Monoclonal antibodies against murine immune interferon (IFN-y) were produced by fusing the murine nonsecreting myeloma cell line P3.X63.Ag8.653 with spleen cells from rats immunized with IFN-y-containing supernatants obtained by stimulating a T-cell lymphoma, L12-R4, with phorbol 12-myristate 13-acetate. Supernatants from a twicecloned hybridoma were found to neutralize and to adsorb in depletion experiments up to 27 units of mouse IFN-y but not equivalent amounts of mouse leukocyte or fibroblast IFNs. The AN-18.17.24 monoclonal antibody neutralized to the same extent mouse IFN-y from different sourcesnamely, (i) concanavalin A-stimulated spleen cells, (ii) alloantigen-stimulated spleen cells, and (iii) monkey fibroblasts transfected with the cloned gene of murine IFN-y. Moreover, the monoclonal antibody displayed species specificity, since it did not neutralize IFN-y of human origin. Binding inhibition experiments with murine IFN-y preparations exposed to enzymatic or physicochemical degradation demonstrated that the protein moiety and not the carbohydrate residues were responsible for the binding to the Activation of T lymphocytes by alloantigens (1, 2), mitogens (3), and conventional antigens (4) induces the release of an interferon (IFN) type classified as immune (IFN-y) that acts on a variety of somatic and immunorelated target cells in a genetically unrestricted manner (5). Some in vitro and in vivo studies indicate that IFN-y has a greater antiproliferative effect on neoplastic cells than do leukocyte and fibroblast IFNs (IFN-a and IFN-p, respectively) (6).Recently, genes of both human and murine IFN-y have been successfully cloned by recombinant DNA technology. The results of these experiments show that the molecular weights of human and mouse IFN-'y are 17,100 and 15,900, respectively (7-9). A limit of this kind of technology, however, is that it does not take into account the possible posttranslational modifications of the synthesized molecules. In fact, as far as IFN-y is concerned, discrepancies on molecular weights have been reported in studies using conventional physicochemical techniques for purification (10) as opposed to those using recombinant DNA technology (7-9).Monoclonal antibodies have proved to be valuable reagents for the unambiguous identification, quantitative analysis, and large-scale purification of a variety of proteins (11). Whereas different monoclonal antibodies have already been produced and successfully used for the purification and biological characterization of IFN-a and -,/ (12-15), few and conflicting reports exist in the case of monoclonal antibodies against human 16).In previous studies we established in vitro a murine T-cell lymphoma line producing, upon stimulation with phorbol 12-myristate 13-acetate (PMA), huge amounts of homogeneous IFN-y apparently not contaminated by other known lymphokines (17, 18). Taking advantage of this system, we now have produced a rat monoclonal antibody recognizing murine IFN-y but not murine IFN-a or -,3. MATERIALS AND METHODSIm...

The addition to mixed-leukocyte reactions of monoclonal antibodies to interferon-gamma abrogated alloantigen recognition and induction of cytotoxic T lymphocytes by inducing early and highly effective suppressor T lymphocytes. This inhibitory activity was not confined to in vitro models, since daily injection of the antibodies into CBA/J mice blocked the usual rejection of allogenic tumor cells.

The effects of interferon (IFN)-gamma or IFN-alpha/beta on virus yield, (2'-5')oligo(A) synthetase activation, H-2 antigen expression and proliferation of T lymphocytes have been investigated. Under the culture conditions used, vesicular stomatitis virus or Semliki Forest virus replication in T cells was not impaired by the addition of IFN-gamma, whereas it was completely inhibited by the addition of IFN-alpha/beta. In contrast, B cell lines, macrophage-transformed cell lines and fibroblasts were fully protected by both IFN-gamma as well as IFN-alpha/beta following virus infection. The lack of sensitivity of T lymphocytes to the antiviral effects of IFN-gamma was not due to absence of specific membrane receptors, since in saturation binding experiments with 125I-labeled murine IFN-gamma most T cell lines displayed a number of binding sites and a degree of affinity comparable to those found on B cells, which are fully sensitive to IFN-gamma antiviral activity. Analysis of IFN-induced dsRNA-dependent (2'-5')oligo(A) synthetase activity, one of the biochemical markers for cellular responses to IFN, showed that it was not induced in T lymphocytes after IFN-gamma treatment, whereas IFN-alpha/beta induced high levels. Both IFN-gamma and IFN-alpha/beta enhanced H-2 antigen expression on T cells as well as on cells of different histological type. Moreover, when IFN-gamma was tested for its antiproliferative activity on T cells, it was found to consistently potentiate the response of these cells to mitogens or growth factors, rather than inhibit their proliferation. Taken as a whole these results suggest that on T lymphocytes IFN-gamma should not be regarded as an antiviral agent, but rather as a modulator of T cell growth and functional differentiation, transducing intracellular signals dissimilar to those observed with target cells of different origin.

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The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by doublestranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A). synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa