François Romagné
email: francois.romagne@innate-pharma.fr

Eric Vivier

Natural killer cell-based therapies

Allotransplantation of natural killer (NK) cells has been shown to be a key factor in the control and cure of at least some hematologic diseases, such as acute myeloid leukemia or pediatric acute lymphocytic leukemia. These results support the idea that stimulation of NK cells could be an important therapeutic tool in many diseases, and several such approaches are now in clinical trials, sometimes with conflicting results. In parallel, recent advances in the understanding of the molecular mechanisms governing NK-cell maturation and activity show that NK-cell effector functions are controlled by complex mechanisms that must be taken into account for optimal design of therapeutic protocols. We review here innovative protocols based on allotransplantation, use of NK-cell therapies, and use of newly available drug candidates targeting NK-cell receptors, in the light of fundamental new data on NK-cell biology.

Introduction

Natural killer (NK) cells are the front-line troops of the immune system that help to keep you alive while your body marshals a specific response to viruses or malignant cells. They constitute about 10% of circulating lymphocytes [START_REF] Vivier | Innate or adaptive immunity? The example of natural killer cells[END_REF] and are on patrol constantly, always on the lookout for virus-infected or tumor cells, and when detected, they lock onto their targets and destroy them by inducing apoptosis while signaling danger by releasing inflammatory cytokines. By using NK cells that do not need prior exposure to their target, the innate immune system buys time for the adaptive immune system (T cells and B cells) to build up a specific response to the virus or tumor. Recent advances in understanding this process have led to the hope that NK cells could be harnessed as a therapy for cancers and other diseases, and we shall outline recent progress in understanding NK-cell biology that brings this approach into the realm of clinical trials.

Considerable advances have been made in understanding the molecular mechanisms governing NK-cell activation, which are assessed by the cells' ability to lyse different targets and/or secrete inflammatory cytokines such as interferon gamma (IFN-g) when in their presence. NK-cell activation is the result of a switch in the balance between the positive and negative signals provided by two main types of receptors. The receptors NKG2D, NKp46, NKp30, NKp44, the activating form of KIR (killer cell immunoglobulin-like receptor), known as KIR-S, and CD16 provide positive signals, triggering toxicity and production of cytokines. Although some of the ligands of these receptors remain unknown, the discovery of NKG2D ligands (MICA and the RAET1 family) and the NKp30 ligand (B7H6) suggests that such receptors recognize molecules that are seldom present on normal cells but are induced during infection or carcinogenesis. It is worth noting that CD16 recognizes antibody-coated target cells through their Fc portion, the receptor that mediates antibody-dependent cellular cytotoxicity, an important mechanism of action of therapeutic monoclonal antibodies (mAbs). The function of KIR-S, a family of activating receptors with a lot of homology with inhibitory KIRs (KIR-L) including the sharing of some ligands, remains largely unknown.

In the normal state of affairs, there are checks and balances to keep NK cells from attacking normal cells: activating ligands are rare on normal cells and there are inhibitory receptors on NK cells (Figure 1). The most studied inhibitory receptors are a family of immunoglobulin (Ig)-like receptors with two (KIR2DL1 and KIR2DL2/3) or three (KIR3DL1) Ig-like domains, and immunoreceptor tyrosine-based inhibition intracellular motifs (ITIMs), which transduce negative signals [START_REF] Vivier | Natural killer cell signaling pathways[END_REF]. The ligands of these receptors are well characterized and each consist of large families of major histocompatibility complex (MHC) class I gene variants (alleles) sharing structural determinants. KIR2DL1 and KIR2DL2/3 molecules recognize MHC-C alleles with a lysine or an asparagine at position 80 (collectively termed C2 alleles and C1 alleles, respectively), whereas KIR3DL1 recognizes MHC-B alleles sharing a Bw4 epitope, representing about half of the overall MHC-B alleles. Another receptor, NKG2A, recognizes HLA-E, an MHC class I-like molecule, loaded mostly with peptides derived from other class I molecules [START_REF] Parham | MHC class I molecules and KIRs in human history, health and survival[END_REF]. The expression of these molecules is variegated, and an individual NK cell will express either one or several inhibitory receptors. In combination, these receptors are sensors of the presence of MHC class I molecules on target cells and inhibitors of NK function. An integrated, although simplified, view of NK-cell activation is that NK cells quantitatively integrate positive and negative signals provided by cancer cells or infected cells, which express NK-stimulatory ligands de novo, while often down-modulating MHC class I to avoid detection by T cells.

