HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Beq, S. Articles by Israël, N. Search for Related Content PubMed PubMed Citation Articles by Beq, S. Articles by Israël, N.

IL-7 Induces Immunological Improvement in SIV-Infected Rhesus Macaques under Antiviral Therapy¹

Stéphanie Beq^2,*,, Marie-Thérèse Nugeyre^2,*, Raphaël Ho Tsong Fang, David Gautier, Roger Legrand^¶, Nathalie Schmitt^*, Jérôme Estaquier, Françoise Barré-Sinoussi^*, Bruno Hurtrel, Rémi Cheynier^3, and Nicole Israël^4,*

^* Unité de Régulation des Infections Rétrovirales, Institut Pasteur, Paris, France; Cytheris, Vanves, France; Unité de Recherche et d’Expertise Physiopathologie des Infections Lentivirales, Institut Pasteur, Paris, France; Unité des Virus Lents, Institut Pasteur, Paris, France; and ^¶ Service de Neurovirologie, Département de Recherches Médicales, Commissariat à l’Energie Atomique, Fontenay-aux-Roses, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Despite efficient antiretroviral therapy (ART), CD4⁺ T cell counts often remain low in HIV-1-infected patients. This has led to IL-7, a crucial cytokine involved in both thymopoiesis and peripheral T cell homeostasis, being suggested as an additional therapeutic strategy. We investigated whether recombinant simian IL-7-treatment enhanced the T cell renewal initiated by ART in rhesus macaques chronically infected with SIV_mac251. Six macaques in the early chronic phase of SIV infection received antiretroviral treatment. Four macaques also received a 3-wk course of IL-7 injections. Viral load was unaffected by IL-7 treatment. IL-7 treatment increased the number of circulating CD4⁺ and CD8⁺ memory T cells expressing activation (HLA-DR⁺, CD25⁺) and proliferation (Ki-67⁺) markers. It also increased naive (CD45RA^brightCD62L⁺) T cell counts by peripheral proliferation and enhanced de novo thymic production. The studied parameters returned to pretreatment values by day 29 after the initiation of treatment, concomitantly to the appearance of anti-IL-7 neutralizing Abs, supporting the need for a nonimmunogenic molecule for human treatment. Thus, IL-7, which increases T cell memory and de novo renewal of naive T cells may have additional benefits in HIV-infected patients receiving ART.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Infection with HIV or SIV leads to severe T lymphopenia and general immune dysfunction. Several mechanisms, including direct and indirect T cell killing, disrupted peripheral homeostasis, and impaired central de novo production, contribute to lymphopenia (1, 2, 3). Combinations of antiretroviral treatments targeting various viral proteins/functions considerably slow disease progression, allowing considerable decreases in viral load and, in most patients, significant increases in peripheral CD4⁺ T cell counts (4). However, although such treatments generally decrease levels of virus production to undetectable levels, they often fail to reverse lymphopenia and do not permit the restoration of specific antiviral immune responses (5, 6, 7, 8).

IL-7, which is constitutively produced by the bone marrow, the thymus, mucosal lymphoid tissues, and lymph nodes (9), plays a crucial role in T cell homeostasis. This cytokine is implicated in thymopoiesis, in which it sustains thymocyte proliferation and survival (10, 11). It also regulates peripheral naive T cell survival by modulating production of the antiapoptotic molecule Bcl-2 (12) and sustaining peripheral T cell expansion in response to antigenic stimulation in mice (13, 14, 15).

HIV-infected patients generally have high plasma IL-7 concentrations. The increase in IL-7 production generally occurs in the first few weeks of infection and persists throughout disease progression. The inverse correlation between plasma IL-7 concentrations and CD4⁺ T cell counts (16, 17, 18) suggests either a feedback mechanism for restoring peripheral T cell counts in lymphopenic patients or an increased IL-7 availability in lymphopenic hosts (15, 16, 19). Moreover, recent studies suggest that plasma IL-7 concentration might be a good predictive marker of CD4⁺ T cell restoration under therapy (20, 21, 22).

Previous studies in macaque models have demonstrated that exogenous IL-7 induces the expansion of naive and memory peripheral T cell populations in both healthy and SIV-infected animals (23, 24) However, the possible effects of modifying thymic production in response to IL-7 injections remain unclear.

Thymic output can be estimated by quantifying the signal joint T cell receptor excision circle (sjTREC),⁵ generated during TCR locus deletion, which usually precedes TCR-chain rearrangement (25, 26). However, as recently demonstrated (26), thymic function can be estimated more accurately in HIV-infected humans by quantifying precursor T cell proliferation. Indeed, such proliferation has a direct impact on both the number of cells subjected to thymic selection and the dilution of DJTRECs (byproducts of TCR-chain rearrangement). This dilution effect can be followed by measuring, in PBMC, the sj:TREC ratio (26) In contrast to sjTREC frequency, which decreases during T cell proliferation, the sj:TREC ratio is not affected by homeostatic or Ag-induced peripheral T cell proliferation. This parameter is therefore a reliable marker for thymic function, even in HIV-infected patients and SIV-infected macaques, demonstrating variation in naive or memory T cell proliferation.

In this study, we administered recombinant simian IL-7 (rsIL-7) to SIV-infected rhesus macaques under antiretroviral therapy (ART). We first checked that IL-7 therapy did not counteract the decrease in plasma viral load obtained under ART. We then analyzed the impact of exogenous IL-7 on T cell activation and proliferation under ART. We also demonstrated that IL-7 treatment directly influenced naive T cell homeostasis by enhancing thymic function.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals and virus infection

Six young rhesus macaques (2 years old), housed and cared for in accordance with European guidelines, were included in this study. These animals were demonstrated to be seronegative for SIVmac, simian T cell leukemia virus type 1, simian retrovirus type 1 (type D retrovirus), and herpesvirus B. All of the animals were inoculated with 10 AID₅₀ (50% animal infective doses) of the pathogenic SIVmac251 isolate (provided by A.-M. Aubertin, Institut National de la Santé et de la Recherche Médicale Unité 544, Strasbourg, France).

