Reduction of death receptor 5 expression and apoptosis of CD4 + T cells from HIV controllers

Clinical Immunology (2014) 155, 17–26

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

Reduction of death receptor 5 expression and apoptosis of CD4 + T cells from HIV controllers Lucie Barblu a,1 , Nikaïa Smith b,1 , Stéphanie Durand c , Daniel Scott-Algara d , Faroudy Boufassa e , Jean-François Delfraissy f,g,h , Andrea Cimarelli c , Olivier Lambotte f,g,h,1 , Jean-Philippe Herbeuval b,⁎ a

CNRS UMR 8147; Université Paris Descartes, Paris, France CNRS UMR 8601, Faculté des Saints-Pères, Université Paris Descartes, Paris, France c Department of Human Virology, Ecole Normale Supérieure de Lyon; INSERM, U758; Université Lyon1, France d Unité de régulation des infections virales, Institut Pasteur, Paris, France e INSERM U1018, Bicêtre, Prais, France f INSERM, U1012, Bicêtre, Paris, France g AP-HP, Department of Internal Medicine and Clinical Immunology, Bicêtre Hospital, Bicêtre, Paris, France h Université Paris-Sud, Bicêtre, France b

Received 30 May 2014; accepted with revision 30 July 2014 KEY WORDS HIV infected controllers; DR5; TRAIL; Apoptosis; CD4 + T cell

Abstract TNF-related apoptosis ligand (TRAIL) induces apoptosis of HIV-1-exposed CD4 T cells expressing the death receptor 5 (DR5) in vitro and has been associated with reduced CD4 T cell number in viremic HIV-1-infected patients. Alterations of the TRAIL/DR5 apoptotic pathway could be involved in the absence of massive CD4 T cell depletion in HIV-1-infected controllers (HIC). We studied here apoptosis of CD4 T cells from HIV-infected progressors and controllers. Reduced apoptosis of CD4 T cells from HIC was observed upon HIV stimulation. This lower apoptosis correlated with a deficiency of DR5 cell surface expression by CD4 T cells upon HIV-1 stimulation. The significant lower apoptosis observed in CD4 T cells after HIV exposure, associated with lower expression of membrane DR5 could explain the better survival of HIV-specific CD4 T cells from HIV controllers. The levels of DR5 cell surface expression on CD4 T cells could represent a new prognostic marker. © 2014 Elsevier Inc. All rights reserved.

Abbreviations: (HIC), HIV-1 infected controllers; (IFN-α), interferon alpha; (pDC), plasmacytoid dendritic cell; (TRAIL), TNF-related Apoptosis-Inducing Ligand; (DR5), Death Receptor 5. ⁎ Corresponding author at: CNRS – UMR8601, Université Paris Descartes, Faculté des Saints-Pères, 45 rue des Saints-Pères, 75006 Paris, France. Fax: + 33 1 42 86 83 83. E-mail address: [email protected] (J.-P. Herbeuval). 1 Authors contributed equally to the work.

http://dx.doi.org/10.1016/j.clim.2014.07.010 1521-6616/© 2014 Elsevier Inc. All rights reserved.

