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The Journal of Immunology, 2007, 178: 5340-5350.
Copyright © 2007 by The American Association of Immunologists, Inc.

Potential Role for IL-7 in Fas-Mediated T Cell Apoptosis During HIV Infection1

Caroline Fluur*, Angelo De Milito{dagger}, Terry J. Fry{ddagger}, Nancy Vivar*, Liv Eidsmo*, Ann Atlas§, Cristina Federici{dagger}, Paola Matarrese{dagger}, Mariantonia Logozzi{dagger}, Eva Rajnavölgyi, Crystal L. Mackall{ddagger}, Stefano Fais{dagger}, Francesca Chiodi* and Bence Rethi2,*

* Department of Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden; {dagger} Department of Drug Resistance and Evaluation, Istituto Superiore di Sanità, Rome, Italy; {ddagger} 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 Immunology, Medical and Health Science Center, Faculty of Medicine, University of Debrecen, Debrecen, Hungary


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ligation of Fas molecules in the membrane of cells sensitive to apoptosis initiates several signaling pathways, which can lead to apoptosis through either a direct caspase-cascade or the involvement of the mitochondrial apoptotic machinery (1). During the course of the immune response, activated T lymphocytes become progressively more sensitive to Fas-mediated apoptosis due to the up-regulation of both Fas and Fas ligand (FasL)3 expression (2, 3), the polarization of Fas molecules on the cell surface (4), a process that requires the association of Fas to the actin cytoskeleton (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 following HIV infection. Fas expression on T cells, the levels of membrane-bound and soluble FasL molecules, and sensitivity to apoptosis are all increased in parallel with disease progression in HIV-infected individuals (8, 9, 10, 11, 12, 13, 14, 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, 19, 20), suggesting that lymphopenia predisposes to Fas-mediated cell death.

IL-7 is a stromal cell-derived cytokine acting on peripheral naive and memory cells as an essential survival factor (21, 22, 23, 24, 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 concentration was detected in correlation with the level of CD4+ T cell depletion (29, 30, 31, 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 increased 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 sensitivity 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 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-mediated 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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
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/µl). The ethical committee at the Karolinska Institute (Stockholm, 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 Institutional Review Board of the National Cancer Institute, and patients or their guardians provided informed consent. Patients 1, 3, and 4 had Ewing’s sarcoma and patient 2 had alveolar rhabdomyosarcoma. Postchemotherapy blood samples were obtained upon hemopoietic recovery from at least 6 cycles of cyclophosphamide-based chemotherapy as previously reported (37).

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 contained 90–97% CD3+ lymphocytes as measured by FACS analysis. Separation 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 x 106 cells/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 µg/ml anti-CD3 Abs (BD Pharmingen) or by the combination of 5 µg/ml PHA (Sigma-Aldrich) and 50 U/ml IL-2 (PeproTech).

Animal studies

The effect of IL-7 on the Fas expression of T cells was tested in cynomolgus 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 (National Academy of Sciences, 1996), and the U.S. Department of Agriculture through the Animal Welfare Act (Public Law 91-579), and these studies 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 µg/kg/day (n = 2 animals), 200 µg/kg/day (n = 2 animals), or 500 µg/kg/day (n = 3 animals).

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{alpha} 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% paraformaldehyde. Alternatively Fas was labeled using polyclonal mouse anti-Fas Ab (Santa Cruz Biotechnology) followed by FITC-labeled anti-mouse IgG after 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 Biosciences), specific for the active form of the enzyme.

Measurement of IL-7 in serum

IL-7 concentrations were determined by Quantikine high sensitivity immunoassay (R&D Systems) according to the manufacturer’s recommendations.

Apoptosis detection

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 µg/ml to some cultures. After 24 h, cells were labeled with FITC-conjugated Annexin V (BD Pharmingen) and measured by flow cytometry.

Immunohistochemistry

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 (Invitrogen 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) analysis

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 unlabelled 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/FL3D and 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 (Qiagen). 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 (Applied Biosystems). Relative expression level of Fas mRNA in cultured T cells was compared with the expression in freshly isolated samples by the 2–{Delta}{Delta}CT method (40).

