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Originally published online as doi:10.1189/jlb.0706431 on March 30, 2007

Published online before print March 30, 2007
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(Journal of Leukocyte Biology. 2007;82:142-151.)
© 2007 by Society for Leukocyte Biology

Protection of CD8+ T cells from activation-induced cell death by IL-18

Wen Li, Shin-ichiro Kashiwamura, Haruyasu Ueda, Atsuo Sekiyama and Haruki Okamura1

Laboratory of Host Defenses, Institute for Advanced Medical Sciences and Hyogo College of Medicine, Hyogo, Japan

1 Correspondence: Laboratory of Host Defenses, Institute for Advanced Medical Sciences and Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. E-mail: haruoka{at}hyo-med.ac.jp


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ABSTRACT
 
Role of IL-18 on proliferation and survival of CD8+ T cells, activated by immobilized anti-CD3 antibody (anti-CD3), was examined. Proliferation and survival of activated T cells, especially that of CD8+ T cells, were impaired by IL-18 deficiency [IL-18 knockout (KO)]. After 3 days of culture with anti-CD3, the number of living CD8+ T cells from IL-18KO mice was ~25% of that from wild-type (WT) mice but was increased to the same level as WT cells by the addition of IL-18. The expression of IL-18 receptors (IL-18Rs), particularly IL-18Rß chain, in naïve CD8+ T cells was very low but elevated after stimulation with anti-CD3. Blockade of IL-18R by anti-IL-18R antibody on activated WT CD8+ T cells resulted in reduction of living cells, suggesting that IL-18 promotes survival of proliferating CD8+ T cells. Levels of Bcl-2 in activated IL-18KO CD8+ T cells were lower than those in WT cells but were raised by exogenous IL-18. Blockade of IL-18R on WT CD8+ T cells decreased the expression of surface markers CD122 and CD94, which are related to cell viability, and the expression of these markers was increased by exogenous IL-18 in IL-18KO cells. These results suggest that IL-18 acts directly on activated CD8+ T cells through IL-18Rs and promotes their survival to expand the population.

Key Words: cell survival • cell division • IL-18 receptors


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INTRODUCTION
 
The balance of activation-induced proliferation and death of effecter cells is a key point in the homeostatic expansion of T cells. Resting T cells are susceptible to apoptosis and short-lived in culture. However, stimulation of T cells through TCR/CD3 in the presence of cytokines, such as IL-2, IL-4, IL-7, and IL-12, results in clonal expansion [1 2 3 4 5 6 ]. Roles of these molecules in the homeostasis of T cells are complex and sometimes paradoxical; for example, IL-2 is necessary for proliferation and survival of CD4+ T cells but is also a prerequisite for activation-induced cell death (AICD) [1 , 2 ]. Regulation of proliferation and AICD of activated T cells is closely associated with immune/inflammatory responses.

IL-18, a member of IL-1 family, was discovered originally as an IFN-{gamma}-inducing factor produced by macrophages but later found to be able to augment production of various cytokines including Th2 cytokines [7 8 9 ]. IL-18 also activates NK cells, enhances CTL activity, and induces Fas/Fas ligand on lymphocytes [8 , 9 ], indicating that IL-18 is involved in host defenses against infection and in pathogenesis of various diseases. IL-18 has been shown to be involved in activation, differentiation, and clonal expansion of CD4+ T cells, promoting their short-term survival [8 9 10 ]. However, relatively little is known about the action of IL-18 on CD8+ T cells. It has been reported recently that IL-18 induces Type I CD8+ effecter T cells efficiently with strong CTL activity dependently on CD4+ T cells in MLC [11 ]. It is likely that IL-18 acts directly on CD8+ T cells, as IL-18 receptor {alpha} (IL-18R{alpha}) is expressed on activated CD8+ T cells [12 , 13 ].

In T cells, IL-18 activates various transcription factors such as NF-{kappa}B dependently on MyD88, IL-1R-associated kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), P38 MAPK, and ERK [14 , 15 ]. Recently, it has also been shown that IL-18 enhances a prosurvival signal, PI-3K/Akt, in myocardial cells, some cancer cells [14 , 16 ], and neutrophils [17 ]. These results indicate that IL-18 is involved in survival of various types of cells.

Here, we present data suggesting that IL-18 plays a role in expansion and survival of activated CD8+ T cells with specific functions involved in immune/inflammatory responses.


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MATERIALS AND METHODS
 
Mice
Female C57BL/6 mice (8–12 weeks old) were purchased from Seac Yoshitomi (Fukuoka, Japan), and IL-18-deficient [IL-18 knockout (KO)] mice on a C57BL/6 background were generated and backcrossed to C57BL/6 mice as described [18 ]. All mice were bred and maintained in our animal facility in accordance with the guidelines for the care and use of experimental animals in Hyogo College of Medicine (Japan). All experiments were conducted with the approval of the Animal Care Committee of Hyogo College of Medicine.

