Dalil Hannani

Etienne Merlin

Françoise M Gabert

David Laurin

Laurence Chaperot

Merlin Etienne

Francois Demeocq

Justyna Kanold

Photochemotherapy Induces a Faster Apoptosis of Alloreactive Activated T Cells Than of Nonalloreactive Resting T Cells in Graft Versus Host Disease

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Abstract:

Background:

Extracorporeal photopheresis (ECP) is a powerful therapy currently used to treat various haematological disorders as in graft versus host disease (GVHD). Clinical data clearly demonstrate its efficacy and immunomodulation towards the pathogenic T cells. However, ECP mechanism of action is still poorly understood.

Monocytes represent up to 30% of the total amount of treated cells and are known to play an important role in adaptive immunity. However, data from previous reports analyzing the effect of PUVA (psoralen and UV-A irradiation) on their functions are heterogeneous. In this study, we focused on the effect of PUVA on human monocytes functions in adaptive immunity.

Design and Methods:

Purified human monocytes were treated in vitro by PUVA. We measured their kinetic of apoptosis following the treatment. We also determine if their phenotype and functionalities were modified. Finally we assessed the functionalities of PUVA-treated monocytes-derived dendritic cells (DC).

Results:

PUVA treatment sentenced purified monocytes to die in six days and immediately altered their migratory capacities without impairing their ability of endocytosis. It also up-regulated co-stimulatory molecules and production of inflammatory cytokines upon activation and consequently stimulated allogeneic or autologous T cells as efficiently as untreated monocytes. Moreover, PUVA-treated monocytes retained their ability to differentiate into fully functional DC that maturated and stimulated T cells as well as normal DC.

Conclusions:

Our data demonstrate that monocytes undergo apoptosis and loose a part of their migratory capacity following ECP and the surviving cell functionalities are not impaired, suggesting that monocytes have a minor effect on ECP-mediated immunomodulation.

Introduction

Extracorporeal photopheresis (ECP), a therapy initially developed for the treatment of cutaneous T-cell lymphoma (CTCL) [START_REF] Miller | Review of extracorporeal photopheresis in early-stage (IA, IB, and IIA) cutaneous T-cell lymphoma[END_REF], is now currently employed for various hematological disorders involving pathogenic T-lymphocyte clones such as in GVHD [START_REF] Marshall | Technology insight: ECP for the treatment of GvHD--can we offer selective immune control without generalized immunosuppression?[END_REF]. In latter, ECP proved very effective with impressive clinical results [START_REF] Dall'amico | Extracorporeal photochemotherapy: a new therapeutic approach for allograft rejection[END_REF].

Moreover, ECP is a safe and well-tolerated therapy. Briefly, ECP process consists of the withdrawal of a large amount of PBMC by apheresis (between 10 to 15 billions) followed by incubation with the photoactivable drug 8-MOP (8-methoxypsoralene) and UV-A irradiation (PUVA). Depending on the method used (on-line or off-line system), PUVA treated cells are reinfused back to the patient within 1 to 3 hours .

Patients usually receive 5 to 50 or even more procedures depending on their clinical response to the treatment. Although ECP procedure is rather simple but its mechanisms of action seem more complex and still remain enigmatic [START_REF] Marshall | Technology insight: ECP for the treatment of GvHD--can we offer selective immune control without generalized immunosuppression?[END_REF][START_REF] Plumas | Mechanisms of action of extracorporeal photochemotherapy in the control of GVHD: involvement of dendritic cells[END_REF]. The direct effect of ECP on the treated cells is the formation of covalent linkage between DNA and 8-MOP induced by the UV-A light [START_REF] Kanne | Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8methoxypsoralen and 4,5'8-trimethylpsoralen[END_REF]. The treated cells, including pathogenic T cells, become unable to proliferate in vitro and undergo apoptosis after several hours [START_REF] Jacob | Quality control for the validation of extracorporeal photopheresis process using the Vilbert-Lourmat UV-A irradiation's system[END_REF][START_REF] Yoo | Apoptosis induction of ultraviolet light A and photochemotherapy in cutaneous T-cell Lymphoma: relevance to mechanism of therapeutic action[END_REF][START_REF] Truitt | Photochemical treatment with S-59 psoralen and ultraviolet A light to control the fate of naive or primed T lymphocytes in vivo after allogeneic bone marrow transplantation[END_REF].

This apoptosis is well characterized, however, it cannot explain the efficacy of ECP because only a small proportion of pathogenic T cells is treated during the process. Indeed, in GVHD, like in CTCL, the pathogenic T cells are mainly located in periphery, so they are not directly affected by the 8-MOP/UV-A exposure. It has been proposed that in humans the immune system could downmodulate the pathogenic Tlymphocyte reactivity(4). In animal models, the injection of PUVA-treated cells to healthy animal has been shown to induce an immune response that impedes, after induction, the development of the pathology studied [START_REF] Truitt | Photochemical treatment with S-59 psoralen and ultraviolet A light to control the fate of naive or primed T lymphocytes in vivo after allogeneic bone marrow transplantation[END_REF][START_REF] Ullrich | Photoinactivation of T-cell function with psoralen and UVA radiation suppresses the induction of experimental murine graft-versus-host disease across major histocompatibility barriers[END_REF][START_REF] Perez | Inhibition of antiskin allograft immunity induced by infusions with photoinactivated effector T lymphocytes--the congenic model[END_REF][START_REF] Maeda | Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells[END_REF]. However, in these studies the PUVA treated cells were taken from spleen, lymph nodes or in vitro cultured T-cells, whereas in human they are usually PBMC. Therefore, the immunomodulation and effectors identified in these studies could be different than those involved in human ECP. Among the treated cells, monocytes are a major group since they represent up to 30% of PBMC [START_REF] Garban | Extracorporeal chemophototherapy for the treatment of graft-versus-host disease: hematologic consequences of short-term, intensive courses[END_REF]. It is well known that monocytes play an important role in adaptive immunity as they modulate immune responses and differentiate into dendritic cells [START_REF] Randolph | Antigen presentation by monocytes and monocytederived cells[END_REF]. However, data from previous reports analyzing the direct effect of PUVA treatment on the functions of monocytes are heterogeneous [START_REF] Yoo | Apoptosis induction of ultraviolet light A and photochemotherapy in cutaneous T-cell Lymphoma: relevance to mechanism of therapeutic action[END_REF][START_REF] Legitimo | Psoralen and UVA light: an in vitro investigation of multiple immunological mechanisms underlying the immunosuppression induction in allograft rejection[END_REF][START_REF] Tambur | Extracorporeal photopheresis induces lymphocyte but not monocyte apoptosis[END_REF][START_REF] Renzo | Extracorporeal photopheresis affects interleukin (IL)-10 and IL-12 production by monocytes in patients with chronic graftversus-host disease[END_REF][START_REF] Rao | In vitro experiments demonstrate that monocytes and dendritic cells are rendered apoptotic by extracorporeal photochemotherapy, but exhibit unaffected surviving and maturing capacity after 30 Gy gamma irradiation[END_REF]. In this study, we analyzed the kinetic of monocytes apoptosis after PUVA treatment and the changes in their functional characteristic involved in adaptive immunity. We also evaluated the effects of PUVA treatment on antigen-presenting function of monocytes and on their ability to differentiate into functional DC.