Statistical analysis

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 expression, or FRET efficiency in differently treated sample groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-7 induces elevated Fas expression on naive and memory T lymphocytes

Following Ag-specific T cell activation, T cells become gradually sensitive for Fas-mediated apoptosis and down-regulate the IL-7R{alpha} (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, 30, 31, 32, 33) and T cells show an increased sensitivity for activation-induced cell death (8, 9, 10, 11, 12, 19, 20). These observations led us to analyze whether long-term exposure of peripheral 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 concentrations of IL-7 and Fas expression was analyzed on these cells on days 1, 2, and 5. Surface expression of Fas was dramatically enhanced 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 measured 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 combination 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 analyzing 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 expression (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 difference 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 indirectly 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.


Figure 1
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  		FIGURE 1. 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 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.

 
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 intracellular compartments and/or stabilization of Fas in the cell membrane, instead of an increasing Fas production.

Other cytokines involved in T cell apoptosis regulation, including IL-2, IL-4, or IL-15, share the common {gamma}-chain of their receptors with IL-7. We investigated whether these cytokines, similarly 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 general role of {gamma}-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 association with the actin cytoskeleton, is a key intracellular mechanism 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 molecules in the cell membrane. Immunofluorescent staining and microscopic analysis showed that Fas receptors are polarized on a high number of naive and memory T cells following IL-7 treatment, 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-mediated CD43-actin association (42). Upon IL-7 treatment we detected a CD43-Fas colocalization on cells that showed Fas polarization (Fig. 2b), suggesting the presence of a similar molecular organization process on the surface of IL-7- and anti-CD3treated 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 demonstrated the IL-7 induced colocalization of these molecules. Similarly 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 polarized expression of these molecules. This expression pattern has been shown to be associated with the acquisition of susceptibility to Fas-mediated apoptosis in long-term activated T cells (5, 6).


Figure 2
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  		FIGURE 2. Fas polarization and Fas-ezrin colocalization on T cells 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 µg/ml anti-CD3 for 5 days and then the T cells were separated and analyzed similarly as the IL-7-treated cells. b, Cell surface localization 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 µg/ml) and IL-2 (50 U/ml) for 6 days. Results are calculated from three independent experiments.

 
Increased Fas expression of T cells upon IL-7 treatment in macaques

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 µg/kg/day (n = 2 animals), 200 µg/kg/day (n = 2 animals) or 500 µg/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 µg/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 vivo as observed in cell cultures. Injections of 50 µg/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.


Figure 3
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  		FIGURE 3. IL-7 treatment increases Fas expression of T cells in macaques. Healthy juvenile cynomolgus monkeys were s.c. injected with human IL-7 once daily from days 1 to 10 at doses of 50 µg/kg/day (n = 2 animals), 200 µg/kg/day (n = 2 animals), or 500 µg/kg/day (n = 3 animals). Fas expression on peripheral blood CD4+ (a) and CD8+ (b) T cells was analyzed before treatment, on days 11, 21, and 27.

 
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 during 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 measurement of Fas expression on various T cell subsets. Consistently with previous reports, serum IL-7 concentration and Fas expression on T cells increased in parallel with CD4+ T cell depletion (Fig. 4, a and b).


Figure 4
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  		FIGURE 4. Increased serum IL-7 concentration is associated with elevated Fas expression of T cells in HIV-infected individuals. Serum IL-7 concentration (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{alpha}+ (d) and IL-7R{alpha}– (e) T cell pools separately. A significant correlation was found between serum IL-7 concentration and Fas levels on IL-7R{alpha}+ T cells. On the contrary, there was no association between Fas expression of the IL-7R{alpha}– T cells and IL-7 concentration. Fas expression was analyzed on naive (CD3+CD45RA+IL-7R{alpha}+) (f), effector (CD3+CD45RA+IL-7R{alpha}–) (g), and memory (CD3+CD45RA–) (h) T cells. Because the memory pool contained several IL-7R{alpha}– cells, we also analyzed Fas expression on the CD3+CD45RA–IL-7R{alpha}+ and on the CD3+CD45RA–IL-7R{alpha}– subsets (i and j, respectively). We detected a correlation between serum IL-7 concentration and Fas expression on naive, total memory, or IL-7R{alpha}+ memory T cells.

