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

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 α-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-CD3ε, 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

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 × 10⁵ cells/well (96-well microplates) or 1.5 × 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-γ concentrations, respectively.

Measurement of IFN-γ concentrations

IFN-γ concentrations were measured by ELISA. Mouse IFN-γ ELISA kits were purchased from Genzyme (Cambridge, MA), and our own ELISA system was prepared using two types of anti-mouse IFN-γ 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-γ 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 α-chain was accomplished by incubating up to 1 × 10⁶ cells with biotinylated-anti-IL-2R α-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).

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-γ 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-γ 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.

• Download figure
• Open in new tab
• Download powerpoint

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.

• Download figure
• Open in new tab
• Download powerpoint

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.

IL-18 responsiveness and IL-18R expression are induced depending on the presence of IL-12 during stimulation with anti-CD3 plus anti-CD28

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 3⇓A 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. 3⇓B). 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. 3⇓B, middle panel), which is consistent with the results of Figure 3⇓A. 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 3⇓B (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.

• Download figure
• Open in new tab
• Download powerpoint

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).

We also examined whether IL-18R is induced on CD8⁺ and CD4⁻CD8⁻ T cells following simultaneous stimulation with anti-CD3/anti-CD28 and IL-12. CD8⁺ or CD4⁺ T cells were depleted along with the removal of B cells and APC from lymph node cells, yielding a CD4- or CD8-enriched T cell population. A CD4⁻CD8⁻ cell population was prepared from spleen cells by depleting CD4⁺ and CD8⁺ T cells, B cells, and APC. The CD4⁺, CD8⁺, and CD4⁻CD8⁻ populations were stimulated with anti-CD3/anti-CD28 in the presence of rIL-12 or rIL-2 (control) for 48 h, and the expression of IL-18R on these three populations gated for CD4⁺, CD8⁺, and CD4⁻CD8⁻, respectively, is shown in Figure 4⇓. The results show that CD4⁺ T cells failed to express IL-18R whereas CD8⁺ and CD4⁻CD8⁻ populations expressed comparable levels of IL-18R. The IL-18R levels on these two populations were weaker than those induced by sequential stimulation with anti-CD3/anti-CD28 and rIL-12 shown in Figure 3⇑. However, the expression of IL-18R was apparently positive compared with the groups that included rIL-2 instead of rIL-12.

• Download figure
• Open in new tab
• Download powerpoint

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.

Induction of IL-18 responsiveness in resting T cells following stimulation with anti-CD3/anti-CD28 in the presence of IL-12

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-γ 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-γ in response to IL-18 although they produced moderate amounts of IFN-γ following IL-12 stimulation. In contrast, anti-CD3/anti-CD28-activated T cells in the presence of IL-12 produced large amounts of IFN-γ in response to rIL-18. The levels of IFN-γ 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-γ 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-γ 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.

• Download figure
• Open in new tab
• Download powerpoint

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-γ 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-γ 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. 6⇓A). These sorted CD4⁺ and CD8⁺ populations were stimulated with different concentrations of rIL-18 or 1000 pg/ml rIL-12 (Fig. 6⇓B). The results show that CD8⁺ and CD4⁺ populations exhibit, respectively, high and low levels of IFN-γ production in response to IL-18. It should also be noted that there is a marked difference in IL-12-stimulated IFN-γ production between CD8⁺ and CD4⁺ T cell populations.

• Download figure
• Open in new tab
• Download powerpoint

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-γ produced in the culture SNs was determined by ELISA (B). The results were representative of three similar experiments.

Because CD4⁻CD8⁻ T cells are present more numerously in spleens than in lymph nodes, we isolated a CD4⁻CD8⁻ cell population from spleen cells by depleting CD4⁺ and CD8⁺ T cells together with B cells and APC (Fig. 7⇓). This population consisted of approximately 50% CD3⁻NK1.1⁺ cells and 40% CD3⁺ (NK1.1⁺ or NK1.1⁻) cells (Fig. 7⇓). Following stimulation with anti-CD3/anti-CD28 in the presence of IL-12 or IL-2, most cells in either group remained CD4⁻CD8⁻, but the proportion of CD3⁺NK1.1⁻ cells increased in both groups, especially in the anti-CD3/anti-CD28 plus IL-12 group (Fig. 7⇓). These two groups of cells were stimulated with rIL-18. As shown in Figure 8⇓, anti-CD3/anti-CD28-plus-IL-2-activated CD4⁻CD8⁻ cells produced moderate amounts of IFN-γ in response to IL-18. In contrast, stimulation of anti-CD3/anti-CD28-plus-IL-12-activated CD4⁻CD8⁻ cells with IL-18 resulted in strikingly high levels of IFN-γ production. The levels were, in fact, one order higher than those induced in CD8⁺ T cells as shown in Figure 6⇑. Activated CD4⁻CD8⁻ cells were also found to be high responders to IL-12 compared with activated CD8⁺ T cells shown in Figure 6⇑. Thus, these results indicate that compared with the CD4⁺ T cell subset, the CD8⁺ T cell subset has a larger capacity to produce IFN-γ in response to IL-18, but CD4⁻CD8⁻ cells can exhibit much more potent IL-18 responsiveness.

• Download figure
• Open in new tab
• Download powerpoint

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).

• Download figure
• Open in new tab
• Download powerpoint

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.