Determination of cell-free interleukin 2 receptor level in the serum of normal animals and of animals bearing IL-2 receptor positive tumours with high or low metastatic capacity

Serum levels of cell-free interleukin-2 receptors were elevated above normal in mice bearing the IL-2R positive T-cell lymphoma Eb or its highly metastatic variant ESb. Although ESb cells expressed less IL-2R molecules than Eb cells on their cell surface, serum receptor levels were raised more quickly in ESb than in Eb tumour bearing animals. Elevated IL-2R serum levels were a sensitive tumour marker in animals bearing the aggressive variant ESb but not in animals bearing the low metastatic line Eb. Peritoneal ascites tumour-bearing animals had higher serum IL-2R levels than corresponding animals with subcutaneously growing tumours. Thus, serum IL-2R levels in tumour-bearing animals were dependent on the tumour line and influenced by the site and mode of tumour growth.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. IV. Antigenic differences between a metastasizing variant and the parental tumor line revealed by cytotoxic T lymphocytes.

The syngeneic cytotoxic T-cell response against a metastasizing murine lymphoma variant was investigated and compared with the response against the non-metastasizing parental tumor line Eb. Anti-tumor cytotoxicity was not detectable in a 4-h 51Cr release assay in spleens taken directly from tumor-bearing animals (primary CMC). After restimulation in vitro (secondary CMC) however, high anti-tumor cytotoxic activity was detected. This activity was mediated by immune T lymphocytes as shown by its sensitivity to treatment with anti-Thy 1.2 serum and complement. Ten cells of the metastasizing tumor ESb, inoculated subcutaneously, were sufficient to raise a local tumor and metastases and to induce cytotoxic T memory cells in the spleens. In contrast, about 104 cells were required to raise a local tumor and to induce splenic cytotoxic T memory cells, when the parental tumor Eb was tested. The specificity studies of the anti-tumor cytotoxic activity demonstrated that cytotoxic T cells could distinguish unrelated, chemically induced syngeneic tumors and also recognize antigenic differences between the parental tumor Eb and its variant ESb. Eb and ESb tumor cells were recognized as carrying distinct antigens at the responder cell level, the stimulator cell level and the target cell level. The in vivo significance of these findings is discussed.     

Anti-tumor effects of interleukin-2 and interleukin-1 in mice transplanted with different syngeneic tumors

We have studied the anti-tumor effects of human recombinant IL-2, alone or in association with LAK cells, in mice transplanted subcutaneously (s.c.) with the following syngeneic tumors: highly metastatic Friend leukemia cells (FLC), nonmetastatic FLC, lymphoma RBL-5 cells and HeJ16 fibrosarcoma cells. In these tumor models, peri-tumoral injections of IL-2 were more effective in inhibiting tumor growth than a systemic treatment. Although s.c. IL-2 treatment resulted in marked inhibition of tumor growth in mice injected s.c. with highly metastatic FLC, it was not effective in inhibiting growth of FLC in the liver and spleen. IL-2 therapy was more effective at increasing survival time in mice transplanted with non-metastatic FLC or with RBL-5 cells. In mice transplanted with HeJ16 fibrosarcomas, s.c. IL-2 treatment resulted in highly significant anti-tumor effect and survival of 70% of tumor-injected mice. No general correlation was found between in vitro sensitivity or resistance to the cytolytic activity of LAK cells and the anti-tumor effects observed in vivo. Subcutaneous injection of IL-1 beta in mice transplanted with highly metastatic FLC resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. Combined treatment of IL-1 beta and IL-2 produced a synergistic anti-tumor effect: 60% of mice injected with highly metastatic FLC survived. Combined IL-1/IL-2 treatments exerted no anti-tumor activity either in DBA/2 mice injected with antibody to Thy 1.2 antigen or in nude mice, indicating that T cells play important roles during IL-1/IL-2 therapy. In vitro treatment of FLC with IL-1 beta resulted in a slight inhibition of cell multiplication, whereas even high doses of IL-2 did not affect FLC multiplication. Our results indicate that local combined treatments with IL-1 and IL-2 can induce potent, host-dependent (T cell-mediated) anti-tumor effects against highly malignant tumors.     

