ARTICLE
Auteur(s) : Harmonie Perdreau1,2, Erwan
Mortier1,2, Grégory Bouchaud1,2,
Véronique Solé1,2, Yvan Boublik3, Ariane
Plet1,2, Yannick Jacques1,2
1Inserm, UMR 892, Centre de Recherche en Cancérologie
Nantes/Angers, Groupe de Recherche Cytokines et Récepteurs en
Immuno-Cancérologie, Nantes, France
2Université de Nantes, IFR 26, Nantes, France
3CNRS, UMR 5237, Centre de Recherche en Biochimie
Macromoléculaire, Montpellier, France
accepté le 30 Juillet 2010
Interleukin-15 (IL-15) is a cytokine that was originally
described as a soluble factor mimicking IL-2 functions in vitro
[1]. Despite a functional redundancy initially demonstrated in
vitro, subsequent experiments have indicated that IL-2 and IL-15
exert complementary actions in vivo. Although both cytokines
play pivotal roles in innate and adaptive immunity, the major role
of IL-2 now appears one of limiting T cell responses and promoting
the development of regulatory T cells, whereas IL-15 appears to be
critical for the development of NK and NK-T cells, the initiation
of T cell division, and the survival of memory T cells [2-4].
Both cytokines belong to the four-α-helix-bundle family, their
membrane receptors sharing two subunits: the IL-15Rβ (CD122) and
the common γ (γc or CD132) chains [5]. The
IL-15Rβ/γc receptor, such as that expressed by most
resting T and NK cells, is a common intermediate-affinity receptor
that can be activated by nanomolar concentrations of IL-2 or IL-15.
The high-affinity IL-2 and IL-15 receptors chains (IL-2Rα or CD25,
and IL-15Rα) confer cytokine specificity and enhance affinity for
cytokine binding. The trimeric, high-affinity receptors can be
activated with picomolar concentrations of either cytokine [6],
whereas the single chains IL-2Rα and IL-15Rα bind respectively IL-2
with a low affinity (Kd = 10 nM), and IL-15 with a
high affinity (Kd = 0.100 nM) [7].
The IL-15Rβ and γc chains bind intracellular
signaling complexes, and signal through three major pathways:
Jak/STAT, MAPK, and PI3K/Akt [8-10]. The private α receptors
are not thought to play a major role in cell signaling. However,
the IL-15Rα cytoplasmic domain has been described as interacting
with TRAF2 and Syk kinase signaling molecules [11, 12], although
the role of these associations has not been well established for
IL-15 function in vivo.
IL-15 signals can also be delivered through an original
mechanism called trans-presentation, in which IL-15Rα, expressed at
the surface of IL-15 producer cells (dendritic cells, macrophages
and epithelial cells), presents IL-15 in trans to responder cells
(NK or memory CD8+ T cells) bearing the
IL-15Rβ/γc receptor [13-15]. This specificity is due to
the capacity of the α chain to bind IL-15 with a high affinity in
the absence of the IL-15Rβ and γc chains. As a
co-stimulatory event occurring at the immunological synapse, IL-15
trans-presentation now appears to be a dominant mechanism for IL-15
action in vivo [14], and seems to play a major role in tumor
immunosurveillance [16].
A soluble form of the human IL-15Rα (sIL-15Rα) is naturally
released from IL-15Rα-expressing cells by a shedding process
involving matrix metalloproteinases. This sIL-15Rα is able to bind
IL-15 with high affinity, and efficiently blocks the proliferation
driven by the high-affinity IL-15Rα/β/γc signaling
receptor in vitro [17]. Notably, sIL-15Rα preserves the capacity to
trans-present IL-15, and high concentrations of soluble complex
IL-15/IL-15Rα can support NK cell activation in vitro and in vivo
[18, 19].
We previously engineered a fusion protein, RLI, comprising the
IL-15Rα binding domain linked to IL-15 [20]. This molecule is able
to bind the IL-15Rβ/γc receptor with a high affinity
[20, 21]. RLI highly stimulated the mobilization of NK cells in a
mouse model, deficient for the trafficking of these cells [22]. The
highly agonistic activity of RLI on the development and the
differentiation of NK cells was demonstrated in vivo in an HIS
mouse model [23]. In the B16-F10 melanoma model, RLI inhibited the
development of lung and liver metastases, and also reduced
metastatic progression in a model of HCT-116 human colorectal
cancer in the nude mouse. The antitumoral effect of RLI was
abolished by in vivo depletion of NK cells [24].
