IFN-gamma and Fas/FasL are required for the antitumor and antiangiogenic effects of IL-12/pulse IL-2 therapy.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Administration of interleukin 12 with pulse interleukin 2 and the rapid and complete eradication of murine renal carcinoma.

BACKGROUND: Interleukin 2 (IL-2) and interleukin 12 (IL-12) are potent immunoregulatory cytokines that exhibit antitumor activity. Preliminary evidence suggests that combined administration of IL-2 and IL-12 may yield greater antitumor activity than that observed with either agent alone. PURPOSE: We evaluated the ability of combination regimens of IL-2 and IL-12 to induce regression of established primary and metastatic murine renal carcinoma (Renca) tumors. METHODS: BALB/c mice were given either subcutaneous or intrarenal injections of 10(5) Renca cells; tumor cell injections were given to 10-12 mice for each treatment group. Mice bearing subcutaneous primary tumors were treated with chronic IL-2 (300,000 IU given on a daily basis) or pulse IL-2 (300,000 IU given twice daily one day per week) alone, IL-12 alone (0.5 micrograms given on a daily basis), or IL-12 in combination with either chronic or pulse IL-2. Mice with metastatic tumors (arising from intrarenal implants; animals were nephrectomized to remove the primary tumors) were treated with IL-12 plus or minus pulse IL-2; in these experiments, IL-12 was given at doses of either 0.5 or 1.0 micrograms. In most experiments, treatment was continued for at least 3 weeks. Two-sided statistical tests were used to evaluate the data. RESULTS: Most mice with subcutaneous Renca tumors treated with the combination of IL-12 and chronic IL-2 died of treatment-related toxic effects within 7-14 days. In contrast, treatment with IL-12 plus pulse IL-2 was well tolerated, and six of 10 mice experienced complete tumor regression; none of the mice treated with either IL-12 alone or pulse Il-2 alone experienced a curative response. Seven of eight and nine of nine mice with metastatic tumors experienced complete tumor regression after treatment with 0.5 micrograms IL-12 plus pulse IL-2 or 1.0 microgram IL-12 plus pulse IL-2, respectively; two of 12 mice treated with pulse IL-2 alone and 10% or less of mice treated with IL-12 alone were cured of metastatic tumors (with 0.5 micrograms IL-12, none of 10 mice; with 1.0 micrograms IL-12, one of 10 mice). Five of 10 mice with metastatic tumors treated with a short-course regimen of IL-12 and pulse IL-2 (two pulses of IL-2 flanking 5 days of 0.5 micrograms IL-12) experienced complete tumor regression, while only one of the 12 mice treated with IL-2 alone and none of the mice treated with IL-12 alone experienced complete tumor regression. Virtually all curative response frequencies obtained with IL-12 and pulse IL-2 combination regimens differed significantly (P < .05) from those obtained with corresponding single-agent treatments. CONCLUSIONS: IL-12 administered in combination with pulse IL-2 induced rapid and complete regression of primary and metastatic Renca tumors and displayed greater antitumor activity than that observed with either IL-12 or IL-2 alone.

Interleukin 12 primes macrophages for nitric oxide production in vivo and restores depressed nitric oxide production by macrophages from tumor-bearing mice: implications for the antitumor activity of interleukin 12 and/or interleukin 2.