Glycoconjugates of murine tumor lines with different metastatic capacities. II. Diversity of glycolipid composition

A syngeneic tumor system in DBA/2 mice consisting of a methyl-cholanthrene-induced, weakly metastatic lymphoma, L5178YE (= Eb), its spontaneous strongly metastatic variant, L5178YES (= ESb), and an unrelated, methylcholanthrene-induced, metastasizing tumor, MDAY-D2, were used to study the relationship between metastatic behavior and composition of GSLs. The D-1-14C galactose and D-1-14C glucosamine-labelled neutral GSL and gangliosides of these tumor cells, and additionally ConA-stimulated spleen T cells from normal mice, were analysed by thin-layer chromatography. Unlabelled GSLs of the tumors were also characterized by (HPLC) after perbenzoylation. Results obtained with the radioactively labelled GSLs correlated with those of the HPLC analysis of unlabelled GSLs. All tumors contained neutral GSL of the ganglio-series. Weakly metastatic tumor Eb showed neutral GSL patterns comparable to those from ConA-stimulated spleen cells, whereas strongly metastatic tumors ESb and MDAY-D2 had an enhanced expression of lactosylceramide. Gangliosides of metastatic ESb and MDAY-D2 had a higher degree of polarity than those of weakly metastatic Eb. Eb cells expressed primarily GM1. Metastasizing ESb and MDAY-D2 had significantly higher amounts of GM3, GM2 and GD1a. An unusual ganglioside, IV3GalNAc-GM1, was found in MDAY-D2 cells and ConA blasts. When the extent of label was compared in neutral GSLs and gangliosides, metastasizing ESb and MDAY-D2 were more heavily labelled in the ganglioside fraction (62%, 58%) than Eb (39%). ESb and MDAY-D2 also contained larger amounts of gangliosides than Eb.     

Cytogenetic changes during tumor progression towards invasion,metastasis and immune escape in the Eb/ESb model system

Related tumor lines which represent different stages in their progression towards metastatic capacity were investigated and compared at the chromosomal level. The parental low-metastatic tumor line (L5178Y/Eb) was derived from a long-term transplanted, chemically induced T-cell lymphoma of the DBA/2 mouse. The cytogenetic analysis included this Eb line, a spontaneous high metastatic variant thereof which expressed a distinct tumor-associated transplantation antigen (ESb TATA⁺), and an immunoresistant TATA-negative variant of the latter (ESb TATA^?). All three cell lines were characterized by a near-diploid chromosome count and by some common chromosomal markers derived from Nos. 6, 13 and 16 Large-scale chromosomal rearrangments resulted in the formation of eight marker chromosomes in Eb cells, 16 in ESb TATA⁺ cells and 18 in ESb TATA^? cells. Tumor progression in this system showed a tendency to monosomies, which could bring the corresponding genes to a hemizygous state and possibly to a release from repression. Chromosome 15 was trisomic in Eb cells, monosomic in ESb TATA⁺ cells and hardly detectable in ESb TATA^? cells. The Ig heavy-chain gene-carrying region of both chromosomes No. 12 was found in translocation with chromosomes Nos. 5, 13 and 14 (Eb cells) and with Nos. 1 and 17 (ESb cells). ESb TATA^? cells differed from ESb TATA⁺ cells at four different chromosomes (Nos. 5, 8, 14 and 15).     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. VI. Similar specificity patterns of protective anti-tumor immunity in vivo and of cytolytic T cells in vitro.

