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Tumor necrosis can be induced in transplanted mouse methylcholanthrene-induced sarcoma by a tumor necrosis factor in the serum of mice infected with bacillus Calmette-Guérin and given bacterial endotoxin. Sera from normal mice, endotoxin-treated mice, and mice infected with bacillus Calmette-Guérin do not contain this factor. A 20- to 30-fold purification of the serum factor has been achieved by (NH4)2SO4 fractionation, Sephadex G-100 and G-200 gel filtration, and preparative polyacrylamide electrophoresis. Tumor necrosis factor is not bacterial endotoxin. It migrates with alpha-globulins, is made up of at least four subunits, and has a molecular weight of about 150,000. The active factor is a glycoprotein that contains sialic acid and galactosamine.

TNF-resistant lines of L cells can be derived from TNF-sensitive populations by repeated exposure to TNF, and these resistant L cells, in contrast to sensitive L cells and other types of cells, lack demonstrable cell surface receptors for TNF. We have now found that TNF-resistant L cells produce a factor that is cytotoxic for L cells and has the following distinguishing characteristics of mouse TNF: it is a protein of 43 kD, composed of 16 kD subunits, that competes with TNF for receptor binding, induces hemorrhagic necrosis of the TNF-sensitive mouse sarcoma Meth A, has synergistic cytotoxic action with interferon, and its activity is neutralized by antibody to TNF. The two conclusions of this study are that cells selected for TNF resistance spontaneously produce a molecule resembling macrophage TNF, and that cells of nonhematopoietic origin are capable of producing TNF.

A factor with tumor necrosis factor (TNF) activity produced by the LuKII human lymphoblastoid cell line [designated TNF(LuKII)] was purified sequentially by using controlled-pore glass, lentil lectin-Sepharose, and procion red agarose chromatography, yielding TNF with a specific activity of 1.5 X 10(7) units per mg of protein and an isoelectric point of approximately equal to 6.7. Purified TNF(LuKII) fractionated by NaDodSO4/PAGE under reducing as well as nonreducing conditions was found to contain seven protein bands of Mr 80,000, 70,000, 43,000, 25,000, 23,000, 21,000, and 19,000. The proteins of Mr 80,000 and 70,000 could not be dissociated into lower molecular weight components. Peptide mapping analysis and immunoblotting analysis revealed that the seven protein bands in the purified TNF(LuKII) preparations are related. After fractionation of TNF(LuKII) by NaDodSO4/PAGE under reducing conditions, TNF activity was recovered from the regions of Mr 70,000 and 19,000-25,000. Purified human TNF(LuKII) (i) produces hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay; (ii) has the same pattern of reactivity as mouse TNF (cytotoxic/cytostatic/no effect) on a panel of human cancer cell lines; and (iii) has its anticellular effect potentiated by interferon, also a feature of mouse TNF.

Mouse tumor necrosis factor (TNF) was purified from serum through a series of steps, and each step was monitored for L-cell cytotoxicity in vitro and tumor-necrotizing activity in vivo. The two activities copurified and could not be dissociated. Purified mouse TNF has a specific activity of 2.2 X 10(7) (L-cell assay in the absence of actinomycin D) and 1 microgram causes necrosis of the standard TNF-sensitive sarcoma Meth A. TNF has a Mr of 39,000 +/- 2000 by gel filtration and a Mr of 16,000-18,000 by NaDodSO4/PAGE. Both molecular weight forms display cytotoxic and necrotizing activities. TNF has a pI of 3.9 and is destroyed by trypsin, protease, elastase, and alpha-chymotrypsin but not by neuraminidase or papain. These characteristics of nonrecombinant mouse TNF clearly resemble those of recombinant human and mouse TNF.

Tumor necrosis factor, lymphocyte-activating factor, and enhanced levels of type I interferon were found in serum samples taken 2 h after mice infected with Plasmodium vinckei subsp. petteri received a small intravenous injection of endotoxin. These three mediators are among those released when mice receive an endotoxin injection 2 weeks after Mycobacterium bovis BCG or Corynebacterium parvum have been administered. There is indirect evidence that this wider range of mediators is also released in P. vinckei subsp. petteri-infected mice given parenteral endotoxin. A recent report that endotoxin is detectable in the plasma of malaria-infected mice and children implies that these mediators may also be released in the acute phase of the natural infection. We propose that these macrophage-derived mediators may be important in the glucocorticoid antagonism, bone marrow depression, fever, hypergammaglobulinemia, splenomegaly, elevation of serum amyloid A, consumptive coagulopathy, and shock syndrome with associated organ damage which can accompany malaria. The intraerythrocytic parasite death seen at crisis in some malarias, as well as the subsequent development of specific protective immunity, may also depend on these mediators.

