level of expression was consistently less than that observed on freshly explanted CD4 T cells (compare Fig. 3 A with Fig. 1 A) and CD4 T cells cultured in IL-2 alone (com- pare Fig. 2 A, M1 with Figs. 3 A and 1 A). However, the addition of IL-12 to anti–CD3-stimulated cultures led to marked upregulation of IL-18R expression (Fig. 3 B), while the addition of IL-18 alone did not result in en- hanced IL-18R expression (Fig. 3 C). IL-18 did synergize with IL-12 to enhance IL-18R expression (Fig. 3 D). Resting CD4 T cells failed to produce IFN- on expo- sure to IL-12, IL-18, or the combination of IL-12/IL-18 (data not shown). However, after anti-CD3 induced T cell activation, large amounts of IFN- were produced in re- sponse to IL-12/IL-18 irrespective of the priming condi- tions (Fig. 3, E–H). More importantly, the upregulation of IL-18R expression seen when T cells were cultured with IL-12 correlated with the ability of IL-18 to induce IFN- production in the absence of IL-12 (Fig. 3, F and H). T cells primed with IL-2 (Fig. 3 E) or IL-18 (Fig. 3 G) did not produce detectable IFN- when cultured with IL-18 alone. Regulation of IL-18R Expression by IFN- . As IFN- is induced by IL-12, it was possible that IL-12–mediated upregulation of the IL-18R was secondary to IFN- production. To test this, we stimulated T cells from IFN- / mice with anti-CD3 and IL-2. After 4 d of culture, CD4 T cells from IFN- / mice expressed levels of IL- 18R that were similar to wild-type C57BL/6 mice (Fig. 4, A and B). Therefore, the mere absence of IFN- does not lead to downregulation of IL-18R expression. How- ever, IL-12–dependent upregulation of IL-18R expres- sion was severely impaired in the absence of IFN- . A ma- jority of CD4 T cells from IFN- / mice failed to upregulate IL-18R expression when cultured with anti- CD3 and IL-12 (M1; Fig. 4, C and D). Addition of IFN- to these cultures restored IL-12–driven upregulation of 146 Regulation of IL-18 Receptor Expression Figure 2. Stimulation with IL-12 or IL-18 alone does not lead to upregula- tion of IL-18R expression on CD4 IL-18R int cells. Spleen cells from C57BL/6 mice were cultured for 96 h in IL-2 media (A) supplemented with 20 ng/ml IL-12 (B), 20 ng/ml IL-18 (C), or both IL-12 and IL-18 (D). Cells were subsequently harvested and analyzed for IL-18R expression by FACS®. Figure 3. IL-12 in concert with TCR stimulation results in upregula- tion of IL-18R expression on CD4 T cells. CD4 T cells from C57BL/6 mice were stimulated with plate-bound anti-CD3 for 2 d in IL-2 alone (A and E), or with IL-2 and IL-12 (B and F), IL-18 (C and G), or IL-12 and IL-18 (D and H). Cells were then washed and placed into fresh IL-2 media with or without cytokines for an additional 2 d. Cells were then harvested and stained for IL-18R expression (A–D). Alterna- tively, the cells were restimulated with either IL-12, IL-18, or both IL-12 and IL-18 for 48 h and supernatants tested for IFN- production (E–H).