Céline Coppard

PhD Dalil Hannani

PhD Marion Humbert

Virginie Gauthier

Joël Plumas

PhD Etienne Merlin

PhD Françoise M Gabert

PhD Laurence Chaperot
email: laurence.chaperot@efs.sante.fr

Joel Plumas

in GVHD: New insights in extracorporeal photopheresis

Keywords: Extracorporeal photopheresis, Damage-Associated Molecular Patterns, apoptosis, Graft versus Host Disease, dendritic cells

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In vitro PUVA treatment triggers calreticulin exposition and HMGB1 release by dying T lymphocytes

Abbreviations

1-Introduction

Extracorporeal photopheresis (ECP) is an autologous cell therapy in which leukocytes collected by cytapheresis are incubated with 8 methoxy-psoralen (8-MOP), irradiated with UVA, before reinfusion to the patient. ECP is used to treat diseases with circulating pathogenic T cells, such as cutaneous T cell lymphoma (CTCL) [START_REF] Alfred | The role of extracorporeal photopheresis in the management of cutaneous t-cell lymphoma, graft-versus-host disease and organ transplant rejection: A consensus statement update from the uk photopheresis society[END_REF], Graft-versus-Host Disease (GVHD) [START_REF] Kitko | Extracorporeal photopheresis in prevention and treatment of acute gvhd[END_REF][START_REF] Radojcic | The role of extracorporeal photopheresis in chronic graft-versus-host disease[END_REF], and various T-cell mediated disorders (organ transplant rejection (4), auto-immune [START_REF] Adamski | Extracorporeal photopheresis for the treatment of autoimmune diseases. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF] or inflammatory diseases [START_REF] Fowler | Extracorporeal photopheresis for the treatment of crohn's disease[END_REF]). Despite its widespread use, ECP mechanisms of action have long been a matter of debate [START_REF] Heshmati | Updating ecp action mechanisms[END_REF]. Indeed, ECP can be considered both as a immunizing or a tolerizing therapy, depending on the pathology considered, and recently, a new organization was funded focused on this unique cell therapy [START_REF] Raval | Extracorporeal photopheresis and personalized medicine in the 21st century: The future's so bright![END_REF].

PUVA treatment (Psoralen -i.e. 8-MOP +UVA irradiation) induces T cell apoptosis. It has been shown that PUVA-treated T lymphocytes -including tumoral, allo-reactive or autoimmune T cells -undergo apoptosis within 24 to 48 hours, with an accelerated kinetics for activated cells [START_REF] Hannani | Photochemotherapy induces a faster apoptosis of alloreactive activated t cells than of nonalloreactive resting t cells in graft versus host disease[END_REF]. During ECP, only a fraction of total circulating leukocytes are treated, and thus only relatively few pathogenic T cells are directly affected, indicating that direct elimination of the apoptotic pathogenic T cells cannot be the sole mechanism responsible for therapeutic efficacy [START_REF] Marshall | Technology insight: Ecp for the treatment of gvhd--can we offer selective immune control without generalized immunosuppression? Nature clinical practice[END_REF]. This treatment nevertheless leads to potent and prolonged clinical responses with dampening of pathogenic T cell activity, suggesting systemic ECP-induced immune-modulation, and highlights the central role of the presence of pathogenic T cells for treatment efficacy.

Several hypotheses have been put forward to explain ECP mechanisms of action: ECP is shown to be tolerogenic in the GVHD or auto-immunity context, while inducing anti-tumor responses in the CTCL context. Monitoring of regulatory T cells frequencies during ECP treatment has been performed in several trials. Some reports describe an increase in circulating Treg percentages over the course of several months during ECP treatment [START_REF] Biagi | Extracorporeal photochemotherapy is accompanied by increasing levels of circulating cd4+cd25+gitr+foxp3+cd62l+ functional regulatory t-cells in patients with graft-versus-host disease[END_REF][START_REF] Rao | Cutaneous t cell lymphoma and graft-versus-host disease: A comparison of in vivo effects of extracorporeal photochemotherapy on foxp3+ regulatory t cells[END_REF][START_REF] Quaglino | Reciprocal modulation of circulating cd4+cd25+bright t cells induced by extracorporeal photochemotherapy in cutaneous t-cell lymphoma and chronic graft-versus-host-disease patients[END_REF][START_REF] Schmitt | Extracorporeal photophoresis augments function of cd4+cd25+foxp3+ regulatory t cells by triggering adenosine production[END_REF], predominantly in the context of GHVD; however, stable or decreasing Treg frequencies were reported in other pathologic contexts [START_REF] Quaglino | Reciprocal modulation of circulating cd4+cd25+bright t cells induced by extracorporeal photochemotherapy in cutaneous t-cell lymphoma and chronic graft-versus-host-disease patients[END_REF][START_REF] Dieterlen | Indication-specific immunomodulatory effects of extracorporeal photopheresis: A pilot study in heart transplanted patients[END_REF]. In most cases, Treg numbers display large inter-individual heterogeneity [START_REF] Dieterlen | Immunological monitoring of extracorporeal photopheresis after heart transplantation[END_REF], and do not necessarily correlate with clinical responses [START_REF] Denney | Regulatory t cells in chronic graft-versus-host disease after extracorporeal photopheresis: Correlation with skin and global organ responses, and ability to taper steroids[END_REF]. In the context of CTCL treatment, it has been suggested that ECP could induce a cytotoxic response directed towards the malignant clone [START_REF] Atta | The role of extracorporeal photopheresis in the treatment of cutaneous t-cell lymphomas. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF]. Whether immunizing or tolerizing, ECP should involve dendritic cells as central antigen-presenting cells [START_REF] Edelson | American council on ecp (ace): Why now?[END_REF]. Using the Therakos device, Edelson et al. found that PUVA-treated monocytes differentiate into dendritic cells (MoDC), via interactions involving platelets, integrins, plasma proteins and the plastic vessel [START_REF] Berger | Rapid generation of maturationally synchronized human dendritic cells: Contribution to the clinical efficacy of extracorporeal photochemotherapy[END_REF][START_REF] Gonzalez | Integrin-driven monocyte to dendritic cell conversion in modified extracorporeal photochemotherapy[END_REF][START_REF] Durazzo | Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: Initiation via direct platelet signaling. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF], these ECP-induced DC being efficient immunizing agent. In a recent study in murine model, it was shown that ECP was able to drive anti-cancer immune response, while using PUVAtreated tumor cells, in combination with untreated PBMC and platelets [START_REF] Ventura | Extracorporeal photochemotherapy drives monocyteto-dendritic cell maturation to induce anticancer immunity[END_REF], mixed together, passed d v d " s z " d cocultured overnight before intreavenous injection to tumor-bearing mice. In this model, treatment efficacy relies on extracorporeal device induced dendritic cells presenting Ag from PUVA-apoptotic malignant cells to prime CD4 and CD8 anti-tumor T lymphocytes. Besides these immunogenic ECP-induced DC, it was also shown that PUVA stimulates GILZ expression on MoDC, and down-regulation of CD80 and CD86 leading to immunosuppressive moDC function [START_REF] Futterleib | Activation of gilz gene by photoactivated 8methoxypsoralen: Potential role of immunoregulatory dendritic cells in extracorporeal photochemotherapy[END_REF], and it could be interesting to consider modification of ECP apparatus to better control this polarization [START_REF] Edelson | American council on ecp (ace): Why now?[END_REF]. In our settings, free of platelets, most PUVA-treated monocytes became apoptotic, with a delayed kinetics compared to T cells, their apoptosis occurring 5 to 6 days following PUVA treatment [START_REF] Hannani | Photochemotherapy induces the apoptosis of monocytes without impairing their function[END_REF]. Moreover, the few surviving monocytes did not differentiate spontaneously into MoDC, while displaying no compromised functionality and in particular without acquiring immunosuppressive properties [START_REF] Hannani | Photochemotherapy induces the apoptosis of monocytes without impairing their function[END_REF].

As a working model, it can be hypothesized that upon phagocytosis of pre-apoptotic PUVA-treated cells, the re-infused APC or the resident APC initiate a specific immunomodulatory program, targeting pathogenic T cells without inducing generalized immunosuppression.

