Induction of cytotoxic T lymphocyte responses against hepatitis delta virus antigens which protect against tumor formation in mice.

The cellular immune response is a crucial defense mechanism against hepatotropic viruses and in chronic viral hepatitis prevention. Moreover, hepatitis delta virus (HDV) immunogenicity may be an important component in the development of prophylactic and therapeutic vaccines. Therefore, we evaluated the immunogenicity of the small (HDAg) or large delta antigen (LHDAg) to be used as a DNA-based vaccine. We immunized different mouse haplotypes, determined cellular immune responses, and tested protection of animals against tumor formation using syngeneic tumor cells stably expressing the delta antigens. Both LHDAg and HDAg primed CD4+ and CD8+ T cell immunity against both forms of delta antigens. CD8+ T cell frequencies were about 1% and antigen-specific CD8+ T cells remained detectable directly ex vivo for at least 35 days post-injection. No anti-delta antibody responses could be detected despite multiple detection systems and varied immunization approaches. We observed protection against syngeneic tumor formation and growth in mice immunized with DNA plasmids encoding secreted or intracellular forms of HDAg and LHDAg but not with recombinant HDAg establishing the generation of significant cellular immunity in vivo. Both CD4+ and CD8+ T cells were required for antitumoral activity as determined by in vivo T cell depletion experiments. The results indicate that DNA-based immunization with genes encoding LHDAg and HDAg induces strong T cell responses and, therefore, is an attractive approach for the construction of therapeutic and prophylactic T cell vaccines against HDV.

Prevention of lethal graft-versus-host disease following allogeneic bone marrow transplantation in mice by short course administration of LF 08-0299.

We investigated the ability of LF 08--0299, a new immunosuppressive compound, to prevent murine graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). A short term LF 08--0299 treatment at optimal dosage protected more than 75% of recipient mice from lethal GVHD induced either across minor antigens alone or the full H2 barrier. Furthermore, LF 08--0299 still prevented lethal GVHD when treatment was delayed to 10 days post-BMT. Long-term LF 08--0299-treated survivors were free of clinical signs of GVHD, and histopathologic examination of liver, skin, and intestines was normal, demonstrating that recipient mice did not develop chronic GVHD. We assessed the immunocompetence of long-term surviving recipient mice. Results from MLR and CTL assays were weak whereas responses against unrelated H2 antigens were reduced but still preserved. Moreover, in vivo transfer experiments demonstrated that spleen cells from long-term survivors were unable to induce lethal GVHD in irradiated recipients of host origin, while spleen cells injected in irradiated recipients of a host-unrelated H2 were fully competent to induce a lethal GVHD. Together these results indicate that stable chimeric recipient mice were specifically tolerant to host antigens. We further showed that while LF 08--0299 can protect recipient mice from lethal GVHD, it also preserved a graft-versus-leukemia effect when mice were inoculated with P815 tumor cells. These data suggest that LF 08--0299 may be a novel pharmaceutical agent that would prevent GVHD in human unrelated bone marrow transplantation.

Increased surface expression of class I MHC molecules on immunogenic cells derived from the xenogenization of P815 mastocytoma cells with 8-methoxypsoralen and long-wavelength ultraviolet radiation.

In a previous study we demonstrated that the treatment of the highly tumorigenic cell line, P815, with 8-methoxypsoralen and long-wavelength ultraviolet radiation resulted in the production of several immunogenic clones (tum-). Mice inoculated with the tum- cells survived much longer than mice inoculated with the original tumorigenic cells (tum+). It was suggested that the increased survival of mice treated with the tum- clones arose as a result of an increased antigenicity derived from the phototreatment. In this report we show that the tum- cells have a greater density of class I MHC molecules on their surface (50-157% compared to P815). Class I MHC density on the cell surface is required to elicit targeted cytotoxic responses. These results can be considered in terms of human class I MHC assays which show that many human tumor cells have a reduced expression of class I MHC. Because other DNA damaging agents have also been shown to enhance class I expression, it is suggested that in addition to the cytotoxic effects of these agents, other pleiotropic effects must be considered. Photochemotherapy may phenotypically alter cells so that the enhanced expression of class I MHC molecules on the surface of phototreated cells may be associated with the clinical responses observed in cutaneous T cell lymphoma patients.

[The synergistic action of lipopolysaccharide and muramyl dipeptide in the immunotherapy of DBA/2 mice with mastocytoma P815]. / Sinergichnoe deistvie lipopolisakharida i muramildipeptida pri immunoterapii myshei DBA/2 s mastotsitomoi P815.

The intratumoral administration of lipopolysaccharide (LPS) and muramyl dipeptide (MDP) in combination, but not separately, resulted in necrosis and rejection of subcutaneous P815 mastocytoma nodules in DBA/2 mice with 30 to 40% survival. Previous sensibilization of animals by LPS + MDP, treatment by indomethacin, cyclophosphamide or syngeneic lymphocytes did not augment the immunotherapeutic action of LPS + MDP combination. Reinoculation of P815 cells into cured DBA/2 mice 8 months after the disappearance of the primary tumor led to rejection of new nodules with 50% survival rate. In LPS + MDP immunotherapy of these tumors two stages may be distinguished by a thrombo-necrotic stage and that of development of immunity.