In an attempt to analyze mechanisms of immunity against tumor metastases, protective anti-tumor immunity in vivo was compared with cytotoxic T-cell activity in vitro in a well-defined syngeneic tumor model system. The system consists of a chemically induced parental tumor cell line (Eb) with little or no metastatic potential and a spontaneous variant thereof (ESb) with pronounced metastatic properties. Tumor protection experiments revealed the presence of tumor-associated transplantation antigens (TATAs) on both Eb and ESb tumor cells. TATAs of Eb and ESb were found to be distinct and non-cross-reactive. One of several unrelated tumors, however, RL♂1, expressed TATAs which cross-reacted with those of Eb. Protective immunity against the non-metastasizing tumor was much stronger than that against the metastasizing variant. Furthermore, the optimal procedures for induction of immunity in vivo were strikingly different for each tumor. Tumor-specific cytotoxic T lymphocytes (CTLs) were obtained after sensitization in vivo with viable tumor cells and restimulation in vitro for 4–5 days with mitomycin-C-treated autologous tumor cells. Both anti-Eb and anti-ESb CTLs showed high cytolytic activity in a 4-h ⁵¹Cr release assay against the autologous tumor lines. The target antigens recognized by these cells were similar to the TATAs as defined in the protection experiments. (1) The target antigens of Eb and ESb were distinct and non-cross-reactive. (2) Only one of 14 unrelated syngeneic and allogeneic tumors expressed a target antigen which cross-reacted with that of Eb. (3) This tumor was the radiation-induced BALB/c lymphoma RL♂1 which also cross-reacted at the level of the TATAs. The correlations between protective immunity obtained in vivo and cytolytic T cells induced in vitro suggest that cytolytic T cells can recognize TATAs and may thus play an important role in the establishment of protective immunity.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. I. Tumor invasiveness in vitro and metastasis formation in vivo

A syngeneic model system for the study of tumor metastases and cell-mediated immunity is described. The system consists of two related, chemically induced murine lymphomas, the non-metastasizing parental line Eb and its metastasizing variant ESb. An unrelated, chemically induced tumor (MDAY) is included for specificity controls. Serological typing revealed that both Eb and ESb were of T lymphoid origin and expressed the H-2K and H-2D molecules of the host strain DBA/2. By various electron microscope techniques, morphological differences were observed between the two cell lines. In comparison to Eb cells, ESb tumor cells had a more polymorphic nucleus with many in vagi-nations of the nuclear envelope and a more prominent expression of microvilli on the cell surface. An in vitro organ culture test for tumor invasiveness, presented here for the first time in a syngeneic murine system, revealed that ESb but not Eb tumor cells had the ability to attach to and invade normal tissue. Accordingly, ESb tumor cells showed higher malignancy in vivo. This was apparent from their higher tumorigenicity and their ability to disseminate and metastasize and to kill recipient mice more quickly. Upon histological examination of the local primary tumors a striking difference was noticed with regard to the degree of infiltration by host-derived mononuclear cells, mostly histiocytes. The non-metastasizing tumor Eb was heavily infiltrated while tumor ESb contained only a few of these cells. The differences between the tumor lines ESb and Eb are considered in the light of their possible relevance for metastases in general. The etiology of the two tumors is discussed in particular with respect to their relatedness.     

Tumor metastases and cell-mediated immunity in a model system in DBA/2 mice. X. Immunoselection of tumor variants differing in tumor antigen expression and metastatic capacity

We previously described morphological, functional and antigenic differences between a chemically-induced DBA/2 lymphoma, Eb, and a spontaneous variant, ESb, which arose in 1968 and had highly increased metastatic capacity. Now we present evidence that the two cell lines, in spite of the differences observed, are still related. (1) Shifts from Eb to ESb can be reproduced after 11 years. They occur during routine i.p. transplantation, especially when high cell numbers are passaged. (2) ESb variant cells can be recovered from Eb tumor populations after immunoselection in vivo with specific anti-Eb cytolytic T lymphocytes (CTL). (3) Parental-type Eb cells can be isolated from ESb tumor populations after immunoselection in vitro with specific anti-ESb CTL Ten ESb cells mixed with 10(6) Eb cells and inoculated s.c. caused a shift in the mortality curve suggesting that a possible contamination of the Eb population with pre-existing ESb variant cells was less than 1 in 100,000.     

