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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. [ABSTRACT FROM AUTHOR]

Background: Covalent attachment of a 40-kd branched-chain polyethylene glycol moiety to interferon alfa-2a results in a compound (peginterferon alfa-2a) that has sustained absorption, a slower rate of clearance, and a longer half-life than unmodified interferon alfa-2a. We compared the clinical effects of a regimen of peginterferon alfa-2a with those of a regimen of interferon alfa-2a in the initial treatment of patients with chronic hepatitis C.Methods: We randomly assigned 531 patients with chronic hepatitis C to receive either 180 microg of peginterferon alfa-2a subcutaneously once per week for 48 weeks (267 patients) or 6 million units of interferon alfa-2a subcutaneously three times per week for 12 weeks, followed by 3 million units three times per week for 36 weeks (264 patients). All the patients were assessed at week 72 for a sustained virologic response, defined as an undetectable level of hepatitis C virus RNA (<100 copies per milliliter).Results: In the peginterferon group, 223 of the 267 patients completed treatment and 206 completed follow-up. In the interferon group, 161 of the 264 patients completed treatment and 154 completed follow-up. In an intention-to-treat analysis in which patients who missed the examination at the end of treatment or follow-up were considered not to have had a response at that point, peginterferon alfa-2a was associated with a higher rate of virologic response than was interferon alfa-2a at week 48 (69 percent vs. 28 percent, P=0.001) and at week 72 (39 percent vs. 19 percent, P=0.001). Sustained normalization of serum alanine aminotransferase concentrations at week 72 was also more common in the peginterferon group than in the interferon group (45 percent vs. 25 percent, P=0.001). The two groups were similar with respect to the frequency and severity of adverse events, which were typical of those associated with interferon alfa.Conclusions: In patients with chronic hepatitis C, a regimen of peginterferon alfa-2a given once weekly is more effective than a regimen of interferon alfa-2a given three times weekly. [ABSTRACT FROM AUTHOR]

Objectives: This study compared the efficacy and safety of peginterferon alpha-2a 135 microg/wk, peginterferon alpha-2a 180 microg/wk and interferon alpha-2a in patients with chronic hepatitis C., Methods: A total of 639 patients received peginterferon alpha-2a 135 microg or 180 microg once weekly, or interferon alpha-2a 3 MIU thrice weekly for 48 wk., Results: Sustained virological responses were significantly higher with peginterferon alpha-2a than with interferon alpha-2a 3 MIU (28% in the 135 microg and 180 microg peginterferon alpha-2a groups vs 11% with interferon alpha-2a, p = 0.001). The proportion of patients with clinically significant histological improvement was lower in the peginterferon alpha-2a 135 microg (48%) than the 180 microg group (58%, p = 0.035 vs peginterferon alpha-2a 135 microg), but similar to that in the interferon alpha-2a group (45%, p = 0.820 vs peginterferon alpha-2a 135 microg and p = 0.017 vs peginterferon alpha-2a 180 microg, respectively). The overall safety profiles were similar for the three treatments. In patients with chronic hepatitis C, peginterferon alpha-2a 135 microg/wk and 180 microg/wk produced similar sustained virological response rates, both of which were significantly higher than that achieved with interferon alpha-2a thrice weekly. A significantly higher proportion of patients treated with the 180 microg dose of peginterferon alpha-2a had clinically significant histological improvement.

The injection of a mixture of bona fide T cell epitopes can lead to the occurrence of immunodominance, meaning that the immune response is focused on the recognition of a single epitope or a small portion of the epitopes injected. We have previously demonstrated that the administration of rIL-12 can counteract immunodominance in BALB/c mice. In this study, we show that the administration of rIL-12 to HLA-A2.1 transgenic mice (A2k(b) mice) abrogates specifically the immune response against HLA-A2.1-restricted HIV epitopes in the spleen. This lack of immune response is most probably due to a transient depletion of B cells, T cells, macrophages, and dendritic cells in this organ. Therefore, our study explains the mechanism of immunosuppression by rIL-12 in vivo.

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.

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.

Background: Numerous animal model studies have examined the ability of genetically engineered tumor cells to release cytokines and to elicit an immune memory against the parental tumor. Often only a single cytokine is studied, and few comparative studies have been conducted., Purpose: We evaluated the antitumor efficacy of adenocarcinoma cells engineered to release interleukin (IL)-12 in a mouse model system. The efficacy of this cytokine was compared with that of other cytokines released by engineered adenocarcinoma cells and that of exogenous IL-12 administered both locally and intraperitoneally., Methods: BALB/cAnCr mice were inoculated with syngeneic parental mammary adenocarcinoma (TSA) cells in quantities sufficient to lead to tumors in all inoculated mice. TSA cells engineered to release IL-12 (TSA-IL12) were also injected into normal and selectively immunosuppressed BALB/cAnCr mice. Tumor incidence, growth, and rejection patterns were evaluated by the measurement of neoplastic masses and by the study of the histologic and ultrastructural features of the tumor site. The effects of local or intraperitoneal administration of recombinant IL-12 (rIL-12) on tumor-bearing animals were also studied., Results: Most mice rejected TSA-IL12 cells through a CD8-positive, T-lymphocyte-dependent reaction associated with macrophage infiltration, vessel damage, and necrosis. The systemic immunity of mice that had rejected TSA-IL12 cells to a subsequent challenge with parental TSA cells was less efficient than that elicited by TSA cells engineered to release IL-4 or IL-10 but equivalent to that elicited by TSA cells engineered to release IL-2, IL-7, and interferon alfa. Compared with TSA cells engineered to produce other cytokines, TSA-IL12 cells were the most efficient in curing mice with established TSA tumors; injection of 0.1 million proliferating cells contralaterally to the tumor growth area cured five of 15 mice bearing 1-day-old tumors; injection of the same dose of proliferating cells into the tumor growth area cured two of 20 tumor-bearing mice. However, two 5-day courses with a nontoxic dose (0.1 microgram) of rIL-12 given intraperitoneally cured a similar proportion of these animals (six of 20). Only two of 20 mice with 7-day-old TSA tumors were cured by vaccination with proliferating TSA-IL12 cells, whereas 24 of 30 mice with such tumors were cured by intraperitoneal administration of rIL-12., Conclusions: TSA cells engineered to release IL-12 are rejected by most mice; the ensuing immune memory for TSA parental cells, however, was less efficient than that elicited by proliferating TSA cells engineered to release other cytokines (e.g., IL-4, IL-10, and possibly interferon gamma). The immune reaction elicited by TSA-IL12 cells was the most efficient in curing mice with established TSA tumors; notably though, the same or a better cure rate was obtained with rIL-12 given intraperitoneally.