There has been considerable interest in stimulation of NKcell activity in recent years because of genetic studies, both in preclinical and clinical settings, showing that it can increase tumor immunosurveillance and eradicate established hematological diseases such as acute myeloid leukemia (AML), as well as some viruses [START_REF] Terme | Natural killer cell-directed therapies: moving from unexpected results to successful strategies[END_REF]. In mouse models, the expression of NK-stimulatory NKG2D ligands not only induces short-term rejection of tumors, but also induces a protective adaptive immune response [START_REF] Diefenbach | Rae1 and H60 ligands of the NKG2D receptor stimulate tumour immunity[END_REF]. Similarly, mice genetically deficient in NKG2D are more susceptible to spontaneous cancer than wild-type mice [START_REF] Guerra | NKG2Ddeficient mice are defective in tumor surveillance in models of spontaneous malignancy[END_REF]. In humans, the development of allotransplantation, a clinical procedure involving transplantation of genetically nonidentical cells (routinely used in AML), shed light on the role of NK cells and particularly the role of inhibitory receptors in this process. For certain donor recipient pairs, genetic differences in MHC class I genes between the donor and the recipient cause the KIR-expressing cells NK cells sense interacting cells via their activating and inhibitory receptors. The density of ligands for these receptors dictates whether or not this interaction will lead to NK-cell activation and hence cytotoxicity and/or cytokine secretion. MHC, major histocompatibility complex; KIR, killer cell immunoglobulin-like receptor.

from the donor to not recognize their inhibitory MHC class I ligands in the recipient, leaving a subpopulation of donor NK cells free from inhibition, referred to as "alloreactive" NK cells. For example, a donor NK-cell subpopulation expressing only KIR2DL1 transplanted in C1/C1 homozygotes or KIR2DL2/3 NK cells transplanted in C2/C2 individuals do not find their cognate inhibitory ligands and become alloreactive. In haploidentical MHCmismatched hematopoietic stem cell transplantation (HSCT)-a situation where one MHC haplotype is similar between donor and recipient whereas the other is fully mismatched-that absence of inhibition due to the KIR-MHC incompatibility results in major differences in the clinical outcome [START_REF] Ruggeri | Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants[END_REF][START_REF] Ruggeri | Donor natural killer cell allorecognition of missing self in haploidentical hematopoietic transplantation for acute myeloid leukemia: challenging its predictive value[END_REF][START_REF] Ruggeri | Role of natural killer cell alloreactivity in HLA-mismatched hematopoietic stem cell transplantation[END_REF]. Clinical benefit correlates with the presence in the recipient of these disinhibited alloreactive NK cells from the donor, which are effective against recipient tumor cells. In viral infections, particular combinations of NK-activating receptors or KIR and their ligands are protective. Presence of the activating receptor KIR3DS1 and its putative ligand HLABw4-I80 has been shown to be a key factor in preventing HIV infection from leading to full-blown AIDS [START_REF] Alter | Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes[END_REF][START_REF] Alter | HLA class I subtype-dependent expansion of KIR3DS1+ and KIR3DL1+ NK cells during acute human immunodeficiency virus type 1 infection[END_REF][START_REF] Carrington | KIR-HLA intercourse in HIV disease[END_REF]. In hepatitis C, KIR2DL3 homozygosity and HLA-C1 homozygosity are beneficial in both early eradication of infection and response to standard treatment (type I IFN + ribavirin) [START_REF] Khakoo | HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection[END_REF][START_REF] Vidal-Castiñeira | Effect of killer immunoglobulin-like receptors in the response to combined treatment in patients with chronic hepatitis C virus infection[END_REF]. Homozygosity of KIR2DL3 and HLA-C1 alleles has been reported to lead to lower levels of NK inhibition than other pairs of KIR ligand combinations [START_REF] Ahlenstiel | Distinct KIR/HLA compound genotypes affect the kinetics of human antiviral natural killer cell responses[END_REF][START_REF] Moesta | Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3[END_REF], suggesting that this underlies the enhanced response to hepatitis C. However, as KIR can also be expressed by some T-cell subsets, it remains to be firmly established whether NK cells are responsible for these effects. Nevertheless, the results of these studies suggest that we should extend the design of NK cell-based therapies to diseases other than cancer, such as infections and inflammation.

We will review here the recent advances that could help with the design of proper protocols and therapies and advance the use of NK cells in the clinic, starting with allotransplantation (transplantation between genetically different individuals of the same species). This will be followed by a discussion of the cell therapy procedures that are being developed, and the pharmacological agents that are currently or could be used in clinical trials to take advantage of the activity of NK cells.

Lessons from transplantation

Since the initial data from haploidentical HSCT, a number of retrospective studies in allostransplantation have been published, sometimes leading to differing clinical outcomes [START_REF] Witt | The influence of NK alloreactivity on matched unrelated donor and HLA identical sibling haematopoietic stem cell transplantation[END_REF]. These conflicting results may be explained in the light of new findings in NK-cell physiology and maturation.