Prevention of metastatic spread by postoperative immunotherapy with virally modified autologous tumor cells. III. Postoperative activation of tumor-specific CTLP from mice with metastases requires stimulation with the specific antigen plus additional signals

DBA/2 mice transplanted with the high metastatic syngeneic lymphoma variant ESb can be successfully treated by a combination of surgery and active-specific immunotherapy (ASI). The ASI procedure was applied postoperatively and involved vaccination either (1) with inactivated ESb tumor cells which had been modified by incubation with a low dose of Newcastle Disease Virus (NDV) or (2) with mutagenesis-derived immunogenic tumor variants. The analysis of the immune status of spleens from tumor-bearing or tumor-immune mice revealed important differences. The activation of tumor-specific cytotoxic T-lymphocyte precursors (CTLP) from mice with metastasis required stimulation with the specific antigen plus additional helper factors (IL-2 or NDV). The importance of second signal immune stimulation for the activation of sensitized tumor-specific CTLP in tumor-bearing mice is underlined.     

Qualitative differences in position of sialylation and surface expression of glycolipids between murine lymphomas with low metastatic (Eb) and high metastatic (ESb) potentials and isolation of a novel disialoganglioside (GD1 alpha) from Eb cells

Glycolipids of murine lymphoma cell lines with low metastatic (Eb) and high metastatic (ESb) potentials have been investigated. The Eb cell line was characterized by a high quantity of gangliotriaosylceramide (Gg3), gangliotetraosylceramide (Gg4), GM1b, and a new type of disialoganglioside, termed GD1 alpha. In contrast, the high metastatic ESb cell line was characterized by the absence of these glycolipids and instead by the presence of GM3, GM2, GM1a, GD1a, and GD1b gangliosides. A clear cell surface reactivity with monoclonal antibody anti-Gg3 (2D4) was observed only in Eb cells. Thus, Eb cells are distinct from ESb cells in their ability to add the GalNAc residue to LacCer, supplying Gg3 for synthesis of a series of glycolipids via an asialogangliotetraosyl pathway, while ESb cells are capable of synthesizing GM3, which initiates synthesis of ganglio-series gangliosides GM2, GM1a, GD1a, and GD1b. While disialogangliosides of ESb cells were identified as GD1a and GD1b, a disialoganglioside isolated from Eb cells was characterized as having a novel structure (referred to as GD1 alpha) as follows: (formula; see text) Thus, Eb and ESb cells are clearly different in their qualitative sialylation patterns, i.e., the position of sialic acid residues. Cell surface labeling with galactose-oxidase/NaB[3H]4 revealed a high exposure of Gg3 and Gg4 at the Eb cell surface, while both labels were absent in ESb cells. In contrast, ESb cells showed a substantial label at GM1a, which was greatly enhanced after sialidase treatment.     

Differential induction of the two early genes c-jun and c-fos in weakly and strongly metastatic murine lymphoma cell lines

Induction of the 2 early-response genes c-jun and c-fos was investigated in the weakly metastatic T-lymphoma Eb line and the related strongly metastatic lymphomacrophage ESb line to find possible correlations with their different in vitro and in vivo phenotypes. The response of c-jun was elicited by the protein kinase-C activators TPA and A23187 in ESb but not in Eb cells. A much lower response of c-fos was also found in Eb than in ESb cells, in this case by means of scrum and the cAMP elevator forskolin. However, both TPA and the calcium ionophore A23187 were similarly effective in inducing fos-mRNA in both cell lines. The uncoupling of c-jun and c-fos induction in Eb, but not in ESb cells, as well as the uncoupling of c-fos response to different stimulators, point to a differential activation of these 2 early-response genes by the main signal transduction pathways in the 2 cell types. The coordinate/uncoordinate availability of the fos/jun heterodimer may confer distinct regulatory patterns on different target genes in ESb/Eb cells. Activation of these genes may underlie the distinct differentiation phenotypes and in vivo behavior of Eb/ESb cells. © Wiley-Liss, Inc.     