Reagents
Recombinant murine IL-18 was kindly supplied from Glaxo-Smithkline-Beecham Pharmaceutical Co. Ltd. (Research Triangle Park, NC, USA). Monoclonal anti-CD3 (Clone 145-2C11), neutralizing anti-IL-18R (Clone 112624), and IL-1{alpha}, IL-1-ß, IL-2, and anti-Bcl-2 (Clone YTH-10C4) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Rat anti-FcRII/III (Clone 2.4G2 ascites), PerCP-conjugated streptavidin (Cat. 340130), labeled antibodies to CD4 (Clone L3T4), CD8 (Clone LY-2), CD94 (Clone 18d3), CD122 (IL-2R/IL-15R, Clone TM-ß1), and CD3 (Clone 145-2C11), and Annexin-V/propidium iodide (PI) for flow cytometry staining were purchased from BD PharMingen (San Jose, CA, USA). Biotinylated antibodies to IL-18R{alpha} and IL-18Rß were purchased from R&D Systems, Inc. CellTraceTM CFSE cell proliferation kit was purchased from Molecular Probes, Inc. (Eugene, OR, USA).

Preparation and culture of spleen cells and CD8+ T cells
Spleens were resected from mice, and cells were processed into suspension, passed through 70 mm pore nylon mesh filters to remove debris, and depleted of RBC with ACK lysis buffer (pH 7.2). Cells were cultured in 12-well plates (1x106/2 ml/well) in PRMI-1640 medium containing 10% FCS and standard supplements in a 5% CO2 incubator at 37°C. T cells were stimulated by incubating on plates preincubated with anti-CD3 (2.5 µg/ml) at 4°C for 16 h. In the experiments examining IL-18 effect on T cells activated by anti-CD3 plus anti-CD28 (2.5 µg/ml), T cells were enriched from spleen cells by passing through a nylon-wool (NW) column. To measure the viability focusing on CD8+ T cells, CD8+ T cells were selected positively by a magnetic bead labeled with anti-CD8 (Clone LY-2, MACS, Miltenyi Biotec Inc., Auburn, CA, USA), and over 95% of these cells were CD8+CD3+ T cells, which were confirmed by flow cytometry. Removal of CD4+ T cells and B cells was done using magnetic beads coated with anti-CD4 (L3T4) and anti-CD19 (6D5). The resultant preparation contained CD8+ T cells up to 80%, as analyzed by flow cytometry.

Measurement of viability of cells
The number of living cells was analyzed by the trypan blue exclusion test or by flow cytometry using Annexin-V/PI staining.

Flow cytometric analysis
For the analysis of cell surface molecules, cells were stained with FITC-, PE-, PerCP-, APC-, and biotin-conjugated antibodies against CD3, CD4, CD8, CD94, CD122, and IL-18R{alpha} and -ß for 20 min at 4°C. Flow cytometric analysis was conducted by a FACSCalibur flow cytometer, and the data were analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA).

For the analysis of frequency of cell division, a single-cell suspension was prepared as described in the manual in the CellTraceTM CFSE cell proliferation kit, and 1 µl 5 mM stock CFSE solution was added to a milliliter of cell suspension. Pulse-labeling of cells was performed by brief CFSE exposures at appropriate times for the determination of cell-cycle kinetics. Frequency of cell division was measured by CFSE dilution using flow cytometric analysis.

Western blot analysis
Approximately 5 x 106 cells were lysed by incubation with M-PER® mammalian protein extraction reagent for 15 min on ice (Pierce, a Perbio Science Co., Rockford, IL, USA). The protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The lysate (10 µg of protein) was electrophoresed in a 10% SDS polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Hybond-P, Amersham Bioscience, Little Chalfont, UK), preincubated with PBS containing 2% nonfat dry milk to prevent nonspecific binding using a Trans-Blott semi-dry transfer cell (Bio-Rad Laboratories). Then, membranes were incubated with anti-Bcl-2YTH-10C4 mAb overnight, washed with PBS containing 0.5% Tween-20, and incubated with a HRP-linked F(ab')2 fragment. Specific bindings of antibodies were detected by the ECL chemiluminescence reagent (Amersham Bioscience), and chemiluminescence images were captured using LAS-1000 photo-image analyzer (Fuji Photo Film Co., Ltd., Tokyo, Japan).

Statistical analysis
Data are expressed as mean ± SD and analyzed by SPSS to determine statistical difference between groups using the Student’s t test or Bonferroni multiple comparisons test. Significant difference was accepted at P < 0.05.