All procedures with animals were performed after anesthesia with ketamine (Imalgène 1000; Mérial).

Antiretroviral and IL-7 treatment

All animals were injected with antiretroviral treatment at day 70 after infection, during the early asymptomatic chronic phase of infection, to mimic human tritherapy treatment. The antiretroviral treatment consisted of AZT (GlaxoSmithKline; 4.5 mg/kg twice per day), 3TC (GlaxoSmithKline; 2.5 mg/kg twice per day), and ddI (Videx; Bristol Myers; 3 mg/kg twice per day).

rsIL-7 was originally cloned by Cytheris from Macaca mulatta and filed as Q95J83 in the "Swiss Prot" data bank; this sequence was further confirmed to be identical with the Papio papio (baboon) IL-7 sequence filed as Q8HZN1 in the same bank (27).

The rsIL-7 injections were performed after a significant decrease (2 log of viral load expressed in number of RNA copies per milliliter) in viral load due to antiretroviral treatment. Four infected animals treated with antiretroviral drugs for 34 days received injections s.c. every 2 days for 26 days with 100 µg/kg recombinant macaque IL-7 (rsIL-7) derived from Escherichia coli. IL-7 was supplied by Cytheris (CYT 01 008). These four infected macaques continued to receive ART during the period of rsIL-7 treatment. The antiretroviral treatment was stopped 17 days after the end of rsIL-7 treatment. The four infected macaques treated with both antiretroviral drugs and rsIL-7 were macaques 3, 4, 5, and 6. During the same period, the two control macaques, macaques 1 and 2, received only antiretroviral drugs. All of the animals were treated with antiretroviral drugs for 77 days.

TREC quantifications

Primers specific for each of the sjTREC (Rec-J), nine DJ TRECs (D1-J1S2 to D2-J2S4), and the human CD3-chain of the gene (see Table I) were defined on macaque germline sequences (GenBank accession nos.: L43137, L43138, DQ005631, and DQ005632). Parallel quantification of each deletion circle and of the CD3- amplicon was performed for each sample, using LightCycler technology (Roche Diagnostics) as previously described (28). Briefly, PBMCs were lysed by incubation in Tween 20 (0.05%), Nonidet P-40 (0.05%), and proteinase K (100 µg/ml) for 30 min at 56°C, and then 15 min at 98°C. Multiplex PCR amplification was performed for sjTREC or each of the nine DJ TRECs, together with the CD3-chain, in a final volume of 100 µl (10 min initial denaturation at 95°C, then 22 cycles of 30 s at 95°C, 30 s at 60°C, 2 min at 72°C) using outer 3'/5' primer pairs. PCR conditions in the LightCycler experiments, performed on 1/100th of the initial PCR products, were as follows: 1 min initial denaturation at 95°C, then 40 cycles of 1 s at 95°C, 10 s at 60°C, 15 s at 72°C; fluorescence measurements were performed at the end of elongation. TREC and CD3 LightCycler quantifications were performed in independent experiments, using the same first-round serial dilution standard curve. This highly sensitive nested quantitative PCR assay made it possible to detect one copy in 10⁵ cells for each DNA circle. The sjTRECs and the nine DJ TRECs were quantified in triplicate for all the samples studied. The sum of DJ TREC frequencies (DJ TRECs) was estimated to be 13 times the mean of the nine measured DJ TREC frequencies. The sj:TREC ratio corresponds to the sjTREC frequency divided by the sum of DJ TREC frequencies (sj/TREC = sjTRECs/DJ TRECs).

View this table: [in this window] [in a new window]   Table I. Primers and probes for the quantification of sjTREC, DJTRECs, and CD3-chain

rsIL-7 was run on a 15% polyacrylamide gel containing SDS and then transferred to Immobilon-P membrane (Millipore). The membrane was incubated in blocking buffer (PBS containing 0.1% Tween 20 and 5% skimmed milk powder) for 45 min, and incubated with macaque serum at a dilution of 1/200 in this buffer for 1 h. We used 10 µg of anti-human IL-7 Ab as a positive control (R&D Systems). Membranes were washed with blocking buffer. Specifically bound Ab was detected by incubation with rabbit anti-monkey or anti-goat IgG peroxidase-conjugate (Sigma-Aldrich) at a dilution of 1/16,000 in blocking buffer. Western blots were then washed and developed using the Amersham ECL system according to the manufacturer’s protocol.

Quantification of plasma IL-7 levels

IL-7 was quantified in the plasma using the IL-7 Quantikine HS kit according to the manufacturer’s conditions (R&D Systems).

Abs used for immunostaining

Cells were immunostained with the following conjugated mAbs: CD20-PE (clone L27), HLADR-PE (clone L243), CD8-PerCP (clone SK1), CD4-FITC or CD4-PE (clone M-T477), CD4-PerCP (clone SK3), CD62L-PE (clone SK11), CD25-PE (clone 2A3) from BD Biosciences; CD45RA-FITC (clone 2H4), CD127-PE (clone R34.34) from Beckman Coulter; and Ki-67-FITC (clone Ki-67) from DakoCytomation; and IgG1, k-FITC, PE, and PerCP (clone MOPC-21), as negative controls.

Lymphocyte immunophenotyping and flow cytometry analysis

EDTA-treated blood cells were incubated for 15 min with the conjugated mAbs. For intracellular labeling, cells were permeabilized with the Cytofix/Cytoperm kit (BD Biosciences) before incubation with mAb Ki-67. Erythrocytes were lysed with the Beckman Coulter lysing kit according to the manufacturer’s instructions. Samples were then washed and fixed in PBS supplemented with 1% paraformaldehyde, and analyzed by flow cytometry in an XL-4C machine (Beckman Coulter).

