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Department of Molecular Biology, Molecular Immunology Unit, Flanders Interuniversity Institute for Biotechnology and University of Ghent, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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c subunit of the IL-2R. Yet, the
requirement of IL-15 for an IL-15R
chain for high affinity
interaction and the separate cellular sources of IL-2 and IL-15 suggest
divergent activities for both cytokines. We compared the
growth-inducing and proapoptotic or antiapoptotic activities of IL-15
and IL-2 on mature CD4+ T lymphocytes in the presence or
absence of TCR occupancy. We found that the nature of IL-15 activity
was critically dependent on the activation status of the T cells. In
the absence of TCR triggering, IL-15 did not exert the growth factor
activity of IL-2, but induced a quiescent phenotype, characterized by
maintenance of the cells in the G0/G1 phase of
the cell cycle and down-regulation of CD25, CD71, and CD95 expression.
In the presence of appropriate TCR engagement, the IL-15-induced
quiescent T cells were resistant against TCR-induced cell death and
proliferated strongly. IL-2-treated cells, on the contrary, were
sensitized to cell death, resulting in a negative feedback on cellular
expansion and weak proliferative responsiveness. Consecutive action of
IL-15 during the distinct phases of an in vitro immune response
markedly increased the cell output of a second antigenic stimulation,
as compared with IL-2. These results imply that during immune
reactivity in vivo, IL-15 may take over from the transiently available
IL-2 the role of survival factor but not of growth factor, hence
promoting the long term maintenance of resting, Ag-experienced
CD4+ T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-2Rß
knockout mice, these animals suffer from severe lymphoproliferative
diseases and autoimmunity. This phenotype is a consequence of the
inability to activate the Fas/Fas ligand
(FasL)3 death pathway (3),
which is the main effector mechanism for maintenance of peripheral
lymphoid homeostasis (reviewed in 6 . In addition to TCR-induced
death, apoptosis induced by deprivation from growth factor is a second
general mechanism for restoration of cellular homeostasis after
subsidence of an immune response (7). The transient nature of IL-2
production by activated T cells, critically dependent on growth factor
for their survival, adds to the deletion of superfluous effector cells
once the Ag has been cleared successfully. Therefore, IL-2
contributes to both the development and the conclusion of a primary
immune response.
IL-15 is a cytokine that was cloned from CV-1/EBNA (8), a simian kidney
epithelial cell line, and from the human T cell leukemia cell line
HUT-102 (9). Although IL-15 does not show sequence homology with IL-2,
both cytokines share many biologic functions. IL-15 induces
proliferation of the CD8+ T cell clone CTLL-2 and of
phytohemagglutinin-activated CD4+ and CD8+
human peripheral blood T lymphocytes (8). IL-15, like IL-2, promotes
differentiation and growth of human B cells (10), induces development
of NK cells (11), activates cytotoxic activity of NK cells (12), and is
a chemoattractant for T lymphocytes (13). These overlapping activities
are not surprising, since both cytokines use the same IL-2Rß and
c-chain for binding and signal transduction (14).
Nevertheless, the composition of the high-affinity receptor of both
cytokines differs, because IL-15 uses a specific IL-15R -chain that
is structurally similar to IL-2R, but does not bind IL-2 (15). As a
consequence, a differential regulation of both -chains, as reported
by Kumaki et al. (16), may determine the reactivity to either cytokine.
Also, IL-15 and IL-2 differ in their cellular source of production.
IL-15 mRNA is most abundantly found in fibroblast and epithelial cell
lines, placenta, skeletal muscle, and activated peripheral blood
monocytes (8). Activated T lymphocytes, however, do not produce any
IL-15 protein (17) but are the exclusive source of IL-2. The transient
and local nature of IL-2 production, as opposed to the presumed
persistent and systemic availability of IL-15, provides a mechanism in
vivo for the distinct activities of these cytokines. Furthermore, it
has recently been demonstrated that IL-15, but not IL-2, protects
against Fas-mediated apoptosis in the liver, spleen, and thymus of mice
treated with an IL-15 IgG2b fusion protein (18). This result indicates
that both cytokines also exert different functions. Therefore, it is
reasonable to hypothesize that IL-15 has a distinct place in the
regulation of T cell responses.
In the present study, we followed the pro- and antiapoptotic as well as growth-inducing activities of IL-2 and IL-15 during the course of an in vitro CD4+ T cell response. The fate of the T cells was followed during TCR activation, after subsidence of activation when the cells became devoid of autocrine growth factor, and finally, during rechallenge with Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 (H-2b) mice were purchased from the Broekman Instituut (Eindhoven, The Netherlands). All mice were used at the age of 9 to 14 wk.
