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AIDS:
doi: 10.1097/QAD.0b013e328349a437
Basic Science

Despite an impaired response to IL-7, T cells from HIV-positive patients proliferate normally in response to IL-15 and its superagonist, RLI

Pacheco, Yovanaa; Solé, Véroniqueb; Billaud, Ericc; Allavena, Clotildea,c; Plet, Arianeb; Ferré, Virginiea; Garrigue-Antar, Laureb; Raffi, Françoisa,c; Jacques, Yannickb; McIlroy, Doriana

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Author Information

aEA 4271 Laboratoire d’Immunovirologie et Polymorphisme Génétique, Université de Nantes

bINSERM U892

cService d’Infectiologie, Hôtel-Dieu, Nantes, France.

Correspondence to Dorian McIlroy, PhD, Université de Nantes, 1 rue Gaston Veil, 44000 Nantes, France. Tel: +33 2 40 41 28 39; e-mail: dorian.mcilroy@univ-nantes.fr

Received 24 November, 2010

Revised 27 May, 2011

Accepted 7 June, 2011

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Abstract

Objective: In phase I/II trials, IL-7 immunotherapy has been shown to expand CD4+ T cells. However, expression of the IL-7 receptor α-chain, CD127, is reduced on CD4+ T cells from HIV-positive patients, and defects in CD127 signaling have also been reported. To refine and improve cytokine immunotherapy, it is important to identify stimuli that can restore proliferation of CD4+ cells with defective responses to IL-7.

Design: Observational study comparing viremic HIV-positive patients with HIV-negative controls.

Methods: Peripheral blood mononuclear cells were cultured in the presence of 1 nmol/l IL-2, IL-7, IL-15 or RLI (an IL-15Rα/IL-15 fusion protein). Proliferation of different T-cell subsets was assessed by carboxyfluorescein succinimidyl ester fluorescence. Expression of CD127 on CD4+ T-cell subsets was also analyzed.

Results: In HIV-positive patients, CD127 expression was correlated with CD4+ T-cell count in the

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(R2 = 0.36; P < 0.01) and

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(R2 = 0.45; P < 0.001) populations, whereas CD127 expression on

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cells was significantly reduced in HIV-positive individuals compared with controls (P = 0.001) independently of CD4+ T-cell count. In patients with high CD4+ T-cell counts, proliferation in response to IL-7 was significantly reduced only in

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cells (P < 0.05). RLI, and to a lesser extent IL-15, induced strong proliferation of

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cells from both HIV-positive patients and controls. Neither agent stimulated proliferation of

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or

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cells.

Conclusion: In HIV-positive patients,

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cells are deficient in both CD127 expression and proliferation in response to IL-7. RLI and IL-15 specifically induced proliferation of

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cells, suggesting that they may have a unique potential to complement IL-7 immunotherapy.

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Introduction

Over the last 15 years, combination antiretroviral therapy (ART) has dramatically reduced the risk of clinical disease progression in HIV-infected patients. Current first-line treatment regimes can effectively suppress viral replication to undetectable levels in more than 90% of patients, and the availability of new drug families has improved the management of patients with drug-resistant virus. In some patients receiving ART, however, the recovery of CD4+ T-cell counts is incomplete despite complete suppression of viral replication. In particular, patients who delay therapy until their CD4+ cell count decreases to less than 200 cells/μl rarely achieve complete recovery of CD4+ T-cell numbers [1]. These patients would benefit from adjuvant therapies aimed at improving immune reconstitution.

As cytokines of the common γ-chain family have an essential role in T-lymphocyte homeostasis, they have been proposed as adjuvant therapies for HIV infection to accelerate the recovery of CD4+ T cells during antiretroviral therapy or to enhance the activity of HIV-specific CTL. Because of their stimulatory and antiapoptotic properties, three cytokines, interleukin-2 (IL-2), interleukin-7 (IL-7) [2] and interleukin-15 (IL-15) [3], have been advocated as potential adjuvants to ART.

The first cytokine to be studied as a therapeutic in HIV infection was IL-2, which showed proliferative and antiapoptotic effects on CD4+ T lymphocytes in vitro and in vivo[4,5]. Nevertheless, results from two large randomized controlled trials in HIV-infected patients receiving IL-2 in combination with ART did not show clinical benefits compared with ART alone, despite a substantial and sustained increase in CD4+ cell count [6].

