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Originally published online as doi:10.1189/jlb.0807530 on July 23, 2008

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(Journal of Leukocyte Biology. 2008;84:1047-1056.)
© 2008 by Society for Leukocyte Biology

IL-15-induced CD8+CD122+ T cells increase antibacterial and anti-tumor immune responses: implications for immune function in aged mice

Akira Motegi*, Manabu Kinoshita*, Akihito Inatsu{ddagger}, Yoshiko Habu*, Daizoh Saitoh{dagger} and Shuhji Seki*,1

* Department of Immunology and Microbiology, National Defense Medical College,
{dagger} Division of Traumatology, National Defense Medical College Research Institute, and
{ddagger} Department of Laboratory Medicine, National Defense Medical College Hospital, Tokorozawa, Japan

1 Correspondence: Department of Microbiology, National Defense Medical College, 3-2 Namiki, Tokorozawa 359-8513, Japan. E-mail: btraums{at}ndmc.ac.jp


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ABSTRACT
 
We previously proposed that mouse CD8+CD122+ T cells and human CD57+ T cells, which increase with age and exhibit potent IFN-{gamma} production, represent a double-edged sword as they play critical roles in host defense and the lethal IL-12/LPS-induced generalized Shwartzman reaction (GSR). However, our proposal was based solely on comparisons of young and old mice. In this study, we attempted to increase CD8+CD122+ T cells in young mice with exogenous IL-15 and confirm their countervailing functions in young mice. After young mice (6 weeks) were injected with IL-15, they showed significant increases in CD8+CD122+ T cells in the liver and spleen. Liver CD8+CD122+ T cells from IL-15-pretreated mice had a potent capacity to produce IFN-{gamma} after IL-12 injection or Escherichia coli infection. IL-15-pretreated mice showed increased survival to E. coli infections and enhanced anti-tumor activities against liver metastatic EL4 cells, as well as an exacerbation of the GSR. Correspondingly, liver CD8+CD122+ T cells produced more perforin than CD8+CD122– T cells in EL4-inoculated mice. Unexpectedly, comparable IL-15 treatment did not induce further increases in CD8+CD122+ T cells in aged mice and did not enhance their defenses against bacterial infection or tumor growth. Interestingly, however, nontreated, aged mice (50 weeks) showed twofold higher IL-15 levels (but not TNF or IFN-{gamma}) in liver homogenates compared with young mice. Our results further support that CD8+CD122+ T cells, which are increased physiologically or therapeutically by IL-15, are involved in antibacterial immunity, anti-tumor immunity, and the GSR.

Key Words: generalized Shwartzman reaction • infection • cytotoxicity • EL4 • liver immunity • innate immunity


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INTRODUCTION
 
Mouse CD8+CD122+ T cells, as well as human CD57+ T cells (CD56–CD57+CD8+ T cells), increase in the elderly and may be intimately involved with age-related changes of immune responses as a result of their high potential to produce IFN-{gamma} [1 2 3 4 ]. Mouse CD8+CD122+ T cells play a critical role in the age-associated augmentation of the generalized Shwartzman reaction (GSR) [5 ]. Human CD57+ T cells are also closely tied to the age-associated augmentation of Shwartzman reaction-like responses in human PBMC [6 ]. Elderly patients are more susceptible to septic shock, often making their prognoses worse than that of younger patients [7 8 9 10 11 ]. These age-associated increases in mouse CD8+CD122+ T cells and human CD57+ T cells are one of the major reasons why these aging mammals are susceptible to septic shock.

However, it does not appear that these age-associated increases in CD8+CD122+ T cells have exclusively harmful effects on host defense, as they may also positively contribute to host defense. As these cells have a potent IFN-{gamma}-producing capacity, we have focused on IFN-{gamma}-mediated cellular immunity. {alpha}-Galactosylceramide ({alpha}-GalCer), a synthetic ligand for NKT cells, has a potent anti-tumor activity via activation of NKT cells as well as NK and CD8+ T cells [12 , 13 ]. We recently found that {alpha}-GalCer induced the proliferation of CD8+CD122+ T cells with anti-tumor function in mouse liver [14 ]. We therefore speculated specific involvements of CD8+CD122+ T cells, not only in anti-tumor activity but also in host defense against bacterial infections.

IL-15 is a growth factor that uses the β- and {gamma}-chains of the IL-2R for signal transduction in conjunction with the {alpha}-chain of the IL-15-specific receptor [15 , 16 ]. CD122 is an IL-2R β-chain that is a functional receptor for IL-15 and controls the proliferation and survival of CD8+CD122+ T cells [17 ]. Consistent with this, it was reported that in vivo injection of IL-15 potently and selectively stimulated CD44high CD8+ T cells in mice, and most of the CD8+CD122+ T cells have high CD44 expression [18 ]. We therefore attempted to increase CD8+CD122+ T cells in young mice to numbers comparable with those seen in aged mice using repeated stimulations with exogenous IL-15. We then examined the effects of these increased numbers of CD8+CD122+ T cells for host reactions to endotoxin shock, bacterial infection, and tumor progression/invasion.