Material and Method

Medium and Reagents

The cell culture medium used throughout these experiments was RPMI 1640 Glutamax (Invitrogen, Paisley, UK) supplemented with 1 mmol/L sodium pyruvate (Sigma-Aldrich, l'Isle d'Abeau, France), non-essential amino acids (Invitrogen), 20ng/ml gentamicin (Invitrogen) and 10% heat inactivated human AB serum (referred as complete medium).

Immunophenotyping was performed by using Fluorescein Isothiocyanate (FITC)-, Phycoerythrin (PE)-, PE-Cyanin5 or 7 (PC5 or PC7)-and Allophycocyanin (APC)conjugated and purified mouse monoclonal antibodies from Beckman Coulter (Marseille, France). IgG2a isotypic control, IFNγ and IL-4 antibodies were purchased from BD Biosciences (Pont de Claix, France).

Cells were stained with IgG1, IgG2a, CD11a, CD14, CD40 , CD54, CD58, CD80, CD83, CD86, CD209, HLA-I, HLA-II and HLA-DR markers. DioC6 (3,3-dihexyloxacarbocyanine iodide) and OVA-FITC were purchased from Invitrogen. Lucifer Yellow was purchased from Sigma-Aldrich. The 7-Aminoactinomycin D (7AAD) viability dye was purchased from Beckman Coulter. The 8-methoxypsoralen (8-MOP) was provided by our cell therapy unit. Flow cytometric analyses were performed using a 4 colors FACScalibur flow cytometer and the CellQuest-Pro software (BD Biosciences).

Cell purification

Blood samples were collected from three healthy volunteers who gave their written informed consent. The peripheral blood mononuclear cells (PBMC) were separated by density gradient on lymphocyte separation medium (Eurobio, Les Ulis, France).

Monocytes and T cells were purified according to the manufacturer instructions by using the human monocyte enrichment cocktail RosetteSep and from PBMC using the human T cells enrichment cocktail EasySep kit (Stem Cell Technologies Inc, Vancouver, Canada) respectively. Cells were cryopreserved in liquid nitrogen until use.

Monocyte-derived dendritic cells (moDC) generation

Purified monocytes were seeded at 0.5x10 6 cells/ml in a complete medium in the presence of 50U/ml of GM-CSF (Leucomax, Shering Plough,France) and 10 ng/ml of IL-4 (Tebu, Le Perray-en-Yvelines, France) as previously described [START_REF] Chaperot | Differentiation of antigen-presenting cells (dendritic cells and macrophages) for therapeutic application in patients with lymphoma[END_REF]. Both cytokines were added at day 0 and additional IL-4 at day 3. After 6 days immature moDCs were harvested and verified as CD14 -CD209 + by flow cytometry.

Experimental Psoralen/UV-A treatment (PUVA)

Purified monocytes were seeded at 1x10 6 cells/ml in a complete medium. Cells were incubated for 15 min at 37°C with 200ng/ml of 8-MOP in the dark and were exposed to 2 J/cm² 365nm wavelength UV-A radiation (Bio Sun, Vilbert-Lourmat). After PUVA treatment, cells were washed in a complete medium and then cultured in the same medium at 37°C in 5% atmosphere CO2.

Cell Viability

Apoptotic cells were quantified by DioC6/7AAD staining as described previously [START_REF] Manches | In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas[END_REF].

DioC6 was used to monitor mitochondrial transmembrane potential disruption.

Monocytes (1x10 6 cells in 1ml) were incubated with DioC6 at a final concentration of 1 nM for 15 min at 37°C. Cells were washed in PBS and then analyzed by flow cytometry.

Percentage of specific apoptosis was calculated by taking into account of the number and the viability of cells as follows:

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Endocytosis

Endocytosis of monocytes was assayed by Lucifer Yellow and Ovalbumine-FITC after PUVA treatment. 2x10 5 monocytes in 200µl of RPMI with 10% Fetal Calf Serum (FCS) medium were incubated with 1 mg/ml of Lucifer yellow or 0.1mg/ml of FITCconjugated ovalbumine in polypropylene tubes (BD Bioscience) for 3 hours at 37°C in 5% CO2 to allow endocytosis. Negative control was performed at 4°C. For ovalbuminmacropinocytosis -negative control, cells were incubated at 37°C in the presence of 10µg/ml of Cytochalasin D (Sigma-Aldrich). After 3 hours, cells were centrifuged twice in HBSS with 2% FCS and then the pellet was incubated with CD11b antibody. Phagocytic cells were determined as CD11b-FITC/Lucifer Yellow (FL-2) or Ova-FITC/CD11b-PE double positive cells by flow cytometry.

