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Development of CD8⁺ Effector T Cells Is Differentially Regulated by IL-18 and IL-12

Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We investigated the effects of IL-18 on the development of CD8⁺ effector T cells in DBA/2 anti-BDF₁ whole spleen cell MLC and compared the results with those of IL-12. Addition of IL-18 to the MLC resulted in a twofold increase in CD8/CD4 ratios compared with the control cultures when cells were expanded in IL-2-containing medium following MLC. Purified CD8⁺ T cells recovered from the IL-18-stimulated MLC produced 20- to 30-fold more IFN- after secondary stimulation with C57BL/6 spleen cells or anti-CD3 mAb, and exhibited strong allospecific CTL activity. Neither IL-18 nor IL-18-supplemented culture supernatants from DBA/2 anti-BDF₁ MLC induced type I CD8⁺ effector T cells when purified CD8⁺ T cells were used as responder cells in primary MLC. Furthermore, CD4⁺ T cell depletion from the responder cells abrogated the IL-18-induced increase in secondary IFN- production by CD8⁺ T cells, suggesting that IL-18-induced type I effector CD8⁺ T cell development was CD4⁺ T cell dependent. In marked contrast, adding IL-12 to primary MLC decreased CD8/CD4 ratios by 50% and suppressed secondary IFN- production and CTL activity by CD8⁺ T cells regardless of concentration, whereas Th1 development was promoted by IL-12. Moreover, both IL-12 and IL-18 efficiently induced type I CD8⁺ effector T cells in C57BL/6 anti-BDF₁ MLC. These findings show that IL-18 plays an important role in the generation of type I CD8⁺ effector T cells, and further suggest that functional maturation of CD8⁺ T cells is differentially regulated by IL-18 and IL-12.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-18 is a recently cloned cytokine that was identified originally as a factor having potent IFN--inducing activity on T cells and NK cells 1, 2 . Besides its IFN--inducing activity, IL-18 has been shown to augment NK cell activity and to induce the proliferation of T cells. The effects of IL-18 on the activation of murine CD4⁺ T cells committed to the Th1 or Th2 responses have been investigated 3 . IL-18 serves as a costimulatory factor for Th1 clones stimulated with Ag on Ag-presenting B cells, immobilized anti-CD3 mAb, Con A, or IL-2 to augment IFN- production and to induce the IL-2R -chain expression and proliferation of the Th1 clones. However, IL-18 has little or no effect on the type II cytokine production and proliferation of Th2 clones stimulated with anti-CD3 mAb or Ag. These functions are similar to those of IL-12 4, 5 , although IL-18 and IL-12 are completely different molecules in terms of their primary structures, receptor binding, and signal transduction pathways 6 .

Interestingly, IL-18 and IL-12 together exert a synergistic effect on IFN- production by Th1 cells 3, 7, 8 . IL-18, as well as IL-12, is released endogenously through interaction between Th1 cells and APCs in the presence of specific Ags 3, 9 . Furthermore, it has been shown that IL-18, unlike IL-12, does not drive Th1 development, but potentiates IL-12-driven Th1 development 6, 10 . These results suggest that both IL-18 and IL-12 are required for significant expression of the Th1 phenotype, and they further imply that IL-18, by itself or in concert with IL-12, plays an important role in the regulation of Th1-type immune responses.

In spite of extensive investigation concerning the effects of IL-18 on the activation and differentiation of CD4⁺ T cells, few studies regarding the effects of IL-18 on the activation of CD8⁺ T cells are found in the literature. Recently, Tomura et al. showed that CD8⁺ T cells stimulated with anti-CD3 and anti-CD28 mAbs in the presence of IL-12 exhibited higher levels of IL-1R-related protein (a component of the IL-18R) expression and IFN- production than CD4⁺ T cells 11 . However, it remains unclear whether IL-18 induces the development of CD8⁺ T cells into effector CTLs under physiologic conditions. Understanding the factors generating a Tc1-type, as well as Th1-type, immune response could lead to therapeutic approaches for infectious disease, allergy, neoplasia, and systemic autoimmune disease.

In a manner similar to CD4⁺ T cell subsets, it has been established that CD8⁺ effector T cell subsets can be divided into Tc1 and Tc2 subsets on the basis of their cytokine secretion profiles upon Ag stimulation 12, 13 . Tc1-type cells preferentially secret the type I cytokines, IFN- and IL-2, whereas Tc2-type cells secret the type II cytokines, IL-4 and IL-10. Both Tc1 and Tc2 subsets possess cytolytic function in vitro 12, 14 . It is well known that IL-12 induces the Tc1 development of naive CD8⁺ T cells and potentiates the cytotoxic activity of CTLs 15, 16, 17 .

To determine the effect of IL-18 on the generation of CD8⁺ effector T cells, we investigated whether exogenous IL-18 is able to activate CD8⁺ T cells in MLC. Responder spleen cells from DBA/2 mice were cultured with mitomycin C-treated stimulator spleen cells from BDF₁ mice (DBA/2 anti-BDF₁ MLC). The results were compared with those obtained from MLC supplemented with IL-12. It is well known that the injection of DBA/2 spleen cells into BDF₁ mice causes chronic graft-vs-host disease (GVHD)² characterized by B cell hyperactivation, auto-Ab formation, and lupus-like disease 18 . On the other hand, the injection of C57BL/6 spleen cells into BDF₁ mice results in acute GVHD characterized by augmented anti-host (BDF₁) CTL activity and subsequent immunodeficiency 18, 19 . Importantly, donor anti-host CTLs are not detected in mice with chronic GVHD. Moreover, spleen cells from DBA/2 mice show a ninefold lower anti-BDF₁ CTL activity and a twofold lower number of CD8⁺ T cells than C57BL/6 mouse spleen cells 20 .