Interleukin-12 (IL-12) is a recently described immunoregulatory cytokine with potent therapeutic activity in various preclinical models of infectious or malignant disease. As part of our ongoing evaluation of potential mechanisms accounting for the potent antitumor activity of IL-12, we have investigated the influence of IL-12 administration on total serum nitrate/nitrite (NO(x)(-)) levels and the production of nitric oxide (NO) by peritoneal macrophages from normal and tumor-bearing mice. We report here that IL-12 administration to either normal or tumor-bearing mice for periods of time ranging from 7-19 days induced progressive increases in serum NO(x)(-) levels and primed peritoneal macrophages for NO production on subsequent exposure to lipopolysaccharide or IL-2 ex vivo. Treatment of resident peritoneal macrophages or the macrophage cell line ANA-1 with IL-12 alone or IL-12 in combination with various other stimuli failed to induce NO production, suggesting that the effects of IL-12 occurred via an indirect mechanism. Furthermore, we have shown that not only was the production of NO by macrophages from untreated long-term, tumor- bearing mice suppressed compared with control mice treated with vehicle or IL-12, but also that IL-12 administration overcame this suppression and delayed tumor growth. Lastly, we have shown that administration of weekly pulses of IL-2 in combination with IL-12 additively enhanced the priming of macrophages for NO production ex vivo and delayed tumor growth far more effectively than either agent alone. These observations and reports in the literature regarding the potential influence of NO on development of the immune response and on the regulation of tumor growth and vascularization suggest that NO may play a significant role in the antitumor activity of IL-12 and IL-2.

Induction of transplantable mouse renal cell cancers by streptozotocin: in vivo growth, metastases, and angiogenic phenotype.

Interleukin-2-based regimens of biological therapy have shown some clinical promise for the treatment of kidney cancer in humans, although the mechanisms responsible for tumor regression occurring in these patients remain unclear. Preclinical insight into these mechanisms is limited by a paucity of orthotopic animal models of kidney cancer. We have used streptozotocin, an antibiotic and diabetogenic nitrosamine compound derived from Streptomyces achromogenes to induce new kidney tumors in BALB/c mice. Single or multiple doses of streptozotocin induced kidney tumors in up to 25% of mice by 50-90 weeks of age, with up to 18% characterized as renal cell carcinomas (RCCs). Several transplantable lines were obtained from the RCCs, and one of these lines was subsequently cloned. The initial tumor isolates and sublines were histologically reconfirmed to be RCCs, and all grew progressively but slowly (mean survival times, 57 to >100 days) in vivo after intrarenal implant. None of the primary isolates or sublines revealed mutations in either the VHL or Ras genes, although karyotype analysis and chromosome painting revealed the consistent presence of a submetacentric chromosome resulting from the fusion of chromosomes 16 and 19. Biological characterization of these tumors revealed several features analogous to the growth of human kidney cancers, including a propensity for the formation of lung metastases and high vascularity. This hypervascularity is evident by both gross and microscopic analysis and correlates with the expression of several proangiogenic genes. Overall, the features of orthotopic transplantability, slower in vivo growth (relative to the rapid growth rates of other transplantable mouse kidney tumors), propensity for lung metastases, and hypervascularity may make these tumors valuable models for the study of new therapeutic strategies based on antineovascular agents and antitumor cytokines.

Antitumor activity of interleukin 12 in preclinical models.

Interleukin 12 (IL-12) is a heterodimeric cytokine with a number of biological effects that are consistent with its potential role as an antitumor agent. The antimetastatic and antitumor activities of IL-12 have been demonstrated in a number of murine tumor models. Both the inhibition of established experimental pulmonary or hepatic metastases and a reduction in spontaneous metastases have been achieved by treatment with murine IL-12. Systemic treatment of mice bearing subcutaneous tumors with IL-12 results in tumor growth inhibition, prolongation of survival, and, in some models, tumor regression. The antitumor effect of IL-12 in these models is dose-dependent and can be initiated against well-established tumors. Mice cured of their tumor by IL-12 treatment are specifically immune to rechallenge with the same tumor. A series of experiments have demonstrated that both T-cells and interferon-gamma (IFN-gamma) induction are necessary for the optimal antitumor effects of IL-12. However, the antitumor efficacy of IL-12 has not been observed after exogenous administration of murine IFN-gamma, suggesting that additional factors may be important for the antitumor effects of IL-12. In several tumor models, IL-12 is more active or has a larger therapeutic window than either IL-2 or IFN-alpha, two cytokines with demonstrated antitumor activity against human malignancies. Combining IL-12 with other cytokines or chemotherapeutic drugs can improve antitumor effects.