Immunopotentiation by a new antitumor polysaccharide, DMG, a degraded D-manno-D-glucan from Microellobosporia grisea culture fluid.

The immunopharmacological behavior of DMG, an antitumor polysaccharide, was studied in mice. DMG administered ip or sc stimulated peritoneal macrophages to produce high levels of interleukin-1 activity, which can amplify successive immune responses. DMG dose-dependently and schedule-dependently increased the cellular immune response against allogeneic tumor cells and the humoral immune response to sheep erythrocytes. DMG also enhanced nonspecific antitumor effector functions, such as natural killer activity of spleen and peritoneal cells, and the cytostatic activity of peritoneal macrophages. Peritoneal macrophages activated by ip or sc injection of DMG exhibited high cytostatic activity, especially after exposure in vitro to lymphokine supernatants containing macrophage activation factor. Moreover, granulocyte/macrophage colony-stimulating activity in the serum increased 2-10 hr after DMG administration. Thus, DMG potentiated antigen-specific immunological functions and nonspecific functions of host defense systems against cancer both qualitatively and quantitatively.

Immunotherapy of cancer with cell wall skeleton of Myocabacterium bovis-Bacillus Calmette-Guérin: experimental and clinical results.

Adjuvant and antitumor activities of CWS prepared from cells of mycobacteria, nocardia, and corynebacteria were examined. Oil-attached CWS of M. bovis BCG (BCG-CWS) stimulated the generation of cell-mediated cytotoxic effector cells in mice. Tumor growth was suppressed in mice inoculated intradermally with a mixture of oil-attached CWS and living tumor cells. Systemic and specific tumor immunity was demonstrated in mice in which tumor growth was suppressed. Tumor growth was also suppressed by oil-attached CWS of BCG or N. rubra in autochthonous autografts of spontaneous mammary adenocarcinoma and methylcholanthrene-induced fibrosarcoma in mice. The intravenous injection of oil-attached BCG-CWS prevents the appearance of lung cancer in rabbits by the instillation of chemical carcinogens. It was also shown that treatment with oil-attached BCG-CWS was able to elevate the immunologically depressed state of tumor-bearing mice to a normal level, as determined by a cell-mediated cytotoxicity assay that empolyed chromium release as the standard. Preliminary results suggest that oil-attached BCG-CWS is useful as an immunotherapeutic agent for both lung cancer and for malignant melanoma, leukemia, Hodgkin's disease, and other neoplastic diseases and that this agent operates without any significant complications.

Effect of cell-wall skeleton of Mycobacterium bovis BCG on cell-mediated cytotoxicity in tumor-bearing mice.

Effect of oil-attached BCG cell-wall skeleton (BCG-CWS) on cell-mediated cytotoxicity in tumor-being mice was investigated using the allograft system. It was found that the treatment with oil-attached BCG-CWS was able to elevate the immunolgically depressed state of tumor-bearing mice to a normal level as determined by chromium release assay.

Chemical and immunological studies on the cell walls of Propionibacterium acnes strain C7 and Corynebacterium parvum ATCC 11829.

The chemical and immunological properties of the cell walls prepared from the cells of anaerobic coryneforms, Propionibacterium acnes C7 and Corynebacterium parvum ATCC 11829, were partially investigated. The cell walls prepared from P. acnes C7 and C. parvum ATCC 11829 were composed of fatty acids, polysaccharides consisting glucose, galactose and mannose and mucopeptides consisting mainly of alanine, glutamic acid, alpha, epsilon-diaminopimelic acid, glycine, muramic acid and glucosamine. As the fatty acid constituents of the cell wall of P. acnes C7, iso-pentadecanoic acid and iso-heptadecanoic acid were detected as major components. Both cell walls prepared from P. acnes C7 and C. parvum ATCC 11829 showed potent adjuvant activity on the formation of circulating antibody and development of delayed type hypersensitivity in vivo and on the primary immune response to sheep erythrocytes in vitro, however, could not augment helper function of carrier-primed T cells and on the development of cell-mediated cytotoxicity to mastocytoma P815-X2 cells in C57BL/6J mice. It is also shown that the cell walls of P. acnes C7 and C. parvum ATCC 11829 act on mouse spleen cells as mitogen.

Demonstration of organ differences in peripheral B cell populations through the use of deficient fetal bovine serum.

Deficient fetal bovine serum (FBS) was used in culture to distinguish differences in the B cell populations of peripheral lymphoid organs. Culture medium supplemented with deficient FBS supported the induction of primary humoral immune responses to heterologous erythrocyte antigens in murine Peyer's patch and lymph node cultures, but not in spleen cultures obtained from the same unimmunized mice. This difference was not due to a difference in thymus-derived (T) cell or adherent cell activity, or to the production of stimulating factors by Peyer's patch or lymph node cells. In view of the finding that spleen cells from mice immunized with erythrocyte antigens support immune responses in deficient FBS, we suggest that murine Peyer's patches and lymph nodes contain antigen-experienced B cells, whereas spleens contain predominately antigen-inexperienced B cells. In contrast, spleen and Peyer's patch cells cultured with deficient FBS can be induced to mediate specific cytotoxic allograft responses which are similar in magnitude to responses observed in normal FBS. Deficient FBS may provide a useful tool for distinguishing between B cells on the basis of prior antigenic encounter, and for examining the induction of T cell responses in the absence of B cell responses.