E Merlin
email: e_merlin@chu-clermontferrand.fr

N Goncalves-Mendes

Dalil Hannani

Anne De

La Torre

M C Farges

H Laroye

F Demeocq

J Kanold

M P Vasson

A De La Torre

Extracorporeal photochemotherapy induces arginase 1 in patients with graft versus host disease

Keywords:

HAL is

Introduction

Extracorporeal photochemotherapy (ECP) is a cellular therapy that has demonstrated efficacy in a number of Tcell-mediated diseases, such as T-cell cutaneous lymphoma, allograft rejection, and severe and/or steroidresistant graft-versus-host-disease (GVHD) [START_REF] Heshmati | History of ECP[END_REF]. The process consists in an extracorporeal exposure of peripheral blood mononuclear cells to UVA-photoactivated 8-methoxypsoralen (8-MOP) and their subsequent reinfusion into the patient [START_REF] Edelson | Photopheresis: a new therapeutic concept[END_REF]. ECP induces immediate lymphocyte and delayed monocyte apoptosis [START_REF] Hannani | Photochemotherapy induces the apoptosis of monocytes without impairing their function[END_REF], and it is widely suspected that its therapeutic effects are related to the infusion of apoptotic lymphocytes and tolerogenic dendritic cells [START_REF] Maeda | Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells[END_REF]. The role of interleukin 10-induced synthesis (IL-10 is a potent anti-inflammatory and regulatory cytokine) by treated and untreated monocytes could be decisive [START_REF] Di Renzo | Extracorporeal photopheresis affects interleukin (IL)-10 and IL-12 production by monocytes in patients with chronic graft-versus-host disease[END_REF][START_REF] Maeda | Experimental extracorporeal photopheresis inhibits the sensitization and effector phases of contact hypersensitivity via two mechanisms: generation of IL-10 and induction of regulatory T cells[END_REF], together with the generation of CD4+CD25+Foxp3+ regulatory T cells (Tregs) [START_REF] Maeda | Intravenous infusion of syngeneic apoptotic cells by photopheresis induces antigen-specific regulatory T cells[END_REF], although other signaling pathways are probably involved. In particular, ECP triggers significant metabolic changes, some involving fatty acids [START_REF] Wiswedel | Detection of enhanced monohydroxyeicosatetraenoic acid and F2-isoprostane levels in human plasma samples after extracorporeal photoimmunotherapy[END_REF]. Thus, one possibility is that the effects of ECP are dependent on the patient's metabolic status. GVHD is a multiorgan syndrome in which sustained inflammatory response, organ involvement (such as cholestasis, mouth ulcerations and colitis) together with recurrent infections can lead to impaired nutritional status. Moreover, some of our preliminary data suggest a significant decrease in free arginine rates in cases of gastro-intestinal involvement (unpublished data).

Two forms of GVHD are distinguished based on chronological, clinical and pathophysiological features.

Classically, acute GVHD occurs prior to 100 days after hematopoietic cell transplantation and involves the skin, the gastrointestinal tact and/or the liver. Chronic GVHD is a pleiomorphic syndrome with onset generally occurring between 3 and 24 months after allogeneic hematopoietic cell transplantation. The highly variable clinical manifestations have features resembling autoimmune and other immunologic disorders [START_REF] Filipovich | National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report[END_REF]. They frequently involve the skin, liver, eyes, mouth, upper respiratory tract, esophagus, and less frequently involve serosal surfaces, lower gastrointestinal tract, female genitalia, and fascia. The biological mechanisms leading to chronic GVHD are not as well understood as those leading to acute GVHD. However, it is well establish that Donor T cells are critical for the development of both acute and chronic GVHD [START_REF] Toubai | GVHD pathophysiology: is acute different from chronic?[END_REF]. Schematically, target-tissue damage in acute GVHD is caused by cytopathic donor T cells that respond to the genetically disparate host polymorphic antigens, which are presented by host and/or donor antigen-presenting cells (APCs) [START_REF] Toubai | GVHD pathophysiology: is acute different from chronic?[END_REF]. The T cell response is amplified by several pro-inflammatory cytokines (such as IL-1, IL-6, IL-8 and tumor necrosis factor α) released in response to the conditioning regimen-induced epithelial injury [START_REF] Ferrara | The pathophysiology of acute graft-versus-host disease[END_REF]. .This T cell response can be counter-regulated by costimulatory inhibitors (CTLA-4 and ICOS) as well as regulatory cell populations (CD4_CD25hi Treg subset and NKT cells) [START_REF] Socie | Acute graft-versus-host disease: from the bench to the bedside[END_REF]. Convserly, chronic GVHD is thought to result from an "auto-reaction" against non-polymorphic antigens [START_REF] Toubai | GVHD pathophysiology: is acute different from chronic?[END_REF]. This reaction is caused by CD4_ T cells generated de novo from donor cells hematopoietic cells in a setting of thymic dysfunction caused by both conditioning regimen and previous acute GVHD. The mechanisms leading to organ injury involve both B cells and T cells, and are mediated at least by the secretion of TGF ß and agonistic antibodies against platelet-derived growth factor receptor (PDGFR). The role of regulatory T cells is much less clear than in acute GVHD [START_REF] Martin | Biology of chronic graft-versus-host disease: implications for a future therapeutic approach[END_REF]. L-arginine is an amino acid thought to become essential after injury. It plays a central role in several functions of the immune system [START_REF] Moinard | Involvement of glutamine, arginine, and polyamines in the action of ornithine alpha-ketoglutarate on macrophage functions in stressed rats[END_REF]. In leukocytes, L-arginine can be metabolized by two pathways, under the control of two key enzymes, i.e. inducible nitric oxide synthase (iNOS) and arginase 1 leading to the modulation of available extracellular L-arginine and to the production of metabolites. Free L-arginine concentration in the environment was the first parameter shown to modulate T cell activation and function: arginine is known to enhance lymphocyte proliferation and IL2 utilization [START_REF] Ochoa | Effects of L-arginine on the proliferation of T lymphocyte subpopulations[END_REF], whereas L-arginine starvation impairs T-lymphocyte function, at least partly by triggering the internalization of the CD3 zeta chain and impairing cell cycle progression by interfering with the cyclin D3 and cdk4 pathway [START_REF] Rodriguez | L-arginine availability regulates T-lymphocyte cell-cycle progression[END_REF]. Hence, L-arginine depletion under the action of arginase is known to contribute to tumor-induced immune suppression [START_REF] Zea | Arginase-producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion[END_REF], as well as the counterregulation of inflammatory purulent reactions in humans [START_REF] Munder | Suppression of T-cell functions by human granulocyte arginase[END_REF] as well as the maternal unresponsiveness toward the semi-allogenic fetus [START_REF] Kropf | Arginase activity mediates reversible T cell hyporesponsiveness in human pregnancy[END_REF].

Aside of extracellular arginine concentration, the products of arginine metabolites are also involved in immune regulation. Inducible NO Synthase leads to the production of nitric oxide and citrulline, whereas the action of arginase results in the production of ornithine, proline and polyamines [START_REF] Cynober | Arginine metabolism in mammals[END_REF]. Depending on the predominant metabolic pathway, these products can mediate multiple biological responses. The increase of intracellular NO resulting from iNOS activity can mediate normal signaling or be cytostatic and cytotoxic, particularly at high concentration, in responses to cytokines [START_REF] Bronte | L-arginine metabolism in myeloid cells controls T-lymphocyte functions[END_REF]. When arginase and iNOS are concomitantly activated, the action of NOSynthase in the condition of low arginine level leads to the production of peroxynitrites (ONOO-) and triggers lymphocyte apoptosis [START_REF] Bronte | L-arginine metabolism in myeloid cells controls T-lymphocyte functions[END_REF]. Arginase activity not only leads to arginine depletion but also to the production of proline and ornithine which can be utilized to synthesize polyamines (putrescine, spermidine and spermine). Ornithine and polyamines are known to exert some immunomodulatory effects, in particular in the setting of allogeneic MLR [START_REF] Susskind | Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine[END_REF]. Although the immunological role of spermine is not clear, a negative correlation has been found between blood spermine levels in patients with breast cancer and the impairment of IL12 synthesis in vivo. Moreover, it has been shown that in vitro exposure of dendritic cells (DCs) to spermine may impair their stimulatory function. [START_REF] Della Bella | Altered maturation of peripheral blood dendritic cells in patients with breast cancer[END_REF] To our knownledge, nothing is known about the activity of arginase in patients with GVHD. On the other hand, NO is suspected to be increased in patients with GVHD [START_REF] Choi | Plasma nitric oxide is associated with the occurrence of moderate to severe acute graft-versus-host disease in haemopoietic stem cell transplant recipients[END_REF], but at present, the full range of effects of NO during GVHD is not yet fully described. The immunosuppressive effect of NO in acute GVHD is well established, but it is not clear whether this plays a deleterious or a protective role [START_REF] Bobe | Nitric oxide mediation of active immunosuppression associated with graft-versus-host reaction[END_REF].