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1. Introduction The majority of HIV-1-infected individuals progress to AIDS, which is characterized by high viral load and massive CD4 T cell depletion [1,2]. However, a subset of HIV-1-positive individuals does not progress to AIDS and spontaneously maintains an undetectable viral load [3]. This rare patient population is defined as HIV-1 controllers (HIC) or elite controllers [4,5], and represents less than 1% of HIV-1-infected patients [6,7]. We and others have shown that immunity of HIV-1 controllers is defined by a unique polyfunctional HIV-1-specific CD8 T cell response, ex vivo cytotoxicity against infected CD4 T cells and a strong ability to proliferate in response to HIV-1 proteins [7–9]. HIV-1-specific CD4 T cells and the pool of central memory CD4 T cells are also preserved despite immune activation due to HIV-1 infection [10,11]. The majority of HIC is also defined by the absence of massive CD4 T cell depletion, even after 10 years of infection [12]. However, the mechanisms involved in protection against HIV-1 disease progression have not been elucidated yet. TNF-related apoptosis ligand (TRAIL), a TNF-α family member [13], induces apoptosis by binding to its death receptors, DR4 and DR5 [14,15]. Previous reports showed that TRAIL selectively induces apoptosis of human HIV-1-exposed CD4 T cells in vitro [16] and participates ex vivo to the CD4 T cell depletion observed in HIV-1-infected hu-PBL-NOD-SCID mice [17]. We previously reported that HIV-1 specifically upregulated DR5 expression on CD4 T cell membranes making them prone to TRAIL-mediated apoptosis [18]. Moreover, the percentage of CD4 T cells co-expressing TRAIL and DR5 were elevated in the blood of viremic patients (VIR) [18] and were reduced by successful highly active anti-retroviral therapy (HAART) [19]. As HIV controllers generally maintain high CD4 T cell levels and extremely low viral loads [7,20], we studied whether the TRAIL pathway was modified in this rare subset of HIV-infected patients compared to viremic progressors. We demonstrate in this report that TRAIL expression by CD4 T cells from HIC was not altered in response to HIV-1 activation. However, we show that CD4 T cells from HIC do not undergo apoptosis but show a deficiency of DR5 cell surface expression after HIV-1 stimulation. This observation constitutes a major difference between CD4 T cells from HIC compared to viremic progressors. DR5 silencing of HIV-1-activated CD4 T cells from healthy donors significantly abolished cell death, validating that the DR5 defect is an important component in the resistance to HIV-1-induced apoptosis in HIC.

2. Materials and methods 2.1. Blood samples Thirteen HIV-1 controllers (HIC) enrolled from the French ANRS CO18 HIV Controller cohort, were selected on the following characteristics: HIV-1-infected patients, never treated with any antiretroviral treatment, with a follow-up longer than 10 years and with more than 90% of plasma HIV-1 RNA measurements lower than 400 copies/mL (Amplicor Monitor, Roche Diagnostics, Meylan, France). Fourteen viremic chronically-infected untreated patients (VIR) were selected at the Bicêtre University Infectious Diseases Clinic, and 15 healthy HIV-1-seronegative

L. Barblu et al. blood bank donors (HD) were obtained from Etablissement Français du Sang (convention # 07/CABANEL/106, France). The characteristics of the patients are given in Table 1. The study was promoted by the Agence Nationale de Recherche sur le Sida (ANRS, EP36-V).

2.2. Ethics statement All participants gave written informed consent prior to blood sampling. Experimental procedures have been approved by the Bicêtre and Necker Hospital Ethical Committees for human research (Comité de Protection des Personnes CPP Ile de France VII, number 05–22, decision 12/18/2008) and were done according to the European Union guidelines and the Declaration of Helsinki.

2.3. Preparation of noninfectious AT-2 HIV HIV-1MN (X4-tropic) were produced and inactivated with 1 mM AT-2 for 18 hours at 4 °C, as described. Noninfectious AT-2 HIV-1 retains functionally intact envelope glycoproteins and interacts with cell receptors. Microvesicles, isolated from uninfected cell cultures matched to the cultures to produce the virus, were used as negative controls (mock).

2.4. Isolation and culture of blood leukocytes All the study was made using fresh blood samples. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation using lymphocyte separation medium (Cambrex, Gaithersburg, MD). CD4 T cells from HD, HIC and VIR PBMC were purified by using the negative selection purification kit “CD4 + T Cell Isolation Kit II, human“(Miltenyi Biotech, Bergisch Gladbach, Germany). Cells were cultured overnight in RPMI medium 1640 (Invitrogen, Gaithersburg, MD) containing 10% FBS (HyClone, Logan, UT) and 1% Pen-Strep-Glut (Invitrogen) in the presence of AT-2 HIV-1 (MN) at 60 ng/mL p24CA equivalent or in the presence of anti-CD3 and anti-CD28 (Invitrogen).

