Figure 7. Regulation of IL-18R expression during Th1/Th2 differentiation. CD4 T cells from 5CC7 trans- genic mice were primed with irradiated APCs and cyto- chrome C under Th1, Th null, or Th2 conditions. After 1 wk in culture, T cells were rechallenged for 6 h with PMA and ionomycin and then stained for intracellular IFN- and IL-4 to confirm polarization of T cells into Th1 (A), Th (B), and Th-2 (C) phenotypes. Alternatively, T cells were rechallenged for 2 d with APCs and antigen, but in the absence of cytokines. Cells were analyzed for IL- 18R expression both before (D–F) and after (G–I) re- challenge. Also, Th1 (J), Th (K), or Th2 (L) cells were restimulated with the indicated cytokine and levels of IFN- determined by ELISA. with IL-2 alone also led to downregulation of IL-18R expression. In contrast to the findings of Yoshimoto et al. (23), we found that culture of CD4 T cells with the com- bination of anti-CD3 and IL-12 led to a pronounced in- crease in IL-18R expression on the CD4 IL-18R int population. A possible difference between the results pre- sented here and those of others is that in our 4-d culture system with anti-CD3 and cytokines, T cells were cultured with immobilized anti-CD3 for only the first 2 d to reduce cell death as the result of repeated stimulation via the TCR. Culture of CD4 T cells with anti-CD3 and IL-18 failed to induce upregulation of the IL-18R . However, IL-18 could synergize with IL-12 to induce IL-18R upregula- tion in the presence of anti-CD3. Collectively, these results are most consistent with a model in which IL-12 dictates IL-18 responsiveness in CD4 T cells. Further evidence of this is supported by the observation that the ability of IL-12 to upregulate IL-18R expression is abrogated in Stat4 / mice (data not shown). The capacity of IL-12 to induce upregulation of the IL- 18R was completely IFN- dependent, as T cells from IFN- / mice did not upregulate their IL-18R when stimulated with anti-CD3 and IL-12. Although reconstitu- tion of these cultures with IFN- restored IL-18R ex- pression, IFN- itself did not enhance IL-18R expression in the absence of IL-12. Thus, IFN- increases T cell re- sponsiveness to IL-12 mostly likely via its effects on induc- tion/maintenance of expression of the IL-12R 2 chain. The detection of IL-18R on resting CD4 T cells prompted us to examine the factors responsible for the ab- sence of the IL-18R on Th2 clones. We have demon- strated that IL-4 rapidly downregulates the expression of the IL-18R during short-term stimulation of naive CD4 T cells with anti-CD3. When CD4 T cells were primed under Th2 conditions, marked downregulation was seen after 1 wk of culture and after 3 wk of culture under Th2 conditions, there was no detectable IL-18R expression (data not shown). There was no observable downregulation of IL-18R when resting T cells were cultured with IL-4 (data not shown) in the absence of TCR stimulation, sug- gesting that TCR-mediated signals are also required. The effect of IL-4 on IL-18R expression was even more dra- 150 Regulation of IL-18 Receptor Expression