Whereas UV exposure is known to upregulate the activity of several enzymes such as ornithine decarboxylase (ODC, involved in the metabolism of ornithine and polyamines) in skin cells [START_REF] Lee | Melatonin reduces ultraviolet-B induced cell damages and polyamine levels in human skin fibroblasts in culture[END_REF], to date, nothing is known about the combined effects of UVA and 8-MOP on arginine metabolism in human leukocytes during ECP.

Moreover, given the impact of L-arginine availability on immune response, one possibility is that the response to ECP could be affected by L-arginine content in the cell environment. We therefore investigated the effects of ECP on arginine metabolism in the context of GVHD and in healthy donors, and determined whether the modulation of arginine availability can modify leukocyte response after ECP treatment.

Our work shows that ECP significantly impacts the metabolism of arginine toward the production of ornithine but not nitric oxide, and that under ECP condition arginine availability is an important factor for the modulation of TNFα in patients with GVHD.

Material and methods

Patients and healthy donors

The study enrolled 6 patients (4 males and 2 females aged 9 to 56, median 15) diagnosed with acute (n=3) or chronic (n=3) GVHD and 6 healthy donors (3 males and 3 females aged 24 to 35, median 28). Acute GVHD was graded according to Glücksberg criteria [START_REF] Glucksberg | Clinical manifestations of graft-versus-host disease in human recipients of marrow from HL-A-matched sibling donors[END_REF], and the severity of chronic GVHD was assessed as proposed by Shulman [START_REF] Shulman | Chronic graft-versus host syndrome in man: a long-term clinicopathologic study of 20 Seattle patients[END_REF]. Complete response was defined as resolution of all organ manifestations. Partial response was defined as greater than 50 percent response of organ involvement. Absence of response was defined as stable or progressively worsening GVHD and/or the inability to taper other medication by at least 50 percent. The patients had undergone different immunosuppressive treatments but had not responded (table 1). Blood donations in this study conformed to institutional review board-approved protocols, and informed consent was obtained.

PUVA exposure / Extracorporeal Photochemotherapy

ECP was performed using the Vilber-Lourmat procedure, as previously described [START_REF] Kanold | Photopheresis in pediatric graft-versus-host disease after allogeneic marrow transplantation: clinical practice guidelines based on field experience and review of the literature[END_REF]. Briefly, mononuclear cells were isolated from patients by cytapheresis (Cobe Spectra, Gambro BCT, Bourg-la-Reine, France) then diluted in normal saline solution to obtain a final hematocrit <1%, and 8-methoxypsoralen was added at 200 ng/ml. After a 3-minute incubation, cells were transferred into a UV-A transparent bag (Macopharma) and exposed to UV-A light (340 nm) at 2 J/cm 2 for 10 minutes (UVA-matic, Vilber-Lourmat, France). ECP sessions were performed three times a week (with a one-day interval between two ECP sessions) for three weeks, and then gradually reduced for those patients who were stabilized or who showed improvement. For each session, apheresis duration was 120 minutes. This allowed to collect between 1.6x10 7 and 8.2 x10 7 lymphocytes/kg (for patients with acute and chronic GVHD, respectively).

PBMC sampling and preparation

Two cell samples were collected from patients: one peripheral blood sample was collected by venous puncture in EDTA from patients immediately prior to apheresis (ie before ECP), and a second cell sample was collected from the buffy coat bag just prior to reinfusion (ie after ECP). Blood samples were also collected from healthy donors by venous puncture, but cytapheresis were not performed for healthy volunteers. PBMC were isolated by density gradient centrifugation over Ficoll-Histopaque (Sigma-Aldrich). The isolated cells were then washed twice in phosphate buffered saline (PBS, Gibco). Cells from the healthy volunteers were then separated into two fractions, and one was cultured without additional treatment ("non-treated") while the other fraction was treated by PUVA exposure before being cultured ("ECP-treated"). The PMBC from the patients were cultured as control "non-treated" cells.

Media and cell cultures

"Non-treated" and "ECP-treated" mononuclear cells were cultured (37°C, 5% CO2) for 48 h in arginine-free and nitrate-free RPMI (1x10 6 cells/ml) which was supplemented with L-arginine at 0, 50, 100, 200 or 1000 µM. 10% dialyzed fetal calf serum, 4mM L-glutamine and 100 UI/l penicillin-streptomycin were added. The cells were stimulated by phytohemagglutinin (5µg/ml). After 48 h culture, the supernatant was removed by centrifugation and stored at -80°C until NO and amino acid analysis. PBMC were washed in PBS then assessed for apoptosis and mRNA quantification of cytokines.

For arginase 1 and iNOS expression, 1x10 6 cells/ml were cultured (37°C, 5% CO2) for 24 h in Minimal Essential Medium with 10% dialyzed fetal calf serum, 4mM L-glutamine and 100 UI/l penicillin-streptomycin.

The cells were stimulated by LPS (1µg/ml).

Supernatant amino acid analysis

For amino-acid analysis, after deproteinization using sulfosalicylic acid, 30 µl of the supernatant was used for HPLC analysis with an ion-exchange column on a Dionex System. After post-column derivatization with ninhydrin, amino acids were detected at 570 nm. Data was collected and processed using Chromeleon® software, as previously described [START_REF] Minet-Quinard | Pre-and postoperative aminoacidemia in breast cancer: a study vs. matched healthy subjects[END_REF].

Supernatant NO analysis

Nitric oxide (NO) production was measured as nitrite plus nitrate using the Griess method after incubation with nitrate reductase (Cayman, France), as previously described [START_REF] Berard | Total parenteral nutrition enriched with arginine and glutamate generates glutamine and limits protein catabolism in surgical patients hospitalized in intensive care units[END_REF].

Supernatant cytokine analysis

IL-10, TNFα and IFNγ assays were performed simultaneously using Cytometric Beads Array (CBA) kits on EPICS apparatus (Becton Dickinson Biosciences Pharmingen, San Diego, CA, USA) following the manufacturer's instructions.

Apoptosis

FITC-conjugated annexin-V coupled with 7-aminoactinomycin D (Beckman-Coulter) was used to detect apoptosis. Cells were washed twice in PBS then stained with FITC-libeled Annexin-V, 7-aminoactinomycin D (in a 100 µl of labeling solution) and incubated for 10 min in the dark according to the manufacturer's protocol.

Then, 400 µl of buffer was added, and stained cells (> 10,000 events) were analyzed by flow cytometry (Beckman-Coulter EPICS). This staining makes it possible to differentiate cells in early apoptosis (annexin + 7-AAD -), late apoptosis (annexin + 7-AAD +) and necrotic cells (annexin -7-AAD+) from viable cells (annexin -7-AAD -).

Real-Time RT-PCR quantification of the mRNA levels of cytokines and enzymes involved in arginine metabolism

PBMC total RNA was extracted using Trizol Reagent (Invitrogen) according to the manufacturer's instructions.

