Profound Enhancement of the IL-12/IL-18
Pathway of IFN- γ Secretion in Human CD8+
Memory T Cell Subsets via IL-15
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J Immunol 2007; 178:4786-4792; ;
doi: 10.4049/jimmunol.178.8.4786
http://www.jimmunol.org/content/178/8/4786
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References
Ronald B. Smeltz
The Journal of Immunology
Profound Enhancement of the IL-12/IL-18 Pathway of IFN-␥
Secretion in Human CD8ⴙ Memory T Cell Subsets via IL-151
Ronald B. Smeltz2
uman memory CD8⫹ T cell subsets with distinct phenotypes and effector functions have been characterized,
and these T cell subsets are referred to as central memory (TCM)3 cells (CD62LhighCCR7highCD45RAlow) and effector
memory (TEM) cells (CD62LlowCCR7lowCD45RAlow) (1, 2). A
second population of CD45RA⫹ T effector memory cells (TEMRA)
(CD62LlowCCR7lowCD45RAhigh), is typically observed only
among CD8⫹ T cells, and the frequency of TEMRA cells increases
with viral infections, homeostatic proliferation, and tumor responses (3–9). Functionally, CD8⫹ TEM and TEMRA cells demonstrate rapid effector functions such as cytokine production (i.e.,
IFN-␥) and cytotoxicity (1, 2). Although human TCM cells do not
exhibit equivalent effector functions, they possess greater proliferative capacity and IL-2 production. Further understanding of the
functional properties of these memory T cell subsets is necessary
for vaccine development. Evaluation of effector functions (i.e.,
IFN-␥) is typically assayed after TCR stimulation. However, T
cells also secrete IFN-␥ in a TCR-independent manner after stim-
H
Department of Microbiology and Immunology, Medical College of Virginia, Virginia
Commonwealth University, Richmond, VA 23298
Received for publication October 16, 2006. Accepted for publication January
26, 2007.
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 by funds provided by Virginia Commonwealth University
and Department of Microbiology and Immunology, as well as a grant from the
Thomas F. and Kate Miller Jeffress Memorial Trust.
2
Address correspondence and reprint requests to Dr. Ronald B. Smeltz, Department
of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980678, Richmond, VA 23298. E-mail address:
[email protected]
3
Abbreviations used in this paper: TCM, central memory T cell; TEM, effector memory T cell; TEMRA, CD45RA⫹ T effector memory; ␥c, common ␥-chain; CD62L,
CD62 ligand.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
ulation with the proinflammatory cytokines IL-12 and IL-18 (10 –
13). This TCR-independent, cytokine-dependent pathway of
IFN-␥ production results from the synergistic actions of IL-12 and
IL-18 (13) and is an important aspect of chronic inflammation, as
well as the early innate immune response of CD8⫹ T cells before
the development of Ag-specific immunity (14 –16).
IL-15, a member of the common ␥ chain (␥c) family of cytokines, is important for the development and maintenance of CD8⫹
T cell memory (17–19). Paradoxically, studies have also shown
that IL-15 induces CD8⫹ T cell differentiation and effector functions (20 –24). For example, the increased availability of IL-15 and
IL-7 after total body irradiation/lymphodepletion (due to the removal of cytokine “sinks”) results in increased effector functions
of adoptively transferred antitumor CTLs. Thus, the role of IL-15
is not limited to the development of memory. In this study, we
determined whether IL-15 impacted the IL-12-IL-18 pathway of
IFN-␥ secretion in human CD8⫹ T cell subsets. We report that
both IL-15 and IL-7 significantly increase the number of IFN-␥⫹
CD8⫹ T cells, as well as IFN-␥ secretion, obtained with IL-12IL-18 stimulation. This result is in contrast to stimulation with
IL-15 or IL-7 alone, which failed to induce significant IFN-␥.
The effect was mediated mostly by IL-15, with no synergy between IL-15 and IL-7. We also report that the ␥c cytokines
IL-15 and IL-7 increased sensitivity to IL-12-IL-18 stimulation
by reducing the concentrations of IL-12-IL-18 required to induce IFN-␥. Enhanced IL-12-IL-18-induced IFN-␥ was a functional property of terminally differentiated TEM/TEMRA cells,
and down-regulation of CD62 ligand (CD62L) expression by
TCM cells correlated with the acquisition of IL-12-IL-18-induced IFN-␥ production. We propose that in addition to promoting the development of T cell memory, IL-15 synergizes
with inflammation to enhance CD8⫹ T cell effector functions.
These results have important implications in clinically relevant
situations in which ␥c cytokines and concomitant inflammation
may synergize to enhance T cell functions in vivo.