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RESULTS
 
Reduced survival of IL-18KO spleen cells activated by immobilized anti-CD3 and restoration by exogenous IL-18
To clarify the IL-18 action on activated T cells, we first examined the effect of endogenous IL-18 on the number of T cells developing in vitro. Spleen cells from wild-type (WT) and IL-18KO mice (survival rate determined by PI staining, >95%) were cultured at 1 x 106 cells/well (2 ml/well in 12-well plates) for 3 days with immobilized anti-CD3, and the living cells were counted. The number was 84.8 ± 20.3 x 104/well for IL-18KO and 216.5 ± 18.6 x 104/well for WT spleen cells (Fig. 1A ). The addition of IL-18 to the culture of IL-18KO cells resulted in increases in the number of living cells in a dose-dependent manner (Fig. 1B) . Exogenous IL-18 also augmented the number of living spleen cells from WT mice (Fig. 1C) . The addition of anti-IL-18R-blocking antibody (anti-IL-18R) to the culture of WT cells resulted in a decrease in the number of living cells, and the addition of isotype rat IgG2 had no effect (Fig. 1D) . These results suggest that IL-18 boosts the expansion of CD3/TCR-stimulated T cells. As IL-18 belongs to the IL-1 family, we examined whether other members of the IL-1 family have similar activity on spleen cells. Exogenously added IL-1{alpha}, IL-1ß, and IL-2, a classical growth factor, failed to augment the number of living cells in IL-18KO spleen cells (Fig. 1E) . IL-2 failed to increase viability of the cells when added at higher concentration than 10 ng/ml (data not shown).


Figure 1
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  		Figure 1. Reduction of living IL-18KO spleen cells activated with immobilized anti-CD3 and restoration by exogenous IL-18. (A) The number of living spleen cells from WT and IL-18KO mice after incubation on anti-CD3-bound plates for 3 days. The starting concentration of cells was 1 x 106cells/2 ml/well in 12-well plates. Living cells were analyzed by the trypan blue dye exclusion test. Open bar, WT; solid bar, IL-18KO. Values were expressed as mean ± SD (n=4). **, P < 0.01. (B) Dose effect of IL-18 on the number of living IL-18KO cells stimulated with anti-CD3. IL-18KO spleen cells were cultured for 3 days in the presence of various doses of IL-18, and the number of living cells was measured. There was significant difference in the number of living cells at the concentration of more than 30 ng/ml. **, P < 0.01 (n=4). (C) Effect of IL-18 on WT cells. P = 0.077 (n=4). There was no significant difference. (D) Effect of IL-18R blockade in WT cells. Spleen cells from WT mice were incubated with anti-CD3 in the presence of anti-IL-18R (2.5 µg/ml), and 85% inhibition of IL-18R-mediated induction of IFN-{gamma} production was obtained at 2 µg/ml. Isotype IgG2a was used for control (2.5 µg/ml); **, P < 0.01; n = 4. (E) Comparison of IL-18 with other members of the IL-1 family and IL-2. IL-18KO cells were cultured in the presence of IL-1{alpha} (0.01–10 ng/ml), IL-1ß (0.01–10 ng/ml), IL-2 (0.1–10 ng/ml), or IL-18 (100 ng/ml) for 3 days, and living cells were counted (**, P < 0.01; n = 4).

IL-18 promotes survival of living CD8+ T cells
Next, we analyzed the proportions of living CD4+ and CD8+ T cells in WT and IL-18KO spleen cells and compared the proportions in unstimulated cells with those in activated T cells (Fig. 2 ). The results showed that the percentage of living CD8+ T cells and CD4+ T cells measured by CD8/CD3/PI or CD4/CD3/PI staining was 17 ± 3.5% and 24 ± 5% in resting cells from WT and IL-18KO mice before culture with anti-CD3 (Fig. 2A) . As reported previously [2 , 3 , 6 ], the percentage of CD8 T cells was augmented after anti-CD3 stimulation in WT as well as in IL-18KO cells; however, although the percentage of living CD8+ T cells was 35.7 ± 3% in the WT cells, it was 24.8 ± 4% in the IL-18KO cells (Fig. 2B) . As the absolute number of living cells and the percentage of CD8+ T cells after culture with immobilized anti-CD3 were smaller in IL-18KO cells than those in WT cells (Fig. 1A and 1B) , the absolute number of living CD8+ T cells in IL-18KO cells was far lower than that in WT cells (approximately one-fourth). Conversely, almost no significant difference was observed in the percentage of CD4+ T cells between WT and IL-18KO cells (Fig. 2B) . Consequently, there was much difference in the CD4:CD8 ratio between WT (0.48) and IL-18KO cells (0.81).


