Potential Role for IL-7 in Fas-Mediated T Cell Apoptosis
During HIV Infection1
Caroline Fluur,* Angelo De Milito,†Terry J. Fry,‡Nancy Vivar,* Liv Eidsmo,* Ann Atlas,§
Cristina Federici,†Paola Matarrese,†Mariantonia Logozzi,†Eva Rajnavo ¨lgyi,¶
Crystal L. Mackall,‡Stefano Fais,†Francesca Chiodi,* and Bence Rethi2*
IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels
occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis,
we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression
on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments
to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression
on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-
infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7
treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to
Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T
cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the
increased sensitivity of T cells for activation-induced apoptosis. The Journal of Immunology, 2007, 178: 5340–5350.
ing the course of the immune response, activated T lymphocytes be-
come progressively more sensitive to Fas-mediated apoptosis due to
the up-regulation of both Fas and Fas ligand (FasL)3expression (2,
3), the polarization of Fas molecules on the cell surface (4), a
process that requires the association of Fas to the actin cytoskel-
eton (5, 6), and the down-regulation of FLICE/FLIP, an inhibitor
of caspase-8 activation (7). In contrast to activated T cells, resting
T lymphocytes are characterized by low levels of Fas expression
and by resistance to Fas-mediated apoptosis. The Fas pathway has
been strongly implicated in the process of T cell depletion follow-
ing HIV infection. Fas expression on T cells, the levels of mem-
igation of Fas molecules in the membrane of cells sensi-
tive to apoptosis initiates several signaling pathways, which
can lead to apoptosis through either a direct caspase-cascade
brane-bound and soluble FasL molecules, and sensitivity to apo-
ptosis are all increased in parallel with disease progression in HIV-
infected individuals (8–15). Although apoptosis can be induced
directly by HIV-encoded proteins in HIV infection, continuous
activation of the immune system by chronic viral infection might
itself result in the acceleration of lymphocyte apoptosis (16, 17).
Interestingly, non-HIV-related lymphopenic conditions have also
been associated with an increased Fas expression and sensitivity to
activation-induced apoptosis (18–20), suggesting that lymphope-
nia predisposes to Fas-mediated cell death.
IL-7 is a stromal cell-derived cytokine acting on peripheral na-
ive and memory cells as an essential survival factor (21–25). IL-7
also promotes T cell activation (26) and memory formation (27,
28). In the serum of HIV-infected subjects, an increased IL-7 con-
centration was detected in correlation with the level of CD4?T
cell depletion (29–32). A similar association between CD4?T cell
depletion and increased serum IL-7 concentration was also found
in patients with other types of T cell depletion (30, 33). The in-
creased concentration of IL-7 found in T cell-depleted individuals
is considered to be a homeostatic response to T cell depletion,
which may accelerate thymic output and promote peripheral T cell
survival and proliferation (22, 29, 30). In addition to the extensive
data on T cell maintenance by IL-7, it has also been suggested that
IL-7 might participate in the up-regulation of Fas expression on
naive T lymphocytes (34, 35). Another study described IL-7 as a
stimulator of Fas-induced apoptosis in T cell cultures infected with
HIV-1 (36). These observations suggest that chronically elevated
IL-7 concentration may have a role in inducing increased sensi-
tivity to Fas-mediated apoptosis during HIV infection; however,
no previous studies have demonstrated the association of high IL-7
levels and high Fas expression in vivo.
In this study, we show that IL-7 primes peripheral T cells to
Fas-mediated apoptosis through stimulating Fas accumulation at
the cell surface and by inducing a polarized organization of Fas
molecules in the membrane. Accordingly, IL-7 treatment induced
increased susceptibility of T cells to Fas-induced apoptosis. The
*Department of Microbiology and Tumor Biology Center, Karolinska Institutet,
Stockholm, Sweden;†Department of Drug Resistance and Evaluation, Istituto Supe-
riore di Sanita `, Rome, Italy;‡Pediatric Oncology Branch, Center for Cancer Research,
National Cancer Institute, Bethesda, MD 20892;§Department of Medicine, Infectious
Diseases Unit, Karolinska University Hospital, Solna, Sweden;¶Institute of Immu-
nology, Medical and Health Science Center, Faculty of Medicine, University of De-
brecen, Debrecen, Hungary
Received for publication August 25, 2006. Accepted for publication January 17, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by the Swedish Medical Research Council, the Swedish
International Development Cooperation Agency, Department of Research Coopera-
tion, the Swedish Royal Academy of Science, the Division of Intramural Research
Programs of the National Cancer Institute, National Institutes of Health, and the
Hungarian National Research Fund (OTKA T043420). B.R. is supported by a Euro-
pean Community Marie Curie Training and Mobility Program Fellowship and by the
Hungarian State Eotvos Fellowship. Angelo De Milito was supported by the fellow-
ship of the Swedish Medical Research Council.
2Address correspondence and reprint requests to Dr. Bence Rethi, Microbiology and
Tumor Biology Center, Karolinska Institutet, Nobels va ¨g 16, S-17177 Stockholm,
Sweden. E-mail address: Bence.Rethi@ki.se
3Abbreviations used in this paper: FasL, Fas ligand; FRET, fluorescence resonance
energy transfer; MFI, mean fluorescence intensity.
The Journal of Immunology
role of IL-7 in Fas induction was further supported by showing Fas
up-regulation in macaques upon IL-7 treatment and by the finding
that Fas expression and apoptosis sensitivity in naive and memory
T cells of HIV-infected individuals correlated with serum IL-7
concentrations. Our results suggest a role for IL-7 in the Fas-me-
diated regulation of T cell homeostasis with important implications
in HIV infection in which disease progression is associated with an
elevated IL-7 concentration, increased Fas and FasL expression,
and also with an increased apoptosis sensitivity of T lymphocytes.
Materials and Methods
Patient samples and cell cultures
Blood samples were obtained from healthy blood donors and from 18 HIV-
1-infected patients (8 men, 10 women, mean CD4?T cell count is 342 ?