CD4+ T cell clone
The influenza A/H3 hemagglutinin (HA)-specific and H-2b-restricted CD4+ murine T cell clone T-HA was developed in our laboratory by initial immunization of C57BL/6 mice with 10 µg bromelain-cleaved hemagglutinin (BHA) and 0.1 ml Ribi adjuvant (Ribi Immunochem Research, Hamilton, MT) and a second immunization with 3 µg BHA after 3 wk. Five days after this boost immunization, lymph nodes were isolated, and 3 x 107 cells were stimulated in vitro with 0.5 µg/ml BHA in 25-cm2 culture flasks. On day 4, 10 U/ml murine IL-2 (from PMA-stimulated EL4.IL-2 cells) was added to the cultures. After 2 additional biweekly restimulations with 0.5 µg/ml BHA and APC, a pool of optimally HA-reactive T lymphocytes was obtained. T-HA cells were maintained long term in vitro by biweekly restimulation in 25-cm2 culture flasks with 10 ng/ml BHA and 7 x 107 syngenic spleen cells from C57BL/6 mice (3000 rad gamma-irradiated). On day 2, 30 IU/ml of human IL-2 was added, after which T cells were further cultured and expanded by medium renewal and addition of IL-2 every 4 days. T-HA cells were cultured in 12.5 mM HEPES-buffered RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% FCS, 2 mM GlutaMAX-I (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 5 x 10-5 M 2-ME.
Cytokines
Human rIL-15 was purchased from PeproTech (London, U.K.) and had a sp. act. of 2 x 106 U/mg. Human rIL-2 was produced in our laboratory and had a sp. act. of 1.3 x 107 IU/mg as determined in a CTLL-2 assay (1 IU corresponds to 77 pg). These cytokine preparations were used throughout this study.
Isolation of viable cells and IL-2 or IL-15 pretreatment
T-HA cells were harvested from cultures by incubation in nonenzymatic cell dissociation buffer (Sigma Chemical, St. Louis, MO). Dead cells were removed by centrifugation on a Histopaque-1077 density gradient (Sigma-Aldrich, Irvine, U.K.) for 25 min at 2000 rpm. Routinely, cultures contained less then 2% dead cells after density gradient centrifugation. Recovered cells were washed three times before further use. For IL-2 or IL-15 pretreatment, 2–5 x 105 viable T-HA cells were cultured for 48 h in 24-well flat-bottom tissue culture plates in the presence of variable concentrations of IL-2 or IL-15.
Analysis of viable and dead cell populations
Viable cell numbers were determined in a hemocytometer on the basis of trypan blue exclusion. Apoptosis was analyzed by addition of 30 µM propidium iodide (PI; ICN Pharmaceuticals, Costa Mesa, CA) to harvested cells; the percentage of PI+ cells was measured with an EPICS 753 flow cytometer (Coulter Electronics, Luton, U.K.), equipped with an argon-ion laser emitting at 488 nm, after gating out cell debris. PI fluorescence was detected at 610 to 630 nm. Additionally, the percentage of apoptotic cells was also determined by forward light scatter analysis (not shown). In all experiments, data obtained by the latter method correlated well with the PI dye uptake data.
Abs and reagents
For immunofluorescence, rat anti-mouse CD25 (clone PC 61), rat anti-mouse CD71 (clone R217 17.1.3, kindly provided by Dr. G. Leclercq, Ghent, Belgium), and hamster anti-mouse CD95 (clone Jo2; PharMingen, San Diego, CA) were used as primary Abs. Anti-CD25 and anti-CD71 binding was detected with a FITC-conjugated goat anti-rat IgG (Sera-Lab, Crawley Down, U.K.). FITC-conjugated anti-hamster IgG (clone G70-204; PharMingen) was used as secondary Ab for anti-CD95. Purified anti-CD3 mAb (145-2C11; kindly provided by Dr. G. Leclercq) was used at a concentration of 10 µg/ml in PBS to coat flat-bottom microwells (30 µl/well) for 2 h at 37°C. Unbound Ab was removed before adding cells. The mitochondrial transmembrane potential was measured by addition of 1 µM rhodamine 123 (Molecular Probes, Eugene, OR) to the cells for 30 min and subsequent flow cytometric analysis of the fluorescence intensity.