Reversal of ferritin-mediated immunosuppression by levamisole: a rationale for its application to management of the acquired immune deficiency syndrome (AIDS).

Ferritin is a complex polypeptide which functions primarily as an iron-storage protein. Ferritin may also play a role in the modulation of immune function. It is known to suppress several global measures of the immune response. Specifically, ferritin may mask and/or down-regulate expression of cell surface molecules important in T-cell activation and effector functions. These interactions may become pathologically significant in conditions where marked hyperferritinemia occurs, most notably malignancies and the acquired immunodeficiency syndrome (AIDS). Levamisole appears to possess immunomodulatory properties and be capable of disrupting the interaction of ferritin with T lymphocytes. This activity may be therapeutically useful in conditions of ferritin excess, such as progressive human immunodeficiency virus (HIV) infection and its associated opportunistic complications.

The potential role of serum ferritin in the pathogenesis of acquired immune deficiency syndrome (AIDS).

Elevated serum ferritin levels have been observed in several disease states including various malignancies, inflammatory states, and the acquired immunodeficiency syndrome (AIDS). This discussion will examine the normal sequence of events in T cell activation and proliferation, as well as the various defects in these events, and monocyte/macrophage and NK cell activity seen in AIDS patients. Further, the potential role of a serum suppressor factor as a contributor to the profound immunosuppression seen in AIDS will be discussed, as will evidence suggesting that ferritin may be this factor. A model is presented to explain mechanisms by which ferritin might suppress immune function and further studies to elaborate these mechanisms are proposed.

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer.

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.

IFN-gamma-dependent delay of in vivo tumor progression by Fas overexpression on murine renal cancer cells.

The role of Fas in the regulation of solid tumor growth was investigated. Murine renal carcinoma (Renca) cells were constitutively resistant to Fas-mediated killing in vitro, but exhibited increased expression of Fas and sensitivity to Fas-mediated killing after exposure to IFN-gamma and TNF. Transfected Renca cells overexpressing Fas were efficiently killed in vitro upon exposure to anti-Fas Ab (Jo2). When Fas-overexpressing Renca cells were injected into syngenic BALB/c mice, there was a consistent and significant delay in tumor progression, reduced metastasis, and prolonged survival that was not observed for Renca cells that overexpressed a truncated nonfunctional Fas receptor. The delay of in vivo tumor growth induced by Fas overexpression was not observed in IFN-gamma-/- mice, indicating that IFN-gamma is required for the delay of in vivo tumor growth. However, there was a significant increase of infiltrated T cells and in vivo apoptosis in Fas-overexpressing Renca tumors, and Fas-overexpressing Renca cells were also efficiently killed in vitro by T cells. In addition, a strong therapeutic effect was observed on Fas-overexpressing tumor cells by in vivo administration of anti-Fas Ab, confirming that overexpressed Fas provides a functional target in vivo for Fas-specific ligands. Therefore, our findings demonstrate that Fas overexpression on solid tumor cells can delay tumor growth and provides a rationale for therapeutic manipulation of Fas expression as a means of inducing tumor regression in vivo.

Intradermal delivery of IL-12 naked DNA induces systemic NK cell activation and Th1 response in vivo that is independent of endogenous IL-12 production.