Reverse transcription was conducted from 1 µg of total RNA, giving rise to 20 µl of cDNA as previously described [START_REF] Goncalves-Mendes | Placental expression of SCOspondin during mouse and human development[END_REF]. One µl was used for the PCR performed in real-time on a Lightcycler Instrument (Roche Diagnostics). Sample mRNA copy numbers were extrapolated from standard curves obtained with serially diluted purified PCR products. 18S RNA was used as internal standard, so the results were expressed as fold induction. IL-10, Interferon γ, Tumor Necrosis Factor-α, Arginase-1 and inducible NOSynthase mRNAs were detected using the primer probes reported in Table 2.

Results

Before ECP exposure, arginine is differentially metabolized in circulating leukocytes from ECP-treated GVHD patients and from healthy donors

We investigated whether the metabolism of arginine was similar in cells harvested from ECP-treated patients as in cells from healthy donors. After a culture in nitrite-free medium, we measured the products of arginine catalysis that had been release into the supernatant. After a 48 hr-culture, NO production was lower in cells from patients with GVHD compared with cells from healthy donors (Figure 1, p<0.01). Moreover, the L-ornithine concentration in the supernatant was significantly higher in cells from patients treated by ECP for a GVHD than cells from healthy donors (p<0.01). This shows that in leukocytes from patients who were previously treated by ECP for a GVHD, arginine is preferentially metabolized toward the production of ornithine.

ECP induces the synthesis of L-ornithine but not nitric oxide

To assess whether the differences of arginine metabolism between patients and donors could be imputed to ECP, we measured the impact of ECP on the production of arginine metabolites. Cells were treated by ECP or not and then cultured during 48h. At the end of the culture, L-ornitine concentration in the supernatant has been evaluated. For both patients and healthy donors, L-ornithine concentration was significantly higher for ECPtreated cells (figure 2A) than for non-treated cells. This suggests that ECP activates the arginase pathway in treated-leukocytes. Through competition for arginine, the arginase can also modulate the production of nitric oxide [START_REF] Toubai | GVHD pathophysiology: is acute different from chronic?[END_REF]. In leukocytes, the production of NO by iNOS is accompanied by the equimolar production of citrulline. Cells from both GVHD patients and donors showed similar NO and citrulline production with and without ECP treatment. Taken together, these results suggest that in ECP-treated leukocytes, arginine is metabolized through the arginase pathway, and does not serve as a precursor for the synthesis of N0.

ECP upregulates arginase expression.

In leukocytes, ornithine is produced from arginine by the enzyme arginase 1, whereas NO° is produced by inducible NOSynthase (iNOS). We found that arginase 1 but not iNOS was up-regulated in ECP-treated cells as compared with non-treated cells, in healthy volunteers and in patients with GVHD (figure 2B). These data are in line with those presented in figure 2A.

ECP affects cytokine synthesis

The effect of ECP on cytokine expression is still unclear. Some authors have reported an increase of Th1 cytokines [START_REF] Tokura | Treatment of T lymphocytes with 8-methoxypsoralen plus ultraviolet A induces transient but biologically active Th1-skewing cytokine production[END_REF] whereas other observed an increase of Th2 [START_REF] Bladon | Early reduction in number of T cells producing proinflammatory cytokines, observed after extracorporeal photopheresis, is not linked to apoptosis induction[END_REF]. The discrepancies are attributed in part to the cells origin (healthy donors or patients). One well-established impact of ECP in the setting of GVHD is its upregulating effect on the synthesis of IL10 [START_REF] Di Renzo | Extracorporeal photopheresis affects interleukin (IL)-10 and IL-12 production by monocytes in patients with chronic graft-versus-host disease[END_REF][START_REF] Craciun | Increased production of interleukin-10 and interleukin-1 receptor antagonist after extracorporeal photochemotherapy in chronic graft-versus-host disease[END_REF], while TNFα is down-regulated. IL10 is a suppressive and regulatory cytokine which is involved in the triggering of Tregs [START_REF] Allan | CD4+ T-regulatory cells: toward therapy for human diseases[END_REF], and which is able to down-regulate the expression of TNFα [START_REF] Haddad | Redox-and oxidant-mediated regulation of interleukin-10: an antiinflammatory, antioxidant cytokine?[END_REF]. We evaluated ECP impact on cytokine synthesis by mRNA expression level in GVHD patients. After 48 hr culture with PHA, ECP induced a significant increase (up to 1.5-fold) in IL-10 mRNA expression and a decrease of TNFα and IFNγ mRNA expression (0.2 and 0.4-fold, respectively; figure 3).

After ECP, L-arginine availability impacts the production of ornithine but not NO

Arginine content of the culture medium is known to regulate the expression of some of the proteins of the metabolism of arginine, such as iNOS [START_REF] El-Gayar | Translational control of inducible nitric oxide synthase by IL-13 and arginine availability in inflammatory macrophages[END_REF]. To assess the impact of extracellular L-arginine concentration on the metabolism of L-arginine in an ECP setting, cells from GVHD patients and healthy donors were treated by ECP then cultured for 48 hours in an arginine and nitrate-free RPMI 1640 supplemented with defined amounts of Larginine (0, 50, 100, 200 and 1000 µmol/l) and then stimulated with PHA. In both GVHD patients and healthy donors, the final post-ECP supernatant concentration of arginine was about 50% lower than the initial concentration. Ornithine increase was strikingly dependent on arginine availability (figure 4A). Conversely, the concentration of L-arginine in the cell culture medium did not impact the final concentration of citrulline and NO in the supernatant. This indicates that under ECP conditions, the excess of arginine is only transformed in ornithine, and does not lead to an increase of NO synthesis, supporting data shown in figure 2.

After ECP, L-arginine availability impacts the expression of cytokine mRNA from GVHD patient mononuclear cells without impacting the triggering of apoptosis

We have shown that after ECP, increasing the availability of extracellular arginine leads to a dose-dependent increase of its derivative L-ornithine. Ornithine is known to exert some immunomodulatory effects; in particular it is able to inhibit the alloreactive proliferation in the setting of MLR [START_REF] Susskind | Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine[END_REF]. To test whether the increase of free L-arginine impacts the immunological response of mononuclear cells to ECP via L-ornithine, we measured the synthesis of cytokine mRNAs after 48 hours in a culture media containing different amounts of L-arginine (0, 50, 100, 200 and 1000 µmol/l). Enriching the culture medium with L-arginine resulted in a significant dosedependent decrease in TNFα mRNA transcription levels (figure 4B). IL10 and IFNγ mRNA transcription levels were significantly affected at concentrations ranging between 0 and 100 µM, but this effect plateaued out at above 100 µM. This difference in mRNA expression level could not be related to a variation of apoptosis, since the arginine content in the medium has no impact on the percentage of apoptotic cells after ECP (figure 4C).

Discussion

The metabolism of some free amino acids is known to play a central role in several immune system functions.

We therefore investigated whether ECP induces significant changes in the extracellular concentration of amino acids after a 48-hr culture of PBMC. Our study shows that ECP significantly impacts the metabolism of arginine in leukocytes by upregulating the arginase pathway but not NO.

In leukocytes, arginine is metabolized either to ornithine by arginase or to citrulline and NO via iNOS, and the prevalent view is that these 2 pathways are antithetic [START_REF] Bronte | L-arginine metabolism in myeloid cells controls T-lymphocyte functions[END_REF]. Cells from ECP-treated GVHD patients produced less NO than those cells from healthy donors. This result is quite unexpected, since NO is known to be higher in plasma from patients with acute GVHD than in patients without GVHD [START_REF] Choi | Plasma nitric oxide is associated with the occurrence of moderate to severe acute graft-versus-host disease in haemopoietic stem cell transplant recipients[END_REF]. Moreover, before ECP (but after that all but one patient had undergone several previous sessions) cells from GVHD patients produced more ornithine than cells from the healthy donors. Taken together, these data show that arginine is metabolized through different pathways in circulating leukocytes from ECP-treated GVHD patients than in healthy donors.