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MATERIALS AND METHODS
 
This study was conducted according to the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College (Japan).

Mice and reagents
Male C57BL/6 (B6) young mice (6 weeks old, 20 g) and aged mice (50 weeks old, 30 g) were purchased from SLC Japan Inc. (Hamamatsu, Japan). LPS (Escherichia coli 0111: B4) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). E. coli strain B (ATCC 11303, Sigma Chemical Co.) was grown in brain heart infusion broth (Difco Co. Ltd., Detroit, MI, USA). Recombinant mouse IL-12 and human IL-15 were purchased from R&D Systems (Minneapolis, MN, USA). Human IL-15 is available for mouse in vivo and in vitro studies [18 ].

Pretreatment with IL-15
Pretreatment with IL-15 was performed by i.p. injections of IL-15 (10 µg/kg) on alternate days (Days –7, –5, –3, and –1 prior to the start of experiments). Sham treatment was also performed by i.p. injections of PBS on alternate days.

Induction of the GSR, E. coli challenge, and EL4 inoculation
As described in our previous reports [5 , 19 ], the mice were i.p.-injected with IL-12 (25 µg/kg) as a priming injection and were subsequently i.v.-challenged with LPS (2.5 ng/kg) 24 h later. Young and aged mice were i.v.-challenged with 5 x 1010 CFU/kg E. coli to examine the effect of IL-15 pretreatment. Aged mice were also i.v.-challenged with 7.5 x 109 CFU/kg E. coli to examine the effect of pretreatment with anti-NK1.1 antibody or anti-IL-2Rβ antibody. Young and aged mice were i.v.-inoculated with 1 x 104 EL4 cells.

In vivo cell depletion of NK/NKT cells and CD8+CD122+T cells
Anti-NK1.1 antibody (PK 136; 500 µg/mouse) or anti-mouse IL-2Rβ antibody (TMβ-1; 500 µg/mouse) was i.v.-injected into mice 10 days and 7 days before the IL-12 priming. Twelve hours after the last antibody injection, pretreatment with IL-15 was started. In the E. coli challenge model, anti-NK1.1 antibody or anti-IL-2Rβ antibody was also i.v.-injected into mice 5 and 3 days before bacterial challenge. The depletion of NK/NKT cells (~85% depletion) by anti-NK1.1 antibody and the depletion of NK/NKT cells and CD8+CD122+T cells (85% depletion) by anti-mouse IL-2Rβ antibody were confirmed by flow cytometry as follows. As the injected, specific antibodies might remain on the surfaces of the respective lymphocytes, they cannot be stained using the same antibodies. Liver mononuclear cells (MNC) from mice pretreated with either antibody were stained with anti-CD44 antibody in conjunction with anti-CD4 antibody or anti-CD8 antibody. NK cells and T cells with intermediate TCR levels (NKT cells and CD8+CD122+T cells) express CD44high, and conventional T cells (with high TCR levels) express CD44low [20 ]. It was confirmed that ~90% of NK cells and 85% of CD4+NKT cells were depleted by anti-NK1.1 antibody. In addition to the depletion of NK cells and CD4+NKT cells, 85% of CD8+CD122+T cells were depleted by anti-mouse IL-2Rβ antibody.

Isolation of MNC
Mice were killed humanely using lethal ether anesthesia, and the livers and spleens were removed. Liver MNC were prepared essentially as described [19 , 21 , 22 ]. Briefly, livers were minced and suspended in HBSS containing 0.05% collagenase (Wako, Osaka, Japan) and then shaken for 20 min in a 37°C water bath. The specimens were passed through a 200-gauge stainless steel mesh. After washing, cells were resuspended in an osmolarity- and pH-adjusted 33% Percoll solution containing 100 U/ml heparin and centrifuged at 500 g for 20 min at room temperature. After RBC lysis, liver MNC were resuspended at 2.5 x 106 cells/ml in 10% FBS RPMI-1640 medium. Splenocytes were also passed through a stainless steel mesh and then treated with RBC lysis solution.

Cell culture
The MNC obtained from liver and spleen were stained with Turk’s solution (Wako) and counted by light microscopy. After counting, 5 x 105 MNC from liver or spleen in 200 µl 10% FBS RPMI-1640 medium were cultured in 96-well flat-bottom plates in 5% CO2 at 37°C. To examine IFN-{gamma} production, MNC obtained from mice 6 h after IL-12 priming were cultured for 18 h. To examine TNF production, MNC obtained from mice 24 h after IL-12 priming were cultured with LPS (10 µg/ml) for 24 h. The supernatants were stored at –80°C.

