Intratumoral IL-12 gene transfer improves the therapeutic efficacy of IL-12 but not IL-19.

We have compared the therapeutic activity of IL-12 and IL-18 in mice carrying IL-2 gene-transduced syngeneic sarcoma Mc12. The IL-2 gene-transduced sarcoma has previously been utilized as an irradiated, genetically modified tumour vaccine. Murine recombinant IL-12 was capable of suppressing growth of the IL-2 gene-modified sarcoma Mc12 in syngeneic mice more efficiently than growth of the parental Mc12 sarcoma. In contrast, murine recombinant IL-18 could neither inhibit growth of the parental Mc12 sarcoma, nor suppress growth of its IL-2 gene-modified transfectant. These results suggest that although both of these cytokines are functionally related and participate in the induction of IFN gamma production as well as in cell-mediated immune cytotoxicity, in the murine sarcoma system only IL-12 is therapeutically active and exerts its therapeutic effect in concert with the IL-2 gene. Thus, intratumoral IL-2 gene transfer improves the therapeutic efficacy of IL-12; administration of recombinant IL-12 should therefore be considered as adjuvant in IL-2 gene therapy with irradiated, genetically modified tumour vaccines.

Irradiation of genetically modified plasmacytoma vaccines results in upregulation of CD80 molecule expression, IL-2 production and higher therapeutic efficacy of the vaccines.

It has been found previously that irradiated, IL-2 gene-modified plasmacytoma (X63-m-IL-2) vaccines are more efficient in the therapy of the parental (X63-Ag8.653) plasmacytoma than live plasmacytoma vaccines. In this communication, we have demonstrated that irradiation of murine IL-2-producing plasmacytoma vaccines resulted in upregulation of CD80 molecule expression and IL-2 production. The expression of MHC class I antigens was not altered. The upregulation of the CD80 membrane molecule expression in X63-m-IL-2 cells was higher after irradiation with 150 Gy than after irradiation with 50 Gy. Comparable upregulation of the CD80 molecule expression has also been demonstrated after irradiation of the parental murine X63-Ag8.653 plasmacytoma cells. The results indicate that upregulation of the CD80 molecule expression and enhanced IL-2 production in irradiated X63-m-IL-2 cells was responsible for the higher therapeutic effectiveness of the irradiated plasmacytoma vaccine.

Clinical ineffectiveness of IL-2 and/or IFN alpha administration after autologous PBSC transplantation in pediatric oncological patients.

Clinical impact of s.c. administration of IL-2 and/or IFN alpha was studied in 23 pediatric patients with Hodgkin lymphoma (IFN alpha group) and sarcoma, non-Hodgkin lymphoma, peripheral neuroepitelioma, neuroblastoma, and embryonic carcinoma (IL-2 + IFN alpha group) after autologous PBSC transplantation. Expression of CD3, CD4, CD8, CD25, CD38, CD56, CD71, CD122, and HLA-DR antigens, serum level of the soluble IL-2R alpha, and NK activity against K562 cell line were evaluated in 11 patients representative for both types of immunotherapy. T and, more markedly, NK cell proliferation, induction of activation markers on the surface of T and NK subsets, and elevation of sIL-2R alpha concentrations were seen in the IL-2 + IFN alpha subgroup. In the IFN alpha subgroup, the total number of lymphocytes and expression of activation markers remained unchanged, but the number of CD8+ T cells increased at the expense of CD4+ T and NK cells during the therapy. Cytotoxic activity against K562 cells was not influenced by the immunotherapy in either subgroup. No significant clinical benefit of the immunotherapy was seen in these patients compared to 27 control patients with relevant diagnoses who did not receive immunotherapy.

CD80 and IL-2 signals cooperate in the regression of tumors transplanted in congenitally athymic mice.