165 cells/?l). The ethical committee at the Karolinska Institute (Stock-
holm, Sweden) approved the studies involving patient samples. Fifteen
patients were on combination therapy, two patients were treatment naive,
and one patient had interrupted treatment for 1.5 years. Viral loads ranged
from ?50 to 71,000 copies/ml.
Blood samples were also collected from cancer patients enrolled on an
investigational trial of immunotherapy. The trial was approved by the In-
stitutional Review Board of the National Cancer Institute, and patients or
their guardians provided informed consent. Patients 1, 3, and 4 had Ew-
ing’s sarcoma and patient 2 had alveolar rhabdomyosarcoma. Postchemo-
therapy blood samples were obtained upon hemopoietic recovery from at
least 6 cycles of cyclophosphamide-based chemotherapy as previously re-
PBMCs were separated by Ficoll gradient centrifugation (Lymphoprep;
Nyegaard). For cell cultures T lymphocytes were separated using the Pan
T cell Isolation kit (Miltenyi Biotec). The selected cell populations con-
tained 90–97% CD3?lymphocytes as measured by FACS analysis. Sep-
aration of naive and memory T cells was done using CD45RO-MicroBeads
or CD45RA-MicroBeads, respectively (Miltenyi Biotec) in combination
with the Pan T cell Isolation kit. Cells were cultured in RPMI 1640 with
L-glutamine containing 10% FCS and antibiotics at cell concentration of
1 ? 106cells/ml. Human rIL-2, IL-4, IL-7, and IL-15 (PeproTech) were
added to T cell cultures at the concentrations and conditions indicated in
each experiment. In some experiments T cells were activated with 5 ?g/ml
anti-CD3 Abs (BD Pharmingen) or by the combination of 5 ?g/ml PHA
(Sigma-Aldrich) and 50 U/ml IL-2 (PeproTech).
The effect of IL-7 on the Fas expression of T cells was tested in cynomol-
gus macaques. Human IL-7 used in this study was manufactured by the
Biopharmaceutical Development facility (Frederick, MD), as previously
described (38). All animals were housed and handled in accordance with
standards of the American Association for the Accreditation of Laboratory
Animal Care, the Guide for the Care and Use of Laboratory Animals (Na-
tional Academy of Sciences, 1996), and the U.S. Department of Agricul-
ture through the Animal Welfare Act (Public Law 91-579), and these stud-
ies were approved by the National Cancer Institute Animal Care and Use
Committee. Healthy juvenile cynomolgus monkeys were s.c. injected with
human rIL-7 once daily for 10 days at doses of 50 ?g/kg/day (n ? 2
animals), 200 ?g/kg/day (n ? 2 animals), or 500 ?g/kg/day (n ? 3
Flow cytometric analysis of T cells
For measurement of Fas expression FITC- or PE-conjugated anti-Fas (BD
Pharmingen) or the appropriate isotype control mAbs (BD Pharmingen)
were used. Fas expression was measured on naive and memory CD4?
and CD8?T cells using FITC-labeled CD4- and CD8-specific mAbs,
fluorescein-conjugated anti-CCR7, and PE-labeled anti IL-7R? mAbs
(R&D Systems); CyChrome-conjugated anti-CD45RA and anti-CD3
mAbs; and FITC-conjugated anti-CD3 and Alexa Fluor 647-labeled anti-
CD3 mAbs (BD Pharmingen). Stained cells were fixed in 2% paraformal-
dehyde. Alternatively Fas was labeled using polyclonal mouse anti-Fas Ab
(Santa Cruz Biotechnology) followed by FITC-labeled anti-mouse IgG af-
ter preparing the cells using the Intrastain fixation and permeabilization kit
(DakoCytomation). Fluorescence intensities were measured by FACSort
and data analyzed by CellQuest software (BD Biosciences). Caspase-3
activation was measured using the FITC-labeled C92-605 Ab (BD Bio-
sciences), specific for the active form of the enzyme.
Measurement of IL-7 in serum
IL-7 concentrations were determined by Quantikine high sensitivity immuno-
assay (R&D Systems) according to the manufacturer’s recommendations.
Anti-Fas (clone CH-11; Nordic Biosite), anti-CD3 (BD Pharmingen), or
control IgM (Sigma-Aldrich) Abs were coated for 3 h in 24-well plates at
room temperature. T cells were placed onto the precoated wells at 106
cell/ml. Cycloheximide (Sigma-Aldrich) was added at the concentration of
10 ?g/ml to some cultures. After 24 h, cells were labeled with FITC-
conjugated Annexin V (BD Pharmingen) and measured by flow cytometry.
IL-7-treated and nontreated T cells were incubated for 2 h at 37°C on glass
chamber slides (Nunc) precoated with poly-L-lysine (Sigma-Aldrich) and
fixed with 80% methanol for 10 min at ?20°C. Cells were then treated with
a protein block (DakoCytomation) for 1 h. Samples were incubated with a
polyclonal rabbit anti-Fas Ab (Santa Cruz Biotechnology) and a mouse
mAb against ezrin (Sigma-Aldrich) or CD43 (BD Pharmingen) in 20%
protein block for 1 h at room temperature. After washing with PBS, cells
were incubated with anti-mouse IgG labeled with Alexa Fluor 488 (In-
vitrogen Life Technologies) and anti-rabbit IgG labeled with Cy3 (The
Jackson Laboratory) in 20% protein block for 1 h at room temperature.
Samples were mounted with mounting medium (Vector Laboratories) and
analyzed with a Leica microscope. Images were captured with a chilled
3-CCD camera (Hamamatsu).
Flow cytometric fluorescence resonance energy transfer (FRET)
We applied FRET analysis to demonstrate colocalization of ezrin and Fas.
