Figure 5. IL-4 induces downregulation of IL-18R in response to stimulation with anti-CD3. CD4 T cells from wild-type C57BL/6 (A–I and K) or IL-4 / (J and L) mice were stimulated for 2 d with plate- bound anti-CD3 and IL-2 alone (A, E, I, and J), or with IL-2 and IL-4 (B, F, K, and L), IL-10 (C and G), or TGF- (D and H). 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 (A–D and I–L). Alternatively, cells were restimulated with IL-12, IL-18, or both IL- 12 and IL-18, and supernatants were tested for IFN- production. pression was significantly enhanced in the absence of Stat6 (Fig. 6, C and F). To correlate the differences in receptor expression with the ability of IL-18 to induce IFN- pro- duction, anti-CD3–primed T cells from both BALB/c and Stat6 / mice were restimulated with either immobilized anti-CD3 or cytokine and subsequently stained for intra- cellular IFN- . T cells from Stat6 / mice produced more IFN- in response to anti-CD3 stimulation, as well as the combination of IL-12 and IL-18 (Fig. 6, G–K). Impor- tantly, IL-18–induced IFN- production was significantly enhanced in Stat6 / mice (13 vs. 7%, Fig. 6 J), which is consistent with the higher levels of IL-18R expression observed on Stat6 / mice (Fig. 6 D). These results suggest that IL-4 and Stat6 are critical regulators of both IL-18R expression and IL-18 responsiveness. CD4 T Cells Primed under Th1 Conditions Upregulate IL- 18R . Since we found that all naive CD4 T cells ex- pressed the IL-18R , it was of interest to examine the ki- netics of regulation of expression during the differentiation of naive CD4 T cells into Th1 and Th2 cells. For this purpose, we used T cells from 5CC7 transgenic mice on the RAG-2 / background to minimize the number of previously activated T cells in the starting population. Th1 cells were generated by culturing purified CD4 T cells with APCs, antigen, IL-12, and anti–IL-4 as described pre- viously (21). After 1 wk in culture, the cells were restimu- lated with antigen and APCs. Intracellular cytokine staining revealed that 45% of 1-wk (1 ) Th1 cells expressed IFN- (Fig. 7 A). No IL-4 could be detected in this population. 1-wk Th1 cells expressed high levels of IL-18R before restimulation (Fig. 7 D), but these levels increased upon stimulation with APCs and antigen (Fig. 7 G). Upon re- stimulation, Th1 cells secreted large amounts of IFN- after culture with IL-12 and IL-18, but also secreted significant amounts of IFN- when cultured with IL-18 alone (Fig. 7 J). The cells were generated by priming with peptide, APCs, and IL-2 in the presence of anti–IL-4, anti–IL-12, and anti–IFN- antibodies. Although these cells prolifer- ated vigorously when restimulated with peptide and APCs, they failed to differentiate into Th1 or Th2 cells as judged by the lack of intracellular staining for IFN- or IL-4 (Fig. 7 B). The level of IL-18R on Th cells after 1 wk of culture and restimulation was similar to that seen on un- stimulated CD4 T cells (Figs. 7, E and H, and 1 A). Thus, neither IL-12 nor IFN- is required for maintenance of the basal level of IL-18R expression during the 1-wk priming period. These results are consistent with those obtained with anti-CD3–stimulated T cells from IFN- / (Fig. 4) and IL-12 / (data not shown) mice. Interestingly, Th cells failed to produce IFN- when restimulated with IL- 12 or IL-18 alone, but did respond to the combination of IL-12 and IL-18 (Fig. 7 K). CD4 T Cells Primed under Th2 Conditions Rapidly Down- regulate IL-18R Expression. The results obtained with Th1 cells (Fig. 7) are consistent with the observation that terminally differentiated Th1 cells express high levels of IL- 18R . To understand the dynamics of IL-18R during Th2 differentiation, TCR transgenic CD4 T cells were cultured in the presence of antigen, APCs, IL-4, anti–IL- 12, and anti–IFN- . After 1 wk of priming, 22% of the cells expressed intracellular IL-4, but not IFN- , when re- stimulated with antigen and APCs (Fig. 7 C). However, expression of the IL-18R was markedly downregulated after 1 wk of priming under Th2 conditions (Fig. 7, F and I). More impressively, Th2 cells failed to secrete any de- tectable IFN- when cultured with IL-12, IL-18, or both IL-12 and IL-18 (Fig. 7 L). Thus, signaling via IL-12 and IL-18 receptors is extinguished even after 1 wk of priming under Th2 conditions. 148 Regulation of IL-18 Receptor Expression