2.5. Flow cytometry Purified CD4 T cells were incubated for 20 min at 4 °C with FITC-conjugated anti-DR4, −DR5 (Alexis, San Diego, CA), anti-DcR1 (Alexis), anti-DcR2 (Alexis), anti-TWEAK-R (Alexis), TOPRO III (Invitrogen) or Annexin-V (MBL International, Woburn, MA), PE-conjugated anti-TRAIL and APC-Cy7-conjugated Table 1

Male Median Median Median Median Median

Characteristics of the patients studied.

age year of HIV diagnosed CD4 T cell count (/mm3) years of viral follow-up RNA viral load (copies/mL)

HIV controllers N = 13

Viremic patients N = 14

9 46 1988 748 11 b 50

5 40 2006 384 1.5 86156

Reduced Death Receptor 5-mediated CD4 T Cell Apoptosis in HIV controllers anti-CD14 (BD Biosciences, San Jose, CA), Vioblue-conjugated anti-CD4 (Miltenyi) or with appropriate isotype-matched control antibodies (at 5 μg/mL each) in PBS (Sigma, Saint Louis, MO) and Fc-Receptor blockers (BD, Biosciences). Cells were washed twice in ice-cold PBS or Annexin V buffer, and FACS analysis was performed on a FACS Canto (7 colors) using FACS Diva software (BD Biosciences) and analyzed with FlowJo software (Treestar, Ashland, OR).

2.6. SiRNA T cell transfection CD4 T cells were seeded at 106 cell/mL in 96 well/plates and incubated at 37 °C. 3 μl of Hiperfect transfection reagent (Qiagen) were added to appropriate siRNA concentration and adjusted at 100 μl with serum-free medium. Then, the solution was gently mixed and incubated at room temperature during 30 minutes. After incubation, the mix was added to cells in culture. Finally, cells were incubated at 37 °C overnight with or without HIV-1. Control was performed using scramble siRNA, and transfection efficiency was tested by FACS using Alexa-488 tagged scramble siRNA (all from Qiagen). Apoptosis was determined using Annexin-V staining and analyzed by flow cytometry.

2.7. Statistical analysis Experiments were repeated at least four times. P values (P) were determined using a two-tailed Student's t test or Mann–Whitney. P b 0.05 was considered statistically significant. Unvaried distributions of flow cytometry data were performed by probability binning, in 300 bins using FlowJo software [21]. Correlation between DR5 expression and CD4 T cell counts was done using Pearson's correlation.

3. Results

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expressing mTRAIL was multiplied by 2.6 in HD (P = 0.0010), 1.4 in VIR (P = 0.0013) and 3.3 in HIC (P = 0.0005) (Fig. 1E).

3.2. Expression of DcR1, DcR2, Tweak-R and DR4 We tested the expression of TRAIL decoy receptors DcR1 and DcR2, TRAIL death receptor DR4, and TNF-related weak inducer of apoptosis receptor (Tweak-R) both ex vivo and under stimulation in vitro. CD4 T cells weakly expressed DcR1, DcR2 and DR4 ex vivo, and we did not observe any statistical difference in these receptors expression between HD, VIR and HIC (Supplementary Fig. 1A). We then tested the expression of DR4, DcR1 and DcR2 by purified CD4 T cells after HIV or T cell specific activation (CD3-CD28) (Supplementary Fig. 1B). We found that DcR1 and DcR2 were neither significantly expressed nor regulated in any group. Similarly, DR4 expression ex vivo was low (Supplementary Fig. 1A), and was not statistically increased by HIV-1 and CD3-CD28 in CD4 T cells from HD, VIR and HIC (Supplementary Fig. 1B). Tweak-R was also weakly expressed by freshly purified CD4 T cells from each group of patients (Supplementary Fig. 1A) and was not statistically upregulated, neither by HIV-1 nor by CD3-C28 activation (Supplementary Fig. 1B). Thus, none of these molecules seemed to be involved in the limited CD4 T cell depletion observed in HIC patients. We also studied the Fas pathway that had been shown to be involved in CD4 T cell death during HIV disease. We observed that the level of Fas expression on CD45RO CD4 T cell was high in all group of patients and was not increased by HIV exposure. Furthermore, Fas expression was increased on CD45RA CD4 T cells under HIV stimulation in healthy donors, viremic and controller patients (Supplementary Fig. 2). Thus, we did not observe any alteration of Fas expression in HIV controllers.