A preclinical evaluation of the immunogenicity of various preparations of interferon-alpha (IFN-alpha) was performed with in vitro and in vivo animal models. The distribution of genes for IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c in various cell populations and the response of human T cell clones to IFN-alpha peptides were investigated. The immunogenicity of IFN-alpha in IFN-alpha 2b transgenic mice and factors that influence the immunogenicity of IFN-alpha in normal mice were also studied. The genes for IFN-alpha 2a and IFN-alpha 2b were found in KG-1 cells, whereas IFN-alpha 2b and IFN-alpha 2c genes were present in Namalwa cells. No difference in proliferation of human T cells, T cell lines, or T cell clones could be obtained with IFN-alpha peptides. In transgenic mice bearing the human IFN-alpha 2b gene, no antibody response was obtained following immunization with either IFN-alpha 2a or IFN-alpha 2b. Normal mice immunized with either IFN-alpha 2a or IFN-alpha 2b produced equivalent titers of antibodies, which cross-reacted with both IFNs. Studies evaluating the relative immunogenicity of IFN-alpha in normal mice demonstrated that a number of treatment and host variables can modulate immunogenicity of IFN-alpha preparations.

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.

Immunodominance (ID) of T cell epitopes is a well-documented phenomenon that might have profound significance in the evolution of T cell responses to pathogens, tumors, autoantigens and vaccines. With the intention of developing vaccines composed of several cytotoxic T cell (CTL) epitopes, we injected mice with peptide mixtures containing two to five CTL epitopes and observed clear patterns of ID. In a first case, ID strictly correlated with the competitor activity of the individual peptides for H-2Kd, whereas in a second case, the absence of correlation between ID and competitor activity, binding affinity, half-life of the peptides in serum, induction of proliferation in vitro and the individual immunogenicity of the peptides, suggested to us that ID of co-injected CTL epitopes can be determined both at the peptide level (binding affinity to H-2Kd) and at the T cell level. This hypothesis is supported by our finding that interleukin-12 strongly modulates ID when it is not correlated with MHC binding.

Interleukin-12 (IL-12) is a cytokine that exerts immunoregulatory effects on T cells and natural killer cells, playing a unique role in promoting type 1 T helper cell responses and, thereby, cell-mediated immunity. IL-12 has been shown to exert striking therapeutic effects at nontoxic doses in mouse tumor models and in mouse models of a variety of infectious diseases and airway inflammation. In mouse tumor models, the therapeutic effects of IL-12 have been shown to result from its immunoenhancing activity, requiring T cells and IFN-gamma. Administration of IL-12 can result in antiangiogenic effects that may also contribute to its antitumor activity in some models. Enhanced antitumor effects may be achieved by administering IL-12 in combination with certain other cytokines or with radiotherapy or chemotherapy. The striking therapeutic effects of IL-12 in these preclinical models have led to the initiation of clinical trials to examine the potential therapeutic activity of IL-12 in human cancer patients and in patients with human immunodeficiency virus infection or with chronic hepatitis B or C virus infections.

Despite considerable advancement in anticancer therapy, minimal residual disease (MRD) is still a major problem in the clinical management of cancer, including lymphoma. In this report, we have studied the antitumor effects of interleukin-12 (IL-12) against an aggressive liver metastatic murine RAW117-H10 lymphoma. Our results using three different doses of IL-12 (0.175, 0.35 and 0.7 micrograms/mouse) showed that a 0.35 micrograms dose is the most efficacious against lymphoma grown in intact mice. Furthermore, we have evaluated the therapeutic effects of IL-12 against residual lymphoma in a transplantation setting. BALB/c mice were treated with high-dose therapy (HDT) and transplanted with syngeneic bone marrow cells added with a known number of RAW117-H10 lymphoma cells to mimic the clinical situation of MRD. The mice were then treated with IL-12 (0.25 micrograms/mouse/day) alone or IL-12 plus activated cytotoxic effector cells. Our results showed that IL-12 had a significant (P < 0.05) antitumor therapeutic effect against liver metastatic lymphoma grown in intact mice as well as in lymphoma-bearing mice treated with HDT followed by stem cell transplantation as determined by survival period. The therapeutic effect of IL-12 was also demonstrated by a very significant decrease (P < 0.05) in the tumor burden in livers from the IL-12-treated mice. Mice that were treated with IL-12 following HDT and hematopoietic stem cell transplantation had a significant decrease in circulating white blood cells (P < 0.05), a significant increase in spleen weight and cellularity (P < 0.05), and hematopoietic progenitor cells (P < 0.05), a significant increase in the number of splenocytes expressing IL-2 alpha-chain receptor (P < 0.05), and an increase in the frequency of natural killer cells in their spleens. These studies suggest that cytokines such as IL-12 may have the potential to mediate antitumor effects against residual lymphoma without compromising lymphohematopoietic recovery.

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.

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.