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Human memory CD8ⴙ T cell subsets, termed central memory and effector memory T cells, can be identified by expression of
CD45RA, CD62 ligand (CD62L), and CCR7. Accordingly, functional differences have been described for each subset, reflecting
unique roles in immunological memory. The common ␥-chain cytokines IL-15 and IL-7 have been shown to induce proliferation
and differentiation of human CD8ⴙ T cell subsets, as well as increased effector functions (i.e., cytokines, cytotoxicity). In this study,
we observed that addition of IL-15 or IL-7 to cultures of human CD8ⴙ T cells profoundly enhanced the IL-12-IL-18 pathway of
IFN-␥ production. Importantly, IL-15 and IL-7 lowered the threshold concentrations of IL-12 and IL-18 required for induction
of IFN-␥ by 100-fold. Comparison of IL-15 and IL-7 demonstrated that IL-15 was superior in its ability to enhance IL-12-IL18-induced IFN-␥, without evidence of a synergistic effect between IL-15 and IL-7. We also observed that IL-15- and IL-7mediated enhancement of IL-12-IL-18-induced IFN-␥ production was a functional property of effector memory CD8ⴙ T cells.
Despite a lack of association between cell division and acquisition of IL-12-IL-18-induced IFN-␥, down-regulation of CD62L
expression correlated well with increased IL-12-IL-18-induced IFN-␥. Purified central memory T cells stimulated with IL-15 and
IL-7 down-regulated CD62L and acquired potent IL-12-IL-18-induced IFN-␥ similar to effector memory T cells. Thus, in addition
to its known role in development of T cell memory, IL-15 may amplify memory CD8ⴙ T cell effector functions by increasing
sensitivity to proinflammatory cytokine stimulation. The Journal of Immunology, 2007, 178: 4786 – 4792.
The Journal of Immunology
4787
Materials and Methods
Isolation of PBMC and purification of CD8⫹ T cells
Buffy coats from healthy human donors were provided by Virginia Blood
Services (Richmond, VA). PBMC were obtained by Ficoll-Paque separation, followed by repeated washes with HBSS. PBMC were cultured overnight in tissue culture flasks at 37°C in RPMI 1640 medium containing
10% FBS before functional assays (complete RPMI 1640). For some experiments, CD8⫹ T cells were purified from nonadherent PBMC using the
human CD8⫹ T cell Negative Isolation kit/MACS (Miltenyi Biotec) and
LS columns according to the manufacturer’s instructions. Cell purities
were ⬎90% CD8⫹CD3⫹CD56⫺.
CFSE labeling
⫹
Subsequent to column purification, CD8 T cells were labeled with CFSE
by incubating T cells at 50 million cells/ml in 5 ␮M CFSE/HBSS for 5 min
at room temperature. Labeling was quenched by addition of an equal volume of neat FBS, followed by three washes in FBS-containing HBSS.
Trypan blue exclusion was used to confirm the viability of the T cells after
CFSE labeling.
PBMC were cultured in 24-well plates at 2 million cells per well. Alternatively, purified CFSE-labeled CD8⫹ T cells were cultured at 2 million
cells/ml. Cytokine-stimulated cultures received 25 ng/ml recombinant human IL-15 or IL-7, as well as various concentrations of recombinant human
IL-12 and IL-18 (R&D Systems). For anti-CD3-stimulated cells, wells
were precoated with 10 ␮g/ml anti-human CD3 Ab (HIT3; BioLegend) and
washed extensively before addition of cells. Alternatively, soluble antiCD3 was added to PBMC cultures at 0.5 ␮g/ml. Cells were cultured in
complete RPMI 1640 at 37°C for 24, 48, and 72 h before collection and
preparation for FACS analysis.
FACS analysis
Approximately 8 –10 h before cells were harvested for FACS, monensin
solution (BioLegend) was added to allow the accumulation and detection
of intracellular cytokine. Cultured cells were collected and washed with
PBS containing 2% FBS. Before addition of fluorescent Abs, cells were
incubated with purified anti-CD32 Ab (BioLegend) to reduce nonspecific
binding via FcRs. Cells were sequentially incubated with FITC anti-human
CD62L (BD Pharmingen), PE anti-human CD45RA (BioLegend), energycoupled dye anti-human CD8␣ (Beckman Coulter), and either PE-Cy7 antihuman CCR7 or PE-Cy7 anti-human CD3 (BD Pharmingen). Cells were
washed with PBS/FBS to remove unbound Ab. For intracellular cytokine
staining, fluorescently labeled cells were first fixed with 4% formaldehyde.
Fixed cells were washed and permeabilized using 1⫻ Perm/Wash buffer
(BioLegend), then incubated with allophycocyanin anti-human IFN-␥ or an
appropriate isotype control diluted in Perm/Wash buffer (BioLegend).
Cells were washed and finally resuspended in PBS/FBS. Five-color FACS
analysis of CD3⫹CD8␣high cells was performed on the Beckman Coulter
FC500 instrument with data analysis using the CXP software package.