Proliferation assays
Cells, cultured under the conditions indicated, were harvested and washed three times to remove cytokines. Cytokine-induced proliferation was measured by incubating 1 x 104 T-HA cells with serial dilutions of IL-2 or IL-15. [3H]TdR (Amersham Life Science, Amersham, U.K.) was added at 0.5 µCi/well for the last 8 to 12 h of incubation. Cells were harvested on glass fiber filters, and [3H]TdR incorporation was measured by liquid scintillation in a TopCount (Packard Instrument, Meriden, CT). All results are means of triplicate cultures. Ag-induced proliferation was determined with 200 ng/ml BHA and 2 x 105 irradiated C57BL/6 spleen cells (as a source of APC) in 96-well flat-bottom microtiter plates. Cultures were pulsed with [3H]TdR for the last 12 h of an 84-h assay period. Results shown are the means of triplicate wells. Cocultures of T cells and APC without Ag were always included in the experiments as a control on the Ag dependency of the response. Proliferation of these cultures never exceeded 1000 cpm (not shown).
Cell cycle analysis
T-HA cells were harvested, washed once in cold PBS, and lysed in Krishan’s reagent (0.05 mg/ml PI, 0.02 mg/ml ribonuclease A, 0.3% Nonidet P-40, 0.1% sodium citrate). Cell nuclei were analyzed for DNA content by flow cytometry; the distribution of cells along the distinct stages of the cell cycle was calculated with Para1 software (Coulter Electronics).
Semiquantitative RT-PCR
mRNA isolation was conducted with a MicroFastTrack kit (Invitrogen, San Diego, CA). cDNA was synthesized in the presence of RNase block (Stratagene Cloning Systems, La Jolla, CA) after the addition of oligo(dT) primer (Boehringer, Mannheim, Germany) and incubation at 37°C with Superscript II reverse transcriptase (Life Technologies). The primers used for PCR amplification were 5'-CAGCTCTTCCACCTGCAGAAGG-3' and 5'-CAATATTCCTGGTGCCCATGAT-3' (murine FasL, 597-bp fragment), as well as 5'-TGGAATCCTGTGGCATCCATGAAAC-3' and 5'-TAAAACGCAGCTCAGTAACAGTCCG-3' (murine ß-actin, 348-bp fragment). The PCR reaction mixture contained 1.5 mM MgCl2, 0.4 mM dNTP, 200 nM primers, and 0.5 U of Goldstar Taq polymerase (Eurogentec, Seraing, Belgium). Samples were amplified during 35 cycles (FasL) or 30 cycles (ß-actin) (1 min denaturation at 94°C, 2 min annealing at 58°C, and 1 min extension at 72°C) in a Peltier Thermal Cycler-200 (MJ Research, Watertown, MA). In each PCR, water was included as a negative control. For semiquantitative RT-PCR, four twofold dilutions of each cDNA sample were amplified. PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining.
Cell membrane labeling by means of an aliphatic fluorescent molecule
T-HA cells were harvested and washed twice in serum-free medium. Cells (1 x 106–107) were resuspended in 1 ml of diluent A and stained with the membrane stain PKH2-GL (2 µM; Sigma Chemical) following the manufacturer’s instructions. Stained cells were washed twice with serum-containing medium and were incubated overnight in their culture medium to allow dissociation of excess dye from the membrane. In mixed cultures of PKH-2GL-stained T-HA cells and splenocytes, percentages of viable and apoptotic T-HA cells were obtained by flow cytometric analysis of PI- and PI+ cells, respectively, emitting green fluorescence (525 nm).
Experiments with freshly isolated spleen cells
Spleen cells (8 x 108) were prepared from the
spleens of naive, 8-wk-old C57BL/6 mice and were activated in
25-cm2 tissue culture flasks with 1 µg/ml soluble
anti-CD3 mAb (145-2C11). After 24 h, excess Ab was removed,
and cells were further cultured for 72 h without addition of
exogenous cytokine. Following this stimulation period, cultures were
harvested, and CD4+ T cells were isolated by immunomagnetic
cell sorting. A negative selection procedure, using an Ab mixture
designed for the enrichment of murine CD4+ T cells
(StemSep; Stem Cell Technologies, Vancouver, Canada), was followed
according to the manufacturer’s instructions. Recovered cells
(7.5 x 106) were further cultured for 10 days and
supplemented (every fourth day) with their respective cytokines
(none, 10 ng/ml IL-15, or 10 ng/ml IL-2). Viable cell numbers
were determined on day 14, based on trypan blue dye exclusion. For
restimulation, we used 1 µg/ml soluble anti-CD3 mAb and the
immortalized macrophage cell line Mf4/4 (19). Before use, Mf4/4 cells
were activated for 24 h with 400 U/ml IFN- to enhance
expression of costimulatory molecules. The cells were then treated for
90 min with 30 µg/ml mitomycin C (Duchefa, Haarlem, The Netherlands)
to block their proliferation, thus avoiding interference with
proliferation measurements from the restimulated lymphocytes.