Flow cytometric analysis of liver MNC
A three-color immunofluorescence analysis was performed. Liver MNC were incubated for 10 min at 4°C with Fc block (2.4G2; BD PharMingen, San Diego, CA, USA) to prevent nonspecific binding. The cells were stained with FITC-conjugated anti-mouse CD8 mAb (eBioscience, San Diego, CA, USA), PE-conjugated anti-mouse CD122 (IL-2Rβ) mAb (eBioscience), and PE-Cy5-conjugated anti-mouse β-TCR mAb (eBioscience). CD8+CD122+ T cells were positive for CD8, CD122, and β-TCR staining. The fluorescently tagged cells were analyzed using an EPICS XL (Beckman Coulter, Hialeah, FL, USA).

Intracellular staining with IFN-{gamma} or perforin
Prior to intracellular staining for IFN-{gamma}, liver MNC were incubated with BD GoldiStop (0.7 µl/ml, BD PharMingen) for 2 h. Thereafter, the cells were incubated with Fc blocker and then were stained with FITC-conjugated anti-mouse CD8 mAb and PE-conjugated anti-mouse CD122 mAb. Following this, the cells were incubated with BD Cytofix/Cytoperm solution (BD PharMingen) at 4°C for 20 min and then washed with BD Perm/Wash solution (BD PharMingen). The cells were then incubated with biotin-conjugated anti-mouse IFN-{gamma} mAb (eBioscience) or isotype control mAb (rat IgG1, eBioscience) at 4°C for 30 min followed by streptavidin-PE-Cy5-conjugated mAb (eBioscience). After washing, the cells were analyzed using an EPICS XL (Beckman Coulter). For intracellular perforin staining, the cells were also incubated with Fc blocker and then stained with PE-conjugated anti-mouse CD122 mAb and PE-Cy5-conjugated anti-mouse CD8 mAb (eBioscience). The cells were incubated as above with BD Cytofix/Cytoperm solution and then washed with BD Perm/Wash solution. After this, the cells were incubated with anti-mouse perforin mAb (rat IgG2a, KM585, Kamiya Biochemical Co., Seattle, WA, USA) [23 , 24 ] or isotype control mAb (rat IgG2a, eBioscience) at 4°C for 30 min followed by FITC-conjugated anti-rat IgG2a (eBioscience).

In vitro depletion of CD8+CD122+ T cells by cell sorter and cell culture
For in vitro depletion of CD8+CD122+ T cells, whole liver MNC were stained with anti-CD8 mAb, anti-CD122 mAb, and anti-β-TCR mAb. The CD8+CD122+ T cells were depleted from the liver MNC using an EPICS ultra fluorescent cell sorter (Beckman Coulter), and the purity (greater than 95%) of the sorted cells was confirmed by an EPICS XL (Beckman Coulter). Thereafter, 5 x 105 sorted cells or whole (control) MNC in 200 µl medium were cultured with IL-12 (25 ng/ml) in 96-well flat-bottom plates in 5% CO2 at 37°C for 24 h to examine IFN-{gamma} production.

Anti-tumor cytotoxicity assay
As described previously [14 ], EL4 lymphoma cells (B6 origin) were used as target cells (2x103 cells/well), which when labeled with 100 µCi Na251CrO4, were incubated for 4 h at 37°C in 96-well round-bottom microtiter plates (total volume of 100 µl) with the liver or spleen MNC obtained from IL-15- or PBS-pretreated mice. Cytotoxicity was calculated as the percentage of released radioactivity after correcting for spontaneous release, which was <15% of maximal release.

Assay for IFN-{gamma}, TNF, and alanine aminotransferase (ALT) in sera or culture supernatants and IL-15 in tissue homogenates
As shown in our previous studies [5 , 19 ], serum IFN-{gamma} levels peak at 6 h after IL-12 priming, and serum TNF levels peak at 1 h after a subsequent LPS challenge in mice. Thus, blood samples were obtained from the retro-orbital plexus 6 h after IL-12 priming and 1 h after subsequent LPS challenge to measure IFN-{gamma} and TNF levels at their respective peaks. The sera were stored at –80°C until assayed. The IFN-{gamma} and TNF levels in sera or culture supernatants were measured by cytokine-specific ELISA kits (Endogen, Woburn, MA, USA). The sera were typically diluted tenfold with assay buffer prior to cytokine measurement. For tissue homogenates, liver and spleen were removed from young and aged mice and used to produce 1 mL homogenized suspension in PBS. The homogenates were centrifuged at 400 g for 15 min, and the supernatants were stored at –80°C until assayed. IL-15 levels in tissue homogenates were measured by cytokine-specific ELISA kits (R&D Systems). Serum ALT levels were measured using the Fuji Dri-Chem system (Fuji Film, Tokyo).

