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Differential Capacities of CD4⁺, CD8⁺, and CD4^-CD8^- T Cell Subsets to Express IL-18 Receptor and Produce IFN- in Response to IL-18¹

Michio Tomura^*, Seiji Maruo^*, Jie Mu^*, Xu-Yu Zhou^*, Hyun-Jong Ahn^*, Toshiyuki Hamaoka^*, Haruki Okamura, Kenji Nakanishi, Steven Clark^§, Masashi Kurimoto^¶ and Hiromi Fujiwara^2,*

^* Biomedical Research Center, Osaka University Medical School, Osaka, Japan; Department of Bacteriology and Department of Immunology, Hyogo College of Medicine, Hyogo, Japan; ^§ Genetics Institute Inc., Cambridge, MA 02140; and ^¶ Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-12 and IL-18 have the capacity to stimulate IFN- production by T cells. Using a T cell clone, we reported that IL-18 responsiveness is generated only after exposure to IL-12. Here, we investigated the induction of IL-18 responsiveness in resting CD8⁺, CD4⁺, and CD4^-CD8^- T cells. Resting T cells respond to neither IL-12 nor IL-18. After stimulation with anti-CD3 plus anti-CD28 mAbs, CD8⁺, CD4⁺, and CD4^-CD8^- T cells expressed IL-12R, but not IL-18R, and produced IFN- in response to IL-12. Cultures of T cells with anti-CD3/anti-CD28 in the presence of rIL-12 induced IL-18R expression and IL-18-stimulated IFN- production, which reached higher levels than that induced by IL-12 stimulation. However, there was a substantial difference in the expression of IL-18R and IL-18-stimulated IFN- production among T cell subsets. CD4⁺ cells expressed marginal levels of IL-18R and produced small amounts of IFN-, whereas CD8⁺ cells expressed higher levels of IL-18R and produced more IFN- than CD4⁺ cells. Moreover, CD4^-CD8^- cells expressed levels of IL-18R comparable to those for CD8⁺ cells but produced IFN- one order higher than did CD8⁺ cells. These results indicate that the induction of IL-18R and IL-18 responsiveness by IL-12 represents a mechanism underlying enhanced IFN- production by resting T cells, but the operation of this mechanism differs depending on the T cell subset stimulated.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
IFN-, which is produced mainly by the Th1 type of lymphocytes (1), exhibits a number of immunoregulatory effects, including the capacity to stimulate the activation of CTL (2, 3) and macrophages (4, 5, 6, 7). Through these effects, it is becoming increasingly evident that this cytokine has an important role in the manifestation of T cell-mediated inflammatory responses.

Like IL-2 production, IFN- production by Th1 cells is induced following stimulation of their TCR and costimulatory receptor, CD28 (8, 9). Simultaneous ligation of TCR and CD28 molecules with anti-CD3 and anti-CD28 mAbs in the absence of APCs leads to significant levels of IFN- production (10, 11). However, a number of studies (12, 13, 14) have revealed that an APC-derived cytokine strikingly stimulates T cells for IFN- production; IL-12 has been shown to induce the production of IFN- by T cells and NK cells (12, 13, 14). More recently, another cytokine, designated IFN--inducing factor (now termed IL-18), was described to exhibit a more potent IFN--inducing capacity than IL-12 (15). Regarding the functional relationship between these two cytokines, our previous study (16) demonstrated that IL-12 and IL-18 exhibit a synergistic effect on IFN- production by a particular T cell clone (IL-12-responsive T cell clone). It was also shown that IL-12 allows T cells to respond to IL-18 by inducing the expression of IL-18R (16). While this represented a mechanism underlying synergy between IL-12 and IL-18 in enhanced IFN- production by a single unique T cell clone, it remained to be solved whether it holds true for various subsets of fresh resting T cells, i.e., CD4⁺, CD8⁺, and CD4^-CD8^- T cells.