In the present study, we used RLI as a tool for studying
trans-presentation. In order to compare IL-15 cis- and
trans-presentation modes, we analyzed cytokine receptor expression,
cytokine binding, and signaling responses in a T cell line
expressing both IL-15Rα/β/γc and
IL-15Rβ/γc.
Methods and materials
Cytokines and reagents
Recombinant human IL-15 (rIL-15) was purchased from Peprotech, Inc.
(Rocky Hill, NJ, USA), and recombinant human IL-2 (rIL-2) was
purchased from Chiron (Emeryville, CA, USA). RLI fusion protein was
produced in baculovirus-Sf9 cells using Bac-to-Bac expression
system (Invitrogen), and was purified on an anti-FLAG-agarose
affinity column (Sigma-Aldrich), essentially as described
previously [21]. Monoclonal mouse anti-human IL-15 (MAB247),
polyclonal goat anti-human IL-15Rα (AF247), polyclonal goat
anti-human IL-2Rβ (AF224-NA), and PE-conjugated donkey anti-goat
IgG (F0107) were obtained from R&D Systems (Abington, UK). The
control isotype IgG goat was purchased from Santa-Cruz
Biotechnology. Monoclonal mouse anti-FLAG M2 conjugated to
peroxidase was purchased from Sigma-Aldrich (St Louis, MO, USA).
Polyclonal rabbit and mouse antibodies anti-phospho-STAT5 (#9351),
anti-phospho-STAT3 (#9131), anti-phospho-Akt (#9271), anti-Akt
(#9272), anti-phospho-p44/42 MAPK (Erk 1/2) (#9106), and
anti-p44/42 MAPK (Erk 1/2) (#9102) were obtained from Cell
Signaling Technology. Monoclonal mouse antibodies anti-STAT5
(610191), and anti-STAT3 (610190) were purchased from BD
Transduction Laboratories, and monoclonal mouse anti-actin antibody
(MAB1501R) was acquired from Millipore.
Cells and media
The Kit225 T lymphoma human cell line [25] was cultured in
RPMI-1640 medium (Sigma-Aldrich), containing 6% heat-inactivated
FCS (Gibco), 2 mM glutamine, and 325 pM human
rIL-2. This cell line was maintained at 37°C, in a humidified, 5%
CO2 atmosphere.
Proliferation assays
The proliferation-inducing activity of rIL-15 and RLI was assessed
using [3H]-thymidine incorporation by Kit225 cells as
described previously [26]. To measure the residual proliferative
response of Kit225 cells after rIL-15 or RLI treatment, Kit225
cells were maintained for three days with 500 pM rIL-15 or RLI.
Cells were washed, starved for 24 h in cytokine-deprived
medium, and plated at 104 cells in 100 μL of
cytokine-deprived medium. After 48 h, residual radioactivity
was measured by [3H]-thymidine incorporation.
Western blot analysis
Exponentially growing Kit225 cells were washed and serum-starved to
reduce basal phosphorylation (16 h in cytokine-deprived medium and
3 h in serum-free medium supplemented with 0.5% BSA). After
stimulation with rIL-15 or RLI under various conditions at 37°C,
cells were suspended in ice-cold, phosphate-buffered saline (PBS,
pH 7.4). Cell pellets were lysed by addition of ice-cold lysis
buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% glycerol, 1% NP-40, 20
μM Na3VO4, 10 mM NaF, 1 mM EDTA,
0.4 mM Pefablock with aprotinin and leupeptin at 1 μg/mL). After
incubation on ice for 20 min, samples were centrifuged (13,000
rpm, 15 min, 4°C), and protein concentration was determined with a
BC Assay Kit (Uptima) using BSA as standard. Fifty μg of total
protein cell lysates were analyzed on 10% SDS-PAGE and 4-12%
Bis-Tris Gels (Invitrogen), and the resolved proteins were
transferred to Immobilon-P PolyVinylidene DiFluoride (PVDF)
membranes (Millipore, Bedford, MA, USA). Membranes were blocked
with 5% milk, 0.05% Tween-20 in PBS for 1 h at room
temperature. Subsequently, membranes were immunoblotted with
specific antibodies according to their technical data sheet. After
incubation with secondary HRP-conjugated anti-mouse/anti-rabbit
antibody (Roche, Mannheim, Germany) for 1 h at room
temperature, visualization of specific proteins was conducted with
a chemiluminescence system using BM Chemiluminescence Blotting
Substrate (Roche), according to the manufacturer's instructions.