Another potential molecule for HIV treatment is IL-7. Recent stage I/II trials have confirmed that IL-7, used in conjunction with ART, can increase CD4+ T-cell counts in HIV-positive patients [7,8]. In contrast to IL-2, both naive and memory CD4+ T cells proliferate in response to IL-7, supporting the view that clinical responses to IL-7 therapy will be better than those observed with IL-2. Nevertheless, some questions remain concerning the potential efficacy of IL-7 therapy in HIV infection, due to the perturbation of the IL-7/IL-7r system in HIV-positive patients. In particular, several studies have reported downregulation of the α chain of the IL-7 receptor, CD127, on CD4+ T cells of HIV-infected patients [9–11] and defects in IL-7R signaling [12–14]. As IL-7Rα expression is more severely reduced in patients with advanced disease [9,10], IL-7 may not be so effective in the patients who would most benefit from adjuvant therapy to aid CD4+ T-cell reconstitution.

Finally, the possibility that IL-15 could aid immune reconstitution in HIV-positive patients has also been investigated, because of its positive effects on innate and adaptive immune responses. IL-15 is known to have a major role in the homeostasis and functional maturation of natural killer (NK) and CD8+ cells [15,16] and, more recently, its importance in the homeostatic proliferation of memory CD4+ cells has also been recognized [17,18]. Furthermore, the functional response of T cells to IL-15 does not seem to be impaired in HIV-positive patients [19,20]. Compared with other cytokines, the activation of target cells by IL-15 is unusual, as IL-15 signaling involves trans-presentation of the IL15rα–IL-15 complex on APC to the IL-2/IL-15r βγ dimer expressed by T or NK lymphocytes [21]. In a therapeutic context, the levels of endogenous IL-15rα expression may limit the efficacy of exogenously added IL-15, and, in order to resolve this problem, two groups have independently developed recombinant fusion proteins consisting of IL-15 and different fragments of the IL-15rα extracellular domain [22,23] that act as IL-15 superagonists.

The aim of our study was to compare the effects of IL-2, IL-7, IL-15 and IL-15 receptor α-sushi domain-linker–IL-15 (RLI) fusion protein [22] on CD4+ and CD8+ lymphocytes from HIV-infected patients in order to evaluate the therapeutic potential of RLI in HIV infection. The inhibition of spontaneous apoptosis and the induction of proliferation by these different molecules were analyzed in peripheral blood mononuclear cells (PBMCs) from untreated HIV-infected patients. The proliferative response of naive, central-memory and effector-memory T-cell subsets was also measured. Overall our results showed that CD127 expression and proliferative responses to IL-7 were reduced in CD4+ effector-memory (EM) cells from HIV-positive patients, and that IL-15 and RLI specifically induced proliferation of this CD4+ subpopulation.

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Patients and methods

Patients

This study enrolled 30 antiretroviral untreated HIV-infected adults, 23 with high CD4+ T-cell count (>500 cells/μl, and a drop of less than 300 cells/μl in the preceding year, CD4HI group) and seven patients with low CD4 cell count (<200 cells/μl at presentation, or showing a reduction of >300 cells/μl CD4+ T cells over the preceding year, CD4LO group). However, due to the limited volume of blood drawn, it was not possible to perform all analyses on each patient included in the study. Age, peripheral blood CD4+ T-cell counts and viral load in the CD4HI and CD4LO patient groups are shown in Table 1.

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All patients gave informed consent and provided 20 ml blood samples on the occasion of their regular hospital visits. CD4+ lymphocyte counts and plasma viral load were obtained from the hospital database and incorporated into the study data after anonymization. The study was authorized by the Nantes Hotel Dieu Hospital ethical review board. Regular blood donors enrolled at the Etablissement Français du Sang provided HIV-negative control samples. These samples were donated anonymously and baseline clinical data concerning donors was not provided.