Apoptosis is usually a silent physiological process inducing tolerance [START_REF] Morelli | The immune regulatory effect of apoptotic cells and exosomes on dendritic cells: Its impact on transplantation[END_REF]. During apoptosis, dying s ss " -" s s, w phagocytes. Phosphatidylserine (PS), normally found on the inner leaflet of the plasma membrane, is exposed at the cell surface of apoptotic cells. PS is directly recognized by several receptors, such as stabilin 2, BAI1 and Tim-4, or indirectly by MerKT or v5 integrin [START_REF] Penberthy | Apoptosis and engulfment by bronchial epithelial cells. Implications for allergic airway inflammation[END_REF]. These receptors can cooperate to mediate an immunologically silent removal of dying cells by phagocytes, with secretion of anti-inflammatory cytokines, leading to regulatory T cells differentiation/ expansion and active tolerance induction. Some of these PS receptors belong to the scavenger receptors (SR) family [START_REF] Prabhudas | A consensus definitive classification of scavenger receptors and their roles in health and disease[END_REF][START_REF] Prabhudas | Standardizing scavenger receptor nomenclature[END_REF], which are predominantly expressed by myeloid cells. They mediate endocytosis, phagocytosis, adhesion, or signaling leading to the elimination of non-self or altered-self targets. In addition to their patternrecognition and phagocytic functions, SR also regulate inflammatory signaling. On the other hand, it has become evident that apoptosis can also be immunogenic, depending on death inducers and environmental context [START_REF] Kroemer | Death, danger, and immunity: An infernal trio[END_REF]. For example, anthracyclines, oxaliplatin or gamma-radiation induce the surface expression or secretion of DAMPs (damage-associated molecular patterns), such as calreticulin (CRT), heat shock proteins (HSP), high mobility group box 1 (HMGB1), or ATP. Calreticulin, a chaperone found in the endoplasmic reticulum (ER), rapidly translocates to the plasma membrane following ER stress induced during some forms of apoptosis [START_REF] Obeid | Calreticulin exposure dictates the immunogenicity of cancer cell death[END_REF][START_REF] Zitvogel | Immunogenic tumor cell death for optimal anticancer therapy: The calreticulin exposure pathway. Clinical cancer research : an official journal of the[END_REF]. Early exposure of CRT is a hallmark of Immunogenic Cell Death (ICD). CRT is recognized by CD91 expressed by phagocytes, allowing apoptotic cell phagocytosis. ATP released during apoptosis is recognized by purinergic receptors, activating the NLRP3 inflammasome and stimulating the release of IL-1 and IL-18 by phagocytes [START_REF] Kepp | Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy[END_REF]. The nuclear protein HMGB1 released extracellularly during late apoptosis or necrosis acts as an alarmin, mediating phagocyte chemo-attraction and activation upon its recognition by TLR2, TLR4 and RAGE.

In order to better characterize ECP mechanisms of action, we set up an in vitro model of alloreactive T cells in mixed lymphocyte reactions to mimic alloreactive T cells in GVHD, and analyzed whether in vitro PUVA (ivPUVA) treatment could induce their expression or secretion of ICD signals. We also studied the ability of ivPUVA-treated alloreactive T cells to trigger DC maturation and analyzed the subsequent DC-induced T cell polarization.

2-Materials and methods

Medium and Reagents

Cultures were performed in RPMI 1640 Glutamax supplemented with 1 mmol/L sodium pyruvate, nonessential amino acids, 20 ng/mL gentamycin, 50µM beta-mercapto-ethanol, and 10% heat inactivated fetal calf serum (referred to as complete medium).The 8-Methoxy-psoralen used for ivPUVA treatment was from Sigma-Aldrich.

Immunophenotyping was performed by using antibodies directed towards: CD3, HLA-DR (BD Biosciences), calreticulin, HSP-90 and HSP-70 (AbCam). CD209, CD14, CD40, CD80 and the FITC-AnnexinV and 7AAD kit were purchased from Beckman Coulter.

Flow cytometric experiments and analyses were performed using an 8-color FACSCanto II flow cytometer with the Diva or the Flow Jo software (BD Biosciences).

Cells, mixed lymphocytes reactions and antigen-presenting cell (APC) generation Blood samples were collected from adult healthy volunteers, and patients with extensive chronic GVHD who gave their informed consent in accordance with the Declaration of Helsinki (cell collections declared to the French Health ministry (GRE-DC-2008-787, NCT 00824954, CPP Sud Est I : ref CPP JV-TV/2008-273 authorization). Cord blood not suitable for cord blood banking was obtained from Bourgogne Franche-Comte EFS (GRE-DC-2011-1487). PBMC were isolated by density gradient on lymphocyte separation medium, and cryopreserved until use, by standard freezing methods (37.5% FCS, 10% Dimethyl sulfoxide). T cells and monocytes enrichment cocktails EasySep kit (Stem Cell Technologies Inc) were used for cell separation.

Mixed lymphocyte reaction (MLR), a simple and efficient in vitro model for the study of T-cell activation and proliferation, was used to obtain alloreactive-activated T lymphocytes. PBMC from healthy donors were co-cultured during 6 days with irradiated (-irradiation, 30Gy) allogeneic PBMC (1:1 ratio), 96-round bottomed plates. Dead cells were removed by density gradient on lymphocyte separation medium. CD25 was expressed by less than 10% of T cells purified from PBMC, whereas it was expressed by more than 50% of the T cells after MLR (data not shown). Therefore we considered purified T cells extracted from fresh PBMC as 'resting T cell', whereas T cells obtained after MLR were considered as 'alloreactive activated T cells'. Activated T cells were purified after the MLR with EasySep kit (Stem Cell Technologies Inc).

Monocyte-derived dendritic cells (MoDC) were generated by culturing monocytes in complete medium supplemented with 10% FCS in presence of GM-CSF (500 U/mL, Miltenyi) and IL-4 (10 ng/mL, Miltenyi) for 6 days. IL-4 was added at day 2 and 5 of culture, and MoDC were harvested at day 7. Macrophages were generated by a 7-day culture of monocytes with GM-CSF (500 U/mL). These cultures were performed in low attachment 25cm 2 flasks (Corning).

In vitro PUVA (ivPUVA) Treatment PBMC or purified T cells were seeded at 1.10 6 cells/mL in complete medium, in 24-well culture plates. Cells were incubated for 15 min at 37°C with 200 ng/mL of 8-MOP and exposed to 2 J/cm 2 365-nm UVA radiation (Bio Sun, Vilbert-Lourmat), as previously described [START_REF] Hannani | Photochemotherapy induces a faster apoptosis of alloreactive activated t cells than of nonalloreactive resting t cells in graft versus host disease[END_REF][START_REF] Hannani | Photochemotherapy induces the apoptosis of monocytes without impairing their function[END_REF]. After ivPUVA treatment, cells were washed and cultured in complete medium at 37°C in 5% CO2 atmosphere.

Calreticulin and HSP expression

Flow cytometry was used to measure surface expression of calreticulin, HSP-70 and HSP-90. Cells were gated on CD3 + 7-AAD negative cells to focus on T lymphocytes that retained their membrane integrity.

HMGB1, ATP and cytokine secretion assays

Culture supernatants were harvested and stored at -20°C. HMGB1 was measured by ELISA (IBL international). ATP content was measured with Bioluminescent somatic cell assay kit (Sigma-Aldrich). Cytokines (IL-6, IL-8, IL-10, TNF, IL-12p70, IL-1, IL-4, IL-17, or IFN) were quantified in culture supernatants by cytometric bead array (CBA, BD Biosciences).

Phagocytosis and MoDC maturation assay

For phagocytosis, APC (macrophages or MoDC) were incubated for 1 to 4 hours at 37°C or 4°C with CFSE (Life technologies)-labeled purified activated or resting T lymphocytes (ratio 1:1, culture tubes, Falcon), treated or not by ivPUVA. Phagocytosis was stopped (washing at 4°C in PBS), APC were labeled (MoDC with CD209, and macrophages with CD14), T cells with BV421-CD3. After washing, CFSE fluorescence was analyzed on gated APC. CD3-positive APC were excluded from the analysis as they had likely formed conjugates with T cells but had not necessarily internalized cellular material, as previously described by Lui et al. [START_REF] Lui | Preparation of purified lymphoma cells suitable for therapy[END_REF].

MoDC maturation was measured after 24h and 48h of coculture with purified activated or resting T lymphocytes (ratio 1:1), treated or not by ivPUVA. Poly I:C (25µg/ml, Invivogen) was used as positive control. Cells were harvested, and stained with CD209, CD40 and CD80 antibodies. Mean fluorescence intensity of CD40 and CD80 was quantified on gated CD209+ cells, and expressed as fold increase in comparison with MoDC incubated in medium alone.

Naive T cell proliferation and polarization

Purified activated or resting T lymphocytes were treated or not by ivPUVA, and cultured for 24h to allow DAMPs expression and release. Autologous MoDC were then added (5,000 cells of each subset in 100µL, 96-well round bottomed plates) for 24h to allow MoDC maturation. Purified allogeneic CD4+ naive T cells from cord blood were then added to these co-cultures (50,000 naive T cell/well, APC: naive T cell ratio = 1:10, final culture volume : 200µL,). Proliferation was measured after 6 days by 3 H-Thymidine incorporation (addition of 1µCi 3 H-Thymidine (PerkinElmer) during the last 18 hours of culture). In another setting, cells were stimulated at day 6 for 5 hours with PMA (5ng/mL) and ionomycin (5µg/mL) (both from Sigma Aldrich), and supernatants were harvested to measure their cytokine contents.

Statistical analysis

Data are presented in percentages, concentrations or fold changes, with mean and standard deviation shown. All statistical analyses were performed with the GraphPad prism software. Mann and Whitney U test, or ANOVA analysis was used, followed by Tukey multiple comparison tests to compare the different groups. Significance level was set at p<0.05 (*), p<0.01 (**) or p<0.001 (***).