We report in this study that IL-18 efficiently induces the development of type I CD8⁺ effector T cells with strong CTL activity via a CD4⁺ T cell-dependent pathway in DBA/2 anti-BDF₁ MLC. In marked contrast, IL-12 suppressed the maturation of CD8⁺ T cells to IFN--producing CTLs, whereas it promoted the maturation of IFN--producing CD4⁺ effector T cells. However, both IL-18 and IL-12 efficiently induced the development of type I CD8⁺ effector T cells in C57BL/6 anti-BDF₁ MLC. These findings indicate that IL-18 plays an important role in the development of CD8⁺ effector T cells, and further provide evidence for differential regulation of CD8⁺ effector T cell development between IL-18 and IL-12.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

DBA/2J (H-2^d), C57BL/6 (H-2^b), and BDF₁ (H-2^b/d) mice were obtained from Charles River Japan (Kanagawa, Japan). Females, aged 2–4 mo, were used throughout these experiments.

Medium

The culture medium used in these experiments was RPMI 1640 containing 10% FCS, 5 x 10^-5 M 2-ME (Life Technologies, Grand Island, NY), 60 µg/ml penicillin, and 50 µg/ml streptomycin.

Cytokines and Abs

Murine rIL-18 was generated by expression in Escherichia coli and purification, as previously described 1, 3 . The purity of the rIL-18 was >95%, as verified by SDS-PAGE analysis, and the endotoxin content was <1 ng/mg, as measured by Limulus amebocyte lysate assay. Murine rIL-12 was expressed in Chinese hamster ovary cells cotransfected with rIL-12 p35 and p40 cDNAs. The cytokine was purified from the supernatants of the Chinese hamster ovary cultures by chromatography. The sp. act. of IL-12 was 5.5 x 10⁶ U/mg, as determined by the induction of IFN- production by murine spleen cells 3 . SDS-PAGE analysis indicated that the IL-12 was >95% pure, and the endotoxin content was <30 ng/mg. Human rIL-2 was purchased from Genzyme (Boston, MA).

Hamster anti-mouse CD3 mAb (145-2C11) and biotinylated anti-CD3 mAb were purchased from Cedarlane Laboratories Limited (Ontario, Canada). Rat anti-mouse IL-2R -chain mAb (3C7), rat anti-mouse CD8a mAb (53-6.7), and anti-mouse I-A^d mAb (AMS-32.1) were purchased from PharMingen (San Diego, CA). Anti-L3T4 mAb (GK1.5) was obtained from Becton Dickinson (San Jose, CA). Goat anti-rat IgG (nonreactive with mouse IgG) and goat anti-mouse IgG were purchased from Cappel (Aurora, OH). Rabbit anti-mouse IFN- polyclonal Ab (pAb) was prepared in our laboratory and used as a purified IgG fraction 21 . The neutralizing activity of rabbit anti-mouse IFN- pAb was 5.94 x 10⁴ neutralizing U/mg. Rabbit anti-Cryj II pAb (Abs against Japanese cedar pollen allergen, Cryj II) was prepared in our laboratory and used as an isotype-matched control for rabbit anti-mouse IFN- pAb. Sandwich ELISAs were used to determine IFN- and IL-10 levels. The Abs for plate coating and the biotinylated secondary mAbs were as follows: for IFN-, rabbit anti-mouse IFN- pAb and biotinylated rat anti-mouse IFN- mAb (XMG1.2; PharMingen); for IL-10, rat anti-mouse IL-10 mAb (JES5-2A5; PharMingen) and biotinylated anti-mouse IL-10 mAb (SXC-1; PharMingen).

Purification of CD8⁺ T cells

Purification of CD8⁺ T cells was conducted as follows. To remove dendritic cells and macrophages, single cell suspensions from DBA/2 mouse spleens were incubated for 1 h at 37°C on a FCS-coated plastic plate. After incubation, nonadherent cells were recovered by washing the surface of the plate with medium. To remove B cells, recovered nonadherent cells were incubated with anti-mouse I-A^d mAb for 30 min on ice, washed twice, and seeded on a goat anti-mouse Ig-coated plate for 1 h at 4°C, and then the nonadherent cells were recovered by gentle washing. To recover CD8⁺ T cells by positive selection, nonadherent cells were incubated with rat anti-mouse CD8 mAb for 30 min on ice, washed twice, and seeded on a goat anti-rat IgG-coated plate for 1 h at 4°C. After the incubation period, nonadherent cells were removed by gentle washing. The remaining adherent CD8⁺ T cells were incubated in culture medium at 37°C overnight. During this incubation, the cells detached from the plate. Purified CD8⁺ T cells were washed twice and used for primary MLC as responder cells. The purity of the resulting purified CD8⁺ T cells (85–90%) was determined by flow cytometry before each experiment (CD4⁺, <2%). In some experiments, CD8⁺ T cells (>85%) were purified by negative selection. Briefly, CD8⁺ T cells were prepared from anti-L3T4 mAb-injected DBA/2 spleen cells passed through a nylon wool column, followed by depletion of B cells using anti-I-A^d mAb and goat anti-mouse Igs.