IL-12 is a heterodimeric cytokine that promotes cell-mediated immunity through its regulatory effects on T and NK cells. In some murine infectious disease models, IL-12 was shown to be produced endogenously in response to infection, and the exogenous administration of IL-12 to mice with either infectious diseases or tumors has resulted in significant therapeutic effects. IL-12 was protective early in the disease process, as well as against established disease. However, the biologic activities of IL-12 that are beneficial in the host response to these infectious diseases and malignancies can also be deleterious in certain disease states. Thus, IL-12 has considerable potential for the treatment of a variety of human disorders if used under the appropriate conditions. Likewise, antagonists of IL-12 may have a role in controlling diseases with pathologies that are mediated through immune mechanisms.

Although interleukin-12 (IL-12) has marked antitumor activity against the murine Renca renal cell carcinoma in vivo, no antiproliferative activity with IL-12 was observed against these tumor cells in vitro; in contrast, interferon-gamma (IFN-gamma) had growth inhibitory activity. Since one of the properties of IL-12 is its ability to stimulate production of IFN-gamma, the role of IFN-gamma in mediating the antitumor activity of IL-12 was evaluated. Substantially diminished antitumor activity was observed in mice injected with IL-12 and neutralizing antibody to murine IFN-gamma compared with mice receiving IL-12 alone, indicating that IFN-gamma was required for the optimal antitumor efficacy of IL-12. However, several lines of investigation suggest that the antitumor effect of IL-12 is not mediated solely through the induction of IFN-gamma. Exogenous administration of IFN-gamma to Renca tumor-bearing euthymic mice resulted in less antitumor efficacy than that which could be obtained with IL-12. In addition, the antitumor effect of IL-12 was reduced in nude mice compared with euthymic mice, but an approximately 10-fold higher level of serum IFN-gamma was induced in nude than in euthymic mice. Thus, these results indicate that induction of high serum levels of IFN-gamma is not sufficient to mediate the antitumor efficacy of IL-12.

IL-12 is a heterodimeric cytokine that promotes cell-mediated immunity through its regulatory effects on T and NK cells. IL-12 produced endogenously in response to various microbial agents likely plays a role in the host response to infection by intracellular pathogens, and administration of rIL-12 to mice has bee shown to have dramatic therapeutic effects in a number of tumor models and models of infectious diseases. The relatively long serum half-life of IL-12 compared to other lower molecular weight cytokines such as IL-2 should permit more flexibility in dose scheduling. At doses which are efficacious in murine tumor models, IL-12 has been well tolerated. Phase I clinical trials with IL-12 in the treatment of human malignancies have recently been initiated. The results of such studies are required to determine whether the therapeutic potential IL-12 has displayed in murine disease models can be translated into clinical utility in man.

Recent evidence supports the critical and proximate role of IL-12 in regulating both T and NK cell function during inflammation. In these studies, we evaluated the in vivo antitumor activity of murine IL-12 in murine adenocarcinoma and sarcoma models using both systemic and peritumoral administration. Antitumor effects were consistently demonstrated both in models of microdisease, in which IL-12 treatment was initiated soon after tumor inoculation (1 to 5 days), and in animals bearing large established tumors (7 to 14 days). Treatment with IL-12 markedly prolonged survival and, in most cases, caused complete tumor regression. Significant reduction in pulmonary metastases after systemic treatment was observed when treatment was delayed for 10 days after tumor inoculation. Increases in serum IFN-gamma, TNF-alpha, and nitrogen oxides were demonstrated, exceeding those observed with IL-2 treatment. Systemic administration of anti-IFN-gamma Abs before IL-12 treatment nearly completely abrogated the antitumor effect in experiments using subcutaneous tumors or pulmonary metastases. Depletion of the individual T cell subsets CD4 and CD8 by systemic administration of mAbs diminished the effectiveness of IL-12 when administered in combination. An infiltrate composed primarily of CD8+ + cells was demonstrated by using immunohistochemical analysis of tumors after IL-12 treatment. Minimal apparent toxicity was demonstrated at effective doses (0.1 to 1.0 microgram/day) of IL-12. These results indicate that IL-12 is an effective and minimally toxic antitumor agent in murine tumor models and leads to an immune-mediated rejection involving, at least in part, IFN-gamma, CD4+, and CD8+ cells. Human clinical trials of IL-12 for the treatment of malignancy are supported by these studies.

IL-12 is a cytokine that promotes cell-mediated immunity by promoting Th1-type cytokine responses, enhancing the lytic activity of NK/LAK cells, augmenting specific CTL responses, and inducing the production of IFN-gamma. On the other hand, IL-12 suppresses the development of Th2-type cytokine responses and humoral immunity, particularly IgGl and IgE responses. It is likely that IL-12 normally plays an important role in the host defense against intracellular microbial pathogens. In addition, the administration of rIL-12 to mice has been shown to have potent therapeutic effects in several tumour and infectious disease models. IL-12 has been shown to be more efficacious than IL-2 in several murine tumour models, and toxicology studies suggest that it may have a substantially better therapeutic index. In addition, the long serum half-life of IL-12 relative to other cytokines will allow more flexibility in dosing schedules. However, future clinical trials are required to determine whether the efficacy of IL-12 seen in these experimental models is predictive for its use as an immunomodulatory drug in humans.