3-Results

ivPUVA induces apoptosis in resting and allo-reactive activated T cells

In order to partially recapitulate the GVHD situation, mixed lymphocyte reactions (MLR) were used to generate activated alloreactive T cells in vitro. Apoptosis was measured on CD3+ T lymphocytes, 24 and 48 hours after ivPUVA treatment of resting or activated cells. Following ivPUVA treatment, resting and activated T cells exposed PS (Annexin-V labeling), and lost membrane integrity (7-AAD incorporation), demonstrating the induction of apoptotic cell death (Figure 1A). After a 24hincubation, 29% and 71% of resting and activated T cells died (Annexin-V positive, and/or 7AAD positive). These percentages increased at 48h, reaching 46 and 90%, respectively (Figure 1B). The death kinetics was more rapid, and the percentage of apoptosis was statistically higher (Annova test, p<0.01, Tuckey post-test) for ivPUVA-treated activated alloreactive T cells (i.e. 'pathogenic' T cells) compared to ivPUVA-treated resting T cells, recapitulating the T cell behavior observed in GVHD patients [START_REF] Hannani | Photochemotherapy induces a faster apoptosis of alloreactive activated t cells than of nonalloreactive resting t cells in graft versus host disease[END_REF], and validating MLR-activated alloreactive T cells as an in vitro model of GVHD.

ivPUVA induces DAMPs expression in human alloreactive and tumoral T cells

To explore the immunogenicity of apoptotic ivPUVA-treated cells, DAMPs expression and secretion were analyzed. Resting and alloreactive activated cells were treated by ivPUVA, and the expression of calreticulin was measured by flow cytometry 24 and 48 hours post-treatment (Figure 2A). Calreticulin exposure was analyzed only on T cells which retained their membrane integrity (7-AAD negative cells). Indeed, most 7AAD+ cells appeared CRT positive, but in this case it is not possible to differentiate external or intracellular expression (not shown). In resting 7AAD-negative T cells, 9% of cells expressed calreticulin 24h following ivPUVA treatment, and 15% after 48h. A significantly higher percentage of calreticulin expression was measured on activated T cells following ivPUVA treatment: 14% and 27% of calreticulin expressing cells were detected at 24 and 48 hours, respectively (Figure 2B, p<0.01, Mann and Whitney U test). Of note, CRT was not detected on T cells 5 hours after ivPUVA treatment (not shown).

As comparison, ivPUVA-treated samples from two GVHD patients were analyzed, and HLA-DR expression was used to identify activated (alloreactive) T cells (Figure 3A,B). A slightly higher percentage of calreticulin-expressing T cells was detected on activated (45 and 59%) compared to resting cells (31 and 47%). In another pathological context, we found that ivPUVA-treated T cells from 3 CTCL patients also up-regulated calreticulin (9 to 37%) 24 hours after ivPUVA treatment (Supplementary figure 1A,B). HMGB1 was measured in the supernatant of resting or alloreactive activated cells following ivPUVA treatment. HMGB1 was detected in resting cell supernatants after 24h (mean: 26 ng/ml), and 48 hours (29 ng/ml) post-treatment (Figure 2C). The release of HMGB1 following ivPUVA-treatment was higher in the supernatant of alloreactive-activated cells (35 and 54 ng/ml, after 24 and 48 hours, respectively). Both HSP-70 and HSP-90 were induced on activated T cells 48 hours following ivPUVAtreatment (as measured by flow cytometry analysis of surface expression-supplementary Figure 2), but ATP (measured by a bioluminescence assay and HPLC), was undetectable in all samples (data not shown).

Altogether, ivPUVA-treatment induces DAMPs expression (CRT and HSP70 and 90) or release (HMGB1) by T cells in different contexts, and at higher levels when treated T cells are activated (i.e. pathogenic alloreactive T cells).

Antigen-presenting cells phagocytize ivPUVA-treated, dying cells and partially mature

The capacity of APC (macrophages and DC differentiated from monocytes) to phagocytize resting and activated ivPUVA-treated cells was evaluated by flow cytometry (Figure 4A,B). Phagocytosis was measured from 30 min to 4 hours, and determined by the percentage of CFSE positive cells (T cells were stained by CFSE prior to the assay) among CD3-negative CD14-or CD209-positive APC (to exclude APC and T cell conjugates). After 4 hours, 20% and 42% of the macrophages had phagocytized resting T cells and activated T cells, respectively (Figure 4C), indicating that macrophages engulfed alloreactive-activated ivPUVA-treated T cells more efficiently and more rapidly than resting T cells. A similar result was obtained when using MoDC (Figure 4D), with 12% of ivPUVA-treated resting T cell phagocytosis observed after 4 hours, compared to 39% for alloreactive activated T cells. Microscopic examination of the APC showed that whole T cells were engulfed, ruling out transfer of microvesicles or trogocytosis (not depicted). The more efficient phagocytosis of activated compared to resting T cells may be related to the observed differential CRT exposure by these ivPUVA-treated T cells.

The capacity of ivPUVA-treated cells to induce MoDC maturation was evaluated by measuring CD40 and CD80 on MoDC following 48-hour co-cultures. Resting T cells, treated or not by ivPUVA, did not induce MoDC maturation. Conversely, activated T cells induced an up-regulation of CD40 and CD80 expression on MoDC (2.5 and 1.5 fold increase, respectively), however ivPUVA-treated, activated T cells did not trigger DC maturation. Furthermore, the secretion of inflammatory cytokines (IL-12, IL-6, TNF-) was not altered compared to DC alone, and IL-10 secretion induced by incubation with activated T cells was abrogated when the T cells were ivPUVA-treated (supplementary Figure 3).

ivPUVA-induced dying cells do not modulate naive T cells proliferation or polarization

The ability of MoDC to stimulate and polarize naive T cells from allogeneic cord blood was measured after pre-incubation with autologous ivPUVA-treated resting or activated T cells (ratio 1:1, 24h preincubation) (Figure 5A). MoDC alone efficiently induced the proliferation of naive T cells (mean: 56,782cpm). When MoDC were pre-incubated with resting T cells, whether treated or not by ivPUVA, proliferation of allogeneic naive T cell was similar to the proliferation observed in the control condition (medium= MoDC alone i.e. not pre-incubated with any T cells). In line with the upregulation of costimulatory molecules on MoDC induced by activated T cells, a higher proliferation of naive T cells was induced in this condition (85,023cpm); however, MoDC pre-incubated with ivPUVAtreated, activated T cells induced a comparable T cell proliferation as in the control condition (Figure 5B). Naive T cell polarization was measured upon co-culture with MoDC pre-incubated with resting or activated ivPUVA-treated, or non-treated T cells. Increased levels of TNF, IFN and IL-4 were detected in the supernatants of co-cultures with MoDC and untreated resting or activated T cells. However, no such secretion was observed when T cells were treated by ivPUVA. Similar levels of IL-10 were detected in all co-cultures, albeit heterogeneously (Figure 5C).

4-Discussion

Photopheresis, a safe and efficient therapy initially used to treat CTCL, has been approved for several T cell-mediated diseases, including GVHD, solid organ transplant rejection and some auto-immune disorders. However the precise mechanisms of action of ECP remain elusive, hampering its wider implementation. ECP is particularly interesting in that it is not a global immunosuppressive therapy, s v s' [START_REF] Suchin | Extracorporeal photochemotherapy does not suppress t-or b-cell responses to novel or recall antigens[END_REF]. This study aimed at dissecting the interaction between ECP-treated T cells and APC, and in particular it evaluated whether immunogenic features of cell death are induced upon ivPUVA treatment, potentially leading to a specific immunomodulatory response directed toward pathogenic T cells [START_REF] Hannani | Extracorporeal photopheresis: Tolerogenic or immunogenic cell death? Beyond current dogma[END_REF]. Apoptotic cell death accompanied by Calreticulin exposure, HMGB1 release and ATP secretion has been described as immunogenic, eliciting dendritic cell maturation and Th1 polarized adaptive immune responses. Such phenomenon has been mostly described in murine models, where cancer cell lines treated by doxorubicin, mitoxantrone or photodynamic therapy (PDT) were shown to be immunogenic.