The present study investigated how responsiveness to IL-12 and IL-18 is induced in resting T cells. The results demonstrate that purified resting T cells fail to respond to either IL-12 or IL-18 while all subsets (CD4⁺, CD8⁺, and CD4^-CD8^-) of anti-CD3/anti-CD28-activated T cells expressed IL-12R and produced IFN- in response to IL-12. The expression of IL-18R and IL-18 responsiveness were induced when resting T cells stimulated with anti-CD3 and CD28 were exposed to IL-12 but not to IL-2. Importantly, the levels of both IL-18R and IFN- production as a marker of IL-18 responsiveness were found to differ greatly among the T cell subset; CD4⁺ T cells expressed marginal levels of IL-18R and produced little IFN- in response to IL-18 whereas CD8⁺ T cells exhibited higher levels of IL-18R expression and IFN- production. While the IL-18R levels expressed on CD4^-CD8^- T cells were comparable to those on CD8⁺ T cells, these cells produced one order larger amounts of IFN- than did CD8⁺ T cells. These results indicate that IL-18 responsiveness is induced in various subsets of T cell receptor-triggered T cells, but the magnitude of responsiveness is determined by the levels of IL-18R induced by IL-12 as well as the intrinsic properties (IL-18R-mediated signaling) of each T cell subset.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 mice were purchased from Shizuoka Laboratory Animal Center, Hamamatsu, Japan and used at 7 to 9 wk of age.

Reagents

Murine rIL-12 purified from the supernatants (SN)³ of Chinese hamster ovary cells transfected with p35 and p40 (IL-12 subunits) cDNA plasmids was from Genetics Institute (Cambridge, MA). Murine rIL-18 was obtained by expression of the murine IL-18 cDNA in Escherichia coli and purification by chromatography as described (17). Murine rIL-2 was kindly provided by Shionogi (Osaka, Japan). Anti-CD3 (145-2C11) (18), anti-CD28 (Pv-1) (19), anti-IL-2R -chain (7D4) (20), anti-I-A^d/b (34-5-3S) (21), anti-CD4 (American Type Culture Collection (Rockville, MD) clone GK1.5), anti-CD8 (ATCC clone 2.43), and anti-murine IL-12 (C17.8) (22) mAbs were purified from culture SNs or ascitic fluids of the respective hybridomas. Anti-murine IL-18 polyclonal Ab was purified from immunized rabbit sera. Phycoerythrin (PE)-conjugated anti-CD4, PE-conjugated anti-NK1.1, FITC-conjugated anti-CD3, and FITC-conjugated anti-CD8 mAbs were obtained from PharMingen (San Diego, CA). Biotinylated mouse anti-rat lgG and biotinylated goat anti-rabbit lgG were purchased from Jackson ImmunoResearch (West Grove, PA). PE-conjugated streptavidin and RED670-conjugated streptavidin were from Becton Dickinson (Mountain View, CA) and Life Technologies (Gaithersburg, MD), respectively. Normal rat lgG and rabbit lgG (controls) were purchased from BioMeda (Foster City, CA) and Jackson ImmunoResearch, respectively.

Preparation of T cell subsets

Negative selection. Lymph node or spleen cells were depleted of B cells and Ia⁺ APC by immunomagnetic negative selection as follows: Cells were allowed to react with anti-I-A^d/b mAb and then incubated with advanced magnetic particles bound to goat anti-mouse lg (Advanced Magnetic, Cambridge, MA). A T cell population depleted of anti-I-A^d/b-labeled and/or surface Ig⁺ cells was obtained by removing cell-bound magnetic particles with a rare earth magnet (Advanced Magnetic). CD4⁺ and/or CD8⁺ T cells were further depleted by incubating this population with anti-CD8 and/or anti-CD4 mAb followed by magnetic particles conjugated to goat anti-rat IgG (Advanced Magnetic). Purity of the resulting populations was checked by flow cytometry. The purity of CD4⁺, CD8⁺, and CD4^-CD8^- subsets was consistently >97%, >97%, and >95%, respectively.

Positive selection. Cells were labeled with superparamagnetic microbeads conjugated to goat anti-rat IgG mAb (Miltenyi Biotec, Sunnyvale, CA). Labeled cells were separated from unlabeled cells by magnetic cell sorting using the MiniMACS (Miltenyi Biotec) according to the procedure described in detail (23). The magnetic cells were retained in a MiniMACS column inserted into a MiniMACS magnet while the nonmagnetic cells passed through. Labeled cells were eluted after the column was removed from the magnet.