Cells activation and Cytokine production

Monocytes (1x10 6 ) were cultured in 1 ml of complete medium and activated with 1µg/ml LPS (Sigma-Aldrich) for 24hours. Immature moDCs were produced as described above. At day 6, they were activated with 1µg/ml LPS and 100ng/ml IFNγ (Peprotech Neuilly sur Seine, France) for 48 hours.

Culture supernatants from activated monocytes and moDCs were collected after 24 and 48 hours respectively. The contents were analyzed for IL-1, IL-6, IL-8, IL-10, IL-12 p70 and TNFα by Cytokine Bead Array (CBA) inflammation kit (BD Biosciences) according to the manufacturer's recommendations with FACScan (BD Bioscience).

Data were analyzed with CBA software (BD Biosciences).

Transwells chemotaxis assay

Migration assays were carried out by using transwells (6.5mm diameter, COSTAR, Cambridge, MA, USA) with 5.10 5 cells/well. Control or treated PBMCs were washed, preincubated for 1 h at 37°C in RPMI with FCS 2 % and then placed for 2 h in 5 µm pore size inserts at 37°C. The migrating monocytes were harvested and immunostained with CD14-FITC. The results were shown as the percentage of monocytes which have migrated relative to the input number of monocytes. The chemokines used were MCP-1/CCL2 (CCR2), MIP-1β/CCL4 (CCR5), RANTES/CCL5 (CCR1) and SDF-1α/CXCL12 (CXCR4), at 100, 50, 50 and 100 ng/mL respectively (R&D systems, MN, USA).

Antigen specific T cell amplication

Autologous monocytes (1x10 6 /ml) were pulsed with 10µM of the influenza M1 peptide (GILGFVFTL; HLA-4 of control or treated Flu-monocytes were co-cultured with 1x10 5 T cells for 7 days. PE -Flu-M1/HLA-A2 tetramers (Beckman Coulter, France) were used for the staining of the M1 specific CD3+CD8+ T cells according to the manufacturer's recommendations.

Mixed lymphocyte reaction

Responding T lymphocytes and stimulating monocytes were purified from PBMC of two healthy volunteers. Unidirectional MLR were conducted in quadruplicate of 96well flat-bottom plates (BD Biosciences) by mixing 5x10 4 responding purified CD3+ T cells and 5x10 3 4 monocytes or moDC +/-PUVA (ratio responding/stimulator cells from 1/1 to 1/0.1) in a final volume of 200µl [START_REF] Plumas | Mesenchymal stem cells induce apoptosis of activated T cells[END_REF]. After five days, [ 3 H]thymidine was added to each well and cell were harvested after 18 h later (Cell harvester :

Inotech IH 280 Berthold) [ 3 H]thymidine incorporation was measured by a liquid scintillatrion counter, TopCount NXT (Perkin Elmer).

T cell polarization

A mixed lymphocyte reaction was performed as described above. After 6-day culture, cells were harvested and T cells were restimulated with 0.5ng/ml of PMA and 5ng/ml of Ionomycine (Sigma-Aldrich) for 6 hours. The secretion pathway was inhibited by using Monensine after 1 hour. At the end of the culture, cells were permeabilized by cytofix/cytoperm Kit (BD Bioscience) allowing the intracellular staining of IFNγ and IL-4 (BD Bioscience). Analysis was performed by using a

FACSCanto II cytometer (BD Bioscience).

Statistical analysis

The unpaired student's T-test or two-way ANOVA were used to determine if the differences observed between control and treated conditions were significant or non-significant. A value of p < 0.05 was considered significant.

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Results

Monocytes undergo apoptosis after exposure to PUVA

First, we analyzed the kinetic of PUVA induced apoptosis, a direct effect of the treatment due to the formation of photo adducts [START_REF] Kanne | Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8methoxypsoralen and 4,5'8-trimethylpsoralen[END_REF]. In this study we used DioC6 instead of the annexin-V staining method [START_REF] Manches | In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas[END_REF] because the numerous platelets sticked on monocytes were highly bound to annexin-V leading to a non specific labelling [START_REF] Silverstein | Thrombospondin binds to monocytes-macrophages and mediates platelet-monocyte adhesion[END_REF][START_REF] Thiagarajan | Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets[END_REF]. We cultured PUVA-treated monocytes for 6 days in the presence or absence of GM-CSF used here as a monocyte-survival factor. At day 0, more than 90% of monocytes were alive. Six days after PUVA treatment in presence of GM CSF about 80% of them were dead (Figure 1A). The observation of PUVA-monocytes after May-Grünwald-Giemsa staining clearly showed an apoptotic features (cell shrinkage, apoptotic bodies, nuclear condensation) as compared to untreated monocytes. Moreover, since monocytes have the ability to engulf cell debris or apoptotic bodies, we have also taken into account the absolute number of viable cells during the culture to calculate the percentage of specific apoptosis. The results presented in figure 1B showed that specific apoptosis was continuously increasing in the days following the treatment. One day after treatment, the specific apoptosis was low (around 20%) whereas at day 6 it reached up to 80%. The presence of GM-CSF did not change this kinetic suggesting that the sensitivity of monocytes to cell death was not modified by survival factors. Apoptosis induced by PUVA in monocytes has a slow kinetic and thus surviving treated cells may still be functional until day 6.

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The main functions of monocytes are not altered by the PUVA treatment except their migratory capacities

We then analyzed the effect of PUVA treatment on the capacity of monocytes to perform endocytosis. Capture of Lucifer Yellow (LY) was used as a model of pinocytosis and FITC-ovalbumine (FITC-OVA) as a model of macropinocytosis. After 3-hours incubation at 37°C, more than 90% of the monocytes efficiently engulfed LY and FITC-OVA (Figure 2). The PUVA treatment neither affected the percentage of positive cells nor the intensity of labeling, meaning that the endocytic ability of monocytes was not altered.

As monocytes died slowly following PUVA treatment, it can be assumed that once they are injected back to the patient these cells could still be able to migrate in periphery or in the lymphoid organs and/or to stimulate T-cells. So, we evaluated the expression of molecules involved in these processes on control or PUVA -monocytes.