Initially, alloreactive NK cells were simply defined by having KIRs that were only incompatible with the host MHC, and several studies have identified such alloreactive NK cells that are effective against AML blasts. However, it has been shown in normal mice that NK cells with only inhibitory receptors incompatible with self MHC class I alleles do arise physiologically (i.e., not after transplantation) but are partially functionally disabled [START_REF] Raulet | Self-tolerance of natural killer cells[END_REF]. Hence, NK cells undergo a complex maturation process that necessitates the interaction of their inhibitory receptors with their ligands, in order to be fully functional against class I negative cells (recognition of missing self; see [START_REF] Raulet | Self-tolerance of natural killer cells[END_REF] for review). The precise molecular mechanisms and localization of this process remain largely unknown in mice but were shown to be dynamic and reversible [START_REF] Elliott | MHC class I-deficient natural killer cells acquire a licensed phenotype after transfer into an MHC class I-sufficient environment[END_REF][START_REF] Joncker | Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment[END_REF]. It has since been confirmed in humans that NK cells with only MHCincompatible KIR cells do exist in normal individuals but, as in mice, they are partially functionally disabled [START_REF] Anfossi | Human NK cell education by inhibitory receptors for MHC class I[END_REF][START_REF] Cooley | A subpopulation of human peripheral blood NK cells that lacks inhibitory receptors for self-MHC is developmentally immature[END_REF], indicating that human NK cells also undergo education much like mouse NK cells. This leads to a revision of the concept of alloreactivity: KIR mismatch is necessary to induce activity against MHC-positive cells (we will refer to these cells as potentially alloreactive) but not entirely sufficient, as they must have undergone an education process. It follows that functional assays must be performed to demonstrate activity and define truly alloreactive cells.

These new findings may lead to reconciliation of the conflicting data from allogeneic HSCT. Allogeneic HSCT (from a nonidentical donor) is a complex clinical procedure, with considerable differences in the nature and origin of the graft, as well as in pregraft treatments (conducted to remove recipient hematopoietic cells and thereby allow the graft to implant) and postgraft treatments (to prevent graft-versus-host disease [GVHD] caused by donor T cells). Generally, there are two main scenarios. In the first, haploidentical grafts consisting of high doses of highly purified donor CD34positive hematopoietic stem cells, with very few mature cells, are injected after very intense conditioning regimens of the host to avoid graft rejection (there is virtually no postgraft treatment as the graft is highly T cell-depleted) (Figure 2). Truly alloreactive NK cells have been consistently found ex vivo following such transplantation, in an activated state resulting from missing-self recognition, and this scenario is associated with an improved clinical outcome [START_REF] Moretta | Killer Ig-like receptor-mediated control of natural killer cell alloreactivity in haploidentical hematopoietic stem cell transplantation[END_REF]. Unfortunately, such haploidentical procedures also require profound immunosuppression of the host, and the treatmentrelated morbidities caused by infection are high, so such procedures are not used widely. In the second scenario, allogeneic HSCT can be matched except at a given HLA-B or HLA-C allele, and require much less conditioning pregraft, but more immunosuppressive treatment postgraft to avoid GVHD. Such protocols vary widely depending on the laboratories, both in terms of pregraft and postgraft treatments and cell content in the graft (mature cell content and origin of graft consisting of either bone marrow cells or mobilized peripheral cells). Not surprisingly, such protocols vary widely in the KIR mismatch effect, with outcomes to match: beneficial, neutral, or even pejorative. Taking into account the new findings on NK-cell physiology, the current prevailing hypothesis is that in haploidentical HSCT, the harsh conditioning regimen and high CD34-positive cell content allow the donor NK cells to mature with a recognition of the "self" MHC type on the donor hematopoietic cells, and therefore become truly alloreactive against residual recipient blast cells, whereas normal host tissues are spared because of lack of NK-stimulatory ligand expression [START_REF] Pende | Anti-leukemia activity of alloreactive NK cells in KIR ligand-mismatched haploidentical HSCT for pediatric patients: evaluation of the functional role of activating KIR and redefinition of inhibitory KIR specificity[END_REF][START_REF] Haas | NK-cell education is shaped by donor HLA genotype after unrelated allogeneic hematopoietic stemcell transplantation[END_REF]. In nonhaploidentical situations, education of NK cells on donor HLA may be lacking in some graft preparation and pregraft regimens, which might account for the neutral effects seen (cells remain potentially alloreactive). Conflicting results in nonhaploidentical situations [START_REF] Giebel | Survival advantage with KIR ligand incompatibility in hematopoietic stem cell transplantation from unrelated donors[END_REF][START_REF] Davies | Evaluation of KIR ligand incompatibility in mismatched unrelated donor hematopoietic transplants. Killer immunoglobulin-like receptor[END_REF] may be also explained by different treatments resulting in different T-cell levels in grafts and consequently different levels of GVHD [START_REF] Cooley | Donors with group B KIR haplotypes improve relapse-free survival after unrelated hematopoietic cell transplantation for acute myelogenous leukemia[END_REF]. This hypothesis is further supported by protocols where the graft origin is cord blood, a situation with few mature T cells in the graft, which results in a beneficial outcome [START_REF] Willemze | Eurocord-Netcord and Acute Leukaemia Working Party of the EBMT: KIR-ligand incompatibility in the graft-versus-host direction improves outcomes after umbilical cord blood transplantation for acute leukemia[END_REF].