3.3. Ex vivo and in vitro expression of membrane DR5 on CD4 T cells from HIC, HD and VIR

3.1. Apoptosis and TRAIL expression in CD4 T cells We first compared HIV-1-mediated apoptosis of CD4 T cells from 15 HD, 13 HIC and 14 VIR. HIV-1 induced 31% (P = 0.003) and 40% (P = 0.001) of Annexin V expression by CD4 T cells from HD and VIR, respectively. In contrast, apoptosis was not statistically increased in CD4 T cells from HIC after HIV-1 exposure (Fig. 1A). We verified that Annexin V positive cells from HD and HIC were in apoptotic stage by using the marker of late stage of apoptosis Topro III (Fig. 1B). We noticed that the vast majority of Annexin V positive cells were also Topro III positive indicating that cells were in late stage of apoptosis. To explain these differences, we focused on the role of TRAIL pathway. Level of membrane TRAIL expression (mTRAIL) was similar between cells from HD and HIC. Indeed, mTRAIL was expressed by 19% and 22% of freshly purified CD4 T cells from HD and HIC, respectively (Figs. 1C and D). In contrast, number of CD4 T cells expressing TRAIL was higher in VIR (48%) than in HD (P = 0.006) and HIC (P = 0.02). However, in vitro exposure to HIV-1 significantly increased the number of CD4 T cells expressing mTRAIL from all groups of patients (Fig. 1D). Indeed, the number of CD4 T cells

We thus examined membrane expression of DR5 (mDR5) that we previously reported to be strongly involved in HIV-1-induced CD4 T cell apoptosis [18,22]. Purified CD4 T cells from HD and HIC express barely detectable ex vivo mDR5 (2% and 4%, respectively), in contrast to high mDR5 levels on CD4 T cells from VIR (38%) (Fig. 2A). The percentage of DR5 expressing CD4 T cells was statistically higher in VIR than in HD (P = 0.002) and HIC (P = 0.0003). Thus, we cultured in vitro CD4 T cells from the different groups overnight and quantified DR5 expression. In vitro exposure to HIV-1 significantly increased the proportion of CD4 T cells expressing mDR5 from HD (7% to 26%, P = 0.001). The number of mDR5 expressing CD4 T cells from VIR was high but was not statistically increased by HIV-1 (37% to 39%, P = 0.7), probably due to the high level of mDR5 expression ex vivo (Fig. 2B). However, mDR5 levels on CD4 T cells from VIR, quantified by mean fluorescence intensity (MFI), were significantly increased (32 to 62 MFI, P = 0.04) upon exposure to HIV-1 (Fig. 2C). Thus, we found here that HIV did not increase the number of DR5-expressing CD4 T cells from VIR after in vitro stimulation but increased the quantity of membrane DR5 molecules per cell.