Here we have shown that ECP induces ornithine synthesis via arginase 1. Hence, one possibility is that the difference in leukocyte arginine metabolism between patients and healthy donors results from the systemic effect of the previous sessions of ECP. In our study, GVHD patients naive from ECP are lacking as controls. It should be underlined that cells from patient 6 (harvested before the first ECP session) produced less ornithine than the other cells, both before (2 µmol/l) and after (16 µmol/l) ECP exposure. Thus, the effect of ECP on arginine metabolism could be cumulative. This needs to be investigated by serial assessments in GVHD patients starting prior to first exposure to ECP. Moreover, it is necessary to perform further serial analyses on a larger patient number. Thus, our results have to be considered preliminary, and additional studies are warranted.

There is growing evidence that ECP acts in part through the generation of CD4+CD25+Foxp3+ Tregs [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], but the exact mechanism of Tregs triggering is not known. Recent data have shown a pivotal role for NO in the suppression of Foxp3 and other Treg-specific molecules [START_REF] Brahmachari | Suppression of regulatory T cells by IL-12p40 homodimer via nitric oxide[END_REF]. As arginase and iNOS are antagonistic pathways, our study suggests the hypothesis that ECP could promote Tregs by interfering in vivo with the release of NO through the activation of arginase pathway. To our knowledge, nothing is known about arginase activity in patients with GVHD. In humans, the modulation of T-cell response by arginase-induced arginine depletion is an important immunoregulatory pathway [START_REF] Bansal | Arginine availability, arginase, and the immune response[END_REF]. The expression and regulation of arginase in human leukocytes was thought to be limited to polymorphonuclear cells (PMN) [START_REF] Munder | Suppression of T-cell functions by human granulocyte arginase[END_REF] but there is increasing evidence that mononuclear cells are also involved. PMN contamination in our culture wells may have been an explanation for this production of ornithine; however, after apheresis and density gradient centrifugation, PMN contamination was less than 1% of cells, as shown by standard-cytology white blood cell differential count. Moreover, arginaseproducing myeloid-derived suppressor cells (MDSC) have recently been well characterized in the context of renal cell carcinoma. Although they look like polymorphonuclear cells, these myeloid CD11b+CD14-arginaseproducing cells migrate within the monocyte gate on FACS [START_REF] Zea | Arginase-producing myeloid suppressor cells in renal cell carcinoma patients: a mechanism of tumor evasion[END_REF]. Hence, given that we separated cells based on their density, the presence of MDSC in our experiment cannot be ruled out. Secondly, in patients with hepatocellular carcinoma, arginase-producing MDSC belong to the mononuclear cells, express CD14 and are capable of inducing IL-10 and regulatory CD4+CD25+Foxp3+ T cells [START_REF] Hoechst | A new population of myeloid-derived suppressor cells in hepatocellular carcinoma patients induces CD4(+)CD25(+)Foxp3(+) T cells[END_REF] (two pathways involved in the mechanism of action of ECP). In mice, UV-induced immune tolerance to a specific hapten was shown to involve IL-10-secreting myeloid CD11b+/CMH II+ cells, which share phenotypic characteristics with these myeloidderived suppressor cells [START_REF] Hammerberg | Active induction of unresponsiveness (tolerance) to DNFB by in vivo ultraviolet-exposed epidermal cells is dependent upon infiltrating class II MHC+ CD11bbright monocytic/macrophagic cells[END_REF]. Taken together, these data support the hypothesis of a role for arginase-producing suppressive cells in the mechanism of action of ECP.

In mice, the metabolism of arginine in leukocytes is strikingly dependent on the cytokines present in the microenvironment [START_REF] Bronte | L-arginine metabolism in myeloid cells controls T-lymphocyte functions[END_REF]. Induction of iNOS is controlled mainly by Th1 cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), whereas Arg1 is induced by Th2 cytokines including IL-4, IL-13, transforming growth factor-ß (TGF-ß) and IL10 [START_REF] Peranzoni | Role of arginine metabolism in immunity and immunopathology[END_REF]. In humans, the regulation of arginine metabolism in leukocytes looks more complex. Whereas iNOS is expressed mainly by the presence of inflammatory mediators, the pathways regulating arginase-expressing cells remain less clear. It has been suggested that arginase expression is constitutive in and limited to the granulocytic population. However, other cell populations, such as alternativelyactivated macrophages, also exhibit this enzymatic activity [START_REF] Babu | Alternatively activated and immunoregulatory monocytes in human filarial infections[END_REF]. Moreover, increased arginase activity in human mononuclear cells has been demonstrated after severe trauma, and correlates with the plasma level of IL-10 [START_REF] Ochoa | Arginase I expression and activity in human mononuclear cells after injury[END_REF].

Here we have shown that the link between arginase and IL10 also exists in leukocytes after ECP. ECP is known to decrease the number of TNFα and IFNγ-producing T-cells in patients with GVHD, while IL-10 secretion is at least preserved after ECP exposure [START_REF] Bladon | Early reduction in number of T cells producing proinflammatory cytokines, observed after extracorporeal photopheresis, is not linked to apoptosis induction[END_REF]. IL-10 is known to downregulate TNFα [START_REF] Haddad | Redox-and oxidant-mediated regulation of interleukin-10: an antiinflammatory, antioxidant cytokine?[END_REF] by promoting the degradation of its mRNA. Hence, the observed decrease in TNFα should reflect the induction of IL-10. IL-10 has recently emerged as an anti-inflammatory and anti-oxidant cytokine that inhibits the release of free oxygen radicals and suppresses the increase of nitrite concentration [START_REF] Haddad | Redox-and oxidant-mediated regulation of interleukin-10: an antiinflammatory, antioxidant cytokine?[END_REF]. Hence, the combined effects of ECP on ornithine, NO and IL-10 production could be of interest in clinical situations where excessive NO synthesis by iNOS is deleterious (such as in sepsis) [START_REF] Mcgown | Beneficial effects of statins on the microcirculation during sepsis: the role of nitric oxide[END_REF][START_REF] Zhu | Spermine protects mice against lethal sepsis partly by attenuating surrogate inflammatory markers[END_REF].

Another finding of our study was that increasing the extracellular concentration of arginine impacts the level of cytokine mRNA expression. The physiological plasma arginine concentration ranges between 80 and 120 µM. In patients with GVHD, especially in cases of gastro-intestinal involvement, this concentration can drop to 50 µM.

Of note, we observed a significant and linear dose-dependent impact of arginine concentration variation between 0 to 100 µM. Above this physiological value, the observed effect only impacts TNFα mRNA. The observed effect of arginine on TNFα downregulation is intriguing, and requires further investigations. TNFα is known to upregulate the synthesis of NO through the induction of iNOS. However, little is known about the potential of Larginine to regulate TNFα synthesis. In J774 cells, endogenous NO production has been shown to enhance TNF-synthesis [START_REF] Deakin | The modulation of IL-6 and TNF-alpha release by nitric oxide following stimulation of J774 cells with LPS and IFN-gamma[END_REF]. Moreover, TNFα synthesis is down-regulated by spermidine (one of the three polyamines, which are synthesized from ornithine downstream the arginase pathway) [START_REF] Yu | Suppression of TNF-alpha production by S-adenosylmethionine in human mononuclear leukocytes is not mediated by polyamines[END_REF]. In our study ornithine was dramatically increased after ECP, whereas NO was not affected. Hence, the TNFα downregulation we observed could result from several mechanisms (increase of IL10 and ornithine derivatives) which reflect the global reorientation of leukocytes toward a "tolerogenic state" after ECP. TNFα is clearly recognized as deleterious in GVHD settings [START_REF] Roy | The tissue expression of cytokines in human acute cutaneous graft-versus-host disease[END_REF]. Hence, our data suggest that specific therapeutic intervention aimed at enhancing arginine availability for ECP-treated cells could prove useful. This intervention could be conceived at the global patient level, or limited to the irradiation bag.