 
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{alpha}– T cell subset in HIV-infected subjects (43, 44, 45), we analyzed Fas expression on the IL-7R{alpha}+ and IL-7R{alpha}– T cell subsets. Consistent with the predicted insensitivity of the IL-7R{alpha}– T cells for IL-7 (43), serum IL-7 levels correlated with Fas expression of IL-7R{alpha}+ (Fig. 4d) but not with Fas expression of the IL-7R{alpha}– 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 of individual cells. Therefore we analyzed Fas levels in different T cell subsets, such as in naive CD45RA+IL-7R{alpha}+ (Fig. 4f), effector CD45RA+IL-7R{alpha}– (Fig. 4g), and memory (Fig. 4h) and in the CD45RA–IL-7R{alpha}+ or CD45RA–IL-7R{alpha}– memory T cells (Fig. 4, i and j, respectively). Measurement of the IL-7R{alpha} expression allowed us to discriminate CD45RA+ naive and effector T cells as we previously showed a massive down-regulation of IL-7R{alpha} on the CD45RA+CCR7– effectors both in HIV-infected and noninfected individuals (43). Our results showed that the increased serum IL-7 levels were associated with the increasing Fas expression on the IL-7R{alpha}+ naive and memory subsets. In contrast, we could not detect a correlation between IL-7 concentration and the Fas expression by IL-7R{alpha}– 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 concentration 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 proportion 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. Altogether, the correlation between serum IL-7 levels and Fas expression 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 possible 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/µl. 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 patients tested (Fig. 5a). Increased IL-7 levels were accompanied by increased Fas expression on CD45RA+IL-7R{alpha}+ naive and CD45RA–IL-7R{alpha}+ memory T cells (Fig. 5b), although the difference 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.


Figure 5
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  		FIGURE 5. Elevated serum IL-7 levels are associated with Fas up-regulation on T cells in patients receiving cancer chemotherapy. a, Serum IL-7 levels in four patients receiving cancer chemotherapy are shown before and after therapy. b, Fas expression of naive and memory T cells isolated from peripheral blood before and after chemotherapy is shown.

 
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 sensitivity 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 compound 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 massive increase in the apoptosis of IL-7-treated T cells in the presence 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 sensitivity 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).


Figure 6
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  		FIGURE 6. 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 with or without 25 ng/ml IL-7 for 5 days. The cells were exposed to 1 µg/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 µg/ml cycloheximide, by the anti-Fas Ab cross-linked with coated anti-IgM (2.5 µg/ml) or by the combination of coated anti-Fas and anti-CD3 Abs (5 µg/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 µg/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 µg/ml) and IL-2 (50 U/ml) for 5 days was tested after exposing the cells to 1 µg/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 µg/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.

 
We were interested in whether IL-7 treatment resulted in a comparable 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 induce massive T cell loss through the activation of endogenous apoptotic pathways, whereas high IL-7 concentration, on the contrary, predispose T cells to Fas-mediated apoptosis without inducing apoptosis directly.

To identify which T cell subsets show sensitivity for Fas-induced apoptosis upon IL-7 treatment, we analyzed apoptosis of CD4+ and CD8+ T cells. Following 5 days of IL-7 treatment, 1 µg/ml anti-Fas Ab induced similar level of apoptosis in these subsets (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{alpha}+) T cell subsets was then analyzed. Serum IL-7 concentrations were determined from the same blood samples. The ratio of cells undergoing apoptosis correlated 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 expression of Fas and sensitivity of naive and memory T cells for Fas-mediated killing. Apoptosis sensitivity and surface Fas levels correlated within the memory pool, although we did not find a significant correlation in the case of naive T cells (Fig. 7b).


Figure 7
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  		FIGURE 7. Correlation of serum IL-7 levels and Fas-mediated apoptosis sensitivity of T cells isolated from HIV-infected patients. The anti-Fas-induced apoptosis of the CD45RA+IL-7R{alpha}+ naive and the CD45RA–IL-7R{alpha}+ 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 µg/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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Increased circulating IL-7 levels have been described in HIV-infected 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, 30, 31, 32, 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{alpha} 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 regeneration of the T cell pool by promoting maintenance and proliferation 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 antiapoptotic 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, 30, 31, 32), high Fas expression on T cells, and increased sensitivity to Fas-induced apoptosis (8, 9, 10, 11, 12, 13, 14, 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-mediated apoptosis.