Pathological examinations
Mice were humanely killed, and the lungs, livers, and kidneys were removed. Livers and kidneys were immersed in 20% formalin for 2 days. Lungs were also immersed in 20% formalin for 2 days after gentle intratracheal instillation of 20% formalin with a pressure of ~10 cm H2O. Slides were prepared and stained with H&E.

Statistical analysis
Statistical analyses were performed using the Stat View 4.02J software package (Abacus Concepts, Berkeley, CA, USA). Survival rates were compared by the Wilcoxon signed-rank test. Comparison between two groups was done using Student’s t-test. Multiple group comparisons used one-way ANOVA, followed by a Bonferoni post-hoc test. Results are given as the mean ± SE. P < 0.05 was considered statistically significant.


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RESULTS
 
IL-15 pretreatment induces CD8+CD122+T cells in the livers and spleens of young mice and augments the GSR
Young mice were i.p.-injected with IL-15 or PBS on alternate days for 7 days prior to experimental analyses. Subsequently, liver and spleen MNC were obtained to examine the proportions of CD8+CD122+ T cells. Pretreatment with IL-15 significantly increased the proportions of CD8+CD122+ T cells in the MNC from liver and spleen (Fig. 1 ).


Figure 1
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  		Figure 1. Proportions of CD8+CD122+ T cells in liver and spleen MNC after IL-15 or PBS pretreatment in young mice. Liver and spleen MNC were obtained from young mice after IL-15 or PBS pretreatment (see Materials and Methods) and stained with FITC-conjugated anti-mouse CD8 mAb, PE-conjugated anti-mouse CD122 mAb, and PE-Cy5-conjugated anti-mouse β-TCR mAb. After gating on β-TCR+ cells, CD8+CD122+ T cells were identified by positive staining for CD8 and CD122. Percentages in the upper-right quadrants are CD8+CD122+ T cells in the liver or spleen. Percentages in the lower-right quadrants are CD8+CD122– cells. Representative data are shown for three separate experiments with three or four mice per group. Percentages shown are mean ± SE. *, P < 0.01, versus CD8+CD122+ T cells from PBS-pretreated young mice.

The GSR was induced by IL-12 priming and subsequent LPS challenge in the IL-15- or PBS-pretreated young mice. IL-15 pretreatment decreased survival remarkably after LPS challenge (Fig. 2A ). The IL-15-pretreated mice showed significant elevations of serum IFN-{gamma} levels 6 h after IL-12 priming and serum TNF levels 1 h after LPS challenge (Fig. 2B) . They also showed a significantly higher serum ALT level after LPS challenge, suggesting hepatocyte injury (Fig. 2B) .


Figure 2
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  		Figure 2. Effect of depletion of CD8+CD122+ T cells on the GSR in young mice. The GSR was induced by IL-12 priming and subsequent LPS challenge (after 24 h) in IL-15- or PBS-pretreated young mice (see Materials and Methods). (A) Survival of IL-15-pretreated young mice in the GSR. (B) Serum IFN-{gamma} levels at 6 h after IL-12 priming and serum TNF levels at 1 h and ALT levels at 24 h after LPS challenge in IL-15-pretreated mice (see Materials and Methods). (A and B) Results are mean ± SE from three separate experiments; five mice per group. (C) The effect of depletion of CD8+CD122+ T cells on the survival and (D) serum IFN-{gamma} (6 h after IL-12 priming) and TNF (1 h after LPS challenge) levels of IL-15-pretreated young mice in the GSR. (C and D) CD8+CD122+ T cells and NK/NKT cells were depleted using the anti-IL-2Rβ antibody; NK/NKT cells were only depleted using the anti-NK1.1 antibody. Results are pooled from two separate experiments; five mice per group. (D) Results are mean ± SE. *, P < 0.01; {dagger}, P < 0.05, versus other groups; {ddagger}, P < 0.01, versus IL-15-pretreated mice.

Next, we examined the effects of depletion of CD8+CD122+ T cells on the GSR in the IL-15-pretreated mice. NK/NKT cells and CD8+CD122+ T cells were depleted in the mice using the anti-IL-2Rβ antibody. NK/NKT cells, but not CD8+CD122+ T cells, were depleted using the anti-NK1.1 antibody. Subsequently, these mice were pretreated with IL-15, and the GSR was induced. Although the depletion of NK/NKT cells did not significantly increase the survival of IL-15-preteated mice, depletion of NK/NKT cells and CD8+CD122+ T cells increased their survival significantly (Fig. 2C) . Depletion of NK/NKT cells suppressed serum IFN-{gamma} levels after IL-12 priming in the IL-15-preteated mice, and the additional depletion of CD8+CD122+ T cells further suppressed their IFN-{gamma} levels (Fig. 2D) . Although depletion of NK/NKT cells did not affect serum TNF levels after LPS challenge, the additional depletion of CD8+CD122+ T cells did suppress TNF levels significantly (Fig. 2D) . These findings suggest that IL-15-induced CD8+CD122+ T cells play crucial roles for the occurrence of GSR.

