A Programmed Switch from IL-15- to IL-2-Dependent
Activation in Human NK Cells1
Anne-He ´le `ne Pillet, Florence Bugault, Jacques The `ze, Lisa A. Chakrabarti, and Thierry Rose2
IL-2 and IL-15 differentially control the development, activation and proliferation of human NK cells, although they share common
signal-transducing receptor chains CD122 and common ?. To explore this issue, we analyzed in detail the kinetics of cytokine receptor
expression, cytokine binding, and signaling responses in human NK cells treated with common ?-chain family cytokines. We provide
evidence for the sequential expression of IL-15R? and IL-2R? at the surface of cytokine-stimulated human NK cells, independent of the
cytokine used for stimulation (IL-2, IL-15, or IL-7). Binding experiments confirmed the switch of high-affinity receptor from IL-15R to
IL-2R between 18 and 48 h after stimulation. Consequently, phospho-STAT5 signaling responses to IL-15 were efficient in human NK
cells pretreated with cytokines for 18 h, but were abolished at 48 h. Functional NK cell responses to IL-15, including IFN-? secretion
and CD107a expression, followed a similar pattern, indicating the physiological relevance of the cytokine receptor switch. Importantly,
IL-15 complexed to soluble IL-15R? preserved the capacity to activate cytokine-stimulated human NK cells at 48 h, suggesting that
human NK cells remained competent for IL-15 trans-presentation, while they had become refractory to free diffusible IL-15. These
findings define a common cytokine receptor expression program, which increases human NK cell sensitivity to free IL-15 in early
activation and redirects responses toward IL-2 and trans-presented IL-15 at later stages. Such a program may prevent excessive human
NK cell activation by effectors of innate immunity and regulate the transition between the innate and adaptive stages of immune
responses. The Journal of Immunology, 2009, 182: 6267–6277.
control is mediated through a potent cytotoxic activity against cells
bearing altered or foreign MHC molecules (1, 4). NK cells syn-
thesize and accumulate large amounts of cytolytic proteins such as
perforin and granzymes that can be released to induce the death of
target cells (5). In addition, NK cells modulate the initiation of
adaptive immune responses through cytokine/chemokine secretion
and direct interactions with APCs (6, 7). In particular, activated
NK cells secrete IFN-?, which plays a key role in triggering the
innate immune response against pathogens, in the activation and
maturation of colocalized monocytes and dendritic cells (DC),3
and in the early differentiation of a Th1-adaptive response after
Development, survival, proliferation, and effector functions of
NK cells are critically dependent on cytokines of the common
?-chain (?c) family (2, 9). In vitro studies indicate that IL-2, IL-15,
and IL-7 can all support NK cell activation (10). IL-15 is essential
for NK cell differentiation from T/NK progenitor cells (11) and for
NK cell function (2, 12–14). IL-2 also potentiates NK cell function
atural killer cells are large granular lymphocytes that
play a key role in innate immune responses against vi-
rus-infected cells and tumor cells (1–3). This immune
and has been used as an immunotherapeutic agent to promote NK
cell antitumor activity (9). IL-2 and IL-15 share a common inter-
mediate- affinity receptor formed by the association of two chains,
IL-2/IL-15R? (CD122) and the ?cor CD132), with a Kdin the
nanomolar range. Specificity is conferred by a third receptor chain,
IL-2R? (CD25) or IL-15R?, which together with CD122 and ?c
constitute the tripartite high-affinity receptor with a similar Kdof
12 pM for both cytokines, whereas the single chains IL-2R? and
IL-15R? bind their respective cytokine with very distinct Kdof 19
nM for IL-2 and 30 pM for IL-15 (10). The cytoplasmic domains
of CD122 and ?cbind intracellular signaling complexes, while the
private ? subunits of the receptors are not considered to play a
major role in signaling. IL2-R? has a short cytoplasmic domain of
13 residues and is not known to bind signaling molecules. IL-15R?
has a 43-residue cytoplasmic domain that can associate with the
TRAF2 and Syk signaling molecules, although these associations
remain of unknown significance (2, 15–17). Importantly, the pri-
vate subunits of the IL-2 and IL-15 receptors are key in conferring
receptiveness to their cognate cytokine, as exemplified by the very
similar phenotypes of mice with targeted inactivation of the IL-2 and
IL-2R? genes (lymphoproliferation) and of mice inactivated for the
IL-15 and IL-15R? genes (lack of NK cell development) (10).
A specific property of IL-15R? expressed at the surface of IL-15
producer cells, e.g., dendritic cells, monocytes, or stromal fibro-
blasts, is its capacity to bind IL-15 with high affinity in the absence
of the CD122 and ?creceptor chains (18). The IL-15/IL-15R?
complex at the surface of the producer cells can then be “trans-
presented” to a responder cell bearing the intermediate-affinity re-
ceptor, thus reconstituting a tripartite high-affinity receptor com-
plex (9, 17, 19–23). Trans-presentation results in an efficient and
long-lasting signal due to the tight intercellular junction and the
capacity of the IL-15/IL-15R? complex to recycle at the cell sur-
face after internalization (19, 24). IL-15 trans-presentation is par-
ticularly relevant during NK/dendritic cell (DC) interactions,
which link the innate and adaptive stages of immune responses
(23, 25). Indeed, NK cells activate or lyse DC depending on their
Institut Pasteur, De ´partment d’Infection et d’Epide ´miologie, Unite ´ d’Immuno-
ge ´ne ´tique Cellulaire, Paris, France
Received for publication June 13, 2008. Accepted for publication March 17, 2009.
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.
1This work was supported by the Institut Pasteur. A.-H.P. is the recipient of a fel-
lowship from the Ministe `re de l’Education Nationale et de la Recherche.
2Address correspondence and reprint requests to Dr. Thierry Rose, Unite ´
d’Immunoge ´ne ´tique Cellulaire, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris,
Cedex 15, France. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: DC, dendritic cell; ?c, common ?-chain; MFI,
mean fluorescence intensity.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
activation status, while in return DC activate NK cells and promote
their cytotoxic activity (6, 7). These studies have led to an increas-
ing appreciation of the role of NK cells in shaping the initiation of
DC-dependent adaptive responses.