125I-labeled TNF(LuKII) (tumor necrosis factor) binds specifically to human and mouse cell lines sensitive to the cytotoxic effect of TNF, but not to cells made resistant to TNF. TNF-sensitive cells have cell surface receptors with a high affinity for TNF(LuKII). Mouse TNF competes with TNF(LuKII) for receptor binding. Scatchard analysis of the binding data yielded linear plots and suggests that TNF(LuKII) binds to homogeneous receptor sites. The number of TNF(LuKII) receptors on two TNF-sensitive cell lines is 200-300 per cell and the affinity constant of the receptor for TNF(LuKII) is approximately 1 X 10(-10) M.

Human cell lines of hematopoietic origin were tested for production of tumor necrosis factor (TNF). B-cell lines transformed by Epstein-Barr virus release a factor (referred to as hTNF) that is cytotoxic for mouse L cells sensitive to mouse TNF but not for L cells resistant to mouse TNF. Exposure to 4 beta-phorbol 12 beta-myristate 13 alpha-acetate augmented production of hTNF. hTNF activity was not found in supernatants of cell lines of T-cell, monocytic, or promyelocytic origin. Partially purified hTNF has a molecular weight of approximately 70,000, has no interferon activity, is acid labile, is destroyed by heating at 70 degrees C for 1 hr, induces cross-resistance to mouse TNF in vitro, and causes hemorrhagic necrosis of Meth A mouse sarcoma in the standard in vivo mouse TNF assay. Tests with a panel of 23 human cancer cell lines showed that hTNF is cytotoxic for 7 cell lines, cytostatic for 5, and has no effect on 11. Comparative studies with human alpha, beta, and gamma interferons indicated that sensitivity to hTNF and interferon can be distinguished. Combined treatment with hTNF and alpha or gamma interferon resulted in a synergistic cytotoxic effect.

R24 is an IgG3 mouse monoclonal antibody that identifies GD3, a prominent ganglioside on the surface of melanoma cells and other cells of neuroectodermal origin. Twelve patients with metastatic melanoma were treated with R24 at three dose levels, 8, 80, or 240 mg/m2, over a period of 2 weeks. Peak antibody levels in the serum were dose related and ranged from less than 0.1 to 62 micrograms/ml. Inflammatory reactions (urticaria, pruritus, erythema, subcutaneous ecchymoses) were observed around tumor sites in patients treated at doses greater than or equal to 80 mg/m2. Tumor biopsies during and after treatment showed lymphocyte and mast cell infiltration, mast cell degranulation, and complement deposition. Side effects were mild and were readily controlled by antihistamines. Major tumor regression has been observed in three patients.

Spleen cell transfer studies were done in BALB/c strain mice in an attempt to define the role of θ-antigen-bearing lymphoid cells in immune responses to SE. Incubation with alloantiserum to θ-C3H and rabbit C' virtually completely abolished the ability of the cells to transfer both primary and secondary (IgM and IgG) responses to 650 R irradiated recipients. Normal thymus cells partially reconstituted the ability of such treated cells to transfer the primary but not the secondary response. The results are interpreted as showing immunological memory for SE in the θ-bearing thymus-derived cells. Incubation of the spleen cells with alloantiserum to the PC.1 antigen present on antibody-forming cells did not significantly affect the ability to transfer either primary or secondary response.

ALLOANTIGENS expressed on one cell type but not on another provide the basis for homograft reactions directed against particular tissues. The Sk alloantigens of mice are expressed on epidermal cells but not on haemopoietic cells1. Thus C57B1/6 (B6) mice that have been lethally irradiated and restored with (B6 × A)F1spleen and bone marrow cells [abbreviated to (B6 × A)-to-B6 chimaeras] invariably reject (B6 × A) and A strain skin although they are permanent haemopoietic chimaeras. Rejection is accompanied by the production of antibody which is cytotoxic for A strain epidermal cells but not for A strain haemopoietic cells of any type2. The antigen thus recognized, Sk.1, is absent from all tissues of B6 mice; B6 epidermal cells carry the alternative alloantigen Sk.2.