Pathological examinations revealed that IL-15-pretreated mice had coagulation necrosis in the liver (Fig. 3A , arrows), septum thickness, and mild intra-alveolar edema in the lung (Fig. 3B) and moderate tubular injury in the kidney (Fig. 3C , arrows). These findings were much less prominent in PBS-pretreated mice.


Figure 3
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  		Figure 3. Pathological findings in the GSR for young mice. The GSR was induced by IL-12 priming and subsequent LPS challenge in young mice (see Materials and Method). For IL-15-pretreated young mice, representative images are shown for (A) liver, (B) lung, and (C) kidney. Images for PBS-pretreated young mice are for (D) liver, (E) lung, and (F) kidney. Arrows indicate coagulation necrosis of the liver (A) or moderate tubular injury of the kidney (C). Images are representative of two separate experiments for three mice per group with similar results. H&E; original magnification, x200 (A and D); x100 (B and E); and x300 (C and F).

IL-15 pretreatment enhances IFN-{gamma} production by liver MNC of IL-12-primed mice and also enhances TNF production by liver and spleen MNC after in vitro LPS stimulation
Liver and spleen MNC were obtained from the IL-15- or PBS-pretreated mice 6 h after IL-12 priming and then cultured for 18 h to determine IFN-{gamma} production. Liver MNC from IL-15-pretreated mice produced larger amounts of IFN-{gamma} than cells from PBS-pretreated mice, although spleen MNC from IL-15- and PBS-pretreated mice did not produce large amounts of IFN-{gamma} (Fig. 4A ). Liver and spleen MNC were also obtained from IL-15- or PBS-pretreated mice 24 h after IL-12 priming and then cultured with LPS for 24 h to determine TNF production. Liver and spleen MNC from IL-15-pretreated mice produced significantly larger amounts of TNF than MNC from PBS-pretreated mice (Fig. 4B) .


Figure 4
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  		Figure 4. Effects of IL-15 pretreatment on cytokine production by liver and spleen MNC from young mice. (A and B) Liver and spleen MNC were obtained from IL-15- or PBS-pretreated young mice after IL-12 priming during peak production for IFN-{gamma} and TNF (see Materials and Methods for priming and cell culture schedules). (A) IFN-{gamma} production by liver and spleen MNC. (B) TNF production by liver and spleen MNC. (A and B) Results are mean ± SE from three separate experiments with three to four mice per group. (C) CD8+CD122+ T cells were depleted from liver MNC using a cell sorter after IL-15 pretreatment (see Materials and Methods). Whole (control) MNC from IL-15-pretreated mice were passed through the sorter but not actively sorted. IFN-{gamma} production by whole and CD8+CD122+ T cell-depleted liver MNC. Results are mean ± SE from two separate experiments with three to four mice per group. (D) Intracellular staining with IFN-{gamma} in the liver MNC of the nontreated control mice (left panel) and the mice after IL-12 priming in the IL-15-pretreated mice (right panel). After staining with anti-CD8 mAb and anti-CD122 mAb, intracellular IFN-{gamma} staining of the liver MNC was evaluated. Isotype control (rat IgG1) was shown as a shaded area. Comparisons among CD8+CD122+ T cells, CD8+CD122– T cells, and CD8–CD122+ (NK/NKT) cells are shown in right panel. *, P < 0.01; {dagger}, P < 0.05, versus the other group.

Liver MNC were also obtained from IL-15-pretreated mice to deplete CD8+CD122+ T cells using a cell sorter. Only control whole MNC from IL-15-pretreated mice were passed through the sorter. Afterwards, the cells were cultured with IL-12 for 24 h. Although passing MNC through a cell sorter, with or without active sorting, tends to inhibit their ability to produce IFN-{gamma}, depletion of CD8+CD122+ T cells from liver MNC significantly decreased IFN-{gamma} production compared with that of whole MNC (Fig. 4C) , suggesting that IL-15-induced CD8+CD122+ T cells produce a large amount of IFN-{gamma}.

We also examined intracellular IFN-{gamma} staining of CD8+CD122+ cells by flow cytometry. Liver MNC were obtained from the IL-15-pretreated mice 1 h after IL-12 priming and the nontreated young mice as controls. CD8+CD122+ cells showed higher staining intensity for intracellular IFN-{gamma} than CD8+CD122– cells, although their intensity was lower than that of CD8–CD122+ cells, namely NK/NKT cells (Fig. 4D) . Nontreated control mice did not show any positive staining of intracellular IFN-{gamma} in all analyzed cell populations (Fig. 4D) .