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 molecules to a polarized site of the cell surface is known to be a prerequisite for Fas-mediated apoptosis of chronically activated T cells (4, 6), we analyzed whether IL-7 influences the surface pattern 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 cytoskeleton (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 leading to high Fas expression upon IL-7 treatment possibly include the increased relocation of Fas molecules from intracellular compartments 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 produced in the circulation, IL-7 levels in the blood might be obviously lower than the IL-7 concentration in solid tissues at the site of production. In addition, the extracellular matrix-mediated concentration of the cytokine has also been shown as a mechanism that increases IL-7 availability for the responding cells (48, 49, 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 characterized 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 differentiation stage of T cells had a strong impact on the effectivity of IL-7 to induce sensitivity for Fas-mediated killing. Analysis 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 increased IL-7 concentration on T cell regeneration might be coupled with a negative feedback mechanism through Fas molecules in lymphopenic individuals. Chronically elevated IL-7 concentration 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 supported by two lines of experimentation. First, we showed that injection 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 correlated 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 expression of T cells within non-HIV-induced lymphopenic conditions 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 {gamma}-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 consequence 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-mediated or mitochondrial apoptotic pathways in vitro (17). Interestingly, 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 bystander T cells (53).

Taken together, the presence of apoptosis-sensitizing viral compounds together with high FasL availability in HIV-infected individuals 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 expression 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, 19, 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 critical step toward increasing the efficiency of IL-7 in T cell restoration during the natural course of infection or upon IL-7 immunotherapy.


   Acknowledgments
 
We thank Kerstin I. Falk and Rigmor Thorstensson for helpful discussions, and Francesco Lozupone and Horvath Gabor for help with FRET analysis.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Medical Research Council, the Swedish International Development Cooperation Agency, Department of Research Cooperation, 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 European Community Marie Curie Training and Mobility Program Fellowship and by the Hungarian State Eotvos Fellowship. Angelo De Milito was supported by the fellowship of the Swedish Medical Research Council. Back

2 Address correspondence and reprint requests to Dr. Bence Rethi, Microbiology and Tumor Biology Center, Karolinska Institutet, Nobels väg 16, S-17177 Stockholm, Sweden. E-mail address: Bence.Rethi{at}ki.se Back

3 Abbreviations used in this paper: FasL, Fas ligand; FRET, fluorescence resonance energy transfer; MFI, mean fluorescence intensity. Back

