Interleukin-12 (IL-12) is known to upregulate production of interferon-gamma (IFN-g) by activated T cells; however, the effects of IL-12 on expression of other cytokines are less well defined. In this project, we are examining the effects of recombinant human IL-12 (rHuIL-12) on production of multiple cytokines, including IFN-g, IL-2, IL-4, IL-10 and IL-13, by purified normal human CD3+ T cells. Although resting T cells are largely nonresponsive to IL-12, anti-CD3-activated T cell blasts are strongly responsive, as demonstrated by the ability of IL-12 to induce STAT4-mediated DNA-binding activity. We found that activation of purified human T lymphocytes on immobilized anti-CD3 mAb induces rapid expression of TNF-alpha and IL-2 mRNA, and more gradual increases in mRNA levels for IFN-g and IL-10. Changes in mRNA expression levels were measured by RNase protection assay using primer template sets that enable simultaneous analysis of multiple cytokine genes. We found that IL-12 markedly upregulates expression of IFN-g and IL-10, and down-regulates production of IL-2. Inhibition of IL-2 production by IL-12 correlates directly with increased production of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalized IL-2 production in IL-12-treated T cells, demonstrating that the inhibitory effects of IL-12 are IL-10-mediated. Thus, we have found that IL-12 simultaneously upregulates production of IFN-g and IL-10, and, by a direct IL-10-dependent pathway, feedback inhibits production of IL-2. The fact that IL-12 differentially regulates synthesis of IFN-g and IL-2 in T cells demonstrates that IL-12 does not globally enhance expression of all Th1-type lymphokines. Furthermore, the ability of IL-12 to upregulate production of IL-10 provides a mechanism for limiting the IL-2-dependent clonal expansion of activated T cells, and defines a novel cytokine regulatory pathway that is inducible by IL-12. In future studies, we plan to further define the effects of IL-12 on production of other cytokines, particularly the Th2-type cytokines IL-4 and IL-13 by activated T cells, and to compare the effects of IL-12 with that of other macrophage-derived immunoregulatory cytokines, including IL-1, IL-18 and TNF. We will also further explore the functional consequences of increased IL-10 production in IL-12-treated T cells. The results of these studies will expand our current understanding of the actions of IL-12 on immune effector cells. This information may be useful in interpreting any therapeutic activity induced by IL-12 in clinical trials that are now underway. It may also provide insight to the physiological basis for the systemic toxicity associated with high dose IL-12 therapy in cancer patients. Furthermore, analysis of the effects of IL-12 on cytokine and cytokine receptor gene expression in vitro may facilitate identification of useful markers of IL-12 bioactivity that could be useful in monitoring the potency of recombinant IL-12 in human recipients.