Quantification of serum viral loads

Real-time quantitative RT-PCR was performed as previously described with minor modifications, to determine serum viral load (24). Briefly, standard RNA was obtained from the pGEM-5Zf(+) GAG plasmid (provided by A.-M. Aubertin, Institut National de la Santé, Strasbourg, France). The plasmid was linearized with SpeI (New England Biolabs) and used for in vitro transcription, with an in vitro transcription kit and T7 RNA polymerase (Ambion). The resulting transcripts were treated with RNase-free DNase and purified on a Chroma SPIN-100 column (Clontech). Standard dilutions of RNA were aliquoted and immediately frozen at –70°C. RNA from serum samples was purified with the TRI Reagent BD Kit (Molecular Research Center).

PBMCs culture

PBMC from healthy donors were purified on a Ficoll-Hypaque gradient and cultured in medium supplemented with 10% FCS, 1% phosphatidylserine (10,000 U/ml penicillin and 10 mg/ml streptomycin), and 2 mM L-glutamine (Invitrogen Life Technologies). Recombinant human IL-7 (rhIL-7) was added as required, at a concentration of 1 µg/ml. Plasma from one macaque was added to the culture at a final concentration of 1 µg/ml IL-7. After 24 h, the PBMCs were labeled with CD127-PE and analyzed by flow cytometry in an XL-4C machine (Beckman Coulter).

Statistics

Statistical analysis (Pearson’s correlation test, r and p values) was performed using the Vassar College web site. r values 0.3 or –0.3, and p values 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
rsIL-7 transiently increases T cell counts without affecting viral load

Six juvenile rhesus macaques were infected with a primary isolate of SIVmac251. Following acute infection and the establishment of viral set point, the animals received a combination of three antiretroviral drugs: 3TC, AZT, and ddI. On day 34 of ART, when viral load had stabilized, two animals (animals 1 and 2) were maintained on the same regimen while the other four animals (animals 3, 4, 5, and 6) received ART plus 100 µg of rsIL-7 per kg of body weight every other day, for 26 days. Antiretroviral treatment was stopped in all the animals on day 17, after the last rsIL-7 injection. This particular IL-7 regimen was chosen on the basis of previous pharmacodynamic studies in monkeys showing full receptor occupancy 24 h after single-dose injection, and reappearance of CD127 after 48 h, reflecting receptor availability (Cytheris, unpublished observations).

Despite heterogeneous initial viral loads (10⁴ to 10⁶ copies/ml at set point), all of the animals displayed a drastic decrease in peripheral viral load in response to ART (Fig. 1). Viral loads reached undetectable levels for macaques 1 and 5 and were reduced by 3 to 5 log in the other four animals. Macaques 3, 4, 5, and 6 received rsIL-7 on day 104 after infection, in addition to ART. IL-7 did not affect viral load, during or after treatment or before the end of ART. In contrast, serum viral load rapidly increased in all macaques following the cessation of ART, reaching pretreatment values within a few days. Peripheral CD4⁺ T cell counts rapidly decreased in four infected animals (by a factor of 2 to 3 within 63 days for macaques 1, 3, 4, and 5) and decreased only slightly in the other animals (macaques 2 and 6). ART limited the decrease in peripheral CD4⁺ T cell counts in all treated animals. Treatment with IL-7 was immediately followed by a large increase in CD4⁺ T cell counts in macaques 3 (from 1000 to 3000 cells/µl) and 4 (from 2000 to 5000 cells/µl), with preinfection values reached in both cases, and by a less dramatic increase in macaque 5 (from 1200 to 2500 cells/µl). The increase in peripheral CD4⁺ T cell counts following rsIL-7 treatment was transient in all animals and decreased rapidly before the end of rsIL-7 treatment.

View larger version (43K): [in this window] [in a new window]   FIGURE 1. rsIL-7 transiently increases CD4+ and CD8+ cell counts without affecting viral load. Six young rhesus macaques were infected with the pathogenic SIVmac251 isolate. They all received ART for 70 days after infection (light gray area). Two animals (nos. 1 and 2) were maintained under this treatment, whereas the remaining four animals (nos. 3–6) were treated with ART plus 100 µg/kg rsIL-7 every other day for 26 days (dark gray area). The indicate changes in viral load, and • and correspond to counts of CD4+ and CD8+ T cells, respectively.

Changes in CD4⁺ and CD8⁺ T cell counts during rsIL-7 treatment can be used to classify the animals according to their response to treatment: responder animals (macaques 3 and 4) displayed significant increases in circulating T cell levels, whereas macaque 5 responded less strongly (poorer responder) and macaque 6 did not respond at all (nonresponder) to IL-7 treatment.

rsIL-7 increases proliferation of CD4⁺ and CD8⁺ subsets concomitantly with activation

Having demonstrated an increase in peripheral CD4⁺ and CD8⁺ T cell counts in response to IL-7 treatment, we investigated whether this increase was due to peripheral T cell activation. We assessed the ongoing proliferation of CD4⁺ and CD8⁺ T cells by evaluating the expression of Ki-67, a nuclear Ag expressed during and shortly after cell proliferation. Animals 3, 4, and, to a lesser extent, 5 displayed large increases in the percentages of CD4⁺ and CD8⁺ T cells undergoing proliferation during IL-7 treatment (Fig. 2). This effect, like the increase in absolute T cell counts, was transient, with a return to baseline either during or immediately after the end of the IL-7 treatment period. In parallel, we analyzed the expression of activation markers on T cells. In responders (macaques 3 and 4), the number of CD25 and HLA-DR-expressing CD4⁺ T cells increased transiently during IL-7 treatment (by factors of 3.49 and 3.48, respectively, for CD25 and 4.88 and 3.32, respectively, for HLA-DR in macaques 3 and 4). In contrast, in the CD8⁺ T cell population, CD25 expression remained stable, whereas the number of HLA-DR-expressing cells increased in macaques 3 and 4 (by factors of 3.76 and 4.29, respectively). IL-7-induced T cell activation was less pronounced in macaque 5 and was entirely absent in macaque 6. Thus, in responders, rsIL-7 transiently increased the activation of peripheral T cells, concomitant with increased proliferation.