IL-2 and IL-15 signal through three major pathways: Jak/
STAT, MAPKs, and PI3K/Akt (26–29). It should be noted that
at high cytokine concentrations, the intermediate-affinity recep-
tor recapitulates signaling events transduced through high-af-
finity receptors (9, 30). Activation of the Jak/STAT pathway
depends on conformational rearrangement of receptor chains,
leading to phosphorylation of receptor-associated Jak1 and Jak3
kinases, which in turn phosphorylate STAT5a and STAT5b and
to a lesser extent STAT1b and STAT3 transcription factors. The
phosphorylated STATs dimerize, diffuse into the nucleus, and
activate the transcription of multiple target genes (26). To date,
no difference has been characterized in NK cells between the
patterns of protein phosphorylation induced after IL-2 or IL-15
stimulation (31). However, these two cytokines clearly induce
different effects on NK cells (10, 32–34). IL-15 has mostly pro-
survival effects (35) and induces a stronger proliferative re-
sponse than IL-2 (36, 37). In contrast, IL-2 induces NK cell
apoptosis under some conditions in vivo (35). The molecular
basis for these functional differences still remains a matter of
debate (9, 32). Several mechanisms have been proposed to dif-
ferentially control IL-2 and IL-15 action, including cytokine
compartmentalization (10), differential expression of the high-
affinity receptors (9), and differential activation of cell growth
and survival pathways (38).
To address this issue, we investigated the kinetics of expression
of all of the chains comprising the IL-2 and IL-15 receptors, as
well as the binding characteristics and signaling responses of the
receptor complexes expressed by human NK cells upon activation.
We demonstrate a tight temporal regulation of IL-15R? and IL-
2R? expression in cytokine-stimulated NK cells, resulting in an
initial and transient responsiveness to free IL-15, which is replaced
at later stages by a dependency on IL-2 and trans-presented IL-15.
Thus, NK cells activation can initially be sustained by a variety of
IL-15-producing cells, but is restricted at a later stage to effectors
of the adaptive responses that produce IL-2 and/or trans-present
IL-15. This programmed switch in cytokine responsiveness may
have profound consequences on the regulation of NK cell activity
in innate and adaptive immune responses.
Materials and Methods
Surface expression of cytokine receptor chains in human NK
Venous blood was obtained from healthy volunteers through the Etablisse-
ment Francais du Sang, Centre Cabanel (Paris, France). PBMC were pu-
rified by density gradient centrifugation on Lymphoprep solution (Axis-
Schield). For cytokine activation, PBMC resuspended at 106/ml in RPMI
1640 medium (Cambrex) complemented with 5% FBS (complete medium)
in 24-well plates were treated with 5 nM IL-2 (Chiron-Novartis) or 2 nM
IL-15 (Abcys) and cultured for 12, 24, or 48 h in 5% CO2at 37°C. For flow
cytometry analysis with labeled Abs, PBMC were harvested and resus-
pended in 50 ?l of FACS buffer (PBS with 0.02% sodium azide and 5%
FBS) and labeled for 1 h at 4°C with Abs to CD56-allophycocyanin, CD3-
allophycocyanin-AF750 (eBioscience), CD122-PE (BD Biosciences), and
CD25-FITC (DakoCytomation). Alternatively, PBMC were labeled with
CD56-FITC, CD3-allophycocyanin-AF750 (eBioscience), CD132-PE (BD
Biosciences), and IL-15R? (goat IgG1; R&D Systems), with a donkey
anti-goat IgG-FITC Ab (R&D Systems) added as a secondary reagent,
CD127-PE (R&D Systems), and anti-?crat TUGh4-PE (BD Pharmingen).
Receptor expression was measured by flow cytometry on CD3?CD56?
NK cells on a Cyan LX cytometer (DakoCytomation). Data were acquired
with Summit version 4.1 software (DakoCytomation) and analyzed with
FlowJo version 8.3.3 software (Tree Star).
Purification of NK cells
CD3?CD56?NK cells were prepared from human PBMC by separation on
magnetic beads (NK purification kit; Miltenyi Biotec). The enriched NK
cell population contained ?95% CD3?CD56?cells. The recovered NK
cells were not activated as controlled by the absence of CD69 and CD25
Western blot analysis of mature and immature ?cchains
Purified NK cells were harvested, washed twice in PBS, and lysed in 0.5%
Triton X-100 buffer (50 mM Tris-HCl (pH7.4), 5 mM EGTA, 5 mM
EDTA, 30 mM NaF, 20 mM sodium pyrophosphate, 1 mM Na3VO4, 1 mM
PMSF, and 10 ?M leupeptin). Two hundred micrograms of proteins from
NK cell lysates was denatured for 5 min at 100°C in SDS-loading buffer
(62.5 mM Tris-HCl, 10% glycerol, 2% SDS, bromphenol blue, and 5%
2-ME). Proteins were separated by electrophoresis on an 8% SDS-poly-
acrylamide gel and transferred onto a Hybond-ECL nitrocellulose mem-
brane (Amersham Biosciences) overnight at 4°C. The membrane was
saturated with BSA, incubated with either anti-?crat TUGh4 (BD Pharm-
ingen) or goat anti-?cpolyclonal Abs (R&D Systems), and washed in TBS/
0.5% Tween 20 buffer before being incubated with HRP-coupled anti-rat or
anti-goat Abs (Amersham Biosciences). Proteins were then revealed by
ECL-plus Western blotting detection reagents (Amersham Biosciences).
125I-labeled IL-2- and IL-15-binding assays
Solutions of IL-2 (Chiron-Novartis) or IL-15 (Abcys) at 1 ?g/ml dissolved
in 100 ?l of 100 mM sodium phosphate buffer were radiolabeled with 10
?l of125I (0.5 mCi; MP Biomedicals) by the chloramine-T method (Riedel-
de-Hae ¨n) to a specific activity of 734 ? 106cpm/mg for IL-2 and 1920 ?
106cpm/mg for IL-15.
Purified NK cells were harvested and washed twice in cold 100 mM
glycine/150 mM NaCl buffer (pH 2.7) to release bound cytokines, then
washed twice in PBS with 0.02% sodium azide and 10 mg/ml BSA (pH
7.4) and centrifuged after each wash for 5 min at 4°C. Binding experiments
were conducted on 2–10 ? 106cells per experiment. NK cells resuspended
in a final volume of 80 ?l were incubated for 60 min in binding buffer (PBS
with 0.02% sodium azide and 10 mg/ml BSA) in the presence of various
amounts of125I-labeled IL-2 or IL-15 (from 1 pM to 250 nM). A 100-fold
molar excess of cold IL-2 or IL-15 was then added for 10 min at 4°C,
followed by 2 ml of cold binding buffer. Cell pellet and supernatant ra-
dioactivities were measured four times for 1 min on a multi-crystal LB
2111 gamma counter (Berthold Technologies). Kdvalues and the number
of binding sites were obtained by fitting the binding curves using Origin 6.0
software (Microcal) and the results are displayed as Scatchard plots for
convenience as previously described (39).