In truly matched transplantation, there are no obvious reasons for alloreactive cells to develop as the MHC of donor and recipient are the same and the maturation of NK cells should spare all host cells (either normal or NK-stimulating ligand-expressing cells). Surprisingly, even in completely MHC-matched transplantation, in particular in T-depleted grafts, functionally alloreactive NK cells have been reported, with an improved outcome for patients homozygous for HLA-C1 or HLA-C2, for example [START_REF] Yu | Breaking tolerance to self, circulating natural killer cells expressing inhibitory KIR for non-self HLA exhibit effector function after T cell-depleted allogeneic hematopoietic cell transplantation[END_REF][START_REF] Sobecks | Survival of AML patients receiving HLA-matched sibling donor allogeneic bone marrow transplantation correlates with HLA-Cw ligand groups for killer immunoglobulin-like receptors[END_REF]. In the same vein, it has been recently demonstrated in a large retrospective study that the KIR genotype alone influences clinical outcome, with the presence of KIR2DL3 and/or absence of KIR2DL2 and KIR2DS2 being less favorable, opening the way for the selection of donors based on KIR genotype in matched allotransplantation [START_REF] Cooley | Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia[END_REF]. The functional basis of such observations is still incompletely understood: during NK-cell reconstitution from stem cells, KIR expression is variegated, and potentially alloreactive cells appear, but, as mentioned above, if such reconstitution was equivalent to normal NK-cell maturation, they should be functionally impaired and tolerant to self. It is possible that during hematopoietic reconstitution and in certain allograft protocols, the cytokine milieu, strength of inhibitory interaction and presence of different activating genes (depending on KIR genotype), and absence of T-cell interaction (T cell-depleted grafts) favor the maturation of truly alloreactive NK cells despite the presence of matched inhibitory receptors. Indeed, new studies describing NK-cell maturation point to the fact that the hyporesponsiveness of NK cells is very subtle and malleable, influenced by cytokines and probably genotype, and reversible [START_REF] Elliott | MHC class I-deficient natural killer cells acquire a licensed phenotype after transfer into an MHC class I-sufficient environment[END_REF][START_REF] Joncker | Mature natural killer cells reset their responsiveness when exposed to an altered MHC environment[END_REF]. In summary, although many studies strongly suggest the efficacy of KIR-mismatched NK cells, definitive studies are needed to optimize the clinical settings. We need a better understanding of NK-cell development and function after matched allogeneic transplantation, depending on the specific allotransplantation protocol used, to take full advantage of the alloreactive potential of NK cells.

Cell therapy protocols in development

The current view arising from the results of allotransplantation studies is that NK cells, and particularly allogeneic KIR-mismatched NK cells, are effective, at least in adult AML and pediatric acute lymphocytic leukemia, but that the effect may depend on NK-cell maturation/ activation state. One way to better control NK-cell functional status (as well as the ratio of NK cells to target cells) would be to generate large quantities of these cells in vitro and inject them either as a therapeutic regimen alone or after allotransplantation.

Historically, crude, short-term (1-2 days) interleukin (IL)-2-activated cells were used as graft material (lymphokine-activated killer [LAK] cells), and although this was enriched in NK cells, the LAK cells were mostly T cells, and the cellular content was poorly defined and variable (see [START_REF] Suck | Emerging natural killer cell immunotherapies: large-scale ex vivo production of highly potent anticancer effectors[END_REF] for review). Initial attempts to work with purified preparations of NK cells led to promising results, although with a limited number of patients. Autologous HSCT followed by injection of purified, short-term IL-2-stimulated KIR-mismatched NK cells in multiple myeloma patients destroyed multiple myeloma blasts in vitro, did not lead to graft failure, and the NK cells survived at least a few days [START_REF] Shi | Infusion of haploidentical killer immunoglobulin-like receptor ligand mismatched NK cells for relapsed myeloma in the setting of autologous stem cell transplantation[END_REF]. In a protocol not involving HSCT, purified short-term IL-2-activated haploidentical NK cells were injected into AML and other hematologic cancer patients after mild conditioning to avoid rejection of injected NK cells. This study showed that injected NK cells survived in the host for a few days and were well tolerated [START_REF] Miller | Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer[END_REF]. Although the number of patients is still too limited to draw firm conclusions, encouraging clinical signs of activity were seen in the above protocols. As neither protocol reached any doselimiting toxicity, these findings suggest that it may be possible to inject higher cell numbers if cell sources or ex vivo expansion procedures improve.

These initial results have prompted several groups to embark on the large-scale expansion of highly purified, GMP ("good manufacturing practice") grade NK cells after longer-term in vitro expansion. NK-cell purification by magnetic beads is followed by IL-2 expansion with or without feeder cells. Protocol for generation of single KIR-positive cells has also been designed but is not yet ready to be applied to large-scale clinical trials [START_REF] Siegler | Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients[END_REF]. Functional studies using NK cells against leukemic cells or NK infusion in xenogenic models have demonstrated, however, that the cells generated are very active. Some of the protocols have reached smallscale phase I clinical trials and have demonstrated that high numbers of these infused NK cells are safe in humans [START_REF] Barkholt | Safety analysis of ex vivo-expanded NK and NKlike T cells administered to cancer patients: a phase I clinical study[END_REF][START_REF] Fujisaki | Expansion of highly cytotoxic human natural killer cells for cancer cell therapy[END_REF].