In conclusion, we have shown that leukocyte exposure to psoralen and UVA during ECP significantly reorients the metabolism of arginine toward the production of ornithine via arginase 1 but not NO, concomitantly with an increase of IL10 and a decrease of TNFα. Moreover, the ECP-triggered downregulation of TNFα was significantly enhanced after arginine enrichment of the medium. This underlines the central role for arginine metabolism in immune tolerance induced by ECP in patients with GVHD.

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. Arginine is differentially metabolized in circulating leukocytes from ECP-treated GVHD patients and from healthy donors. Mononuclear cells of 6 healthy donors and 6 patients on therapy by ECP for a GVHD were cultivated for 48 hours on nitrate-free RPMI 1640 (arginine content: 1148 µmol/l) and stimulated with 5 µg/ml phytohemagglutinin. Ornithine and NO were measured in the supernatant by HPLC and the Griess reaction, respectively. Data are given as means ± SEM. Wilcoxon paired test. ** p<0.01

Table 1 .

 1

	1	14	M	ALL	Acute	3	SL	St -CyA -MMF	43	15	101	S	Partial
	2	9	M	ALL	Acute	2	S	St -CyA	51	5	69	S	Complete
	3	56	M	MDS	Acute	3	SL	St -CyA -MMF	67	3	27	-	No
	4	45	F	AML	Chronic	E	SL	St -tacrolimus	212	8	250	S	Partial
	5	16	M	ALL	Chronic	E	SLG	No	137	22	47	-	No
	6	12	F	AML	Chronic	Lim	S	No	554	1	64	S	Complete

Patient characteristics. Abbreviations: ALL: acute lymphoblastic leukemia; AML: acute myeloblastic leukemia; E: extensive; G: gastro-intestinal tract; Lim: limited; L: liver; MDS: myelodysplastic syndrome; S: Skin; St: steroids; CyA: cyclosporin A; MMF: mycophenolate mofetil; S: skin. Acute GVHD was graded according to Glücksberg criteria (réf), and chronic GVHD according to Shulman criteria

[START_REF] Shulman | Chronic graft-versus host syndrome in man: a long-term clinicopathologic study of 20 Seattle patients[END_REF]

Table 2

 2 Primers used to quantify mRNA levels by Real-Time RT-PCR.

	Primers are listed 5'-3. F, Forward; R, reverse.


Laura Abad Galán

José M Andrés Castán

Clément Dalinot

Marqués Pablo Simón

Philippe Blanchard

Olivier Maury

Clément Cabanetos

Tangui Le Bahers

Cyrille Monnereau

Theoretical and experimental investigation on the intersystem crossing kinetics in benzothioxanthene imide luminophores, and their dependence on substituents effects

In spite of their remarkable luminescence properties, benzothioxanthene imide (BTXI an imide containing rylene chromophores) derivatives have been largely overlooked compared to their perylene bisimide and naphthalene bisimide counterparts. Thus their detailed photophysics are much less understood. In this paper, we show how relatively simple structural modifications of the backbone of BTXIs can lead to impressive variations in their Inter-System Crossing kinetics. Thus, through rational engineering of their structure, it is possible to obtain a triplet formation quantum yield that reaches unity, making BTXI a promising class of compounds for triplet-based applications (photodynamic therapy, electroluminescence, etc.).

Introduction

Rylene imide derivatives are a very intriguing subclass of extended conjugated dyes which have been thoroughly studied for various organic electronic applications, 1,2 such as electroluminescent devices, 3,4 field effect transistors, 5,6 photovoltaics [7][8][9][10] or luminescent tags for bioimaging. [11][12][13] Their success mostly results from the interplay of outstanding spectroscopic properties such as high luminescence quantum yields, 14 a large range of structure dependent accessible emission wavelengths, 15 high thermal and photostability 16 and associated to a moderate synthetic cost compatible with large scale syntheses. 17,18 Within this rylene family, the benzothioxanthene imide derivatives (BTXI) constitute a largely overlooked class of chromophore, [19][20][21][22][23] especially when compared to perylene bisimide. Recently, we have demonstrated that because of their relative synthetic ease, and the possibility to introduce a variety of functional groups on the -position of the naphthyl unit with regards to the sulphur atom (position 5 in Figure 1)position of the naphthalimide moieties, BTXI derivatives could be used as relevant building blocks to prepare extended p-functional molecular systems. 24,25 As for other rylenes, the photophysical properties of BTXI derivatives are clearly remarkable. As a matter of fact, in spite of its apparent structural simplicity, the BTXI core in its most simple form (BTXI, Figure 1) combines large extinction coefficient in the blue part of the electromagnetic spectrum, along with near unity emission quantum yields in the yellow-green domain. 26,27 Moreover, in the course of these preliminary studies on the spectroscopic properties of BTXI derivatives, we identified that the excited state photophysics of this class of chromophore were extremely sensitive to their substitution pattern. 28 In particular, singlet-to-triplet Inter-System Crossing (ISC) seemed to be consistently increased upon simple substitution with a variety of groups. Quite remarkably, this evolution was not limited to halogen substituents, as often observed in organic molecules owing to the so-called heavy atom effects. Strong substituents effects were also observed with generally more innocent functional groups such as nitro, amino, or diphenylamino. A similar observation has also been reported by Zhang et al. on a particular extended BTXI derivative. 29,30 Besides, we demonstrated that simple asfunctionalised molecules could efficiently sensitise the production of singlet oxygen, with potential applications in photocatalysis or photodynamic therapy (PDT). 31,32 Triggered by this initial observation, we initiated a more detailed investigation of the mechanisms responsible for such dramatic dependence of ISC kinetics on the substitution parameters. We were particularly eager to study whether we could amplify this effect by appropriate modification of the substitution pattern, beyond simple tuning of the position 5. In order to do so, we initiated a coupled theoretical and experimental study to elucidate the molecular features that impact the spin-orbit coupling (SOC) in this class of compounds. Thus, in addition to the molecules previously reported, where only modification of the position 5

was considered, we investigated other possible modifications: complementary substitution on the bay position (position 11) were investigated; we also looked at the effect of oxidation of the sulfur heteroatom of the thioxanthene moieties, leading to the thioxanthene dioxide (Figure 1). For all synthesised compounds, we monitored in detail the effect of substitution on the spectroscopic and photophysical features of the chromophore (energy of the absorption and emission, fluorescence quantum yield, singlet oxygen generation quantum yield). Additionally, most of the studied molecules were investigated by computational modeling, mostly aiming at rationalizing the kinetics of ISC on the basis of the calculated spin-orbit coupling matrix and difference in energy between the first singlet excited states and triplet excited states. As a result, three main conclusions can be drawn from this study: (i) chemical modifications on the BTXI core are extremely efficient to tune the extent of ISC within the molecule, allowing to reach a quantum yield in triplet formation that approaches unity; (ii) the influence of the substitution on the ISC kinetics does not exclusively relies on classical heavy atom effects, but to a more complex interplay, involving molecular distortion and (iii) this last effect, and its influence on the ISC kinetics, depends strongly on the nature of the substituents.

