Induction of leukemia regression in mice by immunotherapeutic transfer of T-lymphocytes.

The erythroleukemia induced in susceptible mice by Friend virus (FV) is a progressive, lethal disease. A variant strain of Friend virus (regressing FV) produces a histopathologically identical leukemia except that the disease spontaneously regresses in 50% of leukemic mice. Normal T-cell and macrophage function are required for regression to occur and in animals that are going to regress, specifically reactive T-cells are found in the spleen. Passive transfer of sensitized T-cells from regressing FV immunized or regressed mice caused regression of the conventional lethal leukemia induced by FV. To expand the effector cell populations, characterize them and improve their therapeutic efficacy, sensitized T-cells were cultured in vitro. The T-cells, isolated from regressed or immunized mice, were grown and expanded in vitro with interleukin 2 and antigen (mitomycin C treated regressing FV-infected cell lines). The T-cells demonstrated high levels of in vitro cytotoxicity against FV antigens but exhibited no blastogenic response to the same antigens. When fully FV-induced leukemic mice (14 days post virus inoculation; spleen weight, greater than 0.75 g) were given one injection of 5 X 10(6) in vitro cultured T-cells and no other treatment the mice experienced permanent regressions of their disease. From T-cell cultures depleted of specific cell populations with monoclonal antisera, helper Lyt-1+ cells were shown to be responsible for permanent regressions (cures), whereas cytotoxic Lyt-2+ cells caused temporary leukemia remissions. This model thus provides an experimental system of highly effective passive cellular immunotherapy against an autochthonous, fully developed leukemia, requiring no adjunctive treatment for activity.

Mechanism of macrophage reversal of Friend erythroleukemia: macrophage regulation of normal and leukemic erythropoiesis.

The acute erythroleukemia induced in mice by the anemia-inducing strain of the Friend virus complex is caused to regress by normal macrophages. We examined the possibility that reversal of leukemia is related to a macrophage regulatory function in erythropoiesis. We found that the ability of macrophages to induce leukemia regression correlates with nonimmunological, in vivo suppression of normal and susceptible leukemic erythroid progenitors. The macrophage effect on erythropoiesis appears to be due to changes in a humoral regulator, related to but independent of erythropoietin. The results suggest a novel regulatory system for erythropoiesis, operative in vivo, and involving macrophages as accessory or suppressor cells. This regulation appears to be disrupted in erythroleukemic mice, but can be restored, and the disease can be made to regress by treatment with normal macrophages.

Immunotherapeutic approaches to leukemia: the use of the Friend virus-induced erythroleukemia model system.

We have developed a model system to study immunologically mediated regression of leukemia based on Friend virus-induced erythroleukemias. This system has been used to evaluate the immunotherapeutic activity of macrophages, specifically reactive T-cells (CTL/RFB), lymphokine-activated killer cells and interleukin 2, and tumor necrosis factor alpha and interferon-gamma. In the present studies, CTL/RFB were evaluated for their ability to prevent disease recurrence. Animals with the regressing strain of Friend virus at Day 39 post virus were treated with either one or two injections of 5 x 10(6) CTL/RFB. Animals given one or two injections of CTL/RFB had a significantly lower rate of recurrence than did untreated animals. The helper T-cell component of CTL/RFB was implicated in causing leukemia regression. Interleukin 1 alpha and tumor necrosis factor alpha, multifunctional cytokines with similar biological activities, were evaluated for their ability to suppress leukemic erythroid colony-forming cells and induce regression. Interleukin 1 alpha suppressed the conventional strain of, but not the polycythemia-inducing strain of, Friend virus-leukemic late erythroid colony-forming units and caused only a temporary regression of disease, while tumor necrosis factor alpha suppressed both forms of the disease and with multiple inoculations could cause permanent disease regressions. This system provides an excellent model for examining the efficacy of immunotherapy of leukemias with various mediators and effector mechanisms.

Lymphokine-activated killer cell plus recombinant interleukin-2 therapy of erythroleukemia in mice.