View larger version (50K): [in this window] [in a new window]   FIGURE 2. rsIL-7 concomitantly increases the proliferation and activation of CD4+ and CD8+ subsets. T cell activation, as quantified by the expression of HLA-DR (•) and CD25 (), was assessed in CD4+ (left panel) and CD8+ (right panel) T cells in ART- (macaques 1 and 2) or ART + IL-7-treated (macaques 3–6) animals. Peripheral T cell proliferation was estimated by evaluating Ki-67 expression (). Periods of ART treatment are highlighted in light gray, with the dark gray bars indicating IL-7 treatment.

We analyzed the effects of rsIL-7 treatment on the expansion of naive and memory T cell populations, by carrying out FACS analysis of the surface expression of CD45RA and CD62L. In this analysis, naive T cells were defined as CD45RA^bright, CD62L⁺ T cells, with all other T cell populations defined as memory T cells. These memory T cells included CD45RA^–CD62L^– central (T_CM) and CD45RA^lowCD62L⁺ effector (T_EM) memory T cells, as defined by Seder and Ahmed (29).

In responders, the number of memory T cells in the CD4⁺ and CD8⁺ subsets increased considerably during the IL-7 treatment period (Fig. 3). CD4⁺ and CD8⁺ memory T cell counts increased by a factor of 2.2 to 3.5 with respect to pretreatment values, during treatment with rsIL-7. At the peak of the IL-7 response, memory T cells accounted for 40 and 70% of circulating CD4⁺ T cells in macaques 3 and 4, respectively, whereas memory CD8⁺ T cells substantially outnumbered naive cells (70 and 80%).

View larger version (46K): [in this window] [in a new window]   FIGURE 3. Naive (CD45brightCD62L+) and memory CD4+ and CD8+ T cell counts increased in rsIL-7-treated animals. The frequency of naive () and memory () T cells, as defined by the expression of CD45RA and CD62L surface Ags, was followed in CD4+ (left panel) and CD8+ (right panel) peripheral T cell subsets during treatment with either ART alone (macaques 1 and 2) or ART+ IL-7 (macaques 3–6). Periods of ART treatment are highlighted in light gray, with the dark gray bars corresponding to IL-7 treatment.

In macaque 5, the expansion of peripheral T cell subsets observed under rsIL-7 therapy remained more limited (Fig. 3). CD45RA^brightCD62L⁺ naive T cells, which steadily declined in number following SIV infection, displayed a significant increase in number in both the CD4⁺ and CD8⁺ subsets under rsIL-7 therapy (by factors of 3.6 and 2.7, respectively). During the same period, similar variations were observed in the memory CD4⁺ and CD8⁺ subsets.

The observed expansions of the naive and memory T cell populations were only transient, with both the frequencies and absolute counts of all the subsets concerned returning to pre-IL-7 treatment values before the end of the IL-7 treatment period or by day 139.

The nonresponder (macaque 6) showed no significant change in the naive or memory CD4⁺ and CD8⁺ subpopulations.

Thus, under efficient ART, IL-7 therapy has, in some macaques, a rapid impact on T cell numbers leading to a transient expansion of CD4⁺ and CD8⁺ CD45RA^brightCD62L⁺ naive and memory T cells.

rsIL-7 induces the central renewal of naive T cells by increasing thymic function

IL-7 is the principal chemokine responsible for the proliferation of intrathymic T cell precursors (30). This mechanism is required to increase the number of double-positive cells subjected to positive and negative selection, thymic output (30). We investigated changes in thymic function during IL-7 treatment in SIV-infected macaques. We first analyzed sjTREC frequency in peripheral blood samples (Fig. 4a). During the first 2 mo of infection, sjTREC frequency decreased slightly in macaques 1 and 4. IL-7 injection during antiretroviral treatment did not generate readily interpretable results, in contrast to what has been reported for rhesus macaques infected with a SIVmac-nef isolate (31). However, this parameter is strongly influenced by peripheral T cell proliferation, which changes markedly during IL-7 treatment. We therefore estimated the effect of IL-7 treatment on thymic production by assessing the intrathymic proliferation of precursor T cells, through quantification of the sj:TREC ratio, which is totally independent of peripheral T cell proliferation (26). Thymic function is initially reduced during primary SIV infection (Fig. 4b), but this effect is less marked than that in HIV-infected humans (26). During ART and IL-7 treatment, the sj:TREC ratio rapidly increased in most of the animals (Fig. 4b). This increase was particularly large in the animals that responded well (macaques 3 and 4), suggesting that this effect was enhanced by IL-7 treatment. Moreover, the increase in sj:TREC ratio, during the ART and IL-7 treatment period, correlated directly with variations in CD4⁺, CD8⁺, and total naive T cells counts in IL-7-treated and untreated animals (Fig. 4, c–e: r = 0.93, p = 0.007; r = 0.92, p = 0.007; and r = 0.86, p = 0.02). In particular, macaque 6 (, Fig. 4), for which T cell counts continued to decline during IL-7 treatment, showed no rebound in intrathymic precursor T cell proliferation. In contrast, the sj:TREC ratio increased for macaques 3, 4, and 5, and this increase was accompanied by the maintenance or enhancement of circulating naive T cell numbers, in both the CD4 and CD8 compartments. Thus, IL-7 treatment increases thymopoiesis.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. Analysis of thymic function in rsIL-7-treated animals. Changes in sjTREC (a) and sj:TREC ratio (b) between baseline and day 63 (pre-ART) are presented on the left part of a and b. Changes in these parameters between days 63 and 118 (at peak rsIL-7 activity) are presented on the right part of these panels. Each animal is represented by a different symbol: macaque 1, ; macaque 2, ; macaque 3, •; macaque 4, ; macaque 5, ; and macaque 6, . The correlations between variations of sj:TREC ratio and fluctuations in number of naive T cells, naive CD4+ T cells, or naive CD8+ T cells between days 63 and 118 are shown in c, d, and e, respectively. Pearson’s correlation coefficients and their associated probabilities are shown for each graph.