20 In contrast, the number of mDR5 expressing CD4 T cells (Fig. 2B) and mDR5 levels (Fig. 2C) from HIC was not statistically increased after in vitro exposure to HIV-1

L. Barblu et al. (5% versus 6% of DR5+ CD4 T cells and 4 versus 4.5 MFI, respectively). Those latest results highlighted a negligible induction of DR5 membrane expression under HIV stimulation

Reduced Death Receptor 5-mediated CD4 T Cell Apoptosis in HIV controllers in CD4 T cells from HIC patients (Fig. 2D). The grey solid histograms showed DR5 staining in mock samples from HD, VIR and HIC and allowed the comparison with HIV stimulated samples from HD, VIR and HIC. We can then notice that only VIR patients express DR5 on mock CD4 T cells, suggesting an ex vivo expression of DR5. Thus, to verify whether the lack of mDR5 induction in CD4 T cells from HIC was due to an HIV response deficiency or was a characteristic of HIC cells, we also tested CD4 T cells activator anti-CD3-CD28 antibodies. Anti-CD3-CD28 and HIV-1 stimulation induced mDR5 expression by CD4 T cells from HD and VIR. Similarly to HIV-1 stimulation, anti-CD3-CD28 activation did not significantly induce mDR5 expression by CD4 T cells from HIC (Fig. 2E). A hypothesis could be that CD4 T cells from HIC are refractory to these activators. Therefore, we tested the expression of a well-defined activation marker, HLA-DR, on CD4 T cells from the different groups of patients ex vivo and in vitro after HIV and anti-CD3-CD28 stimulations. HIV and CD3-CD28-mediated activation of CD4 T cell led to the induction of HLA-DR in a similar manner in HD and in HIC (Supplementary Fig. 3). Thus, the lack of mDR5 induction in CD4 T cells from HIC cannot be due to a lack of T cell activation but seems to be a characteristic of these cells.

3.4. DR5 silencing experiments To determine whether the low membrane expression of DR5 observed in CD4 T cells from HIC could mediate resistance to HIV-1 induced apoptosis, we silenced DR5 mRNA in CD4 T cells from HD. We first highlighted the efficiency of T cell transfection by using Alexa-488 scramble RNA (Fig. 3A) compared to a non-stained scramble RNA. We observed that 70% of CD4 T cells were positively transfected (Fig. 3A) and that transfection did not induce any significant CD4 T cell apoptosis. Thus, freshly isolated CD4 T cells from HD were transfected with scramble or DR5 siRNA. Cells transfected with DR5 siRNA showed massive downregulation by 60% of DR5 expression compared to scramble transfected cells (P = 0.004) (Fig. 3B). Furthermore, we showed that DR5 siRNA transfected CD4 T cells from HD become resistant to HIV-1-induced apoptosis due to the lack of membrane DR5 expression (Fig. 3C). Thus, DR5 siRNA transfected cells were more resistant to HIV-1-induced apoptosis than untransfected HD CD4 T cells or scramble siRNA treated HD (Figs. 3C–D). We verified that scramble and DR5 siRNA by themselves did not induce apoptosis of CD4 T cells (Fig. 3C). Taken together, these results demonstrate that the lack of HIV-induced DR5 expression at the surface of CD4 T cells from HIC and/or DR5 siRNA treated HD directly correlates with drastic reduction of HIV-1-mediated apoptosis.

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3.5. Ex vivo correlation of DR5 expression and CD4 count Finally, we tested whether a reduced mDR5 expression ex vivo could be associated with higher CD4 T cell count. We observed a negative correlation between mDR5 expression ex vivo and CD4 T cell count from HIC (r2 = 0.6878, p = 0.0005) and VIR (r2 = 0.4535, p = 0.0083) patients (Fig. 4). These clinical correlations support that limited mDR5 expression is associated with higher CD4 T cell count.