The antitumor efficacy of recombinant murine interleukin-1 alpha (rMuIL-1 alpha) was evaluated either alone or in combination with recombinant human hybrid interferon alpha A/D (IFN-alpha A/D) against the murine B16 F10 malignant melanoma. Treatment of subcutaneous tumor-bearing mice intraperitoneally with rMuIL-1 alpha resulted in a dose-dependent inhibition of tumor growth with the greatest activity obtained with the maximum tolerated dose of rMuIL-1 alpha (10 micrograms per treatment). Augmented tumor inhibition comparable to that seen in mice treated with a high dose of rMuIL-1 alpha was observed in subcutaneous tumor-bearing mice injected with the combination of IFN-alpha A/D and a low dose of rMuIL-1 alpha. Similar inhibition of subcutaneous tumor growth was obtained in T-cell-deficient nude or natural killer cell-deficient beige mice. In contrast, treatment of mice bearing B16F10 experimental pulmonary metastases with rMuIL-1 alpha resulted in no decrease in the number of metastases, and rMuIL-1 alpha did not potentiate the antimetastatic activity of IFN-alpha A/D. A synergistic induction of IL-6 was induced in mice treated with the combination of rMuIL-1 alpha plus IFN-alpha A/D but the level of IL-6 induced was not correlated with inhibition of tumor growth because this elevation of IL-6 was not observed in tumor-bearing nude mice. No direct antiproliferative activity was demonstrable in vitro against B16 F10 cells with rMuIL-1 alpha, IL-6, or rMuIL-1 alpha plus IL-6, and addition of these cytokines did not enhance the antiproliferative activity of IFN-alpha A/D.(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin-12 (IL-12) is a newly characterized cytokine that has a unique heterodimeric structure. It was initially cloned from B lymphoblastoid cell lines, but the majority of IL-12 is produced by macrophages/monocytes following appropriate stimulation. IL-12 can (1) enhance the cytolytic activity of a number of effector cells including T cells, natural killer (NK) cells, lymphokine activated killer (LAK) cells, and macrophages, (2) increase proliferation of activated NK and T cells, (3) induce production of cytokines, such as interferon gamma, (4) stimulate the induction of TH1 cells, (5) upregulate a number of cell surface molecules, (6) inhibit IgE secretion, and (7) act as a synergistic factor for hematopoietic stem cells. Based on these potent immunomodulatory activities, IL-12 has been evaluated in several disease models for parasitic infections and malignancies. Marked activity of IL-12 against both Leishmania and Toxoplasma has been reported. Likewise, antimetastatic and antitumor activity, including tumor regression, has been observed against a number of murine malignancies treated with IL-12 using doses that result in little toxicity. The results suggest that IL-12 may be a useful cytokine for the treatment of a number of diseases.

It has recently been demonstrated that in vivo administration of murine interleukin 12 (IL-12) to mice results in augmentation of cytotoxic natural killer (NK)/lymphocyte-activated killer cell activity, enhancement of cytolytic T cell generation, and induction of interferon gamma secretion. In this study, the in vivo activity of murine IL-12 against a number of murine tumors has been evaluated. Experimental pulmonary metastases or subcutaneous growth of the B16F10 melanoma were markedly reduced in mice treated intraperitoneally with IL-12, resulting in an increase in survival time. The therapeutic effectiveness of IL-12 was dose dependent and treatment of subcutaneous tumors could be initiated up to 14 d after injection of tumor cells. Likewise, established experimental hepatic metastases and established subcutaneous M5076 reticulum cell sarcoma and Renca renal cell adenocarcinoma tumors were effectively treated by IL-12 at doses which resulted in no gross toxicity. Local peritumoral injection of IL-12 into established subcutaneous Renca tumors resulted in regression and complete disappearance of these tumors. IL-12 was as effective in NK cell-deficient beige mice or in mice depleted of NK cell activity by treatment with antiasialo GM1, suggesting that NK cells are not the primary cell type mediating the antitumor effects of this cytokine. However, the efficacy of IL-12 was greatly reduced in nude mice suggesting the involvement of T cells. Furthermore, depletion of CD8+ but not CD4+ T cells significantly reduced the efficacy of IL-12. These results demonstrate that IL-12 has potent in vivo antitumor and antimetastatic effects against murine tumors and demonstrate as well the critical role of CD8+ T cells in mediating the antitumor effects against subcutaneous tumors.

Although several murine macrophage (m phi) cell lines from different sites have previously been obtained by in vitro infection with the J2 murine retrovirus, which carries the v-raf and v-myc oncogenes, it was not possible to immortalize thioglycolate-elicited peritoneal macrophages (Pm phi s) by this in vitro procedure. A technique utilizing in vivo injection of the J2 virus has been developed to overcome this problem. The J2 virus immortalized Pm phi s in a very efficient manner in vivo because no exogenous growth factors were required for the in vitro proliferation of these cells and numerous continuous cloned cell lines were readily established. In contrast, Pm phi s obtained from uninfected mice or Pm phi s infected in vitro with the J2 virus did not proliferate. The in vivo immortalized cells had many of the morphological and functional characteristics of m phi s. Analysis of two of the clones, PMJ2-PC and PMJ2-R, demonstrated intracellular expression of the product of the v-raf gene, presence of m phi-associated cell surface antigens, interleukin-6 secretion induced by lipopolysaccharide, and biological response modifier-induced cytotoxic activity against tumor cells. In addition, one of the clones, PMJ2-PC, constitutively expressed major histocompatibility complex (MHC) class II antigens, and in the other clone, PMJ2-R, MHC class II antigens expression was induced by recombinant murine interferon-gamma. This method of utilizing the J2 virus in vivo represents a novel technique for obtaining hematopoietic cell lines from cells that are difficult to immortalize in vitro.

