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...
Cytotoxic lymphocyte maturation factor (CLMF) is a disulfide-bonded heterodimeric lymphokine that (i) acts as a growth factor for activated T cells independent of interleukin 2 and (ii) synergizes with suboptimal concentrations of interleukin 2 to induce lymphokine-activated killer cells. We now report the cloning and expression of both human CLMF subunit cDNAs from a lymphoblastoid B-cell line, NC-37. The two subunits represent two distinct and unrelated gene products whose mRNAs are coordinately induced upon activation of NC-37 cells. Coexpression of the two subunit cDNAs in COS cells is necessary for the secretion of biologically active CLMF; COS cells transfected with either subunit cDNA alone do not secrete bioactive CLMF. Recombinant CLMF expressed in mammalian cells displays biologic activities essentially identical to natural CLMF, and its activities can be neutralized by monoclonal antibodies prepared against natural CLMF. Since this heterodimeric protein displays the properties of an interleukin, we propose that CLMF be given the designation interleukin 12.The molecular cloning and expression of recombinant cytokines has made possible both significant advances in our understanding of the molecular basis of immune responses and the development of new approaches to the treatment of disease states. As an example, recombinant interleukin 2 (recombinant IL-2) has been shown to be capable of causing regression of established tumors in both experimental animals (1) and in man (2); however, its clinical use has been associated with significant toxicity (2). One potential approach to improving the therapeutic utility of recombinant cytokines is to use them in combination (3,4 MATERIALS AND METHODScDNA Cloning. A subline of NC-37 cells selected for its ability to produce high levels of CLMF (7), NC-37.98, was induced with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 for 16 hr. Poly(A)+ RNA was isolated, and random hexamer-primed cDNA libraries were established in phage AgtlO by standard procedures. Mixedprimer polymerase chain reaction (PCR) using controlled ramp times (8) was performed as follows. PCR primers contained all possible codons and were 14 or 15 nucleotides long ( Fig. 1) with a 5' extension of 9 nucleotides containing an EcoRI site for subcloning. Degeneracies varied from 1 in 32 to 1 in 4096; 0.5-4 pmol per permutation of forward and reverse primer was used in a 50-to 100-,lI PCR mixture with 40 ng of cDNA made from NC-37.98 cells that had been activated by culture with 10 ng of PMA and 25 ng of calcium ionophore A23187 per ml for 16 hr (40-kDa subunit) or with 3 ,tg of human genomic DNA (35-kDa subunit). PCR cycling parameters were as follows. Initial denaturation was at 95°C for 7 min. Low-stringency annealing was performed by cooling to 37°C over 2 min, incubating 2 min at 37°C, heating to 72°C over 2.5 min, extending at 72°C for 1.5 min, heating to 95°C over 1 min, and denaturing at 95°C for 1 min. This cycle was repeated once. Thirty standard cycles (40-kDa subun...
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