Resistant C57BL/6 mice infected with Leishmania major are self-healing, whereas susceptible BALB/c mice fail to contain cutaneous infection and subsequently undergo fatal visceral dissemination. These disparate outcomes are mediated by dissimilar expansions of T helper type 1 (Th1) and Th2 CD4+ T lymphocyte subsets in vivo during cure and progression of disease. Because interleukin 12 (IL-12) has potent T cell growth and interferon gamma (IFN-gamma) stimulatory effects, we studied its effect on CD4+ T cell differentiation during murine leishmaniasis. Treatment with recombinant murine (rMu)IL-12 during the first week of infection cured 89% of normally susceptible BALB/c mice, as defined by decreased size of infected footpads and 1,000-10,000-fold reduced parasite burdens, and provided durable resistance against reinfection. Cure was associated with markedly depressed production of IL-4 by lymph node cells cultured with antigen or mitogen, but preserved or increased production of IFN-gamma relative to untreated mice. IL-4 and IFN-gamma mRNA associated with CD4+ T lymphocytes isolated from infected lymph nodes showed similar reciprocal changes in response to rMuIL-12 therapy. A single injection of anti-IFN-gamma monoclonal antibody abrogated the protective effect of rMuIL-12 therapy and restored Th2 cytokine responses. We conclude that rMuIL-12 prevents deleterious Th2 T cell responses and promotes curative Th1 responses in an IFN-gamma-dependent fashion during murine leishmaniasis. Since BALB/c leishmaniasis cannot be cured with rMuIFN-gamma alone, additional direct effects of IL-12 during T cell subset selection are suggested. Because rMuIL-12 is uniquely protective in this well-characterized model of chronic parasitism, differences in IL-12 production may underlie heterogenous host responses to L. major and other intracellular pathogens.

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...

At least two subsets of CD4+ T helper cell lymphocytes termed Th1 and Th2 exist in the mouse and probably in humans. They are characterized by the secretion of different lymphokines and by their functional behavior. Dysregulated expansion of one or the other subset may be one reason for the development of certain diseases. Thus, it is of importance to define the signals involved in the differentiation and activation of the two Th cell subsets. It is known and has been confirmed in this report that the cytokine interleukin (IL)-1 acts on Th2 cells but not on Th1 cells. We now report that a previously identified cytokine which was provisionally termed T cell stimulating factor is identical with IL-12 and exhibits a reciprocal behaviour to IL-1. IL-12 has several effects on Th1 cells. It can induce the proliferation of certain Th1 cells in combination with IL-2. Synthesis of interferon (IFN)-gamma by Th1 cells can be triggered by IL-2 plus IL-12. In contrast to the IFN-gamma production observed after T cell receptor (TcR) CD3 stimulation of Th1 cells with lectin Concanavalin A the IFN-gamma production induced by IL-12 + IL-2 is insensitive to the immunosuppressive drug cyclosporin A. Furthermore, IL-12 enhances the TcR/CD3-induced synthesis of IFN-gamma of several Th1 clones. Finally, IL-12 (+IL-2) induces homotypic cell aggregation of Th1 clones. This type of cell aggregation depends on the participation of LFA-1 and ICAM-1 molecules. In all activation systems with Th1 cells no effect of IL-1 was demonstrable. In contrast, only IL-1 but not IL-12 served as a co-stimulatory signal for several Th2 cell lines activated via the TcR/CD3 complex.

Gamma interferon (IFN-y) is produced in response to circulating lipopolysaccharide (LPS) and contributes to the lethality of endotoxic shock To address the cellular source of IFN-y production in vivo, T cells and B cells were magnetically purified from C57BL/6 mouse spleens 5 h following endotoxin injection. IFN-,y RNA was abundant in splenic CD4+ and CD8+ T cells and in a T-and B-cell-depleted population of splenocytes

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