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Cell preparation and culture

PBMCs from heparinized blood were separated by Ficoll density gradient centrifugation. Ten million fresh cells were used to perform apoptosis and proliferation assays analyzing CD4+ subpopulations and the remainder were cryopreserved in 50% FCS, 40% RPMI and 10% DMSO and stored in liquid nitrogen. Cryopreserved cells were used for bulk CD4+ and CD8+ lymphocyte proliferation assays and analysis of CD127 expression. In some experiments, T cells were purified by negative selection using the Pan T-cell Isolation kit (Miltenyi, Bergisch Gladbach, Germany) according to the manufacturer's instructions. PBMCs were cultured at 37°C 5% CO2 in 96-well plates at a concentration of 106 cells per ml and purified T cells were cultured at 2–3 × 106 cells/ml in U-bottomed plates, in culture medium (RPMI medium 1640 supplemented with glutamine penicillin-streptomycin and 10% FCS). The following cytokines were added at the beginning of the culture: IL-2 (1 nmol/l), IL-7 (1 nmol/l), IL-15 (10, 1 and 0.1 nmol/l) or RLI (10, 1 and 0.1 nmol/l).

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Apoptosis assay

After 3 days, apoptosis in CD4+ and CD8+ lymphocytes was analyzed by Annexin V binding. Briefly, 105 cells were recovered, CaCl2 was added to 2.5 mmol/l and cells were incubated with anti-CD4-PE-Cy7, anti-CD8-APC and Annexin V-PE for 20 min at room temperature. Cells were washed once with wash buffer (10 mmol/l HEPES, 140 mmol/l NaCl, 2.5 mmol/l CaCl2) and fixed in wash buffer 0.75% formaldehyde before flow cytometry analysis.

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Proliferation assay

Cellular proliferation was assessed using the CellTrace Carboxyfluorescein Succinimidyl Ester (CFSE) Cell Proliferation Kit (Invitrogen, Carlsbad, California, USA). Four million freshly isolated cells were labeled with 1 μmol/l CFSE in PBS 0.1% BSA for 10 min at 37°C. Staining was stopped by adding 8 ml of cold culture medium (RPMI medium 1640 supplemented with glutamine, penicillin–streptomycin and 10% FCS) and then the cells were washed twice, incubated 5 min on ice and washed twice. Labeled cells were stimulated by cytokines and plated as described above.

After 7 days culture, cells were incubated with the following monoclonal antibody combinations: CD4-PE/CD8-APC or CD4-PE/CD27-APC/CD45RA-PECy7 (BD Biosciences, Pont de Claix, France) for 20 min at room temperature. Cells were washed once with PBS and fixed with PBS 0.75% formaldehyde before acquisition on a FACSCalibur instrument (BD Biosciences). Data were analyzed using FlowJo software, and expressed as the percentage of cells that had divided at least once by day 7 of culture.

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CD127 expression

Frozen PBMCs were thawed, resuspended in PBS 0.1% BSA and then stained with the following monoclonal antibody combinations: CD4-PE/CD27-APC/CD45RA-PEcy7/CD127-FITC or CD4-PE/CD27-APC/CD45RA-PEcy7/IGg2a-FITC. After staining, cells were washed once then fixed in PBS 0.75% formaldehyde before analysis on a FACSCalibur instrument (BD Biosciences). Data were analyzed using FlowJo software and expressed as percentage of cells staining positive for CD127. The following monoclonal antibodies (BD Biosciences) were used: anti-CD4-PE (clone SK3), anti-CD45RA-PECy7 (clone L48), anti-CD127-FITC (clone L128) and IgG2a-FITC (clone MOPC-21).

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Statistics

Two group comparisons (HIV-positive patients versus HIV-negative controls) were carried out using the Wilcoxon Rank Sum test. With paired data, Wilcoxon's Signed Rank test was used. Three group comparisons (HIV-negative controls, HIV-positive CD4HI patients and HIV-positive CD4LO patients) were performed using one-way ANOVA. To compare effects of the four different cytokines in different patients, we used repeated measures ANOVA followed by posthoc tests as indicated on http://www.graphpad.com/quickcalcs/posttest1.cfm. Correlations were calculated using the Spearman correlation test. Calculations were performed using GraphPad software version 5.00 and a difference or a correlation was considered significant when P was less than 0.05. EC50 values were calculated using the NCGC Curve Fit software (http://ncgc.nih.gov/pub/openhts/curvefit/).