Pretreatment with IL-15 increases survival and serum IFN-{gamma} levels after E. coli challenge in young mice but not in aged mice
IL-15- and PBS-pretreated young mice were i.v.-challenged with 5 x 1010 CFU/kg E. coli. Pretreatment with IL-15 significantly increased survival after bacterial challenge and also increased serum IFN-{gamma} levels at 6 h after challenge (Fig. 5 A and B ). We also examined intracellular IFN-{gamma} staining of liver MNC from IL-15-pretreated mice at 1 h after E. coli challenge. CD8+CD122+ cells showed higher intracellular IFN-{gamma} staining intensity than CD8+CD122– cells, and the intensity was lower than that of CD8–CD122+ cells (NK/NKT cells; Fig. 5C ).


Figure 5
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  		Figure 5. Effect of IL-15 pretreatment on E. coli challenge in young and aged mice. The survival (A) and serum IFN-{gamma} levels (B) after E. coli challenge in IL-15- or PBS-pretreated young mice (see Materials and Methods). Sera were obtained at the indicated times to measure IFN-{gamma} levels. (C) Intracellular staining with IFN-{gamma} in liver MNC after E. coli challenge in the IL-15-pretreated young mice. Intracellular IFN-{gamma} staining evaluated as described in Figure 4D . Isotype control is shown as shaded area. Comparisons among CD8+CD122+ cells, CD8+CD122– cells, and CD8–CD122+ (NK/NKT) cells are shown in the right panel. The survival (D) and serum IFN-{gamma} levels (E) after E. coli challenge in IL-15- or PBS-pretreated, aged mice. Data were pooled from two separate experiments with five mice per group (A, B, D, and E). (B and E) Results are mean ± SE. (C) Representative data are shown from two separate experiments with three mice per group with similar results. *, P < 0.05, versus PBS control.

We next examined the effect of IL-15 pretreatment on aged mice, which were i.p.-injected with IL-15 or PBS on alternate days for 7 days and were subsequently i.v.-challenged with 5 x 1010 CFU/kg E. coli. In contrast with young mice, aged mice did not show the IL-15-induced enhancement of survival after bacterial challenge. In addition, no significant difference in serum IFN-{gamma} level was observed after bacterial challenge between the IL-15- and PBS-pretreated, aged mice (Fig. 5 D and E) .

Aged mice do not induce CD8+CD122+T cells in liver or spleen after IL-15 treatment, although aged mice have significantly higher levels of IL-15 in liver
We examined the effect of IL-15 treatment on the proportions of CD8+CD122+T cells in liver and spleen MNC of aged mice. In contrast with young mice, treatment with IL-15 did not increase the proportions of CD8+CD122+ T cells in the liver or spleen MNC of aged mice (Fig. 6A ). As IL-15 is difficult to detect at the protein level [18 , 25 , 26 ], we could not detect significant amounts of IL-15 in the sera or culture supernatants of MNC from young or aged mice. Instead, we examined IL-15 levels in tissue homogenates of liver and spleen, which were removed from nontreated young and aged mice and homogenized to measure IL-15 at the organ level. The IL-15 levels were significantly higher in the liver homogenates from aged mice than from young mice (Fig. 6B) , although no significant differences in TNF or IFN-{gamma} levels were found between the aged and young mice (data not shown).


Figure 6
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  		Figure 6. (A) The proportion of CD8+CD122+ T cells in liver and spleen MNC after IL-15 pretreatment in aged mice (see Materials and Methods). Percentages in the upper-right quadrants are for the CD8+CD122+ T cells in the liver or spleen β-TCR+ T cells, and the percentages in the lower-right quadrants are for the CD8+CD122– cells in the β-TCR+ T cells. Representative data were shown in each group from three separate experiments with three or four mice per group. Result percentages are mean ± SE. (B) IL-15 levels in the tissue homogenates of the livers and spleens from young and aged mice (see Materials and Methods). Results are mean ± SE from three separate experiments with two or three mice per group. *, P < 0.01, versus other groups. (C) The effect of depletion of CD8+CD122+ T cells on the survival after E. coli challenge in aged mice. CD8+CD122+ T cells and NK/NKT cells were depleted using the anti-IL-2Rβ antibody; NK/NKT cells were only depleted using the anti-NK1.1 antibody. Results are pooled from three separate experiments; five mice per group. *, P < 0.01, versus other groups; {dagger}, P < 0.05, versus NK1.1 antibody-pretreated mice.

We also examined the effects of CD8+CD122+ T cell depletion in aged mice during bacterial infection. NK/NKT cells and CD8+CD122+ T cells were depleted in aged mice using the anti-IL-2Rβ antibody. NK/NKT cells, but not CD8+CD122+ T cells, were also depleted using the anti-NK1.1 antibody. Thereafter, these aged mice were i.v.-challenged with 7.5 x 109 CFU/kg E. coli. Depletion of NK/NKT cells significantly decreased the survival of aged mice after E. coli challenge. An additional depletion of CD8+CD122+ T cells by anti-IL-2Rβ antibody further decreased their survival (Fig. 6C) . These results suggest that CD8+CD122+ T cells from aged mice are effective against bacterial infection.