FACS sorting of memory CD8⫹ T cell populations
After overnight adherence and purification of CD8⫹ T cells by MACS,
CD8⫹ T cells were incubated with purified anti-CD32 Ab/PBS/2% FBS to
block nonspecific binding to FcRs. Cells were then stained with antiCD62L FITC, anti-CD8 PE, and anti-CD45RA allophycocyanin (BD
Pharmingen) Abs in PBS/2% FBS on ice for 30 min. Cells were washed
twice with cold PBS/2% FBS and then resuspended in complete medium
for FACS sorting. High-speed, single cell FACS sorting was performed at
the National Institutes of Health (Bethesda, MD) on a BD FACSAria
equipped with a four-way sorter, in which naive (CD62L⫹CD45RA⫹),
TCM (CD62L⫹CD45RA⫺), TEM (CD62L⫺CD45RA⫺), and TEMRA
(CD62L⫺, CD45RA⫹) subsets were sorted to ⬎98% purity. Sorted cells
were allowed to rest overnight in complete RPMI 1640 medium before
culture with cytokines.
Statistical analysis
One-way ANOVA, as well as Tukey-Kramer multiple comparison posttest,
were performed using data from all donors analyzed by GraphPad InStat
version 3.0a for Macintosh (www.graphpad.com). A value of p ⬍ 0.01 was
considered significant.
Results
Effect of ␥c cytokines IL-15-IL-7 on the IL-12-IL-18 pathway of
IFN-␥ production
Previous studies showed that culture of human CD8⫹ T cells with
IL-15 and IL-7 induced proliferation and acquisition of effector
functions (6, 25). To evaluate function with respect to IL-12-IL18-induced IFN-␥, we measured the ex vivo response of CD8⫹ T
cells to these cytokines, as well as the effect of IL-15 and IL-7 on
this pathway. We routinely observed a low but detectable number
of IFN-␥⫹ CD8⫹ T cells after stimulation with IL-12 and IL-18
compared with the number of cells found from stimulation with
medium alone. However, this difference was not statistically significant. Stimulation with IL-15 and IL-7 failed to induce a significant number of IFN-␥⫹ T cells relative to medium, which is in
contrast to reports that IL-15 can directly induce IFN-␥ (26). However, the addition of IL-15 and IL-7 to IL-12-IL-18-stimulated
cultures (referred to as Quadra stimulation) profoundly increased
the number of IFN-␥⫹ cells (Fig. 1). T cells expressing IFN-␥ after
anti-CD3 stimulation were used as a comparison. Among six donors tested, 45– 85% of CD8␣highCD3⫹CD56⫺ T cells were
IFN-␥⫹ after addition of IL-15 and IL-7 to cultures ( p ⬍ 0.001 for
Quadra stimulation relative to medium) (Fig. 1).
The ␥c cytokines IL-15 and IL-7 lower the threshold required
for IL-12-IL-18-induced IFN-␥ production
Due to the significant increase in the number of IFN-␥⫹ CD8⫹ T
cells with the addition of IL-15-IL-7 to IL-12-IL-18-stimulated
cultures, we performed titration experiments to determine whether
IL-15-IL-7 lowered the concentrations of IL-12 and IL-18 required
to induce IFN-␥. Indeed, after 48 h of stimulation, we observed
that addition of IL-15-IL-7 to cultures reduced the threshold required for IL-12-IL-18-induced IFN-␥ by 2 logs (Fig. 2). Addition
of 10 ng/ml IL-12 and 50 ng/ml IL-18 induced a modest increase
in the number of IFN-␥⫹ CD8⫹ T cells, and this response was
undetectable at lower concentrations of IL-12-IL-18. Conversely,
stimulation with IL-15-IL-7 did not induce a significant number of
IFN-␥-producing CD8⫹ T cells. However, addition of IL-15-IL-7
lowered the concentrations of IL-12 and IL-18 required to achieve
a significant IFN-␥ response to as low as 0.1 ng/ml IL-12 and 1
ng/ml IL-18 (Fig. 2c). Data summary obtained from six healthy
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Functional assays
FIGURE 1. The ␥c cytokines IL-15 and IL-7 profoundly enhance the
IL-12-IL-18 pathway of IFN-␥ production in human CD8⫹ T cells. PBMC
were cultured for 48 h in complete medium, IL-15-IL-7 (25 ng/ml each),
IL-12-IL-18 (10 and 50 ng/ml, respectively), both IL-15-IL-7 and IL-12IL-18 (Quadra stimulation), or anti-CD3. Cells were stained for surface
CD8 and intracellular IFN-␥ expression and analyzed by FACS. Data from
six healthy donors were compiled and analyzed using one-way ANOVA
with Tukey-Kramer multiple comparison posttest (InStat3 software). Data
represent the mean percentage of CD8⫹ T cells that are IFN-␥⫹ based on
intracellular cytokine secretion detection, and error bars show SD among
donors indicated.