We used mixed lymphocyte reactions to generate alloreactive T lymphocytes, as in these cultures thousands of alloreactive CD4+ and CD8+ T cell clones are amplified [START_REF] Emerson | Defining the alloreactive t cell repertoire using high-throughput sequencing of mixed lymphocyte reaction culture[END_REF], generating large quantities of alloreactive T cells. In line with our hypothesis, we found that ivPUVA-treated T cells exposed CRT at their surface. CRT exposure was detected at the surface of early apoptotic cells following ivPUVA treatment in several models (primary T cells, alloreactive activated T cells from GVHD patients or in vitro-generated alloreactive T cells, and CTCL cells). CRT expression by human cells in the context of ICD has been reported on a melanoma cell line following infection with oncolytic vaccinia viruses [START_REF] Heinrich | Immunogenicity of oncolytic vaccinia viruses jx-gfp and tg6002 in a human melanoma in vitro model: Studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic t lymphocytes[END_REF], and on a bladder carcinoma cell line following hypericin-based photodynamic therapy [START_REF] Garg | Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like hsp70 and calreticulin[END_REF][START_REF] Garg | A novel pathway combining calreticulin exposure and atp secretion in immunogenic cancer cell death[END_REF]. CRT exposure at the surface of human acute myeloid leukemia blasts has also been correlated to enhanced autologous T cell responses [START_REF] Wemeau | Calreticulin exposure on malignant blasts predicts a cellular anticancer immune response in patients with acute myeloid leukemia[END_REF], independently of chemotherapy treatment. Detection of CRT at the surface of various kinds of ivPUVA-treated T-cells is very interesting and intriguing, and to our knowledge it is the first evidence of CRT expression on pre-apoptotic non-malignant human primary cells. Upon ivPUVA treatment, calreticulin exposure was accompanied by HMGB1 release, and both DAMPs were detected at higher levels with alloreactive-activated T cells than with resting T cells. It is worth noting that in our in vitro allogeneic model non-treated activated T cells expressed higher levels of CRT than resting T cells, as well as for HLA-DR positive cells from GVHD patients, thus confirming the expression of CRT at the surface of activated T cells [START_REF] Arosa | Calreticulin is expressed on the cell surface of activated human peripheral blood t lymphocytes in association with major histocompatibility complex class i molecules[END_REF]. In our hands, the supernatant of ivPUVA-treated cells did not contain ATP. This cannot be explained by ATP degradation by ectonucleotidases such as CD73 or CD39 (43) since we did not detect any ADP or AMP in ivPUVA-treated cells supernatants (not shown). In hypericin-based PDT, CRT exposure and HMGB1 release were accompanied with ATP secretion by dying cells [START_REF] Garg | A novel pathway combining calreticulin exposure and atp secretion in immunogenic cancer cell death[END_REF]; on the contrary, ATP secretion was not detected in the oncolytic virus-induced cell death model [START_REF] Heinrich | Immunogenicity of oncolytic vaccinia viruses jx-gfp and tg6002 in a human melanoma in vitro model: Studying immunogenic cell death, dendritic cell maturation and interaction with cytotoxic t lymphocytes[END_REF], despite dendritic cell maturation being induced in both instances.

Since ivPUVA-treatment of T cells induced CRT expression at the surface of dying cells, we asked whether APC could phagocytize ivPUVA-treated T cells. PUVA-treated cells re-infused to the patient first go into the lungs, before relocating to the spleen and liver (44), hence we focused our analysis on phagocytosis by the tissue-resident macrophages and DC, and used in vitro culture of monocytes with GM-CSF or GM-CSF+IL4 as in vitro model for these APC. For the first time, we provide evidence of ivPUVA-treated cell phagocytosis by macrophages and DC. Both phagocytes engulf ECP-induced apoptotic cells with a similar kinetics and efficacy. Interestingly, following ivPUVA-treatment, phagocytosis of activated compared to resting T cells was higher, in correlation with their higher level of CRT expression. Phagocytosis was highly efficient, and such efficient phagocytosis by DC has also been described for hypericin-PDT treated murine cells, and was mediated partially by CRT exposure [START_REF] Garg | Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like hsp70 and calreticulin[END_REF].

In our model, despite expression of several DAMPs by dying cells, DC co-cultured with ivPUVAtreated cells did not mature. This result is in agreement with the observations published by Lamioni et al, who found that DC in contact with ECP-treated PBMC from patients did not up-regulate costimulatory molecule expression, but rather up-regulated HLA-DR molecules and displayed a tolerogenic phenotype [START_REF] Lamioni | The immunological effects of extracorporeal photopheresis unraveled: Induction of tolerogenic dendritic cells in vitro and regulatory t cells in vivo[END_REF]. While remaining in an immature or semi-mature state, DC co-cultured with ivPUVA-treated cells did not secrete significantly different levels of inflammatory or antiinflammatory cytokines compared to immature DC. The absence of IL-1 secretion (not shown) is in line with the lack of ATP release by dying cells, as in the context of ICD extracellular ATP is a key factor promoting the release of IL-1 and IL-18 by APC [START_REF] Kepp | Consensus guidelines for the detection of immunogenic cell death[END_REF]. It is noteworthy that DC in contact with live activated T cells up-regulate CD40 and CD86, a phenomenon that could be related to DC licensing by, among others, members of the TNF family and IFN secretion by T cells [START_REF] Summers Deluca | Fine-tuning of dendritic cell biology by the tnf superfamily[END_REF]. Strikingly, such up-regulation did not occur anymore when activated T cells were treated by ivPUVA, and despite HMGB1 release, these cells did not induce DC maturation.

In line with the absence of DC maturation induced by contact with ivPUVA-treated cells, there was no difference in the proliferation of naive T lymphocytes in allogeneic MLR induced by immature DC, and DC pre-incubated with ivPUVA-treated T lymphocytes, whether activated or not. Neither was there a difference in T cell polarization (Th1, Th2 or Treg) when measured at the MLR endpoint. Here again, these data indicate that apoptosis induced by ivPUVA treatment impaired the alloreactive activated T cell ability to prime DC for T cell activation. One limit of our study may be the use of monocyte-derived dendritic cells. Indeed, DCs comprise several subsets, with different roles in the presentation of antigens derived from pathogens, vaccines, and self-tissues. Monocytes are able to differentiate into DC when properly stimulated in vitro, and monocyte-derived-dendritic cells obtained after culture with GM-CSF and IL-4 are often used as surrogate for dendritic cells since they can be easily generated in large numbers [START_REF] Sallusto | Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha[END_REF]. They may represent the equivalent of the inflammatory dendritic cells identified in mice [START_REF] Boltjes | Human dendritic cell functional specialization in steady-state and inflammation[END_REF], and may present different functionality compared to conventional myeloid or plasmacytoid dendritic cells. These GM-CSF+IL-4 moDC could be different from the platelet-induced dendritic cells derived from monocytes, as observed in the ECP Transimmune device [START_REF] Durazzo | Induction of monocyte-to-dendritic cell maturation by extracorporeal photochemotherapy: Initiation via direct platelet signaling. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF]. The in vitro model we chose here recapitulates the main steps of the process, but may not reflect all aspects of ECP as performed in the clinics.

It may be interesting to evaluate in an in vivo setting where and when re-injected PUVA-treated T cells encounter APC, as only a narrow window of time may exist during which PUVA-activated pathogenic T cells can cross-talk with APC and induce their maturation, while at the same time expressing eat-me signals allowing their capture by these APC [START_REF] Hannani | Extracorporeal photopheresis: Tolerogenic or immunogenic cell death? Beyond current dogma[END_REF]. In the experimental transimmunization device developed by Edelson et al., ECP efficacy relies on extracorporealdifferentiated DC that are incubated overnight with PUVA-treated malignant cells, allowing the capture, processing and presentation of tumor-Ag to T cells, a device that seems attractive for improving ECP process. In our in vitro setting, the observed partial release of DAMPs by ivPUVAtreated cells fails to induce DC maturation; however, during ECP procedure as it is performed in clinics, it would be very interesting to decipher which are the APC involved in T cell priming, since they could originate from ECP-treated monocytes, or be the non-treated resident APC rendered immunogenic by the inflammatory milieu (due to systemic disease-associated immune activation).

In conclusion, we show here that ivPUVA-treatment of T lymphocytes induces the expression of CRT and the secretion of HMGB1 by dying cells, but no secretion of ATP. This unique apoptotic phenotype promotes very efficient phagocytosis of apoptotic cells by macrophages and DC but is not followed by DC maturation and T cell activation in our experimental settings. Further in vivo evaluations are still necessary to decipher the precise mechanisms of action of ECP, preferably in an inflammatory context recapitulating the clinical situation.

Additional supporting information

Additional supporting information are found in the supplementary figure file (pdf)

  , heat shock proteins 70 and 90 ivPUVA, in vitro treatment of cells with 8 methoxy-psoralen plus ultra violet A irradiation MLR, Mixed lymphocyte reactions MoDC , monocyte-derived dendritic cell PBMC, peripheral blood mononuclear cells PDT , photodynamic therapy PS, Phosphatidylserine PUVA, 8 methoxy-psoralen plus ultra violet A irradiation SR, scavenger receptors Treg , regulatory T cells

Figure 1 :

 1 Figure 1: PUVA-induced resting and activated T cell apoptosisThe effect of PUVA treatment on the apoptosis of resting and alloreactive-activated T lymphocytes was measured by flow cytometry. A) Example of AnnexinV/7AAD analysis gated on CD3+ T lymphocytes, for non-treated (NT) or PUVA-treated alloreactive activated T cells. In this setting, early apoptotic cells are AnnV+/7AADneg, late apoptotic cells are 7AAD+ cells. B) Mean and standard deviation of the percentages of dead cells (7AAD+ and/or AnnV+ cells) measured after 24 and 48h hours in 22 (resting cells) and 16 (alloreactive-activated cells) independent experiments (healthy v s ' s).

