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Overexpression of IL-15 In Vivo Increases Antigen-Driven Memory CD8⁺ T Cells Following a Microbe Exposure¹

Toshiki Yajima^*,, Hitoshi Nishimura^*, Ryotaro Ishimitsu^*, Taketo Watase^*, Dirk H. Busch, Eric G. Pamer, Hiroyuki Kuwano and Yasunobu Yoshikai^2,*

^* Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya, Japan; First Department of Surgery, Gunma University School of Medicine, Maebashi, Japan; Sections of Infectious Diseases and Immunology, Yale University School of Medicine, New Haven, CT 06520; Infectious Disease Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, Immunology Program, Sloan-Kettering Institue, New York, NY 10021

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
To elucidate potential roles of IL-15 in the maintenance of memory CD8⁺ T cells, we followed the fate of Ag-specific CD8⁺ T cells directly visualized with MHC class I tetramers coupled with listeriolysin O (LLO)_91–99 in IL-15 transgenic (Tg) mice after Listeria monocytogenes infection. The numbers of LLO_91–99-positive memory CD8⁺ T cells were significantly higher at 3 and 6 wk after infection than those in non-Tg mice. The LLO_91–99-positive CD8⁺ T cells produced IFN- in response to LLO_91–99, and an adoptive transfer of CD8⁺ T cells from IL-15 Tg mice infected with L. monocytogenes conferred a higher level of resistance against L. monocytogenes in normal mice. The CD44⁺CD8⁺ T cells from infected IL-15 Tg mice expressed the higher level of Bcl-2. Transferred CD44⁺CD8⁺ T cells divided more vigorously in naive IL-15 Tg mice than in non-Tg mice. These results suggest that IL-15 plays an important role in long-term maintenance of Ag-specific memory CD8⁺ T cells following microbial exposure via promotion of cell survival and homeostatic proliferation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antigen-specific memory T cell responses are of vital importance in establishment of protective immunity against microbial infection. Upon encounter with a pathogenic microbe, Ag-specific T cells proliferate and differentiate into activated effector T cells. Most of the activated T cells die by apoptosis (1), but the few that survive become memory cells and persist for a long period of time, sometimes throughout the life of an animal (2, 3, 4). It has recently been reported that naive T cells directly acquire a memory phenotype in the absence of overt antigenic stimulation (5, 6, 7, 8, 9). The prolonged survival of this type of memory CD8⁺ T cell requires low-level TCR signaling from contact with a self-MHC/peptide ligand but neither IL-2 nor costimulation via CD28 (6, 7). In contrast, Ag-driven memory CD8⁺ T cells can persist even in the absence of MHC class I (10, 11). Recent studies have suggested that cytokines such as IL-15 seemed to be involved in the proliferation and survival of the Ag-driven memory CD8⁺ T cells, especially central memory phenotype in the CD8⁺ T cells (5, 12, 13, 14). However, direct evidence for the involvement of IL-15 in the maintenance of Ag-driven memory CD8⁺ T cells has not yet been obtained.

IL-15 uses - and -chains of IL-2R for signal transduction and thus shares many properties of IL-2 despite having no sequence homology with it (15, 16, 17, 18). Similar to IL-2, IL-15 promotes activation, proliferation, and cytokine release of various subsets of T, NK, and B cells (19, 20, 21). However, in contrast to IL-2, which accelerates activation-induced cell death in CD8⁺ T cells, IL-15 maintains the homeostasis of memory phenotype CD8⁺ T cells (22, 23). We have previously constructed transgenic (Tg)³ mice expressing IL-15 cDNA encoding a secretable isoform of the IL-15 precursor protein under the control of an MHC class I promoter, and we found that the IL-15 Tg mice, producing IL-15 constitutively, had markedly increased numbers of memory-type (CD44^highLy6C⁺) CD8⁺ T cells in the periphery lymphoid tissue (22). IL-15 Tg mice showed resistance against infection with Salmonella choleraesuis, Listeria monocytogenes, or Mycobacterium bovis accompanied by marked increases in memory CD8⁺ T cells (22, 24, 25). We have also reported that IL-15 Tg mice showed increased CD8⁺ Tc1 cell responses producing IFN- following multiple immunization with OVA/CFA (26). Thus, our IL-15 Tg mice may be useful for determining molecular mechanisms whereby IL-15 play a role in generation and/or maintenance of Ag-driven memory CD8⁺ T cells.