Briefly, cells were fixed and permeabilized using 2% paraformaldehyde for
24 h and 0.05% Triton X-100 for 10 min at 4°C, respectively. After two
washings in cold PBS the cells were labeled with mAbs tagged with donor
(PE) or acceptor (Cy5) dyes. Ezrin staining was performed using unla-
belled mouse anti-ezrin Abs (clone 18; Transduction Laboratories) and a
saturating amount of PE-labeled anti-mouse IgG (Sigma-Aldrich). Fas was
detected by anti-Fas (clone C-20; Santa Cruz Biotechnology) Ab followed
by biotinylated anti-rabbit IgG and then saturating concentrations of
streptavidin-Cy5 (both from BD Pharmingen). Cells were analyzed with a
dual-laser FACSCalibur. For determination of FRET efficiency, changes in
fluorescent intensity of donor plus acceptor labeled cells were compared
with the emission signal from cells labeled with donor-only and acceptor-
only fluorophores. All data were corrected for background by subtracting
the binding of the isotype controls. Efficient energy transfer resulted in an
increased acceptor emission on cells stained with both donor and acceptor
dyes. The FRET efficiency (ET) was calculated according to Riemann et al.
(39) using the formula: ET ? (FL3DA? FL2DA/a ? FL4DA/b)/FL3DA,
where A is the acceptor and D is donor, a ? FL2D/FL3Dand b ? FL4A/
FL3A. As a further control, the cross-reactivity among all the different
primary and secondary Abs was also assessed.
Detection of Fas mRNA in T cells
Cellular RNA was isolated from T cells using the RNeasy Mini kit (Qia-
gen). Reverse transcription was performed with the High Capacity cDNA
Archive kit (Applied Biosystems). For the real-time PCR we used the
human Fas Assay on Demand kit and the Transferrin Receptor assay as
endogenous control, both from Applied Biosystems. The cycling reactions
were performed with a 7700 ABI PRISM Sequence Detector System (Ap-
plied Biosystems). Relative expression level of Fas mRNA in cultured T
cells was compared with the expression in freshly isolated samples by the
Statistical analyses were performed using the SigmaStat program. Linear
regression analysis was applied to study the relationship between serum
IL-7 concentration, CD4?T cell depletion, Fas expression, and apoptosis.
Paired t test was used for the analysis of apoptosis sensitivity, Fas expres-
sion, or FRET efficiency in differently treated sample groups.
5341The Journal of Immunology
IL-7 induces elevated Fas expression on naive and memory T
Following Ag-specific T cell activation, T cells become gradually
sensitive for Fas-mediated apoptosis and down-regulate the IL-
7R? (27), suggesting that IL-7 and Fas act at different stages of T
cell differentiation. However, in lymphopenic individuals, IL-7
levels are increased (29–33) and T cells show an increased sensi-
tivity for activation-induced cell death (8–12, 19, 20). These ob-
servations led us to analyze whether long-term exposure of pe-
ripheral T cells to IL-7 would influence Fas expression and
apoptosis sensitivity. Peripheral T cells isolated from healthy
blood donors were cultured in the presence of various concentra-
tions of IL-7 and Fas expression was analyzed on these cells on
days 1, 2, and 5. Surface expression of Fas was dramatically en-
hanced upon incubation of the T cells with IL-7 for 5 days (Fig.
1a). In response to 25 ng/ml IL-7, the whole population of T cells
became Fas-positive and expressed 4- to 8-fold higher levels of
Fas than T cells cultured in the absence of IL-7 (measured in 10
independent experiments). Culturing T cells in the absence of IL-7
did not influence the level of Fas expression. Fas expression
showed a slow increase on the cell membrane during the 5 days of
culture in the presence of IL-7, with minimal or no response mea-
sured after 1 or 2 days (Fig. 1a). The slow kinetics of Fas up-
regulation indicates the requirement for chronically elevated
IL-7 concentration for Fas induction. Earlier studies have also
shown increased Fas expression in response to IL-7, but only in
the naive T cell pool and not on memory T cells, and in one of
these studies IL-7 modulated Fas expression only in combina-
tion with IL-2 (34, 35). In contrast to these studies we detected
a massive Fas up-regulation on naive (CCR7?CD45RA?), central
memory (CCR7?CD45RA?), and effector memory (CCR7?
CD45RA?) T cells in the presence of IL-7 (Fig. 1b). When ana-
lyzing freshly isolated T cells or cells cultured without IL-7, naive
T cells were typically Fas-low/negative, the central memory pool
contained cells with heterogeneous Fas expression, whereas the
effector memory cells were characterized by homogenous Fas ex-
pression (Fig. 1b, left). In response to IL-7, T cells from all subsets
became Fas-positive and all subsets showed a robust increase in
Fas surface density (Fig. 1b, right). We found no significant dif-
ference in the effect of IL-7 on Fas expression of CD4?or CD8?
T lymphocytes (data not shown). Upon 5 days of IL-7 treatment,
we did not detect any sign of an accelerated T cell differentiation
from CD45RA?toward CD45RO?memory stage and we did not
detect T cell proliferation or increased apoptosis that might indi-
rectly result in the relative increase of Fas-positive T cells in the
cell cultures (data not shown). These findings together with the
increased surface Fas level detected in all analyzed T cell subsets
support a direct effect of IL-7 on Fas surface density.
To identify the mechanism responsible for IL-7 mediated Fas
induction, we analyzed whether IL-7 modulates the transcription
of fas. Levels of Fas mRNA measured after 2 or 5 days of IL-7
exposure did not significantly change (Fig. 1c and data not shown),
indicating a posttranscriptional Fas regulation by IL-7. To clarify
whether IL-7 level influences Fas synthesis in T cells we analyzed
Fas levels in permeabilized T cells. Compared with the difference
in Fas expression observed on the cell surface between IL-7-
treated and nontreated T cells we observed a much-reduced effect
of IL-7 (5-fold vs 1.5-fold, n ? 3 experiments) when we analyzed
Fas expression in the whole cell (Fig. 1d). These findings suggest
that IL-7 induces the redistribution of Fas molecules from intra-
cellular compartments and/or stabilization of Fas in the cell mem-
brane, instead of an increasing Fas production.
