J. Bubeník

Antigen-mediated macrophage adherence inhibition

Abstract Antigen-mediated macrophage adherence inhibition (MAI) was studied in inbred rats immunized with various transplantation, tumour-specific and protein antigens. A macrophage-rich suspension of peritoneal cells (PC) was obtained from the peritoneal cavity of immunized and control animals by washing. The adherence to glass of PC was specifically inhibited by the addition of the antigens used for sensitization of PC donors or by related (cross-reacting) antigens but not with unrelated antigens. The MAI seems to be due to the direct interaction of the respective antigen with a corresponding PC receptor and not due to the humoral factor released from immune lymphocytes of PC population upon contact with the specific antigen.

Local administration of cells containing an inserted IL-2 gene and producing IL-2 inhibits growth of human tumours in nu/nu mice

We have prepared a retroviral expression construct, pPS-IL-2, in which human IL-2 cDNA has been inserted into the polylinker region, and have used the retroviral vector to introduce the functional IL-2 gene into a fibroblast cell line, RAT-1. Peritumoral administration of IL-2-producing RAT-1 cells into congenitally athymic (nu/nu) mice carrying subcutaneous transplants of human carcinoma cells inhibited the growth of the human tumour xenografts.

Immunotherapy of cancer using local administration of lymphoid cells transformed by IL-2 cDNA and constitutively producing IL-2.

Peritumoral administration of X63-m-IL-2 cells transformed by IL-2 cDNA and constitutively producing large quantities of recombinant IL-2 mediated regression of X63-Ag8.653 plasmocytoma and MC14 sarcoma transplanted in syngeneic mice. Injections of the IL-2-producing cells containing an inserted, modified IL-2 gene effectively inhibited tumour growth also in allogeneic recipients. Activation of murine spleen cells in vitro by co-cultivation with X63-m-IL-2 cells gave rise to lymphokine-activated killer (LAK) cell populations cytotoxic for the X63-Ag8.653 plasmocytoma and MC14 sarcoma. The results suggest that peritumoral administration of lymphoid cells transformed with IL-2 cDNA and constitutively producing IL-2 in the immediate tumour vicinity is sufficient for the effective activation of local IL-2-dependent tumour defence mechanisms and, therefore, can be considered a novel, genetic approach to the immunotherapy of cancer.

Antigenic characteristics of the interaction between Rous sarcoma virus and mammalian cells. Complement-fixing and transplantation antigens.

Abstract Antigenic characteristics of mouse fibrosarcomas induced by three strains of Rous sarcoma virus (Schmidt-Ruppin-, Prague- and Bryan standard-RSV) were studied by complement-fixation (CF) and transplantation tests. Approximately 50% of the induced tumors contained CF antigen specific for the avian sarcoma-leukosis group of viruses when examined with high-titer rabbit antiserum against sodium lauryl sulfate-treated avian myeloblastosis virus. Common specific transplantation antigen was found in all but one tumor. No correlation was observed between the presence of transplantation and CF antigen. Four tumors were positive in both CF and transplantation tests. Three tumors were positive in transplantation but negative in CF tests. One tumor was positive in CF tests, but negative in transplantation tests. Injection of CF positive mouse tumors in chickens caused chicken Rous sarcomas. No Rous sarcomas appeared in chickens injected with CF negative tumors. The only exception to this rule was a tumor induced by Bryan standard strain of RSV. This tumor contained CF antigen but gave negative results after injection in chickens. The possible significance of antigenic markers for studies of different types of virus-cell interaction is discussed.

Growth inhibition of a MC-induced mouse sarcoma by TCGF (IL 2)-containing preparations

SummarySupernatants from Con A-stimulated rat spleen cell cultures containing T cell growth factor inhibited growth of a transplantable 3-methylcholanthrene-induced sarcoma in syngeneic mice. The tumour-inhibitory effects were dependent on the concentration of T cell growth factor and repeated injections of the supernatants.

Tumour inhibitory effects of TCGF/IL-2/-containing preparations

SummarySupernatants from ConA-stimulated rat spleen cell cultures and from cultures of PMA-stimulated murine lymphoma subline EL-4TF were found to contain TCGF and to inhibit growth of a transplantable, MC-induced sarcoma MC11 in syngeneic mice. Tumour-inhibitory effects of the supernatants were dependent on local and repeated administration. Prior to use of the supernatants obtained from PMA-stimulated EL-4TF cell cultures, the dialysable PMA had to be removed; contamination with PMA was found to abolish the tumour-inhibitory effect of the supernatants and to produce enhancement of tumour growth. A significant tumour-inhibitory effect has also been obtained with partially purified TCGF prepared from culture supernatants of cloned EL-4TF cells by ammonium sulphate precipitation, ion-exchange (FPLC) chromatography, and AcA 44 Ultrogel filtration.

Induction of cytotoxicity of lymphocytes from tolerant donors by antibodies to target cell alloantigens.