To this end, we followed the fate of Ag-specific CD8⁺ T cells directly visualized with MHC class I tetramers coupled with listeriolysin O (LLO)_91–99 in IL-15 Tg mice after L. monocytogenes infection. We found that the number of LLO_91–99-specific CD8⁺ T cells had increased significantly at 3 and 6 wk after infection in IL-15 Tg mice. Both cell survival and homeostatic proliferation of Ag-specific memory CD8⁺ T cells are suggested to be involved in persistence of Ag-specific memory CD8⁺ T cells in IL-15 Tg mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

IL-15 Tg mice (C57BL/6 background, H-2^b, Ly5.2), which were constructed using originally described IL-15 cDNA, have been described previously (22). IL-15 Tg mice with C57BL/6 background were backcrossed onto the BALB/c (H-2^d) background more than eight times. Age- and sex-matched BALB/c (H-2^d) mice were obtained from Japan SLC (Hamamatsu, Japan). (IL-15 Tg x BALB/c)F₁ mice, (C57BL/6 x BALB/c)F₁ mice, and B6-Ly5.1 mice (H-2^b, Ly5.1) were bred in our laboratory. Mice were maintained under specific pathogen-free conditions and were offered food and water ad libitum. All mice were used at 6–8 wk of age.

Microorganism

L. monocytogenes, strain EGD, was used in all experiments. Bacterial virulence was maintained by serial passages in BALB/c mice. Fresh isolates were obtained from infected spleens grown in tryptic soy broth (Nissui Pharmaceutical, Tokyo, Japan), washed repeatedly, resuspended in PBS, and stored at -70°C in small aliquots. Mice were inoculated i.p. with various doses of viable L. monocytogenes in 0.2 ml of PBS on day 0. The spleen and liver were removed and separately placed in homogenizers containing 2 ml of HBSS. These samples were spread on trypto-soya agar plates, and colonies were counted after incubation for 24 h at 37°C.

Abs and reagents

FITC-conjugated anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-Ly6C (AL-21), and anti-IFN- (XMG1.2); PE-conjugated anti-CD8 (53-6.7), anti-CD44 (IM7), anti-CD62L (MEL-14), and anti-CD25 (7D4); CyChrome-conjugated anti-CD8 (53-6.7), and anti-CD4 (RM4-5); and biotin-conjugated anti-Ly5.1 (A20) were purchased from BD PharMingen (San Diego, CA). CyChrome and allophycocyanin-conjugated streptavidin were also obtained from BD PharMingen. CFSE was purchased from Molecular Probes (Eugene, OR).

Generation of H2-K^d tetramers

MHC-peptide tetramers for staining of epitope-specific cells were generated as recently described (27, 28). Briefly, purified H chain and ₂-microgobulin were dissolved in 8 M urea and diluted in a refolding buffer containing high concentrations of synthetic peptide LLO_91–99 (29) or the Janus kinase (JAK)1 self-peptide (30) to generate monomeric, soluble H2-K^d-peptide complexes. Biotinylation and tetramerization of the heterodimer were performed as described by Altman et al. (27). The monomeric complexes were tetramerized by the addition of PE-labeled streptavidin (BD PharMingen) at a molar ratio of 4:1.

Flow cytometry analysis

The cells were incubated with saturating amounts of FITC-, PE-, CyChrome-, and biotin-conjugated mAbs for 30 min at 4°C. To detect biotin-conjugated mAbs, cells were stained with CyChrome or allophycocyanin-conjugated streptavidin. For staining of epitope-specific CD8⁺ T cells using tetrameric H2-K^d-peptide complexes, cells were incubated at 4°C for 20 min in unconjugated streptavidin (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO) and Fc-block (2.4G2), followed by triple staining with FITC-CD44, CyChrome-CD8, and PE-conjugated tetrameric H2-K^d/peptide complex (0.2–0.5 mg/ml) for 30 min at 4°C. The cells were analyzed using an FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).