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Results

Effects of IL-2, IL-7, IL-15 and RLI on bulk CD4+ and CD8+ T cells

PBMCs from 15 HIV-positive patients with CD4+ T-cell count more than 500 cells/μl (CD4HI group), seven HIV-positive patients with CD4+ T-cell count less than 200 cells/μl (CD4LO group) and nine HIV-negative controls were labeled with CFSE and then cultured in the presence of IL-2, IL-7, IL-15 or IL-15 agonist, RLI. On day 7, cells were harvested and stained for CD4 and CD8 expression and the percentage of CD4+ and CD8+ cells that had undergone division was determined (Fig. 1a). Compared with culture in the absence of cytokines, IL-2, IL-7, IL-15 and RLI all induced significant proliferation of CD8+ lymphocytes (Fig. 1b), although the response to IL-2 was weaker than that observed with the other cytokines. Significant proliferation of CD4+ lymphocytes was observed only in response to IL-7 (Fig. 1c). For each culture condition, the percentage of proliferating cells was then compared between patient groups. The response to IL-2 did not differ significantly between groups, either in CD4+ or CD8+ lymphocytes. Furthermore, no reduction in the response to IL-7 was detected in HIV-positive patients, including those in the CD4LO group, compared with HIV-negative controls. Proliferation of CD8+ T cells in response to IL-15 or RLI was lower in patients from the CD4LO group compared with the CD4HI group or HIV-negative controls (P < 0.05 by Student's t-test), although this difference was not significant when the three groups were compared by ANOVA.

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In a subset of patients (n = 9 CD4HI and n = 4 CD4LO) and controls (n = 7), the effects of different cytokines on the level of apoptosis observed after 3 days of culture were compared (Fig. 2). Spontaneous apoptosis of CD4+ and CD8+ T cells was greater in PBMCs from HIV-positive patients compared with HIV-negative controls (P < 0.005 for CD8+ lymphocytes and P < 0.05 for CD4+ lymphocytes by Student's t-test). All of the cytokines tested reduced the levels of spontaneous apoptosis in CD4+ and CD8+ cells from HIV-positive patients (P < 0.05 by ANOVA), with no significant differences between the four molecules. However, the level of apoptosis observed in CD8+ lymphocytes from HIV-positive patients remained significantly greater than that seen in HIV-negative controls after culture with IL-7 or IL-15 (P < 0.05 by Student's t-test). Although these two cytokines did significantly inhibit apoptosis in CD8+ lymphocytes from HIV-positive patients, this effect was not sufficient to reduce apoptosis to normal levels.

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IL-7rα expression and proliferative response in CD4+ T-cell subsets

We next analyzed expression of the IL-7 receptor α-chain, CD127, and the proliferative response of CD4+ T-cell subsets in HIV-positive patients compared with HIV-negative controls. As shown in Fig. 3a, CD127 expression was reduced in CD45RACD27 CD4+ effector-memory cells from HIV-positive patients, but not in CD45RA+CD27+ CD4+ naive (N) or CD45RACD27+ CD4+ central memory (CM) cells. In the studied patients, CD127 expression on

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cells was not related to CD4+ T-cell count (Fig. 3e) or viral load (not shown). However, the percentage of CD127+ cells among

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and

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was strongly correlated to the CD4+ T-cell count, patients with low CD4+ counts having reduced CD127 expression in both

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and

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subsets (Fig. 3c and d). Because not all CD4+ subsets showed the same relationship between CD127 expression and disease progression, the correlation between CD127 on total CD4+ lymphocytes and CD4+ T-cell count, although discernible, was weak (Fig. 3b).

In terms of functional responses to IL-7, the proliferation of

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and

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cells from HIV-positive patients in the CD4HI group was comparable to that seen in HIV-negative controls (Fig. 4d and e). However, the proportion of

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cells that divided in response to IL-7 was reduced from 13% in HIV-negative controls to 5% in HIV-positive patients (P < 0.05, Fig. 4f). Unlike IL-7, which stimulated proliferation of all CD4+ T-cell subsets, IL-15 and RLI specifically induced proliferation of

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cells. Furthermore, proliferative responses of

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cells in response to IL-15 and RLI were not reduced in HIV-positive patients compared with HIV-negative controls.