4. Discussion Among the different mechanisms potentially involved in CD4 T cell depletion, TRAIL pathway may participate to death of infected but also HIV-exposed uninfected CD4 T cells ex vivo, and in vivo [18,23–25]. Since HIC patients do not exhibit massive CD4 T cells depletion, we determined whether the TRAIL pathway was involved in resistance to the massive T cell death characteristic of HIV-infected patients who progress to AIDS. Thus, we studied expression and regulation of membrane TRAIL and its death receptors [DR4 and DR5] ex vivo and in vitro. We found that CD4 T cells from HIC did not express TRAIL on their membrane ex vivo, although in vitro HIV-1 exposure induced mTRAIL expression on these cells. Simian models showed that antigen-presenting cells (APC) from AIDS-resistant species do not express membrane TRAIL in contrast to AIDS-susceptible macaques [26]. Plasmacytoid dendritic cells (pDCs) are the principals ex vivo producers of IFN-α at peak acute infection in lymphoid tissues. Several studies have shown a rapid down-regulation of IFN-α response in SIV-infected sooty-mangabey in early chronic infection contrasting with a sustained pDC type I IFN response in macaques [27,28]. Thus, because IFN-α is the main regulator of TRAIL, absence of IFN-α may explain absence of mTRAIL on APC in chronic infection for AIDS resistant sooty-mangabey. In human, we recently demonstrated that in vitro stimulation of pDCs from HIC resulted in high levels of IFN-α protection [29]. Thus, our results of mTRAIL expression on CD4 T cells from HIC ex vivo and after HIV exposure demonstrate that TRAIL regulation and expression are not affected in these patients. The extremely low viral replication in HIC in chronic phase leads probably to limited pDCs activation and IFN-α production, insufficient for strong mTRAIL expression on CD4 T cells. However, in primary infection, viral replication was detected in HIC, and CD4 T cells depletion could have occurred because of normally mTRAIL induced by HIV on CD4 T cells from HIC [30].

Figure 1 Apoptosis and TRAIL expression in CD4 T cells. A. Flow cytometry analysis of CD4 T cells. Purified CD4 T cells from healthy donors [HD], viremic progressors [VIR] and HIV-1 controller patients [HIC] were cultured overnight in the absence or presence of HIV-1. Annexin-V staining revealed that cells from HD [n = 15] and VIR [n = 14] exposed to HIV-1 were undergoing apoptosis in contrast to CD4 T cells from HIC [n = 13]. B. Dot-plot analysis of DR5/Topro III expression by CD4 T cells from mock and HIV-1 exposed CD4 T cells from one HD and one HIC patient. (Representative of n = 3 experiment) C. Percentage of mTRAIL expressing CD4 T cells purified from HD [n = 15], VIR [n = 14] and HIC [n = 13]. D. TRAIL expression profile of HIV-1-stimulated CD4+ T cells. CD4+ T cells isolated from HD, VIR and HIC were phenotyped for TRAIL expression after HIV-1 exposure (Red histogram) compared to mock CD4+ T cells (Grey histogram) by FACS. (Representative of n = 3 experiment). E. Fold increase index analysis of mTRAIL expression by CD4 T cells from HD [n = 15], VIR [n = 14] and HIC [n = 13] after overnight HIV-1 exposure.