Analysis of intracellular cytokine synthesis

The spleen cells from infected mice were harvested, washed, and suspended at 10⁶ cells/ml in complete culture medium, and then were incubated for 4 h at 37°C in the presence of 10 µg/ml brefeldin A (Sigma-Aldrich), 5 µg/ml LLO_91–99, or JAK1 peptide. These cells were harvested, washed, and incubated for 30 min at 4°C with PE-conjugated anti-CD44 mAb and CyChrome-conjugated CD8 mAb. After surface staining, cells were subjected to intracellular cytokine staining using the Fast Immune Cytokine System according to the manufacturer’s instructions (BD Biosciences). The cells were washed and fixed in 1000 µl of FACS lysing solution (BD Biosciences) for 10 min at room temperature and were then washed again, resuspended in 500 µl of FACS permeabilizing solution (BD Biosciences), and incubated for 10 min at room temperature. After washing, the cells were stained with FITC-conjugated IFN- mAb or FITC-conjugated isotype control rat IgG (BD PharMingen) for 30 min at room temperature, and the fluorescence of the cells was analyzed using a flow cytometer.

Before staining for intracellular Bcl-2, cells were stained for cell surface Ags as describe above. After washing, cells were fixed and permeabilized with above solution. Cells were stained with either FITC-conjugated hamster anti-mouse Bcl-2 mAb (3F11) or its isotype FITC-conjugated control Ab to hamster (BD PharMingen).

Adoptive transfer assays

Nylon wool-enriched spleen T cells were incubated with appropriate dilutions of FITC-conjugated anti-I-A^d, IgM, and biotinylated anti-DX-5, -CD11c, and -TCR mAbs, and were washed twice in HBSS. The cells were then incubated with anti-FITC microbeads, streptavidin microbeads, and anti-CD4 mAb microbeads for 15 min at 4°C. CD8⁺ T cells were enriched to >90% by negative selection using LD⁺ depletion columns (Miltenyi Biotec, Bergisch Gladbach,Germany). Enriched CD8⁺ T cells (1 x 10⁷ cells) were adoptively transferred into recipient mice via tail vein inoculation. At 12 h after adoptive transfer of these cells, mice were i.p. challenged with a lethal dose of L. monocytogenes (1 x 10⁶ CFU) and 3 days later the number of bacteria in the peritoneal cavity, spleen, and liver were counted. In an another experiment, purified CD8⁺ T cells from Ly5.1-B6 mice infected with L. monocytogenes 7 days previously were suspended at a concentration of 1–5 x 10⁷/ml in PBS and then labeled with CFSE at a concentration of 5 mM for 10 min. CFSE-labeled CD8⁺ T cells were inoculated i.v. into naive (IL-15 Tg x BALB/c)F₁ mice or naive (C57BL/6 x BALB/c)F₁ mice. After 6 wk, transferred Ly5.1⁺ T cells were analyzed using a flow cytometer.

RT-PCR

LLO_91–99-specific CD44⁺CD8⁺ T cells were sorted from IL-15 Tg or non-Tg mice on day 7 or 21 after L. monocytogenes infection using FACSVantage (BD Biosciences). The first-strand cDNA synthesized from the mRNA was amplified using 10 pmol of each primer specific for murine -actin, CCR7, CXCR3, Bcl-2, Bcl-XL, or caspase-8/Fas-associated death domain protein-like IL-1-converting enzyme inhibitory protein (FLIP). The specific primers were as follows: -actin sense, 5'-GGAATCCTGTGGCATCCATGAAAC-3'; antisense, 5'-TAAAACGCAGCTCAGTAACAGTCCG-3'; CCR7 sense, 5'-GAGATGCTCACTGGTCAGTG-3'; antisense, 5'-CTACGGGGAGAAGGTTGTGG-3'; CXCR3 sense, 5'-CAACATCAACTTCTATGCAG-3'; antisense, 5'-AGGATATGGGCATAGCAGTA-3'; Bcl-2 sense, 5'-TGGCCTTCTTTGAGTTCGGT-3'; antisense, 5'-AGCCTCCGTTATCCTGGATC-3'; Bcl-XL sense, 5'-CCGGAGAGCGTTCAGTGATC-3'; antisense, 5'-TCAGGAACCAGCGGTTGAAG-3'; and FLIP sense, 5'-GTCACATGACATAACCCAGATTGT-3'; and antisense, 5'-GTACAGACTGCTCTCCCAAGCACT-3'. The PCR products were separated on 1% agarose gels, transferred to a GeneScreen Plus filter (NEN, Boston, MA), and hybridized with ³²P-labeled oligo probes. The oligonucleotide probes were as follows: -actin, 5'-TTCTGCATCCTGTCAGCAAT-3'; CCR7, 5'-CGCCGATGAAGGCATACAAG-3'; CXCR3, 5'-CTCACCTGCATAGTTGTATG-3'; Bcl-2, 5'-CCGGTTCAGGTACTCAGTCA-3'; Bcl-XL, 5'-CTGCATCTCCTTGTCTACGC-3'; and FLIP, 5'-CTAAGGAATGTAAGTAGGGA-3'.