Experimental

Synthesis

All reagents and chemicals from commercial sources were used without further purification. Solvents were dried and purified using standard techniques. Flash chromatography was performed with analytical-grade solvents using Aldrich silica gel (technical grade, pore size 60 Å, 230-400 mesh particle size). Flexible plates ALUGRAM® Xtra SIL G UV254 from MACHEREY-NAGEL were used for TLC. Compounds were detected by UV irradiation (Bioblock Scientific). NMR spectra were recorded with a Bruker AVANCE III 300 (1H, 300 MHz and 13C, 75MHz) or a Bruker AVANCE DRX500 (1H, 500 MHz; 13C, 125 MHz). Chemical shifts are given in ppm relative to TMS and coupling constants J in Hz. IR spectra were recorded on a Bruker spectrometer Vertex 70 and UV-vis spectra with a Perkin Elmer 950 spectrometer. Matrix Assisted Laser Desorption/Ionization was performed on MALDI-TOF MS BIFLEX III Bruker Daltonics spectrometer using dithranol as matrix. High resolution mass spectrometry (HRMS) was performed with a JEOL JMS-700 B/E. Synthetic procedures to prepare BTXI, BTXI-Br, BTXI-BrBr, BTXI-I, BTXI-BrI, BTXI-NO2 and BTXI-NPh2 are accessible from our early reported publications. 24,28,33 General procedure for the synthesis of BTXI-SO2 derivatives: a solution of m-Chloroperbenzoic acid (mCPBA) (4 eq) in CH2Cl2 was added dropwise on a stirred solution of BTXI (1 eq) in CH2Cl2. After a night of stirring at room temperature, the reaction was quenched with a saturated solution of Na2S2O3. The organic phase was then subsequently extracted with dichloromethane, washed with a saturated solution of NaHCO3 before being dried with MgSO4 and finally concentrated under vacuum.

BTXI-SO2:

The crude was purified by column chromatography on silica gel (eluent: CH2Cl2) affording a pale yellow solid in quantitative yield (100% yield). 1

BTXI-SO2-NO2:

The crude was purified by column chromatography on silica gel (eluent: CH2Cl2) affording a pale yellow solid in 74% yield. 1 H NMR (300 MHz, CDCl3): δ (ppm) 8.87 (d, J = 8.0 Hz, 1H), 8.78 (s, 1H), 8.73 (d, J = 8.0 Hz, 1H), 8.31 (dd, J = 7.8, 1.4 Hz, 1H), 8.24 (dd, J = 7.8, 1.0 Hz, 1H), 7.85 (td, J = 7.8, 1.4 Hz, 1H), 7.77 (td, J = 7.8, 1.0 Hz, 1H), 5.10 -4.90 (m, 1H), 2.32 -2.13 (m, 2H), 2.01 -1.81 (m, 2H), 0.90 (t, J = 7.5 Hz, 6H). 13

BTXI-SO2-I:

The crude was purified by column chromatography on silica gel (eluent: CH2Cl2) affording a pale yellow solid in 31% yield. 1

BTXI-SO2-BrBr:

The crude was purified by column chromatography on silica gel (eluent: CH2Cl2) affording a pale yellow solid in 19% yield. 1

Photophysical measurements

Absorption spectra were recorded on a JASCO V-650 spectrophotometer in diluted solution (ca. 10 -5 or 10 -6 mol L -1 ), using spectrophotometric grade solvents. Emission spectra were measured using Horiba-Jobin-Yvon Fluorolog-3 fluorimeter. The steady-state luminescence was excited by unpolarised light from a 450 W xenon continuous wave (CW) lamp and detected at an angle of 90° for measurements of dilute solutions (10 mm quartz cuvette) by using a Hamamatsu R928. Spectra were corrected for both excitation source light-intensity variation and emission spectral responses. Luminescence quantum yields Q were measured in diluted solutions with an absorbance lower than 0.1, by using the following Equation 1:

(1)
where A(λ) is the absorbance (or optical density) at the excitation wavelength, n the refractive index of the solvent and D the integrated luminescence intensity. "r" and "x" stand for reference and sample, respectively. Here, the reference is coumarin-153 in methanol (Qr = 0.45). 34 Excitations of reference and sample compounds were performed at the same wavelength. The reported results are the average of 4-5 independent measurements at various absorbances (comprised between 0.01-0.1) for both sample and reference. The plot of the integrated luminescence intensity vs. absorbance gives straight line with excellent correlation coefficients and the slope S can be determined for both sample (x) and reference (r). Equation 1becomes Equation 2.

(2)

For singlet oxygen quantum yield determination φΔ, the principle is exactly the same except that the singlet oxygen luminescence emission band (D) is integrated for both sample (x) and reference (r) compounds. A(λ) is the absorbance (or optical density) at the excitation wavelength. In this case it is very important that both experiments are conducted in the same solvent at exactly the same excitation wavelength (nx = nr). The reported results are the average of 4-5 independent measurements at various absorbances (comprised between 0.01-0.1) for both sample and reference. The plot of the integrated singlet oxygen luminescence intensity vs. absorbance gives straight line with excellent correlation coefficients and the slope S can be determined for both sample (x) and reference (r). In the present case, the reference is phenalenone (φΔr = 0.98 in dichloromethane). 35 Excited state lifetime analyses were measured at the maximum emission wavelength using a 390nm nanoLED by means of the time correlated single photon counting (TCSPC) method of diluted dichloromethane solutions at room temperature.

Computational details

Molecular calculations were carried out with the Gaussian16 code. [START_REF] Frisch | Gaussian 16 Rev. C[END_REF] The global hybrid functional PBE0 was used both for ground state and excited state geometry optimisations [START_REF] Adamo | [END_REF] This functional was chosen because of its frequently reported accuracy for modelling the localised electronic transitions in organic molecules. 38 Structural optimisations and subsequent frequency calculations for both the ground and excited states were performed using an all electron Pople triple zeta basis set with one polarisation function on all atoms and one diffuse function of heavier atoms, known as 6-311+G(d,p), for H, C, N, O, S and Br atoms. 39 For I atoms, the aug-cc-pVDZ basis set along with the associated pseudo-potential was used. Bulk solvent effects were included using the Polarizable Continuum Model (PCM) of Tomasi and co-workers. 40 Default radii (from the UFF, scaled by 1.1) were used. Transitions in absorption were simulated by computing the first 10 transitions in TD-DFT at the same level of theory. The fluorescence energies were obtained by relaxing the first excited state geometry in TD-DFT at the same level of theory.

The Dalton 41 program was used to compute the SOC between the two first triplet states (namely T1 and T2) and the S1 state at the S1 optimised geometry using the quadric-response TD-DFT at the B3LYP/PCM level with the aug-cc-pVDZ basis set adapted for the Douglas-Kroll calculations. 42 The Spin-Orbit Coupling was computed using the Douglas-Kroll Hamiltonian along with the spin-orbit mean field approach. 43 Vibrationally resolved spectra were obtained, after performing frequency calculations for the ground and excited states, using the FC classes program. 44 The reported spectra were simulated at 0K using convolution Gaussian function presenting a FWHM of 0.05 eV. 25 overtones for each mode and 20 combination bands were used as a maximum of 10 10 integrals for each class.

Results and discussion

Synthesis BTXI, BTXI-Br, BTXI-BrBr, BTXI-I, BTXI-BrI, BTXI-NO2 and BTXI-NPh2 were prepared according to our early reported preparations. 24,28,33 Sulfone derivatives (BTXI-SO2, BTXI-SO2-NO2, BTXI-SO2-Br, BTXI-SO2-BrBr and BTXI-SO2-I) were simply afforded by reacting the corresponding functionalized BTXI with m-Chloroperbenzoic acid (mCPBA) in dichloromethane, overnight and at room temperature. 20