Moreover, for an optimal T-lymphocytes stimulation, monocytes are required to present peptide associated to HLA molecules and to provide costimulation signal via molecules such as CD40 and CD86 in the immune synapse reinforced by adhesion molecules such as CD11a, CD54 and CD58. These molecules were constitutively expressed on monocytes at various levels (data not shown). Twenty-four hours following PUVA treatment, we did not observe any significant change of their expression in comparison to untreated cells (figure 3A et 3B). Also, the main chemokine receptors expressed by monocytes were not modified by the treatment (figure 3C). Interestingly, despite an absence of a differential chemokine receptors expression, PUVA treatment significantly (p<0.01) impaired the monocytes migration towards CCL2 and CCL4 but not to CCL5 and CXCL12 (Figure 3D). These data suggest that reinfused treated monocytes have lost a part of their capacities to patrol in the body.

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We next analyzed the production of inflammatory or inhibitory cytokines by monocytes known to modify the immune response like IL-1b, TNF--10.

Monocytes were treated or not by PUVA and then activated or not by LPS for 24h (figure 4A). Without activation, there was no cytokine production. The production of these cytokines after activation with LPS was affected neither positively nor negatively by the PUVA treatment. This suggests that PUVA does not modify the ability of monocytes to secrete cytokines in response to an infection or others stimulatory signals.

The APC function of the cells can be tested by performing a mixed lymphocyte reaction [START_REF] Chaperot | Differentiation of antigen-presenting cells (dendritic cells and macrophages) for therapeutic application in patients with lymphoma[END_REF]. Indeed, this assay reflects the global stimulatory capacities of APC taking into account the expression of molecules involved in the immunological synapse (HLA, costimulatory, adhesion molecules) and the cytokines released. As presented in figure 4B, after 6-day culture, the T-cell proliferation increased according to the number of allogeneic monocytes added in the culture. A similar Tcells proliferation was observed when PUVA-treated monocytes were used as APC.

Moreover, the T cell polarization induced by either control or PUVA -monocytes was similar in both conditions (figure 4C). So, it appears that PUVA does not affect the allogeneic stimulatory potential of monocytes.

We next analyzed in autologous setting the impact of PUVA on the capacity of monocytes to induce antigen specific T-cells. Influenza M1 peptide pulsed monocytes were used to stimulate autologous T cells (Figure 4D). Following 7 day culture, the frequency of Flu-specific T cells reached to up to 15% in control condition and was not affected in the treated one.

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All together, these data indicate that immediately after PUVA the allogeneic and antigen-specific stimulation capacities of monocytes are not modified. By contrast, PUVA-treated monocytes seem to partially loose their migratory capacity.

The PUVA treatment does not impair the differentiation of monocytes into functional dendritic cells

Since dendritic cells are potent inducers of immune response and monocytes are one of their precursors, we asked if the PUVA treatment could alter or induce their differentiation into functional DC.

The differentiation of monocytes into DC was induced by 6-day culture with GM-CSF and IL-4. This differentiation can be easily evidenced by the concomitant decrease of CD14 and an increase of CD209 (DC-SIGN) expression. At day 0, monocytes were CD14+CD209-and after culture they become CD14-CD209+ DC (figure 5A). We then evaluated the impact of the PUVA treatment on this differentiation. As the PUVA treatment induce apoptosis in monocytes, we performed analysis only on the surviving cells for 6-day culture. Upon culture with IL-4 and GM-CSF, PUVA treatedmonocytes lost CD14 while they acquired CD209 with the same kinetic as control monocytes (figure 5B, upper panels). In addition, in the absence of cytokines, monocytes did not turn into DC as evidenced by the persistent expression of CD14

and the low level of CD209 (figure 5B, lower panels), whatever were the conditions.

We then wondered if DC generated from treated-monocytes were functional by stimulating them with LPS in the presence of IFNγ for 48h. We analyzed their activation by measuring the up-regulation of CD83, CD86 and CD40 and by production of cytokines. As presented in figure 5C, DC derived from treatedmonocytes up-regulated the expression of these activation markers to the same extent as non-treated monocytes. Regarding cytokine production, IL1b and IL-10

were not detected whatever was the condition. However, it seems that the levels of IL-6, IL-12 and TNFα were slightly, but not significantly, lower in PUVA-monocytes derived DC condition (figure 5D). Moreover, when control and PUVA-moDC were used as stimulating cells for allogenic T cells, they both induce the same IL-4+/IFNγ+ positive cells ratio (figure 5E). These results suggest that the functions of PUVAmonocytes derived DC remain preserved.

Discussion

ECP is a cell therapy commonly used for the treatment of diseases caused by pathogenic T cells like CTCL [START_REF] Miller | Review of extracorporeal photopheresis in early-stage (IA, IB, and IIA) cutaneous T-cell lymphoma[END_REF] or GvHD [START_REF] Marshall | Technology insight: ECP for the treatment of GvHD--can we offer selective immune control without generalized immunosuppression?[END_REF]. However, its overall mechanism of action remains unclear. Several studies demonstrate the clinical beneficial effects of ECP, however the immune pathway leading to this ECP-mediated immunomodulation remains to be elucidated.

