Overexpression of IL-15 In Vivo Increases
Antigen-Driven Memory CD8 + T Cells
Following a Microbe Exposure
This information is current as
of June 14, 2017.
Toshiki Yajima, Hitoshi Nishimura, Ryotaro Ishimitsu,
Taketo Watase, Dirk H. Busch, Eric G. Pamer, Hiroyuki
Kuwano and Yasunobu Yoshikai
J Immunol 2002; 168:1198-1203; ;
doi: 10.4049/jimmunol.168.3.1198
http://www.jimmunol.org/content/168/3/1198
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References
Overexpression of IL-15 In Vivo Increases Antigen-Driven
Memory CD8ⴙ T Cells Following a Microbe Exposure1
Toshiki Yajima,*† Hitoshi Nishimura,* Ryotaro Ishimitsu,* Taketo Watase,* Dirk H. Busch,‡
Eric G. Pamer,§ Hiroyuki Kuwano,† and Yasunobu Yoshikai2*
A
ntigen-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– 4). It has recently been reported that naive T cells
directly acquire a memory phenotype in the absence of overt antigenic stimulation (5–9). 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, 12–14). 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
*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
Received for publication September 26, 2001. Accepted for publication November
29, 2001.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
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;
[email protected]
Copyright © 2002 by The American Association of Immunologists
homology with it (15–18). Similar to IL-2, IL-15 promotes activation, proliferation, and cytokine release of various subsets of T,
NK, and B cells (19 –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
(CD44highLy6C⫹) 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 LLO91–99-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
Mice
IL-15 Tg mice (C57BL/6 background, H-2b, 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-2d) background more than eight times. Age- and sex3
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.
0022-1767/02/$02.00
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To elucidate potential roles of IL-15 in the maintenance of memory CD8ⴙ T cells, we followed the fate of Ag-specific CD8ⴙ 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 LLO91–99-positive memory CD8ⴙ T cells were significantly higher at 3 and 6 wk after
infection than those in non-Tg mice. The LLO91–99-positive CD8ⴙ T cells produced IFN-␥ in response to LLO91–99, and an
adoptive transfer of CD8ⴙ T cells from IL-15 Tg mice infected with L. monocytogenes conferred a higher level of resistance against
L. monocytogenes in normal mice. The CD44ⴙCD8ⴙ T cells from infected IL-15 Tg mice expressed the higher level of Bcl-2.
Transferred CD44ⴙCD8ⴙ 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 CD8ⴙ T cells following microbial exposure via
promotion of cell survival and homeostatic proliferation. The Journal of Immunology, 2002, 168: 1198 –1203.
The Journal of Immunology
1199
Abs and reagents
RT-PCR
FITC-conjugated anti-CD44 (IM7), anti-CD69 (H1.2F3), anti-Ly6C (AL21), and anti-IFN-␥ (XMG1.2); PE-conjugated anti-CD8␣ (53-6.7), antiCD44 (IM7), anti-CD62L (MEL-14), and anti-CD25 (7D4); CyChromeconjugated anti-CD8␣ (53-6.7), and anti-CD4 (RM4-5); and biotinconjugated 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).
LLO91–99-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⬘-GGAATCCTGT
GGCATCCATGAAAC-3⬘; antisense, 5⬘-TAAAACGCAGCTCAGTAA
CAGTCCG-3⬘; CCR7 sense, 5⬘-GAGATGCTCACTGGTCAGTG-3⬘;
antisense, 5⬘-CTACGGGGAGAAGGTTGTGG-3⬘; CXCR3 sense, 5⬘CAACATCAACTTCTATGCAG-3⬘; antisense, 5⬘-AGGATATGGGCAT
AGCAGTA-3⬘; Bcl-2 sense, 5⬘-TGGCCTTCTTTGAGTTCGGT-3⬘; antisense, 5⬘-AGCCTCCGTTATCCTGGATC-3⬘; Bcl-XL sense, 5⬘-CCGGA
GAGCGTTCAGTGATC-3⬘; antisense, 5⬘-TCAGGAACCAGCGGTTGA
AG-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⬘-TTCTG
CATCCTGTCAGCAAT-3⬘; CCR7, 5⬘-CGCCGATGAAGGCATACA
AG-3⬘; CXCR3, 5⬘-CTCACCTGCATAGTTGTATG-3⬘; Bcl-2, 5⬘-CCG
GTTCAGGTACTCAGTCA-3⬘; Bcl-XL, 5⬘-CTGCATCTCCTTGTCTAC
GC-3⬘; and FLIP, 5⬘-CTAAGGAATGTAAGTAGGGA-3⬘.