Jak3 immunoprecipitation and Western blot analysis of Jak and
NK cell lysates containing 200 ?g of proteins in lysis buffer were incubated
with 4 ?l of anti-Jak3 (rabbit Ab C-21 Santa Cruz Biotechnology) for 2 h
at 4°C with agitation. Protein G-Sepharose beads (Amersham-GE Health-
care) washed in lysis buffer were then added for 1 h at 4°C. After four
washes in lysis buffer, samples were denatured for 5 min at 100°C in
SDS-loading buffer and proteins were separated by SDS-PAGE (8%) and
transferred on nitrocellulose membranes overnight at 4°C. The membrane
was saturated with BSA, incubated with either anti-p-Jak, anti-Jak1 (mAb
Q19), anti-Jak3 (rabbit Ab C-21), anti-STAT5a (rabbit Ab L-20) (all Abs
from Santa Cruz Biotechnology) or anti-p-STAT5a (pY694; BD Bio-
sciences) and washed in TBS/0.5% Tween 20 buffer before being incu-
bated with HRP-coupled anti-mouse Ab (Amersham Biosciences). Proteins
were revealed by ECL-plus reagents (Amersham Biosciences).
Intracellular STAT5 phosphorylation assay
In brief, 106PBMC cultured in 1 ml of complete medium at 37°C in 5%
CO2were initially stimulated with 150 pM IL-15, IL-2, or IL-7 and then
restimulated 18 or 48 h later by adding 0, 10, 20, 100, or 500 pM of either
IL-2 or IL-15. To mimic IL-15 trans-presentation, IL-15 was complexed at
a 1:1 ratio with soluble IL-15R? (recombinant human soluble IL-15R?
(sIL-15R?):Fc chimera; R&D Systems) and used at the same concentra-
tions as free IL-15. In these experiments, cells were pretreated with human
plasma to saturate FcRs before addition of the IL-15/sIL-15R?:Fc com-
plex. Cells were harvested 15 min after stimulation, fixed with 1.6% para-
formaldehyde, permeabilized in 90% methanol as described previously
(40), and labeled with anti-CD3-allophycocyanin-AF750 (DakoCytoma-
tion), anti-CD56-PE (Immunotech and Beckman Coulter) for NK cell de-
tection and with anti-p-STAT5-AF647 (BD Biosciences) to detect the
6268SWITCH FROM IL-15- TO IL-2-DEPENDENT NK CELL ACTIVATION
STAT5 molecules phosphorylated at Y694. Analysis was carried on a cyan
flow cytometer, on a minimal number of 500 000 events in the NK cell gate.
The mean fluorescence intensity (MFI) of p-STAT5 after cytokine stimulation
was determined by subtracting the p-STAT5 MFI after the 18 or 48 h pre-
treatment from the p-STAT5 MFI after cytokine restimulation. Each experi-
ment included fluorescence minus one controls in which the p-STAT5 mAb
was replaced by an isotypic control Ab (mouse IgG1?; BD Biosciences).
Analysis of intracellular IFN-? production and cell surface
In brief, 106PBMC were prestimulated with cytokines for 18 or 48 h
following the same protocol as for the STAT5 phosphorylation assay. Cells
were then incubated for 1 h at 37°C in 5% CO2with brefeldin A (Sigma-
Aldrich) at a final concentration of 0.5 ?g/ml to prevent exocytosis of
expressed cytokines stored in vesicles. Six hours after restimulation, cells
were permeabilized with a staining buffer set (eBioscience) and labeled
with anti-CD-3-allophycocyanin-AF750 (eBioscience), CD-56-allophyco-
cyanin (Beckman Coulter), anti-IFN-?-PE-Cy7, and anti-CD107a-AF488
(eBioscience). Cells were analyzed by flow cytometry on a minimal num-
ber of 500,000 events in the NK cell gate. The MFI of IFN-? and CD107a
after cytokine stimulation were corrected by subtracting baseline values
after 18 or 48 h of cytokine pretreatment from the MFI postcytokine
IL-15 activates quiescent NK cells more efficiently than IL-2
We monitored the induction of CD69, an early activation marker,
in NK cells treated for 48 h with increasing concentrations of the
cytokines IL-2 and IL-15. Surface expression of CD69 was mea-
sured by flow cytometry in CD3?CD56?NK cells from five
healthy blood donors (Fig. 1). CD69 was induced more efficiently
by IL-15 than by IL-2, with maximum expression on NK cells
reached at 2 nM IL-15 instead of 5 nM IL-2. In addition, the point
of half-transition was 150 pM for IL-15 and 800 pM for IL-2,
indicating that IL-15 was 5- to 6-fold more potent than IL-2 at
activating quiescent NK cells. The maximum CD69 expression
level was comparable for both cytokines at high concentrations, as
expected for cytokines targeting the same intermediate-affinity re-
Rapid induction of the intermediate-affinity receptor at the
surface of NK cells
The expression kinetics of individual IL-2R and IL-15R chains
were followed on human NK cells from six healthy blood donors
after in vitro stimulation with IL-2 or IL-15. CD122 was consti-
tutively expressed at high levels by most quiescent NK cells
(?90%) (Fig. 2A). After IL-2 and IL-15 stimulation, the percentage
man PBMC from five healthy blood donors were cultured in the presence
of different concentrations of IL-2 or IL-15 for 48 h. CD3?CD56?NK
cells were then analyzed by flow cytometry for the expression of the early
activation marker CD69. CD69 MFI is plotted vs IL-2 and IL-15 concen-
trations in a log scale. Error bars indicate SDs.
CD69 induction in IL-2- and IL-15-treated NK cells. Hu-
cells. Human PBMC were cultured for 72 h in the presence of either
IL-2 (5 nM) or IL-15 (1 nM). CD3?CD56?NK cells were analyzed by
flow cytometry 12, 24, 48, and 72 h after stimulation. A and C, Results
from a representative donor showing the expression of CD122 (A) and
?c(C) assayed 12 h after cytokine stimulation. Fluorescence intensity
distribution for CD122 and ?care shown as solid gray-filled histograms.
The profile obtained with the IgG-PE isotypic control is plotted as a
solid line. B and D, The MFI of CD122 (B) and ?c(D) from six donors
are reported as a function of the duration of cytokine treatment. Bars
represent SDs (n ? 6).
Expression of CD122 and ?cin cytokine-stimulated NK
6269The Journal of Immunology
of CD122-expressing cells remained high (Fig. 2B) and did not vary
significantly over 3 days from six healthy blood donors.
The ?c(CD132) was not detectable at the surface of quiescent
NK cells (Fig. 2C) from 10 healthy blood donors. However,
stimulation by IL-2 or IL-15 led to the rapid induction of ?c
surface expression in the whole NK cell population, with a max-
imum reached as early as 12 h after stimulation (Fig. 2D). Sur-
face expression of ?cthen persisted at high levels for up to 3
days. Thus, cytokine stimulation led to persistently high ex-
pression of the two chains comprising the intermediate- affinity
receptors CD122 and ?c.