Background: Combination therapy with systemically administered interleukin-2 (IL-2) and interferon alpha (IFN-alpha) has resulted in long-term objective remissions in 30% of patients with metastatic renal cell carcinoma (RCC), but toxic effects are clinically significant., Purpose: We have thus investigated an alternative therapeutic approach--continuous intratumoral production of IL-2 and/or IFN-alpha by a cytokine-transfected human RCC tumor cell line., Methods: Plasmid vectors were used to transfect the R11 RCC line with the genes for human IL-2 and/or IFN-alpha by the calcium phosphate precipitation method. Biologic characteristics of the cytokine-transfected tumor cells were determined by assays of thymidine incorporation and cytotoxicity, fluorescence-activated cell-sorter analysis, Northern blotting, and in vivo studies in C3Hf/Sed/Kam mice rendered T-cell deficient., Results: The transfected cell lines produced the following amounts of cytokine per 10(6) cells per day: R11-IL-2 (220 U), R11-IFN-alpha (10,240 U), and R11-IL-2 + IFN-alpha (95 U + 1270 U, respectively). Gamma irradiation did not eliminate cytokine secretion. Morphology and growth rates were identical to those for the parental R11 cell line, except for IFN-alpha-producing clones, which showed significant growth inhibition. All cytokine-producing cells demonstrated increased susceptibility to cell killing by peripheral blood leukocytes (PBL). IFN-alpha producers exhibited enhanced HLA antigen expression and suppressed c-myc messenger RNA expression; when cocultured in vitro, they induced similar changes in parental R11 cells. IL-2 producers could stimulate growth and cytotoxicity of naive (i.e., freshly isolated, uncultured) and activated PBL. All cytokine-producing cells lost their tumorigenicity, as evidenced by failure to grow in the T-cell-depleted mice. When co-injected at a local site but not at a distant site, these cells prevented growth of parental R11 cells. Histologic examination of the injection sites revealed a substantial influx of macrophages. Intraperitoneal administration of IL-2 and/or IFN-alpha could not, however, prevent growth of the parental R11 tumors., Conclusion: Local production of high concentrations of IL-2 and IFN-alpha at the tumor site is more effective in preventing tumor growth than systemic administration., Implication: Continuous local delivery of cytokines via transfer of cytokine genes into tumor cells for use as live cancer vaccines is a novel strategy for manipulation of host-mediated antitumor immune response in patients with advanced RCC.

Continuous alveolar macrophage (AM) and tumor-infiltrated (TIM) cell lines have been generated from C57B16J mice by in vitro infection with the J2 retrovirus carrying the v-raf and v-myc oncogens. Four cloned AM cell lines (AMJ2-C8, AMJ2-C10, AMJ2-C11, AMJ2-C20) and 3 cloned TIM cell lines (TIMJ2-C4, TIMJ2-C7 and TIMJ2-C15) were expanded for further characterization. Flow cytometry detected the product of the raf gene in the cytoplasm of all these cell lines. Studies on the tumoricidal properties of these AM and TIM cell lines demonstrated differences in their response to a panel of known macrophage activators. Four of these cell lines (AMJ2-C8, AMJ2-C10, TIMJ2-C7 and TIMJ2-C15) were activated following exposure to recombinant murine interferon gamma (rMuIFN-gamma) but not lipopolysaccharide (LPS) or muramyl dipeptide (MDP). AMJ2-C20 was only activated by incubation with rMuIFN-gamma plus LPS. AMJ2-C11 and TIMJ2-C4 are the cell lines that most closely resembled the response pattern of the parental AM and TIM, since they could be activated by either the combination of rMuIFN-gamma plus LPS or rMuIFN-gamma plus MDP. Constitutive expression of MHC-class-II antigens was low on AMJ2-C11 or TIMJ2-C4 but was increased following exposure to rMuIFN-gamma. Neither cell line secreted substantial amounts of IL-1 or TNF but both secreted large amounts of IL-6. Thus these cell lines could be powerful tools to study AM and TIM activation and cytotoxicity.

Murine macrophages from different anatomical sites were compared for their ability to become tumoricidal and to secrete interleukin-1 (IL-1) and tumor necrosis factor (TNF) following stimulation in vitro by several biological response modifiers (BRM). Peritoneal macrophages (PM), alveolar macrophages (AM), and tumor-infiltrating-macrophages (TIM), isolated from B16F10 melanoma colonies in the lung, were incubated overnight with BRM [recombinant murine interferon gamma (rMulFN-gamma), lipopolysaccharide (LPS), muramyl dipeptide (MDP)], either alone or in combination. PM exhibited an increased cytotoxic response following incubation with LPS or rMuIFN-gamma but not with MDP. Both AM and TIM were induced to become tumoricidal following incubation with rMuIFN-gamma plus LPS or rMuIFN-gamma plus MDP but not after stimulation with any BRM alone; the level of cytotoxicity obtained with TIM incubated with rMuIFN-gamma plus LPS was slightly lower than that observed with PM or AM, while with rMuIFN-gamma plus MDP both AM and TIM had lower cytotoxicity than PM. Secretion of IL-I and TNF was observed in PM stimulated with LPS or MDP but not with rMuIFN-gamma. Likewise, secretion of IL-I by AM or TIM was also induced with LPS, although less than that obtained with PM. AM stimulated with LPS secreted larger amounts of TNF than PM while TIM secreted very low amounts of TNF. However, this result may be a consequence of the enzymatic isolation procedure used to obtain TIM since TNF secretion was also impaired in LPS-stimulated normal lung macrophages isolated by a similar enzymatic procedure, or enzyme-treated PM. Our results suggest that TIM obtained from lung metastases share certain functional characteristics with normal AM and respond to BRM in like manner with respect to induction of tumoricidal activity and cytokine secretion.

The mechanisms that allow circulating basement membrane antibodies (Ab) to interact with the alveolar basement membrane (ABM) inducing Goodpasture's hemorrhagic pneumonitis are unknown. In laboratory animals the ABM is inaccessible to phlogogenic amounts of ABM Ab unless the permeability of the unfenestrated alveolar endothelium is increased. This study was designed to test the hypothesis that in the mouse polypeptide mediators, generated by activated lymphoid cells or cells infected by viruses, contribute to the pathogenesis of passive Goodpasture's hemorrhagic pneumonitis. In naive mice that received rabbit ABM Ab, these bound to the glomerular basement membrane but not to the ABM and their lungs were normal. In the lungs of mice injected with human recombinant IL-2 and IFN-alpha specific binding of ABM IgG, C3, and fibrinogen to the ABM, diffuse and severe erythrocyte extravasation, and accumulation of mononuclear and polymorphonuclear leukocytes were constantly observed. ABM Ab and IL-2 or ABM Ab and IFN-alpha did not produce comparable effects. Mice injected only with IL-2 and IFN-alpha had enlarged, edematous lungs without pulmonary hemorrhages. The results show that the synergism of IL-2 and IFN-alpha convert the lung into a preferential target for AMB Ab, suggesting that cytokines may have a role in the pathogenesis of human Goodpasture's pneumonitis.

Requirement for macrophages in in vitro augmentation, by interferon (IFN), polyinosinic-polycytidylic acid (poly I:C), or Corynebacterium parvum, of rat, mouse, and human natural killer (NK) activities was examined. Several differences were seen among the species. Mouse NK activity demonstrated some lability at 37 degrees C and a strict macrophage requirement for in vitro production of IFN, and augmentation of NK activity was demonstrated by either poly I:C or C. parvum. In contrast, human peripheral blood leukocytes (PBL) depleted of monocytes by adherence on nylon wool demonstrated NK activity, which was not labile but rather increased substantially upon overnight culture at 37 degrees C alone or with poly I:C or C. parvum. Monocyte-depleted human PBL also produced IFN in these cultures. The pattern of reactivity seen with rat spleen cell cultures was different from either that of mouse and human cells. This pattern of reactivity had no lability at 37 degrees C and had a macrophage requirement for IFN production and NK cell augmentation upon culture alone or with poly I:C but not with C. parvum. These results indicated some major differences among species in the regulation of NK activity in vitro and the requirement for macrophages for the in vitro production of IFN. A better understanding of these differences will be helpful in choosing appropriate models for in vitro and in vivo studies of NK cell activity.

Antibodies reactive with effector cells were shown to augment the cytotoxicity of spleen cells from athymic nude and euthymic mice. The addition of alloantibody to the assay or pretreatment of the effector cells with alloantibody resulted in increased cytotoxicity against the human cell K562, a relatively poor target for spontaneous mouse NK activity. When monoclonal antibodies were tested, cytotoxicity was markedly increased by some antibodies, such as anti-H-2, anti-la, and anti-Thy 1.2, while others had no effect. The degree of augmentation of cytotoxicity was dependent on the concentration of antibody added. Nylon-wool-nonadherent nu/nu splenic effector cells mediated the antibody-induced cytotoxicity and anti-asialo GMI plus complement abolished activity, indicating that the cells mediating the cytotoxicity were NK cells and not mature T cells, B cells or macrophages. When spleen cells from mice having different levels of NK activity were evaluated in this system, the magnitude of augmentation by antibody correlated with the level of spontaneous NK activity and no increased cytotoxicity was found with cell populations that had low spontaneous NK activity. Testing a panel of target cells, showed that certain human and mouse cell lines, with low to moderate susceptibility to spontaneous NK activity, were sensitive to antibody-induced NK-cell-mediated cytotoxicity whereas others were completely resistant. Both Fc-IgG receptor-positive and -negative cell lines were susceptible target cells. These results indicate that antibodies reactive with murine NK cells can increase their cytolytic activity.

A series of monosaccharides were tested for their ability to inhibit the effector phase of macrophage-mediated cytolysis against two susceptible murine tumor target cells, L5178Y and RL male I. Two monosaccharides, D-mannose and N-acetyl-D-galactosamine, were found to decrease cytotoxicity consistently in a dose-dependent manner. However, D-mannose preferentially inhibited lysis of RL male I target cells with little effect on lysis of L5178Y target cells, while the reverse was found with N-acetyl-D-galactosamine. Neither monosaccharide interfered with the activation of macrophages by polyinosinic:polycytidylic acid. Natural killer cell activity was decreased by a 25 mM concentration of D-mannose but not by N-acetyl-D-galactosamine, although increasing concentrations of N-acetyl-D-galactosamine were inhibitory. Neither monosaccharide affected cytotoxicity by alloimmune T cells. Cytotoxicity of macrophage-like cell lines against tumor target cells was also decreased by monosaccharides but the pattern of inhibition was different from that seen with activated macrophage effector cells. Both D-mannose and N-acetyl-D-galactosamine inhibited glucose oxidation by activated macrophages but only D-mannose significantly decreased protein synthesis of activated macrophages. These results indicate that monosaccharides can inhibit macrophage-mediated cytotoxicity in a selective manner with the pattern dependent on the tumor target cell used in the assay.

Inoculation of thioglycollate-elicited peritoneal exudate cells (PEC) into C57BL/6 mice reduced the rate of lung clearance of several intravenously (i.v.) injected murine tumor cells, and increased by up to 100-fold the number of artificially-induced metastatic lung nodules produced by the i.v. injection of B16 melanoma or Lewis lung carcinoma (3LL) tumor cells. Maximum effects were observed when PEC were injected either before, or shortly after, tumor cells. Modulation of lung clearance or metastasis formation was observed only with PEC and not with a variety of other cells, such as splenocytes, thymocytes P815 mastocytoma cells, or several macrophage-like cell lines (PU5-1.8 and IC-21). Lysates of PEC were as efficient in reducing lung clearance and augmenting metastasis formation as were intact viable PEC. Lysates of other cell types, including P815 and the macrophage-like cell lines, were unable to produce these effects. PEC populations, enriched for macrophages by adherence to plastic or by percoll density gradient sedimentation, also increased the number of B16-induced artificial metastasis, implicating the macrophage as the cell responsible for these observations.