Microorganism
Generation of H2-Kd 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 LLO91–99 (29) or the
Janus kinase (JAK)1 self-peptide (30) to generate monomeric, soluble H2Kd-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 biotinconjugated mAbs, cells were stained with CyChrome or allophycocyaninconjugated streptavidin. For staining of epitope-specific CD8⫹ T cells
using tetrameric H2-Kd-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-Kd/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 ␮g/ml brefeldin A (SigmaAldrich), 5 ␮g/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 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 ␮l of FACS lysing
solution (BD Biosciences) for 10 min at room temperature and were then
washed again, resuspended in 500 ␮l 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-Ad, IgM, and biotinylated anti-DX-5,
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
Kinetics of LLO91–99-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 ⫻ 105 CFU of L. monocytogenes, tetrameric MHC molecule
folding with the LLO91–99 peptide, the immunodominant epitope
recognized by H2-Kd-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-Kd/LLO91–99
tetramers, whereas only a few CD8⫹ T cells in IL-15 Tg mice were
stained with H2-Kd/LLO91–99 tetramers on day 7 after infection.
The absolute numbers of H2-Kd/LLO91–99 peptide-positive CD8⫹
T cells in the splenocytes were 2.8 ⫾ 0.4 ⫻ 105 cells in non-Tg
mice and 2.3 ⫾ 0.9 ⫻ 105 cells in IL-15 Tg mice (Fig. 2). Thus,
the number of LLO91–99-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
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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.
-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 ⫻ 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 ⫻ 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 ⫻ 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 ⫻ BALB/c)F1 mice or naive
(C57BL/6 ⫻ BALB/c)F1 mice. After 6 wk, transferred Ly5.1⫹ T cells were
analyzed using a flow cytometer.
matched BALB/c (H-2d) mice were obtained from Japan SLC (Hamamatsu,
Japan). (IL-15 Tg ⫻ BALB/c)F1 mice, (C57BL/6 ⫻ BALB/c)F1 mice, and
B6-Ly5.1 mice (H-2b, 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.
1200
Ag-SPECIFIC MEMORY CD8⫹ T CELLS IN IL-15 Tg MICE
IL-15 in vivo does not augment the generation of CD8⫹ effector T
cells recognizing L. monocytogenes-specific epitope, but increases
the number of Ag-driven memory CD8⫹ T cells after L. monocytogenes infection.
Listeria-specific memory CD8⫹ T cells in IL-15 Tg mice
function to protect against L. monocytogenes infection
stage after infection because memory CD44⫹CD8⫹ T cells other
than those specific for LLO91–99 were markedly increased in IL-15
Tg mice at this stage.
In contrast, the number of LLO91–99-specific CD8⫹ T cells was
significantly higher on day 21 after infection than those in non-Tg
mice (Figs. 1 and 2; p ⬍ 0.05). Although the numbers declined by
day 40 after infection in both non-Tg mice and IL-15 Tg mice, it
remained at high in IL-15 Tg mice. Most of the LLO91–99-specific
CD8⫹ T cells on day 21 or 40 after L. monocytogenes infection
expressed CD122 and Ly6C but not activation markers such as
CD25 and CD62L, indicating that most are of memory phenotype
(data not shown). These results suggest that overexpression of
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. ⴱ, p ⬍ 0.05, significantly different from the value for
non-Tg mice.
Expression of antiapoptotic proteins in Ag-driven memory
CD8⫹ T cells in IL-15 Tg mice
Antiapoptotic molecules play a critical role in regulating cell survival and apoptosis of memory T cells (32). We next sorted
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 ⫻ 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
␮g/ml brefeldin A and 5 ␮g/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 CyChromeconjugated 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.
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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 ⫻ 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.
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 LLO91–99, 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 LLO91–99 in vitro was consistent with that of CD8⫹ T cells
directly stained with H2-Kd/LLO91–99 tetramers in non-Tg or
IL-15 Tg mice, respectively (Figs. 1 and 3).
To elucidate the protective role of CD8⫹ T cells in IL-15 Tg
mice, adoptive transfer experiments were conducted in normal
mice using splenic CD8⫹ T cells from IL-15 Tg mice or non-Tg
mice infected with L. monocytogenes 21 days previously. The
CD8⫹ T cells from infected IL-15 Tg mice conferred a higher level
of protection on normal mice against a lethal challenge with L.
monocytogenes compared with those from the infected non-Tg
mice ( p ⬍ 0.01, Fig. 4). These results suggest that the Ag-specific
CD8⫹ Tc1 cells that increase in IL-15 Tg mice following L. monocytogenes exposure can serve to protect against a challenge with L.
monocytogenes.