The ECP treatment has been shown to induce T-cell apoptosis [START_REF] Yoo | Apoptosis induction of ultraviolet light A and photochemotherapy in cutaneous T-cell Lymphoma: relevance to mechanism of therapeutic action[END_REF][START_REF] Enomoto | Extracorporeal photochemotherapy (photopheresis) induces apoptosis in lymphocytes: a possible mechanism of action of PUVA therapy[END_REF]. Regarding monocytes, previous reports have presented heterogeneous data about the sensitivity of these cells to apoptosis [START_REF] Yoo | Apoptosis induction of ultraviolet light A and photochemotherapy in cutaneous T-cell Lymphoma: relevance to mechanism of therapeutic action[END_REF][START_REF] Legitimo | Psoralen and UVA light: an in vitro investigation of multiple immunological mechanisms underlying the immunosuppression induction in allograft rejection[END_REF][START_REF] Tambur | Extracorporeal photopheresis induces lymphocyte but not monocyte apoptosis[END_REF][START_REF] Renzo | Extracorporeal photopheresis affects interleukin (IL)-10 and IL-12 production by monocytes in patients with chronic graftversus-host disease[END_REF][START_REF] Rao | In vitro experiments demonstrate that monocytes and dendritic cells are rendered apoptotic by extracorporeal photochemotherapy, but exhibit unaffected surviving and maturing capacity after 30 Gy gamma irradiation[END_REF]. We first analyzed the kinetic of monocytes apoptosis after the treatment. We also calculated the specific apoptosis by taking into account the absolute number of cells and their viability accessed with DioC6. This method appears to be more appropriate than the others because the phagocytic activity of monocytes by removing dead cells could alter the results leading to underestimate apoptosis. Moreover, the detection of apoptosis by the commonly used annexin-V marker is unsuitable in this case because it binds to the numerous platelets [START_REF] Thiagarajan | Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets[END_REF] sticked onto monocytes membranes. We show that it takes 6 days for monocytes to undergo apoptosis whereas it takes only 3 days for T cells ((7, 23), unpublished results). So, following PUVA treatment monocytes apoptosis was slow compared to T cell apoptosis. This enhanced resistance of monocytes could be explained by the fact that, contrary to T cells, monocytes do not proliferate and hence could be less sensitive to apoptosis induced by PUVA [START_REF] Kanne | Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8methoxypsoralen and 4,5'8-trimethylpsoralen[END_REF]. Interestingly, GM-CSF did not protect the cells from apoptosis, suggesting that the apoptotic process is definitely engaged following the treatment.

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We next analyzed whether the PUVA treatment could alter the main functions of monocytes in adaptive immunity. We evaluated monocytes properties just after the treatment as soon as possible because of the ongoing apoptosis and the treated cells are rapidly infused back to the patient during the therapeutic process. We observed that the treatment did not change the expression of specific homing receptors on monocytes but inhibit their migratory capacities toward CCL2 and CCL4 suggesting that they could not patrol in the body after reinfusion avoiding monocytes to play their role. Concerning the influence of the PUVA treatment on cytokine secretion, in contrast to other study [START_REF] Vowels | Extracorporeal photochemotherapy induces the production of tumor necrosis factor-alpha by monocytes: implications for the treatment of cutaneous T-cell lymphoma and systemic sclerosis[END_REF], we did not detect any increase in the spontaneous production of TNF-alpha. This discrepancy could be explained by the differences in purification process, indeed, purification by adhesion or positive selection is known to activate monocytes. To avoid this risk, we purified them by a negative magnetic selection. In addition, unlike to some report [START_REF] Renzo | Extracorporeal photopheresis affects interleukin (IL)-10 and IL-12 production by monocytes in patients with chronic graftversus-host disease[END_REF], the PUVA treatment did not lead to the secretion of inhibitory molecules by monocytes such as IL-10. The absence of global inhibitory effect mediated by the PUVA treated monocytes was evidenced by their ability to induce a consistent allogeneic proliferation of T cells even when the monocytes/T cell ratio was high. Conversely, when this ratio was low, no increase of T-cell proliferation was observed with treated compared to untreated monocytes, suggesting that the treatment did not upregulate the stimulatory activity of monocytes. Also, their antigen-specific T cell amplification capacities were not modified. These observations are consistent with the fact that the expression of costimulatory and adhesion molecules, and the secretion of cytokines were not modified by the treatment. Moreover, the observation that the endocytic capacity of treated monocytes remained unchanged suggests that monocyte main function is not impaired by PUVA treatment at least early after the treatment.

Monocytes are precursors of dendritic cells in vivo but the previous data showed that the effect of PUVA treatment on this differentiation was heterogeneous [START_REF] Rao | In vitro experiments demonstrate that monocytes and dendritic cells are rendered apoptotic by extracorporeal photochemotherapy, but exhibit unaffected surviving and maturing capacity after 30 Gy gamma irradiation[END_REF][START_REF] Berger | Induction of human tumor-loaded dendritic cells[END_REF][START_REF] Legitimo | In vitro treatment of monocytes with 8methoxypsolaren and ultraviolet A light induces dendritic cells with a tolerogenic phenotype[END_REF]. In vitro, monocytes can easily be turned into DC by 6-day culture in the presence of GM-CSF and IL-4. In these conditions, we have shown here that PUVA-treated monocytes differentiated into dendritic cells with the same as non-treated cells. Contrary to a previous report [START_REF] Berger | Induction of human tumor-loaded dendritic cells[END_REF] but confirmed by a recent work [START_REF] Legitimo | In vitro treatment of monocytes with 8methoxypsolaren and ultraviolet A light induces dendritic cells with a tolerogenic phenotype[END_REF], we found that in the absence of added cytokines there was no spontaneous DC differentiation. This could be explain by the absence of plastic interaction in our condition.

Moreover, whether monocytes were treated or not, the generated dendritic cells were able to mature in the presence of LPS as evidenced by the upregulation of costimulatory molecules and cytokines production. Moreover, both control and PUVA-moDC were able to induce the T cell polarization with the same extent, suggesting that they were fully functional and ECP do not modify their immunomodulatory properties. So, our results suggest that the low fraction of monocytes that did not undergo apoptosis after 6-day following PUVA treatment were still able to differentiate into fully functional DC only in the presence of differentiating cytokines. These data are consistent with the anti-tumor response hypothesized by Edelson et al [START_REF] Berger | Induction of human tumor-loaded dendritic cells[END_REF][START_REF] Berger | Transimmunization, a novel approach for tumor immunotherapy[END_REF][START_REF] Edelson | Transimmunization: the science catches up to the clinical success[END_REF].

The demonstration that the main functions of monocytes were not impaired suggests that they have not a direct effect on the ECP-mediated immunomodulation.