Densitometric evaluation of the Western blot data was performed
with ImageQuant Software.
Binding assays and internalization
rIL-15 and RLI were radiolabeled with [125I]-labeled
iodine, using a chloramine-T method, to a specific radioactivity of
approximately 2000 cpm/fmol for IL-15 and 4000 cpm/fmol for RLI.
Kit225 cells were used for binding assays, and these experiments
were performed essentially as described previously [27]. Briefly,
cells were incubated for 1 h at 4°C with increasing
concentrations of labeled rIL-15 or RLI. Non-specific binding was
determined in the presence of a 100-fold excess of unlabeled rIL-15
or RLI, and subtracted from total binding. Regression analysis of
the binding data was accomplished using one-site and two-site
equilibrium binding equations (GraphPad PRISM Software), and data
were plotted in the coordinate system.
For internalization analysis, Kit225 cells were treated mostly
as described previously [20]. In short, cells were equilibrated at
4°C for 1 h with 1 nM labeled rIL-15 or RLI. The
temperature was then switched to 37°C, and, at different time
intervals, two samples were washed and treated for 8 min
either with ice-cold glycine-HCl buffer (0.2 M, pH 2.5) or with
ice-cold PBS. Total ligand binding was determined from the pellet
of the cells treated with PBS, whereas the membrane-bound and
internalized fractions were determined, respectively, from the
supernatant and pellet of cells treated at low pH.
Flow cytometry analysis
Kit225 cells were maintained in culture medium for three days,
washed, and starved for 24 h in cytokine-deprived medium.
Cells were incubated with 500 pM rIL-15 or RLI for 0 to
48 h at 37°C. They were next plated at 0.2 × 106
cells in 100 μL, washed twice with PBS/0.1% BSA and incubated for
1 h at 4°C with 10 μg/mL anti-IL-15Rα, anti-IL-15Rβ or control
isotype IgG antibody. Cells were then washed three times with
PBS/0.1% BSA and incubated for 30 min in the dark at 4°C
with 1.25 μg/mL of PE-anti-goat IgG. They were washed three times
with PBS/0.1% BSA and analyzed on a FACScan fluorocytometer (BD
Biosciences) using FlowJo Software.
Results
IL-15 and RLI present different cell surface receptor
binding characteristics
In order to compare IL-15 cis- and trans-presentation modes, we
chose a human T lymphoma cell line, Kit225, expressing both
hIL-15Rα/β/γc and hIL-15Rβ/γc. Low doses of
IL-15 were used to stimulate the IL-15Rα/β/γc high
affinity receptor (cis-presentation), whereas the RLI fusion
protein previously described [20], was used to mimic
trans-presentation. As expected, [125I]-IL-15 displayed
high and low affinity binding sites corresponding respectively to
its binding to the trimeric IL-15Rα/β/γc receptors
(Kd = 0.037 nM; Bmax = 295 sites/cell), and
dimeric IL-15Rβ/γc receptors (Kd = 19.6 nM;
Bmax = 2784 sites/cell) (figure 1A). By
contrast, [125I]-RLI bound to a single class of high
affinity binding sites (Kd = 0.186 nM; Bmax =
3067 sites/cell) as expected for the dimeric IL-15Rβ/γc
receptors (figure 1B).