The current caveats of such protocols are the complexity of the procedures required, which would make it difficult to increase the scale-up to multicenter clinical studies necessary for larger phase II trials. Indeed, successful transfer of cell therapy protocols (compliant with regulatory standards) to industry and large clinical trials requires a centralized cell culture factory and the use of frozen cells. NK-cell culture protocols do not yet meet this benchmark, but further refinements should solve these issues [START_REF] Berg | Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells[END_REF]. Moreover, alloreactive NK cells should be the most potent cells in cell therapy protocol and their source can be a problem outside the context of allotransplantation. This alone may prevent the use of such cells in larger trials. The development of antiKIRtherapeutic mAbs (see below and Figure 2) that block NK inhibition may allow the use of autologous cells as an easier source of cell material, by inducing alloreactivity of NK cells that would otherwise be MHCtolerant.

Another important problem to be solved is the fate of ex-vivo expanded NK cells after infusion. Indeed, if NK cells from allogeneic donors are used, they may be rejected by the host immune system despite the mild immunosuppression used in some protocols. Even in cases of autologous transplantation or allotransplantation where donor NK cells are not rejected, they may be short lived, and protocols usually involve daily injection of IL-2 to sustain NK levels and activation status [START_REF] Shi | Infusion of haploidentical killer immunoglobulin-like receptor ligand mismatched NK cells for relapsed myeloma in the setting of autologous stem cell transplantation[END_REF][START_REF] Miller | Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer[END_REF]. IL-2 injection may increase NK-cell lifespan and activity (although it has not been formally tested, by comparison with untreated cells) but also can generate outgrowth of Treg cells that may hamper the overall response to the tumor as shown in pilot clinical trials [START_REF] Barkholt | Safety analysis of ex vivo-expanded NK and NKlike T cells administered to cancer patients: a phase I clinical study[END_REF][START_REF] Geller | A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer[END_REF]. The very recent availability of GMP grade IL-15, now in phase I clinical trials by the National Institutes of Health (NIH) [START_REF] Geller | A phase II study of allogeneic natural killer cell therapy to treat patients with recurrent ovarian and breast cancer[END_REF] may circumvent the use of IL-2, providing a better activation signal for NK cells, both in vitro and in vivo, without promoting Treg expansion.

Pharmacological agents in development to modulate NK activity

Although cell therapy protocols can be very useful to characterize NK-cell activity and, if successful, can translate into commercially available products, they remain very difficult and costly to develop on a large scale. It should be easier to move new drugs forward now that therapeutic agents are being tested that aim to stimulate NK-cell activity.

The most advanced compound specifically targeting NK cells is a blocking antiKIR mAb. This mAb, 1-7F9, recognizes KIR2DL1, 2, and 3 and therefore blocks the inhibition imposed by virtually all MHC class I C alleles, allowing it to be tested in all patients whatever their KIR and HLA genotypes. Building on the results of allotransplantation in AML and multiple myeloma patients [START_REF]gov -A phase I study of intravenous recombinant human IL-15 in adults with refractory metastatic malignant melanoma and metastatic renal cell cancer[END_REF], as well as preclinical data showing reconstitution of NK-cell lysis of MHC-positive multiple myeloma and AML blasts in vitro and in preclinical models [START_REF] Kröger | Clinical Trial Committee of the British Society of Blood and Marrow Transplantation and the German Cooperative Transplant Group: Comparison between antithymocyte globulin and alemtuzumab and the possible impact of KIR-ligand mismatch after dose-reduced conditioning and unrelated stem cell transplantation in patients with multiple myeloma[END_REF], clinical trials with 1-7F9 mAb in both diseases have been initiated. Phase I results showed good tolerability in both scenarios (Vey et al., manuscript in preparation), paving the way for phase II trials that are now ongoing. While the monoclonal 1-7F9 should be valuable to block the inhibition of NK cells, other products are now available that can enhance the activation of NK cells. One of the most promising of these is IL-15, which is a key cytokine for NK cells.

In the same vein, it has been shown by several groups that certain drugs, already available in the therapeutic arsenal, can increase the expression of NK-activating ligands on the tumor, and therefore increase NK tumor lysis in vivo. Initially, it was shown that some chemotherapy (5-FU, Ara-C, cisplatin) and radiation or ultraviolet therapy targeting the DNA damage pathway can increase expression of the NK-stimulating ligand NKG2D on tumor cells, and lead to enhanced NK lysis of tumors [START_REF] Romagné | Preclinical characterization of 1-7F9, a novel human anti-KIR receptor therapeutic antibody that augments natural killer-mediated killing of tumor cells[END_REF]. More recently, new drugs targeting proteasome inhibitors, such as bortezomib, which is now registered for the treatment of multiple myeloma, have also been shown to induce NK-stimulatory ligands [START_REF] Gasser | The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor[END_REF][START_REF] Ames | Sensitization of human breast cancer cells to natural killer cell-mediated cytotoxicity by proteasome inhibition[END_REF]. Finally, lenalidomide (Revlimid), a drug which has been shown to be active in multiple myeloma and to have promising preliminary results in other hematological malignancies, has been shown, in addition to having a direct antitumor effect, to upregulate NK-cell function through induction of cytokines [START_REF] Butler | Proteasome regulation of ULBP1 transcription[END_REF] and to induce NK-stimulatory ligands on tumor cells. Some of these drugs, such as bortezomib or chemotherapies [START_REF] Davies | Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma[END_REF][START_REF] Markasz | Effect of frequently used chemotherapeutic drugs on the cytotoxic activity of human natural killer cells[END_REF], can also have inhibitory effects on NK cells so their use must be carefully evaluated, but their clinical availability opens the door to multiple combination possibilities, either sequentially or concomitantly, with cell therapy and antiKIR antibodies. Such combinations are beginning to be tested in the clinic (phase I/II for antiKIR in combination with lenalidomide, and cell therapies in combination with bortezomib [START_REF] Berg | Clinical-grade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells[END_REF]).