Depletion of CD4⁺ T cells in vivo

Ascites fluid (2.5-fold diluted with PBS) containing anti-L3T4 mAb derived from rat GK1.5 B cell hybridoma (a gift from Dr. E. Nakayama, Okayama University Medical School, Okayama, Japan) 22 was injected in 200 µl vol into the retro-orbital sinuses of DBA/2 mice, as previously described. Six days following the mAb treatment, spleen cells were used for primary MLC. Depletion of CD4⁺ T cells (<0.5%) in the spleens was verified by flow cytometry before each experiment.

Mixed lymphocyte cultures

Stimulator cells were prepared by incubating the cells with 50 µg/ml of mitomycin C (Sigma, St. Louis, MO) for 30 min at 37°C. The cells were then washed three times with medium. Responder cells (5 x 10⁶/well) were cultured with mitomycin C-treated stimulator cells (5 x 10⁶/well) in flat-bottom 24-well plates in a total volume of 2 ml at 37°C in a 5% CO₂ humidified atomosphere. Cytokines including IL-18 and IL-12, Abs, or culture supernatants previously collected from other conditioned MLCs were added at the initiation of primary MLC. In this study, 540 pM of either IL-18 or IL-12 was added to the culture. At this concentration, IL-18 exhibited maximum IFN- production in primary MLC (data not shown). After 5 days, supernatants were collected and frozen at -20°C for subsequent IFN- and IL-10 assays, and culture cells were harvested and washed. In experiments in which purified CD8⁺ T cells were used as responder populations, primary MLCs were performed for 6 days. Mitomycin C-treated BDF₁ spleen cells produced low levels of IFN- in response to IL-18 during 5 days of primary MLC (medium alone, <0.6 IU/ml; IL-18 treatment, <6.3 IU/ml; IL-12 treatment, <0.8 IU/ml). To determine the function of CD8⁺ T cells and CD4⁺ T cells, each cell population was purified by positive selection through panning. First, the cells were suspended in culture medium containing 0.05% sodium azide, incubated either with rat anti-CD8 mAb or anti-L3T4 mAb for 30 min on ice, and washed twice with medium containing sodium azide. Then the cells were seeded on a goat anti-rat IgG-coated plate for 1 h at 4°C. Nonadherent cells were removed by gentle washing, and the remaining adherent cells were incubated in culture medium at 37°C overnight to allow detachment from the plate. The resulting purified CD8⁺ and CD4⁺ T cells were washed, counted for viable cells, and used for secondary stimulation and cytotoxicity assays. Flow cytometry revealed that all T cell populations used for the secondary stimulation consisted of >98% CD8⁺ T cells or CD4⁺ T cells. To determine their cytokine production profile, the purified CD8⁺ or CD4⁺ T cells (3 x 10⁴/well) were restimulated with mitomycin C-treated stimulator spleen cells (1 x 10⁶/well) in the presence or absence of IL-2 (10 U/ml), or with immobilized anti-CD3 mAb in 96-well plates. After 2 days (for CD8⁺ T cells) or 3 days (for CD4⁺ T cells) of incubation, the cell-free supernatants were collected and frozen at -20°C until assayed. IFN- production by CD8⁺ T cells and CD4⁺ T cells during the secondary stimulation reached a plateau level on days 2 and 3, respectively.

Cytotoxicity assays

Cytotoxicity was examined in standard ⁵¹Cr release assays. Briefly, the target cells EL-4 (H-2^b) or P815 (H-2^d) were labeled with ⁵¹Cr for 1 h at 37°C. Viable effector CD8⁺ T cells were isolated from responder cells by centrifugation over Ficoll-Hypaque (Sigma). Target cells were then incubated with various numbers of effector cells for 4 h. Supernatants were collected, radioactivity was counted by gamma counter, and specific lysis was calculated according to the following formula: percent specific lysis = [(cpm experimental - cpm spontaneous)/(cpm maximum - cpm spontaneous)] x 100.

Flow-cytometric analysis

The cells (10⁵ cells/tube) were washed three times in PBS containing 1% FCS and 0.05% sodium azide, and then stained first with primary mAbs followed by FITC-conjugated F(ab')₂ fragments of goat anti-rat IgG (PharMingen) or FITC streptavidin (Genzyme) for 30 min each on ice. Isotype-matched control Abs were used for background staining. The cells were washed and resuspended in 500 µl of 1% FCS-PBS before analysis. Stained cells were analyzed on an EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL) after gating on viable cells.

Cytokine assays

The concentrations of cytokines (IFN- and IL-10) in the culture supernatants were determined by two-site sandwich ELISA using the Abs described above. Standard curves were generated using recombinant cytokines. The lower limit of detection was 0.6 IU/ml (1 IU = 7 ng) for IFN- and 0.6 ng/ml for IL-10.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-18 increases percentage of CD8⁺ T cells after primary DBA/2 anti-BDF₁ 5-day MLC, followed by a 2-day expansion culture with IL-2