Alternatively, for determination of susceptibility to
anti-CD3-induced death, freshly isolated, unsorted spleen cells
were activated for 72 h in 24-well plates with 1 µg/ml soluble
anti-CD3 mAb (145-2C11) without exogenous cytokine and were
supplemented on day 3 with 10 ng/ml IL-15 or IL-2. After an additional
8-day culture period, viable cells were isolated on a Histopaque
density gradient and restimulated with plate-bound anti-CD3 mAb (10
µg/ml). Apoptotic cell numbers were determined after 24 h by PI
dye uptake. CD4:CD8 ratios were determined by labeling 1 x
105 cells with 0.5 µg PE-conjugated rat anti-mouse
CD4 mAb (PharMingen) and 0.5 µg/ml FITC-labeled rat anti-mouse
CD8 mAb (clone 53-6.7, kindly provided by Dr. G. Leclercq) and, after
gating out dead cells and debris, analysis of stained populations on a
FACScalibur flow cytometer (Becton Dickinson (Sunnyvale, CA)).
Absolute numbers of CD4+ T cells in the respective cultures
were calculated from the percentages obtained and total viable cell
countings by trypan blue dye exclusion.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The T-HA helper T cell clone was routinely cultured by biweekly
antigenic stimulation followed by addition of exogenous IL-2. This
culture condition has become the standard procedure to propagate
Ag-specific T cell clones for prolonged periods in vitro. Addition of
IL-2 at the time when autocrine production ceases is necessary to
ensure further survival of the activated T lymphocytes in the periods
between a repeated challenge with Ag. However, this IL-2 not only
promotes survival but also supports further expansion of the T cells,
thus keeping the T cells in a semiactivated, proliferative state, not
representative of the in vivo situation in which it is believed that
Ag-stimulated T cells persist as small, resting lymphocytes once the Ag
has been cleared (20). In our study, we replaced IL-2 with IL-15 in the
periods between antigenic stimulation and examined the evolution of
viable cell numbers and occurrence of cell death. T-HA cells, harvested
4 days after stimulation with Ag/APC, were cultured in the presence of
decreasing concentrations of IL-15 (6.6–0.03 ng/ml; 460.0–2.0
pM) or IL-2 (9.0–0.03 ng/ml; 585.0–2.0 pM). After 3 days of
treatment, the absolute numbers of viable cells and the percentage of
apoptotic cells in the various cultures were determined (Fig. 1
). As expected, incubation with IL-2
resulted in a dose-dependent increase in cell numbers (Fig. 1A). In contrast, treatment with IL-15 kept the number of
viable cells stable at 
30,000, which is slightly above the input of
20,000 (Fig. 1B). Numbers of viable cells dropped
dramatically when IL-15 or IL-2 was omitted from the cultures.
Concomitantly, extensive cell death was observed as a consequence of
growth factor deprivation. Addition of IL-15 reduced cell death to
background levels, comparable to IL-2. Even with concentrations as low
as 0.08 ng/ml (6 pM) IL-15, no significant cell death was observed. It
may be noted that the minimal concentration of IL-2 required for a
similar full protection was 30- to 40-fold higher, namely 3 ng/ml
(200 pM). From these data, we conclude that IL-15 induces a survival
signal in Ag-primed CD4+ T lymphocytes that, contrarily to
IL-2, is not accompanied by an increase in cell number.
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IL-15 has been described previously as a factor that induces and
sustains the growth of mitogen-stimulated CD8+ T cells and
also CD4+ T cells, much in the same way as IL-2 does (8).
However, the observed stabilization of cell numbers without an apparent
occurrence of cell death suggests that IL-15 silences DNA synthesis
after TCR-induced stimulatory signals have subsided. To verify whether
these stable cell numbers in IL-15-treated cultures indeed reflect an
IL-15-induced growth arrest, T-HA cells derived from standard IL-2
cultures were assayed for proliferation in response to increasing
concentrations of IL-15 or IL-2. From the results shown in Figure 2A, it is clear that IL-15,
even in concentrations as high as 200 ng/ml (14 nM), does not induce
DNA synthesis. Furthermore, starting from an actively dividing
population, IL-15 induced a gradual transition of T cells to a
nondividing condition, while IL-2 further supported cell proliferation
(Fig. 2B). These data, together with the results from Figure 1
B, demonstrate that IL-15 is a survival factor but not a
growth factor for CD4+ T lymphocytes when TCR aggregation
is absent.