Figure 2
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  		Figure 2. Effect of IL-18 on the survival of CD8+ T cells. (A) The percentage of CD4+ and CD8+ T cells in spleen cells prepared from WT and IL-18KO mice was measured by flow cytometry after staining for CD4/PI/CD3 or CD8/PI/CD3, respectively. Results are expressed as mean ± SD (n=4). (B) The percentage of CD4+ and CD8+ T cells in living spleen cells from WT and IL-18KO mice was measured after 3 days of culture on anti-CD3-bound plates. Results are expressed as mean ± SD (n=4); **, P < 0.01. (C) The number of living cells in NW-enriched T cells stimulated with anti-CD3 and anti-CD28 (2.5 µg/ml) in the presence or absence of IL-18 (100 ng/ml) for 3 days. The number of living cells was measured by the trypan blue exclusion test, and the ratio of CD4+ T and CD8+ T cells was analyzed by flow cytometry after staining for CD4, PI, and CD8. Results are expressed as mean ± SD (n=4); **, P < 0.01. (D) The percentage of living cells in CD8+ T cells. WT and IL-18KO spleen cells were cultured with anti-IL-18R and IL-18, respectively, on anti-CD3-coated plates for 3 days. After sorting by positive selection on MACS, surviving, activated CD8+ T cells were analyzed by flow cytometry after staining with Annexin-V and PI. Results are the representative of three independent experiments.

To further confirm the effect of IL-18 on the viability of activated T cells, the effect of IL-18 was examined using T cells enriched from the spleen of WT mice by a NW column. These cells were incubated with anti-CD3 plus anti-CD28 for 3 days, with or without IL-18, and analyzed for living cells. As shown in Figure 2C , the number of living cells was far greater in the culture with IL-18. As the proportion of CD8+ T cells in the viable cells was also greater in the culture with IL-18 than that in the culture without IL-18, the absolute number of living CD8+ T cells was markedly more in the absolute number.

To confirm that the number of CD8+ T cells was preferentially reduced by the lack of IL-18, spleen cells were stimulated by immobilized anti-CD3 for 3 days and analyzed for CD8+ T cells following positive selection by a magnetic sorter to yield over 95% CD8+CD3+ T cells, as identified by flow cytometry. They were stained with Annexin-V/PI and analyzed for living cells by flow cytometry. The results showed that 36% of CD8+ T cells purified from activated IL-18KO spleen cells were alive as compared with 50% of WT CD8+ T cells (Fig. 2D) . Exogenous IL-18 increased the percentage of living cells in IL-18KO CD8+ T cells from 36% to 59%, and inversely, blockade of IL-18R reduced the percentage of living cells in WT CD8+ cells from 50% to 24% (Fig. 2D) . These results showed that IL-18 promoted survival of activated T cells, preferentially that of CD8+ T cells.

Effect of IL-18 on viability of enriched CD8+ T cells
As IL-18 preferentially promoted survival of CD8+ T cells, spleen cells from WT and IL-18KO mice were depleted of CD4+ T cells and B cells by magnetic sorting, and resultant, enriched CD8+ T cells were used for further examination of IL-18 action on CD8+ T cells activated with immobilized anti-CD3. Flow cytometric analysis showed that the enriched CD8+ T cell preparation contained CD8+/TCR+ T cells (80%), CD122+/TCR– NK cells (8–10%), CD11C+/TCR– dendritic cells (DC; 5–7%), CD4+/TCR+ T cells (<1%), and CD19+ B cells (<1%; Fig. 3A ). The majority (>70%) of enriched CD8+ T cells was the resting type with CD69–/CD44– or low, but 18–24% of them belonged to the memory type with CD69–/CD44high (Fig. 3A) . The enriched CD8+ T cells from WT and IL-18KO mice were cultured with immobilized anti-CD3, and the numbers of living cells were measured at each culture day (Fig. 3B) . In WT and IL-18KO cultures, the number of living cells decreased similarly during the first 2 days and then increased, as reported previously with WT cells [2 3 4 5 6 ]. The increase in the number of living cells in IL-18KO cells on Day 3 was much smaller than that of WT cells (Fig. 3B) . The addition of IL-18 to the culture of IL-18KO cells increased the number of living cells markedly. The addition of IL-18 to WT culture also increased living cells (data not shown). In contrast, blockade of IL-18R by the neutralizing antibody strongly decreased the number of living cells (Fig. 3B) .