Other cytokines involved in T cell apoptosis regulation, includ-
ing IL-2, IL-4, or IL-15, share the common ?-chain of their re-
ceptors with IL-7. We investigated whether these cytokines, sim-
ilarly to IL-7, influence Fas level on T cells. Our results
demonstrated a similar effect of IL-2 and IL-15 on T cell Fas
expression as we detected in the presence of IL-7 (Fig. 1e). IL-4,
in contrast, did not influence Fas expression arguing against a gen-
eral role of ?-chain using cytokines in Fas regulation.
Fas is linked to ezrin and expressed in a polarized manner on
IL-7-treated T cells
The polarization of Fas receptors, through an ezrin-mediated as-
sociation with the actin cytoskeleton, is a key intracellular mech-
anism rendering repeatedly activated T lymphocytes susceptible to
Fas-mediated apoptosis (5, 41). Fas molecules on the activated T
cells are recruited to a characteristic pole of the cell surface, the
uropod, which is mostly involved in cell-cell communication. As
IL-7 induces a morphological polarization of T cells in culture that
results in the development of one or two dominant protrusions
(data not shown) resembling the uropod of activated T cells, we
studied whether IL-7 treatment modulates organization of Fas mol-
ecules in the cell membrane. Immunofluorescent staining and mi-
croscopic analysis showed that Fas receptors are polarized on a
high number of naive and memory T cells following IL-7 treat-
ment, whereas we observed no polarization on untreated naive, and
weak/partial polarization on untreated memory T cells (Fig. 2a). T
cells, which polarized Fas molecules, showed colocalization of the
cytoskeletal component ezrin and Fas (Fig. 2a). As expected, on
anti-CD3-activated T cells we observed a similar polarization of
Fas molecules as it was seen on the surface of IL-7-treated T cells.
Similarly to Fas, CD43 molecules are recruited to the uropod on
the surface of long-term activated T cells through the ezrin-medi-
ated CD43-actin association (42). Upon IL-7 treatment we de-
tected a CD43-Fas colocalization on cells that showed Fas polar-
ization (Fig. 2b), suggesting the presence of a similar molecular
organization process on the surface of IL-7- and anti-CD3-
treated T cells. To further demonstrate colocalization of ezrin
and Fas in T cells exposed to high levels of IL-7 we applied
FRET analysis. Increased FRET efficiency upon IL-7 treatment
between fluorochromes used for labeling Fas and ezrin demon-
strated the IL-7 induced colocalization of these molecules. Sim-
ilarly to IL-7-treated T cells, Fas-ezrin interaction was observed
in activated lymphocytes in line with previously reported data
(5, 6). Altogether, these data showed that the IL-7 induced a
direct interaction between Fas and ezrin molecules and a po-
larized expression of these molecules. This expression pattern
has been shown to be associated with the acquisition of sus-
ceptibility to Fas-mediated apoptosis in long-term activated T
cells (5, 6).
Increased Fas expression of T cells upon IL-7 treatment in
To test the possible stimulatory effect of chronically elevated
IL-7 levels on Fas expression of T cells in vivo, healthy juvenile
cynomolgus monkeys were injected with human IL-7 once daily
for 10 days at doses of 50 ?g/kg/day (n ? 2 animals), 200
?g/kg/day (n ? 2 animals) or 500 ?g/kg/day (n ? 3 animals).
Blood samples were obtained before treatment, on days 11, 21,
and 27 and Fas expression was analyzed on CD4?and CD8?T
cells (Fig. 3). Treatment with 200 or 500 ?g/kg/day of IL-7 for
10 days consistently induced Fas up-regulation on both subsets.
Even though only a small number of animals were used in this
experiment, the consistent Fas up-regulation in response to IL-7
injections indicates a similar effect of IL-7 on Fas expression in
5342ROLE OF IL-7 IN Fas-MEDIATED T CELL APOPTOSIS
for 1, 2, or 5 days with or without IL-7 administrated at different concentrations. Fas expression levels were measured by FACS, and mean fluorescence
intensity (MFI) from one representative experiment of three is shown. b, T cells were cultured for 5 days in the presence of 25 ng/ml IL-7 and then Fas
expression was analyzed on CD3?CCR7?CD45RA?naive, CD3?CCR7?CD45RA?central memory, and CD3?CCR7?CD45RA?effector memory T
cells by FACS. Open histogram represents staining with isotype control Abs. Results are representative of four independent experiments. c, Fas mRNA
levels were analyzed with RT-PCR in T cells cultured with or without 25 ng/ml IL-7 for 5 days. Levels of Fas mRNA were compared with the expression
detected in freshly isolated T cells. d, Fas expression of T cell cultured with or without 25 ng/ml IL-7 for 5 days was compared on the cell surface (top)
and in permeabilized cells (bottom). MFI is indicated. One representative result of three independent experiments is shown. Dashed line histogram
represents stainings with isotype control Ab. e, Fas expression was measured on freshly isolated T cells or on T cells cultured for 1, 2, or 5 days with or
without the cytokines IL-2, IL-4, or IL-15. Fas level was measured by FACS and MFI is shown from one representative experiment of three performed.
Error bars represent standard deviation calculated from three independent experiments.
IL-7 up-regulates Fas expression on the surface of T cells. a, Fas expression was measured on freshly isolated T cells or on T cells cultured
5343 The Journal of Immunology
vivo as observed in cell cultures. Injections of 50 ?g/kg/day of
IL-7 resulted in a slight up-regulation of Fas within the CD8?
T cells, whereas nontreated animals (n ? 2) maintained stable
Fas levels. After cessation of IL-7 therapy, Fas levels on both
CD4?and CD8?T cells returned to baseline within 10 days.
Increased IL-7 concentration is associated with elevated Fas
expression during HIV infection
The IL-7-induced up-regulation of Fas in T cell cultures and in
macaques suggested that the high serum IL-7 levels measured dur-
ing HIV infection might play a role in the elevated Fas expression
and increased T cell apoptosis in HIV-infected patients. To test this
hypothesis, we analyzed serum IL-7 concentration in a group of
HIV-infected individuals (n ? 18) in parallel with the measure-
ment of Fas expression on various T cell subsets. Consistently
with previous reports, serum IL-7 concentration and Fas ex-
pression on T cells increased in parallel with CD4?T cell de-
pletion (Fig. 4, a and b).