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It is thought that after conclusion of a primary immune response,
a fraction of activated effector cells reverts to a resting state and
persists in the animal as a population of small lymphocytes, ready for
a "memory" response in case of reemergence of their Ag (20). We
wondered whether T-HA lymphocytes surviving with IL-15 without cycling
could be phenotyped as small, resting lymphocytes. Therefore, a number
of features generally recognized as parameters for lymphocyte
quiescence were studied. We determined whether the observed growth
arrest took place in a specific phase of the cell cycle. Cell cycle
analysis by flow cytometry revealed that IL-15-treated cells
accumulated in the G0/G1 phase (Fig. 3), indicative of the induction by IL-15
of an arrest in cell cycle entry. Thus, cycling cells treated
with IL-15 are arrested neither immediately nor randomly, which in fact
would be apoptosis inducing, but proceed with their cycle until they
reach G0/G1 and then exit cell cycle
progression in an orderly manner without triggering programmed cell
death (PCD). Additionally, cell size, expression of activation
markers, and the mitochondrial transmembrane potential as indicators of
the metabolic state of the cells were evaluated. IL-15-treated
T-HA cells exhibited all the hallmarks of resting cells: the cells were
small, expressed low levels of the CD25 (IL-2R) and CD71
(transferrin receptor) activation markers, and had a low mitochondrial
transmembrane potential (Fig. 3). In contrast, IL-2-cultured cells were
large blastoid cells with high CD25 and CD71 expression levels and a
high oxidative metabolism, as indicated by the increased mitochondrial
transmembrane potential. Thus, the IL-15-induced arrest in the
G0/G1 phase of T-HA cells is accompanied by
acquisition of a typical quiescent phenotype.
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IL-2, secreted by TCR-activated T lymphocytes, regulates
TCR-induced death by sensitizing T cells to execution triggered by
Fas/FasL interactions (1, 6). This mechanism for TCR-induced cell death
has primarily been demonstrated by pretreating CD4+ T cells
with IL-2 and subsequently activating them with plate-bound
anti-CD3 mAb. We compared IL-2 and IL-15 in their ability to
sensitize to TCR-induced death. In a first type of experiment, T-HA
cells were pretreated for 48 h with either IL-15 or IL-2, then
assayed for their sensitivity to cell death induced by immobilized
anti-CD3 mAb. As described above, cell cultures differed in their
survival and cell cycle status at the moment viable cells were isolated
for CD3 triggering, according to the cytokine added during
pretreatment: IL-15 kept the T-HA cells fully viable but in a
growth-arrested state, high-dose IL-2 (10 ng/ml; 650 pM) induced
vigorous cell cycling, and low-dose IL-2 (0.1 ng/ml; 6.5 pM) resulted
in poor viability (50% survival). As shown in Figure 4A, T-HA cells pretreated with
high or low IL-2 concentrations were susceptible to
anti-CD3-induced death. This cell death, shown as percentages of
cells that had taken up the exclusion dye PI, showed all of the typical
features of apoptosis, namely membrane blebbing, nuclear condensation,
hypoploidy, and disintegration of the cells into apoptotic bodies (not
shown). In contrast to IL-2, IL-15 pretreatment resulted in a strong
resistance to anti-CD3-induced death (Fig. 4A). As
Fas/FasL interaction is believed to be the actual trigger of
TCR-induced death in mature T lymphocytes (21), we evaluated whether
altered levels of Fas or FasL expression in IL-2- or IL-15-treated T-HA
cells underlaid the differential susceptibility to anti-CD3-induced
death. Although Fas expression before activation was slightly lower in
IL-15- than in IL-2-cultured cells, in agreement with the resting vs
activated state of the respective populations, both showed a vigorous
and similar up-regulation of Fas in response to CD3 triggering (Fig. 4B). Also, IL-2 or IL-15 treatment did not significantly
affect FasL mRNA levels before and after activation (Fig. 4C). As a control, ß-actin was amplified to make sure that
equivalent amounts of cDNA were present in all samples. These data
demonstrated that protection against anti-CD3-induced death by
IL-15 was not the consequence of an IL-15-induced impairment to express
Fas or FasL after TCR activation. They suggest, rather, that
IL-15-mediated protection is based on interference with the
Fas/FasL-signaling pathway. It has recently been documented that IL-15
protects CD8+ T cells and B cells against Fas-induced
apoptosis (18). Our results confirm these data and add that
CD4+ T cells also can be protected against TCR-induced
apoptosis by IL-15.