Expression of immature and mature ?cin resting and activated
Because we did not detect ?cby cytometry at the surface of highly
purified quiescent NK cells while we expected this chain required
for the IL-2/IL-15 NK cell response, we further studied the ex-
pression of this chain using several anti-?canalyzed by Western
blot. We used the TUGh4 mAb specific for the mature form of ?c
and the polyclonal Ab G??cpAb that detects both the mature and
immature forms of the ?cas previously described (41). We ob-
served that mature ?cwas detectable neither by flow cytometry
(Fig. 3A) nor by Western blotting from NK cell lysed (Fig. 3B) at
the surface of highly purified quiescent NK cells from more than
10 healthy donors (a representative example is shown in Fig. 3).
However, NK cells stimulated for 12 h with IL-2 or IL-15 showed
a strong surface expression of mature ?cby cytometry (Fig. 3A,
bottom plots) as well as by Western blotting (Fig. 3B, 62- to 64-
kDa band). This confirmed the strong expression of mature ?c
chain 12 h after cytokine stimulation. We next used a polyclonal
Ab (G??cpAb) that detects both the mature and immature forms
of the ?cby Western blotting (Fig. 3C). Interestingly, resting NK
cells contained a significant pool of immature ?c(50–52 kDa).
Stimulation of cultured NK cells by IL-2 or IL-15 for 12 h led to
both the maturation of ?c(apparition of the 62- to 64-kDa band)
and to the accumulation of immature ?c(increase in the 50- to
52-kDa band), suggesting that both maturation and neosynthesis
should contribute to the rapid induction of ?cexpression at the cell
surface. The presence of a preexisting intracellular pool of ?ccan
explain its rapid kinetics of expression at the cell surface upon NK
cell activation, by bypassing the lag phase required for the induced
Sequential expression of IL-15R? and IL-2R? on
cytokine-stimulated NK cells
The expression kinetics of IL-2R? (CD25) and IL-15R? was as-
sessed on NK cells from six healthy donors. CD25 was undetect-
able in most of quiescent NK cells, although a few CD25?cells
were detected among the CD56brightNK cell subset (5% of NK
cells; Fig. 4A). NK cells became predominantly CD25?48 h after
IL-2 and IL-15 stimulation (Fig. 4A). The kinetics of CD25
induction after IL-2 stimulation was slow, with an initial in-
crease detectable after 24 h and a maximum reached after 48 h
of culture (Fig. 4B). The kinetics of CD25 expression after
IL-15 stimulation was slightly more rapid, with a detectable
induction from 24 h and a plateau reached after 48 h. Of note,
the CD25 MFI reached at the plateau (48 h) was three times
higher after IL-15 than after IL-2 stimulation, suggesting a
more potent activation of NK cells by IL-15.
The expression of IL-15R? showed a strikingly different pat-
tern. IL-15R? was expressed at low but detectable levels in resting
NK cells (Fig. 4C). Both IL-2 and IL-15 stimulation caused a
transient induction of IL-15R?, with maximum expression reached
24 h after stimulation. IL-15R? was then rapidly down-regulated
and became undetectable 36 h after stimulation, while unstimu-
lated NK cells maintained low but detectable IL-15R? expression.
Again, IL-15 induced IL-15R? expression three times more effi-
ciently than IL-2 in NK cells. It was noteworthy that IL-15R?
down-regulation was as complete after IL-15 than after IL-2 treat-
ment, even though maximal receptor expression was higher in the
former case. Taken together, these findings suggested a differential
regulation of the high-affinity receptors for IL-15 and IL-2 in ac-
tivated human NK cells.
ing and activated NK lymphocytes. A, Human PBMC were cultured for
12 h in the presence of either IL-2 (5 nM) or IL-15 (1 nM). CD3?CD56?
NK cells were analyzed by flow cytometry 12 h after stimulation. B and C,
Highly enriched NK lymphocytes were cultured either alone or in the pres-
ence of IL-2 (5 nM) or IL-15 (2 nM). After 12 h of culture, cells were
harvested and analyzed by Western blotting. Two reagents were used for
the detection of the ?c. A, TUGh4 mAb recognizes preferentially the ma-
ture ?cform (m?c). B, G??cpolyclonal Ab is directed against the extra-
cellular domain of the ?cand recognizes both the immature (i?c) and ma-
ture forms of the protein. Unstim, Unstimulated.
Differential expression of immature and mature ?cin rest-
6270SWITCH FROM IL-15- TO IL-2-DEPENDENT NK CELL ACTIVATION
Sequential expression of high-affinity IL-2 and IL-15 receptors
at the surface of NK cells
It was important to verify that expression of IL-2R? and IL-15R?
resulted in the formation of high-affinity receptor complexes at the
surface of NK cells. To this goal, we measured the binding con-
stants of125I-labeled IL-2 and IL-15 for their receptors at the sur-
face of purified human NK cells from three healthy donors. The
binding of labeled cytokines from picomolar to micromolar con-
centrations was assayed at 4°C at times 0, 18, and 48 h after pre-
activation of NK cells with IL-2 (5 nM) or IL-15 (2 nM). On
resting cells, high-affinity binding sites were barely detectable
(Fig. 5B), while intermediate-affinity sites were present (Fig. 5A).
At peak stimulation (18 h after cytokine pretreatment for IL-15R
and 48 h for IL-2R), both intermediate- and high-affinity binding
sites were detected. The Kdfor the intermediate-affinity sites were
in the same range for both cytokines, with a value of 0.8 ? 0.2 nM
for IL-2 and 0.4 ? 0.2 nM for IL-15 (Fig. 4A). The numbers of
intermediate-affinity binding sites for IL-2 and IL-15 were com-
parable, compatible with these sites consisting in the same moi-
eties (CD122 and ?c). Intermediate-affinity binding sites preexisted
in quiescent NK cells (700–800 binding sites/cell), likely due to
the capacity of CD122 homodimers to bind cytokines (42). The
number of intermediate- affinity binding sites increased upon ac-
tivation, and reached higher levels after IL-15 activation (1700
sites/cell) than IL-2 activation (1100 sites/cell), consistent with the
more efficient induction of CD122 and ?cexpression by IL-15.
The Kdfor the high-affinity binding sites were almost identical
for both cytokines, with values of 13 ? 2 pM for IL-2 and 12 ?
2 pM for IL-15 (Fig. 5C). Although the affinities of IL-2 and IL-15
for receptors at the surface of activated NK cells were comparable,
the number of high-affinity binding sites differed significantly.
Eighteen hours after IL-2 stimulation, we titrated 1.5-fold more
high-affinity IL-15 binding sites (90 sites/cell) than high-affinity
IL-2 binding sites (60 sites/cell; (Fig. 5D). Similarly, 18 h after
IL-15 stimulation, the number of high-affinity IL-15 binding sites
(160 sites/cell) was ?2.5-fold higher than that of high-affinity IL-2
binding sites (60 sites/cell; Fig. 5D). After 48 h of stimulation by
either cytokine, the high- affinity binding sites for IL-15 became
undetectable (n ? 20 sites/cell). In contrast, 48 h of IL-2 or IL-15
stimulation resulted in maximal induction of high-affinity IL-2
binding sites (220 and 480 sites/cell, respectively; Fig. 5, C and D).