Future directions

Since the initial demonstration that NK-cell therapies are effective in some contexts, there has been a lot of progress in refining protocols, as well as new approaches such as the injection of highly purified, functionally controlled NK cells. Also, new drugs allow the in-vivo manipulation of NK cells by targeting their inhibitory receptors or activating receptors (through drugs driving the expression of ligands of activating receptors on tumor cells). All these tools have now been developed to a point where they can be tested in clinical trials either alone or in combination. Because recent advances have increased our understanding of NK maturation and function, such clinical trials can now be monitored for NK-cell activity and represent attractive possibilities to be translated into successful treatments in the clinic.

Figure 1 .

 1 Figure 1. Natural killer (NK) cell recognition strategies

Figure 2 .

 2 Figure 2. Natural killer (NK) cell-based therapies

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(page number not for citation purposes) F1000 Medicine Reports 2011, 3:9 http://f1000.com/reports/m/3/9

Acknowledgements

The authors thank Corinne Beziers-Lafosse (CIML) for excellent graphic assistance and CIML's antibody and cytometry facilities. EV's lab is supported by grants from the European Research Council (ERC advanced grants), Agence Nationale de la Recherche (ANR), Ligue Nationale Contre le Cancer (Equipe labellisée 'La Ligue'), as well as by institutional grants from INSERM, CNRS, and Université de la Méditerranée to the CIML. EV is a scholar from the Institut Universitaire de France.

Abbreviations

AML, acute myeloid leukemia; GMP, good manufacturing practice; GVHD, graft-versus-host disease; HSCT, hematopoietic stem cell transplantation; IFNγ, interferon gamma; Ig, immunoglobulin; IL, interleukin; KIR, killer cell immunoglobulin-like receptor; LAK, lymphokine-activated killer; mAb, monoclonal antibody; MHC, major histocompatibility complex; MICA, MHC class Irelated chain A; NK, natural killer; RAET1, retinoic acid early transcripts-1.

Competing interests

FR and EV are co-founders and shareholders of Innate Pharma.


David Roux
email: david.roux@univ-avignon.fr

Osama Alnaser

Elnur Garayev

Béatrice Baghdikian

Riad Elias

Philippe Chiffolleau

Evelyne Ollivier

Sandrine Laurent

Mohamed El Maataoui

Huguette Sallanon

Ecophysiological and phytochemical characterization of wild populations of Inula montana L. (Asteraceae) in Southeastern France

Keywords:

Inula montana is a member of the family Asteraceae and is present in substantial numbers in Garrigue country (calcareous Mediterranean ecoregion). This species has traditionally been used for its anti-inflammatory properties as well as Arnica montana. In this study, three habitats within Luberon Park (southern France) were compared regarding their pedoclimatic parameters and the resulting morpho-physiological response of the plants. The data showed that I. montana grows in south-facing poor soils and tolerates large altitudinal and temperature gradients. The habitat conditions at high elevation appear to affect mostly the morphology of the plant (organ shortening). Although the leaf contents of total polyphenols and flavonoids subclass essentially followed a seasonal pattern, many sesquiterpene lactones were shown to accumulate first at the low-elevation growing sites that suffered drought stress (draining topsoil, higher temperatures and presence of a drought period during the summer). This work highlights the biological variability of I. montana related to the variation of its natural habitats which is promising for the future domestication of this plant. The manipulation of environmental factors during cultivation is of great interest due to its innovative perspective for modulating and exploiting the phytochemical production of I. montana.