Interleukin-2 and CD80 transfectants of a methylcholanthrene-induced murine sarcoma Mc12 (Mc12-IL-2 and Mc12-CD80 cells) with similar tumorigenicity in euthymic mice were utilized for experiments designed to investigate a co-stimulatory role of the CD80 molecule in allogeneic, congenitally athymic (nu/nu) mice. The CD80-transfected cells were as tumorigenic in nu/nu mice as the parental Mc12 sarcoma. The IL-2-transfected cells grew only transiently and regressed in all nu/nu recipients during four weeks after challenge with doses up to 5x10(7) cells. The 1:1 mixture of parental Mc12 with Mc12-CD80 cells grew progressively in all inoculated nu/nu mice; in a 1:1 mixture with parental Mc12 cells, Mc12-IL-2 cells were able to cause regressions in approximately 50% of nu/nu mice; the 1:1 mixture of Mc12-IL-2 and Mc12-CD80 transfectants showed only transient growth and regressed during four weeks in all inoculated nu/nu mice. Adoptive transfer of cell-mediated immunity revealed that spleen cells from tumor regressors were capable of transferring the resistance to Mc12 tumor in nu/nu mice. The spleen cells from tumor regressors were not cytolytic when allowed to react in vitro with Mc12, Mc12-IL-2, or Mc12-CD80 target cells. However, when grown in IL-2-containing medium, splenocytes from tumor regressors, but not the splenocytes from tumor progressors, could develop cytolytic activity directed against Mc12 target cells that was comparable to that of the splenocytes from tumor-free controls. These results suggest that the rejection of tumors in nu/nu mice was mediated by IL-2-dependent mechanisms in which the CD80 molecule played a co-stimulatory role; the results also indicate that the ability to be activated by IL-2 and to give rise to cytolytic activity of nu/nu splenocytes from tumor progressors is decreased.

Interleukin 2 gene therapy of residual disease in mice carrying tumours induced by HPV 16.

Experiments were designed to examine the efficacy of IL-2 gene therapy in a surgical minimal residual tumour disease, using moderately immunogenic MK16/1/IIIABC murine cells transformed by activated ras and HPV 16 E6/E7 oncogenes (MK16 cells). Previously we demonstrated that surgical minimal residual tumour disease (SMRTD) could be effectively cured when murine Mc12 sarcoma had been resected and the operated mice were treated with irradiated Mc12 sarcoma cells engineered to secrete IL-2. In this study we performed IL-2 gene therapy of MK16 carcinoma with two types of irradiated MK16-unrelated tumour cell vaccines. One type of vaccine was derived from MHC class I-matched Mc12 sarcoma cells engineered to secrete IL-2 and the other from MHC class I-discordant IL-2 producing plasmacytoma X63-m-IL-2. The vaccines did not share any tumour rejection antigen with the MK16 cells and served exclusively as a local source of IL-2 production. Both vaccines were capable of inhibiting MK16 tumours when administered peritumorally up to 15 days after MK16 tumour challenge. The irradiated MHC class I-matched and IL-2-producing Mc12 sarcoma vaccine was then selected for therapy of MK16 SMRTD. Whereas the recurrence rate in the operated MK16 carcinoma bearers was 80%, so that only 20% of mice were cured by surgery, approximately 65% of the MK16 carcinoma bearers were permanently protected when the surgery was followed by local administration of the IL-2-producing Mc12 sarcoma vaccine.

Granulocyte-macrophage colony-stimulating factor-producing tumour vaccines.

Murine sarcoma MC12 cells were transfected with the gene coding for murine granulocyte-macrophage colony-stimulating factor (GM-CSF). Tumorigenicity of a variety of cell clones with different expression of the inserted gene was assessed. All of the genetically manipulated MC12 cell clones examined were found to be less tumorigenic than the parental MC12 cell population. No correlation was observed between the production of GM-CSF by the clones and their tumorigenicity. It has been found that irradiation of the GM-CSF-producing cells with the dose of 150 Gy did not significantly inhibit the GM-CSF production during the period of 5 days after irradiation. These findings provided us with the rationale for using the irradiated GM-CSF-producing MC12 sarcoma vaccine for therapy. It has further been found than immunosensitivity of the genetically manipulated, GM-CSF-producing tumour targets to the IL-2-activated killer (LAK) cell-mediated cytolysis was significantly increased, as compared to the parental target cell population. Irradiated, GM-CSF-producing tumour vaccines were used for therapy of 3-day-old MC12 sarcoma transplants in syngeneic mice and for therapy of surgically induced minimal residual tumour disease. Neither small tumour transplants, nor tumour residua after surgery were significantly sensitive to the therapy with GM-CSF-producing tumour vaccines.

Interleukin-2 gene therapy of surgical minimal residual tumour disease.