Figure 2 :

 2 Figure 2: Calreticulin expression and HMGB1 release induced by PUVA-treatment of T cellsThe surface expression of calreticulin was measured by flow cytometry, on resting and alloreactiveactivated T lymphocytes, after gating on 7AAD negative cells that retained their membrane integrity. A) Example of CRT expression on non-treated (NT, dark histogram) and PUVA-treated (light grey histogram) alloreactive activated T cells. B) Analysis of surface calreticulin expression on resting and activated T lymphocytes treated or not with PUVA and incubated for 24or 48 hours. C) HMGB1 was measured by ELISA in supernatants from cultures of resting and activated T lymphocytes treated or not with PUVA and incubated for 24 or 48 hours. In B and D, data represent mean+/-SD of n=29 (CRT, 24h), n=18 (CRT, 48h), n=13 (HMGB1, 24h), n=24 (HMGB1, resting cells, 48h), and n=9 (HMGB1, activated cells, 48h) experiments. Statistical differences between the groups were analyzed by Annova.

Figure 3 :

 3 Figure 3: Calreticulin expression upon PUVA treatment of PBMC from GVHD patients PBMC from patients with GVHD were treated or not with PUVA, and incubated for 24h, before measurement of calreticulin expression at the surface of 7AAD negative T cells. A and B) calreticulin expression at the surface of activated (HLA-DR+) and non-activated (HLA-DRneg) T lymphocytes from GVHD patient PBMC. Light grey graphs represent NT sample, and dark graphs represent PUVA treated samples.

Figure 4 :

 4 Figure 4: Phagocytosis by Macrophages and MoDC of PUVA-treated T cells; maturation of MoDC Resting and activated T cells were PUVA-treated, incubated 24h, and then stained with CFSE before being mixed with APC for phagocytosis assay. After incubation, cells were harvested, and phagocytosis was determined as the percentage of CFSE positive cells among CD14 (macrophages) or CD209 (MoDC) positive CD3 negative cells (to exclude APC-T cell conjugates). A and B) representative experiment (1 hour incubation), and C and D) mean of 5 and 6 independent experiments with macrophages (A, C) and MoDC (B, D), respectively. Significant differences between resting and activated PUVA-treated T cell phagocytosis are indicated by stars (Annova). MoDC maturation was quantified my measuring the mean fluorescence intensity of CD40 and CD80 after a 24-hour incubation in the same conditions. E) Fold increase of CD40 and CD80 mean fluorescent intensities normalized to the control condition (MoDC in medium). Bars represent mean+/-SD of 9 experiments. Statistical differences between the groups were analyzed by Annova.

Figure 5 :

 5 Figure 5: Proliferation and polarization of naive T cells induced by MoDC in contact with PUVA-treated T cellsPurified resting and activated T cells were PUVA-treated, incubated 24h, and mixed with autologous MoDC for 24h before addition of allogeneic naive CD4+ T cells, as depicted in A. B) 3H-Thymidine was added for 18h at the end of 6-day incubation, to measure cell proliferation. Negative control: MoDC preincubated with medium; positive control: MoDC preincubated with PolyI:C) C) T cell polarization was evaluated by measuring cytokines (cytometric bead array) secreted in culture supernatants after a restimulation with PMA and Ionomycine. Bars represent mean+/-SD of 4 experiments (B) and 8 experiments (C). Statistical differences between the groups were analyzed by Annova.

Acknowledgements

The authors would like to thank all healthy volunteers and patients for participation in the study, and staff in EFS in Grenoble, Valence, St Ismier and Besançon, who helped our research. We also thank Olivier Manches for correcting our manuscript. This work benefited from support of the French Government (ANRT) with CIFRE funding. This project was supported by Etablissement Français du Sang APR 2013 and Agence de Biomédecine grants.

This work benefited from support of the French Government (ANRT) with CIFRE funding. This project was supported by Etablissement Français du Sang APR 2013 and Agence de Biomédecine grants.


Céline Coppard
email: celine.coppard@hotmail.com

Francis Bonnefoy
email: francis.bonnefoy@efs.sante.fr

Dalil Hannani
email: dalil.hannani@univ-grenoble-alpes.fr

Françoise M Gabert
email: francoise.gabert@ujf-grenoble.fr

Olivier Manches
email: olivier.manches@efs.sante.fr

Joël Plumas
email: j.plumas@pdc-line-pharma.com

Sylvain Perruche
email: sylvain.perruche@efs.sante.fr

Laurence Chaperot
email: laurence.chaperot@efs.sante.fr

Joel Plumas

Photopheresis efficacy in the treatment of rheumatoid arthritis: a pre-clinical proof of concept

Keywords: Collagen-induced arthritis, extracorporeal photopheresis, preclinical study, autoimmunity

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

contained within the treated sample. These data represent a pre-clinical proof of concept supporting the use of ECP in the treatment of RA in Human.

Background

Among autoimmune diseases, rheumatoid arthritis (RA)(1) is a complex common autoimmune disease responsible for progressive disabilities due to synovial inflammation, bone and cartilage destruction associated with systemic disorders. Rheumatoid arthritis development involves environmental factors that trigger the disease in individuals with a predisposing genotype, such as some HLA DR polymorphisms, and it affects 0.5 to 1% of Caucasian individuals in western countries.

During the course of RA, immune cells infiltrate the synovial sublining, half of them being CD4+ memory T cells, and it has been shown that autoreactive T cell frequency is correlated with disease activity [START_REF] Scally | A molecular basis for the association of the HLA-DRB1 locus, citrullination, and rheumatoid arthritis[END_REF]. Type 1 helper T (Th1) cells have long been considered as crucial in RA development, but accumulating evidence support a major role for Th17 cells in RA pathogenesis. Despite major advances in rheumatoid arthritis outcome, not all patients achieve remission, and there is still an unmet need for new therapeutic approaches [START_REF] Smolen | Rheumatoid arthritis[END_REF].

Few clinical trials have assessed the therapeutic effect of extracorporeal photopheresis (ECP) in rheumatoid arthritis. In 1991, 7 patients with RA were treated by ECP, and interesting clinical improvements were reported for 4 of them [START_REF] Malawista | Photopheresis for rheumatoid arthritis[END_REF]. clinical improvements were reported in 1993 for seven ECP-treated RA-patients [START_REF] Hilliquin | Treatment of refractory rheumatoid polyarthritis by extracorporeal photochemotherapy[END_REF], and in 1996, for 12 patients with psoriatic arthritis [START_REF] Vahlquist | Treatment of psoriatic arthritis with extracorporeal photochemotherapy and conventional psoralen-ultraviolet A irradiation[END_REF]. Despite these encouraging results (overall clinical improvement for approximately 50% of patients [START_REF] Kuzmina | Extracorporeal photopheresis as a therapy for autoimmune diseases[END_REF]), the clinical use of ECP for RA and autoimmune diseases treatment remains rare. Indeed, ECP is a cell therapy mostly applied to treat graft versus host diseases, transplant rejection and cutaneous T cell lymphomas. The therapeutic process is based on extraction of mononuclear cells by apheresis, followed by treatment of cells with 8-Methoxy-psoralen (8-MOP) and exposure to ultraviolet A light. This procedure results in crosslinking of DNA pyrimidine bases in all treated cells, leading to their apoptosis. The cells are then reinfused to the patients. Depending on the disease, ECP is thought to trigger an immunomodulation either leading to immunization (in CTCL context) or immunosuppression (in GVHD or transplant rejection) [START_REF] Knobler | Extracorporeal photopheresis: past, present, and future[END_REF][START_REF] Ratcliffe | National Institutes of Health State of the Science Symposium in Therapeutic Apheresis: scientific opportunities in extracorporeal photopheresis[END_REF][START_REF] Plumas | Mechanisms of action of extracorporeal photochemotherapy in the control of GVHD: involvement of dendritic cells[END_REF].

The American council on ECP has recently published a consensus report, describing ECP as a bidirectional therapy, able to induce both immunizing and tolerizing effects [START_REF] Edelson | American council on ECP (ACE): Why now?[END_REF]. ECP seems to be a safe and efficient treatment for diseases that are associated to T cell dysregulations, leading to specific long-lasting immunosuppression, making it attractive to treat autoimmune diseases [START_REF] Adamski | Extracorporeal photopheresis for the treatment of autoimmune diseases. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF]. The central role of T cells in RA pathogenesis, with circulating autoreactive T cells is a strong argument to propose ECP as a therapy for this disease. The lack of randomized clinical trials and the poor understanding of ECP mechanism of action clearly hinder ECP development in the context of autoimmune diseases. In order to evaluate the potential efficacy of ECP in arthritis, we used a well characterized mouse model of collagen-induced arthritis, extensively used for modeling human RA.

In this paper, we show that ECP is efficient in reversing arthritis, by decreasing Th17 cells. These data represent a pre-clinical proof of concept, rationalizing the use of ECP in the treatment of RA, and provide a relevant study model for dissecting ECP mechanism of action in autoimmune diseases.

Material and methods

Mice

Six to eight week old male DBA/1 mice were obtained from Janvier Labs (Le Genest-Saint-Isle, 2015061815254659. These animal experiments comply with the ARRIVE guidelines [START_REF] Kilkenny | Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research[END_REF]. Some of the experiments have been performed at the UMR1098 animal facility (agreement #C25-056-7; Besançon, France) under #02831 project number authorization.