Importantly, we detected a correlation between IL-7 level and
Fas expression on T cells (Fig. 4c). T cells isolated from patients
with elevated circulating IL-7 were characterized by increased Fas
expression. As we and others have recently reported the emergence
of an IL-7R??T cell subset in HIV-infected subjects (43–45), we
analyzed Fas expression on the IL-7R??and IL-7R??T cell sub-
sets. Consistent with the predicted insensitivity of the IL-7R??T
cells for IL-7 (43), serum IL-7 levels correlated with Fas expres-
sion of IL-7R??(Fig. 4d) but not with Fas expression of the IL-
7R??subset (Fig. 4e).
The increase of Fas expression in the T cell pool might reflect an
up-regulation of Fas molecules on the cell surface during HIV
infection or alternatively, it could occur as a result of the increase
in Fas-positive effector and memory subsets among peripheral
blood T cells without a real change of Fas expression at the level
treated with IL-7. a, Naive and memory T cell were separated based on the
lack of CD45RO or CD45RA expression, respectively, and the cells were
cultured with or without 25 ng/ml IL-7 for 5 days. Cells were adhered on
poly-L-lysine covered glass chamber slides, fixed, and then stained with
anti-Fas and anti-ezrin as described in Materials and Methods. Individual
stainings for Fas and ezrin and a merged image of the two molecules are
shown. As a positive control for Fas and ezrin polarization, PBMC were
treated with 5 ?g/ml anti-CD3 for 5 days and then the T cells were sep-
arated and analyzed similarly as the IL-7-treated cells. b, Cell surface lo-
calization of Fas and CD43 was analyzed on T cells pretreated with 25
ng/ml IL-7 for 5 days. c, Fas-ezrin interaction was evaluated by FRET
analysis on freshly isolated T cells, on T cells cultured for 5 days with or
without 25 ng/ml IL-7, and in T cells activated with PHA (5 ?g/ml) and
IL-2 (50 U/ml) for 6 days. Results are calculated from three independent
Fas polarization and Fas-ezrin colocalization on T cells
caques. Healthy juvenile cynomolgus monkeys were s.c. injected with hu-
man IL-7 once daily from days 1 to 10 at doses of 50 ?g/kg/day (n ? 2
animals), 200 ?g/kg/day (n ? 2 animals), or 500 ?g/kg/day (n ? 3 ani-
mals). Fas expression on peripheral blood CD4?(a) and CD8?(b) T cells
was analyzed before treatment, on days 11, 21, and 27.
IL-7 treatment increases Fas expression of T cells in ma-
5344 ROLE OF IL-7 IN Fas-MEDIATED T CELL APOPTOSIS
tration (a) and Fas expression (b) of T cells were analyzed in a set of HIV-infected patients (n ? 18). Both parameters showed correlation with CD4?T
cell count. c, Fas expression of T cells showed correlation with serum IL-7 concentration. Fas expression was also analyzed on the IL-7R??(d) and
IL-7R??(e) T cell pools separately. A significant correlation was found between serum IL-7 concentration and Fas levels on IL-7R??T cells. On the
contrary, there was no association between Fas expression of the IL-7R??T cells and IL-7 concentration. Fas expression was analyzed on naive
(CD3?CD45RA?IL-7R??) (f), effector (CD3?CD45RA?IL-7R??) (g), and memory (CD3?CD45RA?) (h) T cells. Because the memory pool contained
several IL-7R??cells, we also analyzed Fas expression on the CD3?CD45RA?IL-7R??and on the CD3?CD45RA?IL-7R??subsets (i and j, respec-
tively). We detected a correlation between serum IL-7 concentration and Fas expression on naive, total memory, or IL-7R??memory T cells.
Increased serum IL-7 concentration is associated with elevated Fas expression of T cells in HIV-infected individuals. Serum IL-7 concen-
5345 The Journal of Immunology
of individual cells. Therefore we analyzed Fas levels in different T
cell subsets, such as in naive CD45RA?IL-7R??(Fig. 4f), effector
CD45RA?IL-7R??(Fig. 4g), and memory (Fig. 4h) and in the
CD45RA?IL-7R??or CD45RA?IL-7R??memory T cells (Fig.
4, i and j, respectively). Measurement of the IL-7R? expression
allowed us to discriminate CD45RA?naive and effector T cells as
we previously showed a massive down-regulation of IL-7R? on
the CD45RA?CCR7?effectors both in HIV-infected and nonin-
fected individuals (43). Our results showed that the increased se-
rum IL-7 levels were associated with the increasing Fas expression
on the IL-7R??naive and memory subsets. In contrast, we could
not detect a correlation between IL-7 concentration and the Fas
expression by IL-7R??T cells. The correlation between serum
IL-7 levels and Fas expression on both naive and memory T cells
indicates a possible effect of IL-7 on Fas expression level instead
of an indirect association between the increased serum IL-7 con-
centration and the increased prevalence of Fas-positive memory
and effector subsets during disease progression. In fact, we did not
find a significant correlation between IL-7 levels and the propor-
tion of Fas-expressing T cells in HIV-infected patients (p ? 0.10).
In addition, we detected a relatively high ratio of Fas-positive T
cells in most patients (79.8 ? 10.1% of peripheral T cells was
Fas-positive in our cohort), suggesting that the altered surface level
of Fas might be a better indicator for the effect of IL-7 than the
ratio of Fas-expressing T cells in HIV-infected individuals. Alto-
gether, the correlation between serum IL-7 levels and Fas expres-
sion on peripheral naive and memory T cells strongly propose IL-7
as an inducer of Fas expression in HIV-infected individuals.