The Journal of Immunology
LLO91–99-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 LLO91–99specific 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
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.
LLO91–99-specific CD44⫹CD8⫹ T cells on day 7 or 21, respectively. CXCR3, which is expressed by Th1/Tc1 cells (34), was
expressed by both LLO91–99-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 LLO91–99 stimulation. Notably, LLO91–99-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).
To confirm the expression of Bcl-2 at protein level, we stained
Bcl-2 in LLO91–99-specific CD8⫹ T cells after L. monocytogenes
infection. As shown in Fig. 6, expression of Bcl-2 in the LLO91–99
⫹
CD8⫹ T cells of IL-15 Tg mice was higher on days 7 and 9 after
infection compared with that of non-Tg mice. These results suggest that increased Bcl-2 expression plays a role in increase in
number of LLO91–99-specific memory CD8⫹ T cells in vivo in
IL-15 Tg mice following L. monocytogenes infection.
Cell division of memory CD8⫹ T cells in IL-15 Tg mice
The number of memory T cells is maintained by a balance among
cell survival, apoptosis, and proliferation (1– 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.
Discussion
Naive CD8⫹ T cells in the periphery proliferate and differentiate
into effector cells upon TCR engagement with microbial peptideMHC class I complex when microbes invade the body (31). After
the battle against microbes has been won, most of the CD8⫹ effector T cells die by apoptosis due to activation-induced death
and/or withdrawal of growth factors (1). A few cells that escape
apoptosis differentiate linearly into memory-type CD8⫹ T cells,
although the differentiation of effector and memory cells along
separate lineages is not completely precluded (2– 4). It is now
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.
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FIGURE 4. Protection against a challenge with a lethal dose of L.
monocytogenes in mice in which CD8⫹ T cells from L. monocytogenesinfected IL-15 Tg mice had been transferred. CD8⫹ T cells (1 ⫻ 107) from
non-Tg or IL-15 Tg mice infected with 1 ⫻ 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 ⫻ 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. ⴱ, p ⬍ 0.05, significant different
between the values for non-TgCD83 BALB/c and these for immune
non-Tg CD83 BALB/c. ⴱⴱ, p ⬍ 0.01, significant different between the
values for Tg CD83 BALB/c and those for immune Tg CD83 BALB/c or
the values for immune non-Tg CD83 BALB/c and those for immune Tg
CD83 BALB/c.
1201
Ag-SPECIFIC MEMORY CD8⫹ T CELLS IN IL-15 Tg MICE
1202
widely accepted that Ag-driven memory CD8⫹ T cells are maintained independently of MHC class I (10, 11). Therefore, the survival and proliferation are driven by Ag-independent factors such
as cytokines. In this study, we show evidence of an increase in the
number of Ag-driven memory CD8⫹ T cells following a microbial
infection in IL-15 Tg mice, which constitutively produce IL-15
protein in the serum (22). IL-15 may play an important role in the
long-term maintenance of Ag-driven memory CD8⫹ T cell in vivo.
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 LLO91–99-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 LLO91–99-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
Table I. Cell division of memory CD8⫹ T cells
% Cells at Each Division Number
Recipient Mice
Non-Tg
IL-15 Tg
a
b
c
0
63.91 ⫾ 1.89
48.88 ⫾ 2.80b
a
1
2
3
4
23.48 ⫾ 0.74
33.65 ⫾ 1.69b
6.15 ⫾ 1.13
11.98 ⫾ 2.06c
2.11 ⫾ 0.23
4.20 ⫾ 0.62b
1.17 ⫾ 0.14
2.01 ⫾ 0.25b
Values represent means and SD of three individual experiments.
p ⬍ 0.01 by Student’s t test compared with the values for non-Tg mice.
p ⬍ 0.05 by Student’s t test compared with the values for non-Tg mice.
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
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 ⫻ 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.
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 activationinduced 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 CalmetteGuérin 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 activationinduced apoptosis. Recent studies have provided several lines of
evidence for homeostatic proliferation of naive CD8⫹ T cells (5–
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– 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⫹
The Journal of Immunology
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.
1203
24.
25.
Acknowledgments
We thank Dr. K. Kishihara for providing B6-Ly5.1 mice and
K. Itano and A. Nishikawa for their excellent technical assistance.
26.
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