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Comparison of the relative efficacy of IL-15 and RLI

Proliferation of CD8+ lymphocytes and

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cells in response to RLI appeared similar to that seen with IL-15. This was surprising, as RLI showed at least 100-fold greater potency than IL-15 in inducing proliferation of the 32Dβ cell line, which expresses the IL-2/IL-15 βγ receptor (Fig. 5a). To compare these molecules in more detail, the proliferative responses of CD8+ lymphocytes from six HIV-negative donors and

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cells from five HIV-negative donors to different concentrations of IL-15 and RLI were analyzed. Results from one donor are shown in Fig. 5b and the dose–response curves for CD8+ lymphocytes and

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cells are shown in Fig. 5c. As the proliferative responses to IL-15 and RLI varied between donors, data were normalized by defining the 100% response to IL-15/RLI for each donor as the mean of two values: proliferation observed with 10 nmol/l IL-15 and that observed with 10 nmol/l RLI. The concentration of RLI necessary to induce half-maximal proliferation (EC50) was similar (0.3–0.7 nmol/l) in

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cells, CD8+ lymphocytes and the 32Dβ cell line. The mean EC50 for IL-15 was seven-fold higher than that of RLI (P = 0.002 by paired t-test) for

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cells, but only two-fold higher in CD8+ cells (P = 0.02, paired t-test). Although these results show that RLI is more potent than IL-15 in primary human T cells, the response of T cells to IL-15 was much stronger than that observed in 32Dβ cells, indicating the involvement of the IL15Rα-chain in the proliferative responses that we observed in PBMC cultures.

As monocytes present in PBMC cultures express IL-15rα and are able to trans-present IL-15 to T cells, we also studied the responses of CD8+ lymphocytes and

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cells in cultures of purified T cells. Overall, proliferative responses were lower in T-cell cultures than in PBMC cultures, independently of the stimulus (data not shown) and the low levels of

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proliferation observed precluded a meaningful analysis. CD8+ T cells, however, were still able to proliferate in response to IL-15 in the absence of monocytes, although the mean EC50 was increased to 2.3 nmol/l (Fig. 5d).

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Discussion

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The main finding of this study is that IL-15 and RLI can induce proliferation of

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cells in both HIV-negative controls and viremic HIV-positive patients. IL-15 has previously been shown to support the homeostatic proliferation of

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in mice [17], nonhuman primates [24] and humans [25]. To the best of our knowledge, the present work is the first study of this function of IL-15 in HIV-positive patients, although the responses of CD8+ lymphocytes from HIV-positive patients to IL-15 have been extensively documented [26,27]. The ability of IL-15 and RLI to specifically induce proliferation of

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cells is particularly interesting, as clinical experience with IL-7 has shown that

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and

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populations, rather than

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cells, are preferentially expanded by IL-7 therapy [7,8]. Consistent with these clinical results, we found that the

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T-cell subset showed the most severe alterations in both CD127 expression and proliferation in response to IL-7 in the HIV-positive patients studied. However, our observation that proliferation of bulk CD4+ and CD8+ T cells in response to IL-7 was normal in HIV-positive patients was unexpected and is worthy of comment.

Several groups have reported that expression [9–12,14,28] of the IL-7 receptor α-chain, CD127, is reduced in CD4+ and CD8+ T cells from HIV-positive patients. With respect to CD4+ T cells, one group has reported that downregulation of CD127 expression was restricted to

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cells [11], whereas most other reports have documented defects in CD127 expression in both naive and memory CD4+ T cells [9,10,12,14,28]. In these latter studies, however, more pronounced defects were found in either

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cells [12], or more generally, the memory compartment [9,10,14,28]. Differences in the immunological status of the patient cohorts studied appears to be the major factor responsible for these discordant findings, as a correlation between CD127 levels on CD4+ T cells and CD4+ T-cell count has been consistently reported [9,10,12]. In particular, Bazdar et al.[12] found that CD127 expression in the

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and

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subsets was strongly correlated with nadir CD4+ T-cell count, whereas the reduction in CD127 on

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cells from HIV-positive patients was independent of this parameter. Our results confirm this finding, and as most of the patients we studied had CD4+ T-cell counts higher than 500 cells/μl, this explains why we only detected a reduction in CD127 expression in the

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subset when comparing HIV-positive patients to HIV-negative controls.