Introduction

The sessile living strategy of terrestrial plants, anchored to the ground, forces them to face environmental variations. Plants have developed complex responses to modify their morpho-physiological characteristics to counteract both biotic and abiotic factors [START_REF] Suzuki | Abiotic and biotic stress combinations[END_REF][START_REF] Rouached | Plants coping abiotic and biotic stresses: a tale of diligent management[END_REF]. Altitude is described as an integrative environmental parameter that influences phytocoenoses in terms of species distribution, morphology and physiology [START_REF] Liu | Influence of environmental factors on the active substance production and antioxidant activity in Potentilla fruticosa L. and its quality assessment[END_REF]. It reflects, at minimum, a mixed combination of temperature, humidity, solar radiation and soil type [START_REF] Körner | Alpine Plant Life[END_REF]. In addition, the plant age, season, microorganism attacks, competition, soil texture and nutrient availability have been proven to strongly influence the morphology and the secondary metabolite profile of plants [START_REF] Seigler | Plant Secondary Metabolism[END_REF]. Altitudinal gradients are attractive for eco-physiological studies to decipher the mechanisms by which abiotic factors affect plant biological characteristics and how those factors influence species distribution [START_REF] Graves | A comparative study of Geum rivale L. and G. urbanum L. to determine those factors controlling their altitudinal distribution II. Photosynthesis and respiration[END_REF]. For instance, a summer increase of nearly 10% in solar irradiance per 1000 m in elevation has been demonstrated in the European Alps. This increase was also characterized by an 18% increase in UV radiation [START_REF] Blumthaler | Increase in solar UV radiation with altitude[END_REF]. Considering the reliefs of the Mediterranean basin, plants must confront both altitude and specific climate, namely high summer temperatures, infrequent but abundant precipitation, and wind [START_REF] Bolle | Mediterranean Climate: Variability and Trends[END_REF]. Moreover, plants that live at higher elevation must also survive winter conditions characterized by low temperatures and high irradiance. All together, these factors force the plants to develop dedicated short-and long-term phenological, morphological and physiological adaptations [START_REF] Kofidis | Combined effects of altitude and season on leaf characteristics of Clinopodium vulgare L. (Labiatae)[END_REF]. Many of these adjustments are protective mechanisms against photoinhibition of photosynthesis [START_REF] Guidi | Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas[END_REF][START_REF] Sperlich | Seasonal variability of foliar photosynthetic and morphological traits and drought impacts in a Mediterranean mixed forest[END_REF], and most of them involve the synthesis of secondary metabolites [START_REF] Ramakrishna | Influence of abiotic stress signals on secondary metabolites in plants[END_REF][START_REF] Bartwal | Role of secondary metabolites and brassinosteroids in plant defense against environmental stresses[END_REF].

The genus Inula (Asteraceae) includes more than 100 species that are widely distributed in Africa and Asia and throughout the Mediterranean region. These plants have long been collected or cultivated around the world for their ethnomedicinal uses. They synthesize and accumulate significant amounts of specific terpenoids and flavonoids. Secondary metabolites (including sesquiterpene lactones) from Inula spp. have shown interesting biological activities such as antitumor, anti-inflammatory, antidiabetic, bactericidal, antimicrobial and antifungal activities, and these plants have also been used for tonics or diuretics [START_REF] Reynaud | Free flavonoid aglycones from Inula montana[END_REF][START_REF] Seca | The genus Inula and their metabolites: from ethnopharmacological to medicinal uses[END_REF].

The species Inula montana is a hairy rhizomatous perennial (hemicryptophyte) herb with a 10-40 cm circumference and solitary capitulum (5-8 cm diameter) of yellow florets (long ligules) positioned at the top of a ≈20 cm floral stem. It grows at altitudes of 50-1300 m from eastern Italy to southern Portugal and is frequent in Southeast France. This calcicolous and xerophilous plant can be locally abundant, particularly in the Garrigue-type lands [START_REF] Gonzalez Romero | Phytochemistry and pharmacological studies of Inula montana L[END_REF][START_REF] Girerd | Flore du Vaucluse: troisième inventaire, descriptif, écologique et chorologique[END_REF][START_REF] Botanica | Tela Botanica[END_REF]. In the south of France, I. montana was incorrectly called "Arnica" because it was used in old traditional medicine as an alternative drug to the well-known Arnica montana [START_REF] Reynaud | Free flavonoid aglycones from Inula montana[END_REF]. Due to herbivory pressure, loss of habitat and the fact that it is mainly harvested from the wild, A. montana is cited in the Red List of Threatened Species (IUCN). In Europe, more than 50 t of dried flowers are traded each year [START_REF] Sugier | Propagation and introduction of Arnica montana L. into cultivation: a step to reduce the pressure on endangered and highvalued medicinal plant species[END_REF]. Although many efforts are currently underway to domesticate A. montana and to correctly manage its habitats, the opportunity to find an alternative plant would therefore be of considerable interest.

In this context, we have developed a scientific program that aims to rehabilitate, domesticate and test I. montana as an efficient pharmaceutical substitute to A. montana. We have recently published a phytochemical investigation of the contents of leaves and flowers of I. montana [START_REF] Garayev | New sesquiterpene acid and inositol derivatives from Inula montana L[END_REF]. Those data showed new compounds with associated anti-inflammatory activity. Here, we present the results of an ecophysiological study of I. montana that aimed to analyze the putative correlations between its morphology, its phytochemical production (with a focus on sesquiterpene lactones) and the characteristics (edaphic and climatic) of its natural habitats. It was expected that I. montana would face various abiotic stresses according to the large altitude gradient of its habitats. Assessing the response of the plant to its natural growing conditions will be helpful for its future domestication. In addition, a successful identification of environmental levers that could modulate the phytochemical production of this medicinal plant would be of great interest.

