cells express IL-18R . Although it has been proposed that IL-12 alone may upregulate expression of the IL-18R , culture of naive CD4 T cells in IL-12 or IL-18 alone had no effect on IL-18R expression. However, activation of T cells via the TCR in the presence of IL-12, but not IL-18, resulted in marked upregulation of IL-18R expression. The combination of IL-18 and IL-12 further enhanced IL-18R expression during TCR activation. Although IFN- did not directly regulate IL-18R expression, IL-12–mediated upregulation of IL-18R was not ob- served in the absence of IFN- . After activation in the presence of IL-12, the capacity of CD4 T cells to produce IFN- in response to IL-18 alone correlated with their ex- pression of high levels of the IL-18R . In sharp contrast, the combination of TCR ligation and IL-4 led to a marked downregulation of IL-18R on unprimed CD4 T cells. The downregulatory effects of IL-4 on IL-18R were completely dependent on expression of signal transducer and activator of transcription (Stat)6. Collectively, these re- sults demonstrate that IL-18R expression is finely regu- lated during the course of CD4 T cell differentiation along either the Th1 or Th2 pathways. They are most con- sistent with a model in which IL-12/IFN- play a critical function in upregulation of IL-18R expression during differentiation along the Th1 pathway, while IL-4 plays a downregulatory role on IL-18R during Th2 differentia- tion. These data are critical to furthering our understanding of the proposed dual role of IL-18 as a differentiation factor for both Th1 and Th2 responses as well as its possible ther- apeutic use as an adjuvant. Materials and Methods Animals. Female C57BL/6, C57BL/6 IFN- / , C57BL/6 IL-4 / mice, and 5CC7 B10.A/Ai recombination activating gene (RAG)-2 / mice expressing a TCR transgene specific for cytochrome C were obtained from Taconic Farms and used at 4–8 wk of age. Female BALB/c and Stat6 / (C129S2-Stat6) mice were obtained from Jackson ImmunoResearch Laboratories. All animals were housed under specific pathogen-free conditions and provided food and water ad libitum. Animals were main- tained according to National Institutes of Health Animal Care Guidelines. Cell Lines. Short-term polarized T cell lines were established as follows: CD4 T cells were isolated from lymph nodes of 5CC7 RAG-2 / mice by negative selection using FITC-labeled anti-B220, anti-CD8, and anti–I-Ak, followed by anti-FITC magnetic separation. Purity of CD4 population was confirmed by FACS® analysis. CD4 T cells (105 cells per milliliter) were stimulated in vitro for 4–5 d with T cell–depleted, irradiated B10.A APCs (106 cells per milliliter) and 1 M cytochrome C peptide in IL-2–supplemented media containing 10% fetal bovine serum, L-glutamine, antibiotics, and -mercaptoethanol. To es- tablish Th1 lines, IL-12 (10 ng/ml; R&D Systems) and anti–IL-4 (10 g/ml 11B11) were added to cultures. For Th2 lines, IL-4 (1,000 U/ml), anti–IL-12 (10 g/ml C17.8), and anti–IFN- (10 g/ml XMG 1.2) were added. Th “null” cells were established by the addition of anti–IL-12, anti–IL-4, and anti–IFN- anti- bodies. After 4–5 d, cells were washed and resuspended in fresh IL-2 media for an additional 48 h (21). In some experiments, splenic CD4 T cells from wild-type C57BL/6 or IFN- / mice were cultured (4 106 cells per milliliter) with plate-bound anti-CD3 (5 g/ml 2C11; BD PharMingen) and IL-2 (with or without rIL-12, rIL-18, rIL-4, rIL-10, rhTGF- ) for 48 h. Cells were washed, then resuspended in fresh IL-2 media (with or without fresh cytokine added) for an additional 48 h before FACS® analysis. Flow Cytometry. Cells were washed twice in PBS, resus- pended in PBS, and placed (1–2 106 cells) into individual wells of a 96-well V-bottomed Costar plate. The cells were first incu- bated for 5 min at 4 C with 1 g of rat anti–mouse CD16/CD32 (Fc block; BD PharMingen) to block nonspecific binding of goat Ig/anti–IL-18R antibody to Fc receptors. Cells were then incu- bated with either 1 g of goat IgG or 1 g of goat anti-murine IL-18R (R&D Systems) diluted in PBS containing 5% normal mouse serum (PBS-NMS). Anti–IL-18R is an affinity-purified, polyclonal goat-specific IgG antibody that recognizes the extra- cellular domain (peptide 19–326) of recombinant murine IL- 18R . Cells were incubated with the respective antibody for 30 min, 4 C, washed two times with PBS, incubated in PBS-NMS for 10 min, and then stained with SP-biotinylated mouse anti– goat IgG (H plus L) diluted in PBS-NMS (Jackson ImmunoRe- search Laboratories) for 20 min. Cells were subsequently washed two times in PBS and then resuspended in streptavidin-PE (BD PharMingen) diluted in PBS. After a 10-min incubation at 4 C, FITC-labeled anti-CD4 (BD PharMingen) was added directly to wells for an additional 20-min incubation at 4 C. Cells were again washed two times in PBS and resuspended in PBS. 7-AAD was added immediately before FACS® analysis to exclude dead cells from analysis. Analysis was performed using CELLQuest™ software (Becton Dickinson). Data are presented as the difference ( ) between the mean fluorescence intensity (MFI) of the positive stain (anti–IL-18R ) and the MFI of the negative control (goat Ig). Cytokine Assays. T cells that had been primed in vitro for 4–7 d under various polarizing conditions were resuspended in fresh IL-2 medium. Cells (106 cells per milliliter) were cultured in 24-well plates for 48 h with 10 ng/ml IL-12, 30 ng/ml IL-18, or both. Cell-free supernatants were collected and levels of IFN- determined by ELISA (R&D Systems). Alternatively, primed cells were restimulated with either immobilized anti-CD3 (5 g/ml 2C11; BD PharMingen) or IL-12 and IL-18 (as above) for 6–8 h, and monensin was added for the final 4 h of culture. Cells were harvested, permeabilized with saponin/PBS buffer, then stained for intracellular IFN- and IL-4. Results Naive T Cells Express the IL-18R . In contrast to pre- vious studies which only detected the IL-18R on fully differentiated Th1 cells (22), a majority (82 2%) of both CD4 and CD8 splenic T cells from C57BL/6 mice were reactive by FACS® analysis with the polyclonal antibody to recombinant IL-18R (Fig. 1, A and C). In multiple ex- periments (n 5) of this type the MFI of IL-18R ex- pression on freshly explanted CD4 T cells was 143 11. CD4 and CD8 T cells in lymph node expressed levels of IL-18R similar to that seen in spleen (data not shown). We also detected high levels of IL-18R on mature CD4 and CD8 single positive T cells in the thymus, while CD4 CD8 double positive thymocytes expressed rela- 144 Regulation of IL-18 Receptor Expression