Moreover, after reinfusion, our data suggest that they become enable to migrate toward inflammatory sites. We show that the monocytes which are in great number CD14 and CD209 membrane expression on monocytes at day 0, 1, 3 and 6. Data

  among the cells treated by PUVA are sentenced to die like T cells, DC and probably all other cells[START_REF] Holtick | Impact of psoralen/UVA-treatment on survival, activation, and immunostimulatory capacity of monocyte-derived dendritic cells[END_REF]. This injection back to the patient of huge amount PUVA-treated page 21/27 cells may induce indirect effects on immune response. For instance, Craciun et al showed that the treated cells trigger the synthesis of immunosuppressive cytokines by non-treated mononuclear cells[START_REF] Craciun | Increased production of interleukin-10 and interleukin-1 receptor antagonist after extracorporeal photochemotherapy in chronic graft-versus-host disease[END_REF]. This could be responsible for the early clinical efficacy of ECP. It is now necessary to further analyze the impact of the treated cells on the long term immunomodulatory effects induced by ECP.

Figure 1 :Figure 2 :

 12 Figure 1: PUVA treatment induces apoptosis in monocytes

Figure 3 :

 3 Figure 3: Chemokine receptors, MHC molecules and activation and adhesion

Figure 4 :

 4 Figure 4: PUVA treatment does not modify either cytokine production under

Figure 5 :

 5 Figure 5: PUVA treatment neither impairs the differentiation of monocytes into

Acknowledgments

We would like to thank MJ. Richard, H.Egelhofer and the staff of Cell and Tissue therapy Unit of Saint-Ismier for providing us the psoralen molecule. We are also grateful to P. Drillat, F. Bernard, C. Morand, I. Michaud, C. Guitton and the staff of EFS Rhône-Alpes for blood sample collections. We also thank R.Muhamad for critical review of the manuscript.

 [START_REF] Miller | Review of extracorporeal photopheresis in early-stage (IA, IB, and IIA) cutaneous T-cell lymphoma[END_REF]This work was supported by the Etablissement Français du Sang and Association


Alassane Sidibe
email: asidibe@laas.fr

Alexandru Takacs
email: atakacs@laas.fr

Gaël Loubet
email: gael.loubet@laas.fr

Daniela Dragomirescu
email: daniela@laas.fr

Ultra-Compact and High-Efficiency Rectenna for Wireless Sensing Applications in Concrete Structure

Keywords: Rectenna, wireless power transmission, energy harvesting, wireless sensing application, structural health monitoring, concrete

This paper presents an ultra-compact and high efficiency rectenna which can power wireless sensing nodes fully embedded in a concrete structure. The innovative proposed design is optimized in term of efficiency and compactness for the targeted application. The rectenna is compact, which a volume of 56 mm x 32 mm x 10 mm, (0.0004𝛌𝛌 𝟑𝟑 ) and can reach a maximum RF to dc conversion efficiency of 70% at 868 MHz for 10kΩ load and for an illuminating RF power density of 4μW/cm². The use of a metallic cavity increases the rectenna performances and limits the negative impact of the concrete material. Thus, the rectenna into the cavity harvest a dc voltage up to 1.15V when the rectenna is illuminated with a RF power density of 1 μW/cm². Experimental results of the rectenna embedded in the concrete beam confirms the feasibility of powering a wireless sensor by wireless power transmission.

I. INTRODUCTION

The use of Radiofrequency (RF) Energy Harvesting (EH) or alternatively of the RF Wireless Power Transmission (WPT) are not now well considered in harsh environments. The work presented in [START_REF] Loubet | Towards the design of wireless Communication Reinforced Concrete[END_REF] addresses an original concept of wireless smart-node network (WSN) for Structural Health Monitoring (SHM) applications. Researchers also evaluate the potential of RFID technology in concrete structure. Similarly, a concrete quality sensor based on chipless ultra-wideband (UWB) radio frequency identification (RFID) is presented in [START_REF] Ramos | Wireless Concrete Mixture Composition Sensor Based on Time-Coded UWB RFID[END_REF]. In [START_REF] Bartholmai | Transmission Characteristics of RFID Sensor Systems Embedded in Concrete[END_REF], a RFID sensor system was developed for being embedded into concrete structures.

However, several challenges must be investigated as the lifetime of the nodes once embedded and the long-term communication reliability.

To overcome the lifetime challenges, we propose in this paper a unique design of an improved Wireless Power Interface embedded in a concrete structure working in the 868 MHz Industrial, Scientific and Medical (ISM) frequency band. The Wireless Power Interface commonly called a Rectenna (Rectifying antenna) was developed in order to supply a Power Management Unit (PMU) by WPT or EH.

Additionally, a metallic cavity was designed in order to minimize the losses which appears once the rectenna is embedded in the concrete. The two prototypes (rectenna with and without cavity) were tested and presented in section III. Experimental test of the rectenna in the concrete is highlighted in section IV.

II. RECTENNA DESIGN AND MEASUREMENT The proposed rectenna consists of a differential rectifier directly connected to a dipole antenna designed for a 50 Ω input impedance. Next, the entire design was embedded in a metallic cavity in order to increase the harvested dc voltage. We proposed a compact 3D rectenna with a high RF to dc conversion efficiency.

A. Antenna design

A compact antenna was designed and simulated thanks to HFSS software for the targeted application. It is like a RFID dipole antenna with a short-circuited loop as presented in Fig. 1 (a). By adding two capacitive arms, it is possible to form a 3D configuration of the antenna and reduce its size. The dipole was connected to the metallic arms by an electric line. Its position was chosen to tune the resonant frequency of the antenna. Two antennas with different electrical line positions were manufactured namely: DPLC5 (Fig. 1 (b)) and DPLC3 (Fig. 1 (c)). The antennas were manufactured on FR4 substrate (with a thickness of 0.8 mm). Due to the planar configuration of the dipole, an easy way to characterize the antenna was to add a 50 Ω UFL connector. As presented in Fig. 3 (a), a frequency shift of 40 MHz (i.e. 4.75%) between simulation and measurement was observed for DPLC3 and DPLC5. This frequency shift is mainly due to the manual assembling/connecting of the vertical arms. For the final implementation of the rectenna, DPLC5 configuration was chosen.