Stimulation of T cells with anti-CD3 plus anti-CD28 mAb

Anti-CD3 and anti-CD28 mAbs (10 µg/ml each) were coimmobilized to individual wells of 96-well flat-bottom microculture plates (Corning 25860, Corning Glass Works, Corning, NY) in a final volume of 0.1 ml or 24-well culture plates (Corning 25820) in a volume of 1 ml. After 3 h, solutions were discarded and plates were washed with PBS twice. Purified T cells were cultured in 0.2 ml (96-well microplates) or 2 ml (24-well plates) of RPMI 1640 medium supplemented with 10% FBS and 2-ME at 2.0 x 10⁵ cells/well (96-well microplates) or 1.5 x 10⁶ cells/well (24-well plates) in the presence of rIL-12 (1000 pg/ml) or rIL-2 (100 U/ml) in a humidified atmosphere at 5% CO₂ at 37°C. T cell proliferation was determined in 96-well microplates after an 8-h pulse with 20 KBq/well of [³H]TdR. Results were calculated from uptake of [³H]TdR and expressed as the mean cpm ± SE of triplicate cultures. Cells and culture SNs in 24-well culture plates were harvested and assessed for the expression of IL-12R and IL-18R and for IFN- concentrations, respectively.

Measurement of IFN- concentrations

IFN- concentrations were measured by ELISA. Mouse IFN- ELISA kits were purchased from Genzyme (Cambridge, MA), and our own ELISA system was prepared using two types of anti-mouse IFN- mAb (XMG1.2 (Endogen, Cambridge, MA) and biotinylated R4–6A2 (R4–6A2 was purified from R4–6A2 hybridoma and biotinylated in our laboratory)) as well as mouse rIFN- provided by Shionogi. One U/ml in our ELISA system corresponded to approximately 100 pg/ml in Genzyme ELISA kits.

Immunofluorescence and flow cytometry

Cells were stained directly with FITC- or PE-conjugated reagents. The detection of IL-2R -chain was accomplished by incubating up to 1 x 10⁶ cells with biotinylated-anti-IL-2R -chain (7D4) mAb for 20 min, and then incubating with PE-conjugated streptavidin. The detection of IL-12R was performed as previously described (24). Briefly, cells were incubated with 7.5 ng of rIL-12 in 10 µl medium for 60 min at 4°C. Cells were washed and then incubated with 1 µg of rat anti-mouse IL-12 mAb (C17.8) for 30 min at 4°C. After washing, cells were allowed to react with 0.1 µg of biotinylated mouse anti-rat lgG followed by incubation with RED670-conjugated streptavidin. The staining protocol used in detection of IL-12R was applied to the detection of IL-18R. Cells were incubated with 0.4 µg of rIL-18 in 40 µl medium for 60 min at 4°C. Cells were washed and then incubated with 0.1 µg of rabbit anti-mouse IL-18 polyclonal Ab for 30 min at 4°C. After washing, cells were allowed to react with 0.1 µg of biotinylated goat anti-rabbit lgG followed by incubation with PE-conjugated streptavidin. Stained cells were analyzed with a FACSCalibur (Becton Dickinson).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of IL-12, but not IL-18, responsiveness in resting T cells following stimulation with anti-CD3 plus anti-CD28 mAb