Our study was designed to examine the effects of IL-2 gene therapy in a surgical minimal residual tumour disease (SMRTD). Mice were inoculated s.c. with methylcholanthrene (MC)-induced MC12 sarcoma cells. When the tumours reached 8 to 12 mm in diameter, they were excised, either completely ("microscopic SMRTD") or incompletely ("macroscopic SMRTD"). On day 90 after surgery, the tumour recurrence rate in untreated mice with microscopic SMRTD was approximately 30%, whereas in those with macroscopic SMRTD it was 75%. After surgery, experimental mice were treated with 2 types of irradiated, IL-2 gene-modified, IL-2-producing tumour cell vaccine. One type of vaccine was derived from the MC12 sarcoma cells (MC12-1L2/IV-3); the other type was derived from an unrelated X63-Ag8.653 plasmacytoma (X63-m-IL-2). Both types of vaccine failed to cure the macroscopic SMRTD. Whereas the X63-m-IL-2 vaccine was also ineffective in the microscopic SMRTD, the MC12-IL2/IV-3 vaccine was capable of preventing growth in all but one mouse (1164) with microscopic SMRTD when administered 2 to 5 days after surgery. If the vaccination took place 2 days before surgery or later than 5 days after surgery, the therapeutic activity was lost. Vaccination with irradiated parental MC12 cells did not produce any significant benefit compared to the operated-only mice. The protective effect of the MC12-L2/IV-3 vaccine was specific and comparatively long-lasting. Vaccinated mice, which had rejected the MC12 tumour residuum, were capable of rejecting a second inoculum of the MC12 sarcoma cells injected on days 35 to 110 after surgery but succumbed to the growth of 2 other unrelated murine sarcomas carrying different tumour-rejection antigens.

Immunogenicity, immunosensitivity and cell surface adhesiveness of tumour vaccines carrying an inserted CD80 gene.

Cell surface adhesiveness, immunogenicity and immunosensitivity of tumour vaccines modified by the CD80 gene transfection was examined and compared to that of the parental MC12 murine sarcoma. Insertion of the CD80 gene substantially enhanced the adhesiveness of the genetically modified tumour cells to nylon wool non-adherent (T) but not to nylon wool adherent (B) lymphocytes. The increased adhesive interaction could be inhibited by anti-CD80 monoclonal antibody. CD80+ transfectants were more sensitive to the cytotolytic effect of MC12-immune splenocytes and IL-2-activated spleen cells than the parental MC12 sarcoma. Similarly, spleen cells from syngeneic mice immunized with CD80+ transfectants displayed a higher cytolytic activity when allowed to react with MC12 cells than splenocytes from mice immunized with the parental MC12 cells. These results suggest that a positive correlation exists among the expression of the CD80 molecules, T cell adhesion to the genetically modified cells, immunosensitivity of the CD80+ transfectants and the capacity of the transfectants to activate cytolytic, tumour-reactive effector cells in vivo. This correlation provides a rationale for gene therapy based on the construction of CD80- modified tumour vaccines.

Use of cryopreserved lymphocytes for assessment of the immunological effects of interferon therapy in renal cell carcinoma patients.

Experiments were designed to assess whether cryopreserved PBL could be used to monitor the immunological effects of IFN-alpha therapy in renal cell carcinoma (RCC) patients. It was found that programmed freezing and thawing of peripheral blood lymphocytes (PBL) from normal blood donors did not substantially change lymphocyte subset proportions and that cryopreserved PBL were able to proliferate in response to IL-2. It was also possible to activate the cytolytic activity of frozen PBL, and the frozen leukocytes did not lose their ability to secrete IFN-gamma after PHA activation. We have used these findings to investigate the immunological effects of IFN-alpha therapy in RCC patients. Cryopreservation of PBL samples collected from various patients over a period of 9-14 months enabled us to compare the in vitro reactivity of PBL from individual RCC patients repeatedly and under standard conditions. It was found that IL-2 induced proliferative responses of PBL from IFN-alpha non-responders, collected prior to IFN-alpha therapy, were significantly decreased as compared to those from normal blood donors. The proliferative responses of PBL from IFN-alpha responders, collected prior to IFN-alpha therapy, did not substantially differ from normal controls. Culture of PBL from IFN-alpha responders for 3 days in IFN-alpha-containing medium increased their lytic activity towards RCC targets, whereas no such increase was observed with non-RCC targets or using PBL from IFN-alpha non-responders or PBL from normal-blood donors. Enzyme-linked immunospot (ELISPOT) assays performed with cryopreserved lymphocytes from IFN-alpha non-responding RCC patients, collected prior to IFN-alpha therapy, revealed a substantially decreased ability to secrete IFN-gamma, as compared to IFN-gamma secretion of PBL from IFN-alpha responders or normal blood donors.