IL-15-induced CD8+CD122+ T cells in young mice but not in aged mice exhibit potent anti-tumor activity against EL4 cells
We examined the contribution of CD8+CD122+ T cells and the effect of aging on anti-tumor cytotoxicity. Liver and spleen MNC were obtained from IL-15- or PBS-pretreated young mice, and their anti-tumor cytotoxicity was determined using EL4 target cells. Liver MNC from IL-15-pretreated mice showed significantly higher anti-tumor cytotoxicity compared with liver MNC from PBS-pretreated mice. However, no difference was observed between spleen MNC from IL-15- and PBS-pretreated mice (Fig. 7A ). To evaluate in vivo anti-tumor activity, IL-15- or PBS-pretreated young mice were i.v.-inoculated with EL4 cells. IL-15-pretreated mice had significantly prolonged survival after EL4 inoculation compared with the PBS-pretreated mice (Fig. 7B) . We also examined intracellular perforin for liver MNC in the IL-15-pretreated mice after EL4 inoculation. Liver MNC were obtained from the IL-15-pretreated mice 7 days after EL4 inoculation. Although CD8+ cells did not show a remarkable intracellular staining for perforin compared with CD8– cells, a certain proportion of CD8+ cells did show positive staining for intracellular perforin (Fig. 7 C a) . The CD122+ subset showed a markedly higher staining with intracellular perforin in the CD8+ cells than that of the CD122– subset (Fig. 7C b) . We confirmed that most of the perforin-positive CD8– cells were CD122+, namely CD8–CD122+ NK/NKT cells (Fig. 7C b) .


Figure 7
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  		Figure 7. Effect of IL-15 pretreatment on in vitro and in vivo cytotoxicities in young mice. (A) Cytotoxicities of liver and spleen MNC in IL-15-pretreated young mice against EL4 (see Materials and Methods). Cytotoxicity of the MNC was determined in vitro with EL4 cells at the indicated E:T ratios. Results are mean ± SE from three separate experiments with three or four mice per group. (B) Survival after inoculation with EL4 cells in IL-15-pretreated young mice (see Materials and Methods). Results are pooled from two separate experiments with five mice per group. (C) Intracellular perforin staining in liver MNC of IL-15-pretreated young mice. Liver MNC were obtained from IL-15-pretreated mice 7 days after EL4 inoculation. After staining with anti-CD8 mAb and anti-CD122 mAb, intracellular perforin staining of liver MNC was evaluated (a). Fluorescence intensity of intracellular perforin in each cell subset is shown (b). Representative data are shown from two experiments with three or four mice per group with similar results. *, P < 0.01; {dagger}, P < 0.05, versus PBS control.

We next examined the effect of IL-15 pretreatment on the anti-tumor activity in aged mice. Liver and spleen MNC were obtained from IL-15- and PBS-pretreated, aged mice to examine anti-tumor cytotoxicity against EL4 cells. Pretreatment with IL-15 did not enhance the cytotoxicity of the liver or spleen MNC from aged mice (Fig. 8A ). To evaluate in vivo anti-tumor activity, IL-15- and PBS-pretreated, aged mice were i.v.-inoculated with EL4 cells. Pretreatment with IL-15 in the aged mice did not increase their survival after EL4 inoculation (Fig. 8B) .


Figure 8
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  		Figure 8. Effect of IL-15 pretreatment on in vitro and in vivo cytotoxicities in aged mice. (A) Cytotoxicities of liver and spleen MNC in IL-15-pretreated, aged mice against EL4 (see Materials and Methods). Cytotoxicity of the MNC was determined in vitro with EL4 cells at the indicated E:T ratios. Results are mean ± SE from three separate experiments with three or four mice per group. (B) Survival after inoculation with EL4 cells in IL-15- or PBS-pretreated, aged mice (see Materials and Methods). Results are pooled from two experiments with five mice per group.


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DISCUSSION
 
Aging is accompanied by a complex set of changes of the immune system that are collectively termed immune senescence [27 , 28 ], and in particular, immune senescence involving T cell-mediated responses might be critically affected by the involution of the thymus, which drastically curtails production of thymus-dependent T cells. However, this loss of thymic cell output with age does not result in any significant change in the total number of peripheral T cells [29 , 30 ]. The maintenance of peripheral T cell numbers with aging appears to be regulated via a homeostatic compensatory process, which causes a thymus-independent expansion of mature T cells [20 , 31 32 33 ]. Extrathymic T cells have unique properties of more primitive lymphocytes, such as intermediate levels of TCR expression [20 , 31 , 32 , 34 ].