IL-15 and RLI induce different kinetics of cell
surface IL-15R down-modulation and display different kinetics
of internalization
Cell surface expression of IL-15Rα and IL-15Rβ chains was monitored
by flow cytometry following IL-15 or RLI treatment. As shown in
figure 2A,
IL-15 treatment induced a rapid reduction of IL-15Rα cell surface
expression, detectable as early as 15 min, reaching an almost
complete disappearance of expression at longer incubation periods
(24 h). By contrast, upon RLI treatment, IL-15Rα cell surface
expression was almost unchanged during the first hours, but then
decreased slightly up to 24 h. IL-15 treatment did not
significantly affect the IL-15Rβ pool during the 24 h
incubation period, whereas RLI induced a late decrease (figure 2B). These
results show that IL-15 and RLI both induce the disappearance from
the cell membrane of IL-15 receptor chains, but with different
kinetics, as the effect of IL-15 on IL-15Rα is much faster than
that of RLI on IL-15Rβ.
Cytokine internalization was then monitored after equilibration
of Kit225 cells with radio-iodinated cytokines and temperature
switching from 4°C to 37°C (figure 3).
[125I]-IL-15 and [125I]-RLI were both found
to be efficiently internalized (between 40% and 50% maximal
internalization) but [125I]-IL-15 internalization was
very quick (Int50 = 1.1 min) (figure 3A) compared
to that of [125I]-RLI (Int50 = 23.1 min)
(figure 3B). This
difference between [125I]-IL-15 and
[125I]-RLI as regards kinetics of internalization, was
comparable to that observed for the down-regulation of cell surface
IL-15R (figure 2A).
The maximal number of [125I]-IL-15 molecules
internalized per cell was five times lower than the maximal number
of [125I]-RLI molecules internalized per cell (figure 3C),
reflecting the fact that high affinity IL-15Rα/β/γc
accounts for a small proportion of the total IL-15R. This probably
explains why IL-15 induction of IL-15Rβ internalization through
IL-15Rα/β/γc could not be detected (figure 2B).
IL-15 and RLI activate similar signaling pathways,
but with different dose- and time-dependent
patterns
In order to compare the signal transductions induced by IL-15 and
RLI, activation of the Jak/STAT, PI3K/Akt and MAPKs pathways was
monitored by studying the phosphorylation of STAT5, STAT3, Akt, and
p44/42 MAPK (Erk 1/2). Firstly, Kit225 cells were stimulated for
15 min with increasing concentrations of IL-15 and RLI,
ranging from 0 to 1500 pM (figure 4). Both
cytokines were shown to stimulate the three signaling pathways in a
dose-dependent manner, and, for each cytokine, the activation
profiles were identical for all proteins of the signaling cascades.
However, the efficiency of both molecules was somewhat different.
The maximum signaling intensities for IL-15 were obtained at
50 pM, whereas at least 350 pM of RLI were necessary to
reach similar intensities. IL-15 therefore seemed five to seven
times more potent than RLI after a 15 min-incubation period.
We next compared the kinetic of the cell signaling induced by a
maximal concentration of IL-15 (50 pM) or RLI (350 pM) at
different time points between 0 and 48 h (figure 5A, B). Here
again, IL-15 and RLI induced the same activation profiles for all
the signaling proteins: the quantification of densitometric
scanning is shown only for STAT5. The signaling induced by IL-15
was strong, rapid (detectable as soon as 5 min, maximal by
15 min), and transient (strongly down-regulated after
1 h, and almost undetectable after 3-6 h). Signaling
induced by RLI was as strong and rapid as for IL-15, but was far
more persistent, being maintained at high levels until 16 h
before then decreasing although still detectable at 48 h.
We have previously shown that IL-15 and RLI were able to
induce identical proliferative responses by the Kit225 cell line
over 48 h [20], with EC50 values in agreement with
the activation of high-affinity receptors (EC50 ~
10-11 M). We therefore compared the signaling responses
at identical (50 pM) concentrations of both molecules (figure 5C, D) over
48 h. The response to RLI was slower than that to IL-15 and
was, again, more persistent. Calculation of the areas-under-curves
(AUC) indicated that the integrated signals of STAT5 induction from
0 to 48 h were similar (11.4 and 16.2 units × hour after
IL-15 and RLI treatments respectively), whether IL-15 or RLI was
used as a stimulus, which is consistent with the similar 48 h
proliferative responses induced by 50 pM IL-15 or RLI. When
calculating the AUC at different IL-15 or RLI concentrations, they
were found to be proportional to the proliferative responses (data
not shown), suggesting that these responses are based on an
integrative transmission mode rather than on the maximal intensity
of signaling.