Figure 3
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  		Figure 3. Effect of removal of CD4+ T, CD19+ B cells on the survival of activated CD8+ T cells. (A) Cellular component of enriched CD8+ T cells. Spleen cells were depleted of CD4+ T and CD19+ B cells using MACS depletion sorting, and resultant, enriched CD8+ T cells were analyzed for surface markers by flow cytometry. Expression of CD44 and CD69 on CD8+/TCR-ß+ cells was analyzed by gating the CD8+/TCR-ß+ area; CD4, CD19, CD122, and CD11C were examined by gating the CD8–/TCR-ß-area. (B) Enriched CD8+ T cells were cultured with immobilized anti-CD3, and the number of living cells was measured daily by the trypan blue dye exclusion test. Results are expressed as mean ± SD (n=4). There were significant differences among WT and IL-18KO, WT and WT with anti-IL-18R, or IL-18KO and IL-18KO with IL-18 (P<0.05) at Days 3 and 4. (C) Flow cytometric analysis of viability of enriched CD8+ T cells after 3 days of culture with immobilized anti-CD3. The figure is representative of three experiments. (D) Cellular component of enriched CD8+ T cells after 3 days of culture. Expression of CD44 and CD69 was measured by gating the CD8+/TCR-ß+ area.

CD8/PI staining showed that 52.90% of IL-18KO CD8+ T cells were dead and 37.8% alive, as compared with 38.69% of WT cells dead and 56.74% alive (Fig. 3C) . The addition of IL-18 to the culture of IL-18KO cells reduced dead cells to 33.46% and increased living cells to 49.84% (Fig. 3C) . Viability was consistent with the analysis by Annexin-V/PI staining (Fig. 3C) . The number of living cells was 60% in WT cells, 42% in IL-18KO cells, and 62% in IL-18KO cells treated with IL-18. After 3 days of culture, more than 95% of the living cells in the culture of WT cells were CD8+/TCR+ T cells, which was confirmed by flow cytometric analysis, and the majority (>90%) of them was the memory type of CD8+ T cells with CD44high/CD69+ (Fig. 3D) .

Effect of IL-18 on division and survival of CD8+ T cells
It has recently been shown that IL-18 boosts clonal expansion of antigen-stimulated CD4+ T cells dependently of DC as APC [10 ], and in this study, IL-18 was not considered to act as a growth factor. In the present study, it was examined whether IL-18 affects cell division as a growth factor. Enriched CD8+ T cells from WT and IL-18KO mice were labeled with CFSE and cultured with or without anti-CD3 in the presence or absence of IL-18. In the absence of stimulation by immobilized anti-CD3, IL-18 was unable to induce cell division of CD8+ T cells in WT and IL-18KO cultures (Fig. 4A and 4B ). Stimulation by immobilized anti-CD3 promoted cell division of WT and IL-18KO CD8+ T cells, and although the addition of IL-18 to the culture did not alter the frequency of cell division, it enlarged the size of populations, which underwent cell division multiple times (Fig. 4A and 4B) . The addition of anti-IL-18R to the WT culture decreased the population size of cells undergoing cell division in the same way as seen in IL-18KO culture (data not shown).


Figure 4
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  		Figure 4. Effect of IL-18 on division and survival of CD8+ T cells activated by anti-CD3. Enriched CD8+ T cells from WT and IL-18KO spleens were labeled with CFSE, incubated with or without anti-CD3 for 3 days, harvested and stained for CD8/PI/TCR-ß, and analyzed by flow cytometry. From the gate of PI(–) living cells, 10,000 CD8+/TCR-ß+ cells were analyzed for dilution of CFSE, and the histograms were overlaid. Results are the representative of three experiments. (A) WT cultured with IL-18 alone, anti-CD3 alone, and anti-CD3 + IL-18. (B) IL-18KO cultured with IL-18 alone, anti-CD3 alone, and anti-CD3 + IL-18. (C) Comparison of the effect of IL-18 (100 ng/ml) and IL-2 (10 ng/ml) on viability of IL-18KO cells activated with anti-CD3. After 3 days of culture, with or without IL-18 and IL-2, the number of living cells was examined by the trypan blue exclusion test. (D) Effect of IL-18 and IL-2 on cell division. After 3 days of culture with immobilized anti-CD3 in the presence or absence of IL-18 or IL-2, the activated CD8+ T cells were analyzed for dilution of CFSE by flow cytometry using 10,000 living cells.

As IL-2 stimulates proliferation of T cells by promoting cell division, the effect of exogenously added IL-2 was examined using IL-18KO cells enriched for CD8+ T cells. As shown in Figure 4C , exogenous IL-2 failed to augment the number of living cells, and exogenous IL-18 increased it markedly. Moreover, the size of population in CD8+ T cells, which underwent multiple times of cell division in the presence of IL-2, was much smaller than that of cells cultured with IL-18, which was confirmed by the CFSE dilution test (Fig. 4D) .

