Differential effect in vitro of tumor necrosis factor-alpha (TNF) on normal and virus-infected erythroid progenitors from Friend virus (FVA)-infected mice.

In vivo administration of tumor necrosis factor-alpha (TNF) suppresses both normal and Friend virus (FVA)-infected erythroid progenitor cells (CFU-E). To examine the mechanism of erythroid suppression by TNF, we examined TNF's direct effect on normal and virus-infected cells in vitro. Productively infected fibroblast cell lines, fresh acute virus-infected spleen cells, and virus-infected CFU-E were sensitive, whereas uninfected CFU-E were resistant to TNF cytotoxicity in vitro. When FVA-infected erythroblasts were depleted from the spleen cell population in vitro with antivirus antibodies, TNF suppression of the remaining (uninfected) cells was abrogated. In contrast, both normal and virus-infected macrophage progenitor cells and immature erythroid progenitor cells were equally sensitive to TNF cytotoxicity in vitro. Normal erythroblasts had significantly fewer TNF receptors than FVA-infected erythroblasts, which also were morphologically less mature. These results suggest that TNF can differentially suppress late-stage virus-infected erythroid progenitors in vitro.

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.

Negative regulators of in vivo erythropoiesis: interaction of IL-1 alpha and TNF-alpha and the lack of a strict requirement for T or NK cells for their activity.

The macrophage-derived cytokines interleukin 1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) have significant effects on hematopoiesis in vitro and in vivo. Studies in our laboratory have demonstrated that, in vivo, IL-1 alpha and TNF-alpha suppress late stage erythropoiesis while stimulating the macrophage-granulocyte lineage. In the present studies, we have examined the mechanisms of these effects. Normal mice were treated with a single dose of either recombinant murine IL-1 alpha or TNF-alpha (1.25 x 10(6) or 10(5) U/mouse i.p., respectively) with or without pretreatment of the animals with monoclonal anti-murine TNF-alpha antibody at a dose that has been shown to be capable of abrogating endogenous TNF activity induced by lipopolysaccharide (LPS). After 5 days, effects on late-stage erythropoiesis and macrophage formation were measured by determining the number of their progenitors, erythroid colony-forming units (CFU-E) and macrophage colony-forming units (CFU-M), in the spleen. Anti-TNF-alpha antibody treatment significantly abrogated CFU-E suppression by IL-1 alpha but had no effect on the IL-1 alpha-induced stimulation of CFU-M. IL-1 alpha and TNF-alpha suppressed CFU-E in vivo and stimulated CFU-M in the spleens of T-cell- and natural killer (NK)-cell-deficient mice. Neither cytokine suppressed CFU-E colony formation in vitro. These results demonstrate that IL-1 alpha-induced suppression of CFU-E is mediated through induction of TNF-alpha, IL-1 alpha stimulation of CFU-M was independent of TNF-alpha, and the in vivo hematopoietic effects of these cytokines do not strictly require intact T- and NK-cell function for activity.

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.

Identification of T-helper cells as the target of stearic acid-inhibition in primary antibody responses in vitro.

We have previously shown that albumin-complexed stearic acid (18:0) inhibited in vitro primary anti-TNP plaque-forming cell (PFC) responses to trinitrophenyl keyhole limpet hemocyanin (TNP-KLH), but did not affect primary PFC responses to trinitrophenyl lipopolysaccharide (TNP-LPS). The present studies were done to identify the cellular target of fatty acid inhibition. The addition of 18:0 at the initiation of antibody cultures exerted a dose-dependent inhibitory effect on subsequent PFC responses to TNP-KLH, and removal of the fatty acid after 20 h did not reverse its inhibitory effect. Preincubation of isolated T-cells with TNP-KLH and 18:0 resulted in a similar inhibition of subsequent PFC responses, but a preincubation of isolated B-cells had no effect. The addition of 18:0 to the culture system in vitro led to a marked reduction in the level of IL-2 detectable in culture supernatants, and PFC responses could be restored by providing exogenous mouse recombinant IL-2. The addition of antigen-primed T-helper cells to antibody cultures partially abrogated the inhibition by 150 microM 18:0, apparently due to their greater production of IL-2. Lastly, following overnight incubation of unfractionated splenic lymphocytes in the presence of TNP-KLH and [1-14C]-18:0, B-cells were shown to contain nearly 5-fold more radiolabeled oleic acid (18:1) than T-cells. Collectively, these findings implicate T-helper cells as the principle target of 18:0-inhibition of primary antibody responses in vitro, possibly as a result of the inability of T-helper cells to avoid an over accumulation of stearic acid in their membrane phospholipids.