SUMMARY Alloantibodies from Campbell ducks which induce rejection of tolerated skin allografts from Peking ducks in vivo were studied for their capacity to induce cell-mediated cytotoxic reactions in vitro. In the presence of heat-inactivated alloantiserum, Campbell lymphocytes from tolerant ducks became eytotoxic to Peking target cells. Cytotoxicity was assayed either as release of 51Cr from labeled Peking erythrocytes or as reduction the number of fibroblasts growing in microtest plates. The sera were not eytotoxic without addition of lymphocytes, and normal Campbell sera with lymphocytes had no effect. Syngeneic or xenogeneic (chicken) target cells were not affected. Antiseruminduced cytotoxicity was also obtained with lymphocytes from Campbell ducks not tolerant to Peking alloantigens or with human lymphocytes. In 11 of 15 immune sera studied, a good correlation was found between their eytotoxic activity in vitro and their graft-rejecting capacity in vivo. No correlation was observed between the capacity of the sera to induce cell-mediated cytotoxicity in vitro and their capacity to produce Arthus-like reactions in vivo.

Cancer immunotherapy using local interleukin 2 administration

Peri-tumoural administration of human recombinant interleukin-2 (RIL-2) into C57BL/10ScSnPh (B10) mice carrying subcutaneous transplants of syngeneic methylcholanthrene (MC)-induced sarcomas substantially inhibited tumour growth. Experiments were designed to compare the tumour-inhibitory effect of highly purified RIL-2 with that of unpurified human and rat lymphoid interleukin-2 (IL-2) preparations. It was found that the effect of RIL-2 was significantly lower than that of the lymphoid IL-2 preparations. These findings indicate that other lymphokines may participate in the positive results of local IL-2 immunotherapy using unpurified lymphoid IL-2 preparations. However, the admixture of human recombinant interleukin-1 (RIL-1) did not potentiate the immunotherapeutic effects of RIL-2. Sensitivity of MC-induced sarcomas to local RIL-2 immunotherapy was a general phenomenon. The growth of approximately eighty percent (5/6) of the MC-induced sarcomas could be inhibited with local RIL-2 administration. Moreover, direct correlation between the sensitivity of tumours to the tumour-inhibitory effect of RIL-2 in vivo and their susceptibility to the cytolytic effect of RIL-2-activated syngeneic killer spleen (LAK) cells in vitro was observed. This correlation indicates that LAK cells represent the effector cell mechanism responsible for the anti-tumour efficacy of local RIL-2 immunotherapy and that in vitro testing of sensitivity to the LAK cell-mediated cytolysis may be used to detect tumours responding to the local RIL-2 immunotherapy in vivo.

Defect in lectin-induced interleukin 2 production by peripheral blood lymphocytes of patients with invasive urinary bladder carcinoma

The production of interleukin 2 (IL-2) by phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) from 21 patients with transitional cell carcinoma of the urinary bladder (BTCC) and 16 control blood donors was measured with a solid phase enzyme immunoassay based on the dual antibody immunometric sandwich principle. PBMC from patients with invasive BTCC (grade III-IV) showed a defect in the production of IL-2. The concentration of IL-2 in the supernatants of PBMC cultures from these patients was substantially lower (0.4 +/- 0.1 U/ml) than that observed in the supernatants of PBMC cultures from patients with non-invasive BTCC, grade II (1.5 +/- 0.7 U/ml), and from tumour-free controls (1.4 +/- 0.8 U/ml). These results suggest an immune dysfunction based on quantitatively impaired IL-2 production in patients with invasive BTCC and indicate that exogenous IL-2 could be used as an immunological response modifier for the treatment of these patients.

Lymphokine-activated killer (LAK) cells: I. Age-dependent decline of LAK cell-mediated cytotoxicity.

Experiments were designed to assess age-related changes in generation of lymphokine-activated killer (LAK) cells and to test whether these changes can be modified by diets differing in the proportion of polyunsaturated to saturated fatty acids (P/S). Ficoll-Hypaque-isolated spleen lymphocytes of rodent chow-fed, 6-85-week-old C57BL/6 (H-2b), 8-81-week-old C57BL/10 (H-2b) and 6-62-week-old SJL (H-2s) mice were cultured in IL-2-containing medium and examined in 51Cr cytotoxicity assay. Similarly, Ficoll-Hypaque-isolated spleen lymphocytes of 6-36-week-old SJL mice fed diets which differed in the ratio of polyunsaturated/saturated fatty acids were cultured in IL-2-containing medium and assayed for cytotoxicity. Age-related decline of LAK cell-mediated cytolysis was observed in mice of both H-2b and H-2s haplotype. The age-related decline of LAK cell-mediated cytolysis was the consequence of age-related decrease in the rate of LAK cell precursor maturation. SJL mice fed from birth with diets differing in P/S did not differ in LAK cell-mediated cytolysis.