Received for publication August 25, 2006. Accepted for publication January 17, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Krueger, A., S. C. Fas, S. Baumann, P. H. Krammer. 2003. The role of CD95 in the regulation of peripheral T-cell apoptosis. Immunol. Rev. 193: 58-69. [Medline]
  2. Klas, C., K. M. Debatin, R. R. Jonker, P. H. Krammer. 1993. Activation interferes with the APO-1 pathway in mature human T cells. Int. Immunol. 5: 625-630. [Abstract/Free Full Text]
  3. Latinis, K. M., L. L. Carr, E. J. Peterson, L. A. Norian, S. L. Eliason, G. A. Koretzky. 1997. Regulation of CD95 (Fas) ligand expression by TCR-mediated signaling events. J. Immunol. 158: 4602-4611. [Abstract]
  4. Muppidi, J. R., R. M. Siegel. 2004. Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nat. Immunol. 5: 182-189. [Medline]
  5. Parlato, S., A. M. Giammarioli, M. Logozzi, F. Lozupone, P. Matarrese, F. Luciani, M. Falchi, W. Malorni, S. Fais. 2000. CD95 (APO-1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway. EMBO J. 19: 5123-5134. [Medline]
  6. Fais, S., A. De Milito, F. Lozupone. 2005. The role of FAS to ezrin association in FAS-mediated apoptosis. Apoptosis 10: 941-947. [Medline]
  7. Refaeli, Y., L. Van Parijs, C. A. London, J. Tschopp, A. K. Abbas. 1998. Biochemical mechanisms of IL-2-regulated Fas-mediated T cell apoptosis. Immunity 8: 615-623. [Medline]
  8. Silvestris, F., P. Cafforio, M. A. Frassanito, M. Tucci, A. Romito, S. Nagata, F. Dammacco. 1996. Overexpression of Fas antigen on T cells in advanced HIV-1 infection: differential ligation constantly induces apoptosis. AIDS 10: 131-141. [Medline]
  9. Baumler, C. B., T. Bohler, I. Herr, A. Benner, P. H. Krammer, K. M. Debatin. 1996. Activation of the CD95 (APO-1/Fas) system in T cells from human immunodeficiency virus type-1-infected children. Blood 88: 1741-1746.
  10. Boudet, F., H. Lecoeur, M. L. Gougeon. 1996. Apoptosis associated with ex vivo down-regulation of Bcl-2 and up-regulation of Fas in potential cytotoxic CD8+ T lymphocytes during HIV infection. J. Immunol. 156: 2282-2293. [Abstract]
  11. Grelli, S., S. Campagna, M. Lichtner, G. Ricci, S. Vella, V. Vullo, F. Montella, S. Di Fabio, C. Favalli, A. Mastino, B. Macchi. 2000. Spontaneous and anti-Fas-induced apoptosis in lymphocytes from HIV-infected patients undergoing highly active anti-retroviral therapy. AIDS 14: 939-949. [Medline]
  12. Li, Q., L. Duan, J. D. Estes, Z. M. Ma, T. Rourke, Y. Wang, C. Reilly, J. Carlis, C. J. Miller, A. T. Haase. 2005. Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells. Nature 434: 1148-1152. [Medline]
  13. Mitra, D., M. Steiner, D. H. Lynch, L. Staiano-Coico, J. Laurence. 1996. HIV-1 upregulates Fas ligand expression in CD4+ T cells in vitro and in vivo: association with Fas-mediated apoptosis and modulation by aurintricarboxylic acid. Immunology 87: 581-585. [Medline]
  14. Hosaka, N., N. Oyaizu, S. Than, S. Pahwa. 2000. Correlation of loss of CD4 T cells with plasma levels of both soluble form Fas (CD95) Fas ligand (FasL) in HIV-infected infants. Clin. Immunol. 95: 20-25. [Medline]
  15. Katsikis, P. D., E. S. Wunderlich, C. A. Smith, L. A. Herzenberg. 1995. Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals. J. Exp. Med. 181: 2029-2036. [Abstract/Free Full Text]
  16. Badley, A. D., A. A. Pilon, A. Landay, D. H. Lynch. 2000. Mechanisms of HIV-associated lymphocyte apoptosis. Blood 96: 2951-2964. [Abstract/Free Full Text]
  17. Gougeon, M. L.. 2003. Apoptosis as an HIV strategy to escape immune attack. Nat. Rev. Immunol. 3: 392-404. [Medline]
  18. Brugnoni, D., P. Airo, M. Pennacchio, G. Carella, A. Malagoli, A. G. Ugazio, F. Porta, R. Cattaneo. 1999. Immune reconstitution after bone marrow transplantation for combined immunodeficiencies: down-modulation of Bcl-2 and high expression of CD95/Fas account for increased susceptibility to spontaneous and activation-induced lymphocyte cell death. Bone Marrow Transplant. 23: 451-457. [Medline]