Induction of collagen-induced arthritis

DBA/1 mice were immunized by subcutaneous injection at the tail base with 100µl of emulsion of bovine collagen type II (200mg/ml dissolved in 0.5M of acetic acid, MD Bioscience) and Complete Freund adjuvant (4mg of Mycobacterium tuberculosis toxin in 1 ml of incomplete Freund adjuvant, Sigma-Aldrich), 50µl in each side of the tail. Arthritis developed at days 21-28 after collagen immunization. The health status of the animals was carefully monitored, by daily observation of animal behavior and external physical appearance. Animals were also weighed every day. The protocol dictated that mice losing more than 20% of start weight at any time would be sacrificed, however this did not occur. No unexpected adverse event occurred. Arthritis severity was measured by visual evaluation of the paws. Each paw was scale of 0-4, where 0 = normal paw, 1= swelling in one digit, 2= swelling of one or more digit or mild swelling of the entire paw, 3= moderate erythema and swelling of the entire paw, and 4= erythema and severe swelling involving the entire paw (fig 1A). The clinical score for each mouse was the result of the sum of the four paws (maximum score [START_REF] Webb | Prevention and amelioration of collagen-induced arthritis by blockade of the CD28 co-stimulatory pathway: requirement for both B7-1 and B7-2[END_REF]. Food and water were placed directly in the cage for animals with a score higher than 8, to compensate for their mobility impairment. To take into account disease heterogeneity at the time of treatment, mice were stratified according to arthritis score and split into the different treatments groups (n=5 mice per group). The sample size was calculated on the basis of pilot experiments, indicating that in untreated animals the mean arthritis score at day 40 post-collagen injection was 9.6 (SD=0.88). Power calculations indicated that for a mean difference in arthritis score of 2 points to be detected with 90% power at this time point with a one-sided significance level of 5%, 5 mice in each treated group were required (calculation made with R software, epi R package). The average and standard deviation of clinical scores were similar for each group in each experiment (n=5). To standardize the monitoring of the therapeutic effect of ECP on arthritis development, we calculated for each mouse and each day a relative CIA score, by subtracting the value of CIA score at the beginning of the treatment to the measured CIA score. Hence, on day 0, when the first injection was performed, all mice have a relative CIA score of zero. Arthritis score was measured every day, and the relative arthritis score was calculated. If arthritis progressed, the calculated CIA score was positive, and if arthritis regressed the relative CIA score was below zero.

Induction of apoptotis by PUVA treatment

Spleens of the donor mice (arthritic or healthy mice) were harvested and mechanically dissociated.

Erythrocytes were removed with a red blood cell lysis solution (eBiosciences). Spleen cells (10.10⁶ cells/ml) were treated by PUVA during 15 minutes at 37°C with 8-MOP (200ng/ml, Sigma-Aldrich) and irradiated with UV-A (365nm, 2J/cm²). Cells were washed twice with PBS. Irradiated cells (PUVA cells) were reinjected intravenously (10.10⁶ cells in 200µl of PBS) three times every other day. Treatment was initiated when the mean arthritic score reached 7. Mice receiving arthritic mice derived PUVA treated-spleen cells were called « Arthritic PUVA » (A-PUVA) mice while mice receiving healthy mice derived PUVA treated-spleen cells were called « Healthy PUVA » (H-PUVA) mice.

Flow Cytometry analysis

Apoptotic PUVA treated-T cells were stained by anti-mouse CD3-PE, Annexin V and 7AAD (AnnV-FITC/7AAD kit, Beckman Coulter) and measured 5 hours after the treatment by flow cytometry. The percentage of apoptotic T cells includes early (AnnV+/7ADD-) and late (7AAD+) apoptotic T cells. For ex vivo T cell analysis, following mice harvest, spleens were collected and mechanically dissociated.

Spleen cells have been homogenized and T cells were stimulated for 6 hours with PMA (50ng/ml)/Ionomycin (1µg/ml) (Sigma) in presence of monensin (protein secretion inhibitor BD Golgistop, BD biosciences). T cells were stained extracellularly by CD3 AF488 (17A2, Biolegend ), CD4-BV421 (GK1.5, Biolegend) and CD25-PECy7 (PC61.5, eBiosciences), and after fixation and permeabilization (Foxp3 Transcription Factor Staining Buffer, Invitrogen or Cytofix/cytoperm plus, BD Biosciences) , intracellular cytokines and transcription factor (IL-17A-APC (TC11-18H10.1, Biolegend), IFN-γ-PE (XMG1.2, BD Biosciences) and FoxP3-PE (FJK-16s, eBiosciences)) were stained.

Cytokine measurement

Sera of the mice were obtained after retro-orbital sampling and centrifugation (2000g, 10 min).

Cytokines (IL-2, -4, -6, -10, -17A, IFN-γ and TNF-α) were quantified by cytometric bead array (CBA Mouse Th1/Th2/Th17 Cytokine Kit, BD Biosciences). Quantification of cytokines was performed by FACS Canto II cytometer (BD).

Statistical analysis

Statistical analyses were performed by Prism6 software (GraphPad; La Jolla, CA, USA). Statistical significance was determined by indicated adequate tests (Mann Whitney to compare two groups or Kruskal-Wallis test with Dunns post-test for more than two groups). To compare the different curves, a nonlinear regression was performed, and a best fit second order polynomial equation was determined for each curve (y=B0 + B1*x + B2*x 2 ). These curves were then compared using two methods: the extra sum-of-squares F test, and Akaike's information criteria. In addition, curves fitted according to the 4-parameter Baranyi model were compared using Bayesian analysis, as implemented in the function Bayescompare from the R TM software package babar (Lydia Rickett, Matthew Hartley, Richard Morris and Nick Pullen (2015). babar: Bayesian Bacterial Growth Curve Analysis in R. R package version 1.0. https://CRAN.R-project.org/package=babar) . The competing hypotheses were: curves are replicates, and curves' parameters are all different.

Results

ECP treatment reverses arthritis progression

Arthritis was induced by the injection of type II bovine collagen mixed with complete Freund adjuvant. Arthritis symptoms arose around 3 weeks after immunization. The severity of arthritis was monitored every day with the classical established scoring system, based on paw swelling and erythema evolution, as illustrated in figure 1A. Each paw individual score were summed to determine the clinical score of the mouse. As shown in figure 2B, arthritis raw score continuously grows, starting from a score of 3 at 25 days post immunization and reaching a score of 10 at 42 days post immunization.

To determine whether ECP has a beneficial effect in the treatment of arthritis, we have translated the human ECP process to adapt it to mice. In humans, ECP is an autologous procedure where blood cells are extracorporally exposed to PUVA (psoralen + UVA irradiation) before being infused back to the patient. Such procedure cannot be performed in an autologous manner in mice. Thus, we used spleen cells from an arthritic mouse (mean score = 7) as source of cells to be injected to a second arthritic mouse, following PUVA treatment. Spleen cells from donor arthritic mice were harvested, incubated with 8-MOP and irradiated with UV-A (PUVA procedure), in the same conditions as for therapeutic ECP procedure in human. This procedure induced leucocyte apoptosis that was measured by annexin V/7-AAD labeling (figure 2A).

Five hours after PUVA application, 60% of treated leucocytes were apoptotic (AnnV + 7-AA -cells) or underwent secondary necrosis (AnnV + 7AAD + ), p=0.04(figure 2B). PUVA-treated spleen cells obtained from arthritic mice were immediately transferred to another group of arthritic mice. Each recipient arthritic mouse received 3 ECP treatments every other day. As shown in figure 2C, the relative CIA score of untreated mice increased over time, while ECP treated mice recovered from arthritis as evidenced by a decreasing score. The analysis of 13 mice per group pooled from 2 independent experiments demonstrated the ability of ECP to reverse arthritis (figure 2D). Of note, 2 days after the third and last injection, ECP treated mice were no longer able to control arthritis development and clinical scores began to progress again, suggesting the need to continue treating the mice to maintain therapeutic efficacy, as observed in humans. Taken together these data prove that ECP treatment can efficiently induce the regression of collagen induced arthritis in mice.

ECP triggers Th17 decrease in vivo

In order to gain further insights on ECP mechanism of action, we evaluated the impact of such treatment on Th17 and Th1 cells (autoimmune cells described as major players in CIA pathogenesis), as well as on Tregs (that help control disease progression), by flow cytometry analysis, as illustrated in figure 3A. While ECP does not affect either Tregs or Th1 cell frequencies, it significantly decreases the frequency of Th17 cells within the spleen (Figure 3B). No differences were observed in lymph nodes (not shown).

In order to evaluate systemic inflammation, serum cytokines were also assessed. Similar levels of circulating IFN, IL-4, IL-6, IL-10, IL-2 and TNF were observed between untreated and ECP treated mice. Interestingly, serum IL-17 was also decreased under ECP treatment, although the trend did not reach statistical significance (Fig 3C). These data suggest that ECP treatment can modulate IL17 production by Th17 cells.

The presence of arthritogenic T cells within the treated sample is mandatory for ECP efficacy in arthritis

In human, it is currently admitted that ECP can be efficient only in pathologies where circulating pathogenic T lymphocytes are present, thus representing a fraction of the treated cells. To test whether pathogenic T cells were also needed for ECP efficacy in CIA, we compared the therapeutic effect of PUVA-treated splenocytes originating from arthritic (A-PUVA) or healthy mice (H-PUVA).