A purified T cell population was prepared from normal C57BL/6 lymph node cells. Cells were cultured in anti-CD3- and anti-CD28-coimmobilized 24-well culture plates. After 48 h, activated T cells were harvested, washed, re-suspended in fresh medium, and stimulated with various cytokines (Table I). Stimulation with IL-2 and IL-12 induced high and moderate levels of proliferation, respectively, whereas IL-18 elicited only a marginal proliferation. IFN- production was inducible at weak albeit significant levels only by stimulation with IL-12. In contrast to these anti-CD3/anti-CD28-activated cells, freshly prepared T cells failed to proliferate or produce IFN- in response to either type of cytokines (data not shown). Anti-CD3/anti-CD28-activated T cells were tested for the expression of IL-2R, IL-12R, and IL-18R (Fig. 1). These activated T cells were found to express IL-2R and IL-12R, but not IL-18R. Figure 2 shows that IL-12R is induced not only on both CD4⁺ and CD8⁺ T cell subsets prepared from lymph node cells or spleen cells, but also on the CD4^-CD8^- T cell subset from spleen cells although the IL-12R levels are higher in CD4⁺ cells than in CD8⁺ and CD4^-CD8^- cells. These results indicate that the activation of T cells with anti-CD3 and anti-CD28 induces IL-2 and IL-12 responsiveness along with IL-2R and IL-12R expression, but does not induce IL-18 responsiveness/IL-18R expression.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Induction of IL-2R and IL-12R, but not of IL-18R, on resting T cells following stimulation with anti-CD3 plus anti-CD28 mAbs. Purified C57BL/6 lymph node T cells were stimulated with anti-CD3 plus anti-CD28 mAbs for 48 h in 24-well culture plates. Cells were stained for IL-2R, IL-12R, and IL-18R as described in Materials and Methods.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Induction of IL-12R on CD4+, CD8+, and CD4-CD8- subsets. A T cell population (B cell/APC-depleted) was prepared from lymph node or spleen cells and stimulated with anti-CD3/anti-CD28 mAbs. Forty eight hours later, cells were stained for CD4, CD8, and IL-12R. Analysis gates for IL-12R were set on the whole population, CD8- (mostly CD4+ cells), CD4- (mostly CD8+ cells), and CD4-CD8- cells.

Our preceding study demonstrated that IL-12 has the capacity to induce IL-18R expression and IL-18 responsiveness in a particular Th clone (IL-12-responsive) as well as unfractionated naive T cells activated with anti-CD3 plus anti-CD28 (16). Here, we determined whether IL-12 can induce IL-18R on all of the three T cell subsets (CD4⁺, CD8⁺, and CD4^-CD8^-) that have expressed IL-12R after stimulation with anti-CD3 plus anti-CD28. Purified T cells were stimulated with anti-CD3/anti-CD28 for 48 h. Cells harvested were then stimulated with rIL-12 or rIL-2 as control. Figure 3A shows that exposure to rIL-2 fails to induce IL-18R whereas stimulation with rIL-12 results in the induction of IL-18R (upper panels), which confirms our previous results (16). When analysis gates were set on the CD4⁺ and CD8⁺ T cell populations in cell preparations obtained following IL-12 stimulation, it was observed that CD4⁺ T cells and CD8⁺ T cells expressed low and high levels of IL-18R, respectively. However, we found that the population obtained following IL-12 stimulation contains a large proportion (75%) of CD8⁺ T cells and a small proportion (<18%) of CD4⁺ T cells. To more accurately examine the IL-18R expression on CD4⁺ T cells, CD4⁺ T cell- or CD8⁺ T cell-depleted T cell populations were prepared and stimulated with anti-CD3/anti-CD28 followed by exposure to rIL-12 (Fig. 3B). Using a CD4⁺ T cell-depleted fraction as a starting population, 94% enriched CD8⁺ T cells were generated following IL-12 stimulation. An analysis gate was further set on the CD8⁺ cells. These CD8⁺ T cells expressed high levels of IL-18R (Fig. 3B, middle panel), which is consistent with the results of Figure 3A. Seventy-two percent of CD4⁺ T cells were generated from a CD8⁺ T cell-depleted starting population. This population was found to contain 26% of CD4^-CD8^- T cells. The expression of IL-18R on cells gated for CD4⁺ and CD4^- is shown in Figure 3B (upper for CD4⁺ and bottom for CD4^-CD8^-). CD4⁺ T cells again exhibited only weak levels of IL-18R expression. In contrast, CD4^-CD8^- T cells expressed high levels of IL-18R that are comparable with those observed on CD8⁺ T cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Differential levels of IL-18R induced on various T cell subsets stimulated with anti-CD3/anti-CD28 followed by exposure to IL-12. A, Unfractioned lymph node cells were stimulated with anti-CD3/anti-CD28 for 48 h. Cells were then cultured with 100 U/ml IL-2 or 1000 pg/ml IL-12 for 24 h and stained for IL-18R. The expression of IL-18R on the whole fraction of cells or on cells gated for CD4+ or CD8+ is shown. B, CD8+ cell-depleted and CD4+ cell-depleted T cell populations from lymph node cells were stimulated with anti-CD3/anti-CD28, and cells were harvested and then exposed to 1000 pg/ml IL-12. Cells obtained from the CD4+ cell-depleted starting population contained 94% of CD8+ T cells on which an analysis gate for CD8 was set (middle). Cells obtained from the CD8+ cell-depleted starting population contained 72% of CD4+ cells and 26% CD4-CD8- cells. Analysis gates were set on CD4+ (top) and CD4- (CD4-CD8-) cells (bottom).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Induction of IL-18R on CD8+ and CD4-CD8- cells following simultaneous stimulation with anti-CD3/anti-CD28 and IL-12. CD4+ and CD8+ T cell populations were prepared from lymph node cells, and a CD4-CD8- population was obtained from spleen cells. These populations were stimulated with anti-CD3 plus anti-CD28 in the presence of IL-2 (100 U/ml) or IL-12 (1000 pg/ml). The expression of IL-18R on cells gated for CD4+, CD8+, or CD4-CD8- is shown.