Statistical analysis

Data were analyzed by Student’s t test, and a Bonferroni correction was applied for multiple comparison. The value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kinetics of LLO_91–99-specific CD8⁺ T cells in IL-15 Tg mice after L. monocytogenes infection

To directly follow the fate of the L. monocytogenes epitope-specific CD8⁺ T cells in IL-15 Tg mice after an i. p. inoculation with 1 x 10⁵ CFU of L. monocytogenes, tetrameric MHC molecule folding with the LLO_91–99 peptide, the immunodominant epitope recognized by H2-K^d-restricted CD8⁺ T cells (29), was used for staining epitope-specific CD8⁺ T cells. Consistent with our previous finding (24), we found that the bacterial number increased to a maximal level on day 3 in the spleen and liver and thereafter cleared completely by day 10 after inoculation in both non-Tg mice and IL-15 Tg mice and that the bacteria were more rapidly eliminated in IL-15 Tg mice than in non-Tg mice (data not shown). As shown in Fig. 1, a significant number of CD8⁺ T cells expressing a high level of CD44 in non-Tg mice infected with L. monocytogenes 7 days previously were stained with H2-K^d/LLO_91–99 tetramers, whereas only a few CD8⁺ T cells in IL-15 Tg mice were stained with H2-K^d/LLO_91–99 tetramers on day 7 after infection. The absolute numbers of H2-K^d/LLO_91–99 peptide-positive CD8⁺ T cells in the splenocytes were 2.8 ± 0.4 x 10⁵ cells in non-Tg mice and 2.3 ± 0.9 x 10⁵ cells in IL-15 Tg mice (Fig. 2). Thus, the number of LLO_91–99-specific T cells in the spleen of IL-15 Tg mice was similar to that in the spleen of non-Tg mice at the early stage after infection because memory CD44⁺CD8⁺ T cells other than those specific for LLO_91–99 were markedly increased in IL-15 Tg mice at this stage.

View larger version (47K): [in this window] [in a new window]   FIGURE 1. LLO91–99-specific CD8+ T cells in the spleen of IL-15 Tg mice after infection with L. monocytogenes. For staining of epitope-specific CD8+ T cells using tetrameric H2-Kd-peptide complexes, spleen cells from IL-15 or non-Tg mice infected with 1 x 105 L. monocytogenes were incubated at 4°C for 20 min in unconjugated streptavidin (0.5 mg/ml) and Fc-block (2.4G2), followed by triple staining with FITC-CD44, CyChrome-CD8, and PE-conjugated tetrameric H2-Kd/LLO91–99 for 30 min at 4°C. The cells were analyzed using a FACSCalibur flow cytometer, and then the analysis gate was set on CD8+ T cells. Each number indicates the percentage of H2-Kd/LLO91–99-positive cells in CD8+ T cells. Data of a representative are shown from three separate experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Kinetics of absolute number of LLO91–99-specific CD8+ T cells in IL-15 Tg mice following L. monocytogenes infection. The absolute number of LLO91–99-specific CD8+ T cells were calculated by multiplying total spleen cells by the percentage of LLO91–99-specific CD8+ T cells in spleen. Data of a representative are shown from three separate experiments and are expressed as means ± SD of five mice of each group from representative experiment. *, p < 0.05, significantly different from the value for non-Tg mice.