Interestingly, only PUVA-treated arthritic mouse derived-spleen cells were able to control arthritis development, while spleen cells from healthy mouse failed to do so. Statistical analyses comparing the two curves highlighted the significant difference of CIA progression in mice treated with A-PUVA spleen cells compared to mice treated with H-PUVA spleen cells or not treated, and the absence of therapeutic effect with H-PUVA spleen cells.

In line with the clinical efficacy observed in figure 4A, only A-PUVA group displayed a decreased Th17 frequency while H-PUVA treated mice displayed a Th17 rate comparable to untreated mice (Figure 4B). Taking together, these results demonstrate that ECP is able to trigger arthritis control and Th17 decrease only when arthritogenic T cells are contained within the treated sample.

Discussion

The newly developed treatments based on synthetic or biologic disease-modifying anti-rheumatic drugs (DMARD) interfering with the inflammatory process(3), allow 10 to 50% of patients with RA to reach 70% improvement in the American College of Rheumatology response criteria (ACR70); their sequential use allows a good control of RA, and many patients reach remission. However, despite these recent improvements, new therapeutic strategies are needed for primary or secondary nonresponder RA patients who do not reach low disease activity.

ECP can be used to treat T cell-mediated diseases, with great efficacy in various disease contexts, either leading to tolerogenic or immunogenic immune responses [START_REF] Edelson | American council on ECP (ACE): Why now?[END_REF]. ECP has been recently shown to induce anticancer immunity, especially when DC differentiation is initiated during the apheresis process, while tumor cells are rendered apoptotic by the PUVA treatment [START_REF] Ventura | Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity[END_REF]. On the other hand, in allogeneic T cell-mediated diseases such as GvHD,organ transplantation and T cell-mediated autoimmune diseases, ECP can have a tolerizing effect. Since RA is thought to involve Th17 autoreactive T cells, ECP could be a very promising therapeutic option for this debilitating disease.

Unlike conventional immunosuppressive regimen, ECP-induced tolerance seems specific rather than systemic, and does not increase the risk of opportunistic infections or cancer development. This considerable advantage renders ECP very attractive as a therapeutic option for the treatment of autoimmune diseases, but only few clinical trials have assessed its efficacy in the treatment of autoimmune diseases. In RA, the results of published small cases series are unfortunately often controversial [START_REF] Ratcliffe | National Institutes of Health State of the Science Symposium in Therapeutic Apheresis: scientific opportunities in extracorporeal photopheresis[END_REF][START_REF] Adamski | Extracorporeal photopheresis for the treatment of autoimmune diseases. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF], making it crucial to develop robust and easy to handle preclinical animal model to rationally evaluate ECP efficacy in this therapeutic indication. Different animal models of arthritis are currently available, each of them displaying immunologic similarities and differences with human rheumatoid arthritis [START_REF] Bevaart | Evaluation of therapeutic targets in animal models of arthritis: how does it relate to rheumatoid arthritis?[END_REF]. We chose the model of collagen-induced arthritis in DBA1 mice to evaluate ECP efficacy and mechanism of action because this model recapitulates most clinical, histologic and immunologic features of the human disease. Indeed, in this robust model, methotrexate, anti-TNF antibodies or Abatacept (CTLA-4Ig), that are currently used to treat RA patients, have been shown to ameliorate clinical CIA scores [START_REF] Webb | Prevention and amelioration of collagen-induced arthritis by blockade of the CD28 co-stimulatory pathway: requirement for both B7-1 and B7-2[END_REF][START_REF] Williams | Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis[END_REF][START_REF] Neurath | Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression[END_REF], rendering it particularly relevant to analyze new immunomodulatory therapeutic regimen. The CIA model is probably quite stringent, since a pellet of antigen and adjuvant continuously releases the immunogenic trigger, thereby making it difficult to maintain control of the disease in the absence of treatment. In the present study, ECP could induce similar decrease in CIA scores as more established therapies in the same model [START_REF] Webb | Prevention and amelioration of collagen-induced arthritis by blockade of the CD28 co-stimulatory pathway: requirement for both B7-1 and B7-2[END_REF][START_REF] Williams | Anti-tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis[END_REF][START_REF] Neurath | Methotrexate specifically modulates cytokine production by T cells and macrophages in murine collagen-induced arthritis (CIA): a mechanism for methotrexate-mediated immunosuppression[END_REF], even though the procedure was initiated at a more advanced disease stage (CIA score=7); it is unclear whether the transient symptom decrease/ stabilization is due to progressing disease despite continuous immunoregulation, or if the immunological mechanisms responsible for clinical amelioration are themselves transient in nature. It is to be noted that CIA can also be initiated in non-human primates, which could prove useful to test novel therapeutic targets and for pre-clinical development. An exact replica of the autologous ECP procedure performed in humans cannot be performed in mice, but can be approximated in inbred mouse strains.

Here, our results show that ECP can induce the decrease of CIA disease scores as soon as 2 days after the first ECP-treated cell infusion, demonstrating ECP efficacy in arthritis. In humans treated with ECP in the context of GVHD, the mean time to response observed is around 1 month [START_REF] Couriel | Extracorporeal photochemotherapy for the treatment of steroid-resistant chronic GVHD[END_REF][START_REF] Del Fante | Response and survival of patients with chronic graft-versus-host disease treated by extracorporeal photochemotherapy: a retrospective study according to classical and National Institutes of Health classifications[END_REF], and this time is even longer in the context of cutaneous T cell lymphoma treatment [START_REF] Duvic | Extracorporeal photopheresis for the treatment of cutaneous T-cell lymphoma[END_REF]. In mice however, in a recent study, tumor growth was almost completely inhibited very soon after an ECP-like treatment, suggesting a rapid efficacy of ECP in rodents [START_REF] Ventura | Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity[END_REF]. The differences in time to response observed in these different studies and ours may rely on differences due to the pathologies and species considered. It is noteworthy that IL-17 and Th17 play a role in early stages of CIA in mice, and that anti-IL17A antibodies can help control arthritis in CIA mice [START_REF] Zhang | Synergistic effects of interleukin-1beta and interleukin-17A antibodies on collagen-induced arthritis mouse model[END_REF]. In human rheumatoid arthritis, Th17 cells frequency and interleukin-17 levels are found associated with arthritis both at the onset and the progression of the disease [START_REF] Leipe | Role of Th17 cells in human autoimmune arthritis[END_REF], supporting the development of IL-17 blocking agents that are currently being tested in RA [START_REF] Kugyelka | Enigma of IL-17 and Th17 Cells in Rheumatoid Arthritis and in Autoimmune Animal Models of Arthritis[END_REF]. In a clinical trial that used ECP to treat systemic sclerosis patients, the percentage of peripheral Th17 cells, initially high, decreased under treatment [START_REF] Papp | The assessment of immuneregulatory effects of extracorporeal photopheresis in systemic sclerosis: a long-term follow-up study[END_REF]. Interestingly, in our CIA model, we observed that ECP treatment triggers the decrease of IL-17 producing Th17 cells, as well as serum IL-17 cytokine, suggesting that pathogenic Th17 cells are efficiently targeted by ECP immunomodulatory action. The observed effect is significant but moderate, perhaps because this measure was performed 1 week after the last ECP treatment, at a time point where arthritis symptoms were rising again. How Th17 frequency reduction was achieved is unclear, since Th17 levels are controlled by many factors, including among many others: differentiation-inducing and expansion-inducing cytokines (e.g. IL-1b, IL-6, TGFb, IL-21, IL-23 etc), inhibitory cytokines (e.g. IL-10, type I IFN), microbial stimulation, selective T cell death or migration. Possible but still unsubstantiated explanations may involve differential sensitivity of Th17 cells to PUVA-induced cell death, a significant reduction of their proliferation, or modulation of Th17-expanding cytokines. In a mouse model of psoriasis (K5.hTGF-b1 transgenic mice) (26), 4-week PUVA treatment suppressed the IL-23/ Th17 pathway while augmenting the frequencies of Th2 and IL-10 secreting Foxp3+ Treg, in a CTLA-4 dependent manner, and the levels of many inflammatory cytokines were reduced. Whether these features also apply to the present model is not clear, as no modulation of Treg and Th1 cell populations or IL-10 was observed in the periphery in the present study, after a shorter course of treatment.

To date, ECP mechanism of action are poorly understood. In CTCL, it has been hypothesized that ECP triggers anti-tumor specific responses directed toward the tumor T cell clone [START_REF] Ventura | Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity[END_REF][START_REF] Durazzo | Induction of monocyte-todendritic cell maturation by extracorporeal photochemotherapy: initiation via direct platelet signaling. Transfusion and apheresis science : official journal of the World Apheresis Association[END_REF][START_REF] Berger | Induction of human tumor-loaded dendritic cells[END_REF], the ECP procedure being able to trigger monocyte differentiation into DC [START_REF] Ventura | Extracorporeal Photochemotherapy Drives Monocyte-to-Dendritic Cell Maturation to Induce Anticancer Immunity[END_REF]. In our hands however, in a platelet-free process, ECP-treated monocytes do not to differentiate into dendritic cells, and even if their apoptosis is slower than T cell apoptosis, all monocytes end up being apoptotic after a few days [START_REF] Hannani | Photochemotherapy induces the apoptosis of monocytes without impairing their function[END_REF]. In a parallel study, we hypothesized that ECP could induce immunogenic cell death [START_REF] Hannani | Extracorporeal Photopheresis: Tolerogenic or Immunogenic Cell Death? Beyond Current Dogma[END_REF].