These results are in agreement with the kinetics of expression of
IL-2R? and IL-15R? and support the notion of a sequential in-
duction of high-affinity receptors for IL-15 and IL-2 at the surface
of activated NK cells. In addition, comparison of the numbers of
high-affinity binding sites induced after IL-15 and IL-2 treatment
(Fig. 5D, compare top and bottom graphs) confirmed the greater
potency of IL-15 at activating NK cells.
Early phosphorylation events after cytokine stimulation of
quiescent NK cells
To investigate the functional responses of quiescent NK cells to
cytokines, we assayed the activation of the Jak/STAT pathway
after 15 min of stimulation with either IL-2 or IL-15 in six healthy
donors. Purified NK cells were stimulated with each cytokine at
150 pM, lysed, and subjected to immunoprecipitation with a Jak3-
specific Ab. The coimmunoprecipitated signaling complexes were
analyzed by immunoblotting with phospho-specific Abs to p-Jak
and p-STAT5. Both cytokines induced the phosphorylation of Jak1
and Jak3 kinases (Fig. 6A), as well as that of the STAT5 transcrip-
tion factor (Fig. 6B) in quiescent NK cells. However, IL-15 was
more efficient at initiating these early phosphorylation events (Fig.
6, A and B). These findings were consistent with the increased
NK cells. Human PBMC were cultured for 72 h in the presence of either IL-2 (5
nM) or IL-15 (1 nM). The kinetics of IL-2R? and IL-15R? expression was ana-
lyzed by flow cytometry. A and C, Fluorescence intensity distribution of IL-2R?
and IL-15R? in NK cells from a representative blood donor. The expression of
IL-2R? (A) and IL-15R? (C) are represented at peak stimulation, 48 and 18 h,
respectively, after cytokine treatment. Profiles of IL-2R? and IL-15R? expression
are shown as solid gray-filled histograms, while the profile obtained with the
and IL-15R? (D) are reported as a function of the duration of cytokine treatment.
6271The Journal of Immunology
efficiency of IL-15 at inducing CD69 and cytokine receptor chain
expression at the cell surface in response to low cytokine
Time-dependent regulation of IL-2- and IL-15-induced STAT5
phosphorylation in activated NK cells
Functional responses of activated NK cells from six healthy donors
were analyzed by pretreating PBMC with cytokines to reach op-
timal receptor expression, restimulating the cultures with a range
of IL-2 and IL-15 concentrations and measuring the phosphoryla-
tion of STAT5 at Y694 in the CD3?CD56?population. PBMC
were pretreated with 150 pM IL-15, which yielded optimal ex-
pression of IL-15R? at 18 h and of IL-2R? at 48 h.
IL-2 stimulation (500 pM) induced more efficient STAT5 phos-
phorylation in NK cells preactivated for 48 h than for 18 h with
IL-15 (Fig. 7A), a finding compatible with the higher IL-2R? ex-
pression at 48 h (Fig. 4B). Dose-response curves were then plotted
to precisely compare the reactivity of NK cells after 18 and 48 h
of pretreatment with IL-15. After 18 h of pretreatment, NK cells
responses to IL-2 were of low intensity, with an increase in the
p-STAT5 MFI detected at IL-2 doses comprised between 100 and
200 pM (Fig. 7B). In contrast, efficient activation of p-STAT5 was
detected after stimulation with as low as 10 pM IL-2 in NK cells
preactivated for 48 h (Fig. 4C). NK cells cultured in the absence of
IL-15 (open symbols, Fig. 7) did show a detectable response to
IL-2 or IL-15 stimulation, even at the 10 pM concentration, con-
sistent with a limited expression of IL-2R? and IL15R? chains
after 2 days of culture.
Functional responses of preactivated NK cells to IL-15 stimu-
lation clearly differed from those observed with IL-2 stimulation.
NK cells preactivated for 18 h with IL-15 responded optimally to
a second IL-15 stimulation, while cells preactivated for 48 h
showed no detectable STAT5 phosphorylation after a second
IL-15 addition (Fig. 7D). Again, these findings were consistent
with the undetectable expression of IL-15R? at 48 h (Fig. 4D).
Dose-response curves showed that NK cells responded to picomo-
lar concentrations of IL-15 after an 18-h preactivation, when IL-
15R? expression was maximal (Fig. 7E). Interestingly, NK cells
preactivated for 48 h showed an undetectable p-STAT5 response
to picomolar IL-15 stimulation, while NK cells that were cultured
for 48 h in the absence of cytokine did show a response, likely due
to the spontaneous expression of IL-15R? (Fig. 7F). Thus, prior
IL-15 activation inhibited subsequent responses to the same cyto-
kine, defining a negative regulatory loop in the IL-15/IL-15R
These experiments were repeated with NK cells preactivated with
IL-2. The data confirmed that IL-15 functional responses were opti-
mal 18 h after preactivation and down-regulated afterward, while IL-2
responses peaked from the 48-h time point onward (data not shown).
Altogether, the data showed a clear association between the number
of high-affinity IL-2R and IL-15R expressed at the surface of NK
cells and their signaling responses. Importantly, activated NK cells
purified NK cells. A, Scatchard plots are shown in the intermediate-affinity range for IL-2 binding (left) and for IL-15 binding (right) 18 h after activation
by IL-15. Bound:free cytokine ratios are plotted vs bound cytokines per cell. Kdand binding sites were computed from best fits of binding curves. B,
Numbers of intermediate-affinity binding sites for IL-2 (u) and IL-15 (?) are reported as bar graphs at different times (0, 18, and 48 h) after stimulation
with IL-15 (top row) or IL-2 (bottom row). C, Scatchard plots are shown in the high-affinity range for IL-2 binding (left) and for IL-15 binding (right). Kd
values were measured from binding curve fitting at optimal expression of the high-affinity receptors, after a 48-h IL-15 treatment for IL-2 binding
radioassays (left), and after a 18-h IL-15 treatment for IL-15 binding radioassays (right). D, Numbers of high-affinity binding sites for IL-2 (u) and IL-15
(?) are reported as bar graphs at different times (0, 18, and 48 h) after stimulation with IL-15 (top row) or IL-2 (bottom row).
Measurement of IL-2 and IL-15 binding to cytokine-stimulated NK cells. The binding of125I-labeled IL-2 and IL-15 were radioassayed on
6272 SWITCH FROM IL-15- TO IL-2-DEPENDENT NK CELL ACTIVATION
remained responsive to picomolar doses of IL-15 for a limited time
period, while responsiveness to IL-2 increased progressively
IL-15 complexed to IL-15R? retains the capacity to signal in
activated NK cells
IL-15 can act on its target cells as a free soluble molecule or in a
trans-presented form while complexed to IL-15R? at the surface
of producer cells. Since studies in mouse models have proven the
physiological relevance of trans-presented IL-15 (20, 24, 25), we
assessed the reactivity of NK cells from six healthy donors to this
form of “chaperoned” cytokine. To mimic trans-presentation, we
used IL-15 complexed at a 1:1 ratio with the extracellular domain
of IL-15R? (sIL-15R?) fused to an Ig Fc region. This complex
was able to activate quiescent NK cells, as indicated by efficient
STAT5 phosphorylation at doses as low as 10 pM (data not
shown). Importantly, NK cells preactivated for 48 h with IL-15
maintained an efficient response to the IL-15/sIL-15R? complex,
whereas the response to free IL-15 was minimal (Fig. 8A). Dose-
response analysis showed that the IL-15/sIL-15R? complex sig-
naled at the 10 pM concentration and was even more efficient than
IL-2 at inducing STAT5 phosphorylation in NK cells preactivated
for 48 h. In contrast, free IL-15 induced a limited response that
became detectable at concentrations above 100 pM (Fig. 8B).
These finding indicated that the IL-15/sIL-15R? complex could
signal efficiently in NK cells that had down-regulated the endog-
enous IL-15R? chain but still expressed the CD122 chain and ?c.
This suggested that, in the late activation stage, NK cells could still
respond to trans-presented IL-15, while sensitivity to free IL-15
had become minimal.
IL-2 and IL-15 functional responses are controlled by
high-affinity receptor expression
To determine the physiological relevance of our findings, we tested
effector functions of NK cells at different times following cytokine
treatment. We measured the surface expression of CD107a (or
LAMP-1), an endosomal marker which is exposed upon degranu-
lation and which has been associated with NK cell cytotoxic ac-
tivity (43). We also measured intracellular IFN-? production, since
this cytokine is one of the primary mediators of NK cell function.
NK cell functions were analyzed in cells from six healthy donors
18 and 48 h after pretreatment with IL-15 (150 pM). At the 18-h
time point, NK cells responded weakly to IL-2 restimulation (Fig.
9, A and E). In contrast, at the 48-h time point, efficient CD107a
by IL-15 than by IL-2. Purified CD3?CD56?NK cells were treated for 15
min with 150 pM IL-15 or IL-2 and lysed. Protein extracts were immu-
noprecipitated (IP) with a Jak3 specific Ab immobilized on beads and
subjected to immunoblotting with the following reagents: (A) Abs to p-Jak
(top gel), Jak1 (middle gel), and Jak3 (bottom gel); (B) Abs to p-STAT5
(top gel) and to STAT5a (bottom gel). WB, Western blot.
Early phosphorylation events are more efficiently induced
STAT5 phosphorylation in activated NK cells. Human PBMC were pre-
cultured in the presence of 150 pM IL-15 for 18 or 48 h and then restim-
ulated with IL-2 or IL-15 for 15 min. Phosphorylation of STAT5 was
assayed by flow cytometry on the CD3?CD56?NK cell population. A and
D, Representative histograms of p-STAT5 expression after stimulation
with 500 pM IL-2 (A) or 500 pM IL-15 (D). In each case, PBMC were
analyzed after 18 h of culture in the absence of exogenous cytokines (thin
line), 18 h of culture in the presence of IL-15 (thick line), or 48 h of culture
in the presence of IL-15 (shaded histogram). B and C, PBMC precultured
for 18 h (B) or 48 h (C) were stimulated with different amounts of IL-2 (10,
20, 100, 200, and 500 pM) for 15 min before analyzing p-STAT5 expres-
sion in the CD3?CD56?population. MFI of p-STAT5 are plotted vs cy-
tokine concentration. Thick lines represent responses of cells precultured in
the presence of IL-15, while thin lines represent responses of cells precul-
tured in the absence of exogenous cytokine. E and F, PBMC precultured
for 18 h (B) or 48 h (C) were stimulated with different amounts of IL-15
(10, 20, 100, 200, and 500 pM) for 15 min before analyzing p-STAT5
expression in the CD3?CD56?population. Results are represented as
Time-dependent regulation of IL-2- and IL-15-induced
6273 The Journal of Immunology
and IFN-? expressions were detected after stimulation with as low
as 10 pM IL-2 (Fig. 9, C and G).
Functional responses of IL-15-pretreated NK cells to IL-15
stimulation clearly differed from those observed with IL-2 stimu-
lation. At the 18-h time point, NK cells responded optimally to
picomolar concentrations of IL-15 (Fig. 9, B and F), while no
detectable CD107a and IFN-? expression was detected at the 48-h
time point (Fig. 9, D and H). Again, these findings were consistent
with maximal IL-15R? induction 18 h after pretreatment and un-
detectable IL-15R? expression 48 h after pretreatment (Fig. 5D).
NK cultured for 2 days in the absence of IL-15 (E, Fig. 9) did
show a low but detectable response to IL-2 or IL-15 stimulation,
even at the 10 pM concentration, consistent with a spontaneous
induction of IL-2R? and IL15R? chains at low levels in cultured
NK cells. Taken together, these findings showed that NK cell re-
sponses followed the same kinetics as that of IL-2R? and IL-15R?
expression, emphasizing the importance of the cytokine receptor
expression pattern in controlling NK cell function.
?cfamily cytokines induce a common cytokine receptor
expression program on NK cells
To test the generality of our findings, we evaluated IL-2R and
IL-15R expressions in NK cells preactivated by IL-7, another cy-
tokine of the ?cfamily. Phenotyping of NK cells from six healthy
blood donors showed that IL-7 treatment did not change signifi-
cantly the already high expression levels of CD122, but caused a
rapid induction of the ?c, which reached high levels from 12 h
onward (Fig. 10, A and B). CD25 induction was progressive, with
detectable expression at 24 h of IL-7 treatment and slowly increas-
ing levels during the 3-day culture (Fig. 10C). Once again, IL-
15R? expression was transient, with efficient induction at 12 h and
complete down-regulation at 48 h. Thus, the pattern of cytokine
receptor expression induced by IL-7 paralleled that observed after
IL-2 and IL-15 treatments. This notion was reinforced by the fact
activated NK cells. Human PBMC were cultured in the presence of 150 pM
IL-15 for 48 h to achieve complete IL-15R? down-regulation. PBMC were
then restimulated with IL-15 or the IL-15/IL-15R? complex for 15 min and
analyzed for p-STAT5 in the CD3?CD56?NK cell population. A, Rep-
resentative histograms of p-STAT5 expression after restimulation with in-
duced by IL-2, IL-15 (thin line), the complex IL-15/IL-15R? complex
(thick line), or IL-2 (shaded histogram) at the 10 pM concentration (left)
and the 500 pM concentration (right). B, Dose-response curves of
p-STAT5 induction in NK cells precultured in IL-15 for 48 h and restim-
ulated with IL-2 (?), IL-15 (f), or the IL-15/IL-15R? complex (3). Bars
represent the mean values obtained in n ? 6 experiments. Error bars rep-
Persistence of responses to the IL-15/sIL-15Ra complex in
affinity receptor expression levels. Human PBMC were preactivated in the
presence of 150 pM IL-15 for 18 or 48 h and then restimulated with IL-2
or IL-15 for 15 min. Intracellular production of IFN-? and CD107a surface
expression were monitored by flow cytometry on the CD3?CD56?NK cell
population. A, C, E, and G, PBMC preactivated for 18 h (A and E) or 48 h
(C and G) were stimulated with different amounts of IL-2 (10, 20, 100, 200,
and 500 pM) for 6 h before analyzing IFN-? secretion and CD107a ex-
pression in the CD3?CD56?population. MFI of IFN-? and CD107a are
plotted vs cytokine concentration. Thick lines represent responses of NK
cells preactivated with IL-15, while thin lines represent responses of cells
cultured in the absence of exogenous cytokine. B, D, F, and H, PBMC
preactivated for 18 h (B and F) or 48 h (D and H) were stimulated with
different amounts of IL-15 (10, 20, 100, 200, and 500 pM) for 6 h before
analyzing IFN-? and CD107a responses in the CD3?CD56?population.
Results are represented as above.
IL-2 and IL-15 functional responses are controlled by high-
6274 SWITCH FROM IL-15- TO IL-2-DEPENDENT NK CELL ACTIVATION
that all three cytokines caused a progressive down-regulation of
the IL-7R? chain CD127, while untreated NK cells up-regulated
CD127 (Fig. 10E). Thus, ?ccytokines triggered a common cyto-
kine receptor expression program in human NK cells.
This study provides evidence for sequential expression of IL-15R?
and IL-2R? at the surface of cytokine-activated human NK cells.
Treatment with IL-2, IL-15, or IL-7 resulted in a similar receptor
chain expression pattern, which conferred transient responsiveness
to free IL-15, followed by long-lasting responsiveness to IL-2 and
trans-presented IL-15. This sequence of events did not depend on
the initial stimulus, even though IL-15 proved more efficient at
initiating early activation events. Thus, human NK cells show a
programmed response to ?ccytokine stimulation, which may dif-
ferentially control NK cell function in the innate and adaptive
stages of the immune response.
Detailed analysis of receptor chains expression provides clues to
the respective role of IL-2 and IL-15 in human NK cell physiology.
NK cells appear primed to respond quickly and efficiently to an
initial IL-15 stimulus. 1) Resting NK cells constitutively express
low levels of IL-15R? and high levels of the ?-chain CD122. The
only chain needed to reconstitute the high-affinity receptor ?c
shows a rapid kinetics of induction, so that high-affinity receptors
are rapidly available to amplify the IL-15 response. In contrast, the
IL-2R? chain is barely detectable in quiescent NK cells and is
induced with a slow kinetics, resulting in delayed optimal IL-2
responses. 2) IL-15R? reaches a maximum of expression between
16 and 20 h after activation, at a time when ?cis also optimally
induced. As a consequence, the number of high-affinity binding
sites for IL-15 is ?2-fold higher than that for IL-2 one day after
activation (Fig. 5), resulting in more efficient signaling responses
(Fig. 7, B and E). We verified that phosphorylation of the early
effectors Jak1 and Jak3 paralleled that of STAT5 (data not shown),
confirming that differences in IL-15 and IL-2 responses were con-
trolled at the receptor level.
The mechanism initiating cytokine responses in quiescent NK
cells is not entirely elucidated, since ?cis not expressed at detect-
able level at the surface of these cells (44). Commercial Abs to ?c
do not appear sensitive enough to detect low amounts of the pro-
tein. Indeed, we did not detect ?cexpression by flow cytometry
(Fig. 2D) nor by immunoradioassay with a125I-labeled anti-?cAb
with a sensitivity threshold above 60 molecules/cell (data not
shown). However, binding of125I-labeled IL-2 or IL-15 revealed
the presence of ?750 intermediate-affinity receptor sites per qui-
escent NK cells, well above the ?cdetection threshold (Fig. 4A).
We and others have previously shown that CD122 can assemble
into homodimers and bind IL-2 with intermediate affinity, which
would explain the detection of IL-2 binding sites on quiescent NK
cells (42, 45, 46). CD122 homodimers may be able to initiate
signaling, which would then be amplified by the rapid induction of
?c. Alternatively, trace amounts of ?cmay build up intermediate-
affinity receptors by association with abundant CD122 chains
present at the surface of quiescent NK cells and may then trigger
signaling. Once signaling is initiated, the rapid induction of ?c
expression provides an efficient means to increase the number of
active receptors. Several mechanisms ensure the induction of ?c
expression. 1) We have observed that ?cmRNA is expressed at
significant levels in quiescent NK cells and is quickly induced after
activation (data not shown). 2) ?cprotein synthesis is also in-
creased after NK cell activation, as indicated by the increase in
total ?ccontent in activated NK cell protein extracts. 3) ?cproteins
preexist in an immature form in resting NK cells and are rapidly
matured upon activation (Fig. 3). We have previously shown that
a similar intracellular pool of immature ?cis present in resting
CD4?T cells and that this immature form can be rapidly glyco-
sylated and exported to the cell surface upon activation (41). Thus,
the maturation of a starter pool of ?crepresents a conserved mech-
anism which confers cytokine responsiveness to recently activated
leukocytes. Of note, several receptors that play key roles in im-
mune activation, such as MHC molecules, show a similar pattern
of intracellular retention and rapid export to the cell surface upon
activation (47, 48). Once signaling is triggered, the inducibility of
?cmRNA and protein expression help sustain and amplify the
activation, resulting in a positive feedback loop between receptor
induction and receptor signaling.
Triggering of signaling through the intermediate-affinity recep-
tor also induces expression of the IL-15R? and IL-2R? chains, but
with slower and sequential kinetics. Expression of these receptor
gram as other ?cfamily cytokines. Human PBMC were cultured for 72 h
in the presence of IL-7 (1 nM). CD3?CD56?NK cells were analyzed by
flow cytometry 12, 24, 48, and 72 h after stimulation for the expression of
cytokine receptor chains. A, Expression of CD122 (MFI) as a function of
IL-7 treatment duration. B, Expression of ?c(MFI) as a function of IL-7
treatment duration. C, Expression of IL-2R? (CD25 MFI) as a function of
IL-7 treatment duration. D, Expression of IL-15R? (MFI) as a function of
IL-7 treatment duration. E, Human PBMC were cultured for 48 h in the
presence of IL-2 (5 nM), IL-15 (1 nM), and IL-7 (1 nM). CD3?CD56?NK
cells were analyzed by flow cytometry 0, 18, and 48 h after stimulation for
IL-7R? (CD127) expression. The percentages of cells expressing CD127
are reported as a function of time. NS, Not stimulated.