Increased IL-7 concentration is associated with elevated Fas
expression during chemotherapy induced T cell depletion
T cell depletion has been implicated as a critical component in the
increase of serum IL-7 levels not only in HIV infection but also in
other non-HIV-related clinical conditions (30, 33). To test the pos-
sible link between elevated IL-7 levels and Fas expression of T
cells, while excluding the effects of the chronic viral infection on
T cells, we analyzed IL-7 levels and Fas expression in cancer
patients receiving chemotherapy. Upon therapy a massive CD4?T
cell depletion was detected in these patients, the average CD4?T
cell count decreased in the blood from 634 to 133/?l. In parallel
with CD4?T cell depletion, we detected an average of 3.9 ?
1.7-fold increase of serum IL-7 levels (p ? 0.01) in the four pa-
tients tested (Fig. 5a). Increased IL-7 levels were accompanied by
increased Fas expression on CD45RA?IL-7R??naive and
CD45RA?IL-7R??memory T cells (Fig. 5b), although the dif-
ference between baseline and postchemotherapy Fas levels
reached statistical significance only in the case of memory cells
(p ? 0.06 and p ? 0.04 for naive and memory cells, respectively).
We detected Fas up-regulation on both CD4?(p ? 0.02) and
CD8?(p ? 0.006) subsets (data not shown). Despite the small
number of patients we analyzed, our results point toward a possible
scenario in which, upon chemotherapy, the high IL-7 levels
reached following T cell depletion lead to increased Fas expression
of T cells.
IL-7 increases the sensitivity of human T cells for Fas-mediated
apoptosis in vitro
We investigated whether the increased Fas expression and cell
surface polarization induced by IL-7 leads to an increased sensi-
tivity for Fas-mediated apoptosis. To this end, we cultured T cells
with or without IL-7 for 5 days and then exposed these cells to
coated anti-Fas Abs for 24 h. In IL-7-pretreated T cell cultures, we
consistently observed an increase in apoptosis in response to anti-
Fas Abs as compared with control IgM-treated cells but not in
samples cultured without IL-7. The frequency of apoptotic cells
in the anti-Fas-treated samples was 24 ? 3% above background in
the IL-7-pretreated T cell cultures (Fig. 6a). Only a very low level
of apoptosis (2 ? 2%) was detected in freshly isolated T cells
under the same conditions (Fig. 6a). Sensitivity of IL-7-treated and
nontreated T cells for Fas-mediated apoptosis was compared at
various conditions, namely, in the presence of a more intense Fas
cross-linking (anti-IgM-coated before anti-Fas Abs), in parallel
with TCR signaling, and in the presence of cycloheximide, a com-
pound that increases apoptosis sensitivity of several cell types
through the rapid elimination of antiapoptotic factors. Our results
indicated a slightly increased sensitivity to Fas-induced apoptosis
of IL-7-treated T cells in the presence of TCR signaling (between
11 and 21% above anti-Fas stimulus only and we detected a mas-
sive increase in the apoptosis of IL-7-treated T cells in the pres-
ence of cycloheximide (Fig. 6a). The fact that even under these
apoptosis-sensitizing conditions we observed a high level of Fas-
induced apoptosis only in T cells precultured with IL-7 indicates
the high potential of IL-7 in priming T cells for Fas-mediated
apoptosis. In accordance with the IL-7-induced apoptosis sensitiv-
ity observed in T cells upon Fas triggering for 24 h, Fas cross-
linking induced rapid caspase-3 activation in several of the IL-7-
pretreated, but not in untreated T cells (Fig. 6b).
We were interested in whether IL-7 treatment resulted in a com-
parable sensitivity to Fas-mediated apoptosis as the repeated TCR
triggering combined with high IL-2 concentration, a condition that
predisposes T cells to activation-induced cell death. T cells were
stimulated with coated anti-CD3 Abs for 5 days in the presence of
a high IL-2 dose. Approximately 50% of these cells underwent
apoptosis in the presence of anti-Fas Abs, and we detected a high
level of apoptosis (29%) in the control samples as well (Fig. 6c).
These results showed that TCR-triggering and a high dose of IL-2
levels are associated with Fas up-reg-
ulation on T cells in patients receiving
cancer chemotherapy. a, Serum IL-7
levels in four patients receiving can-
cer chemotherapy are shown before
and after therapy. b, Fas expression of
naive and memory T cells isolated
from peripheral blood before and af-
ter chemotherapy is shown.
Elevated serum IL-7
5346ROLE OF IL-7 IN Fas-MEDIATED T CELL APOPTOSIS
with or without 25 ng/ml IL-7 for 5 days. The cells were exposed to 1 ?g/ml CH-11 anti-Fas Ab or to control IgM for 24 h and thereafter the percentage
of apoptotic cells was tested by Annexin V staining. Alternatively, apoptosis was induced in the presence of 10 ?g/ml cycloheximide, by the anti-Fas Ab
cross-linked with coated anti-IgM (2.5 ?g/ml) or by the combination of coated anti-Fas and anti-CD3 Abs (5 ?g/ml). Results were calculated using data
from four independent experiments. ?, p ? 0.01; ??, p ? 0.05 tested for differences between anti-Fas induced vs background apoptosis. b, Caspase-3
activation was analyzed in untreated T cells (?) or in T cells cultured with 25 ng/ml IL-7 for 5 days. The cells were exposed to 1 ?g/ml coated CH-11
anti-Fas Ab or to control IgM, and caspase-3 activation was measured at different time points using flow cytometry. The proportion of T cells characterized
by the presence of active caspase-3 is indicated. c, Apoptosis of T cells activated in the presence of anti-CD3 (5 ?g/ml) and IL-2 (50 U/ml) for 5 days was
tested after exposing the cells to 1 ?g/ml CH-11 anti-Fas Ab or to control IgM for 24 h. The percentage of apoptotic cells was tested by Annexin V staining.
d, Testing whether naive or memory T cells show a different sensitivity to Fas-induced apoptosis upon IL-7 treatment. T cells were cultured for 5 days in
the presence of 25 ng/ml IL-7 and then exposed to coated anti-Fas or control IgM Abs followed by the determination of apoptosis within the
CD3?CD45RA?naive and CD3?CD45RA?memory T cells. Percentage represents Annexin V binding cells within the CD45RA?and CD45RA?
populations. A representative result of three independent experiments is shown. e, Alternatively, naive and memory T cells were separated by negative
selection using magnetic cell sorting before the IL-7 treatment and then the cells were uncultured (?) or cultured with 25 ng/ml IL-7 for 5 days. Apoptosis
was triggered using 1 ?g/ml anti-Fas Abs for 24 h and the percentage of apoptosis cells was determined by FACS using Annexin V staining. Data are
calculated from the results of three independent experiments. ??, p ? 0.05 tested for differences between anti-Fas induced vs background apoptosis. Error
bars represent standard deviation calculated in independent experiments.