In this study four murine IL-12 naked DNA expression plasmids (pIL-12), containing both the p35 and p40 subunits, were shown to induce systemic biological effects in vivo after intradermal injection. Three of the four IL-12 expression vectors augmented NK activity and induced expression of the IFN-gamma and IFN-gamma-inducible Mig genes. Both IL-12 p70 heterodimer and IFN-gamma proteins were documented in the serum within 24 h after intradermal injection of the pIL-12o- plasmid, which also induced the highest level of NK activity in the spleen and liver among the IL-12 constructs. Interestingly, both p40 mRNA expression at the injection site and serum protein levels followed a biphasic pattern of expression, with peaks on days 1 and 5. Subsequent studies revealed that the ability of intradermally injected pIL-12o- to augment NK lytic activity was prevented by administration of a neutralizing anti-IL-12 mAb. Finally, injection of the pIL-12o- into BALB/c IL-12 p40-/- mice also resulted in a biphasic pattern of IL-12 p70 appearance in the serum, and induced IFN-gamma protein and activated NK lytic activity in liver and spleen. These results demonstrate that injection of delivered naked DNA encoding the IL-12 gene mediates the biphasic systemic production of IL-12-inducible genes and augments the cytotoxic function of NK cells in lymphoid and parenchymal organs as a direct result of transgene expression. The results also suggest that these naked DNA plasmids may be useful adjuvants for vaccines against infectious and neoplastic diseases.

Recruitment of hepatic NK cells by IL-12 is dependent on IFN-gamma and VCAM-1 and is rapidly down-regulated by a mechanism involving T cells and expression of Fas.

NK cells have been shown to be important antitumor or antiviral effector cells in the liver. In the present study we have examined the factors that regulate the initial recruitment and subsequent fate of hepatic NK and T cells in mice treated with IL-12 or IL-2. Daily administration of IL-12 caused a rapid initial increase in NK cells followed by a subsequent decrease that coincided with an accumulation of T cells. The recruitment of hepatic NK cells by IL-12, but not the subsequent T cell infiltrate, was abrogated in IFN-gamma(-/-) mice. In contrast, daily administration of IL-2 caused a sustained increase in liver-associated NK cells that was not diminished in IFN-gamma(-/-) mice. The IL-12-induced recruitment in both hepatic NK and T cells was abrogated by in vivo treatment with anti-VCAM-1 mAbs, while treatment with anti-ICAM-1 Abs decreased only the recruitment of T cells in the IL-12-treated mice. The rapid loss of newly recruited hepatic NK cells in IL-12-treated mice did not occur in SCID mice or in B.MRL-Fas(lpr) (Fas-) and B6Smn.C3H-Fasl(gld) (FasL-) mutant mice, suggesting that T cells can actively eliminate hepatic NK cells through a Fas-dependent mechanism. These findings also imply that during the endogenous innate immune response to infectious agents or tumors or in the host response induced by cytokine therapies, the biologic effects of NK cells may be limited by T cell-mediated effects.

Molecular mechanisms of immune-mediated lysis of murine renal cancer: differential contributions of perforin-dependent versus Fas-mediated pathways in lysis by NK and T cells.

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

Primary hepatocytes from mice treated with IL-2/IL-12 produce T cell chemoattractant activity that is dependent on monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2).

The IFN-gamma-inducible proteins monokine induced by IFN-gamma (Mig) and chemokine responsive to gamma-2 (Crg-2) can contribute to IL-12-induced antiangiogenic and leukocyte-recruiting activities, but the extent to which leukocytes vs parenchymal cells in different organs contribute to the production of these molecules remains unclear. The results presented herein show that IFN-gamma-dependent induction of Mig and Crg-2 gene expression can occur in many nonlymphoid organs, and these genes are rapidly induced in purified hepatocytes isolated from mice treated with IL-2 plus IL-12, or from Hepa 1-6 hepatoma cells treated in vitro with IFN-gamma. In addition to depending on IFN-gamma, the ability of IL-12 or IL-2/IL-12 to induce Mig and Crg-2 gene expression in purified hepatocytes also is accompanied by the coordinate up-regulation of the IFN-gamma R alpha and beta-chains, in the absence of IL-12R components. Supernatants of primary hepatocytes obtained from mice treated in vivo with IL-2/IL-12 or from hepatocytes treated in vitro with IFN-gamma contain increased chemotactic activity for enriched human and mouse CD3(+) T cells, as well as mouse DX5(+) NK cells. The hepatocyte-derived chemotactic activity for mouse T cells but not NK cells was ablated by Abs specific for Mig and Crg-2. These results suggest that parenchymal cells in some organs may contribute substantially to initiation and/or amplification of inflammatory or antitumor responses.