The lack of stability of the various effects measured following IL-7 injection prompted us to look for possible immunogenicity of the injected rsIL-7 itself, because this molecule was purified from bacterial cultures. We first analyzed levels of CD127, the IL-7R -chain, on the membrane of peripheral CD4⁺ and CD8⁺ T cells. Mean fluorescence intensity for CD127 decreased rapidly, suggesting that the receptor was masked or internalized during IL-7 treatment (Fig. 5a). However, the mean fluorescence intensity for CD127 returned to preinjection values before the end of IL-7 treatment, demonstrating that the cytokine was no longer able to interact with its receptor. We searched for IL-7-specific Abs in sera of the treated animals. A strong immune response to IL-7 was detected in all macaques, as early as 29 days after the start of IL-7 treatment (Fig. 5b). Ab titers reached 1/100, 1/500, 1/1000, and 1/1500 in the four animals. Finally, the neutralizing activity of the induced IL-7-specific Abs was tested in vitro, using human PBMCs as reporter cells. IL-7 decreased the amount of CD127 detected (Fig. 5c, left panel), but a plasma sample taken from macaque 4 on day 29 abolished the effect of the cytokine (Fig. 5c, right panel), demonstrating the presence of neutralizing Abs against IL-7. This neutralization effect may account for the transient nature of the immunological improvement observed in IL-7-treated macaques.

View larger version (38K): [in this window] [in a new window]   FIGURE 5. rsIL-7-neutralizing Abs. a, Mean CD127 fluorescence intensity (MFI) of CD4+ (left panel) and CD8+ (right panel) T cells. Symbols are as in Fig. 4. b, Detection of anti-IL-7 Abs in the plasma of four IL-7-treated animals (lanes 3–6). Lanes 1–2 represent untreated animals 1 and 2. c, Neutralization of exogenous IL-7 by plasma from IL-7-treated animals. CD127 expression was analyzed on PBMCs alone (gray lines; left and right panels) or cultured with rhIL-7 in the absence (dark line; left panel) or presence (dark line; right panel) of plasma from an immune macaque (macaque 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

In animals that responded well, the peripheral expansion of naive and memory T cell populations coincided perfectly with the observed increase in expression of CD25 and HLA-DR activation markers (Figs. 2 and 3). This is not surprising for the memory T cell compartment, because these cells must receive activation signals triggered by a specific Ag to initiate proliferation. T_CM and T_EM CD8⁺ cells have also been shown to proliferate in vitro in response to IL-7 (38). In the CD4 compartment, IL-7 alone did not seem to be sufficient to drive the proliferation of adult memory T cells (39). However, the situation in vivo may be different because, in SIV-infected macaques under ART, activated memory CD4⁺ T cells are already present in the periphery, and this population of cells can be expanded in response to IL-7 treatment.

CD4⁺ and CD8⁺ peripheral naive T cells also increase in number during IL-7 treatment, albeit to a lesser extent than memory T cells. Indeed as suggested by several in vitro studies, only a fraction of peripheral naive T cells proliferate in response to IL-7 (40). Jaleco et al. (41) demonstrated that naive T cells from human cord blood are much more likely to proliferate in response to IL-7 than naive T cells purified from adult blood, suggesting that recent thymic emigrants (RTE) in the naive T cell subset are more likely to respond to IL-7 by increasing their rate of homeostatic proliferation. These IL-7-responding cells expressed HLA-DR. RTE are activated in the thymus. Mature precursor T cells are activated by cytokines such as TNF and IL-7 before leaving the thymic microenvironment (41). Mature thymocytes have been shown to contain activated NF-B and the activation marker HLA-DR. These cells probably maintain a certain degree of activation during their emigration from the thymus as RTE, until their subsequent differentiation into resting naive T cells in the blood, with the loss of NF-B activity (42).

In contrast to the quantification of sjTREC frequency, which is influenced by both central (thymopoiesis) and peripheral (homeostatic) mechanisms, quantification of the sj:TREC ratio is a reliable marker of thymic production because it depends entirely on the proliferation of intrathymic precursor T cells (26). Using this marker, we demonstrated that IL-7 effectively promotes the in vivo proliferation of precursor T cells within the thymus (Fig. 4). Within this organ, several subpopulations express CD127 and may therefore proliferate in response to IL-7. This is the case for early immature thymocytes, such as CD34⁺CD1⁺ and CD34⁺CD1⁺ triple-negative, CD3^–CD4⁺CD8^– intermediate single positive (ISP), and late mature CD4⁺ and CD8⁺ single-positive cells. Okamoto et al. reported the proliferation of ISP thymocytes in IL-7-treated thymic organ cultures (30). Such proliferation during thymopoiesis is directly responsible for the increase in sj:TREC ratio, as these ISP cells have already rearranged their TCR-chain and proliferate before TCR-chain rearrangement occurs.