The effect of IL-7 on Fas-mediated apoptosis of T cells. a, Fas-mediated apoptosis was tested on freshly isolated T cells or on T cells cultured
5347 The Journal of Immunology
induce massive T cell loss through the activation of endogenous
apoptotic pathways, whereas high IL-7 concentration, on the con-
trary, predispose T cells to Fas-mediated apoptosis without induc-
ing apoptosis directly.
To identify which T cell subsets show sensitivity for Fas-in-
duced apoptosis upon IL-7 treatment, we analyzed apoptosis of
CD4?and CD8?T cells. Following 5 days of IL-7 treatment, 1
?g/ml anti-Fas Ab induced similar level of apoptosis in these sub-
sets (17.2 ? 2.4% and 20.4 ? 2.9% in the case of IL-7-treated
CD4?and CD8?T cells, respectively; n ? 3). We investigated
whether the differentiation stage of T cells has an impact on Fas-
mediated killing. We detected a higher sensitivity for Fas-induced
apoptosis within the CD45RA?memory pool as compared with
naive T cells following IL-7 pretreatment of peripheral T cells
(Fig. 6d). Purified memory T cells cultured with 25 ng/ml IL-7 for
5 days were characterized by an ?2.5-fold higher sensitivity for
Fas-mediated apoptosis (p ? 0.05) than naive cells cultured within
the same conditions (Fig. 6e).
High IL-7 levels are associated with increased sensitivity to
Fas-mediated apoptosis in HIV-infected individuals
We analyzed whether increased levels of IL-7 in HIV-infected
individuals might be coupled with an increased sensitivity of T
cells for Fas-induced apoptosis. PBMC isolated from a group of
HIV-infected individuals (n ? 13) were exposed to plate-bound
anti-Fas Abs for 24 h. Apoptosis within the naive (CD45RA?IL-
7R?and memory (CD45RA?IL-7R??) T cell subsets was then
analyzed. Serum IL-7 concentrations were determined from the
same blood samples. The ratio of cells undergoing apoptosis cor-
related with the serum IL-7 concentration in both the naive and the
memory subsets (Fig. 7a). As our in vitro experiments strongly
suggested that the IL-7-mediated modulation of Fas levels might
be of importance in the regulation of apoptosis sensitivity, we
tested whether there is any association between the surface expres-
sion of Fas and sensitivity of naive and memory T cells for Fas-
mediated killing. Apoptosis sensitivity and surface Fas levels cor-
related within the memory pool, although we did not find a
significant correlation in the case of naive T cells (Fig. 7b).
Increased circulating IL-7 levels have been described in HIV-in-
fected individuals as well as in other lymphopenic patients and the
concentrations of IL-7 in serum correlated with the levels of CD4?
T cell depletion (29–33). Although the mechanism underlying the
elevated IL-7 concentration is not completely understood, whether
it is an increased production or a decreased consumption due to T
cell depletion and IL-7R? down-regulation, the increase of IL-7
has been interpreted as a mechanism that might counteract T cell
depletion (22, 29, 46). High IL-7 levels may stimulate the regen-
eration of the T cell pool by promoting maintenance and prolifer-
ation at various stages of T cell differentiation.
IL-7 acts as a survival factor for resting naive and memory T
cells through maintaining a balanced activity of several antiapop-
totic and proapoptotic members of the Bcl-2 family and through
the regulation of metabolic processes (21). However, little is
known about the effect of IL-7 on Fas-mediated T cell apoptosis.
As elevated IL-7 levels (29–32), high Fas expression on T cells,
and increased sensitivity to Fas-induced apoptosis (8–15, 47) can
be detected in HIV-infected individuals we decided to analyze
whether IL-7 has a regulatory role on Fas expression and Fas-
In this study, we demonstrated that IL-7 acts as a potent inducer
of Fas membrane expression on naive and memory T cells in vitro.
Because the cytoskeleton-mediated recruitment of the Fas mole-
cules to a polarized site of the cell surface is known to be a pre-
requisite for Fas-mediated apoptosis of chronically activated T
cells (4, 6), we analyzed whether IL-7 influences the surface pat-
tern of Fas expression. On T cells, activated through the TCR, the
membrane polarization of Fas molecules requires the interaction of
Fas with ezrin, which connects the Fas molecules to the actin cy-
toskeleton (5). We showed that IL-7 treatment, similarly to the
effect of long-term TCR triggering, induces Fas-ezrin interaction
and Fas polarization on naive and memory T cells.
Based on the findings that upon IL-7 treatment of T cells 1) we
could not detect increased Fas mRNA levels, 2) we detected a
significant increase in Fas levels on the cell surface but only a
minor difference in the total Fas protein level of the cells, and 3)
Fas was organized on the cell surface through the association with
the actin cytoskeleton, we hypothesize that the mechanisms lead-
ing to high Fas expression upon IL-7 treatment possibly include
the increased relocation of Fas molecules from intracellular com-
partments to the cell surface and/or the increased stabilization of
membrane-bound Fas molecules through cytoskeletal interactions.
The concentration of IL-7 used in our experiments is above the
IL-7 levels measured in the blood. However, as IL-7 is not pro-
duced in the circulation, IL-7 levels in the blood might be obvi-
ously lower than the IL-7 concentration in solid tissues at the site
of production. In addition, the extracellular matrix-mediated con-
centration of the cytokine has also been shown as a mechanism
that increases IL-7 availability for the responding cells (48–50).