Spectroscopy

All molecules were studied in diluted dichloromethane solutions and systematically evaluated regarding their absorption, emission wavelength, emission quantum yield and lifetime. The generation of singlet oxygen, as a key parameter of this study, was measured by directly looking at the phosphorescence band of singlet oxygen centered at 1270 nm. Finally, the triplet state of all molecules was also assessed when possible, by checking the phosphorescence spectrum in glass medium (ethanol/methanol 4:1 at 77K) after applying a 0.05 ms delay to filter the short component of emission coming from fluorescent states. (Table 1 1.00 15500 a Measured using Coumarin-153 as reference (ΦF = 0.45 in methanol). b Measured using Phenalenone as reference (ΦF = 0.95 in dichloromethane). c Calculated at the zero-phonon transition correspondently marked with *. All compounds present similarities in their spectral signature: absorption is dominated by a broad yet structured intense band, whose position is slightly affected by the presence and nature of the substituents in position 5, but undergoes drastic evolution upon oxidation of the sulfur atom (vide infra). Emission is characterised by a moderate Stokes shift, which tends to increase upon nitro substitution in both the sulfur and sulfone series. Along with the broadening and complete loss of vibronic structure of the band, this suggests a transition in the character of the emissive state from a local π->π* to a transition of a different nature for both BTXI-NO2 and BTXI-SO2-NO2. This is also apparent, in the case of BTXI-NO2 from the increased emission lifetime despite a drastically lowered emission quantum yield, typical of a forbidden nature of the associated transition. Based on TD-DFT calculations, we can confirm the π->π* and local character of the first S0→S1 transition for almost all the molecules investigated here, characterised by DCT around 2.5 Å for the BTXI series and around 3.1 Å for the BTXI-SO2 series. 45,46 In the specific case of BTXI-NO2 and BTXI-SO2-NO2, a large rotation of the -NO2 group at the S1 state first makes the transition S0→S1 out of the Franck-Condon region resulting in the loss of the band's structure and then changes the nature of the transition from a π->π* to a n->π* with a computed 0.0 oscillator strength explaining the long life time and the low fluorescence efficiency for the nitrated molecules. For interested readers, a comparison between simulated and experimental spectra is given in Supporting Information. Although quantitative in the native BTXI compound, luminescence quantum yield drops with substitution. In the sulfur series, except for BTXI--NO2, a good reverse correlation exists between the decrease in luminescence quantum yield and the increase in singlet oxygen generation (vide infra). On the other hand, in the sulfone series, luminescence is fully quenched for all compounds as shown by the low values of quantum yields and the short excited state lifetimes.

Already reported in one of our previous paper, the brominated BTXI-Br (singlet oxygen generation quantum yield φΔ= 0.11), the diphenylamine substituted BTXI-NPh2 (φΔ= 0.46), and nitro derivative BTXI-NO2 (φΔ= 0.44) were shown to be efficient singlet oxygen sensitizers due to important spin orbit coupling (SOC) (as also manifested form the observation of a distinctive phosphorescence signal at 77K) as it will be confirmed by quantum chemistry later in this article. Herein, we extend our investigation on the effect of halogen substitution on the ISC parameters. First, the bromine atome of the BTXI-Br in was replaced by an iodine atom. It turns out that while this substitution had only marginal effect on the position and shape of the absorption and emission band in the resulting BTXI-I molecule, a significant decrease of the emission quantum yield (from 0.78 to 0.58) and lifetime (from 6.5 to 4.7 ns) was observed, with a concomitant increase in singlet oxygen generation efficiency (from 0.11 to 0.37), thus witnessing a more pronounced effect of the heavier iodine atom as compared to bromine. Introduction of a second halogen atom in the bay position was thereafter investigated, revealing a marked increase in the ISC kinetics along with the heavy atom effect. Compared to BTXI-Br, addition of another bromine substituent (BTXI-BrBr φΔ= 0.23) was indeed less effective in boosting singlet oxygen generation efficiency than substituting the bay position with an iodine (BTXI-BrI φΔ= 0.70). Then, we systematically compared the sulfone-modified analogues of BTXI to their parent molecules. Quite remarkably, oxidation of the sulfur bridge did not only result in a marked change in the position of the absorption and emission maxima (ca. 80-90 nm blue shift of both bands upon oxidation, depending on the nature of the substituents with respect to their reference molecule) but also to a tremendous effect on their ISC efficiencies. While the BTXI features a luminescence quantum yield near unity and does not promote any measurable singlet oxygen generation, its oxidized version, namely the BTXI-SO2 experiences a drastic drop in luminescence efficiency (φem= 0.15) that goes with a burst in singlet oxygen efficiency, reaching an impressive value of 0.72. Even more remarkably, cumulative effect is achieved upon additional substitution of the BTXI core with halogen substituents, as singlet oxygen generation quantum yield reaches unity for BTXI-SO2Br, BTXI-SO2-I and BTXI-SO2-BrBr. In contrast, substitution with -NO2 leads to a less straightforward evolution: the resulting BTXI-SO2-NO2 molecule displays a singlet oxygen generation efficiency (φΔ= 0.57) that lies perfectly in between that of its parent BTXI-NO2 molecule (φΔ= 0.42) and that of BTXI-SO2 (φΔ= 0.72) suggestive of a distinct and perhaps concurrent photophysical mechanism of SOC enhancement in both cases.

Hence, a detailed theoretical study was undertaken to investigate the nature of these mechanisms.

Theoretical investigation of the ISC

From an electronic point of view, ISC processes, occurring between singlet and triplet states, depend on the interplay of two parameters. The latter are directly involved in the equation 3, that describes the density of probability of an ISC transition, considered herein between S1 and T1 states (Fermi Golden rule for a first order transition). 47,48 Hence, the first parameter of importance is the SOC between these two states, which was computed based on the Douglas-Kroll Hamiltonian using the Dalton code. The second parameter influencing the efficiency of the crossing is the energy proximity between the two states. The SOC computed between

Γ 𝑆1-𝑇1 = 2𝜋 ℏ |⟨𝑇 1 |𝐻 ̂𝑆𝑂 |𝑆 1 ⟩| 2 𝛿(𝐸 𝑆1 -𝐸 𝑇1 ) (3)
* the S1 state and the two first triplet states (namely T1 and T2) along with the energy difference between these states are given in Figure 5 and Figure 6.

Figure 5. Computed spin-orbit coupling between the (S1,T1) and (S1,T2) states along with (S1,T1) and (S1,T2) energy difference of the BTXI series at the S1 stable geometry and at the B3LYP/PCM/aug-cc-PVTZ level.

Figure 6. Computed spin-orbit coupling between the (S1,T1) and (S1,T2) states along with (S1,T1) and (S1,T2) energy difference of the BTXI-SO2 series at the S1 stable geometry and at the B3LYP/PCM/augcc-PVTZ level.

As a general observation, variations in singlet oxygen generation efficiency from one molecule to another are generally associated to a computed variation in SOC. This assumption holds true in the BTXI series, the large increase of efficiency of the dihalogenated compound as compared with their monohalogenated counterpart characterized experimentally being well corroborated by calculations. Besides the expected heavy atom effect that generally occurs upon halogen substitution, 49,50 which accounts here for the increase of SOC between BTXI-Br and BTXI-I on the one hand and between BTXI- BrBr and BTXI-BrI on the other, we can ascribe the extent of this phenomenon occurring in dihalogenated compounds to the distortion of the molecule induced by the halogen atom placed in the bay position. This distortion relates to a loss of planarity of the aromatic core, quantified by the Θangle in figure 7, which is much enhanced in dihalogenated compounds (Θ=23.1 and 29.3° in BTXI-BrBr and BTXI-BrI, respectively) compared to their monohalogenated analogues (Θ=2.5° in BTXI-Br). To further and more specifically quantify the influence of distortion from planarity on the ISC, we also computed the SOC on molecules that were forced to be flat (Cs point group imposed in the geometry relaxation) and compared to SOC computed for geometries relaxed without any constrains (Figure 7). [51][52][53] To the best of our knowledge, this is the first time that such a large spin-vibronic coupling is reported for a distortion of an extended rigid π-system.

