Interleukin-12 (IL-12) is a heterodimeric cytokine that plays a central role in promoting type 1 T helper cell (Th1) responses and, hence, cell-mediated immunity. Its activities are mediated through a high-affinity receptor composed of two subunits, designated beta 1 and beta 2. Of these two subunits, beta 2 is more restricted in its distribution, and regulation of its expression is likely a central mechanism by which IL-12 responsiveness is controlled. Studies with neutralizing anti-IL-12 antibodies and IL-12-deficient mice have suggested that endogenous IL-12 plays an important role in the normal host defense against infection by a variety of intracellular pathogens. However, IL-12 appears also to play a central role in the genesis of some forms of immunopathology. Inhibition of IL-12 synthesis or activity may be beneficial in diseases associated with pathologic Th1 responses, such as multiple sclerosis or Crohn's disease. On the other hand, administration of recombinant IL-12 may have utility in the treatment of diseases associated with pathologic Th2 responses such as allergic disorders and asthma.

Interleukin-12 (IL-12) is a cytokine that has regulatory effects on T and natural killer (NK) cells and is composed of two disulfide-bonded subunits, p40 and p35. It was recently reported that supernatants from cultures of mouse IL-12 (moIL-12) p40-transfected COS cells could inhibit IL-12-dependent responses in vitro (Mattner, F., et al., Eur. J. Immunol. 1993. 23: 2202). We have further characterized the nature of the inhibitory substance. Purified mouse p40 produced in a baculovirus expression system was found to consist of two species: the p40 monomer and a disulfide-linked p40 dimer [(p40)2]. The (p40)2 was 25- to 50-fold more active than the p40 monomer in causing specific, dose-dependent inhibition of IL-12-induced mouse concanavalin A (Con A) blast proliferation and could also inhibit IL-12-induced interferon-gamma (IFN-gamma) secretion by mouse splenocytes and IL-12-dependent activation of mouse NK cells. Competitive binding studies on mouse Con A blasts showed that (p40)2 was equally effective as moIL-12 in competing with 125I-labeled moIL-12 ([125I]moIL-12) for binding to mouse Con A blasts. However, in contrast to moIL-12, mouse (p40)2 displayed little ability to compete with 125I-labeled human IL-12 (huIL-12) for binding to high-affinity IL-12 receptors (IL-12R) on human phytohemagglutinin (PHA) blasts and caused little or no inhibition of huIL-12-induced human PHA blast proliferation. Nonetheless, mouse (p40)2 was equally effective as moIL-12 in competing with [125I] huIL-12 for binding to COS cells transfected with the human IL-12R beta subunit and expressing low-affinity IL-12 binding sites. These results suggest that (i) the majority of the structural determinants required for binding of IL-12 to its receptor are contained within the p40 subunit, but p35 is required for signaling, (ii) the p40 subunit of IL-12 interacts with the beta subunit of IL-12R, and (iii) (p40)2 may be a suitable IL-12 antagonist for studying the role of IL-12 in various immune responses in vivo as well as in vitro. Further studies are required to determine whether or not (p40)2 is produced by normal lymphoid cells and is a physiologic regulator of IL-12 activity.

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human Blymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 107 units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.The potential utility of cytokines in the treatment of neoplasia and as immunoenhancing agents has recently been demonstrated in studies using human recombinant interleukin 2 (rIL-2) (1-6). However, the clinical use of rIL-2 has been complicated by the serious side effects that it may cause (2, 3). One approach to improving the efficacy of cytokine therapy while reducing toxicity is to use two or more cytokines in combination. For example, synergistic antitumor activity has been shown to result when rIL-2 is administered to tumor-bearing mice together with recombinant interferon a (rIFN-a) (7,8) or with recombinant tumor necrosis factor a (rTNF-a) (9). The antitumor effects of rIL-2 are thought to be mediated by host cytotoxic effector lymphocytes, which are activated by rIL-2 in vivo (10). rIFN-a (11) and rTNF-a (12, 13) have been shown to synergize with rIL-2 in activating cytotoxic effector cells in vitro as well as to exert synergistic antitumor effects when given in combination with rIL-2 in vivo (7-9). Hence, a cytokine was sought that could synergize with rIL-2 to activate cytotoxic lymphocytes in vitro and thus might also have utility as an antitumor agent when administered in combination with rIL-2 in vivo.Previously we demonstrated that IL-2-depleted lymphokine-containing cell supernatant solutions from cultures of human peripheral blood lymphocytes activated with phytohemagglutinin (PHA) or in mixed lymphocyte cultures contained such a factor, provisionally called cytotoxic lymphocyte maturation factor (CLMF) (14, 15). However, the quantities of human CLMF produced by peripheral blood lymphocytes were too low to permit its purification to homogeneity. Therefore, human lymphoid cell lines were screened for the production of cytokines that could synergize with rIL-2 to a...