4788
HUMAN MEMORY CD8⫹ T CELLS AND IL-15
IL-15-IL-7-mediated enhancement of IFN-␥ production is a
functional property of CD8⫹ TEM/TEMRA subsets
FIGURE 2. IL-15 and IL-7 lower the threshold required for IL-12-IL18-induced IFN-␥ production from peripheral blood CD8⫹ T cells. PBMC
from healthy human donors were cultured for 48 h with medium alone,
IL-12-IL-18 at decreasing concentrations (10/50 ng/ml IL-12-IL-18, respectively (a), and 1/10 ng/ml, 0.1/1 ng/ml (c)), IL-15-IL-7 alone (25
ng/ml each), or IL-15-IL-7 and decreasing concentrations of IL-12 and
IL-18 (Quadra stimulation). Cells were harvested and analyzed for intracellular IFN-␥ production by FACS. The percentage represents IFN-␥⫹
cells among gated CD8⫹ T cells. Data from six healthy donors were compiled and analyzed using one-way ANOVA with Tukey-Kramer multiple
comparison posttest (InStat3 software). Data represent the mean percentage
of CD8⫹ T cells that are IFN-␥⫹ based on intracellular cytokine secretion
detection, and error bars represent SD among donors indicated. A value for
p indicates the significance of cytokine stimulation compared with medium
alone (no stimulation).
donors is shown in Fig. 2 in which 10 ng/ml ( p ⬍ 0.001) (Fig. 2a),
1 ng/ml (Fig. 2b), and 0.1 ng/ml (Fig. 2c) concentrations of IL-12
were tested relative to medium. These data demonstrate that IL15-IL-7 not only increase IL-12-IL-18-induced IFN-␥ production,
but also lower the threshold concentrations required to induce
IFN-␥. Increased responsiveness to IL-12 stimulation may be an
important function of IL-15 in promoting T cell effector functions.
Enhancement of the IL-12-IL-18 pathway of IFN-␥ production is
mediated mainly by IL-15
Data shown in Figs. 1 and 2 were obtained from cultures in which
both IL-15 and IL-7 were included, based on previous research that
showed functional effects of these cytokines on CD8⫹ T cells (i.e.,
proliferation) (6). Because of the magnitude of increase in the
number of IFN-␥⫹ cells, we wanted to determine whether IL-15 or
IL-7 differed with respect to potency in enhancing IL-12-IL-18-
Previous experiments showed that the ␥c cytokines IL-15 and IL-7
could enhance the IL-12-IL-18 pathway of IFN-␥ production in
bulk CD8⫹ T cells. Because human memory CD8⫹ T cell subsets
with different functional capacities have been described, we
wanted to determine whether memory CD8⫹ T cells differed in
their response to these cytokines. We used CD45RA vs CD62L
expression to analyze IFN-␥ production in naive, TCM, TEM, and
TEMRA subsets after 48 h stimulation with either medium, immobilized anti-CD3, IL-12-IL-18, or IL-12-IL-18 and IL-15-IL-7
(Quadra stimulation). With anti-CD3 stimulation, a significant percentage of CD8⫹ TEM were IFN-␥⫹ (Fig. 4c), consistent with
published data (2). In contrast, a significant number of TEMRA
were IFN-␥⫹ after IL-12-IL-18 stimulation (Fig. 4a; 12.5%) in one
representative donor. Importantly, we did not detect any significant
ex vivo response of TCM to IL-12-IL-18 stimulation (Fig. 4a). As
expected, the addition of IL-15-IL-7 had a profound effect on the
overall number of IFN-␥⫹ cells. However, the effect was most
evident in TEM and TEMRA subsets (Fig. 4b; 58 and 74%, respectively). In contrast, TCM did not show a similar increase in IFN-␥
production after 48 h. This is consistent with reports that TCM
exhibit low TCR-induced effector functions ex vivo (1, 27). Thus,
these data show that TEMRA possess IL-12-IL-18-induced IFN-␥
production ex vivo, and that IL-15-IL-7 enhances this production
in both TEM and TEMRA subsets.