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Reduced Death Receptor 5-mediated CD4 T Cell Apoptosis in HIV controllers Because TRAIL impairment could not explain the reduced CD4 depletion in HIV controller, we investigated TRAIL death receptors (DR4 and DR5) expression by CD4 T cells. Ex vivo, CD4 T cells from healthy donors, HIC or VIR did not significantly express DR4. In vitro, HIV exposure of CD4 T cells did not induce substantial DR4 expression in any group of patients, excluding a potential role of this receptor in CD4 T cell death. In contrast, DR5 study revealed that CD4 T cells from viremic patients expressed membrane DR5 ex vivo in contrast to CD4 T cells from HIC patients that did not express significant levels of membrane DR5. Those results are in accordance with the non-progressor simian model in which DR5 was not upregulated ex vivo [19,26]. In vitro, we did not observe a significant induction of DR5 on HIV-exposed CD4 T cells from VIR, probably due to the pre-existing expression of membrane DR5 before stimulation. In contrast, while DR5 expression was significantly increased on cell membrane of HIV-exposed CD4 T cells from healthy donors, we did not observe significant DR5 expression on HIV-exposed CD4 T cells from HIC. To better demonstrate the potential role of the lack of membrane DR5 expression on CD4 T cells, we performed DR5 siRNA experiments. After validation of an efficient siRNA transfection, we silenced DR5 expression in human primary CD4 T cells exposed to HIV-1. In contrast to scrambled transfected cells, DR5 siRNA strongly inhibited membrane DR5 expression by HIV-1 stimulated cells. The consequence of this inhibition was a massive reduction of apoptosis by DR5 siRNA transfected cells. These experiments clearly linked DR5 expression and HIV-1-induced apoptosis of CD4 T cells. In parallel, HIV-mediated apoptosis in vitro was strongly reduced in CD4 T cells from HIC patients that harbored very low mDR5. Confirming our results, it has been shown that successful HAART dramatically reduced mRNA DR5 expression leading to a recover of CD4 count in a macaque model [19]. Interestingly, an inverse correlation between CD4 T cells mDR5 expression and CD4 T cells counts was found in both VIR and HIC suggesting that a reduced mDR5 expression could be associated with a control of CD4 count ex vivo. Our data are therefore consistent with better persistence of CD4 T cells in HIV controllers. Of course, extremely low viral replication limits CD4 T cell death but chronic inflammation is present in HIC and has been correlated with CD4 T cell count decrease [10]. Moreover, significant viral replication was present during primary infection of a few controllers in whom data were available [30]. To limit CD4 T cell depletion, several mechanisms could be involved. Alteration in TRAIL-DR5 apoptotic pathway is shown in this study, but defects in other pathways are also possible. Improvement of CD4 T cells survival could also play a role. Indeed, one study found that

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memory CD4 T cells persisted longer in HIC than in VIR through FOX03a-mediated pathway [31]. We focused this study on late apoptosis of uninfected CD4 T cells that is partially dependent of TRAIL pathway. In this purpose, we measured apoptosis of uninfected CD4 T cell cultured with inactivated AT-2 HIV-1. A recent study showed that infected CD4 T cells died faster than uninfected CD4 T cells from HIC, HD and VIR after 6 days post infection [32]. However, in the same study, uninfected CD4 T cells from HIC died slower than cells from VIR in accordance with our data. In HIV infection, especially in primary infection, a large number of uninfected CD4 T cells die. As a large number of circulating viruses are non infectious [33], AT-2 HIV (a chemically non-replicative inactivated HIV), mimics in vivo situation by inducing CD4 T cell death independently of cell infection. AT-2 CD4 T cell exposure leads to induction of TRAIL and DR5 on CD4 T cells leading to non-infected cell depletion. Interestingly, the role of TRAIL + plasmacytoid dendritic cells has also been suggested in CD4 T cell depletion, but pDC could not induce the lysis of autologous infected p24 positive CD4 T cells [34]. In our study, we did not investigate lysis but apoptosis of activated but not infected CD4 T cells pointing to the TRAIL-DR5 pathway as an important actor. As Fas has been described as an important apoptotic pathway in HIV [35–39], we studied this pathway and found that Fas expression on CD4 T cells from HIC, HD, and VIR was not different ex vivo and after AT-2 HIV activation. Thus, the major difference between HIC and other groups remained DR5 expression. We demonstrate here for the first time, that a reduced DR5 induction after HIV exposure on CD4 T cell membrane may contribute to the absence of massive CD4 T cell depletion in HIC allowing the development and persistence of HIV-specific CD4 T cell [11]. Moreover, DR5 expression on CD4 T cells ex vivo and after in vitro HIV-1 stimulation provides a new biological prognostic marker to determine the potential risk for patients to develop massive immune suppression leading to AIDS. Indeed, clinical data showed that successful HAART decreased DR5 expression on circulating CD4 T cells concomitantly with increased CD4 count and clinical benefit [19]. Our study also opens a new research area on TRAIL/DR5 protein regulation and expression by T cells to identify the mechanism responsible for the limited DR5 membrane expression observed in HIC. Interestingly, it has also been shown that a TRAIL antagonist, called TRAIL short, was inhibiting HIV-induced TRAIL-mediated CD4 T cell death [40]. Thus, it would be interesting to determine whether TRAIL short levels are higher in HIC patients compared to progressors and healthy donors. In perspective, development of protein– protein inhibitors targeting TRAIL/DR5 binding might be a new