RLI induced a prolonged effect on cell proliferation
after cytokine withdrawal
RLI, in contrast to IL-15, was still able to sustain cell signaling
after 5 h of stimulation (figure 5). In order
to further document this persistence, we compared the residual
proliferative responses of Kit225 cells after three days in the
presence of 500 pM IL-15 or RLI, followed by a 24-h
starvation. We first verified that the specific activities of the
cytokines were not affected during the three-day incubation period.
As shown in figure 6, the residual
proliferative response was significantly higher in the case of RLI,
indicating a higher capacity of RLI versus IL-15, to induce
long-term activation.
The sensitivity to RLI is more persistent than that
to IL-15 upon re-stimulation
Having shown that IL-15 and RLI induced different kinetics of
receptor chain down-modulation, cytokine down-regulation and signal
transduction, we examined how this could impact the biological
response upon cytokine re-stimulation. Kit225 cells, pre-treated or
not with IL-15 for 24 h, were subsequently stimulated for
1 h with 50 pM IL-15 or RLI, and analyzed for their level
of STAT5-phosphorylation (figure 7). In the
absence of IL-15 pre-treatment, and in agreement with figure 5C, pSTAT5
signals induced by IL-15 and RLI had similar, strong intensities.
By contrast, cells having experienced a 24 h IL-15
pre-treatment had a markedly reduced secondary response to IL-15,
while the secondary response to RLI was far less affected. This
result is in agreement with a more pronounced down-regulation of
IL-15Rα by IL-15 (figure 2) that mainly
affects IL-15 high-affinity receptors and leaves most of the RLI
receptors unaffected.
Discussion
A number of reports have documented the existence of two modes of
action of IL-15 (cis- and trans-presentation), and their relative
importance in the context of immune activation has been widely
discussed. In this study, we compared, on the same cell line, the
effect of IL-15 cis-presentation on the high affinity
IL-15Rα/β/γc to that of RLI, a protein resulting from
the fusion between hIL-15 and hIL-15Rα that mimics the mechanism of
IL-15 trans-presentation on the IL-15Rβ/γc complex. We
provide evidence that these two modes of action of IL-15 are
associated with different dynamics of receptor activation and
signal transduction.
Kit225 cells expressed two classes of IL-15 binding sites: a
majority (90%) of intermediate affinity (Kd ~
20 nM) receptors corresponding to IL-15Rβ/γc, and a
small proportion of high affinity (Kd = 0.037 nM)
receptors corresponding to IL-15Rα/β/γc. On the other
hand, the RLI fusion protein bound to a number of single class,
high affinity (Kd = 0.186 nM) receptors, corresponding
to the large pool of dimeric IL-15 receptors.
Analysis of IL-15 receptor chain internalization upon IL-15 cis-
or trans-presentation, revealed different behavior. In the context
of cis-presentation, IL-15 stimulation through
IL-15Rα/β/γc induced a quick extinction of cell surface
IL-15Rα, a result consistent with previous reports [28, 29], and
reflecting a rapid IL-15Rα/β/γc internalization.
A concomitant disappearance of cell surface IL-15Rβ chains
could not be detected under our experimental conditions, as
IL-15Rα/β/γc only accounts for 10%
of IL-15Rβ-containing receptors. In the context of
trans-presentation, RLI binding also led to the internalization of
IL-15Rβ/γc, as revealed by the down-regulation of cell
surface IL-15Rβ, but with kinetics that were far slower than those
associated with the down-regulation of IL-15Rα by IL-15
(cis-presentation). Although RLI induced a slight decrease in cell
surface IL-15Rα, the fusion protein did not bind the IL-15Rα chain
(G. B., data not shown). This finding is in agreement with the
notion that IL-15Rα seems to be pre-associated or in close
proximity with IL-15Rβ before cytokine binding. Such molecular
proximity of IL-15Rα with the β and γc chains in lipid
rafts on the surface of Kit225 cells has been suggested by flow
cytometry and confocal microscopic FRET measurements [30].