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, clearly demonstrate that
pretreatment with 10 ng/ml of IL-2 resulted in considerable cell death
48 to 72 h after activation with Ag/APC. T-HA cells precultured
with IL-15, or those that had survived low dose (0.1 ng/ml) IL-2
pretreatment, on the contrary, showed no increase or even a slight
decrease in cell death as compared with initial background levels at
24 h. These data clearly demonstrate that T cells cultured with
IL-15 are desensitized to cell death triggered by appropriate
stimulation with Ag. Also, the fraction of cells that survived in the
cultures with low dose IL-2 (50%) were resistant to TCR Ag-induced
death. In agreement with previous reports (1, 6), high levels of IL-2
raise the susceptibility of the cells.
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Resistance against TCR Ag-induced cell death is expected to result
in an enhanced proliferative response of T-HA lymphocytes upon exposure
to Ag. To verify this, we pretreated T-HA cells with IL-15 or high-dose
IL-2 and stimulated the cells with their Ag presented by appropriate
spleen APC. These Ag-stimulated cultures were set up either without
exogenous cytokine or with the addition of IL-2 or IL-15.
[3H]TdR was added to the cultures after 72 or 120 h.
The data shown in Figure 6A
demonstrate that pretreatment with IL-15 resulted in enhanced
proliferation to Ag as compared with IL-2 pretreated cells, whether or
not IL-2 or IL-15 was added during the restimulation period. This
indicates that the status of resistance to TCR Ag-induced death caused
by IL-15 enhances the potential of T-HA cells to respond to a renewed
Ag challenge. Addition of IL-15 to the cultures during the assay period
further supported the proliferative response. Although this effect was
limited at 72 h, probably due to competition with autocrine IL-2
for IL-2Rß and -chains, it became spectacular when the cells were
cultured for 120 h (Fig. 6B). At this time point,
proliferation in response to Ag subsided. The availability of IL-15,
but not of IL-2, at this time point not only prolonged but also further
increased the response. Clearly, IL-15 not only acts as a survival
factor but, in these conditions, also shows a pronounced growth factor
activity. Correlating these results with the conclusions drawn before,
it may be concluded that IL-15 either induces quiescence or supports
growth, depending on whether TCR cross-linking occurs or not.
Furthermore, this concept reconciles the quiescence-inducing activity
that we have demonstrated herein with previous reports describing IL-15
as a cytokine with growth factor activity for PHA-activated
CD4+ T lymphocytes (8).
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Fresh, unsorted spleen cells from naive C57BL/6 mice were isolated
and polyclonally stimulated in vitro. The stimulus consisted of soluble
anti-CD3 mAb (1 µg/ml), which in the presence of costimulation by
spleen APC, polyclonally activates naive T cells (22). After 24 h,
the remaining anti-CD3 mAb was removed, and the activated cells
were further cultured in the absence of exogenous cytokine. To confirm
that activation occurred, anti-CD3-activated and unstimulated cells
were pulsed with [3H]TdR. Soluble anti-CD3 mAb
induced a strong proliferative response: 25,304 cpm as opposed to 2,581
cpm for unstimulated cells. On day 4, CD4+ cells were
isolated by immunomagnetic cell sorting and further cultured without
cytokine or in the presence of IL-15 (1 ng/ml; 70 pM). After 10 days of
culture in the absence of exogenous cytokine, viable cell numbers had
dropped to 15% of the cell input, while IL-15 maintained cell numbers
at 60% of cell input (Fig. 7A). Cells surviving with
IL-15 (10 ng/ml; 700 pM) appeared as small resting lymphocytes and did
not reveal DNA synthesis (59 cpm), whereas proliferation could be
induced with IL-2 (4,184 cpm with 10 ng/ml (650 pM)). Hence, for
freshly isolated and TCR-activated CD4+ T cells as well,
IL-15 acts as a survival factor and induces quiescence. Next, we
investigated the resistance to TCR-induced cell death, triggered by
immobilized anti-CD3 mAb, in these polyclonally activated T cell
cultures. The CD4+ T cell population maintained throughout
with IL-15 was largely resistant, whereas cells cultured with IL-2
showed extensive cell death (Fig. 7B). Finally,
CD4+ T cells residing in an IL-15-induced resting state
proliferated in response to renewed stimulation with soluble
anti-CD3 and APC, while cells maintained with IL-2 did not (Fig. 7C). Also, the addition of IL-15 to the IL-15 pretreated
cultures further increased the proliferative response, thus confirming
the growth-promoting activity of IL-15 in the presence of TCR
aggregation. These experiments demonstrate that the characteristics
induced by IL-15 in the clonal CD4+ T cell T-HA, namely
long term survival as a resting population, resistance to apoptosis,
and increased responsiveness to TCR restimulation are also acquired by
freshly isolated CD4+ T cells treated with IL-15.