Our results showing Fas up-regulation and polarization on the
cell surface during IL-7 treatment strongly support a stimulatory
role for IL-7 in Fas-mediated T cell apoptosis. Indeed, in parallel
with these events, we found that IL-7-treated cells were charac-
terized by an increased sensitivity for Fas-mediated apoptosis in
vitro. We did not detect a differential effect of IL-7 in Fas induction
or apoptosis sensitivity of CD4?or CD8?T cells. In contrast, the
sis sensitivity of T cells isolated from HIV-infected patients. The anti-Fas-
induced apoptosis of the CD45RA?IL-7R??naive and the CD45RA?IL-
7R??memory T cells was analyzed in a group of HIV-infected individuals
(n ? 13). The percentage of cells undergoing apoptosis in response to
incubation with 1 ?g/ml coated anti-Fas Abs for 24 h was correlated with
the levels of spontaneous apoptosis. The correlation (R) between anti-Fas-
induced apoptosis and serum IL-7 levels (a) and anti-Fas induced apoptosis
and Fas expression in MFI (b) is shown for naive and memory T cells.
Correlation of serum IL-7 levels and Fas-mediated apopto-
5348 ROLE OF IL-7 IN Fas-MEDIATED T CELL APOPTOSIS
differentiation stage of T cells had a strong impact on the effec-
tivity of IL-7 to induce sensitivity for Fas-mediated killing. Anal-
ysis of naive and memory T cells revealed that memory cells are
characterized by a higher level of sensitivity to Fas-mediated cell
death following IL-7 treatment as compared with naive T cells.
Our results indicate that the strong stimulatory effect of the in-
creased IL-7 concentration on T cell regeneration might be cou-
pled with a negative feedback mechanism through Fas molecules
in lymphopenic individuals. Chronically elevated IL-7 concentra-
tion might sensitize T cells for apoptosis in HIV-infected patients
by up-regulating Fas expression and by the cytoskeleton-mediated
polarization of Fas molecules. This hypothesis was further sup-
ported by two lines of experimentation. First, we showed that in-
jection of high dose of IL-7 to macaques for 10 days resulted in
Fas up-regulation on T cells confirming the stimulatory effect of
IL-7 on Fas expression in vivo. Second, serum IL-7 levels corre-
lated with Fas expression on naive and memory T cells in HIV-
infected individuals. In addition, the ex vivo sensitivity of T cells
for anti-Fas-induced apoptosis correlated with serum IL-7 levels
upon HIV-infection. Analysis of IL-7 levels and Fas expression in
a small cohort of patients receiving cancer chemotherapy indicated
that high IL-7 levels might be associated with increased Fas ex-
pression of T cells within non-HIV-induced lymphopenic condi-
tions when the potential influence of chronic HIV infection on Fas
levels is excluded.
Interestingly, we found that in addition to IL-7, at least two
other ?-chain using cytokines, IL-2 and IL-15, might be involved
in the regulation of Fas level on T cells. Similarly to IL-7, the
presence of these cytokines was needed for a longer period (?1–2
days) for Fas induction, and the elevated surface level of Fas might
have been achieved through the relocalization of Fas molecules
from intracellular compartments to the cell membrane, as a major
increase was detected in the surface and not in the total amount of
Fas upon IL-2 or IL-15 treatment (data not shown). Although the
role of these cytokines on the regulation of Fas expression during
HIV infection may require further studies, the source and kinetics
of IL-2 production are different as compared with IL-7. Also, the
serum levels of IL-15 are rather decreased in HIV infection (51)
presumably indicating nonoverlapping roles of IL-2, IL-7, and
IL-15 in Fas regulation.
The levels of membrane-bound and soluble FasL molecules are
increased during HIV infection (9, 13, 14) possibly as a conse-
quence of the chronic immune activation and the presence of HIV
proteins such as gp120, tat, and nef, which are all implicated in
FasL induction (52). In addition to FasL regulation, several HIV-
encoded molecules have been shown to induce apoptosis of both
infected and uninfected T cells by triggering death receptor-medi-
ated or mitochondrial apoptotic pathways in vitro (17). Interest-
ingly, HIV gp120 shares the ability of IL-7 or TCR triggering to
induce Fas-ezrin association on T cells, and this mechanism might
play an important role in inducing apoptosis sensitivity in by-
stander T cells (53).
Taken together, the presence of apoptosis-sensitizing viral com-
pounds together with high FasL availability in HIV-infected indi-
viduals may result in an altered balance between the apoptotic and
antiapoptotic effects of IL-7 on peripheral T cells. In this scenario,
chronically elevated IL-7 levels may lead to Fas up-regulation and
surface polarization and thus to an increased sensitivity of T cells
for Fas-mediated apoptosis, which effects may predominate the
antiapoptotic and proliferative activities of the cytokine. In other
non-HIV-related lymphopenic situations, the increased level of
IL-7 may induce Fas expression on T cells without an increase in
apoptosis because Fas up-regulation in these cases is presumably
not accompanied by chronic immune activation or viral infection
that can result in high FasL expression. The IL-7-induced Fas ex-
pression may, however, play a role in T cell apoptosis detected in
non-HIV-related lymphopenic situations in which peripheral T
cells undergo apoptosis upon in vitro stimulation (18–20).
In summary, our results indicate that T cell depletion, which
follows HIV infection, may act as an accelerator of further T cell
apoptosis through high IL-7 levels and through the IL-7-induced
Fas expression and polarization on T cells. Abrogation of viral
replication and generalized immune activation may thus be a crit-
ical step toward increasing the efficiency of IL-7 in T cell resto-
ration during the natural course of infection or upon IL-7
We thank Kerstin I. Falk and Rigmor Thorstensson for helpful discussions,
and Francesco Lozupone and Horvath Gabor for help with FRET analysis.
The authors have no financial conflict of interest.
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5350 ROLE OF IL-7 IN Fas-MEDIATED T CELL APOPTOSIS