Roles of proliferation and differentiation in acquisition
of IL-12-IL-18-induced IFN-␥ production
We failed to detect significant IL-12-IL-18-induced IFN-␥, or IL15-IL-7-mediated enhancement of this pathway among CD8⫹ TCM
after 48 h of stimulation. However, the ␥c cytokines IL-15 and
IL-7 are known to induce proliferation and subsequent differentiation of memory CD8⫹ T cell subsets. Therefore we wanted to
determine whether there was a correlation between IL-15-IL-7induced proliferation, differentiation, and acquisition of IL-12-IL18-induced IFN-␥ production. CD8⫹ T cells were purified by negative selection and subsequently labeled with CFSE. Labeled
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induced IFN-␥; or alternatively, whether there were synergistic
effects between IL-15 and IL-7. For these experiments, we cultured
PBMC with IL-12-IL-18 in the presence of either IL-15 (Tri-15) or
IL-7 (Tri-7) for 72 h. Again, culture with IL-12-IL-18 (no IL-15 or
IL-7) induced a small but significant number of IFN-␥⫹ CD8⫹ T
cells (Fig. 3a) in three individual donors shown. Similar to the
combination of IL-15 and IL-7 (Figs. 1 and 2), culture with either
IL-15 or IL-7 did not induce significant IFN-␥ production from
CD8⫹ T cells in the absence of proinflammatory cytokines (Fig.
3a). Addition of either IL-15 (Tri-15) or IL-7 (Tri-7) to IL-12-IL18-stimulated cultures significantly enhanced IFN-␥ production.
However, we found that at equivalent concentrations IL-15 was
superior to IL-7 based on the number of IFN-␥⫹ cells, which was
on average 50% more than Tri-7-stimulated cultures. Importantly,
there was no synergistic effect between IL-15 and IL-7, as the
number of IFN-␥⫹ CD8⫹ T cells obtained with Tri-15 stimulation
was equivalent to the number obtained with Quadra stimulation. A
summary of the data obtained from five healthy donors is shown
Fig. 3b ( p ⬍ 0.001 for Quadra and Tri-15 stimulation; p ⬍ 0.05 for
Tri-7 stimulation relative to medium; p ⬍ 0.05 for Tri-15 vs Tri-7
stimulation). There was no statistical difference between Tri-15
and Quadra stimulation. Therefore the effect of IL-15-IL-7 on the
enhancement of the IL-12-IL-18 pathway of IFN-␥ production is
primarily due to IL-15.
The Journal of Immunology
4789
FIGURE 3. Potent effect of IL-15 on enhancement of IL-12-IL-18-induced IFN-␥ production. a, Human PBMC were cultured for 72 h in the
presence of medium alone, IL-12-IL-18 (10 and 50 ng/ml, respectively),
IL-15 only, IL-7 only, IL-15-IL-7, or IL-12-IL-18 with IL-15 (Tri-15 stimulation), IL-7 (Tri-7 stimulation), or both IL-15 and IL-7 (Quadra stimulation). Cells were harvested and analyzed for intracellular IFN-␥ production by FACS. Percentage of IFN-␥⫹ cells among gated CD8⫹ T cells is
presented. Data from three individual donors are shown. b, The results of
five donors are shown. Data represent the mean percentage of CD8⫹ T cells
CD8⫹ T cells were cultured for 72 h with IL-15-IL-7 in the presence or absence of IL-12-IL-18. As shown in Fig. 5, a and b, a
small but significant number of CD8⫹ T cells divided in response
to cytokine stimulation (3.9% medium control vs 18.1% with
Quadra stimulation). We expected greater proliferation based on
previous reports that showed extensive CD8⫹ T cell proliferation
in response to IL-15. We suspected that the 7-day culture period
used in these studies explained the observed differences. To confirm this suspicion, we measured CD8⫹ T cell proliferation after
culture with cytokines for 2, 3, and 6 days. Indeed, we found a
significant difference in the number of cells that had divided after
6 days as opposed to 3 days (Fig. 5, b and c; 18.1 vs 81.8%).
However, IL-15-mediated enhancement of IL-12-IL-18-induced
IFN-␥ was evident as early as 48 h of stimulation, despite limited
proliferation. Therefore we performed experiments to simultaneously measure CFSE dilution (as a function of cell division/
proliferation) and expression of CD62L (as a measure of CD8⫹ T
cell differentiation). As shown in Fig. 5d (a donor different from
donors found in Fig. 5, a– c), purified CD8⫹ T cells cultured with
medium alone exhibited minimal proliferation (3.2%). Gating on
either CD62L⫹ (Fig. 5e) or CD62L⫺ (Fig. 5f) CD8⫹ T cell populations showed no IFN-␥ expression, as expected for unstimulated cells. With Quadra stimulation, a significant increase was
observed in the number of divided cells, and almost all of the
divided cells were CD62L⫺ (24.5% of total CD8⫹ T cells, 95% of
divided CD8⫹ T cells) (Fig. 5g). Although the relative number of
CD62L⫹ CD8⫹ T cells observed between unstimulated and stimulated cultures was comparable, there was a substantial decrease in
the mean fluorescence intensity of CD62L expression in the
CD62L⫹ population, suggesting a transition from CD62L⫹ to
that are IFN-␥⫹ based on intracellular cytokine secretion detection, and
error bars represent SD among donors indicated. A value for p indicates the
significance of cytokine stimulation compared with medium alone (no
stimulation), as well as a comparison between Quadra, Tri-15, and Tri-7
cytokine stimulations.