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To verify the significance for long term T cell responses of the
combined activities exerted by IL-15, a comprehensive experiment was
conducted (Table I). T-HA cells, primed
either by IL-15 or by high dose or low dose IL-2, were given a first
stimulation with Ag/APC and, after subsidence of the response, were
further cultured with the respective cytokine concentrations for 8
additional days. Next, the cultures were analyzed for their
quantitative and qualitative secondary response potential. Starting
from a fixed number of IL-15-treated T-HA cells (1 x
105), the combination of optimal proliferation in response
to Ag/APC stimulation and subsequent long term persistence of the
generated effector cells with IL-15 resulted on day 12 in a 16-fold
increase of T cells available for a renewed Ag/APC response (Table I,
Expt. 1). A similar treatment schedule with high dose IL-2 or low dose
IL-2 raised T cell numbers 7.6- and 1.2-fold, respectively. A
comparable result was obtained in another independent experiment (Table I, Expt. 2). Next, the various cultures were harvested, and equal cell
numbers were examined for their functionality upon a second antigenic
restimulation. As expected from the above experiments, cells cultured
with IL-15 or low dose IL-2 expanded vigorously, resulting in the
accumulation of high numbers of immune effector cells on day 16, as
measured by both cell counting (Table I, Expt. 1) and
[3H]TdR incorporation (Table I, Expt. 2). These different
yields and this differential responsiveness to Ag/APC of the respective
T cell populations were combined in a recovery and reactivity index,
indicative of the strenght of the secondary immune response. As shown
in Table I, these indices are dramatically higher for the cell
populations kept in IL-15. Obviously, the enhancement of availability
and response potential of CD4+ T cells by IL-15 has a
cumulative effect, resulting in strongly enhanced secondary responses.
These features could not be achieved by either dose of IL-2. These
results clearly demonstrate that IL-15, but not IL-2, has the
properties required for generating an efficient secondary T cell
response, thus providing a strong survival signal that allows the long
term persistence of immune effectors in a quiescent state as well as
simultaneously priming these cells for an optimal response when Ag
exposure reoccurs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Perhaps the most important new finding of our study is the ability of IL-15 to keep Ag-experienced T cells in a quiescent condition for prolonged periods by providing the necessary signals for survival in the absence of TCR engagement. Persistence of Ag-experienced T lymphocytes for prolonged periods—according to some studies, even for the life span—after elimination of the pathogen involved, likely requires survival factors from the microenvironment to maintain these T cells, devoid of autocrine growth factors, in a primed but resting state (23). Candidates that have recently been proposed for this helper function are an unidentified, >30-kDa factor secreted by stromal cells (24), which promotes T cell survival without inducing proliferation, and TGFß (25). TGFß can reverse the effector T cell population into a more resting state and synergizes with IL-2 to prevent the induction of apoptosis in T lymphocytes of the Th2 type. Also, IL-4, IL-7, and IL-15, initially described as T cell growth and differentiation factors (8, 26, 27, 28), induce a strong survival signal in T cells deprived of endogenous IL-2 (29). However, this protection was always accompanied by the onset of cell cycle progression, also when IL-15 was used to maintain the cells alive. The fact that the authors used T lymphocytes shortly after activation with PHA possibly explains the observed mitogenic activity of IL-15 in their system. Furthermore, it is unlikely that, after subsidence of an immune response, the levels of IL-2, IL-4, or IL-7 in the animal would be sufficient to support long term survival of Ag-experienced T cells. On the other hand, the previously described widespread distribution of IL-15 mRNA in the placenta, skeletal muscle, kidney, lung, heart, fibroblasts, epithelial cells, and monocytes, but not in T cells (8, 17), agrees with the notion of IL-15 as a microenvironmental factor that also remains available when the immune response has been terminated. This persistent presence of IL-15, combined with our data showing that IL-15 induces quiescence when TCR engagement is absent and simultaneously permits survival in the absence of autocrine growth factor, suggests that IL-15 could be an important cytokine for the survival of descendants of activated CD4+ T lymphocytes as resting memory cells. Although the continuous presence of IL-15 was required to maintain the state of resistance to PCD (data not shown), our data nevertheless demonstrate that very low levels (0.08 ng/ml (6 pM) or less) of the cytokine, likely to be available in different tissues, are sufficient for T cell survival. Recent studies suggesting the importance of IL-4, IL-6, and IL-7 for survival of virgin T cells (30, 31) further support the idea that cytokines play a pivotal role in the long term maintenance of T cells in vivo.