To investigate whether exogenous IL-18 affects the activation of CD4⁺, CD8⁺, or both T cell subsets, DBA/2 spleen cells were stimulated with mitomycin C-treated BDF₁ spleen cells (DBA/2 anti-BDF₁ MLC) in the presence or absence of IL-18, and the ratio of CD8⁺ to CD4⁺ T cells was evaluated after MLC by flow cytometry (Table I). In addition, MLC supplemented with IL-12, which is known to share several biological functions with IL-18, was prepared. On day 5 after the start of MLC, there were no significant differences in the CD8/CD4 ratios between all three MLCs (Table I). When cells recovered from MLC on day 5 were expanded in fresh medium containing 10 U/ml of IL-2 and then further incubated for 48 h, IL-18 treatment resulted in a twofold increase in the CD8/CD4 ratio compared with controls receiving medium only. The actual number of CD8⁺ T cells stimulated with IL-18 increased up to threefold after a 2-day expansion culture with IL-2, although IL-18-primed CD4⁺ T cells increased in number slightly. In contrast, the addition of IL-12 resulted in a twofold lower ratio than the controls. Furthermore, neither CD4⁺ nor CD8⁺ T cells recovered from IL-12-supplemented culture increased in number after culturing with IL-2. The mean fluorescence intensity of CD markers of CD4 and CD8 T cell populations in the presence of IL-18 or IL-12 was similar to that observed in control MLC (data not shown). These results suggest that in the DBA/2 anti-BDF₁ MLC, exogenous IL-18 affects the activation of primarily CD8⁺ T cells rather than CD4⁺ T cells, whereas IL-12 affects the activation of CD4⁺ T cells rather than CD8⁺ T cells.

To assess the effect of IL-18 on the development of CD8⁺ effector T cells, the cells were recovered from the primary MLC of DBA/2 anti-BDF₁ system on day 5. CD8⁺ T cells were then purified by panning and were restimulated with mitomycin C-treated C57BL/6 spleen cells or with immobilized anti-CD3 mAb in the presence or absence of IL-2 to determine their cytokine profile. In addition, the results obtained with IL-18 were compared with those of MLC stimulated with IL-12. The levels of IFN- in primary MLC stimulated with either IL-18 or IL-12 were, respectively, about 10 times or 5 times higher than those in the control MLC (Fig. 1A). When CD8⁺ T cells primed with IL-18 were stimulated with mitomycin C-treated C57BL/6 spleen cells in the secondary MLC, IFN- production markedly increased compared with that produced by CD8⁺ T cells cultured in medium alone regardless of the presence or absence of IL-2 (Fig. 1B). Surprisingly, however, IL-12 treatment led rather to a reduction in secondary IFN- production by CD8⁺ T cells, as compared with that produced by CD8⁺ T cells cultured in medium alone. Similar findings were observed when CD8⁺ T cells recovered from primary MLC were restimulated with immobilized anti-CD3 mAb alone (Fig. 1C). This result eliminates the possibility that increased IFN- production by CD8⁺ T cells primed with IL-18 might be caused by APC-derived factors in the secondary MLC. Although the secondary IFN- production shown in Fig. 1, B and C, was assessed 48 h after secondary stimulation, the levels of IFN- produced by CD8⁺ T cells primed with IL-12 remained lower than those in control cultures even when IFN- production was assessed on day 5 of the secondary stimulation (data not shown). Thus, it seems unlikely that IL-12-mediated suppression of the secondary IFN- production by CD8⁺ T cells is due to a difference in the kinetics of IFN- production. In addition, CD8⁺ T cells primed with IL-18, IL-12, or medium alone did not produce detectable levels of the type II cytokine IL-10, in the secondary MLC (data not shown). These results suggest that type I CD8⁺ T cells were generated during primary MLC supplemented with IL-18.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Differential effects of IL-18 and IL-12 on the development of type I CD8+ effector T cells in DBA/2 anti-BDF1 MLC. DBA/2 spleen cells (5 x 106/well) were stimulated with mitomycin C-treated BDF1 spleen cells (5 x 106/well) in the presence or absence of 540 pM of IL-18 or IL-12. Supernatants were taken on day 5 of primary MLC, and the concentrations of IFN- in the supernatants were assessed by ELISA (A). To determine the function of CD8+ T cells, they were purified by panning from the cells collected from the primary MLC and cultured overnight in normal medium to detach the cells. The purified CD8+ T cells (3 x 104/well) were then restimulated with mitomycin C-treated C57BL/6 spleen cells (1 x 106/well) in the presence (black bars) or absence (open bars) of 10 U/ml of IL-2 (B) or with 5 µg/ml immobilized anti-CD3 mAb (C). After 2 days of secondary stimulation, supernatants were taken and assessed by ELISA for IFN- production. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of five separate experiments.

IL-18 enhances generation of allospecific CD8⁺ CTL activity

CD8⁺ T cells are the classical mediators of cytotoxic activity and, in general, develop into cytotoxic effectors upon activation 23, 24 . Therefore, we decided to determine whether IL-18 can also develop the cytotoxic activity of CD8⁺ T cells in DBA/2 anti-BDF₁ MLC. As shown in Fig. 2, IL-18 strongly augmented allospecific CD8⁺ CTL activity against EL-4 (H-2^b). In contrast, IL-12 significantly down-regulated this activity. This cytolytic function of CD8⁺ T cells was allospecific, since these cells exhibited no lysis against the syngeneic tumor target, P815 (H-2^d) (<5% specific lysis). Taken together, these data indicate that IL-18 induced the development of naive CD8⁺ T cells into an allospecific CTL population. In marked contrast and unexpectedly, IL-12 inhibited CTL development in DBA/2 anti-BDF₁ MLC.

View larger version (22K): [in this window] [in a new window]   FIGURE 2. IL-18 augments allospecific CD8+ CTL activity. DBA/2 anti-BDF1 primary MLC was performed in the presence or absence of 540 pM of IL-18 or IL-12, as indicated in Fig. 1. After 5 days of primary MLC, CD8+ T cells were purified and cultured overnight to detach the cells. The purified CD8+ T cells were then assessed for cytotoxicity against EL-4 (H-2b) in a 4-h 51Cr release assay at the indicated E:T ratios. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of two separate experiments.

