinct TAA were present within the population of melanoma cells. It is unlikely that a sufficient number of transfected cells expressing the product of a single gene specifying TAA would be present to induce an antitumor response in the naive mice.

The data reported in this work raise the possibility that an APC cell line that shares identity at one or more MHC class I alleles with the cancer patient may be readily modified to provide immunologic specificity for TAA expressed by the patient’s neoplasm. Transfection of the cell line with DNA from the patient’s neoplasm may provide a practical alternative to the modification of autologous malignant cells for the purposes of tumor immunotherapy.


   Footnotes
 
1 Supported by U.S. Army Medical Research and Material Command, Grant DAMD 17-96-1-6178. Back

2 Address correspondence and reprint requests to Dr. Edward P. Cohen, Department of Microbiology and Immunology (m/c 790), University of Illinois at Chicago, 835 South Wolcott Ave., Chicago, IL 60612. E-mail address: Back

3 Abbreviations used in this paper: TAA, tumor-associated antigens; neor, neomycin resistance gene. Back

Received for publication August 18, 1997. Accepted for publication November 24, 1997.


   References
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 Materials and Methods
 Results
 Discussion
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