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FIGURE 4. IL-12-IL-18-induced IFN-␥ in CD8⫹ memory T cell subsets.
Human PBMC were cultured with IL-12-IL-18 (10 and 50 ng/ml, respectively)
(a) in addition to IL-15 and IL-7 (b), or with immobilized anti-CD3 only (c)
for 48 h. Cells were harvested and stained for FACS. Percentages (upper right
quadrant) represent the IFN-␥⫹ naive (defined as CD62LhighCD45RAhigh),
TCM (CD62LhighCD45RAlow), TEM (CD62LlowCD45RAlow), and TEMRA
(CD62LlowCD45RAhigh) CD8␣highCD3⫹ T cells. Results of one donor of four
are represented.
4790
HUMAN MEMORY CD8⫹ T CELLS AND IL-15
data show that cell division is not required for IL-15-IL-7 to enhance or induce the IL-12-IL-18 pathway of IFN-␥ production in
memory CD8⫹ T cells. However, cell division could enhance
IFN-␥ production as the number of IFN-␥high cells increased
among divided CD62L⫺ cells relative to nondivided CD62L⫺
cells (data not shown). Furthermore, these data suggest that ␥c
cytokine-induced differentiation of TCM is accompanied by induced responsiveness to IL-12-IL-18 stimulation.
Functional responses of FACS-sorted memory CD8⫹ T cells
CD62L⫺ as the result of IL-15-driven differentiation. When these
two subsets were analyzed for IFN-␥ production, we found that
both CD62L⫹ and CD62L⫺ CD8⫹ T cells were IFN-␥⫹ after 72 h
of culture, regardless of whether the cells had divided or not (Fig.
5, d and g). This is in contrast to what was observed after 48 h of
culture, when TCM did not yet express IFN-␥ (Fig. 4). Nevertheless, only CD62L⫺ T cells expressed IFN-␥high and IFN-␥low (Fig.
5i), whereas CD62L⫹ cells were solely IFN-␥low (Fig. 5h). These
FIGURE 6. Functional responsiveness of
FACS-purified memory CD8⫹ T cell subsets. Column-purified CD8⫹ T cells were stained with fluorescent Abs to CD62L and CD45RA, then
FACS-sorted into individual TCM (CD62Lhigh
CD45RAlow), TEM (CD62LlowCD45RAlow), and
TEMRA (CD62LlowCD45RAhigh) subsets using a
BD FACSAria instrument. Memory subsets were
rested overnight in complete medium, then cultured with medium, IL-12-IL-18 (10 and 50 ng/ml,
respectively), or Quadra stimulation. Supernatants
were harvested after 72 h and tested for IFN-␥ by
ELISA. Results for four healthy human donors are
shown. NT, Not tested.
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FIGURE 5. Lack of correlation between cell division and IL-15-IL-7induced functional responsiveness to IL-12-IL-18. Human CD8⫹ T cells
were purified from PBMC by negative selection and labeled with CFSE.
Cells were cultured in complete medium (a, d, e, and f) or with Quadra
stimulation (b, c, g, h, and i) for either 72 h (a, b, and d–i) or 6 days (c) and
subsequently stained for surface CD62L expression (d and g) and intracellular IFN-␥ production (e, f, h, and i). Results of one representative
donor are shown.
The data presented in Fig. 5 suggest that as TCM differentiate into
TEM/TEMRA over time (based on the down-regulation of CD62L
expression), they acquire responsiveness to IL-12-IL-18 stimulation, However, to definitely show that these are the same cells
undergoing differentiation, we purified CD8⫹ memory T cell subsets before in vitro culture. FACS-purified TCM, TEM, and TEMRA
subsets were cultured for 72 h (postsort) with medium, IL-12-IL18, IL-15-IL-7, or Quadra stimulation. Cells cultured with IL-15IL-7 alone did not secrete significant quantities of IFN-␥ (⬍40
pg/ml; data not shown), consistent with data obtained by intracellular cytokine detection. As expected, culture of TEMRA and TEM
with IL-12-IL-18 resulted in a mean of 1084 and 452 pg/ml IFN-␥
production, respectively (Fig. 6), as shown in four donors studies.
However, addition of IL-15-IL-7 to these cultures profoundly increased IFN-␥ secretion (⬎100,000 pg/ml). Although TCM secreted low amounts of IFN-␥ with IL-12/-IL-18 stimulation, the
addition of IL-15-IL-7 enhanced IFN-␥ production to levels that
were comparable to those of TEM and TEMRA (mean, 97,460 pg/
ml). Therefore, prolonged culture of TCM with IL-15-IL-7 induced
functional responses to IL-12-IL-18 that were similar to those of
TEM and TEMRA. To determine whether TCM resembled TEM/
TEMRA phenotypically after the 3-day culture, we analyzed sorted
TCM by FACS for expression of CD62L and CD45RA. TCM remained negative for CD45RA expression after Quadra stimulation.