  19. Hakim, F. T., R. Cepeda, S. Kaimei, C. L. Mackall, N. McAtee, J. Zujewski, K. Cowan, R. E. Gress. 1997. Constraints on CD4 recovery postchemotherapy in adults: thymic insufficiency and apoptotic decline of expanded peripheral CD4 cells. Blood 90: 3789-3798. [Abstract/Free Full Text]
  20. Fry, T. J., B. L. Christensen, K. L. Komschlies, R. E. Gress, C. L. Mackall. 2001. Interleukin-7 restores immunity in athymic T-cell-depleted hosts. Blood 97: 1525-1533. [Abstract/Free Full Text]
  21. Jiang, Q., W. Q. Li, F. B. Aiello, R. Mazzucchelli, B. Asefa, A. R. Khaled, S. K. Durum. 2005. Cell biology of IL-7, a key lymphotrophin. Cytokine Growth Factor Rev. 16: 513-533. [Medline]
  22. Fry, T. J., C. L. Mackall. 2001. Interleukin-7: master regulator of peripheral T-cell homeostasis?. Trends Immunol. 22: 564-571. [Medline]
  23. Schluns, K. S., W. C. Kieper, S. C. Jameson, L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat. Immunol. 1: 426-432. [Medline]
  24. Tan, J. T., E. Dudl, E. LeRoy, R. Murray, J. Sprent, K. I. Weinberg, C. D. Surh. 2001. IL-7 is critical for homeostatic proliferation and survival of naive T cells. Proc. Natl. Acad. Sci. USA 98: 8732-8737. [Abstract/Free Full Text]
  25. Kondrack, R. M., J. Harbertson, J. T. Tan, M. E. McBreen, C. D. Surh, L. M. Bradley. 2003. Interleukin 7 regulates the survival and generation of memory CD4 cells. J. Exp. Med. 198: 1797-1806. [Abstract/Free Full Text]
  26. Gringhuis, S. I., L. F. de Leij, E. W. Verschuren, P. Borger, E. Vellenga. 1997. Interleukin-7 upregulates the interleukin-2-gene expression in activated human T lymphocytes at the transcriptional level by enhancing the DNA binding activities of both nuclear factor of activated T cells and activator protein-1. Blood 90: 2690-2700. [Abstract/Free Full Text]
  27. Kaech, S. M., J. T. Tan, E. J. Wherry, B. T. Konieczny, C. D. Surh, R. Ahmed. 2003. Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat. Immunol. 4: 1191-1198. [Medline]
  28. Li, J., G. Huston, S. L. Swain. 2003. IL-7 promotes the transition of CD4 effectors to persistent memory cells. J. Exp. Med. 198: 1807-1815. [Abstract/Free Full Text]
  29. Napolitano, L. A., R. M. Grant, S. G. Deeks, D. Schmidt, S. C. De Rosa, L. A. Herzenberg, B. G. Herndier, J. Andersson, J. M. McCune. 2001. Increased production of IL-7 accompanies HIV-1-mediated T-cell depletion: implications for T-cell homeostasis. Nat. Med. 7: 73-79. [Medline]
  30. Fry, T. J., E. Connick, J. Falloon, M. M. Lederman, D. J. Liewehr, J. Spritzler, S. M. Steinberg, L. V. Wood, R. Yarchoan, J. Zuckerman, et al 2001. A potential role for interleukin-7 in T-cell homeostasis. Blood 97: 2983-2990. [Abstract/Free Full Text]
  31. Llano, A., J. Barretina, A. Gutiérrez, J. Blanco, C. Cabrera, B. Clotet, J. A. Esté. 2001. Interleukin-7 in plasma correlates with CD4 T-cell depletion and may be associated with emergence of syncytium-inducing variants in human immunodeficiency virus type 1-positive individuals. J. Virol. 75: 10319-10325. [Abstract/Free Full Text]
  32. Mastroianni, C. M., G. Forcina, G. d’Ettorre, M. Lichtner, F. Mengoni, C. D’Agostino, V. Vullo. 2001. Circulating levels of interleukin-7 in antiretroviral-naive and highly active antiretroviral therapy-treated HIV-infected patients. HIV Clin. Trials 2: 108-112. [Medline]
  33. Bolotin, E., G. Annett, R. Parkman, K. Weinberg. 1999. Serum levels of IL-7 in bone marrow transplant recipients: relationship to clinical characteristics and lymphocyte count. Bone Marrow Transplant. 23: 783-788. [Medline]
  34. Jaleco, S., L. Swainson, V. Dardalhon, M. Burjanadze, S. Kinet, N. Taylor. 2003. Homeostasis of naive and memory CD4+ T cells: IL-2 and IL-7 differentially regulate the balance between proliferation and Fas-mediated apoptosis. J. Immunol. 171: 61-68. [Abstract/Free Full Text]
  35. Managlia, E. Z., A. Landay, L. Al-Harthi. 2005. Interleukin-7 signalling is sufficient to phenotypically and functionally prime human CD4 naive T cells. Immunology 114: 322-335. [Medline]
  36. Lelievre, J. D., F. Petit, D. Arnoult, J. C. Ameisen, J. Estaquier. 2005. Interleukin 7 increases human immunodeficiency virus type 1 LAI-mediated Fas-induced T-cell death. J. Virol. 79: 3195-3199. [Abstract/Free Full Text]