These results suggest that IL-18 does not affect cell division through up-regulation of growth factors such as IL-2 but supports accumulation of newly dividing CD8+ T cells by preventing cell death in the activation with anti-CD3/TCR.

Effect of depletion of IL-18 on division and survival of activated CD8+ T cells
Based on results presented above suggesting that IL-18 did not promote cell division but prevented AICD in activated CD8+ T cells, we investigated the effect of a IL-18R blockade on proliferating CD8+ T cells, which were activated by anti-CD3. Enriched CD8+ T cells from IL-18KO mice were labeled with CFSE and cultured with anti-CD3 in the presence of IL-18. After 2 days of culture, anti-IL-18R-blocking antibody or isotype IgG was added to the culture and incubated for another day, and the cells were analyzed by flow cytometry for the viability and cell division. Seventy-one percent of CFSE-labeled cells were PI-negative, living cells in the culture added with IL-18 and 64% in the culture with control isotype IgG, but only 50% were alive in the culture with anti-IL-18R-blocking antibody (Fig. 5A ). It was found that the cells continued to divide three times further after blockade of IL-18R. Overlaid histogram indicated that the continuation of cell division was not interrupted by IL-18 blockade but that the size of the cell population, which underwent multiple times of division, was smaller in the culture to which anti-IL-18R-blocking antibody was added than that in the control culture.


Figure 5
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  		Figure 5. Effect of removal of IL-18 on cell division and survival of activated CD8+ T cells. (A) Effect of anti-IL-18R on the size of population of newly dividing cells. CFSE-labeled CD8+ T cells from IL-18KO mice were cultured with immobilized anti-CD3 in the presence of IL-18 for 2 days, and anti-IL-18R-blocking antibody or isotype IgG was added to the culture, and incubation continued for 1 day. Cells were harvested, and frequency of cell division of activated CD8+ cells was analyzed by the CFSE dilution test using 10,000 living cells (PI-negative). The histograms were overlaid by Cell Quest System, and the number of newly dividing cells was measured. A representative of three independent experiments is shown. (B) Effect of removal of IL-18 from the culture on the size of population of newly dividing cells. CFSE-labeled CD8+ T cells from IL-18KO mice were cultured with immobilized anti-CD3 for 3 days in the presence of IL-18. Cells were harvested, washed with PBS two times, restimulated with anti-CD3, with or without IL-18 for 1 day, and analyzed for living cells by the trypan blue exclusion test and for frequency of cell division by the CFSE dilution test using 10,000 living cells (PI-negative cells). A representative of three independent experiments is shown.

The effect of IL-18 on the viability of dividing CD8+ T cells was also examined by deprivation of IL-18 from the culture restimulated by anti-CD3 (Fig. 5B) . Enriched CD8+ T cells from IL-18KO mice were cultured with immobilized anti-CD3 in the presence of IL-18 for 3 days, washed, and recultured with anti-CD3 in the presence or absence of IL-18 for another day. The number of living cells decreased rapidly by the restimulation in the culture, with and without IL-18, but the extent was much less in the culture with IL-18 (from 2x106 to 1x106/well) than in the culture without IL-18 (from 2x106 to 0.37x106/well; Fig. 5B ). Flow cytometric analysis showed that CD8+ T cells continued to proliferate by the stimulation with anti-CD3 in the presence or absence of IL-18, but the population of dividing cells decreased more rapidly in the absence of IL-18 (Fig. 5B) . This may be a result of the inhibition of cell division or the accelerated cell death by deprivation of the IL-18 signal. Considering that IL-18 deficiency (Fig. 3C) , IL-18R blockade (Fig. 5A) , or deprivation of IL-18 (Fig. 5B) increased the number of dead cells and reduced the population size of newly divided cells, the accumulation of proliferating CD8+ T cells might be a result of the antagonistic effect of IL-18 on AICD caused by activation with anti-CD3.

Effect of IL-18 on expression of surface markers on activated CD8+ T cells
Respecting the role of IL-18 in protecting activated CD8+ T cells, the expression of IL-18R on resting and activated CD8+ T cell was compared. Activation by anti-CD3 did not change the percentage of IL-18R{alpha}-positive cells significantly but increased levels of expression of IL-18R{alpha} (Fig. 6A ). Resting CD8+ T cells did not express IL-18R-ß, but it was induced strongly by the anti-CD3 activation (Fig. 6B) . The percentage of cells with high levels of IL-18R expression increased with time, when compared with resting CD8+ T cells (Fig. 3D) . Thus, it was shown that CD8+ T cells acquired responsiveness to IL-18 after activation by expressing functional IL-18R.