Increased IFN- production is not required for IL-18 to induce type I CD8⁺ effector T cell development

IFN- has been reported to play an important role in CTL generation 25, 26, 27 . In our experiments, IL-18 strongly induced IFN- production in primary MLC (Fig. 1A). To determine whether increased IFN- was required for the IL-18-mediated generation of type I CD8⁺ effector T cells, neutralizing rabbit anti-IFN- pAbs were added to the primary MLC supplemented with IL-18. Rabbit anti-Cryj II pAbs were used as the control Ab. The addition of 25 µg/ml of anti-IFN- pAb, which can neutralize 3000 IU/ml of IFN-, did not reduce secondary IFN- production by CD8⁺ T cells primed with IL-18, but rather increased the IFN- production (Fig. 3). These results suggest that the effect of IL-18 on the generation of type I CD8⁺ effector T cells does not depend on increased IFN- production in primary MLC.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. IL-18-induced type I CD8+ effector T cell development does not require increased IFN- production. DBA/2 anti-BDF1 primary MLC was performed in the presence or absence of 540 pM of IL-18 with or without rabbit anti-mouse IFN- pAb (25 µg/ml) for 5 days. Rabbit anti-Cryj II pAb (25 µg/ml) was used as control pAb. After 5 days of incubation, CD8+ T cells purified from primary MLC were restimulated with mitomycin C-treated C57BL/6 spleen cells in the presence of IL-2 for 2 days, as indicated in Fig. 1. Secondary production of IFN- was assessed by ELISA. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of three separate experiments.

In our experiments, it remained to be resolved whether IL-18 acts directly on CD8⁺ T cells to induce type I effector T cell development. To address this question, naive CD8⁺ T cells purified from DBA/2 mouse spleen cells by positive selection method using anti-CD8 mAb were used as responder populations in the primary MLC with BDF₁ spleen cells in the presence of IL-18. The levels of IFN- produced by CD8⁺ T cells in the primary MLC supplemented with IL-18 were higher than those produced by CD8⁺ T cells in control MLC (medium alone, <0.6 IU/ml; IL-18 priming, 16.7 ± 1.1 IU/ml). This indicates that IL-18 can act on CD8⁺ T cells to induce IFN- production. However, the CD8⁺ T cells recovered from this MLC secreted much lower levels of IFN- after secondary stimulation than CD8⁺ T cells purified after primary IL-18-stimulated MLC using whole spleen cells as responders (referred to as whole spleen cell MLC) (Fig. 4; 2.6 IU/ml versus 61.4 IU/ml), suggesting that IL-18 by itself cannot directly induce type I effector T cell development of naive CD8⁺ T cells purified before primary MLC. Experiments with naive CD8⁺ T cells purified by negative selection method gave similar results with those of naive CD8⁺ T cells purified by positive selection method with anti-CD8 mAb (data not shown). This excludes the possibility that the response of CD8⁺ T cells purified by positive panning may have been modified by residual anti-CD8 mAb. These results further suggest that the effects of IL-18 on the generation of type I effector CD8⁺ T cells may be mediated by other soluble factors or by cell to cell contact. But in contrast to the whole spleen cell MLC as shown in Fig. 4, purified naive CD8⁺ T cells primed with IL-12 produced substantial levels of IFN- after secondary stimulation, although the levels of IFN- produced were lower than those produced by CD8⁺ T cells derived from IL-18-stimulated whole spleen cell MLC. This shows that IL-12 can, to some extent, directly stimulate the generation of type I CD8⁺ effector T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Neither IL-18 nor IL-18-stimulated culture supernatants of primary MLC induce type I CD8+ effector T cell development of purified naive CD8+ T cells. Whole spleen cells (5 x 106/well) or purified CD8+ T cells (5 x 105/well) from DBA/2 mice were stimulated with mitomycin C-treated BDF1 spleen cells (5 x 106/well) in the presence or absence of 540 pM of IL-18 or IL-12, or culture supernatants (final concentration: 1/2 dilution) of DBA/2 anti-BDF1 MLC. The culture supernatants were collected on day 1, day 3, or day 5 from DBA/2 anti-BDF1 whole spleen cell MLC supplemented with IL-18. CD8+ T cells were purified from primary MLC using whole spleen cells as responder populations on day 5. When purified CD8+ T cells were used as responder cells, they were collected from primary MLC on day 6. The CD8+ T cells were restimulated with mitomycin C-treated C57BL/6 spleen cells with IL-2 for 2 days. Supernatants were collected and IFN- production in secondary MLC was assessed by ELISA. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of three separate experiments. SN indicates culture supernatant.

Depletion of CD4⁺ T cells abrogates IL-18-induced type I CD8⁺ effector T cell development, but maintains IL-12-induced suppression of CD8⁺ T cell function

The results presented in the preceding sections demonstrated that IL-18 alone was unable to induce the development of purified naive CD8⁺ T cells into effector cells. Since it has been shown that CD4⁺ T cell helper function is necessary for the expansion and development of CD8⁺ effector T cells 28, 29, 30, 31 , we noted a possible requirement for CD4⁺ T cells in IL-18-induced type I CD8⁺ effector T cell development and in IL-12-induced suppression of CD8⁺ T cell function in DBA/2 anti-BDF₁ whole spleen cell MLC.

