Figure 6. Stat6 is required for IL-4–mediated downregulation of IL- 18R expression and IL-18–induced IFN- . CD4 T cells from wild- type BALB/c (A–C and G–K) and Stat6 / (D–F and G–K) mice were stimulated for two d with plate-bound anti-CD3 and IL-2 alone (A and D), or with IL-2 and IL-4 (B and E) or IL-12 (C and F). The cells were then washed and placed into fresh IL-2 media with fresh cytokine for an additional 2 d. Cells were then stained for IL-18R expression. Alterna- tively, cells from BALB/c or Stat6 / mice were primed for 7 d with anti-CD3 and IL-2, then restimulated with either anti-CD3 (G), IL-2 (H), IL-12 (I and K), or IL-18 (J and K). Cells were stained for intracellu- lar IFN- and IL-4. Discussion IL-18 has now been well characterized as a costimulatory factor for IL-12–mediated Th1 differentiation. IL-18 may mediate its effects directly by augmentation of IFN- pro- duction (6, 10, 23, 24) or by enhancement of the expres- sion of the IL-12R complex, thereby potentiating the ef- fects of IL-12 (25). While many studies have focused attention on the regulation of the IL-12R during Th cell differentiation, fewer have focused on regulation of the IL- 18R (24, 26–29). It has been widely assumed that the IL-18R is expressed only after T cell activation as the IL- 18R could readily be detected on fully differentiated mu- rine Th1 cells, but not on Th2 cells, either at the mRNA level or at the cell surface (22–24, 26). We have used a polyclonal antibody prepared against residues 19–346 of the extracellular domain of the IL-18R and were readily able to detect IL-18R on almost all CD4 and CD8 T cells. It is highly likely that our ability to detect the IL-18R on naive cells is related to both the specificity of the antibody we have used as well as to our staining protocol. Although the IL-18R was expressed at relatively uni- form levels on the majority of CD4 T cells, we detected a subpopulation of unstimulated CD4 T cells that expressed 5–10-fold higher levels of receptor than the majority of the CD4 population. These cells appeared to be activated/ 149 Smeltz et al. memory T cells based on their coexpression of other mark- ers (CD62Llow, CD44hi, and data not shown). In contrast to the majority of CD4 IL-18R int cells, the CD4 IL- 18R hi cells upregulated expression of the IL-18R on culture with IL-12 alone. It is very likely that the effects of IL-12 alone on IL-18R that have been reported by others (23) are secondary to stimulation of this memory T cell population. Indeed, in our hands fully differentiated Th1 clones will also upregulate IL-18R in response to IL-12 and produce IFN- in the absence of TCR ligation. There- fore, it is unlikely that truly naive cells can respond to IL-12 by upregulating the IL-18R , especially since these cells do not express the IL-12R 2 subunit which is required for IL- 12 signaling. We have not yet studied the regulation of the IL-18R on CD8 T cells in response to IL-12 and it also remains possible that the IL-18R is differentially regulated on CD8 T cells. In the studies of Yoshimoto et al. (23), no effort was made to separate CD4 cells and CD8 cells. Short-term culture of CD4 T cells in IL-2 alone led to downregulation of IL-18R on the CD4 IL-18R int population, and this downregulatory effect was further po- tentiated by stimulation via the TCR. These data are con- sistent with a previous report showing anti-CD3–triggered downregulation of IL-18R expression (23) and with the observations of Ahn et al. (30) that culture of a Th1 clone