Differences in the kinetics of cytokine internalization were
also observed, and correlated well with those found for receptor
internalization. RLI was indeed found to be internalized through
IL-15Rβ/γc at a rate far lower than IL-15 through
IL-15Rα/β/γc (half-time of maximal internalization for
RLI > 20 times higher than for IL-15). The higher residual
proliferative response found in the case of RLI, after cytokine
withdrawal from the supernatant, was also consistent with a slower
internalization rate for RLI. Together, these data showed that the
kinetics of internalization of cytokine-receptor complexes were
much slower in the context of trans-presentation than in the
context of cis-presentation, suggesting differences in the
molecular events involved. This could be linked to a specific role
of IL-15Rα in contributing to a quick internalization of the
IL-15Rα/β/γc complex in the context of cis-presentation.
This could also be explained by the different interactions of
receptor complexes with other membrane molecules, such as MHC I and
II found in the molecular vicinity of IL-15R by FRET analysis [30],
or with the adjacent cytoskeleton, as this was shown recently to be
of major importance to the IL-7/IL-7R complex [31].
A number of studies have contributed to deciphering the
signaling cascades associated with the activation of IL-15
receptors. The intracellular domains of the IL-15Rβ and
γc chains are considered to be the major actors
responsible for the initiation of signal transduction. They lead to
the activation of multiple downstream pathways that include the
Jak/STAT, Ras/MAPK/Erk, and PI3K/Akt pathways [32-34]. This study
showed that these three main pathways were activated by both IL-15
and RLI; a more general analysis using a Phospho-Kinase array kit
(R&D Systems), revealed no qualitative differences between
IL-15- and RLI-induced protein phosphorylation (data not shown).
These results therefore indicate that the same signaling pathways
are activated in response to both cis- and trans-presented IL-15,
which is in agreement with the known, common dependence of their
biological responses on the IL-15Rβ/γc complex.
However, dose-dependent and kinetic analyses of signal
transduction revealed major differences between IL-15 and RLI
activation modes. The efficiency of IL-15 to stimulate the three
signaling pathways after a 15 min-incubation period was found
to be five to seven times greater than that of RLI. For instance,
the maximal effect on STAT5 phosphorylation was observed with
50 pM and 350 pM of IL-15 and RLI respectively. This
difference correlates with that found between the affinity
constants of IL-15 for IL-15Rα/β/γc (Kd =
0.037 nM) and that of RLI for IL-15Rβ/γc (Kd
= 0.186 nM). At these optimal concentrations (50 pM IL-15 and
350 pM RLI), the kinetics of induction of phosphorylation were
rapid and very similar. Overall, these results suggest that the
induction phase of the signaling response is dependent on the
affinity of the IL-15R to both cytokines. At later time points, the
duration of signal transduction was markedly different between
IL-15 and RLI. IL-15-induced signaling disappeared quickly, whereas
signaling persisted and decreased slowly in the case of RLI. These
observations are in agreement with those of Sato et al. [35],
showing, ex vivo, that ribosomal S6 phosphorylation in
CD8+ T cells persisted longer following IL-15
trans-presentation by IL-15Rα+-expressing DC cells than
after stimulation by soluble IL-15 (120 h versus 24 h
respectively). They also correlate with the kinetic differences
that we observed for both ligand internalization (20 times faster
for IL-15 than for RLI) and for down-regulation of
cell surface receptor chains after cytokine stimulation (fast
for IL-15 and slow for RLI), suggesting that the duration of
signaling is related to the time of residence of the
cytokine-receptor complex at the plasma membrane. These kinetic
differences between IL-15 and RLI internalization and signaling
were further analyzed for their impact on downstream biological
effects. At IL-15 and RLI concentrations previously shown to induce
similar proliferative dose-responses at 48 h, similar
integrated signaling intensities over 48 hours were also
found. More generally, the integrated intensities, as measured by
the AUC, were found to be proportional to the concentration of the
cytokine used. This suggests that the cellular proliferative
response is based upon an integrative transmission mode
(proportional to the AUC of signaling) rather than being based on
the kinetics of signaling. However, these observations have to be
confirmed in a more physiological context of trans-presentation
involving an IL-15 trans-presenting cell and a responding cell.