Listeria-specific memory CD8⁺ T cells in IL-15 Tg mice function to protect against L. monocytogenes infection

Besides Th1 response, Tc1 response also plays a critical role in protective immunity against L. monocytogenes infection (31). To determine whether the memory CD8⁺ T cells in IL-15 Tg mice belong to the Tc1 cell population, we used cytokine FACS analysis for expression of CD8, CD44, and intracellular IFN-. As shown in Fig. 3, a significant fraction of CD44⁺CD8⁺ T cells from both groups of mice infected with L. monocytogenes 21 days previously produced IFN- in response to LLO_91–99, and the level of CD8⁺ Tc1 cells producing IFN- was significantly higher in IL-15 Tg mice than in non-Tg mice at this stage. It is notable that the relative number of intracellular IFN--positive CD8⁺ T cells responding to LLO_91–99 in vitro was consistent with that of CD8⁺ T cells directly stained with H2-K^d/LLO_91–99 tetramers in non-Tg or IL-15 Tg mice, respectively (Figs. 1 and 3).

View larger version (39K): [in this window] [in a new window]   FIGURE 3. Intracellular cytokine staining of LLO91–99-specific CD8+ T cells in IL-15 Tg mice infected with L. monocytogenes. The spleen cells from IL-15 Tg or non-Tg mice infected with 1 x 105 L. monocytogenes were harvested, washed, and suspended at 106 cells/ml in a complete culture medium and were then incubated for 5 h at 37°C in the presence of 10 µg/ml brefeldin A and 5 µg/ml LLO91–99 or JAK1 peptide. These cells were harvested, washed, and incubated for 30 min at 4°C with PE-conjugated anti-CD44 mAb, biotin-conjugated CD8 mAb, and then CyChrome-conjugated streptavidin. The cells were stained with FITC-conjugated IFN- mAb for 30 min at room temperature, and the fluorescence of the cells was analyzed using a flow cytometer. The analysis gate was set on CD8+ T cells. Each number indicates the percentage of intracellular IFN--positive cells in CD8+ T cells. Data of a representative are shown from three separate experiments.

View larger version (43K): [in this window] [in a new window]   FIGURE 4. Protection against a challenge with a lethal dose of L. monocytogenes in mice in which CD8+ T cells from L. monocytogenes-infected IL-15 Tg mice had been transferred. CD8+ T cells (1 x 107) from non-Tg or IL-15 Tg mice infected with 1 x 105 L. monocytogenes 21 days previously were adoptively transferred to recipient mice via the tail vein. At 12 h after the transfer, mice were challenged with a lethal dose of L. monocytogenes (1 x 106 CFU), and 3 days later, the number of bacteria in the spleen and liver were counted. Each column and vertical bar represent means ± SD of five mice in each group. *, p < 0.05, significant different between the values for non-TgCD8BALB/c and these for immune non-Tg CD8BALB/c. **, p < 0.01, significant different between the values for Tg CD8BALB/c and those for immune Tg CD8BALB/c or the values for immune non-Tg CD8BALB/c and those for immune Tg CD8BALB/c.

Antiapoptotic molecules play a critical role in regulating cell survival and apoptosis of memory T cells (32). We next sorted LLO_91–99-specific CD44⁺CD8⁺ T cells from non-Tg and IL-15 Tg mice on day 7 or 21 after infection and compared the gene expressions of CCR7, CXCR3, and antiapoptotic molecules such as Bcl-2, Bcl-XL, or FLIP. CCR7, which is expressed specifically in central memory T cells (33), was not expressed in LLO_91–99-specific CD44⁺CD8⁺ T cells on day 7 after L. monocytogenes infection, but its expression was up-regulated in those cells on day 21 after L. monocytogenes infection (Fig. 5). These results are consistent with surface markers for effector and memory on LLO_91–99-specific CD44⁺CD8⁺ T cells on day 7 or 21, respectively. CXCR3, which is expressed by Th1/Tc1 cells (34), was expressed by both LLO_91–99-specific CD44⁺CD8⁺ T cells on days 7 and 21, a finding that is also consistent with CD44⁺CD8⁺ Tc1 cells capable of IFN- production upon LLO_91–99 stimulation. Notably, LLO_91–99-specific CD44⁺CD8⁺ T cells in IL-15 Tg mice on day 7 after infection showed a higher level of Bcl-2 gene expression than those in non-Tg mice did. There were no remarkable differences in gene expression of Bcl-XL and that of FLIP, an inhibitor of the Fas/Fas ligand signaling pathway (35).

