SummaryNatural killer cell stimulatory factor (NKSF), or interleukin 12 (IL 12), is a 70-kD heterodimeric cytokine composed of two covalently linked chains, p40 and p35 . NKSFAL 12 has multiple effects on T and NK cells and was originally identified and purified from the supernatant fluid of EpsteinBarr virus (EBV)-transformed human B lymphoblastoid cell lines. We have produced a panel of monoclonal antibodies against both chains of NKSFAL 12 . Some of these antibodies have neutralizing activity, and several combinations of them have been used to establish sensitive radioimmunoassays detecting the free p40 chain, the free p35 chain, or the p70 heterodimer. Using these reagents, we have determined that most EBV transformed human B lymphoblastoid cell lines constitutively produce low levels of the p70 heterodimer and an excess of the free p40 chain, whereas Burkitt lymphoma-derived, T, myeloid, and many solid tumor-derived cell lines produce neither. Production of both p40 and p70 is increased several-fold upon stimulation of the EBVtransformed cell lines with phorbol diesters . The ability of supernatant fluids from unstimulated and phorbol diester-stimulated cell lines to induce interferon y (IFN-y) production from T and NK cells, one of the effects of NKSFAL 12, parallels the levels of production of the p70 heterodimer, known to be the biologically active form of NKSFAL 12 . Staphylococcus aureus Cowan I strain (SAC) and other stimuli induce accumulation of p40 mRNA and production of both p40 and p70 by peripheral blood mononuclear cells (PBMC) . The producer cells appear to include both adherent cells and nonadherent lymphocytes, possibly B cells. The supernatant fluids from SAC-stimulated PBMC mediate the typical functions of NKSFAL 12 (i.e., IFN-y induction, mitogenic effects on T/NK blasts, enhancement of NK cell cytotoxicity) at concentrations of p70 similar to those at which recombinant NKSFAL 12 mediates the same functions . Moreover, these activities are significantly inhibited by anti-NKSF/IL12 antibodies . The neutralizing anti-NKSF/IL12 antibodies also inhibit 85% of the IFN-y production in response to SAC, an NKSF/IL 12 inducer, and approximately 50% of the IFN-y production in response to non-NKSF/IL12-inducers such as IL-2, phytohemaglutinin, and anti-CD3 antibodies . These results indicate that induced or constitutively produced NKSFAL12 has a major role in facilitating IFN-y production by peripheral blood lymphocytes . Our findings that NKSFAL 12 is both spontaneously produced and inducible in adherent PBMC and lymphocytes suggest that NKSFAL 12 might be a major physiological regulator of T and NK cell function during an immune response and inflammation .i A. D'Andrea and M . Rengaraju contributed equally to this work . 1387J . Exp. Med .

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SummaryNatural killer cell stimulatory factor (NKSF), or interleukin 12 (ID12), is a heterodimeric lymphokine produced by B cells that has multiple effects on T and NK cell functions. NKSF at concentrations as low as 0.4 pM enhances the spontaneous cytotoxic activity of peripheral blood lymphocytes (PBL) against a variety of tumor-derived target cell lines and virus-infected target cells. The combined treatment of PBL with NKSF and ID2 results in a less than additive enhancement of cytotoxicity. NKSF enhances the cytotoxic activity of spontaneously cytotoxic CD16+CD5 -NK cells and does not confer cytotoxic activity to CD16-CD5 + T cells. PBL from patients infected with human immunodeficiency virus (HIV) have significantly lower cytotoxic activity against tumorderived target cells and virus-infected target cells than PBL from control healthy donors. Treatment of PBL from HIV-infected patients with NKSF and/or Ib2 results in an increase of NK cell cytotoxicity against both types of target cells to levels similar to or higher than those of untreated PBL from healthy donors. PBL from HIV-infected patients produce interferon 3' in response to NKSF and/or II.-2, although at levels 5-or 10-fold lower than those produced by PBL from healthy donors. The multiple biological effects of NKSF, its activity at very low molar concentrations, and its ability to synergize with other physiological stimuli suggest that NKSF/ID12 is a lymphokine likely to have physiological importance and considerable therapeutic potential.N 'atural killer cell stimulatory factor (NKSF) 1 is a heterodimeric lymphokine composed of a heavy (p40) and a light chain (p35) covalently linked (1, 2). The NKSF p40 chain belongs to the recently defined cytokine receptor family of proteins and has structural homology with the cellular receptor for II.-6 (3). NKSF has also recently been described as cytotoxic lymphocyte maturation factor (CLMF); the name I1.-12 has been proposed for this lymphokine (4, 5).NKSF was originally purified from the cell-free supernarant fluid of phorbol diester-activated human B lymphoblastoid cell lines. The two genes coding for the p40 and p35 chains 1 Abbreviations used in this paper: n, natural; NKSE natural killer cell stimulatory factor. of NKSF have been molecularly cloned; simultaneous transfection of mammalian cells with the two genes is necessary for the production of biologically active NKSF (2, 5). However, B lymphoblastoid-cell lines produce an excess of free P40 chain, in addition to the biologically active heterodimer (1, 4).NKSF exerts a variety of biological functions on human T and NK cells and possibly on other cell types. In particular, it has been shown to: (a) induce IFN-3' production from both T and NK cells and synergize in this effect with I1.-2, mitogens, phorbol diesters, anti-CD3 antibodies, and allogeneic antigens (1, 2, 6); (b) exert a comitogenic effect on fresh resting T cells together with lectins or phorbol diesters (1, 2); (c) mediate a direct mitogenic effect on activated T or NK cell blasts (5, 7...

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