Since free IL-15 cannot be detected in biological fluids under
physiological conditions, it was suggested that IL-15 acts mainly
as a membrane-associated protein bound to its high affinity IL-15Rα
chain, IL-15Rα behaving as a necessary chaperone for the
trafficking, production and secretion of IL-15 [36]. However,
free-circulating IL-15 can be detected in the serum of patients
with inflammatory and auto-immune diseases, or pathogen infections
[37-39], suggesting that it may also function by cis-activation of
the heterotrimeric IL-15R. A number of reports have documented
this mode of action [30, 40, 41]. A structural model has been
proposed [41] in which the highly flexible nature of the linker
and/or proline-threonine-rich region of IL-15Rα allows the
presentation of IL-15 in both cis- and trans-modes. Thus, IL-15
could be cis-presented, particularly in inflammatory situations.
The early and rapid up-regulation of the expression of the IL-15Rα
chain should allow a fast expansion of antigen-responsive T and NK
cells and a strong immune response [42]. Afterwards, fast IL-15
down-regulation of its IL-15Rα chain, as shown in this study, would
avoid harmful consequences due to excessive activation, including
overproduction of inflammatory cytokines, extensive cell lysis, and
incoherent adaptative cell responses. Pillet et al. [43]
showed in vitro that human NK cell sensitivity to free IL-15
is increased in early activation stages, whereas their response is
redirected at later stages toward IL-2 and trans-presented IL-15.
The authors described a sequential expression of IL-15Rα and
IL-2Rα, which may play a key role in coordinating the innate and
adaptive branches of the immune system. The slower down-regulation
of the IL-15Rβ/γc complex, as highlighted in our
study, would then allow the cell to remain competent for IL-15
trans-presentation, a process that has been shown to be important
for the long-term maintenance of antigen-memory cells.
We have recently shown that levels of a soluble form of IL-15Rα
(sIL-15Rα) are elevated in the serum of head and neck cancer
patients [44], and increased in the serum of patients with Crohn's
disease that respond to infliximab treatment [39]. The sIL-15Rα
protein, generated by proteolytic cleavage or through the
expression of an alternative spliced variant of IL-15Rα [17, 45,
46], can act as a chaperone of IL-15, enhancing its biological
activity. The soluble IL-15Rα could therefore trans-present IL-15
to responding cells without the need for cell-cell contact. In
addition, sIL-15Rα has also been shown to increase the half-life of
IL-15, and could therefore facilitate the diffusion of the cytokine
and its action on remote cells and tissues expressing the
IL-15Rβ/γc receptor [19]. Similarly, IL-6 is known to
bind a naturally occurring, soluble form of the IL-6R chain to form
a complex that can stimulate cells expressing the signal
transducing gp130 protein in the absence of IL-6R [47]. This
mechanism, termed IL-6 “trans-signaling”, is involved in the
maintenance of the disease state of many chronic inflammatory
diseases [48]. In view of our results, it would be interesting to
explore whether IL-6 cis- and trans-signaling also involve
different dynamics of receptor activation.
In summary, our present results demonstrate that IL-15 cis- and
trans-presentation modes lead to different kinetics of receptor
expression, cytokine internalization, and sequential cell
signaling. This controlled distribution of IL-15, spatially and
temporally, may constitute a program limiting the unwanted
consequences of a powerful cytokine. Cis- and trans-presenting
modes could equally play a key role in the coordination between
innate and adaptative immunity. This work provides clues for a
greater understanding of the IL-15 system, and consequently the
design and optimization of immunotherapeutic treatments based on
the use of cytokines as adjuvants.
Acknowledgments
We particularly thank Agnès Quéméner for helpful discussions and
the reading of this manuscript.
Disclosure and financial support. This work was
supported, in part, by INSERM, CNRS, Région Pays de Loire
(grant CIMATH), the Association pour la Recherche sur le Cancer,
and the Ligue Nationale contre le Cancer. The costs of publication
of this article were defrayed in part by the payment of
page charges. This article must therefore hereby be marked
advertisement in accordance with 18 U.S.C. Section 1734, solely to
indicate this fact. None of the authors has any conflict of
interest to declare.
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