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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.

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 (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.

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.

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.

We have examined the prevalence of circulating immune complexes in sera from patients with mycobacterial infections. Sera from 68 percent of patients with active M. tuberculosis and 58 percent of patients with M. intracellulare infections had significantly elevated Clq binding activity (Clq-BA). In general there was a fall in Clq-BA with treatment. Only 15 percent of M. tuberculosis patient with a bacteriological cure, 22 percent of non-tubercular patients with chronic obstructive pulmonary disease, and 3 percent of normal individuals had an elevated Clq-BA. Antibodies to a BCG-derived antigen were demonstrated in most of the individuals studied in all groups but, significantly elevated levels were seen only in patients with mycobacterial infections. In certain patients there appeared to be an inverse relationship between Clq-BA and BCG binding, suggesting that perhaps BCG-related antigens participated in the immune complexes found. Other possible antigen-antibody complexes are discussed.

Suppression of growth of the line-10 guinea pig hepatocarcinoma was achieved after the simultaneous injection of line-10 cells and heat-killed Staphylococcus aureus. Growth of tumor was also suppressed when line-10 cells were injected alone, contralaterally, at the same time as the vaccine mixture. Immunity developed to subsequent line-10 cell challenges but not to other syngeneic tumors. Similar results were obtained with the use of protein A-rich or -deficient strains of S. aureus. Multiple intratumor injections of heat-killed S. aureus were therapeutically effective against 6-day tumors. The antitumor effects of nonviable S. aureus were similar in many ways to those of living BCG or bacterial products suspended in oil droplets.