Suggestive evidence that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage

Two lines of evidence are reported which suggest that the highly metastatic variant ESb of the T-cell lymphoma Eb is derived from spontaneous fusion with a host macrophage. Firstly, ESb cells are shown to express the macrophage differentiation antigen Mac-1 which was not found on Eb cells or on any other tumor cells tested except the macrophage tumor line Pu5. Secondly, the progression from low to high metastatic capacity could be reproduced in vitro following hybridization of thioguanine-resistant Eb cells (EbTGR) with syngeneic bone-marrow-derived macrophages. Two HAT medium-selected hybrid tumor lines (Eb-F1 and Eb-F2) could be established. They were found to express cell surface markers of both parental lines: T lymphoid differentiation antigens from T-lymphoma and macrophage antigens (Mac-1, class II MHC antigens) from the normal cell fusion partner. The antigens were identified on the hybrids and subclones thereof by means of monoclonal antibodies and 3 different detection assays: cytofluorography, complement-dependent cytotoxicity and immunoprecipitation followed by gel electrophoresis. Animals inoculated s.c. with the parental line EbTGR developed local tumors but not metastases and survived for more than 40 days. In contrast, animals inoculated similarly with Eb-F1 or Eb-F2 cells quickly developed metastases in visceral organs and died as early as 10-14 days following inoculation. In many but not all respects, the in vitro-derived T-lymphoma-macrophage hybrids resembled the spontaneous in vivo-derived variant ESb. These findings, together with the presence of Mac-1 antigen on ESb cells, suggest (1) that ESb variant cells may be derived from spontaneous fusion with a host cell, most likely a macrophage and (2) that somatic cell fusion may be an important mechanism of genetic rearrangements leading to metastatic variants. The new highly metastatic tumor lines which were developed under well-defined in vitro conditions, and their subclones, may become very useful tools for studying the contribution of specific genetic traits and of membrane-related structures to various steps of the metastatic process.     

Synergistic anti-tumor effects of combined IL-1/IFN-alpha/beta therapy in mice injected with metastatic Friend erythroleukemia cells

Peritumoral injection of relatively low doses of either mouse interferon (IFN)-alpha/beta (10,000-20,000 units/injection) or of recombinant human interleukin-1 (IL-1) beta (125-250 ng/injection) in mice transplanted s.c. with Friend erythroleukemia cells (FLC) resulted in some inhibition of primary tumor growth, inhibition of liver and splenic metastases and increased survival time. A synergistic anti-tumor effect was observed in mice injected with both IL-1 and IFN-alpha/beta. Highly purified mouse IFN-beta also exerted a synergistic anti-tumor effect when combined with IL-1-beta in mice injected with FLC. The anti-tumor action of IL-1/IFN was markedly reduced in mice treated with antibodies to CD4 antigens. Antibodies to asialo-GM1 also diminished the anti-tumor effect by the combined cytokine treatment. The combined IL-1/IFN therapy was effective in NK-deficient bg/bg mice, although the extent of the anti-tumor response in these mice was less than that observed in bg/+mice.     

Anti-tumor Effects of Interleukin-4 and Interleukin-5 against Mouse B Cell Lymphoma and Possible Mechanisms of Their Action

We investigated the anti-tumor effects of recombinant mouse interleukin (IL)-4 and IL-5 by using a transplantable B cell lymphoma 38C13 cell line as a model. Daily local administration of either IL-4 or IL-5 produced moderate but significant inhibition of the rate of local tumor growth and prolongation of mean survival time (MST) in syngeneic C3H/HeJ mice; these anti-tumor effects appeared to plateau at low doses. Histopathologic and immuno-histochemical examination revealed necrotic changes in the cytokine-treated tumors, associated with infiltration of inflammatory cells such as eosinophils, macrophages, and lymphocytes. The infiltrating lymphocytes were found to be Thy-1.2⁺ T cells. To elucidate the importance of T cells, the rate of tumor growth and the MSTs were compared between athymic T cell-deficient BALB/c nude mice and immunocompetent C3H/HeJ mice. In the nude mice the transplanted tumor grew more rapidly and the MST was shorter than in the normal mice, suggesting a significant contribution of infiltrating T cells in the anti-tumor effects of the interleukins. Lastly, in vitro, growth inhibition of the 38C13 cells was observed in a dose-dependent manner at relatively high concentrations of either cytokine. Therefore, we conclude that both IL-4 and IL-5 have moderate anti-tumor effects against 38C13 B cell lymphoma both in vivo and in vitro , and that the observed in vivo anti-tumor effects are probably mediated both by tumoristatic action of infiltrating cells, such as eosinophils, macrophages and T lymphocytes, and by direct anti-proliferative action of the recombinant cytokines.     