Unfractionated resting T cells stimulated with anti-CD3/anti-CD28 in the presence of rIL-12 or rIL-2 (control) were restimulated with different concentrations of rIL-18 or rIL-12. IFN- production in these cultures was determined by ELISA (Fig. 5). T cells activated with anti-CD3/anti-CD28 in the absence of IL-12 (in the presence of IL-2) failed to produce IFN- in response to IL-18 although they produced moderate amounts of IFN- following IL-12 stimulation. In contrast, anti-CD3/anti-CD28-activated T cells in the presence of IL-12 produced large amounts of IFN- in response to rIL-18. The levels of IFN- production by these T cells were higher than those observed for T cells restimulated with rIL-12 instead of rIL-18. T cells stimulated with anti-CD3/anti-CD28 in the presence of IL-12 produced significant amounts of IFN- even when they were not stimulated subsequently with either cytokine. This is considered to be due to the action of IL-12 used in the first step of T cell activation culture because T cells from cultures containing rIL-2 instead of rIL-12 did not produce IFN- in the following cytokine-free cultures. Thus, the results indicate that anti-CD3/anti-CD28-activated T cells acquire IL-18 responsiveness along with the induction of IL-18R expression following their exposure to IL-12.

View larger version (26K): [in this window] [in a new window]   FIGURE 5. Induction of IL-18 responsiveness in a T cell population stimulated with anti-CD3/anti-CD28 in the presence of IL-12. Purified T cells were activated with anti-CD3 plus anti-CD28 mAbs in the presence of rIL-12 (1000 pg/ml) or rIL-2 (100 U/ml) for 48 h. Cells were resuspended and stimulated with different concentrations of rIL-18 or rIL-12. Culture SNs were examined for IFN- production by ELISA. The results are representative of three similar experiments.

Differential capacities of CD4⁺, CD8⁺, and CD4^-CD8^- T cell populations to produce IFN- in response to rIL-18

We determined whether the IL-18R levels induced on the various T cell subsets correlate with their IL-18 responsiveness. First, a comparison was made between CD4⁺ and CD8⁺ T cell subsets from lymph node cells. A CD4⁺ or CD8⁺ T cell population was prepared by depleting CD8⁺ or CD4⁺ T cells, respectively, together with B cells and APC. These two populations were stimulated with anti-CD3/anti-CD28 in the presence of IL-12 for 48 h. Cells harvested from each culture were approximately 90% CD4⁺ or CD8⁺, but these two populations contained significant percentages of the alternative phenotype of cells and CD4^-CD8^- cells. Therefore, CD4⁺ and CD8⁺ T cell populations with >98% purity were isolated using a cell sorter (Fig. 6A). These sorted CD4⁺ and CD8⁺ populations were stimulated with different concentrations of rIL-18 or 1000 pg/ml rIL-12 (Fig. 6B). The results show that CD8⁺ and CD4⁺ populations exhibit, respectively, high and low levels of IFN- production in response to IL-18. It should also be noted that there is a marked difference in IL-12-stimulated IFN- production between CD8⁺ and CD4⁺ T cell populations.