Murine peritoneal macrophages (pM phi) elicited in vivo by intraperitoneal (IP) inoculation of various agents were tested for their homing/distribution patterns after intravenous (IV) adoptive transfer to syngeneic C57BL/6 recipients. Resident pM phi (RpM phi) obtained from normal mice and pM phi elicited by proteose peptone (PpM phi) or thioglycollate broth (TpM phi) exhibited similar homing patterns following IV transfer. After initial arrest in the lungs, these cells rapidly disseminated to liver and spleen, with minimal or no detectable migration to peripheral lymph nodes, intestine, peritoneum, kidney, heart, or retention in the blood. The pattern of results reflected the properties of pM phi themselves, since highly enriched pM phi populations obtained by treatment of crude peritoneal exudate cells with anti-Thy 1.2 + C, or by fractionation on Percoll density gradients, gave similar results. The distribution of pM phi elicited by Brewer's thioglycollate medium (BTpM phi) was markedly different from other pM phi tested. BTpM phi homed rapidly to the lungs and many remained localized there for at least 72 hr with very little migration to the spleen. The distribution of PpM phi could be altered by activation of these cells in vivo through the IP injection of the pyran copolymer, MVE-2, prior to adoptive IV transfer. Activated PpM phi contained a population of highly differentiated, low density pM phi, separable on density gradients, which arrested in the lungs for appreciably longer periods of time than did PpM phi. These cells exhibited reduced ability for migration to the spleen. Macrophage-like (M phi-like) cell lines did not exhibit migration capability, but rather were rapidly cleared from the circulation in a manner similar to other types of tumor cells.

The ability of adherent peritoneal cells (APC) to inhibit murine natural killer (NK) cell activity was examined. Nylon wool-nonadherent splenic effector cells were incubated overnight with or without different numbers of APC. NK activity was then measured against YAC-1 in a 4-hr 51Cr-release cytotoxicity assay. Proteose peptone-elicited or unstimulated resident APC from normal mice markedly suppressed NK activity of splenic effector cells in the presence or absence of exogenously added interferon. The suppression was dependent on the number of APC added with 10% APC, relative to the number of effector cells, resulting in a greater than 65% inhibition of cytotoxicity. The effector phase of cytotoxicity was not the target of the suppressor cells, because APC did not suppress NK activity when they were present only during the cytotoxicity assay. The addition of APC to alloimmune cytotoxic T cells under similar conditions resulted in no inhibition of cytotoxicity. Both syngeneic and allogeneic APC suppressed NK activity, but several murine macrophage-like cell lines lacked this property. In contrast to APC, incubation of effector cells with adherent spleen cells from normal mice resulted in no inhibition of NK activity. APC from mice injected with C. parvum were less inhibitory for NK activity than normal resident APC. In contrast, C. parvum APC suppressed concanavalin A-induced lymphoproliferation and were directly cytotoxic to tumor target cells in vitro, whereas normal APC lacked these properties. The results indicate that the peritoneum of untreated mice contains suppressor cells that can inhibit the in vitro maintenance and IFN-mediated augmentation of NK activity. In addition, these results indicate a broader spectrum of immune reactivities regulated by APC and suggest that, depending on their level of activation, APC can preferentially inhibit different immune functions.

The effect of a human recombinant hybrid alpha interferon (referred to as rHuIFN-alphaA/D) on pulmonary metastases induced by intravenous injection of B16 F10 melanoma cells in C57BL/6 mice was examined; rHuIFN-alphaA/D has been previously shown to have anti-viral, anti-proliferative and immunomodulatory activities in murine cells. Pretreatment of mice with 4 daily intraperitoneal injections of rHuIFN-alphaA/D resulted in a marked decrease in the number of pulmonary metastases. This inhibition was dose-dependent but was not seen when mice were similarly treated with rHuIFN-alphaA, a human recombinant alpha interferon subtype which is inactive on murine cells. Treatment of mice with rHuIFN-alphaA/D following B16 F10 injection resulted in no significant inhibition of pulmonary metastases. Mice given a similar treatment regimen of rHuIFN-alphaA/D had elevated natural killer (NK) cell activity as measured by in vitro cytotoxicity against YAC-I or in vivo pulmonary clearance of B16 F10 cells. Pretreatment of mice with 10 daily injections of rHuIFN-alphaA/D resulted in decreased NK activity and less inhibition of metastases. Therefore, in this model system, rHuIFN-alphaA/D inhibits metastases when given in the appropriate treatment schedule. Furthermore, the data are consistent with the hypothesis that rHuIFN-alphaA/D-induced inhibition is a consequence of the immunomodulation of NK cells, which prevent the establishment of pulmonary metastases.

The ability of human IgA myeloma immunoglobulins to interact with protein A-containing Staphylococcus aureus was examined. Some IgA1 and IgA2 immunoglobulins bound to S. aureus although others of both subclasses failed to do so. These results were obtained by using both direct binding of radiolabeled immunoglobulins to S. aureus and with inhibition-type assays. Binding was dependent on the Fc fragment of IgA since there was no binding to S. aureus by an F(ab')2 fragment of IgA1. Nonprotein A-containing bacteria did not bind these immunoglobulins and isolated protein A interacted with radiolabeled immunoglobulins. This strongly suggested that protein A was responsible for the observed binding to S. aureus. These data indicate, in contrast to previous reports, that there is no simple relationship between IgA subclass and the capacity to bind to protein A.