In a previous study analyzing the effect of rhIL-7 injection to SIV-infected macaques (24), we demonstrated that this cytokine had no effect on the various hemopoietic populations, with the exception of T cells. This is probably also the case for the macaque cytokine. However, one major difference between the two studies is the transient effect of rsIL-7. This time-limited effect may be due to differences in the injection schedule between the two studies. In the previous study, 2 x 40 µg/kg rhIL-7 was injected daily, whereas we used a single injection every 2 days (100 µg/kg), on the basis of previous pharmacodynamic studies in monkeys. Because the half-life of IL-7 in vivo is 6 h (M. Morre, personal communication), the protocol used here may not be optimal for maintaining stable serum IL-7 concentrations. In addition, despite the macaque origin of the injected IL-7, the animals rapidly developed strong IL-7-specific humoral responses (Fig. 5). It could be tempting to suggest that the rate of anti-IL-7-neutralizing Ab response may lead to the weak or nonresponsiveness in macaque 5 and 6. However, as shown in Fig. 5a, the IL-7R was undetectable, for all the treated animals, at day 118 (2 wk postinitiation of the IL-7 treatment), suggesting that the neutralizing Ab response was, at least, not sufficient at this time point to entirely neutralize IL-7. Moreover, the nonresponder animal (macaque 6) was the last one to recover CD127 expression, suggesting that the anti-IL-7 response was slower in this animal than in the others. Indeed, the end of the transient period of response to IL-7 correlated with the reappearance of surface CD127, reflecting the fact that the cytokine was no longer able to interact with its receptor, because of the presence of anti-IL-7 Abs. The production of a nonimmunogenic IL-7 molecule is being planned and should be tested to verify whether it induces sustained effects.

In conclusion, IL-7, in combination with efficient ART, can help to increase the naive T cell pool by enhancing thymopoiesis and peripheral proliferation. This may help HIV-infected patients to restore their naive T cell compartment, not only quantitatively, but also qualitatively, by increasing the diversity of naive T cells without influencing viral load.

	Acknowledgments

 
We thank Dr. A.-M. Aubertin (Institut National de la Santé et de la Recherche Médicale Unité 544, Strasbourg, France) for providing us with the SIVmac251 isolate and the pGEM-5Zf(+) GAG plasmid; GlaxoSmithKline for providing 3TC; Cytheris for providing rsIL-7; and Drs. D. Scott-Algara and G.-F. Pancino for helpful discussions and careful reading of the manuscript.

	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 Agence Nationale pour la Recherche sur le Sida (ANRS). S.B. was the recipient of a Cytheris-Association Nationale de la Recherche Technique Fellowship, under Convention Industrielle de Formation par la Recherche convention. R.H.T.F. was the recipient of an ANRS Fellowship. N.S. was the recipient of a Sidaction Fellowship.

² S.B. and M.-T.N. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Rémi Cheynier, Unité des Virus Lents, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France. E-mail address: remi{at}pasteur.fr

⁴ This work is dedicated to the memory of Nicole Israël.

⁵ Abbreviations used in this paper: sjTREC, signal joint T cell receptor excision circle; rsIL-7, recombinant simian IL-7; ART, antiretroviral therapy; rhIL-7, recombinant human IL-7; RTE, recent thymic emigrant; ISP, intermediate single positive.