Despite an increase in the number of CD62L⫺ TCM, a majority
remained CD62L⫹. Importantly however, the mean fluorescence
intensity of CD62L expression of those cells that were CD62L⫹
had decreased by 50% (our unpublished observations), suggesting
that cells were actively undergoing differentiation. This observation is similar to that observed for bulk CD8⫹ T cells (Fig. 5).
Therefore, transition from TCM into TEM is accompanied by an
The Journal of Immunology
increase in IL-12-IL-18-mediated IFN-␥ secretion, although complete differentiation into TEM/TEMRA is not a prerequisite for IL12-IL-18 functional responsiveness.
Discussion
agent (see below). A comparison between IL-15 and IL-7 showed
that IL-15 was more potent in enhancing IFN-␥ production, and
there was no synergy between IL-15 and IL-7 (Fig. 3). This latter
result is in contrast to a report showing synergy between IL-15 and
IL-21 (which also uses the ␥c receptor) in TCR-induced IFN-␥
(22). Of particular importance and possible relevance in vivo, we
show that IL-15 and IL-7 decreased the concentrations of IL-12
and IL-18 required for induction of IFN-␥ by 100-fold (Fig. 2).
Although short-term culture of CD8⫹ T cells with IL-15/IL-7 enhanced IL-12-IL-18-induced IFN-␥ in TEM and TEMRA subsets,
prolonged culture with IL-15 and IL-7 (⬎72 h) generated both
IFN-␥high (CD62L⫹) and IFN-␥low (CD62L⫹ and CD62L⫺) populations (Fig. 5). This result is in apparent contrast to findings
published in another report (14), in which murine CD62L⫹ memory T cells expressed IFN-␥ after overnight culture with Leishmania-infected macrophages. It is difficult to reconcile these data because only the number of IFN-␥⫹ cells is shown in (14), not the
relative levels of IFN-␥ secretion among CD62L⫹ and CD62L⫺
cells. In addition, the use of IL-2 instead of IL-15 may change the
kinetics of appearance of IFN-␥ expression in CD62L⫹ cells. Alternatively, other signals generated in response to infection may
alter the immediate T cell response to cytokines. Finally, there
could be a fundamental difference between CD62L⫹ memory T
cells in mice and those found in humans, but we do not yet fully
understand those differences. Surprisingly, cell division was not
required for the appearance of IFN-␥lowCD62Lhigh cells (Fig. 5).
This suggests that memory CD8⫹ T cells, particularly the TCM
subset, can rapidly acquire effector functions before extensive proliferation. Furthermore, complete differentiation of CD62L⫹ TCM
into CD62L⫺ TEM/TEMRA is not a prerequisite for IL-12-IL-18induced IFN-␥. However, by day 3, TCM had significantly downregulated CD62L expression, indicating active differentiation and
transition into TEM/TEMRA. This emphasizes the point that analysis
of TCM should be performed by gating on only the CD62Lhigh cells
because inclusion of cells that are CD62L⫹ but express intermediate levels of CD62L may include cells in the TCM to TEM/TEMRA
transition. Therefore as T cells undergo differentiation from
CD62Lhigh TCM to CD62Llow TEM/TEMRA, they acquire functional
responsiveness to IL-12 and IL-18. Because cell division was not
required for acquisition of IL-12-IL-18-induced IFN-␥, we believe
IL-15-enhanced expression of IL-12 and IL-18 receptors may be
the mechanism for this enhancement. A recent report would support this conclusion (29). In addition, we (our unpublished observations) and others (30) have observed increased IL-18R␣ expression among TEM.