As discussed before in this article, the rotation of the -NO2 group at the S1 state induces a change in the nature of transition from π ->π* to n->π*. This is clearly visible by the large change in the shape of orbitals when computed at the S0 and S1 geometries (see Supporting Information). This excited state reorganisation induced symmetry breaking thus explains both the low fluorescence efficiency of this molecule (vide supra) and the large ISC as the symmetry forbidden n->π* transitions is known to strongly favor the SOC. 54,55 The strong ISC of the BTXI-SO2 series, highlighted by their large φΔ experimentally measured, is also supported by TD-DFT with a strong SOC computed between the S1 and T1 states. For these molecules, the substituent-mediated aromatic distortion also slightly increases the SOC (+33%), but contrary to the dihalogenated BTXI series (BTXI-BrBr and BTXI-IBr), the forced Cs conformation of BTXI-SO2 already provides a strong SOC. In other words, the -SO2 group itself constitutes the main driving force for the ISC process. As mentioned before, a particularly intriguing experimental observation was the discrepancy between the additive effect observed on ISC efficiency when merging -SO2 and halogen substitution (BTXI-Br, φΔ= 0.11; BTXI-SO2, φΔ=0.72; BTXI-SO2-Br, φΔ=0.97) and the corresponding decrease in ISC efficiency between BTXI-SO2 and BTXI-SO2-NO2 (BTXI-NO2, φΔ= 0.42; BTXI-SO2, φΔ=0.72; BTXI-SO2-NO2, φΔ=0.57). While the former is again well explained by distortion phenomena, the latter requires a less straightforward analysis. Interestingly, in the BTXI-SO2 series, the T2 state is particularly close in energy (<0.3 eV) to the S1 state for the BTXI-SO2 and BTXI-SO2-Br compounds. A thermal population of T2 from S1 is thus possible opening a new path for triplet population and finally for singlet oxygen generation. For the BTXI-SO2-NO2, the T2 state is further in energy (>0.4 eV) and thus more difficult to populate by thermal energy that could explain the lower singlet oxygen generation efficiency of this last compound.

Conclusions

The study depicted herein confirms the exceptional potential of BTXI derivatives as triplet state harnesser and singlet oxygen photosensitizers. By playing on the nature and positions of the substituents on the π-conjugated structure, and simultaneously varying the nature and oxidation state of the heteroatoms involved in the heteroaromatic cycles, we were able to manipulate the ISC kinetics to afford in some cases a singlet oxygen generation quantum efficiency reaching unity. A systematic spectroscopic study coupled with computational modelling afforded a mean to study the effect of each individual modification of the skeleton on the global ISC process. We found out that halogenation of the molecule on the positions 5 and 11 rather than just one (5) resulted in much enhanced ISC kinetics, and that iodine were much more effective than bromine as substituents in that regard. The origin of the large increase of the ISC when both 5 and 11 positions are substituted stems from a spin-vibronic coupling itself activated by the distortion of the Pi-system due to the substituent. Even more interestingly, a strong improvement in the ISC could be achieved by simple oxidation of the thioxanthene moieties of the molecule, providing a very simple and efficient chemical mean to drive triplet formation in BTXI derivatives. Finally, combination of both strategies (sulfonation of the BTXI ring and halogenation of both positions) was highly efficient in maximizing ISC processes within the chromophore: as an illustration, a singlet oxygen generation efficiency φΔ= 1 was monitored for two molecules of the series. We believe that this study constitutes a useful guideline for future works aiming at taking advantage of photoinduced triplet state generation in BTXI derivatives, which could find interesting applications in many topics ranging from molecular electronics to biomedicals.

Figure 1 .

 1 Figure 1. Representation of the well-studied perylenebisimide molecule, the BTXI core and the derivatives studied in this report.

  C NMR (75 MHz, CDCl3): δ (ppm) 146.7, 136.1, 134.2, 134.0, 132.1, 131.3, 130.5, 129.1, 129.0, 127.1, 126.9, 125.1, 125.0, 124.5, 58.8, 25.0, 11.4. HRMS (EI): m/z calcd for C23H18N2O6S: 450.0880, found: 450.0886.

  H NMR (500 MHz, CDCl3): δ (ppm) 9.23 (s, 1H), 8.75 (d, J = 8.0 Hz, 1H), 8.61 (d, J = 8.2 Hz, 1H), δ 8.32 (dd, J = 7.8, 1.5 Hz, 1H), 8.27 (d, J = 7.8, 1H), 7.80 (td, J = 7.6, 1.2 Hz, 1H), 7.75 (td, J = 7.6, 1.2 Hz, 1H), 5.00 (m, 1H), 2.28 -2.15 (m, 2H), 1.98 -1.86 (m, 2H), 0.90 (t, J = 7.5 Hz, 6H). 13 C NMR (125 MHz, CDCl3): δ (ppm) 144.6, 139.2, 135.9, 133.7, 132.1, 131.2, 129.8, 129.1, 128.3, 125.8, 125.5, 125.3, 91.0, 58.5, 25.1, 11.4. HRMS (FAB): calcd for C23H18INO4S: 531.0001, found: 531.0002.

Figure 2 .

 2 Figure 2. Normalised absorption spectra of the selected BTXI derivatives in diluted dichloromethane solutions at room temperature.

Figure 3 .

 3 Figure 3. Normalised emission spectra of the selected BTXI derivatives in diluted dichloromethane solutions at room temperature.

Figure 4 .

 4 Figure 4. a) Normalised excitation (λem= 500 nm) (black trace), emission (λexc= 375 nm) at room temperature (red trace) and 77K (blue trace) without (full trace) or with a delay of 0.05 ms delay (dashed trace) of BTXI-SO2-BrBr in a mixture ethanol: (4:1) methanol. b) Phosphorescence band of singlet oxygen observed for BTXI-SO2-BrBr in a diluted dichloromethane solution at room temperature.

1 BTXI 1 BTXI 1 BTXI 1 BTXI 1 BTXI 1 BTXI

 111111 78 eV -SOC = 9.7 cm -1 +0.32 eV -SOC = 74.5 cm -78 eV -SOC = 14.1cm -1 +0.34 eV -SOC = 28.9 cm -14 eV -SOC = 13.7 cm -1 +0.40 eV -SOC = 26.4 cm -14 eV -SOC = 13.7 cm -1 +0.40 eV -SOC = 26.4 cm -96 eV -SOC = 0.4 cm -1 +0.24 eV -SOC = 2.0 cm -74 eV -SOC = 0.9 cm -1 +0.45 eV -SOC = 1.9 cm -

Figure 7 .

 7 Figure 7. (a) Influence of the planarity loss of the aromatic system of the SOC between the S1 and T1 states. (b) Computed ground state and experimental (XRD) dihedral angle quantifying the loss of planearity of the molecule.

  Bredas et al. on molecules designed for TADF application and for distortions induced by the rotation between two parts of the molecule.

  H NMR (300 MHz, CDCl3): δ (ppm) 8.93 (s, 1H), 8.79 (s, 1H), 8.71 -8.62 (m, 1H), 8.43 -8.31 (m, 1H), 7.80 -7.69 (m, 2H), 4.97 (m, 1H), 2.27 -2.11 (m, 2H), 1.99 -1.85 (m, 2H), 0.89 (t, J = 7.5 Hz, 6H). 13 C NMR (75 MHz, CDCl3): δ (ppm) 138.4, 137.6, 135.8, 135.3, 132.7, 132.1, 131.3, 130.9, 130.7, 129.0, 126.2, 125.9, 123.5, 121.7, 58.7, 25.0, 11.3. HRMS (FAB): calcd for C23H17Br2NO4S: 560.9245, found: 560.9238.

Table 1 .

 1 and Figures 2,3,4 and S1-S21). Summary of the main spectroscopic and photophysical data for all studied BTXI derivatives.

		λabs(max) (nm)	λem (nm)	Φf a	τobs (ns)	ΦΔ b	ETriplet (cm -1 ) c
	BTXI	455	510	0.99	7.48	-	-
	BTXI-Br	469	500	0.78	6.49	0.11	14925
	BTXI-BrBr	468	530	0.60	7.15	0.23	14450
	BTXI-I	471	505	0.58	4.74	0.37	14950
	BTXI-BrI	471	535	0.39	4.68	0.70	14350
	BTXI-NO2	486	585	0.01	8.14	0.42	15870
	BTXI-NPh2	483	600	0.33	10.68	0.46	14600-
	BTXI-SO2	379	442	0.15	<1 d	0.72	16250
	BTXI-SO2-NO2	396	495	0.01	<1 d	0.57	-
	BTXI-SO2-Br	386	455	0.06	<1 d	0.97	15550
	BTXI-SO2-BrBr	388	474	0.02	<1 d	1.00	15200
	BTXI-SO2-I	380	446	0.01	<1 d