View larger version (39K): [in this window] [in a new window]   FIGURE 6. Induction of high and low IL-18 responsiveness in the CD8+ and CD4+ T cell subsets. The CD4+ and CD8+ T cell populations were prepared by depletion of CD8+ and CD4+ T cells, respectively, together with B cells and APC from lymph node cells. These two populations were stimulated with anti-CD3/anti-CD28 in the presence of IL-12. From the resulting CD4+- and CD8+-enriched activated T cell populations, CD4+ and CD8+ T cells were further purified by positive selection. Purity of these two populations is shown in A. These were restimulated with rIL-18 or rIL-12, and IFN- produced in the culture SNs was determined by ELISA (B). The results were representative of three similar experiments.

View larger version (44K): [in this window] [in a new window]   FIGURE 7. Phenotypes of CD4-CD8- cells activated with anti-CD3/anti-CD28 in the presence of IL-12 or IL-2. Spleen cells were depleted of B cells, APC, and CD4+ and CD8+ T cells as described in Materials and Methods. This population was stained doubly for CD4 and CD8 as well as for CD3 and NK1.1 (left panel). After stimulation with anti-CD3/anti-CD28 plus IL-12 or IL-2, cells were again stained for cell surface markers (right panel).

View larger version (24K): [in this window] [in a new window]   FIGURE 8. Strikingly high levels of IL-18 responsiveness induced in CD4-CD8- cells following stimulation with anti-CD3/anti-CD28 in the presence of IL-12. Portions of the same activated cells as used for the phenotype analysis in Figure 7 were stimulated with rIL-18 or rIL-12. The results are representative of two similar experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We have previously shown that, using a recently established IL-12-responsive T cell clone, the action of IL-18 depends on the presence of IL-12 or preexposure to IL-12 (16). IL-12 was found to be a particular cytokine capable of generating IL-18 responsiveness through inducing IL-18R expression (16). These observations were made mainly on a single T cell clone (24) and partly observed using unfractionated resting T cells (16). The present study first confirmed that IL-12 contributes to IFN- production by T cells through direct action on T cells and that IL-12 also functions to up-regulate IFN- production by inducing IL-18R expression on T cells and endowing them with IL-18 responsiveness. Next, our results reveal that there is a fundamental difference in the capacity to produce IFN- in response to IL-18 between CD8⁺ and CD4⁺ T cell subsets. CD8⁺ T cells stimulated with anti-CD3/anti-CD28 in the presence of IL-12 expressed high levels of IL-18R and produced moderate amounts of IFN- in response to IL-18. In contrast, similarly treated CD4⁺ T cells expressed only marginal levels of IL-18R and produced much lower amounts of IFN- compared with CD8⁺ T cells. It has been shown that T cells and NK cells produce IFN- in response to IL-12 and that both CD4⁺ and CD8⁺ T cells are primed for IFN- production (13, 25, 26). In contrast to resting T cells, there is no accessory cell requirement for activated T cells and NK cells to respond to IL-12 (13). In the present study, purified CD4⁺ and CD8⁺ T cells that were activated with anti-CD3 plus anti-CD28 expressed high and low levels of IL-12R, respectively. Therefore, differential levels of IL-18R expression on CD8⁺ and CD4⁺ T cells exposed to IL-12 would not be ascribed to a quantitative difference in IL-12R expression but rather to a qualitative difference in IL-12R-mediated intracellular signaling leading to IL-18R expression.

CD4⁺ and CD8⁺ T cells activated with anti-CD3/anti-CD28 were shown to produce smaller and larger amounts of IFN-, respectively, in response to IL-12. However, this does not necessarily represent the difference in the overall capacity to produce IFN- between CD4⁺ and CD8⁺ T cells. Physiologically, T cells are activated through stimulation of TCR and CD28 with their respective ligands on APC (8, 9). IL-12 is produced by APC when APC are stimulated with CD40 ligand (27, 28, 29). However, CD40 ligand expression is restricted to the CD4⁺ T cell subset (30, 31). Therefore, when a separated CD4⁺ or CD8⁺ T cell subset is stimulated with APC capable of producing IL-12 instead of stimulation with anti-CD3/anti-CD28 plus rIL-12, the CD4⁺ T cell subset may be the stronger IFN- producer.