To examine this possibility, CD4⁺ T cells among the responder spleen cells were depleted by injecting anti-L3T4 mAb into DBA/2 mice 6 days before primary MLC. FACS analysis showed that CD4⁺ cell contamination in the spleen cells was <0.5%. Depletion of CD4⁺ T cells substantially reduced secondary IFN- production by CD8⁺ T cells, especially those primed with IL-18 (Fig. 5). This is consistent with previous finding that CD4⁺ T cell help is required for the generation of CD8⁺ effector T cells 28, 29, 30, 31 . On the other hand, even when CD4⁺ T cells were depleted, IL-12 could still suppress secondary IFN- production, suggesting that the IL-12-mediated suppression of CD8⁺ effector T cell development does not depend on the presence of CD4⁺ T cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Requirement of CD4+ T cells for the IL-18-induced type I CD8+ effector T cell development. Whole spleen cells or CD4 cell-depleted DBA/2 mouse spleen cells (5 x 106/well) were stimulated with mitomycin C-treated BDF1 spleen cells (5 x 106/well) in the presence or absence of 540 pM of IL-18 or IL-12. CD4 cell depletion was performed as described in Materials and Methods. CD8+ T cells purified from primary MLC were restimulated with mitomycin C-treated C57BL/6 spleen cells, as indicated in Fig. 1. IFN- production in secondary MLC was assessed by ELISA. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of two separate experiments.

The results presented in the preceding sections revealed that exogenous IL-12 suppressed the development of CD8⁺ effector T cells in DBA/2 anti-BDF₁ whole spleen cell MLC (Figs. 1 and 2). Recent reports showed that high dose IL-12 induces immunosuppression by an unknown mechanism 32, 33, 34, 35 . We therefore investigated the dose response after priming with IL-12 on IFN- production by CD8⁺ T cells in the secondary MLC. As shown in Fig. 6, IL-12, even at very low concentrations (11 and 1.1 pM), did not increase the IFN- production above the levels observed with medium alone. Thus, an inhibitory effect of IL-12 priming was observed on IFN- production by CD8⁺ T cells in the secondary MLC regardless of the concentration of IL-12.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Dose-response effect of IL-12 on inhibition of secondary IFN- production by CD8+ T cells. DBA/2 anti-BDF1 MLC was performed with the indicated concentrations of IL-18 or IL-12. CD8+ T cells purified from the primary MLC were restimulated as indicated in Fig. 1. IFN- production in secondary MLC was assessed by ELISA. The results are expressed as the mean ± SD of triplicate experiments. The data shown are representative of two separate experiments.

Injection of C57BL/6 spleen cells into BDF₁ mice results in acute GVHD, as characterized by the induction of anti-host (BDF₁) CTL, IFN- production, and immunodeficiency 18, 19 . To investigate whether the inhibitory effect of IL-12 on the development of CD8⁺ effector T cells is limited to DBA/2 anti-BDF₁ MLC, we performed C57BL/6 anti-BDF₁ MLC in the presence of IL-18 or IL-12, and compared the results with those of DBA/2 anti-BDF₁ MLC. The ratio of CD8⁺ to CD4⁺ T cells was evaluated after a 5-day MLC followed by a 2-day culture with 10 U/ml of IL-2. As shown in Table II, the CD8/CD4 ratios in all three MLC systems were higher than those observed in the DBA/2 anti-BDF₁ MLC (Table I). In marked contrast to the IL-12-stimulated DBA/2 anti-BDF₁ MLC, the percentage of CD8⁺ T cells was higher than that of CD4⁺ T cells in the IL-12-stimulated C57BL/6 anti-BDF₁ MLC. IFN- and IL-10 production in the primary MLC are shown in Table III. In contrast with the results from DBA/2 anti-BDF₁ MLC, IL-12-induced IFN- production was much higher than that induced by IL-18 in the C57BL/6 anti-BDF₁ MLC. Furthermore, IL-12 induced comparable levels of IL-10 production in both MLC systems, whereas IL-18 did not induce detectable levels of IL-10 production in either system. CD8⁺ T cells recovered and purified after primary MLC were restimulated with mitomycin C-treated DBA/2 spleen cells in the presence or absence of IL-2, and then examined for secondary IFN- production. As expected from the characteristics of mice with acute GVHD that is induced by injection of C57BL/6 spleen cells into BDF₁ mice, CD8⁺ T cells primed with medium alone produced substantial levels of IFN- after secondary stimulation, which were comparable with those produced by CD8⁺ T cells recovered from DBA/2 anti-BDF₁ MLC supplemented with IL-18 (Fig. 7). In marked contrast to the inhibitory effect of IL-12 priming in DBA/2 anti-BDF₁ MLC, both IL-18 and IL-12 induced higher levels of secondary IFN- production than those observed with medium alone (Fig. 7B). Furthermore, CD8⁺ T cells primed with IL-18, IL-12, or medium alone did not produce detectable amounts of IL-10 after secondary stimulation (data not shown). Thus, IL-12 efficiently supported the development of type I CD8⁺ effector T cells in C57BL/6 anti-BDF₁ MLC, suggesting that the difference in the IL-12-mediated development of CD8⁺ effector T cells between the two MLC systems, DBA/2 anti-BDF₁ and C57BL/6 anti-BDF₁, may be due to the reactivity of the responder cells to IL-12.