Figure 6
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  		Figure 6. Effect of IL-18 on expression of surface markers related to cell survival. (A and B) Expression of IL-18Rs. Spleen cells from WT mice were enriched for CD8+ T cells by magnetic sorting. Over 90% cells were CD8+/TCR-ß+ T cells, as determined by flow cytometry. They were cultured for 3 days with immobilized anti-CD3 and analyzed daily for expression of IL-18Rs by flow cytometry. (C) Expression of CD122 in CD8+ T cells. Enriched CD8+ T cells were cultured with immobilized anti-CD3 for 3 days, with or without IL-18. Thin line, WT cells; dotted line, IL-18KO; thick line, IL-18KO with exogenous IL-18. (D) CD94 expression in CD8+ T cells. Solid line, Anti-CD3-activated cells; dotted line, unstimulated CD8+ T cells. Representative results of three or four independent experiments are shown.

Previously, it has been reported that stimulation of TCR/CD3 on T cells up-regulates CD122 (IL-2Rß) expression, which is considered to play a role in protection of T cells against AICD [2 ]. CD94, an inhibitory NK receptor, has also been suggested to be involved in survival of activated lymphocytes [19 ]. We conducted flow cytometric FACS analysis of CD122 and CD94 on CD8+ T cells from WT and IL-18KO mice after stimulation by anti-CD3, with or without IL-18. As expected, expression of CD122 in IL-18KO CD8+ T cells was weaker than that in WT CD8+ T cells, and exogenous IL-18 elevated it to an even higher level than that in WT cells (Fig. 6C) . CD94 was not expressed in resting CD8+ T cells but induced by activation by anti-CD3. The expression of CD94 in activated IL-18KO CD8+ T cells was weak as compared with WT cells but markedly increased by the addition of IL-18 to the culture and suppressed by the addition of anti-IL-18R in WT CD8+ T cells (Fig. 6D) .

Effect of IL-18 on expression of survival signals in CD8+ T cells
Little is known about molecular mechanisms for survival and AICD of CD8+ T cells. Oncosis has been proposed to be involved in AICD of CD8+ T cells [19 ], and the surface markers such as CD94 and CD122 have been shown to be related to the signal pathways for cell survival [2 , 19 ]. Thus, we examined the effect of IL-18 on expression of Bcl-2, a typical molecule, which is concerned with cell survival. Expression of Bcl-2 in WT CD8+ T cells stimulated by immobilized anti-CD3 was reduced by blockade of IL-18R (Fig. 7A and 7B ). Activated IL-18KO CD8+ T cells also expressed Bcl-2, and the addition of IL-18 to the culture resulted in an increase in their expression levels (Fig. 7A and 7B) . These results suggest that IL-18 is involved in the survival of CD8+ T cells through activation of survival signals.


Figure 7
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  		Figure 7. Impaired activation of Bcl-2 in CD8+ T cells from IL-18KO mice. (A) Enriched CD8+ T cells were cultured with anti-IL-18R-blocking antibody for WT and with IL-18 for IL-18KO in the presence of anti-CD3 for 3 days and analyzed for expression of Bcl-2 by Western blotting. The density of bands was analyzed by a LAS-1000 photo-image analyzer. (B) Normalization of Bcl-2 expression using actin as a control.


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DISCUSSION
 
Homeostasis of the peripheral immune/inflammatory system is considered to be maintained mainly by two mechanisms: elimination of expanded lymphocytes by AICD and generation of regulatory cells. In this study, we showed that IL-18 may profoundly influence the size of population of lymphocytes, which have been activated. Spleen cells, particularly CD8+ T cells, from IL-18KO mice were shorter in life than those from WT mice, and exogenously added IL-18 increased the number of viable CD8+ T cells almost to the same number in WT cells (Figs. 1 2 3) . Conversely, blockade of IL-18R on WT CD8+ T cells resulted in reduction of survival of these cells (Figs. 1 2 3) . There was not much difference in the number of CD4+ T cells between WT and IL-18KO cells, and consequently, the CD4:CD8 ratio in IL-18KO cells was much higher than that in WT cells (Fig. 2) . Low levels of IL-18 were detected in the culture medium of WT cells stimulated by anti-CD3, suggesting that the endogenous IL-18 was involved in the promotion of survival in WT cells (data not shown). In the absence of stimulation by immobilized anti-CD3, IL-18 was unable to induce proliferation of CD8+ T cells (Fig. 4A) .