Received for publication July 26, 2005. Accepted for publication October 25, 2005.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Douek, D. C.. 2003. Disrupting T-cell homeostasis: how HIV-1 infection causes disease. AIDS Rev. 5: 172-177. [Medline]
2. Roederer, M., S. C. De Rosa, N. Watanabe, L. A. Herzenberg. 1997. Dynamics of fine T-cell subsets during HIV disease and after thymic ablation by mediastinal irradiation. Semin. Immunol. 9: 389-396. [Medline]
3. Silvestri, G., M. B. Feinberg. 2003. Turnover of lymphocytes and conceptual paradigms in HIV infection. J. Clin. Invest. 112: 821-824. [Medline]
4. Autran, B., G. Carcelain, P. Debre. 2001. Immune reconstitution after highly active anti-retroviral treatment of HIV infection. Adv. Exp. Med. Biol. 495: 205-212. [Medline]
5. Teixeira, L., H. Valdez, J. M. McCune, R. A. Koup, A. D. Badley, M. K. Hellerstein, L. A. Napolitano, D. C. Douek, G. Mbisa, S. Deeks, et al 2001. Poor CD4 T cell restoration after suppression of HIV-1 replication may reflect lower thymic function. AIDS 15: 1749-1756. [Medline]
6. Markert, M. L., A. P. Alvarez-McLeod, G. D. Sempowski, L. P. Hale, J. M. Horvatinovich, K. J. Weinhold, J. A. Bartlett, T. A. D’Amico, B. F. Haynes. 2001. Thymopoiesis in HIV-infected adults after highly active antiretroviral therapy. AIDS Res. Hum. Retroviruses 17: 1635-1643. [Medline]
7. Lange, C. G., M. M. Lederman, J. S. Madero, K. Medvik, R. Asaad, C. Pacheko, C. Carranza, H. Valdez. 2002. Impact of suppression of viral replication by highly active antiretroviral therapy on immune function and phenotype in chronic HIV-1 infection. J. Acquir. Immune Defic. Syndr. 30: 33-40. [Medline]
8. Lange, C. G., H. Valdez, K. Medvik, R. Asaad, M. M. Lederman. 2002. CD4⁺ T-lymphocyte nadir and the effect of highly active antiretroviral therapy on phenotypic and functional immune restoration in HIV-1 infection. Clin. Immunol. 102: 154-161. [Medline]
9. Watanabe, M., Y. Ueno, T. Yajima, Y. Iwao, M. Tsuchiya, H. Ishikawa, S. Aiso, T. Hibi, H. Ishii. 1995. Interleukin 7 is produced by human intestinal epithelial cells and regulates the proliferation of intestinal mucosal lymphocytes. J. Clin. Invest. 95: 2945-2953. [Medline]
10. Suda, T., A. Zlotnik. 1991. IL-7 maintains the T cell precursor potential of CD3^–CD4^–CD8^– thymocytes. J. Immunol. 146: 3068-3073. [Abstract]
11. Maraskovsky, E., L. A. O’Reilly, M. Teepe, L. M. Corcoran, J. J. Peschon, A. Strasser. 1997. Bcl-2 can rescue T lymphocyte development in interleukin-7 receptor-deficient mice but not in mutant rag-1^–/^– mice. Cell 89: 1011-1019. [Medline]
12. Rathmell, J. C., E. A. Farkash, W. Gao, C. B. Thompson. 2001. IL-7 enhances the survival and maintains the size of naive T cells. J. Immunol. 167: 6869-6876. [Abstract/Free Full Text]
13. von Freeden-Jeffry, U., P. Vieira, L. A. Lucian, T. McNeil, S. E. Burdach, R. Murray. 1995. Lymphopenia in interleukin (IL)-7 gene-deleted mice identifies IL-7 as a nonredundant cytokine. J. Exp. Med. 181: 1519-1526. [Abstract/Free Full Text]
14. Peschon, J. J., P. J. Morrissey, K. H. Grabstein, F. J. Ramsdell, E. Maraskovsky, B. C. Gliniak, L. S. Park, S. F. Ziegler, D. E. Williams, C. B. Ware. 1994. Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice. J. Exp. Med. 180: 1955-1960. [Abstract/Free Full Text]
15. Fry, T. J., C. L. Mackall. 2001. Interleukin-7: master regulator of peripheral T-cell homeostasis?. Trends Immunol. 22: 564-571. [Medline]
16. 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]
17. Llano, A., J. Barretina, A. Gutierrez, J. Blanco, C. Cabrera, B. Clotet, J. A. Este. 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]
18. 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]
19. 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]
20. Beq, S., M. T. Rannou, A. Fontanet, J. F. Delfraissy, J. Theze, J. H. Colle. 2004. HIV infection: pre-highly active antiretroviral therapy IL-7 plasma levels correlate with long-term CD4 cell count increase after treatment. AIDS 18: 563-565. [Medline]
21. Boulassel, M. R., G. H. Smith, N. Gilmore, M. Klein, T. Murphy, J. MacLeod, R. LeBlanc, J. Allan, P. Rene, R. G. Lalonde, J. P. Routy. 2003. Interleukin-7 levels may predict virological response in advanced HIV-1-infected patients receiving lopinavir/ritonavir-based therapy. HIV Med. 4: 315-320. [Medline]
22. Resino, S., I. Galan, R. Correa, L. Pajuelo, J. M. Bellon, M. A. Munoz-Fernandez. 2005. Homeostatic role of IL-7 in HIV-1 infected children on HAART: association with immunological and virological parameters. Acta Paediatr. 94: 170-177. [Medline]
23. 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]
24. Nugeyre, M. T., V. Monceaux, S. Beq, M. C. Cumont, R. Ho Tsong Fang, L. Chene, M. Morre, F. Barre-Sinoussi, B. Hurtrel, N. Israel. 2003. IL-7 stimulates T cell renewal without increasing viral replication in simian immunodeficiency virus-infected macaques. J. Immunol. 171: 4447-4453. [Abstract/Free Full Text]
25. Douek, D. C., R. D. McFarland, P. H. Keiser, E. A. Gage, J. M. Massey, B. F. Haynes, M. A. Polis, A. T. Haase, M. B. Feinberg, J. L. Sullivan, et al 1998. Changes in thymic function with age and during the treatment of HIV infection. Nature 396: 690-695. [Medline]
26. Dion, M. L., J. F. Poulin, R. Bordi, M. Sylvestre, R. Corsini, N. Kettaf, A. Dalloul, M. R. Boulassel, P. Debre, J. P. Routy, et al 2004. HIV infection rapidly induces and maintains a substantial suppression of thymocyte proliferation. Immunity 21: 757-768. [Medline]
27. Storek, J., T. Gillespy, 3rd, H. Lu, A. Joseph, M. A. Dawson, M. Gough, J. Morris, R. C. Hackman, P. A. Horn, G. E. Sale, et al 2003. Interleukin-7 improves CD4 T-cell reconstitution after autologous CD34 cell transplantation in monkeys. Blood 101: 4209-4218. [Abstract/Free Full Text]
28. Poulin, J. F., M. Sylvestre, P. Champagne, M. L. Dion, N. Kettaf, A. Dumont, M. Lainesse, P. Fontaine, D. C. Roy, C. Perreault, et al 2003. Evidence for adequate thymic function but impaired naive T-cell survival following allogeneic hematopoietic stem cell transplantation in the absence of chronic graft-versus-host disease. Blood 102: 4600-4607. [Abstract/Free Full Text]
29. Seder, R. A., R. Ahmed. 2003. Similarities and differences in CD4⁺ and CD8⁺ effector and memory T cell generation. Nat. Immunol. 4: 835-842. [Medline]
30. Okamoto, Y., D. C. Douek, R. D. McFarland, R. A. Koup. 2002. Effects of exogenous interleukin-7 on human thymus function. Blood 99: 2851-2858. [Abstract/Free Full Text]
31. Fang, R. H., E. Khatissian, V. Monceaux, M. C. Cumont, S. Beq, J. C. Ameisen, A. M. Aubertin, N. Israel, J. Estaquier, B. Hurtrel. 2005. Disease progression in macaques with low SIV replication levels: on the relevance of TREC counts. AIDS 19: 663-673. [Medline]
32. Smithgall, M. D., J. G. Wong, K. E. Critchett, O. K. Haffar. 1996. IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells. J. Immunol. 156: 2324-2330. [Abstract]
33. Scripture-Adams, D. D., D. G. Brooks, Y. D. Korin, J. A. Zack. 2002. Interleukin-7 induces expression of latent human immunodeficiency virus type 1 with minimal effects on T-cell phenotype. J. Virol. 76: 13077-13082. [Abstract/Free Full Text]
34. Ducrey-Rundquist, O., M. Guyader, D. Trono. 2002. Modalities of interleukin-7-induced human immunodeficiency virus permissiveness in quiescent T lymphocytes. J. Virol. 76: 9103-9111. [Abstract/Free Full Text]
35. Schmitt, N., L. Chene, D. Boutolleau, M. T. Nugeyre, E. Guillemard, P. Versmisse, C. Jacquemot, F. Barre-Sinoussi, N. Israel. 2003. Positive regulation of CXCR4 expression and signaling by interleukin-7 in CD4⁺ mature thymocytes correlates with their capacity to favor human immunodeficiency X4 virus replication. J. Virol. 77: 5784-5793. [Abstract/Free Full Text]
36. Uittenbogaart, C. H., W. J. Boscardin, D. J. Anisman-Posner, P. S. Koka, G. Bristol, J. A. Zack. 2000. Effect of cytokines on HIV-induced depletion of thymocytes in vivo. AIDS 14: 1317-1325. [Medline]
37. Napolitano, L. A., C. A. Stoddart, M. B. Hanley, E. Wieder, J. M. McCune. 2003. Effects of IL-7 on early human thymocyte progenitor cells in vitro and in SCID-hu Thy/Liv mice. J. Immunol. 171: 645-654. [Abstract/Free Full Text]
38. Geginat, J., A. Lanzavecchia, F. Sallusto. 2003. Proliferation and differentiation potential of human CD8⁺ memory T-cell subsets in response to antigen or homeostatic cytokines. Blood 101: 4260-4266. [Abstract/Free Full Text]
39. Dardalhon, V., S. Jaleco, S. Kinet, B. Herpers, M. Steinberg, C. Ferrand, D. Froger, C. Leveau, P. Tiberghien, P. Charneau, et al 2001. IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4⁺ T cells. Proc. Natl. Acad. Sci. USA 98: 9277-9282. [Abstract/Free Full Text]
40. Hassan, J., D. J. Reen. 1998. IL-7 promotes the survival and maturation but not differentiation of human post-thymic CD4⁺ T cells. Eur. J. Immunol. 28: 3057-3065. [Medline]
41. Jaleco, S., S. Kinet, J. Hassan, V. Dardalhon, L. Swainson, D. Reen, N. Taylor. 2002. IL-7 and CD4⁺ T-cell proliferation. Blood 100: 4676-4677. [Free Full Text]
42. Hazan, U., D. Thomas, J. Alcami, F. Bachelerie, N. Israel, H. Yssel, J. L. Virelizier, F. Arenzana-Seisdedos. 1990. Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NF-B translocation but not human immunodeficiency virus 1 enhancer-dependent transcription. Proc. Natl. Acad. Sci. USA 87: 7861-7865. [Abstract/Free Full Text]