An interesting aspect of the present study concerns the induction of IL-18 responsiveness in CD4^-CD8^- cells. The splenic CD4^-CD8^- population contained approximately 40% of CD3⁺ cells. Stimulation of this population with anti-CD3/anti-CD28 resulted in an increase in the proportion of CD3⁺ (CD3⁺NK1.1^-) cells especially when cultures included IL-12. Conversely, the proportion of CD3^-NK1.1⁺ decreased from 50% to less than 10%. Stimulation of the CD4^-CD8^- population with anti-CD3/anti-CD28 in the presence of IL-2 also increased the proportion of CD3⁺NK1.1^- cells along with a decrease in the proportion of CD3^-NK1.1⁺ cells. Of the resultant two populations, the group including IL-12 induced IL-18R but the other group failed to induce the receptor although this group contained more CD3^-NK1.1⁺cells than the former group. Moreover, we have recently observed that while CD3^-NK1.1⁺ cells can be activated in response to the combination of cytokines including IL-18, IL-18R was undetectable on this subset before and after activation (our unpublished observations). Together, it is unlikely that CD3^-NK1.1⁺ cells (9%) in the population prepared following stimulation with anti-CD3/anti-CD28 plus IL-12 express IL-18R. Nevertheless, it will be required to determine whether IL-18R is actually expressed by activated CD3⁺NK1.1^- cells that are the predominant cell type (65%).

Besides IL-12, IL-2 also stimulates production of IFN- from T and NK cells (32, 33). Our results demonstrated that IL-2 failed to induce IL-18R in CD4⁺ and/or CD8⁺ T cells as well as in CD4^-CD8^- cells. In contrast, it is obvious that IL-12 is a representative cytokine capable of inducing IL-18R. However, it remains to be investigated whether IL-12 is absolutely required for inducing IL-18 or whether there are other cytokine substitutes.

More important is that, while CD4^-CD8^- cells activated with anti-CD3/anti-CD28 plus IL-12 expressed levels of IL-18R comparable with those observed on CD8⁺ T cells, the former cells produced much greater (one order) amounts of IFN- in response to IL-18. These observations are similar to those made for IFN- following IL-12 stimulation; namely, despite comparable levels of IL-12R expression on anti-CD3/anti-CD28-activated CD4⁺, CD8⁺, and CD4^-CD8^- cells, the amounts of IFN- produced by these three subsets greatly differed. Thus, similar to a difference in the IL-12-stimulated IFN- production, the marked capacity of CD4^-CD8^- cells to produce IFN- in response to IL-18 may be understood mainly by considering the structure of IL-18R and/or the prominent efficacy of IL-18R-mediated intracellular signaling. IL-18R was recently cloned and shown to be a single chain with the homology to IL-1R (34). The possibility still exists that there are other subunits of the IL-18R that affect IL-18 responsiveness but not ligand binding.

Our results illustrate that IL-12 is capable of inducing IL-18R expression and IL-18 responsiveness in TCR/CD28-stimulated T cells that lead to high levels of IFN- production and that this effect is expressed differently by various T cell subsets: CD4^-CD8^- >>> CD8⁺ > CD4⁺. The magnitude of IFN- production induced by T cells through collaboration between IL-12 and IL-18 is determined not only by the levels of IL-18R induced by IL-12 but also by the intracellular signaling machinery leading to IFN- production, Thus, it would be important to investigate how the operation of such machinery is regulated by external and internal stimuli and whether the IL-12-IL-18 collaboration has the biologic significance in some pathophysiologic conditions.

	Acknowledgments

 
We thank Miss Tomoko Katsuta and Miss Mitsuko Yamanaka for secretarial assistance.

	Footnotes

 
¹ This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.

² Address correspondence and reprint requests to Dr. Hiromi Fujiwara, Biomedical Research Center, Osaka University Medical School, 2–2, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail address:

³ Abbreviations used in this paper: SN, supernatant; PE, phycoerythrin.

Received for publication September 2, 1997. Accepted for publication December 17, 1997.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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