In the literature, some research presents the investigation of antenna embedded in concrete. A microstrip patch antenna operating at 2.5GHz was designed for civil engineering applications [START_REF] Roqueto | A Novel Compact UHF Wideband Antenna for Near Field Electrical Characterization of Steel Fiber Reinforced Concrete[END_REF]. Otherwise in [START_REF] Jeong | UHF RFID Tag Antenna for Embedded Use in a Concrete Floor[END_REF] is presented a design of the UHF RFID tag antenna suitable for embedded use in a concrete floor. This antenna consists of a ceramic path, a parasitic patch and a metal cavity which improve the gain. Consequently, to minimize the impact of the high dielectric constant and high dielectric losses of the concrete material on the antenna/rectenna performances, we proposed to add a top open metallic cavity which covers the antenna (Fig. 2). Its size and the antenna location from the bottom side were optimized in order to increase the antenna gain and to avoid antenna detuning into the concrete. For this implementation, we considered the frequency shift of the antenna as mentioned before. The antenna is placed at 10 cm from the bottom of cavity and the metallic arms was also tune at 7.5 mm. Fig. 3 (b) plots the return loss (S11) of the antenna inside the manufactured cavity.

The radiation pattern of the manufactured antenna was measured in an anechoic chamber. The measured and simulated gain in the E-plane (that is a vertical cut plane orthogonal to the antenna horizontal plane) obtained for the DPLC5 antenna with and without metallic cavity are represented in Fig. 4. The maximum measured gain obtained with DPCL5 antenna is +1.7 dBi. By adding the metallic cavity, the gain is multiplied by 2.5 reaching up to +4.5 dBi.

B. Full Wave Rectifier Design

This section presents the rectifier design optimized to operate at low power densities. Several configurations of rectifiers exist. The proposed design (in Fig. 5) is a doubler rectifier composed by SMS7630_005LF Schottky Diode from Skyworks investigated at low power level. The size considerations allow us to choose a lumped LC (33 nH and 4 pF) matching network. The simulation was done in ADS Software and the circuit was built on FR-4 substrate with a thickness of 0.8 mm. Fig. 6 shows a photo of the manufactured rectifier and its return loss for different input power. The maximal measured RF to dc conversion efficiency and dc voltage of the rectifier is about 35 % and 330 mV for a dc load of 10 kΩ load at 870 MHz with an input power of -15 dBm. The load value was chosen in order to emulate the input impedance of the PMU for the wireless sensors. This one is not the optimal load impedance of the rectifier.

III. IMPLEMENTED RECTENNA

The final design consists to connect the antenna with the rectifier to form the rectenna as previously presented. Two where 𝑃𝑃 𝐷𝐷𝐷𝐷 is the harvested dc power measured across the load and S is the incident EM power density illuminating the rectenna. 𝐴𝐴 𝑒𝑒𝑒𝑒𝑒𝑒 is the antenna effective area computed thanks to the gain of DPLC5 with or without cavity ( 𝐺𝐺 ) and the wavelength ( 𝜆𝜆 ). 𝑃𝑃 𝑡𝑡 is the RF power injected to the patch antenna (Gain 𝐺𝐺 𝑡𝑡 ) positioned at a distance d of 1.5 meter from the rectenna. For accurate measurement, the insertion losses through the RF cable used to connect the RF source to the transmitting (Tx) antenna (about 3 dB) was measured with a power meter and subtracted from the patch antenna gain (about 9.6 dBi). The measured dc voltage of the rectenna as function of frequency is presented in Fig. 7. The resulting dc voltage and the efficiency (at 868 MHz) as function of the illuminating RF power density are presented in Fig. 8. We observe that for higher power density level, RT is more efficient than RMC and reach a maximum efficiency of 70 %. However, RMC maintains a good efficiency (65 % max).

For a power density of 1 μW/cm², the harvested dc voltage is 1300mV and 900mV respectively for the rectenna with (RMC) and without the cavity (RT).

IV. EXPERIMENTA L RESULTS WITH THE CONCRETE BEAM

In order to perform experimentations in the targeted harsh environment, a reinforced concrete beam (size: 135 cm by 35 cm by 35 cm) was designed and manufactured. The experimental setup consists of illuminating the rectenna embedded in the concrete structure with a linearly polarized E-Field as shown in Fig. 9. The RF power transmitter is an Anritsu MG3694B generator connected to a patch antenna though a coaxial cable. The obtained dc voltage was measured by using a dc multimeter. During the measurement, the antenna was placed on the roof at 156cm of the concrete beam (upper side). Otherwise, the rectenna in the concrete cavity was placed in order to match the polarization of the transmitting antenna. The transmitting patch antenna has a gain of +9 dBi. Fig. 10 shows the harvested dc power obtained inside the concrete structure for the RT and the RMC as function of the Effective Isotropic Radiated Power (EIRP). The total distance between the transmitting antenna and the rectenna is 170 cm (with the height of the concrete cover). Uncovered measurement corresponds to the rectenna placed in the concrete without any concrete cover on the top. RT results was obtained with covered concrete at different height positions from the bottom of the concrete cavity. It is obvious that the concrete cavity acts as dielectric resonant cavity and the rectenna position should be optimized inside this cavity. The best results were obtained with RMC positioned inside the covered cavity in the concrete because a dual resonance occurs: (i) first resonance inside the top-open metallic cavity, (ii) the second one in the top-covered concrete cavity. So that, the influence of the concrete material can be reduced by: (i) choosing the appropriate dimensions of the hole in the concrete (it was not possible in our case because the concrete beam was manufactured before the experiments), (ii) inserting the rectenna in a top-open metallic cavity (iii) controlling and optimizing the rectenna position inside the metallic cavity (when used) or in the concrete cavity (when used standalone). An optimization methodology/algorithm by using intensive electromagnetic simulations is under development in order to find the best-fitted size of the concrete and metallic cavity and the optimal position of the rectenna. More results will be available at conference.