This article has been cited by other articles:

				B. Rethi, N. Vivar, S. Sammicheli, C. Fluur, N. Ruffin, A. Atlas, E. Rajnavolgyi, and F. Chiodi Priming of T cells to Fas-mediated proliferative signals by interleukin-7 Blood, August 15, 2008; 112(4): 1195 - 1204. [Abstract] [Full Text] [PDF]

				C. Sportes, F. T. Hakim, S. A. Memon, H. Zhang, K. S. Chua, M. R. Brown, T. A. Fleisher, M. C. Krumlauf, R. R. Babb, C. K. Chow, et al. Administration of rhIL-7 in humans increases in vivo TCR repertoire diversity by preferential expansion of naive T cell subsets J. Exp. Med., July 7, 2008; 205(7): 1701 - 1714. [Abstract] [Full Text] [PDF]

				R. M. Dunham, B. Cervasi, J. M. Brenchley, H. Albrecht, A. Weintrob, B. Sumpter, J. Engram, S. Gordon, N. R. Klatt, I. Frank, et al. CD127 and CD25 Expression Defines CD4+ T Cell Subsets That Are Differentially Depleted during HIV Infection J. Immunol., April 15, 2008; 180(8): 5582 - 5592. [Abstract] [Full Text] [PDF]

				Y. M. Mueller, D. H. Do, S. R. Altork, C. M. Artlett, E. J. Gracely, C. D. Katsetos, A. Legido, F. Villinger, J. D. Altman, C. R. Brown, et al. IL-15 Treatment during Acute Simian Immunodeficiency Virus (SIV) Infection Increases Viral Set Point and Accelerates Disease Progression despite the Induction of Stronger SIV-Specific CD8+ T Cell Responses J. Immunol., January 1, 2008; 180(1): 350 - 360. [Abstract] [Full Text] [PDF]

				A. Vranjkovic, A. M. Crawley, K. Gee, A. Kumar, and J. B. Angel IL-7 decreases IL-7 receptor {alpha} (CD127) expression and induces the shedding of CD127 by human CD8+ T cells Int. Immunol., December 1, 2007; 19(12): 1329 - 1339. [Abstract] [Full Text] [PDF]

				A. Hryniewicz, D. A. Price, M. Moniuszko, A. Boasso, Y. Edghill-Spano, S. M. West, D. Venzon, M. Vaccari, W.-P. Tsai, E. Tryniszewska, et al. Interleukin-15 but Not Interleukin-7 Abrogates Vaccine-Induced Decrease in Virus Level in Simian Immunodeficiency Virusmac251-Infected Macaques J. Immunol., March 15, 2007; 178(6): 3492 - 3504. [Abstract] [Full Text] [PDF]

				A. S. Albuquerque, C. S. Cortesao, R. B. Foxall, R. S. Soares, R. M. M. Victorino, and A. E. Sousa Rate of Increase in Circulating IL-7 and Loss of IL-7R{alpha} Expression Differ in HIV-1 and HIV-2 Infections: Two Lymphopenic Diseases with Similar Hyperimmune Activation but Distinct Outcomes J. Immunol., March 1, 2007; 178(5): 3252 - 3259. [Abstract] [Full Text] [PDF]

				L. Vassena, M. Proschan, A. S. Fauci, and P. Lusso Interleukin 7 reduces the levels of spontaneous apoptosis in CD4+ and CD8+ T cells from HIV-1-infected individuals PNAS, February 13, 2007; 104(7): 2355 - 2360. [Abstract] [Full Text] [PDF]

				L. Swainson, S. Kinet, C. Mongellaz, M. Sourisseau, T. Henriques, and N. Taylor IL-7-induced proliferation of recent thymic emigrants requires activation of the PI3K pathway Blood, February 1, 2007; 109(3): 1034 - 1042. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Beq, S. Articles by Israël, N.