Anti-tumor effects of interleukin-4 and interleukin-5 against mouse B cell lymphoma and possible mechanisms of their action.

We investigated the anti-tumor effects of recombinant mouse interleukin (IL)-4 and IL-5 by using a transplantable B cell lymphoma 38C13 cell line as a model. Daily local administration of either IL-4 or IL-5 produced moderate but significant inhibition of the rate of local tumor growth and prolongation of mean survival time (MST) in syngeneic C3H/HeJ mice; these anti-tumor effects appeared to plateau at low doses. Histopathologic and immuno-histochemical examination revealed necrotic changes in the cytokine-treated tumors, associated with infiltration of inflammatory cells such as eosinophils, macrophages, and lymphocytes. The infiltrating lymphocytes were found to be Thy-1.2+ T cells. To elucidate the importance of T cells, the rate of tumor growth and the MSTs were compared between athymic T cell-deficient BALB/c nude mice and immunocompetent C3H/HeJ mice. In the nude mice the transplanted tumor grew more rapidly and the MST was shorter than in the normal mice, suggesting a significant contribution of infiltrating T cells in the anti-tumor effects of the interleukins. Lastly, in vitro, growth inhibition of the 38C13 cells was observed in a dose-dependent manner at relatively high concentrations of either cytokine. Therefore, we conclude that both IL-4 and IL-5 have moderate anti-tumor effects against 38C13 B cell lymphoma both in vivo and in vitro, and that the observed in vivo anti-tumor effects are probably mediated both by tumoristatic action of infiltrating cells, such as eosinophils, macrophages and T lymphocytes, and by direct anti-proliferative action of the recombinant cytokines.     

Glycoconjugates of murine tumor lines with different metastatic capacities. I. Differences in fucose utilization and in glycoprotein patterns

Since various experimental findings point towards an important role of cell surface carbohydrates - in particular sialic acid - in cancer metastasis, the rationale of this study was to look for possible differences in carbohydrate metabolism and glycoprotein expression in well-defined related tumor lines of different metastatic capacity. The tumor lines analyzed were L5178Y E (=Eb), a low-metastasizing, methylcholanthrene-induced lymphoma of a DBA/2 mouse, and L5178Y ES(=ESb), a spontaneous high-metastatic variant thereof. A non-related, highly metastasizing tumor, MDAY-D2, and ConA-stimulated spleen cells were included in the study. These cell lines were compared for incorporation rates of various labelled carbohydrates and for glycoprotein patterns in SDS-polyacry-lamide gels. Marked differences were observed in the incorporation of ³H-fucose, while the incorporation of ³H-glactose and ³H-mannose was similar inthe different cell lines studied. Only the metastatic variant ESb incorporated ³H-fucose at a rate similar to that of ConA-stimulated T-cell blasts. Eb cells did not incorporate ³H-fucose while MDAY-D2 had a significantly lower ³H-fucose incorporation rate. Separation and purification of the intracellular products of ³H-fucose by gel filtration and high-voltage electrophoresis revealed in Eb cells a block in the synthesis of fucosylated glycoproteins at the step of the fucose-I-P-guanylyttransferase. No apparent defect in the fucose pathway was detectable in MDAY-D2 cells. An Eb → ESb shifted cell line regained the ability to incorporate ³H-fucose. All tumors displayed unique glycoprotein patterns in SDS-PAGE. Labelling with ³H-mannose revealed the most distinct bands, while labelling with ³H-galactose gave fewer and broader bands. Although clonal instability of metastasizing tumor variants has been frequently reported, subclones of Eb and ESb showed characteristics similar to those of the original cell lines with regard to metastatic capacity, fucose metabolism and glycoprotein expression. These results will be discussed in relation to differences in fucose metabolism and in surface expression of fucose as observed in other tumor systems consisting of high- and low-metastatic lines.     