We believe these data to be highly relevant in the treatment of
tumors, where IL-2 and IL-12 have been shown to be efficacious
(31). These cytokines however, have toxicities primarily due to
vascular leak syndrome (32). Theoretically, substitution of IL-15
for IL-2 would permit lower therapeutic doses of IL-12 to be used,
yet still achieve potent T cell effector functions and antitumor
memory. The ␥c cytokines can enhance the effector functions of
tumor-specific CD8⫹ T cells when adoptively transferred into
lymphopenic hosts, and a majority of these effects are attributed to
increased availability of IL-15 and IL-7 due to the absence of cells
that act as cytokine sinks (24). However, irradiation/lymphodepletion can result in transient expression of proinflammatory cytokines, thus creating an environment suitable for increased T cell
effector function by the synergistic actions of ␥c cytokines and
inflammation (33). In addition to adoptive immunotherapy of tumors, IL-15 may play a critical role in the early innate immune
response to pathogens by amplifying the effects of proinflammatory cytokines. Numerous reports have demonstrated that memory
CD8⫹ T cells can secrete IFN-␥ in response to IL-12 and IL-18
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Members of the common ␥c family of cytokines, particularly IL-15
and IL-7, can have multiple effects on T cell function, including
the development and maintenance of memory. Paradoxically,
IL-15 has also been shown to promote differentiation and acquisition of effector functions, and has been implicated in enhancement of antitumor functions in vivo (21, 23). Although some studies suggest that IL-15 directly stimulates IFN-␥ production, this
remains a controversial issue. Culture of human CD8⫹ T cells with
IL-15 led to IFN-␥ production, but only after PMA/ionomycin
restimulation (25). Another study reported that IL-15 induced
IFN-␥ secretion from memory CD8⫹ T cells (26). However,
CD56⫹CD16⫺ cells (which includes NKT cells) were not depleted
from the starting cell population, and 5–10% of the cells were not
memory CD8⫹ T cells. Therefore it is possible that contaminating
NK/NKT cells secreted IFN-␥ when cultured with IL-15. Alternatively, the higher concentration of IL-15 used in (26) could have
induced IFN-␥. We found detection of intracellular cytokine secretion by FACS to be a valuable and necessary approach for studies presented in this experiment because we could gate specifically
on CD8␣highCD3⫹CD56⫺CD16⫺ T cells. Because of the possibility of a small number of contaminating cells in CD8⫹ columnpurified populations (which were 90 –95% pure), we only measured IFN-␥ in culture supernatants from FACS-sorted cells. A
study (28) in mice reported that IL-15 in itself could not directly
induce IFN-␥ secretion, which is in agreement with our observations. We hypothesize that IL-15 is necessary, but not sufficient to
induce CD8⫹ T cell effector functions. We propose that IL-15 (and
possibly other ␥c cytokines) synergize with proinflammatory cytokines (or TCR stimulation) to amplify effector functions, particularly IFN-␥ production. To test this hypothesis in vitro, we cultured CD8⫹ T cells with IL-12 and IL-18, two proinflammatory
cytokines strongly associated with the innate immune response and
chronic inflammation. IL-12 and IL-18 synergize to activate a potent TCR-independent pathway of IFN-␥ production via activation
of multiple elements of the human IFN-␥ promoter (13). This
TCR-independent pathway of IFN-␥ production has been well
documented for both CD4⫹ and CD8⫹ T cells derived from both
humans and mice. We observed that human CD8⫹ TEMRA cells
expressed IL-12-IL-18-dependent IFN-␥ production ex vivo, without any prior in vitro conditioning (i.e., TCR stimulation) (Fig. 4).
This result is in contrast to anti-CD3-induced IFN-␥ production,
which was mainly within TEM. Thus, TEMRA can be functionally
discriminated from TEM and TCM by their ex vivo response to
IL-12 and IL-18 stimulation. We confirmed our results obtained by
FACS analysis of intracellular IFN-␥ by testing culture supernatants of FACS-purified TEMRA (Fig. 6). Furthermore, we found
that addition of IL-15 or IL-7 or both had a significant enhancing
effect on the number of IFN-␥⫹ T cells induced by IL-12 and
IL-18 stimulation (Fig. 1). These results are in contrast to a published report (14) in which a modest effect was observed by addition of IL-15 to murine cultures. The reasons for this discrepancy
could reflect the overnight culture with cytokines, a difference between mouse splenic T cells and human peripheral blood T cells,
or a difference in the potency of IL-15-IL-15R␣ stimulation in
humans and mice. In support of the former, we also observed that
the ability of IL-15 to enhance IFN-␥ production was weak but
detectable at 24 h. Nevertheless, the parallels between the two
studies suggest an important conserved functional response that is
not insignificant, with recent efforts to use IL-15 as a therapeutic
4791
HUMAN MEMORY CD8⫹ T CELLS AND IL-15
4792
induced by bacterial/viral infection, before the development of Agspecific immunity (14, 34, 35). Therefore, IL-15, which is expressed in multiple tissues and whose expression may be linked to
signals generated through TLRs, may help to amplify the response
of T cells to proinflammatory cytokines before their peak expression (36 –39). Thus, the role of IL-15 may serve to not only promote CD8⫹ T cell memory, but also to amplify the responses to
early infection and chronic inflammation. In the case of infection
by a foreign pathogen, this role would be beneficial, whereas in the
case of autoimmunity it could be detrimental. Studies are underway to further characterize this amplification of CD8⫹ T cell
responses.
17.
18.
19.
20.
21.
Acknowledgments
I thank Dr. Dan Conrad (Virginia Commonwealth University) for helpful
discussion and careful reading of this manuscript. I also acknowledge Dr.
Kevin Holmes and Cal Eigsti at the National Institutes of Health for excellent technical assistance and helpful discussion. A special thank you to
Patricia, Corbin, and Makenna, without whom this work would not be
possible.
22.
23.
The author has no financial conflict of interest.
24.
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