The antileukemic effects of lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) therapy were assessed in mice with Friend virus (FV)-induced erythroleukemia. LAK cells were generated by incubating normal spleen cells for 72 hr in the presence of rIL-2 (1000 units/ml). At the time of injection, the LAK cells were cytotoxic in vitro against FV-infected fibroblasts and NK-sensitive and -resistant tumor targets but not normal controls. To determine in vivo activity, fully leukemic mice (spleen weight greater than 0.75 g) were injected with either PBS or LAK cells (10(8) cells/mouse IV at 14 and 17 days post virus) and rIL-2 (10,000 units/mouse IP every 8 hr on days 14 through 18 post virus). More than 70% of the progressively leukemic mice experienced permanent leukemia regressions (disease-free for greater than 100 days) following LAK cell plus rIL-2 therapy. Regressions were characterized by return of spleen and liver weights to normal and elimination of virus-infected erythroid (CFU-E) and macrophage (CFU-C) progenitor cells from spleen and marrow. Leukemic animals treated with either LAK cells alone or IL-2 alone experienced only transient leukemia regressions. These results demonstrate that LAK cell plus rIL-2 treatment can induce permanent regressions in progressively leukemic mice and provide a responsive and manipulable model system to elucidate the mechanisms involved in this form of immunotherapy.

Induction of permanent regression of Friend virus (FV) leukemia by adoptive transfer of T helper and not T cytotoxic cells.

Friend virus (FV) induces a progressive erythroleukemia that can be made to permanently regress by the transfer of in vitro cultured virus-specific T cells (CTL/RFB) without any other adjunctive treatment. To determine the role of T cells in regression, CTL/RFB were enriched for specific T-cell subsets by treatment with monoclonal anti-Lyt2.2 or anti-L3T4 antibody and complement (C'). Pre-treatment of CTL/RFB with anti-Lyt2 antibody and C' did not affect permanent regression incidence, while CTL/RFB depleted of L3T4+ cells induced temporary regressions with all mice recurring. The number of splenic Lyt2+ (CD8+ equivalent) cells was constant irrespective of the leukemic status of the animals. However, the number of L3T4+ cells (CD4+ equivalent) in leukemic mice was three-fold lower than that of normal mice with regressed mice demonstrating a 30% increase in the number of L3T4+ cells compared to normals. Spleen cells from leukemic animals were also unable to produce IL-2 in response to mitogen stimulation. These results indicate that L3T4+ cells are involved in regression of erythroleukemia.

A benzylidene mannopyranoside derivative with antitumor activity in the spontaneous mammary tumor system.

Methyl 2,3,4,6-di-O-benzylidene alpha-D-mannopyranoside (MeDBMP) was synthesized to evaluate its potential as a cytotoxic benzaldehyde liberating agent. MeDBMP was found to cause significant reductions in the sizes of spontaneous mouse mammary carcinomas and to produce long term alterations in their growth characteristics. It was nontoxic at all doses examined and possessed no mutagenic properties in the Ames Salmonella/mammalian-microsome test.

Application of beryllium antibodies in risk assessment and health surveillance: two case studies.

This paper demonstrates that current standards used by the Occupational Safety and Health Administration (OSHA) to establish an area free from potential beryllium contamination may be inadequate. Using the Beryllium Antibody Assay, it was shown that workers exposed to former beryllium work areas, thought to be sanitized and to meet OSHA standards, experienced statistically significant rises in blood beryllium antibody titers. This finding raises the question of whether the equipment currently required to protect workers in beryllium-laden environments is sufficient. The project mission of decommissioning/decontaminating the former nuclear weapons plant at Rocky Flats Environmental Technology Site (RFETS), instituted in 1992, has necessitated development of new technology directed toward safe and responsible cleanup. Challenges have been posed not only by the need to dispose of radioactive and chemical waste, but also by the problem of cleaning up hazardous metals such as the element beryllium. Beryllium was used extensively in research and the manufacture of nuclear weapons components at Rocky Flats for over 40 years. Since inhalation of this element can induce chronic beryllium disease (Eisenbud and Lisson, 1983), an antibody assay was developed to screen workers for internal exposure to beryllium. Exposure is indicated by a titer of antibodies greater than two standard deviations above a normal population control (defined as the mean titer of pooled samples from 51 individuals with no known exposure to beryllium) and a p-value of < 0.05. This paper describes two new applications for the assay: risk assessment and health surveillance. Case study 1 involves a team of three workers who cleaned a beryllium plenum and whose beryllium antibody titers provided a quantitative assessment of their exposure. Case study 2 describes the use of the antibody assay to determine the probable manner in which one worker was exposed to beryllium while performing his duties as an architectural engineer.