View this table: [in this window] [in a new window]   Table III. Comparison of IFN- and IL-10 production between DBA/2 anti-BDF1 MLC and C57BL/6 anti-BDF1 MLC1

View larger version (20K): [in this window] [in a new window]   FIGURE 7. Both IL-18 and IL-12 promote the development of type I CD8+ effector T cells in C57BL/6 anti-BDF1 MLC. DBA/2 anti-BDF1 and C57BL/6 anti-BDF1 primary MLC was performed as indicated in Fig. 1 and Table II, respectively. CD8+ T cells purified from DBA/2 anti-BDF1 MLC or C57BL/6 anti-BDF1 MLC were restimulated with mitomycin C-treated C57BL/6 spleen cells (A) or DBA/2 spleen cells (B), respectively, with or without IL-2. After 2 days of secondary stimulation, supernatants were collected and assayed for IFN- production. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of two separate experiments.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. IL-12-supplemented culture supernatants from DBA/2 anti-BDF1 primary MLC do not inhibit the IL-12-induced augmentation of IFN- production by CD8+ T cells purified from C57BL/6 anti-BDF1 MLC. C57BL/6 spleen cells were stimulated with mitomycin C-treated BDF1 spleen cells in the presence of IL-12 with or without culture supernatants (final concentration: 1/2 dilution) of DBA/2 anti-BDF1 MLC. The culture supernatants were collected on day 5 from DBA/2 anti-BDF1 MLC supplemented with IL-12 or medium (control). Secondary stimulation of purified CD8+ T cells was performed as indicated in Fig. 7 (B). After 2 days of secondary stimulation, supernatants were collected and assayed for IFN- production. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of two separate experiments. SN indicates culture supernatant.

To examine whether CD4⁺ T cells primed with IL-18 or IL-12 differentiated into Th1 cells, CD4⁺ T cells recovered after DBA/2 anti-BDF₁ MLC were restimulated with mitomycin C-treated C57BL/6 spleen cells for 3 days to determine their IFN- production. As shown in Fig. 9, CD4⁺ T cells primed with IL-18 or IL-12 produced substantial amounts of IFN-. Interestingly, IFN- production by CD4⁺ T cells primed with IL-12 was rather higher than in cultures primed with IL-18. Moreover, IL-10 production was not observed after secondary stimulation in any system (data not shown). These results indicate that the addition of either IL-18 or IL-12 efficiently induced Th1 development of naive CD4⁺ T cells in DBA/2 anti-BDF₁ MLC. These results further suggest that IL-12 has a differential effect on the development of CD4⁺ Th1 and type I CD8⁺ effector T cells in immune responses under certain conditions.

View larger version (19K): [in this window] [in a new window]   FIGURE 9. IL-12 induces Th1 development of CD4+ T cells in DBA/2 anti-BDF1 MLC. DBA/2 anti-BDF1 MLCs were performed for 5 days, as indicated in Fig. 1. CD4+ T cells were purified by panning on day 5 and cultured overnight in normal medium to detach the cells. The CD4+ T cells (3 x 104/well) were then restimulated with mitomycin C-treated C57BL/6 spleen cells (1 x 106/well) with or without 10 U/ml IL-2 for 3 days. After incubation, supernatants were collected and assayed for IFN- production. The results are expressed as the mean ± SD of triplicate cultures. The data shown are representative of two separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The IL-18-induced development of CD8⁺ effector T cells and the increase in CD8/CD4 ratios may be accounted for by the suppression of CD4⁺ T cell proliferation by CD8⁺ T cells activated with IL-18. Recently, Noble et al. reported that activated CD8⁺ T cells suppress the proliferation of CD4⁺ T cells and induce CD4⁺ T cell apoptosis through a Fas-dependent mechanism 36 . In fact, in our experimental system, the CD8/CD4 ratios in the responder cells of C57BL/6 anti-BDF₁ primary MLC were higher than those in DBA/2 anti-BDF₁ primary MLC, regardless of the addition of IL-18 or IL-12. These findings correlated well with the levels of IFN- produced by CD8⁺ T cells during secondary culture.

IL-18 alone could not support the optimal development of purified naive CD8⁺ T cells, although it did act on purified naive CD8⁺ T cells to produce low but detectable levels of IFN- during primary MLC. The inability of IL-18 to induce the development of purified naive CD8⁺ T cells into type I effector cells may be due to the reduction in endogenous IFN- in primary MLC compared with that produced in whole spleen cell MLC. In fact, several groups have reported that IFN- stimulates CD8⁺ CTL generation 25, 26, 27 . However, as we have shown in this study, increased IFN- was not responsible for the IL-18-induced generation of effector CD8⁺ T cells, but rather inhibited it. Furthermore, in the case of IL-12, increased IFN- may not be required for allospecific CTL generation, based on a recent report showing that the anti-IFN- Ab could not reduce the CTL activity induced by IL-12, and that large amounts of exogenous IFN- (1000 IU/ml) inhibited this activity 16 . Moreover, IFN- knockout mice could still mount CTL activity against allogeneic target cells 37 , suggesting that IFN- is not essential to induce CTL activity.

CD4⁺ T cells have been reported to play an important role in CD8⁺ CTL induction 28, 29, 30, 31 . In the present study, we demonstrated that IL-18-induced type I CD8⁺ effector T cell development also required CD4⁺ T cell help. This CD4⁺ T cell-dependent mechanism for type I CD8⁺ effector T cell development was not accounted for by the possible existence of soluble factors because the supernatants collected from primary MLC supplemented with IL-18 did not induce CD8⁺ T cell development. Since the depletion of CD4⁺ T cells resulted in an almost complete abrogation of secondary IFN- production by CD8⁺ T cells primed with IL-18, the allospecific type I CD8⁺ effector T cell response induced by IL-18 may be mediated mainly through the interactions between APCs and CD4⁺ T cells. Recently, it has been reported that CD4⁺ T cell help for CD8⁺ CTL generation involves the activation of APCs following CD40-CD40 ligand interactions between CD4⁺ T cells and APCs 38, 39, 40 . IL-18 may facilitate the activation of APCs by up-regulating the expression of CD40 or CD40 ligand.