IL-7 induces the same cytokine receptor expression pro-
6275The Journal of Immunology
chains ensures high-affinity interactions with their respective cy-
tokine ligands, but also extends the time of cytokine residency on
the binding sites (greater kinetic dissociation constant koff). As a
consequence, early signaling events, such as STAT5 phosphory-
lation, and late functional responses, such as CD107a and IFN-?
expression, are more efficient. For instance, we found that IL-2
binding on 200 high-affinity receptors resulted in the same induc-
tion of p-STAT5 as the binding of 1600 intermediate-affinity re-
ceptors (cf Figs. 4 and 7). The sequential expression of IL-15R?
and IL-2R? clearly controlled both signaling and functional NK
cell responses, since receptor expression, STAT5 phosphorylation,
CD107a cell surface expression, and IFN-? production all fol-
lowed the same pattern. Namely, activated NK cells showed op-
timal signaling and functional responses to IL-15 stimulation at the
18-h time point, at the peak of IL-15R? expression, while optimal
responses to IL-2 were achieved later, at 48 h, when IL-2R? ex-
pression was maximal. These results point to a tight temporal reg-
ulation of NK cell functions.
Although several parameters favored an initial responsiveness
of NK cells to IL-15, this phenomenon was transient. Two days
after activation, NK cells became refractory to picomolar IL-15
stimulation, while sensitivity to IL-2 stimulation increased. The
loss of responsiveness to IL-15 was associated to a rapid down-
regulation of IL-15R? expression as measured by flow cytometry
(Fig. 4D). Accordingly, the high- affinity binding sites for IL-15
became undetectable at the surface of NK cells. Since IL-15R?
mRNA levels remained stable at this stage (data not shown), post-
transcriptional regulation likely accounts for the loss of IL-15R?
expression. A possible mechanism is the cleavage of IL-15R? by
the TNF-?- converting enzyme TACE/ADAM17 protease at the
cell surface, a phenomenon shown to generate a soluble inhibitory
form of the receptor (49, 50). Remarkably, IL-15R? down-regu-
lation was drastic, with expression levels at the cell surface lower
in activated NK cells than in resting NK cells. Accordingly, resting
NK cells showed a degree of response to picomolar IL-15 con-
centrations, while activated NK cells remained unresponsive (Fig.
5F). Such a tight negative regulation may serve to avoid deleteri-
ous consequences of excessive NK cell activation. Tight control of
IL-15R? may be particularly important in humans who can show
detectable levels of circulating IL-15, particularly in cases of in-
flammation and autoimmune pathologies (51, 52). This contrasts
with murine models where IL-15 remains mostly undetectable in
the circulation. The presence of free IL-15 in humans emphasizes
the need of keeping IL-15 paracrine effects in check through re-
The fact that IL-15 down-regulates its own receptor has been
reported in murine and human T cells (38, 53). A parallel can be
drawn with the notion of “altruistic receptor down-regulation” es-
tablished by Singer and colleagues (54) for IL-7R whereby acti-
vated T cells down-regulate IL-7R at the same time as they acquire
competency to use other cytokines (such as IL-2), so that compe-
tition with resting T cells for limiting IL-7 resources is minimized.
However, the notion of an early and transient window of induction
of IL-15R? by ?cfamily cytokines is novel and may be more
relevant to the control of NK cell activation than to the maximi-
zation of IL-15 availability. IL-15 is produced by cells of the
monocyte/macrophage lineage, DC, and stromal cells. Production
of this cytokine is markedly increased in APC activated by patho-
gens, a phenomenon that is thought to play a key role in promoting
NK cell activation (25, 55). Activated NK cells, in turn, contribute
to DC maturation through contact-dependent mechanisms and
abundant secretion of cytokines, such as TNF-?, IFN-?, and GM-
CSF (6, 7). The NK/DC cross-talk has consequences on other im-
mune compartments as well, since NK-mediated DC activation
contributes to the initiation of T cell-dependent responses (56). It
ensues that excessive NK cell activation through persistent IL-15
signaling may have numerous unwanted consequences, including
overproduction of inflammatory cytokines, bystander cell lysis, or
perturbed development of adaptive responses.
Loss of IL-15R? at the surface of activated NK cells may render
these cells dependent on stimulation through IL-15/sIL-15R?
complexes trans-presented by APC, resulting in a tighter regula-
tion of the NK activation process. Trans-presentation requires in-
tercellular contact (19, 23), ensuring the coordinated activation of
NK cells and APC. In addition, we showed that NK cells progres-
sively acquire a responsiveness to IL-2, which is produced in
minute amounts by activated DC (57) and mainly originates from
activated T cells. The implications of gaining IL-2 responsiveness
at a late stage of NK cell activation are several. 1) Because IL-2 is
mainly secreted by activated CD4?T cells, it will place NK cell
activation under the control of adaptive immunity. NK cell cyto-
toxic activity will be targeted to sites where foreign Ags and re-
sponding CD4?T cells accumulate, avoiding a generalized NK
cell activation that could prove deleterious in chronic infections. 2)
Since CD4?T cell activation and IL-2 production take place pre-
dominantly within secondary lymphoid organs, activated NK cells
may be recruited to and persist in these sites and influence the
development of adaptive immune responses. IFN-? secretion by
NK cells may facilitate CD4?T cell differentiation toward a Th1
phenotype (8). In addition, activated NK cells have the capacity to
kill immature DC, which may play a key role in maintaining tol-
erance and in terminating immune responses (58). 3) Last, high-
dose IL-2 has been reported in certain studies to induce NK cell
apoptosis (35), which may help terminate NK cell activation
through a negative feedback loop. Thus, IL-2 responsiveness of
NK cells would contribute to the innate/adaptive immunity cross-
talk at multiple levels. Although early IL-15 responsiveness is
probably important in the rapid induction of NK cell innate effector
functions, late IL-2 responsiveness may confer a regulatory role to
NK cells during the adaptive stage of the immune response. Thus,
the sequential expression of IL-15R? and IL-2R? may play a key
role in coordinating the innate and adaptive branches of the im-
In conclusion, this study provides evidence for the sequential
expression of IL-15 and IL-2 high-affinity receptors at the surface
of activated human NK cells. This cytokine receptor expression
program can account for the differential effects of IL-2 and IL-15
on NK cells and may contribute to the tight control of NK cell
activity in humans. These findings are relevant for the optimization
of immunotherapeutic approaches that involve cytokine adminis-
tration by highlighting the time frame relevance for the efficient
concentrations of cytokine to promote NK cell effector functions.
We thank Dr. Nicholas Huntington (Institut Pasteur, Paris, France) for
reagents and critical reading of this manuscript, Dr. Yannick Jacques and
Gregory Bouchaud (Institut de Biologie, Nantes, France), and Dr. Christian
Vosshenrich (Institut Pasteur) for helpful discussions.
The authors have no financial conflict of interest.
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6277The Journal of Immunology