Clearance of IV-injected tumor cells has been correlated with levels of natural killer (NK) cell activity in recipient animals. Studies of in vivo tumor cell clearance strongly suggest a relationship between levels of NK cell activity and antitumor or antimetastatic effector function. This study outlines the applicability of three radiolabels, [125I]iododeoxyuridine, ( [125I]dUrd), indium-111-oxine chelate ( [111In]Ox), and chromium-51 (51Cr), to studies of tumor cell clearance in vivo. The suitability of these labels for analysis of the in vivo migration patterns of normal lymphocytes or thymus-derived T cells cultivated in vitro (CTC) is also discussed. The results indicate that [111In]Ox and 51Cr compare favorably with the more widely used [125]dUrd as radiolabels for the assessment of IV-injected tumor cell clearance from the lungs of mice. The rates of clearance of both [111In]Ox and 51Cr, like that for [125I]dUrd, correlate closely with levels of NK-cell activity of the host. Further studies with [111In]Ox reveal that treatment of recipients with anti-asialo GM1 serum, a regimen known to suppress NK-cell activity, demonstrates the appropriate reduction in isotope clearance from the lungs after NK suppression. However, clearance data obtained by monitoring levels of radioactivity in the liver after IV injection must be viewed cautiously, since the same cells labeled with [111In]Ox and [125I]dUrd had a different pattern of clearance from the liver. The same inconsistencies in clearance were observed when [111In]Ox and [125I]dUrd were injected intrafootpad (i.f.p.). Similar effects were observed when [111In]Ox or 51Cr was applied to studies of CTC migration. Levels of [111In]Ox and 51Cr remained high in the liver after IV injection, while [125I]dUrd was rapidly cleared. Normal spleen or thymic lymphocytes exhibited the expected homing to the spleen after labeling with [111In]Ox, indicating a suitability of this label for migration studies, except possibly in the liver. These results with CTC and normal lymphocytes should be considered during the formulation of immunotherapy protocols based on cell migration data, since the choice of radiolabel can result in widely divergent levels of radioactivity accumulated in some organs, and may not provide an accurate representation of the presence of viable, intact, or functional cells.

A variety of heat-killed bacteria were tested for their capacity to induce regressions of established line 10 hepatocarcinomas in syngeneic guinea pigs. Multiple intralesional injections of heat-killed Escherichia coli, Streptococcus mutans, Listeria monocytogenes, and Propionibacterium acnes resulted in complete regression of the tumor in a majority of guinea pigs. Repeated injections of heat-killed Mycobacterium bovis strain Bacillus Calmette-Guérin caused no regressions. Surviving animals were immune to subsequent challenge with line 10 cells but not L2C cells, another syngeneic tumor.

Animal studies have been carried out to assess the antitumour efficacy of recombinant interleukin-2 (rIL-2) in combination with other cytokines. In several murine tumour models, rIL-2 in combination with recombinant alpha interferon (rIFN-alpha) elicits a potent antitumour response which is often greater than that which can be reached with the individual agents at non-toxic doses. By contrast, recombinant gamma interferon (rIFN-gamma) usually fails to potentiate the antitumour response to rIL-2. Recombinant alpha tumour necrosis factor (rTNF) can synergize with rIL-2 in some circumstances, but, as with the rIL-2/rIFN-alpha combination, the correct regimen is critical for generating a potent response without overt toxicity. Although appropriate cytokine combinations can lead to markedly enhanced tumour infiltration by lymphocytes, it is not clear that only a single type of lymphocyte is invariably involved in the antitumour response or, for that matter, the toxic side effects; nor has the mechanism of action of any of the cytokines in the therapeutic action been unequivocally elucidated. Finally, results of early clinical studies appear to be consistent with results in preclinical models: promising clinical responses to the combination of rIL-2 and rIFN-alpha have already been observed and further study is merited.

The ability of recombinant interferons (IFNs) to modulate recombinant interleukin-2 (rIL-2) augmentation of natural killer (NK)-cell activity and to modulate the generation of activated killer (AK) cells was examined. Incubation of murine spleen cells for 18 hr with either human rIL-2 or a human hybrid recombinant IFN alpha, rHuIFN-alpha A/D, which is active on murine cells, resulted in a dose-dependent increase in NK activity; however, recombinant murine IFN gamma, rMuIFN-gamma, had little activity. A more than additive augmentation of cytotoxicity was obtained when spleen cells were incubated with the combination of rIL-2 and rHuIFN-alpha A/D. Incubation of murine spleen cells with rIL-2 for 3 days resulted in a dose-dependent induction of AK cells which were cytotoxic to an NK-resistant tumor target cell. In contrast to the results observed on NK activity, incubation of spleen cells with rHuIFN-alpha A/D and rIL-2 inhibited AK-cell activity. Partially purified murine IFN-alpha had inhibitory activity comparable to that of rHuIFN-alpha A/D. The addition of rHuIFN-alpha A/D at the initiation of culture of spleen cells with rIL-2 (day 0) resulted in maximal inhibition of cytotoxicity; inhibition was reduced or absent if rHuIFN-alpha A/D was added on day 1 or 2 of culture. The proliferation of spleen cells incubated with rIL-2 was also inhibited by rHuIFN-alpha A/D. Addition of rMuIFN-gamma to spleen-cells and rIL-2 increased the cytolytic activity of AK cells and did not inhibit rIL-2-induced proliferation of spleen cells. Similar data were also obtained with human peripheral blood lymphocytes and recombinant cytokines. Incubation of human peripheral blood lymphocytes with rIL-2 and recombinant human IFN-alpha A (rHuIFN-alpha A) or recombinant human IFN-gamma (rHuIFN-gamma) resulted in a more than additive increase in NK activity. Human AK-cell cytotoxicity was inhibited by rHuIFN-alpha A but enhanced by rHuIFN-gamma. Thus recombinant IFNs have differential effects on rIL-2-induced cytotoxic cells, resulting in augmentation or inhibition of activity, which is dependent on both the type of IFN and the cytotoxic activity examined. These results may have important implications for the potential therapeutic use of combinations of these cytokines.