With respect to receptor function, it is well established that STAT5 phosphorylation in response to IL-7 is diminished in both CD4+ and CD8+ T cells from HIV-positive patients [12–14,29]. Within the CD4+ T-cell population, this defect is more pronounced in the memory subset [14] and, more specifically, in the

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population [12]. On the other hand, in studies that used T-cell proliferation as a measure of functional responses to IL-7, it has proven more difficult to clearly demonstrate a diminution in the responses of cells from HIV-positive patients. Three studies have found that proliferation of T cells in response to IL-7 combined with either anti-CD3 or PHA were significantly reduced in HIV-positive patients compared with controls [29–31]. However, in all three studies, proliferation in response to the TCR/CD3 stimulus alone was also reduced, making it difficult to determine to what extent the response to IL-7 itself was diminished. Two of these studies concluded that the IL-7-dependent component of this proliferative response was reduced in either CD8+ T cells [29] or total T cells [31], whereas one study found that the enhancing effect of IL-7 on CD3-dependent proliferation in naive CD4+ T cells was not significantly different in viremic HIV-positive patients compared with HIV-negative controls [30].

Overall, the results from the literature indicate that measurement of the proliferative response may be a less sensitive indicator of IL-7 receptor function than STAT5 phosphorylation. If this is so, then that would explain why we only detected a significant difference between HIV-positive patients and HIV-negative controls in the

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subset, as this population shows the most severe alteration in both CD127 expression and IL-7 receptor function [12]. Two other methodological limitations of our study should also be noted. First, we used a 7-day CFSE assay in the absence of exogenous antigen to measure proliferation. We interpret the results of this assay as indicating the level of homeostatic proliferation in response to the added cytokines. However, as the HIV-positive patients we studied were not receiving ART, one would expect to observe robust HIV-specific immune responses in these individuals, particularly in CD8+ T cells. Antigenic stimulation in vivo in HIV-positive patients, but not controls, could therefore introduce a systematic bias tending to increase the proliferative responses observed in HIV-positive patients. Second, the high intragroup variability of our results makes type II statistical error more likely, particularly when group size is small, as was the case with the CD4LO patient group (n = 7).

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Finally, we present new data concerning the efficacy of RLI compared with native IL-15. We found that RLI was clearly more effective than IL-15 in inducing proliferation of

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cells, but the difference was less apparent in CD8+ T lymphocytes. In addition, the relatively high efficacy of native IL-15 in stimulating proliferation of primary CD8+ T cells in PBMC cultures could not be attributed to trans-presentation of IL-15 by monocytes, as CD8+ T cells still proliferated in response to IL-15 in cultures of purified T cells. Our results are similar to those reported by Ota et al.[32], who showed that unlike mouse CD8+ T cells, human primary CD8+ T cells do not require trans-presentation of IL-15 for proliferation in vitro. This may be a unique property of human CD8+ T cells, as previous studies in humanized mouse models have shown that RLI is significantly more effective than native IL-15 in inducing human NK-cell proliferation in vivo[33]. As

cells also respond more readily to RLI than to IL-15, our findings indicate that RLI could be more effective than IL-15 as an immunotherapeutic agent aimed at restoring the

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population.

However, as

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cells represent one of the major target cell populations for HIV replication, expanding their numbers would run the risk of increasing viral replication. Indeed, in the macaque-SIV model, administration of IL-15 during acute SIV infection increased setpoint viral load and accelerated disease progression [34]. Therefore, although the combination of RLI or IL-15 immunotherapy with ART might accelerate reconstitution of the

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subset, including mucosal effector cells, in HIV-positive patients, it is unclear whether the potential benefit of such a therapy would outweigh the risk involved.

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Acknowledgements

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The authors would like to thank Dr Lisa Chakrabarti for useful comments on the manuscript. Finally, we would particularly like to thank the patients who agreed to participate in this study.

Author contributions: Y.P. and D.M. performed the experiments and analyzed the data. V.S., A.P. and L.G.-A. prepared and QC-tested RLI. E.B., C.A. and F.R. recruited patients for the study. V.F. analyzed patient clinical and virological data. Y.J. and D.M. designed the study. Y.P. and D.M. wrote the manuscript.

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Conflicts of interest
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This study was supported by a research grant from SIDACTION and by the ARSIID. Y.P. was the recipient of a scholarship awarded by the Région des Pays de la Loire.

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Keywords:

cytokines; HIV; immunotherapy; interleukin-15; interleukin-7; T lymphocytes

© 2011 Lippincott Williams & Wilkins, Inc.

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