Regulation of local host-mediated anti-tumor mechanisms by cytokines: direct and indirect effects on leukocyte recruitment and angiogenesis.

The regulation of tumor growth by cytokine-induced alterations in host effector cell recruitment and activation is intimately associated with leukocyte adhesion and angiogenic modulation. In the present study, we have developed a novel tumor model to investigate this complex series of events in response to cytokine administration. Gelatin sponges containing recombinant human basic fibroblast growth factor (rhFGFb) and B16F10 melanoma cells were implanted onto the serosal surface of the left lateral hepatic lobe in syngeneic C57BL/6 mice. The tumor model was characterized by progressive tumor growth initially localized within the sponge and the subsequent development of peritoneal carcinomatosis. Microscopic examination of the sponge matrix revealed well developed tumor-associated vascular structures and areas of endothelial cell activation as evidenced by leukocyte margination. Treatment of mice 3 days after sponge implantation with a therapeutic regimen consisting of pulse recombinant human interleukin-2 (rhIL-2) combined with recombinant murine interleukin-12 (rmIL-12) resulted in a marked hepatic mononuclear infiltrate and inhibition of tumor growth. In contrast to the control group, sponges from mice treated with rhIL-2/rmIL-12 demonstrated an overall lack of cellularity and vascular structure. The regimen of rhIL-2 in combination with rmIL-12 was equally effective against gelatin sponge implants of rhFGFb/B16F10 melanoma in SCID mice treated with anti-asialo-GM1 in the absence of a mononuclear infiltration, suggesting that T, B, and/or NK cells were not the principal mediators of the anti-tumor response in this tumor model. The absence of vascularity within the sponge after treatment suggests that a potential mechanism of rhIL-2/rmIL-12 anti-tumor activity is the inhibition of neovascular growth associated with the establishment of tumor lesions. This potential mechanism could be dissociated from the known activities of these two cytokines to induce the recruitment and activation of host effector cells. Moreover, this model provides a unique opportunity to study the cellular and molecular mechanism(s) underlying both tumor angiogenesis and leukocyte recruitment to metastatic lesions.

Complete regression of established spontaneous mammary carcinoma and the therapeutic prevention of genetically programmed neoplastic transition by IL-12/pulse IL-2: induction of local T cell infiltration, Fas/Fas ligand gene expression, and mammary epithelial apoptosis.

Using a novel transgenic mouse model of spontaneous mammary carcinoma, we show here that the IL-12/pulse IL-2 combination can induce rapid and complete regression of well-established autochthonous tumor in a setting where the host immune system has been conditioned by the full dynamic process of neoplastic progression and tumorigenesis. Further, this regimen inhibits neovascularization of established mammary tumors, and does so in conjunction with potent local induction of genes encoding the IFN-gamma- and TNF-alpha-inducible antiangiogenic chemokines IFN-inducible protein 10 and monokine induced by IFN-gamma. In contrast to untreated juvenile C3(1)TAg mice in which histologically normal mammary epithelium predictably undergoes progressive hyperplasia, atypical changes, and ultimately transition to overt carcinoma, the current studies also demonstrate a unique preventative therapeutic role for IL-12/pulse IL-2. In juvenile mice, early administration of IL-12/pulse IL-2 markedly limits the expected genetically programmed neoplastic transition within the mammary epithelium and does so in conjunction with enhancement of constitutive Fas and pronounced induction of local Fas ligand gene expression, T cell infiltration, and induction of apoptosis within the mammary epithelium. These events occur in the absence of a durable Ag-specific memory response. Thus, this novel model system demonstrates that the potent therapeutic activity of the IL-12/pulse IL-2 combination rapidly engages potent apoptotic and antiangiogenic mechanisms that remain active during the delivery of IL-12/pulse IL-2. The results also demonstrate that these mechanisms are active against established tumor as well as developing preneoplastic lesions.