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Gene expression of chemokine receptors and antiapoptotic proteins in LLO91–99-specific T cells from IL-15 Tg mice. mRNA was extracted from LLO91–99-specific CD44+CD8+ T cells isolated from IL-15 Tg or non-Tg mice on day 7 or 21 after L. monocytogenes infection. The synthesized first-strand cDNA was amplified by means of the PCR using 10 pmol of each primer specific for murine -actin, CCR7, CXCR3, Bcl-2, Bcl-XL, or FLIP with 2.5 U of rTaq. The PCR products were separated on 1% agarose gels, transferred to a GeneScreen plus filter (NEN), and then hybridized with 32P-labeled oligo probes. Data of a representative are shown from three separate experiments.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Bcl-2 expression on LLO91–99-specific CD8+ T cells in IL-15 Tg mice following L. monocytogenes infection. Spleen cells from non-Tg or IL-15 Tg mice on days 7, 9, 14, and 35 postinfection were stained with anti-CD8 mAb and tetrameric H2-Kd/peptide complex. The cells were stained with either FITC-conjugated hamster anti-mouse Bcl-2 or its isotype control Ab to hamster. The Bcl-2 levels in the gated populations are shown as a single histogram and each number indicates the mean fluorescence intensity. Staining with isotype control Ab was overlaid on each histogram as a dotted line. Data of a representative are shown from three separate experiments.

The number of memory T cells is maintained by a balance among cell survival, apoptosis, and proliferation (1, 2, 3, 4). To elucidate whether cell division is involved in increases in Ag-driven memory CD8⁺ T cells in IL-15 Tg mice, we performed a transfer experiment with CFSE-labeled CD8⁺ T cells from the spleen of non-Tg mice infected with L. monocytogenes 7 days previously into naive non-Tg or IL-15 Tg mice, and we analyzed cell division in vivo 6 wk later. As shown in Fig. 7 and Table I, more CD44⁺CD8⁺ T cells entered the cell cycle in IL-15 Tg mice than in non-Tg mice 6 wk after injection. These results suggest that memory CD8⁺ T cells persist by cell division in IL-15 Tg mice.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Cell division of memory CD8+ T cells in IL-15 Tg mice. CD8+ T cells were sorted from spleen cells of Ly5.1+ mice infected with L. monocytogenes 7 days previously using magnetic cell sorter. The CD8+ T cells were labeled with CFSE, and then 1 x 107 of the CFSE-labeled cells was adoptively transferred to naive IL-15 Tg mice or non-Tg mice. Six weeks later, spleen cells were examined for expression of CD44, CD8, and CFSE using a flow cytometer. The analysis gate was set on Ly5.1+CD8+ T cells. The percentage of cells at each division number is shown in the table. Data of a representative are shown from three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A key issue is the molecular mechanisms whereby IL-15 regulates the size of Ag-driven memory CD8⁺ T cells in the periphery following a microbial infection. It can be speculated that overexpression of IL-15 in vivo may enhance the generation of effector CD8⁺ T cells after L. monocytogenes infection, resulting in an increase in the number of memory CD8⁺ T cells. However, the results of our previous (24) and present studies revealed that the absolute number of LLO_91–99-specific effector CD8⁺ T cells in IL-15 Tg mice was similar or rather smaller than that in non-Tg mice on day 7 after infection, excluding the above-mentioned possibility. The LLO_91–99-specific CD8⁺ T cells from IL-15 Tg mice infected with L. monocytogenes 7 days previously expressed CD69 but not CCR7 mRNA, indicating that these cells are effector cells. The CD8⁺ effector T cells expressed a higher level of Bcl-2 genes than did those in non-Tg mice. Bcl-2 expression is induced via signaling from the common cytokine receptor -chain (36), which is used by IL-15 (37) and prevents apoptosis by both activation-induced cell death and withdrawal of growth factors (1). In fact, annexin V expression in CD44⁺CD8⁺ T cells of IL-15 Tg mice was significantly lower on day 7 after L. monocytogenes infection than that seen in non-Tg mice (data not shown). Therefore, it is likely that overexpression of IL-15 protects the effector CD8⁺ T cells from apoptosis by activation-induced cell death and/or withdrawal of growth factors, resulting in an increased number of memory CD8⁺ T cells. We have recently reported that IL-15 Tg mice showed augmented Tc1 responses against bacillus Calmette-Guérin infection (25) and against multiple immunization with OVA in CFA (26). Augmented Tc1 responses in these reports may be explained by increases in the memory CD8⁺ T cells following OVA or bacillus Calmette-Guérin immunization.