The increased survival of activated CD8+ T cells by IL-18 may be a result of promotion of proliferation and/or protection against AICD. Upon stimulation by immobilized anti-CD3, IL-18 deficiency did not alter the number of times that cells undergo division, which was shown by dilution of CFSE-labeled cells (Fig. 4 , D). Moreover, IL-18R blockade of proliferating WT CD8+ T cells or removal of IL-18 from IL-18-supplemented medium in which IL-18KO CD8+ T cells were grown resulted in the reduction of number of cells undergoing cell division (Fig. 5A and 5B) . However, IL-18R blockade did not affect frequency of cell division in WT cells examined by dilution of CFSE-labeled cells (Fig. 5A) . Conversely, IL-18 deficiency (Fig. 3C) , IL-18R blockade (Fig. 5A) , and IL-18 deprivation (Fig. 5B) increased the number of dead cells and reduced the population size of newly dividing cells. These results indicate that IL-18 does not accelerate cell division but promotes expansion of activated CD8+ T cells by preventing AICD of proliferating cells.

It has been reported recently that IL-18 boosts clonal expansion of antigen-activated CD4+ T cells in vivo, generating a subpopulation of effecter cells [10 ]. In this paper, the authors showed that IL-18 does not act directly on CD4+ T cells but on DC inducing CD134 ligand, which leads to activation of CD134+/CD4+ T cells, although the possibility of IL-18 binding to the CD4+ T cells after the initial contact between these cells is not excluded. In the present experiments, expression of IL-18Rs in naïve CD8+ T cells, particularly the IL-18Rß chain, was negligible or low but elevated by stimulation with anti-CD3, concurrent with the increase of proliferation (Figs. 3 and 6) . Blockade of IL-18R on activated WT CD8+ T cells with anti-IL-18R reduced the number of living cells (Figs. 1 2 3) . These results suggest that IL-18 acts directly on activated CD8+ T cells through IL-18R.

IL-18 has been shown to activate various signal pathways via IL-18Rs, including those generating prosurvival signals such as PI-3K/Akt and IRAK/TRAF6 [8 , 14 15 16 17 ]. In the present study, we found that treatment of activated IL-18KO CD8+ T cells with IL-18 increased expression of Bcl-2, which has been regarded as a prosurvival signal in T cells (Fig. 7A) . In the case of activated WT CD8+ T cells, blockade of IL-18R with anti-IL-18R resulted in a decrease in the expression of Bcl-2 (Fig. 7B) [19 20 21 22 ]. The mechanism of AICD and signal transduction for survival in activated CD8+ T cells are not understood fully [19 , 23 24 25 26 27 28 ]. Our results suggest that IL-18 may prevent AICD of activated CD8+ T cells by activating the pathway involving Bcl-2 through IL-18Rs up-regulated following anti-CD3 stimulation.

In IL-18KO CD8+ T cells, expression of CD122 and CD94 was induced by activation with anti-CD3 and enhanced further by the addition of IL-18 (Fig. 6) . The expression of CD122 was demonstrated to be involved in survival of CD8+ T cells, which is related to the mechanism for autoimmune diseases in mice [2 , 5 , 19 , 29 ]. CD94 is induced in CD8+ T cells committed at the first encounter with antigens through TCR/CD3 [28 29 30 31 ], which may lead to the loss of their cytotoxic activity [30 ] and acquirement of prolonged survival [19 , 28 ]. Moreover, CD122 and CD94 in CD8+ T cells have been shown, not only involved in cell survival but also implicated in regulatory functions [19 , 28 29 30 31 ]. IL-18 may influence not only clonal size but also function of activated CD8+ T cells by augmenting their viability and regulatory function. As production of several cytokines is regulated aberrantly in IL-18KO mice infected by bacteria and reversed by administration of IL-18 [32 ], it is probable that IL-18 plays a role in the regulation of immune/inflammatory responses by promoting expansion of regulatory cell clones.

IL-18 is produced abundantly in various types of cells including epithelial cells. Elucidation of biological activities of IL-18 in differentiating cells will be of help for understanding its pleiotropic, sometimes apparently paradoxical roles of IL-18 in immune diseases. IL-18 may support terminal differentiation of various types of cells up-regulating various genes and at the same time, promote their temporary survival.

In the present study, we show that IL-18 promotes expansion and survival of activated CD8+ T cells. IL-18 may influence immune/inflammatory responses by regulating the size of the CD8+ T cell population with specific functions following exposure to stimuli. IL-18, in combination with other cytokines or growth factors, may be useful for accumulation of cells with specific functions involved in immunotherapy.


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ACKNOWLEDGEMENTS
 
We thank Mrs. Risa Takayasu (Tusji) and Naomi Gamachi for expert technical assistance. We thank Dr. T. Tamaoki for his critical reading of the manuscript and helpful discussions.

Received July 5, 2006; revised March 6, 2007; accepted March 7, 2007.


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