The intermediate-level TCR cells constitutively express the IL-2Rβ chain (CD122). The proportion of CD8+CD122+ T cells with intermediate-level TCR increases progressively with age in the liver and other organs [3 , 35 , 36 ] and has the potential to produce large amounts of IFN-{gamma} [2 , 5 ]. CD8 T cells with a memory phenotype (CD44high), which also produce large amounts of IFN-{gamma} and increase with age in mice, are probably identical to CD8+CD122+ T cells, as CD8+CD122+ T cells are also CD44high as was reported previously [3 , 35 , 36 ]. Although "true" memory CD8+ T cells (CD44intCD122low) are generated/primed by various environmental antigens and are long-lived, the "memory-phenotype" CD8+T cells (CD44highCD122+) might be generated independently of environmental antigens and may be short-lived [37 38 39 40 41 42 ]. This relatively high rate of turnover might indicate that memory-phenotype T cells are not engaged in chronic responses to environmental antigens but are subject to nonantigen-specific ("bystander") stimulation through contact with cytokines, which are released by various stressors such as infections [17 , 18 ].

Young patients—infants, in particular—sometimes tend to be resistant to various shocks, for example, traumatic shock. Proinflammatory cytokines, such as IFN-{gamma}, are crucial for the occurrence of shock. Smaller populations of CD8+CD122+ T cells in mice and CD8+CD57+ T cells in humans (presumably extrathymically developed) [4 ] with a potent IFN-{gamma}-producing capacity might be involved in the tolerance to endotoxin shock in infants [5 , 6 ]. In contrast, infants may be more susceptible to bacterial and viral infections because of their hyporesponsiveness of IFN-{gamma}, resulting from an insufficient number of CD8+CD122+ T cells.

Consistent with these trends, in this study, IL-15-induced CD8+CD122+ T cells rendered young mice susceptible to the GSR, and they are more resistant to E. coli infection. It is well known that NK/NKT cells produce substantial amounts of IFN-{gamma} after IL-12 and/or bacterial stimulation, resulting in an increased susceptibility to the GSR but resistance to bacterial infection. This study demonstrates that CD8+CD122+ T cells produce a larger amount of IFN-{gamma} than CD8+CD122– T cells. Thus, IL-15-induced CD8+CD122+ T cells might play an important role in eliminating bacteria via enhanced IFN-{gamma} production, despite augmenting the GSR.

Aged mice had high endogenous IL-15 levels and also had a large number of CD8+CD122+ T cells. However, their CD8+CD122+ T cells did not seem to be effective against E. coli infection. In a previous study, we showed that CD8+CD122+ T cells from aged mice have a potent IFN-{gamma}-producing capacity [5 ]. In the present study, we also confirmed that depletion of CD8+CD122+ T cells and NK/NKT cells, using an anti-IL-2Rβ antibody, rendered aged mice more susceptible to bacterial infection than by the depletion of NK/NKT cells only using the anti-NK1.1 antibody. These findings suggest that CD8+CD122+ T cells in aged mice, which may be fully up-regulated by endogenous IL-15, still have a certain antibacterial activity. However, other factors involved in immunosenescence may impair host defense against bacterial infection. Further study on this issue is required.

IL-15-induced CD8+CD122+ T cells have a potent anti-tumor activity against EL4 cells in young mice. CD8+CD122+ T cells also showed in vitro anti-tumor cytotoxicity against Yac-1 cells with potent IFN-{gamma} production [2 ]. These CD8+CD122+ T cells increase age-dependently in mice. Nevertheless, the aged mice are more susceptible to malignant tumors as well as infections, although CD8+CD122+ T cells might not be functionally impaired with age [5 ]. Unexpectedly, pretreatment with IL-15 in aged mice did not increase the proportions of CD8+CD122+ T cells and correspondingly, did not up-regulate their anti-tumor cytotoxicity. Interestingly, IL-15 levels were significantly higher in the liver homogenates from aged mice compared with young mice. Several investigators have also reported age-related up-regulation of IL-15 at the protein and mRNA levels [43 , 44 ]. The aged may have already induced CD8+CD122+ T cells with endogenous IL-15 to some limiting level to maximize anti-bacterial and anti-tumor activities. Thus, stimulation with exogenous IL-15 may be ineffective for further induction of CD8+CD122+ T cells in the elderly.

Mammals have dramatically altered T cell-mediated immune responses with aging, possibly resulting from thymic involution. CD8+CD122+ T cells in mice and CD57+T cells in humans, which increase with age, might play crucial roles for host defense in the elderly. Aging mammals might thus attempt to regulate immunity against malignant tumors and bacterial infections during the aging process.

Received August 9, 2007; revised June 17, 2008; accepted June 30, 2008.


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