Acquired immunity in nude mice induced by expression of the IL-2 or IL-4 gene in human pancreatic carcinoma cells and anti-tumor effect generated by in vivo gene transfer using retrovirus.

We have examined the anti-tumor effect in nude mice caused by human pancreatic cancer cells (AsPC-1) modified to secrete IL-2 or IL-4. Loss of tumorigenicity of cytokine-producing, but not wild-type, cells was observed despite their unaltered in vitro proliferation rates; and these anti-tumor effects were dependent on the amount of cytokine released. Wild-type cells inoculated into mice which had rejected IL-2- or IL-4-producer cells showed significant growth retardation, while no retardation was detected when unrelated human colon carcinoma cells were inoculated. Histological examination of regressing IL-2- or IL-4-producing AsPC-1 tumors in nude mice revealed infiltration by CD11b-, but not CD90-, positive cells around the tumors. Treatment of nude mice with anti-asialoGM(1) antibody did not affect loss of tumorigenicity. Mice injected i.p. with IL-2- or IL-4-producing AsPC-1 cells did not die, in contrast to mice inoculated with wild-type cells. Injection of retrovirus-bearing IL-2, but not beta-galactosidase, gene into mice which had wild-type cells in the peritoneal cavity also significantly prolonged survival. Thus, expression of the IL-2 or IL-4 gene in AsPC-1 cells may generate tumor-specific acquired immunity, even in mature T cell-deficient conditions. An anti-tumor response can be induced by in vivo transfer of the IL-2 gene.     

Microvascular injury in pathogenesis of interferon-induced necrosis of subcutaneous tumors in mice

DBA/2 mice were injected sc with cells from the highly malignant Friend erythroleukemia cell (FLC) 3Cl8 subline, which is resistant to mouse interferon alpha/beta, or with the ESb lymphoma. When interferon alpha/beta was injected intratumorally or peritumorally, tumor growth was markedly suppressed, and established vascularized tumor nodules became progressively necrotic. Tumor necrosis was of the coagulation type that usually results from deprivation of blood flow. Morphologic examination of approximately 1,000 blood vessel profiles and approximately 2,000 endothelial cells in 1-micron Epon sections of sc 3C18 FLC tumors showed that interferon treatment resulted in rapid and pronounced vascular endothelial cell damage that preceded tumor necrosis. No inflammatory cell infiltrate was observed. Our results suggest that interferon alpha/beta exerted an antitumor effect in these tumor models by damaging tumor blood vessels, causing disruption of tumor blood flow, which led to ischemic tumor necrosis.     

B7-2 expression above a threshold elicits anti-tumor immunity as effective as interleukin-12 and prolongs survival in murine B-cell lymphoma

The costimulatory molecule, B7-2, is expressed by various lymphomas, but this level of expression is not sufficient to generate effective anti-tumor immunity in vivo. To determine whether up-regulated expression of the costimulatory molecule, B7-2, leads to more effective anti-tumor immunity in vivo, the A20 murine model of B-cell lymphoma was used. A20 tumor cells express major histocompatibility complex (MHC) I and II molecules and moderate constitutive levels of B7-2. While B7-1 and B7-2 have been introduced into tumor cells lacking these molecules, studies have not been conducted to determine whether tumors that constitutively express B7-1 or B7-2 can be made more immunogenic by increasing the expression of these molecules. In this report, A20/B7-2 transfectants expressing greater levels of B7-2 were rejected in syngeneic mice, and systemic immunity against the A20 parental cells was generated. Treatment with the A20/B7-2 variant cells significantly improved the survival of tumor-bearing mice. Coinjection with IL-12 secreting variants did not further augment the anti-tumor immunity observed for B7-2 therapy alone. Both CD8(+) T cells and natural killer (NK) cells mediated the anti-tumor immune response observed in A20/B7-2 immunized mice. In mice that developed tumors after immunization with the A20/B7-2 variant cells, resected tumor cells were shown to express lower levels of B7-2 than the transfected variants. These results suggest that the level of costimulation is important for the generation of anti-tumor immunity and for host survival. In addition, tumors appear to be able to evade the immune response by downregulating the expression of B7-2 below a threshold level.