  37. Zhang, H., K. S. Chua, M. Guimond, V. Kapoor, M. V. Brown, T. A. Fleisher, L. M. Long, D. Bernstein, B. J. Hill, D. C. Douek, et al 2005. Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells. Nat. Med. 11: 1238-1243. [Medline]
  38. Fry, T. J., M. Moniuszko, S. Creekmore, S. J. Donohue, D. C. Douek, S. Giardina, T. T. Hecht, B. J. Hill, K. Komschlies, J. Tomaszewski, et al 2003. IL-7 therapy dramatically alters peripheral T-cell homeostasis in normal and SIV-infected nonhuman primates. Blood 101: 2294-2299. [Abstract/Free Full Text]
  39. Riemann, D., A. Tcherkes, G. H. Hansen, J. Wulfaenger, T. Blosz, E. M. Danielsen. 2005. Functional co-localization of monocytic aminopeptidase N/CD13 with the Fc{gamma} receptors CD32 and CD64. Biochem. Biophys. Res. Commun. 331: 1408-1412. [Medline]
  40. Livak, K. J., T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 25: 402-408. [Medline]
  41. Lozupone, F., L. Lugini, P. Matarrese, F. Luciani, C. Federici, E. Iessi, P. Margutti, G. Stassi, W. Malorni, S. Fais. 2004. Identification and relevance of the CD95-binding domain in the N-terminal region of ezrin. J. Biol. Chem. 279: 9199-9207. [Abstract/Free Full Text]
  42. Fais, S., W. Malorni. 2003. Leukocyte uropod formation and membrane/cytoskeleton linkage in immune interactions. J. Leukocyte Biol. 73: 556-563. [Abstract/Free Full Text]
  43. Rethi, B., C. Fluur, A. Atlas, M. Krzyzowska, F. Mowafi, S. Grutzmeier, A. De Milito, R. Bellocco, K. I. Falk, E. Rajnavölgyi, F. Chiodi. 2005. Loss of IL-7R{alpha} is associated with CD4 T-cell depletion, high interleukin-7 levels and CD28 down-regulation in HIV infected patients. AIDS 19: 2077-2086. [Medline]
  44. Paiardini, M., B. Cervasi, H. Albrecht, A. Muthukumar, R. Dunham, S. Gordon, H. Radziewicz, G. Piedimonte, M. Magnani, M. Montroni, et al 2005. Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals. J. Immunol. 174: 2900-2909. [Abstract/Free Full Text]
  45. Boutboul, F., D. Puthier, V. Appay, O. Pelle, H. Ait-Mohand, B. Combadiere, G. Carcelain, C. Katlama, S. L. Rowland-Jones, P. Debre, et al 2005. Modulation of interleukin-7 receptor expression characterizes differentiation of CD8 T cells specific for HIV, EBV and CMV. AIDS 19: 1981-1986. [Medline]
  46. Fry, T. J., C. L. Mackall. 2005. The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance. J. Immunol. 174: 6571-6576. [Abstract/Free Full Text]
  47. Estaquier, J., M. Tanaka, T. Suda, S. Nagata, P. Golstein, J. C. Ameisen. 1996. Fas-mediated apoptosis of CD4+ and CD8+ T cells from human immunodeficiency virus-infected persons: differential in vitro preventive effect of cytokines and protease antagonists. Blood 87: 4959-4966.
  48. Kimura, K., H. Matsubara, S. Sogoh, Y. Kita, T. Sakata, Y. Nishitani, S. Watanabe, T. Hamaoka, H. Fujiwara. 1991. Role of glycosaminoglycans in the regulation of T cell proliferation induced by thymic stroma-derived T cell growth factor. J. Immunol. 146: 2618-2624. [Abstract]
  49. Borghesi, L. A., Y. Yamashita, P. W. Kincade. 1999. Heparan sulfate proteoglycans mediate interleukin-7-dependent B lymphopoiesis. Blood 93: 140-148. [Abstract/Free Full Text]
  50. Banwell, C. M., K. M. Partington, E. J. Jenkinson, G. Anderson. 2000. Studies on the role of IL-7 presentation by mesenchymal fibroblasts during early thymocyte development. Eur. J. Immunol. 30: 2125-2129. [Medline]
  51. Ahmad, R., S. T. Sindhu, E. Toma, R. Morisset, A. Ahmad. 2003. Studies on the production of IL-15 in HIV-infected/AIDS patients. J. Clin. Immunol. 23: 81-90. [Medline]
  52. Dockrell, D. H.. 2003. The multiple roles of Fas ligand in the pathogenesis of infectious diseases. Clin. Microbiol. Infect. 9: 766-779. [Medline]
  53. Luciani, F., P. Matarrese, A. M. Giammarioli, L. Lugini, F. Lozupone, C. Federici, E. Iessi, W. Malorni, S. Fais. 2004. CD95/phosphorylated ezrin association underlies HIV-1 gp120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4+ T lymphocytes. Cell Death Differ. 11: 574-582. [Medline]



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