Cell division is thought to be required for the long-term maintenance of Ag-driven memory CD8⁺ T cells in vivo (38, 39). The results of our transfer experiments suggest that the transferred CD44⁺CD8⁺ T cells divided more at 6 wk in naive IL-15 Tg mice than in naive non-Tg mice. These results suggest that memory CD8⁺ T cells have a higher rate of homeostatic proliferation in IL-15 Tg mice than in non-Tg mice. IL-15 may play a role in the long-term survival of memory T cells in vivo by cell division of memory CD8⁺ T cells in addition to protection from activation-induced apoptosis. Recent studies have provided several lines of evidence for homeostatic proliferation of naive CD8⁺ T cells (5, 6, 7, 8, 9). Naive CD8⁺ T cells can acquire characteristics of memory T cells in the absence of stimulation with a specific Ag, but by stimulation with self-MHC class I/peptide ligand (6, 7). Therefore, memory CD8⁺ T cells include not only true Ag-experienced cells but also memory cells derived from naive cells via homeostatic proliferation. Additional experiments are needed to elucidate the roles of IL-15 in the homeostasis of memory CD8⁺ T cells directly derived from naive CD8⁺ T cells.

IL-15 mRNA is constitutively expressed by various cells and tissues such as placenta, skeletal muscle, kidney, epithelial cells, synovial cells, and macrophages (21, 40). IL-15 expression is regulated not only at the transcriptional level but also at levels of translation and intracellular trafficking (41, 42, 43, 44, 45, 46). Hence, IL-15 protein was found to be produced only by a limited number of cells such as LPS-stimulated macrophages and bacteria-stimulated epithelial cells, but not by other cells including T cells (41, 47). Masopust et al. (48) have recently reported that Ag-specific memory T cells are maintained preferentially in nonlymphoid tissues such as lamina propria of intestine for the long term. Because IL-15 is thought to be produced abundantly in intestinal epithelium, IL-15 may play a critical role in the long-term maintenance of Ag-driven memory CD8⁺ T cell in the nonlymphoid tissues.

In conclusion, overexpression of IL-15 in vivo shed light on the role of IL-15 in long-term maintenance of memory CD8⁺ T cells in vivo. IL-15 may promote linear differentiation of effector CD8⁺ T cells into memory CD8⁺ T cells through protection from activation-induced cell death by apoptosis and may maintain memory CD8⁺ T cells through induction of cell division. These findings suggest that IL-15 may be useful as an immune adjuvant given with vaccination to enhance its biologic efficacy.

	Acknowledgments

 
We thank Dr. K. Kishihara for providing B6-Ly5.1 mice and K. Itano and A. Nishikawa for their excellent technical assistance.

	Footnotes

 
¹ This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture (JSPS-RFTF9 7L00703, to Y.Y.), by the Yamada Science Foundation, by the Yasuda Medical Research Foundation, and by the Yakult Bioscience Foundation.

² Address correspondence and reprint requests to Dr. Yasunobu Yoshikai, Laboratory of Host Defense, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, Nagoya 466-8550, Japan. E-mail address; yyoshika{at}med.nagoya-u.ac.jp

³ Abbreviations used in this paper: Tg, transgenic; LLO, listeriolysin O; FLIP, caspase-8/Fas-associated death domain protein-like IL-1-converting enzyme inhibitory protein; JAK, Janus kinase.

Received for publication September 26, 2001. Accepted for publication November 29, 2001.

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