Genetically modified tumour vaccines.

Two genes, the gene coding for IL-2 and the gene encoding the CD80 molecule, were inserted into murine sarcoma MC12 cells. Tumorigenicity of a variety of cell clones with different expression of the inserted genes was assessed. Most of the genetically manipulated MC12 cell clones were less tumorigenic than the parental MC12 cell population. Tumorigenicity of the clones declined with increasing production of IL-2 as well as with the increasing expression of the CD80 molecule. When the tumorigenicity of the clones carrying an inserted IL-2 gene was compared with that of the clones carrying an inserted CD80 gene, it was found that the insertion of the IL-2 gene suppresses tumorigenicity more efficiently than insertion of the CD80 gene. Admixture of the IL-2-producing MC12 clones to the tumorigenic CD80(+) MC12 cell doses could completely inhibit the tumorigenicity of the CD80(+) cells. Insertion of the CD80 gene into sarcoma cells substantially enhanced the adhesive interaction between the MC12 sarcoma and syngeneic T lymphocytes.

IFN-alpha therapy of renal-cell carcinoma - defect of lymphocyte sensitivity to mitogenic and activating cytokine signals in patients not-responding to therapy.

The present prospective study was designed to assess whether the renal cell carcinoma (RCC) patients treated with recombinant interferon alpha (IFN alpha), whose tumours respond (responders) and do not respond (non-responders) to IFN alpha therapy, differ with regard to in vitro sensitivity of peripheral blood lymphocytes (PBL) to interleukin 2 (IL-2), IFN alpha, and IFN gamma signals prior to therapy. Twenty-one patients with advanced RCC after nephrectomy, 15 responders and 6 non-responders, were entered into a protocol. The protocol involved isolation and freezing of PBL samples followed by IFN alpha treatment of patients, assessment of proliferative and activating PBL responses, and evaluation of the therapeutic results. Freezing of PBL samples allowed us to compare the in vitro reactivity of PBL from individual RCC patients, repeatedly and under standard conditions. Substantial differences in proliferative responses to the mitogenic IL-2 signal of PBL derived from IFN alpha responders and nonresponders were found. Whereas the IL-2-induced proliferative responses of PBL from normal blood donors and IFN alpha responders were comparable, the proliferative responses of PBL from IFN alpha non-responders were significantly decreased, suggesting an immune dysfunction in non-responders. Cultivation of PBL from RCC patients in medium supplemented with IFN alpha increased the lytic activity of PBL from IFN alpha responders directed against RCC targets; no such increase could be observed with non-RCC targets, with PBL from IFN alpha non-responders, or with PBL from normal blood donors. Detection of phytohaemagglutinin (PHA)-stimulated IFN gamma secretion by PBL at the single cell level using enzyme-linked immunospot (ELISpot) assay revealed that the ability to produce IFN gamma was substantially decreased in IFN gamma non-reponders, as compared to IFN alpha responders and to normal blood donors.

Pathogenesis of Marek's disease in chickens with maternal antibody.

Histopathological lesions were studied in chickens with maternal antibody against MD virus at 14-day intervals after their exposure by contact to HPRS-16 of MD virus in the first-day of life. The first lesions occured 14 days after infection in the liver, kidney, heart, brain and the sciatic nerves. Relatively, the most expressive changes were observed between 56th and 70th day after infection. Lesions were characterized as light to heavy infiltrations formed by immature and mature pleomorphic mononuclear cells. Among these cells myeloid type cells occured too. In some cases expressive tumorous lesions with intensive mitosis were observed mainly in the liver. Intranuclear inclusion bodies were also observed mainly among the proliferating epithelial cells of the proventriculus. 14-days after infection changes in the brain were classified as nonpurulenta encephalitis. Gross lesions characteristic of MD occured the most frequently in the gonads, liver and the kidney. In one case 56 days after infection herpesvirus-like particles were observed by electron microscopy in the tumorous gonads and liver. They measured about 200 to 260 nm in diameter. Degraded and/or aberrant incompleted virus-like particles occured the most often and completed (enveloped) ones with electrondense nucleoids were observed occasionally. It was concluded that MD in chickens with materanal antibody against MD virus shows monophasis progress.