Figure 4. IL-12–mediated enhancement of IL-18R expression is IFN- dependent. Spleen cells from wild-type C57BL/6 (A and C) and IFN- / (B–F) mice were stimulated for 2 d with plate-bound anti-CD3 and IL-2 (A and B) alone, or with IL-2 and IL-12 (C and D), IFN- (E), or IL-12 and IFN- (F). After 2 d, cells were washed and placed into fresh IL-2 media with fresh cytokine for an additional 2 d. Cells were then stained for IL-18R expression. IL-18R ; however, IFN- itself did not enhance IL-18R expression in the absence of IL-12 (M1; Fig. 4, E and F). These data demonstrate that IFN- is required for IL-12– mediated upregulation of IL-18R expression and most likely functions by inducing/maintaining IL-12R 2 ex- pression. However, we consistently observed a minor pop- ulation of T cells that could upregulate their IL-18R in an IL-12–dependent but IFN- –independent fashion (M2; Fig. 4, B and D–F). We suspect these are memory T cells, based on our observations that memory cells can up- regulate their IL-18R expression in response to IL-12 alone (Fig. 2, A–D). IL-4 in Concert with TCR Stimulation Induces Downregula- tion of IL-18R . While previous studies have focused on IL-12 and its ability to augment IL-18 responses, very lit- tle is known about the cytokines which may negatively regulate IL-18R expression. Therefore, we stimulated C57BL/6 CD4 T cells with anti-CD3/IL-2 in the pres- ence of IL-4, IL-10, or TGF- . IL-4 markedly enhanced the downregulation of IL-18R expression observed when T cells were cultured with anti-CD3 and IL-2 (Fig. 5, A, B, I, and K). While the addition of IL-10 had no effect (Fig. 5 C), the addition of TGF- augmented IL-18R expression (Fig. 5 D). Although T cells primed in the pres- ence of IL-4 or IL-10 expressed detectable levels of the IL- 18R , they failed to secrete IFN- in response to IL-18 alone (Fig. 5, F and G). However, these cells were capable of producing significant quantities of IFN- when cultured with both IL-12 and IL-18 (Fig. 5, F–H). Because these studies were performed without concomitant neutraliza- tion of IL-12 and/or IFN- , it is possible that the inability of IL-4 to completely inhibit IFN- induction by IL-12 and IL-18 is due to endogenous IL-12/IFN- . 147 Smeltz et al. Because the addition of exogenous IL-4 was capable of downregulating IL-18R , we suspected that endogenous IL-4 was partially responsible for the downregulation of IL- 18R observed with anti-CD3 stimulation in the absence of any added cytokine (Fig. 5 A). To test this hypothe- sis, we stimulated T cells from IL-4 / mice and measured IL-18R expression. When compared with wild-type C57BL/6 mice, IL-4 / T cells expressed significantly higher levels of IL-18R after anti-CD3 stimulation (Fig. 5, I and J). Levels of IL-18R expression before stimula- tion were comparable for both C57BL/6 and IL-4 / mice (data not shown). However, the addition of exogenous IL-4 resulted in a downregulation of IL-18R that was comparable to that seen with wild-type cells (Fig. 5, K and L). These results confirm a critical role for IL-4 in down- regulation of IL-18R . Requirement for Stat6 in Regulation of IL-18R Expression/ Function on CD4 T Cells. Because IL-4 signals mainly via Stat6, we determined if Stat6 signaling was required for IL-4–induced downregulation of IL-18R after T cell acti- vation. After stimulation with anti-CD3 and IL-2, CD4 T cells from Stat6 / mice expressed higher levels of IL- 18R ( MFI 208) compared with wild-type T cells ( MFI 93; Fig. 6, A and D). This suggests that endoge- nous Stat6 signaling partially contributes to anti-CD3– induced downregulation of IL-18R , which is consistent with the results obtained with IL-4 / mice (Fig. 5, I–K). In contrast to the downregulatory effects of IL-4 seen with wild-type T cells (Fig. 6 B), the addition of IL-4 to cultures of CD4 T cells from Stat6 / mice had no effect on IL- 18R expression (Fig. 6 E) indicating that Stat6 is required for IL-4–mediated downregulation of IL-18R expression. Interestingly, the ability of IL-12 to augment IL-18R ex-