Damage-Associated Molecular Patterns expression by allogeneic activated PUVA-treated T cells was evaluated, and upregulated Calreticulin expression and HMGB-1 secretion were observed [START_REF] Coppard | In vitro PUVA treatment triggers calreticulin exposition and HMGB1 release by dying T lymphocytes in GVHD: New insights in extracorporeal photopheresis[END_REF].

On the other hand, in the context of GVHD, it has been hypothesized that ECP treated DCs, or liverand spleen-resident DC, massively exposed to the reinfused ECP-treated apoptotic cells, acquire tolerogenic properties and will in turn, promote immune tolerance through Treg generation, leading to disease stabilization or regression. The monitoring of regulatory T cells during ECP treatment in human brought controversial results: an increase of Treg has been observed in the context of GHVD at some time points during ECP treatment [START_REF] Biagi | Extracorporeal photochemotherapy is accompanied by increasing levels of circulating CD4+CD25+GITR+Foxp3+CD62L+ functional regulatory Tcells in patients with graft-versus-host disease[END_REF][START_REF] Rao | Cutaneous T cell lymphoma and graft-versus-host disease: a comparison of in vivo effects of extracorporeal photochemotherapy on Foxp3+ regulatory T cells[END_REF][START_REF] Quaglino | Reciprocal modulation of circulating CD4+CD25+bright T cells induced by extracorporeal photochemotherapy in cutaneous T-cell lymphoma and chronic graft-versus-host-disease patients[END_REF][START_REF] Schmitt | Extracorporeal photophoresis augments function of CD4+CD25+FoxP3+ regulatory T cells by triggering adenosine production[END_REF], but in CTCL [START_REF] Quaglino | Reciprocal modulation of circulating CD4+CD25+bright T cells induced by extracorporeal photochemotherapy in cutaneous T-cell lymphoma and chronic graft-versus-host-disease patients[END_REF] or lung transplant rejection [START_REF] Meloni | Peripheral CD4(+)CD25(+) TREG cell counts and the response to extracorporeal photopheresis in lung transplant recipients[END_REF] lower levels of circulating Tregs were observed. Usually, Treg percentages in human diseases are highly heterogeneous [START_REF] Dieterlen | Immunological monitoring of extracorporeal photopheresis after heart transplantation[END_REF], and do not correlate with clinical responses [START_REF] Denney | Regulatory T Cells in Chronic Graft-Versus-Host Disease After Extracorporeal Photopheresis: Correlation With Skin and Global Organ Responses, and Ability to Taper Steroids[END_REF], suggesting that ECP mechanism of action does not directly affect regulatory T cells frequency. In line with these observations, in our preclinical CIA model, the decrease of arthritogenic Th17 cells induced by ECP was not associated with Treg frequency modifications.

In the context of human CTCL treatment, it is currently admitted that ECP efficacy relies on the presence of circulating tumor cells. In "consensus statement update from the UK Photopheresis Society" published in 2017, ECP is recommended in CTCL patients with proven peripheral blood involvement demonstrating a peripheral blood T-cell clone and ⁄ or circulating Sezary cells representing 10% to 20% of peripheral circulating lymphocytes [START_REF] Alfred | The role of extracorporeal photopheresis in the management of cutaneous T-cell lymphoma, graft-versus-host disease and organ transplant rejection: a consensus statement update from the UK Photopheresis Society[END_REF]. In all other ECP indications, no evaluation of correlations between circulating pathogenic T cell presence and clinical efficacy has been performed [START_REF] Knobler | Guidelines on the use of extracorporeal photopheresis[END_REF], such demonstration being particularly challenging. Our data clearly demonstrate that the therapeutic effect in CIA is obtained only when ECP-treated leukocytes originate from arthritic mice, suggesting that the presence of pathogenic T cells within PUVA treated leukocyte samples is mandatory to obtain therapeutic efficacy.

Importantly, these data indicate that the treatment of a fraction of pathogenic T cells provokes a systemic control of untreated pathogenic cells, as illustrated by the decrease of Th17 cells and serum IL-17. This observation is reminiscent of T cell vaccination (TCV) experiments where autoreactive T cells were ex-vivo amplified and gamma-irradiated in order to trigger their apoptosis, before being reinfused back to the patients. Such TCV strategy aims at inducing anti-clonotypic T cell responses, directed toward pathogenic T cell clones, and has shown promising clinical responses [START_REF] Hannani | Extracorporeal Photopheresis: Tolerogenic or Immunogenic Cell Death? Beyond Current Dogma[END_REF]. For instance, a recent Russian clinical trial evaluating the efficacy of TCV with irradiated autologous collagen reactive T-cells in RA has shown a clinical improvement in 87% of treated patients [START_REF] Kugyelka | Enigma of IL-17 and Th17 Cells in Rheumatoid Arthritis and in Autoimmune Animal Models of Arthritis[END_REF].

Interestingly, we have shown that activated T cells undergo faster apoptosis than resting T cells after ECP [START_REF] Hannani | Photochemotherapy induces a faster apoptosis of alloreactive activated T cells than of nonalloreactive resting T cells in graft versus host disease[END_REF]. Since autoreactive T cells are in an activated state, one can speculate that they will very rapidly undergo apoptosis following ECP, emitting immunogenic-cell death-associated signals allowing dendritic cell maturation(14, 42), and will be preferentially captured to serve as a source of Ag, for priming a specific immune response by inducing efficient anti-clonotypic responses against alloreactive T cells and control systemic pathogenic T cells as hypothesized in 2015 by D.

Hannani [START_REF] Hannani | Extracorporeal Photopheresis: Tolerogenic or Immunogenic Cell Death? Beyond Current Dogma[END_REF].

Conclusion:

We demonstrate here that ECP is efficient in a preclinical mouse model of RA, and should be considered as a promising therapeutic option in the course of human rheumatoid arthritis and more broadly of any T cell mediated autoimmune disease. We provide a pertinent pre-clinical in vivo model, recapitulating both human RA-and ECP-related clinical observations, paving the way for dissecting ECP Mechanism of action in autoimmune disorders. We believe that the understanding of ECP mechanisms of action will help with its rational optimization and broaden its clinical applications.

Figure legend

  France), housed at the University of Grenoble animal core facility PHTA (Plateforme de Haute Technologie Animale, agreement #C 38516 10 006 delivered by Direction Départementale de la Protection des Personnes de l'Isère), in individually ventilated cages (5 mice per cage) and fed ad libitum a standard diet and filtered water. All experiments were approved by the local ethic committee as well as French ministry of Research and Innovation, under the number:

Figure 1 :

 1 Figure 1: Typical clinical pattern of collagen-induced arthritis in male DBA/1 mice. Arthritis symptoms started 3 weeks after immunization. A) The severity of arthritis was monitored by

Figure 2 :

 2 Figure 2: ECP treatment efficiently reverses arthritis progressionSpleen cells from arthritic mice were submitted to psoralen+UVA irradiation, resulting in their rapid

Figure 3 :

 3 Figure 3: ECP treatment modifies Th17 frequency Immune cell profile was analyzed in mice treated or not by ECP twelve days after beginning of the

Acknowledgements

We are grateful to F. Blanquet and Dominique Paris, and to all the technical staff of the Grenoble Plateforme de Haute Technologie Animale for expert animal care, and of the Besançon animal facility. We thank all the staff in UMR 1098, and in EFS R&D laboratory, and especially Emmanuelle Grasso and Jean-Paul Molens for their help. We thank Philippe Saas for his support.

Funding

This work was possible thanks to the financial support of EFS-APR 2013 and EFS Rhone-Alpes. Céline

Coppard was recipient of a CIFRE grant from French ANRT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and material

All data generated or analysed during this study are included in this published article

Consent for publication: Not applicable

Competing interests

The authors declare that they have no competing interests Authors' contributions SP, FG, JP and LC designed the study, CC and FB performed the experiments, and SP, CC, LC, FG and DH analyzed the data. LC wrote the article. OM performed statistical analyses and manuscript edition.

Figure 4: ECP efficacy relies on the presence of arthritogenic T cells in the treated sample

Mice with established arthritis were left untreated or were injected 3 times with ivPUVA spleen cells taken from arthritic or healthy mice. A) Mean and SEM of relative clinical score in 15 mice per group from 3 independent experiments. The three curves represent the calculated nonlinear regression, second order polynomial equations and are statistically different for mice treated with ivPUVA spleen cells from arthritic mice (white circles) compared to untreated mice (black squares) or treated with ivPUVA cells from healthy mice (stars). Bayesian analysis using a Baranyi model also indicated that the A-PUVA curve was different from the two others (Bayes factor>1000). B) Th17 cell percentages were determined, and bars represent the mean for 5 mice per groups in one experiment. Kruskal-Wallis test showed significant differences between the 3 groups (p=0.016) and Dunns' post-test was used to compare the 3 groups (* means p<0.05).

Supplementary