Recently, it was shown that high doses of IL-12 inhibit CTL activity, IFN- production, and the proliferation of spleen cells in mice vaccinated with the p53 peptide or syngeneic tumor, or they abrogate the CTL response in mice infected with lymphocytic choriomeningitis virus 32, 33, 34 . Moreover, spleen cells from IL-12-treated normal mice show decreased CTL activity ex vivo 35 . However, the mechanisms underlying the IL-12-induced suppression were not well defined in any of these experiments. In these reports, the in vivo injection of IL-12 into experimental model mice resulted in immune suppression. On the other hand, we have revealed for the first time by in vitro experiments that IL-12 has an inhibitory effect on CD8⁺ T cell function.

Several possibilities are considered for the IL-12-induced inhibition of CD8⁺ T cell function in DBA/2 anti-BDF₁ MLC. For example, IL-12 may indirectly influence CD8⁺ T cells with negative regulatory signals by way of soluble factors or cell to cell contact with other spleen cell populations, including CD4⁺ T cells and macrophages. However, for the two reasons described below, it seems unlikely that soluble inhibitory factors were endogenously induced by exogenous IL-12 to then inhibit CD8⁺ T cell function. First, the levels of IL-10, which inhibits IFN- production, were comparable between the two primary MLCs, DBA/2 anti-BDF₁ and C57BL/6 anti-BDF₁. Second, the culture supernatant from DBA/2 anti-BDF₁ primary MLC did not inhibit the IL-12-induced development of type I CD8⁺ effector T cells in C57BL/6 anti-BDF₁ MLC.

It has been reported that CD4⁺ T cells inhibit CD8⁺ T cell expansion and tumor infiltration in the presence of IL-12, although CD4⁺ T cells do not influence the CTL activity of CD8⁺ T cells 41 . In our in vitro MLC system, however, CD4 cell depletion did not restore the IL-12-mediated suppression of CD8⁺ T cell function. Thus, in the case of IL-12-driven CD8⁺ T cell development, CD4⁺ T cells may not inhibit or be essential for the generation of type I CD8⁺ effector T cells. We also examined whether macrophages were required for the IL-12-mediated suppression of CD8⁺ T cell function. When the DBA/2 spleen cells that had been passed through a Sephadex G-10 column were used as responder cells in MLC with BDF₁ spleen cells in the presence of IL-12, CD8⁺ T cell function could not be restored (data not shown). It has been reported that NK cells also suppress the generation of CD8⁺ T cell cytotoxic activity 42 . Therefore, NK cells may play some role in the IL-12-induced suppressive effect on CD8⁺ T cell function. An alternative explanation for IL-12-induced suppression is that CD8⁺ T cells primed with exogenous IL-12 may express killer cell inhibitory receptors, a new family of MHC class I-specific receptors 43, 44, 45, 46 , and inhibit their own function, including the induction of IFN- production and CTL activity, as a consequence of MHC class I/killer cell inhibitory receptor interaction. Further studies are needed to clarify these possibilities.

Our results, together with findings published elsewhere regarding IL-12-induced suppression 32, 33, 34, 35 , raise the possibility that IL-12 treatment may cause unpredictable consequences in vivo. Therefore, we investigated in a DBA/2 anti-BDF₁ MLC system whether exogenous IL-18 or IL-2 can restore CD8⁺ T cell function suppressed by IL-12. In a preliminary experiment, the addition of IL-18 together with 540 pM of IL-12 to DBA/2 anti-BDF₁ MLC resulted in the restoration of IL-12-mediated immunosuppression and in a substantial augmentation of secondary IFN- production by CD8⁺ T cells in an IL-18 dose-dependent manner (540 pM IL-12, 0.8 IU/ml; 54 pM IL-18 + 540 pM IL-12, 20.8 IU/ml; 540 pM IL-18 + 540 pM IL-12, 49.1 IU/ml). In contrast, IL-2 (10 U/ml) had little effect on the restoration of IL-12-mediated suppression (data not shown). These results suggest that administration of IL-18 can restore the immunosuppression exerted by IL-12 in vivo.

In summary, we demonstrated for the first time that exogenous IL-18 induced the development of naive CD8⁺ T cells into type I effector cells in a CD4⁺ T cell-dependent manner. On the contrary, under certain conditions, the addition of IL-12 inhibited type I CD8⁺ effector T cell development, although IL-12 induced Th1 development. Injection of DBA/2 spleen cells into BDF₁ mice induces a chronic GVHD in which the anti-host (BDF₁) CTL response is defective 19, 20 . Furthermore, Via et al. proposed that anti-host CTL may play an important role in eliminating autoreactive host B cells 20 . The finding that IL-18 augments allospecific CD8⁺ CTL activity in DBA/2 anti-BDF₁ MLC provides a possibility that IL-18 may be useful for the immunotherapy of systemic autoimmune diseases such as chronic GVHD and systemic lupus erythematosus by inducing CD8⁺ CTL. Further in vivo studies are now in progress to confirm this possibility.

	Acknowledgments

 
We thank Dr. M. Micallef for helpful discussions. We are also grateful to L. Keleher for critical review of the manuscript.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Iwao Okamoto, Fujisaki Institute, Hayashibara Biochemical Laboratories, Inc., 675-1 Fujisaki, Okayama 702-8006, Japan. E-mail address:

² Abbreviations used in this paper: GVHD, graft-vs-host disease; pAb, polyclonal Ab.

Received for publication August 11, 1998. Accepted for publication December 11, 1998.

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