Several authors have demonstrated that IL-2, transiently secreted
during immune reactivity, regulates the immune response in a bivalent
way by promoting T cell clonal expansion as well as sensitizing the
cells to TCR-induced cell death triggered by Fas and/or TNF-R55
(reviewed in 1 . The prevention of TCR-induced cell death that we
observed in CD4+ T lymphocytes exposed to IL-15 provides an
escape mechanism from cell death for Ag-primed T cells. The mechanism
by which IL-2 sensitizes T lymphocytes to TCR-induced cell death
remains unclear. Signals that affect cell division are known also to
affect the cell death program. Overexpression of the survival factor
Bcl-2 retards transition from G0 to S phase and represses
TCR-induced death, whereas the opposite effects are observed in T
lymphocytes from Bcl-2-deficient mice (32, 33). Also, mature T cells
expressing a proapoptotic bax transgene show accelerated S phase
entry in response to IL-2 (34). However, the correlation between the
proapoptotic property of IL-2 and its ability to drive T lymphocytes
into the S phase of the cell cycle remains controversial (35, 36, 37). Our
observation that IL-15-treated CD4+ T lymphocytes are
blocked in the G0/G1 phase and are desensitized
to TCR-induced death supports the hypothesis whereby this cell death is
correlated with IL-2-driven cell cycling. Interestingly, our results
show that T cells, once made resistant to TCR-induced cell death by
IL-15, remain protected even when cycling in response to autocrine IL-2
(Fig. 5), indicating that in addition to growth arrest, protection also
involves induction of antiapoptotic proteins or down-regulation of
proapoptotic proteins. In this respect, Bulfone-Paus et al. (18)
recently provided evidence that IL-15-mediated suppression of
anti-Fas induced T cell apoptosis is strictly dependent on RNA
synthesis. Regarding the growth-supporting activity of IL-15 upon TCR
triggering, this observation can be explained by the protective effect
of the cytokine against TCR-induced death, thus abolishing the negative
feedback on autocrine IL-2-driven proliferation. Also, it was recently
described how IL-15 potentiates Con A-induced IL-2 secretion in human T
lymphoblasts (38). We are currently investigating whether a similar
mechanism exists in TCR-activated murine T cells. However, it cannot be
excluded that IL-15 also exerts its growth-promoting activity in a
direct way. A TCR-induced up-regulation (or down-regulation) of
IL-15R as a mechanism for IL-15-induced growth or quiescence seems
unlikely considering that both activities were obtained at similar, low
IL-15 concentrations, hence implicating the high affinity IL-15R in
both functions. Also, both differential activities could be elicited
with simian IL-15 (data not shown), which can bind on murine cells only
when IL-15R is present but not when only the dimeric
IL-2Rßc is available (15). An involvement of IL-15RX,
a recently described new type of IL-15R that is present on mast cells
(39), in the observed IL-15 activities is contradicted by the
inhibitory effect of anti-IL-2Rß mAb (data not shown). This
leaves open the possibility that by some intricate mechanism the
signaling pathway activated by the trimeric IL-15R complex differs in
the absence or presence of TCR cross-linking.
Collectively, our results show that IL-2 and IL-15 differ dramatically in their antiapoptotic and growth-inducing properties. Also, with regard to secondary CD4+ T cell responses, IL-15 elicited an enhancing activity that could not be mimicked by high or low doses of IL-2. These results support the view that IL-15 is an important regulator of CD4+ T cell responses during and after TCR triggering, as distinct from IL-2. Based on the nature of the IL-15 activities we describe herein, we propose that this regulatory role of IL-15 consists of promoting the generation of resting, long-lived CD4+ memory T lymphocytes in vivo. Evidence to support this hypothesis has to come from future in vivo studies evaluating immune memory formation in IL-15-treated animals.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. J. Grooten, Department of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium.
3 Abbreviations used in this paper: FasL, Fas ligand; HA, hemagglutinin; BHA, bromelain-cleaved HA; PCD, programmed cell death; PI, propidium iodide.
Received for publication February 6, 1998. Accepted for publication April 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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