Figure 2 Membrane DR5 expression by CD4 T cells. A. Percentage of freshly purified CD4 T cells from HD (n = 15), VIR (n = 14) and HIC (n = 13) expressing membrane DR5 quantified by flow cytometry. Irrelevant isotype antibody was used as control. B. Flow cytometry analysis of HIV-1-activated CD4 T cells (grey) from HD (n = 15), VIR (n = 14) and HIC (n = 13) expressing membrane DR5 compared to mock conditions (white) after overnight culture. C. Membrane DR5 levels on CD4 T cells from VIR [n = 14] and HIC [n = 13] cultured in absence (white) or presence (grey) of HIV-1 were analyzed by FACS and quantified by MFI. D. Freshly isolated CD4 T cells from healthy (HD), viremic (VIR) and controller (HIC) were phenotyped for DR5 expression (red histogram) by FACS. Mock (grey solid histogram) was used as the control of DR5 staining. (Representative of n = 3 experiment). E. Purified CD4 T cells from HIC, HD and VIR were cultured in the presence of anti-CD3-C28 stimulation. Membrane DR5 expression on CD4 T cells was analyzed by FACS. Mock (grey solid histogram) was used as the control of DR5 expression. Data shown are representative of 3 independent experiments.

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Figure 4 Correlation between CD4 T cell counts and membrane DR5 expression by CD4 T cells was calculated in HIC (green) and VIR (red) patients separately. P values (P) were determined using Mann–Whitney.

therapeutic approach to prevent HIV-1-induced CD4 T cell depletion. Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.clim.2014.07.010.

Conflict of interest statement The author(s) declare that there are no conflicts of interest.

Acknowledgments We thank the Agence Nationale de la Recherche sur le SIDA (ANRS) (grant no. 12186) for its financial support. We greatly appreciate the help of Dr. J. D. Lifson (SAIC-NCI, Frederick, MD) and Julian Bess (SAIC-NCI, Frederick, MD) for providing purified HIV-1MN particles. The authors thank Dr. Laurence Meyer, Pr. Daniel Séréni, Dr. Caroline Lascoux, Dr. Olivier Taulera, Jeannine Delgado, Pr. François Bricaire, Michèle Pauchard, Marie-Thérèse Rannou, Dr. David Zucman, Nadège Velazquez, all the other physicians, nurses, and patients.

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Figure 3 siRNA transfection of CD4 T cells and DR5 silencing. A. Freshly purified CD4 T cells were transfected with unlabelled or Alexa-488 scrambled siRNA. Dot plots show transfection efficiency of CD4 T cells (70%). (Representative of n = 3 experiment). B. CD4 T cells were incubated overnight with scrambled (Scr) or DR5 siRNA. Cells were cultured 24 h in the absence or presence of HIV-1 and DR5 protein levels were quantified. Transfection with DR5 siRNA significantly inhibited DR5 expression on HIV-1-stimulated CD4 T cells in contrast to scrambled (Scr) siRNA. (Representative of n = 3 experiment). C. Dot plots show apoptosis revealed by Annexin-V staining of DR5 expressing CD4 T cells. HIV-1 induced both DR5 expression and apoptosis of CD4 T cells from HD (30%). Inhibition of DR5 expression at the surface of HIV-1-stimulated CD4 T cells using DR5 siRNA dramatically inhibited apoptosis (Annexin-V) mediated by HIV-1 compared to Scr siRNA. Experiment C was repeated 4 times. D. Percentage of Annexin V-expressing CD4 + T cells purified from healthy donors in the presence or absence of HIV, HIV + scramble siRNA and HIV + DR5 siRNA. (Representative of n = 3 experiment).

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