tively low levels (data not shown). Thus, expression of the IL-18R appears to be acquired during the process of T cell differentiation in the thymus. Constitutive expression of the IL-18R was independent of either IL-12 or IFN- as the level of expression of the IL-18R on CD4 cells from IL-12 / or IFN- / mice was identical to CD4 T cells from wild-type mice (data not shown). Analysis of other cell types revealed low levels of IL-18R expression on B220 splenic B cells (Fig. 1 D), while the highest level of IL-18R expression was found on NK1.1 cells (Fig. 1 E). We consistently identified a small population ( 6–10%) of splenic CD4 cells that expressed high levels of the IL- Figure 1. Expression of IL-18R on cells from naive mice: splenic CD4 (A) and CD8 (C) T cells from C57BL/6 mice, CD4 T cells from 5CC7 TCR transgenic mice on RAG-2 / background (B), B220 cells (D), and NK1.1 cells (E) from spleens of C57BL/6 mice. Spleen cells were pooled from naive mice and analyzed for IL-18R expression by FACS®. Analysis was gated on 7-AAD cells only (i.e. viable cells). Numbers in parentheses refer to the difference ( ) between the MFI of the positive stain and the MFI of the isotype (negative) stain. 145 Smeltz et al. 18R (Fig. 1 A, M2, MFI 1,072 286). This IL-18R hi population was absent in the spleens of 5CC7 TCR trans- genic mice on a RAG-2 / background (Fig. 1 B, arrow), suggesting the CD4 IL-18R hi population in the former could be a population of previously activated/memory T cells or CD4 NK1.1 T cells. Further analyses revealed the majority ( 90%) of CD4 IL-18R hi cells are NK1.1 and express markers typically associated with memory/acti- vated T cells (data not shown). Thus, two distinct levels of IL-18R expression (IL-18R int and IL-18R hi) can be detected on CD4 T cells in spleens from normal animals. Stimulation with IL-12 or IL-18 Alone Does Not Lead to Upregulation of IL-18R on CD4 IL-18R int T Cells. Previous reports have suggested that IL-12 in the absence of TCR stimulation was capable of inducing IL-18R ex- pression, thus enabling IL-18 to synergize with IL-12 to enhance IFN- production (23). To evaluate the effects of cytokines on IL-18R expression, we cultured spleen cells from normal C57BL/6 mice for 96 h in IL-2–containing media alone or in IL-2–containing media supplemented with either IL-12 or IL-18. Preliminary studies demon- strated that IL-2 was required to maintain adequate viabil- ity of the cells. Culture with IL-2 alone led to a decrease in IL-18R expression on CD4 IL-18R int cells (Fig. 1 A, M1, MFI 143 21; and Fig. 2 A, M1, MFI 94 15), but did not modify the level of expression of IL-18R on the subpopulation of IL-18R hi cells (Fig. 1 A, M2, MFI 1,088 109; and Fig. 2 A, M2, MFI 1,072 286). However, it should be noted that culture in IL-2 alone did result in a marked increase in the percentage of the IL-18R hi popu- lation from 6–10% to 20–40% of CD4 T cells during the 96-h culture (Figs. 1 A and 2 A). This increase presum- ably reflects the preferential expansion of previously acti- vated/memory T cells in the presence of IL-2. Slight upregulation of IL-18R expression on the CD4 IL-18R int cells was observed in the presence of IL-12 (MFI 102 12), but not IL-18 (Fig. 2, B–D, M1, MFI 88 13). In striking contrast, the minor population of CD4 IL-18R hi cells upregulated IL-18R expression in response to IL-12 (Fig. 2 B, M2, MFI 2,425 1,074), but not IL-18 (Fig. 2 C, M2, MFI 1,029 171). Thus, the pri- mary effects of IL-12 on freshly explanted CD4 T cells from normal animals appear to be via upregulation of the IL-18R on the minor population of CD4 T cells that al- ready express high levels of this receptor and which appear to be memory/activated T cells. IL-12 in Concert with TCR Stimulation Induces Upregula- tion of IL-18R Expression on CD4 T Cells. To exam- ine the effects of TCR stimulation on IL-18R expression, CD4 T cells from naive C57BL/6 mice were stimulated for 2 d with immobilized anti-CD3 in IL-2–containing media, with or without IL-12/IL-18. After 2 d in culture, the cells were harvested, washed, and placed into fresh IL-2 media with fresh cytokines for an additional 2 d. This two- step culture system was used because significant apoptosis was observed when T cells were cultured for 4 d on plate- bound anti-CD3. IL-18R expression was detected on CD4 T cells stimulated with anti-CD3 and IL-2, but the