matic when T cells from IL-4 / mice were used. IL-4 / T cells expressed higher levels of IL-18R after the 4-d culture when compared with wild-type cells; however, the addition of exogenous IL-4 resulted in downregulation of IL-18R to levels similar to wild-type. This is consistent with the notion that endogenous IL-4 contributes to IL- 18R downregulation during anti-CD3 stimulation. Fur- ther support for the role of IL-4 in negative regulation of IL-18R is that Stat6 / T cells, which are impaired in their ability to respond to IL-4, also express higher levels of IL-18R after T cell activation. In addition, the ability of IL-12 to upregulate IL-18R is enhanced in Stat6 / T cells. Surprisingly, the addition of TGF- to either anti- CD3 or antigen-primed cultures led to an increase in IL- 18R expression. TGF- has been shown by others to in- hibit IL-4 production (31), and we hypothesize that the inhibition of IL-4 production by TGF- is responsible for the observed increase in IL-18R expression. In addition to examining the cytokine requirements for expression of the IL-18R during Th1/Th2 differentia- tion, we have also examined their effects on the induction of both IL-12 and IL-18 responsiveness. Restimulation of CD4 T cells that had been cultured for 4 d with anti- CD3, IL-2, and the combination of IL-12 and IL-18, but not either cytokine alone, resulted in production of large amounts of IFN- . More importantly, when CD4 T cells were cultured for 4 d with anti-CD3 and IL-12, they pro- duced significant amounts of IFN- when restimulated with either IL-12 or IL-18 alone. Identical results were ob- served with 1-wk Th1 cells. The capacity to respond to IL- 18 alone correlated with enhanced level of cell surface IL- 18R expression. Although CD4 T cells cultured with anti-CD3 and IL-4 for 4 d retained their capacity to re- spond to the combination of IL-12 and IL-18, 1-wk Th2 cells were completely nonresponsive. This is probably due to the absence of endogenous IL-12 and IFN- in Th2- priming experiments. Similar results have been reported by others (7, 32). The ability of IL-4 to regulate IL-18– induced IFN- production was also evident in T cells from Stat6 / mice, which secreted significantly more IFN- in response to IL-18/IL-12 plus IL-18 when compared with controls. This enhanced production of IFN- correlated with increased levels of IL-18R expression. Although IFN- production in response to IL-18 appeared to corre- late with expression of the IL-18R , we cannot exclude the possibility that increased expression/induction of IL- 18R accounted, in part, for enhanced IFN- production in response to IL-18. Very little information is available concerning the regulation of IL-18R expression (20). Intriguingly, 1-wk Th cells still responded vigor- ously to the combination of IL-12 and IL-18 by producing significant amounts of IFN- . These in vitro findings raise a number of questions about the possible role of IL-12 and IL-18 in inducing IFN- production during the course of an inflammatory response in vivo. As IL-12 and IL-18 may be produced by different cell types under different condi- tions, one possibility is that T cells primed in an IL-12 rich environment will subsequently be able to respond by pro- 151 Smeltz et al. ducing IFN- in response to either cytokine alone in the absence of reactivation by their TCR. Although the in vivo equivalent of our Th cell population has not been directly demonstrated, immunization of mice with autoan- tigens under certain conditions results in a population of primed T cells that only produce IL-2 and not Th1/Th2 cytokines (33–34). It is possible that when these cells are exposed to IL-12 and IL-18 in a paracrine fashion during the course of an infectious insult they would complete their differentiation into pathogenic Th1 cells. Apart from the known effects of IL-18 in synergizing with IL-12 to drive Th1 responses, recent reports have shown that IL-18 can induce Th2-type responses (i.e., IgE, IL-4, IL-13) (12–15). One interpretation of these studies is that IL-18 is a potent inducer of IL-4 and IL-13 production by basophils and mast cells, or possibly CD4 T cells. In- deed, induction of IL-4 production by IL-18 in NK T cells has recently been reported (35), which is consistent with our observation that unstimulated CD4 NKT cells express high levels of IL-18R similar to NK cells (data not shown). Such a systemic production of IL-4 may facilitate the priming of antigen-specific CD4 Th2 cells. Our ob- servation that IL-4 leads to downregulation of the IL- 18R on CD4 T cells is very compatible with such a model, as the absence of the IL-18R on the antigen- responsive T cells would further prevent the potential co- stimulatory effects of IL-18 on IL-12–mediated Th1 differ- entiation. Since this IL-4–mediated downregulation of IL-18R was only observed when T cells were triggered through the TCR, bystander effects of IL-4 on resting cells that express the IL-18R would not occur. Ultimately, the major factor that determines whether IL-18 potentiates Th1- or Th2-type immune responses is likely to be the bal- ance between IL-12 and IL-4 in the microenvironment during T cell priming. The presence of IL-12, as well as the expression of IL-12 receptor components, would allow IL- 18 to synergize with IL-12 in the development of Th1 de- velopment. Conversely, the presence of specialized cell types capable of making IL-4 in response to IL-18 (i.e. mast cells) would favor IL-18 as a potentiator of Th2 responses. The authors would like to thank Dr. William E. Paul for the critical reading of this manuscript. Submitted: 6 March 2001 Revised: 1 June 2001 Accepted: 11 June 2001 References 1. Okamura, H., H. Tsutsi, T. Komatsu, M. Yutsudo, T. Tan- imoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori, K. Akita, et al. 1995. Cloning of a new cytokine that induces IFN- production by T-cells. Nature. 378:88–91. 2. Akira, S. 2000. The role of IL-18 in innate immunity. Curr. Opin. Immunol. 12:59–63. 3. Osaki, T., J.-M. Peron, Q. Cai, H. Okamura, P.D. Robbins, M. Kurimoto, M.T. Lotze, and H. Tahara. 1998. IFN- - inducing factor/IL-18 administration mediates IFN- - and