V. CONCLUSION

In this paper we present a unique and ultra-compact design of rectenna (0.0004λ 3 at 868 MHz) dedicated to RF wireless power transmission in concrete structures. It consists of a doubler rectifier using two SMS7650-005LF Schottky diodes and a 3D compact antenna. The prototypes were fabricated and characterized in anechoic chamber. The 3D compact antenna (DPLC5) of our rectenna exhibits a gain of 1.7 dBi (when measured standalone) and 4.5 dBi when inserted in metallic cavity. The measured rectenna conversion efficiency is about 54% for an illuminating power density of 1.5 µW/cm² and 10kΩ load. Therefore, additional measurements of the rectenna embedded in the concrete structure were performed. With an EIRP of 23.7 dBm, the harvested dc voltage is about 623 mV and 402 mV for RMC and RT (positioned at 13cm from the bottom), respectively.

As compared with the state-of-the-art designs reported in table I, the proposed rectenna is ultra-compact and exhibits excellent performances. The experimental results confirm that the proposed rectenna can be used for wirelessly.

Figure 1 :

 1 Figure 1: Simulation design on HFSS Software (a) and photo of the fabricated antenna DPLC3 and DPLC5.

Figure 2 :

 2 Figure 2: Simulation design and photo of the manufactured prototype of DPL5 embedded in a metallic cavity.

Figure 3 :

 3 Figure 3 : (a) Measured (continuous line) and simulated (dashed line) of the return loss (S11) for DPLC3 (red line) and DPLC5 (blue line). (b) Simulate d (red dashed line) and measurement (blue continuous line) of the return loss (S11) of the antenna DPLC5 embedded in the metallic cavity.

Figure 4 :

 4 Figure 4: Radiation pattern (gain in dB) at 868 MHz in the E-plane: experimental (continuous line) and simulation (dashed line) results for the DPLC5 antenna in the metallic cavity (blue line) and DPLC5 antenna only (red line).

Figure 5 :

 5 Figure 5: Schematic of the doubler rectifier.

Figure 6 :

 6 Figure 6: Measured return loss of the rectifier for different RF input power. The inset shows a photo of the manufactured prototype.

4 .

 4 prototypes were tested, the rectenna without cavity named RT and the rectenna placed in the metallic cavity named RMC. The experimental setup was placed inside an anechoic chamber. It consists on illuminating the rectenna under test positioned at a Far Field region of transmitter with linear Polarization E-Field. The transmitting Patch antenna is connected to an Anritsu MG3694B power generator (placed outside) though a coaxial cable. The harvested dc voltage across a 10 kΩ load is measured by using a Multimeter. The following expression in (1) was used to calculate RF to dc conversion efficiency η: 𝜋𝜋. 𝑃𝑃 𝐷𝐷𝐷𝐷 𝑆𝑆. 𝐺𝐺 𝑟𝑟 . 𝜆𝜆 2 and 𝑆𝑆 = 𝑃𝑃 𝑡𝑡 . 𝐺𝐺 𝑡𝑡 𝑑𝑑 2 .4. 𝜋𝜋[START_REF] Loubet | Towards the design of wireless Communication Reinforced Concrete[END_REF]

Figure 7 :

 7 Figure 7: Measured dc voltage for the two manufactured rectennas as function of the frequency in an anechoic chamber. RMC (red dashed line) and RT (continuous blue line) at a power density of 3.4 μW/cm².

Figure 8 :

 8 Figure 8: Measured RF to dc conversion efficiency (continuous line) and dc voltage (dashed line) as function of the illuminating power densities at 868MHz. RT (blue line) and RMC (red line).

Figure 9 :

 9 Figure 9: (a) Experimental setup of rectenna inside concrete, (b) RMC with its metallic cavity embedded in the concrete. (c) The cover of the concrete beam.

Figure 10 :

 10 Figure 10: Harvested dc power of the RMC and the RT as function of the EIRP of the RF source positioned at 156 cm far away from the top of the concrete beam. RT was fully embedded in the concrete cavity with the cover and positioned at different height from the bottom of cavity.

Table 1 .

 1 Performances comparison of the fabricated rectenna with the state of the art.

	References	Freq.	Diode	Input Power	S	Eff.	G(dBi)	Rectenna size	dc
		(GHz)		(dBm) /EIRP	(µW/cm²)	(%)		(mm x mm x mm)	Power
				(dBm)					(µW)
	[6]	0.868	HSMS285C	--	0.1	30.2	6.43	NR*	12.62
	[7] (2DRRP)	0.868	HSMS285B	--	1	31.5	6.8	150 x 90 x 50 (0.016 𝜆𝜆 3 ) 143.77
	[8]	0.9	HSMS2850	--	1	40	1.3	110 x 110 (0.11 𝜆𝜆²)	47.7
	[9]	1.96	SMS7630	--	200	54.2	NR	60 x 75 (0.19 𝜆𝜆²)	4200
		2.45	SMS7621
	[10]	0.915	SMS7630	-9/ --	1	37	1.87	60 x 60 x 58 (0.0059 𝜆𝜆 3 )	46.6
	[11] In Concrete	5.7	HSMS2862	38.5/ --		75.8	10.1	70 x 50 (1.29 𝜆𝜆²)	10370
	This work
	In anechoic chamber
	RT				1	52.74%	1.7	52 x 32 x 10 (0.0004𝛌𝛌 𝟑𝟑 )	85.7
	RMC	0.868	SMS7630	--	1	51.7%	4.5	130 x 130 x 120	171
	In Concrete structure		005LF
	RT (height = 10cm)			--/ 23.7	1	--	1.7	52 x 32 x 10 (0.0004𝛌𝛌 𝟑𝟑 )	16.22
	RMC			--/ 23.7	1	--	4.5	130 x 130 x 120	38.84