Material and methods

Luberon park

The present study was focused on I. montana populations growing in the French "Parc Naturel Régional du Luberon" (Luberon Park) that is located in southeastern France. The park (185,000 ha) is characterized by medium-sized mountains (from 110 to 1125 m high; mean altitude ≈680 m) that stretch from west to east over the "Vaucluse" and the "Alpes-de-Haute-Provence" regions (Supplemental file). Although the overall plant coverage of Luberon Park belongs to the land type "Garrigue" (calcareous low scrubland ecoregion), there are two significant climatic influences: first, the north-facing shady side is characterized by a cold and humid climate that supports the development of deciduous species such as the dominant white oak (Quercus pubescens). Second, the sunny south-facing side receives eight to ten times more solar radiation. On this side, the vegetation is typically Mediterranean with a majority of green oak (Quercus ilex), Aleppo pine (Pinus halepensis), kermes oak (Quercus coccifera) and rosemary (Rosmarinus officinalis). The ridges of the Luberon Park suffer from extreme climatic variations: windy during all seasons, intense summer sun, cold during the winter, dry atmosphere and spontaneous and intense rains. These conditions limit the spectrum of plant species to those most resistant to these conditions, such as the common juniper (Juniperus communis) and boxwood (Buxus sp.) [START_REF] Gressot | Le parc naturel régional du Luberon[END_REF].

Sites of interest and sampling

Inula montana is present in highly variable amounts over Luberon Park. By exploring the south-facing sides we selected three sites of interest: Murs, Bonnieux and Apt (Supplemental file). At these locations, I. montana forms several small, sparse and heterogeneous groups of tens of plants per hectare. These sites were also selected for their similar presentation as grassy clearings (area from 4 to 9 ha) and for their uniform flatness and slight inclination (≈7%). The linear distance between the 3 sites is 21.4 ± 2 km. The Apt site is 500-600 m higher than both other sites. A preliminary phenological survey showed that the vegetative growth of I. montana extended from early April to late October, consistent with the hemicryptophytic strategy of the plant. Mid-June corresponded to the flowering period, which lasted ≈10 days. Accordingly, samples were synchronously collected from the three habitats at four consecutive periods during 2014: early April (early spring), mid-May (late spring), mid-June (summer) and late October (autumn).

Climatic and edaphic data

The measurements of climate characteristics (standard weather stations, 1.5 m height above soil surface) were accessed from the French weather data provider (meteofrance.fr, 2014, France) and supplemented with agronomic weather station data near each site (climatedata.org, 2014, Germany). The satellite-based solar radiation measurements (Copernicus Atmosphere Monitoring Service (CAMS)) were obtained from the solar radiation data service (soda-pro.com, 2014, MINES ParisTech, France). The measurements of the physical properties of the soils and of the chemical content of the aqueous extracts (cf. Table 1) were subcontracted to an ISO certified laboratory (Teyssier, Bordeaux, France) according to standards. Briefly, 10 g of raw soil were milled, dried (12 h at 45 °C, except for nitrogen determination) and sifted (2 mm grid). Samples were then stirred into 50 ml of demineralized water for 30 min at 20 °C and filtered. Organic matter was measured after oxidation in potassium dichromate and sulfuric acid. NH 4 and NO 3 were extracted with 1 M KCl. Organic matter, NH 4 , NO 3 and water-extractable PO 4 were then determined by colorimetric methods. K, Mg, Ca, Fe, Cu, Mn, Zn and Bo were determined by atomic absorption spectroscopy.

Determination of growth parameters

Plant growth for each period evaluated was determined by using several parameters: fresh and dry weight, water content, leaf area, and height of floral stem at the flowering stage. For each period, ten plants were collected randomly from each of the three sites (Luberon Park). The fresh weight was measured immediately after harvest, and the leaves were scanned to measure their area with the ImageJ software (National Institutes of Health, USA). The collected plants were subsequently dried (80 °C, 24 h) to calculate the water content. Glandular trichome density was assessed on 10 leaves randomly collected at the flowering period from 10 different plants per site. This assessment was performed using a stereomicroscope (Nikon ZX 100, Canagawa, Japan) equipped with fluorescence (excitation 382 nm, emission 536 nm) and digital camera (Leica DFC 300 FX, Wetzlar, Germany). The captured images allowed the quantification of glandular trichomes using ImageJ.

Chlorophyll-a fluorescence measurements

Chlorophyll-a fluorescence was measured in vivo using a portable Handy-PEA meter (Hansatech, Kings Lynn, UK) on 20 plants arbitrarily selected three times per day: in the morning (10:00), at midday (12:00) and in the afternoon (14:00). This was done for each considered time period (season) and for each of the three I. montana habitats. The fluorescence parameters calculated were the maximum quantum yield of primary photosystem II photochemistry (Fv/Fm) and the performance index (PI) according to the OJIP test [START_REF] Strasser | The fluorescence transient as a tool to characterize and screen photosynthetic samples[END_REF]. Both parameters are plant stress indicators and provide indications of the overall plant fitness. The ratio (Fv/Fm) between variable chlorophyll fluorescence (Fv = Fm -F0) and maximum fluorescence (Fm) is the most used parameter to assess plant stress. Initial fluorescence (F0) is obtained from dark adapted samples and maximum fluorescence (Fm) is measured under a saturation pulse [START_REF] Maxwell | Chlorophyll fluorescence -a practical guide[END_REF][START_REF] Rohaçek | Chlorophyll fluorescence parameters: the definitions, photosynthetic meaning, and mutual relationships[END_REF]. PI is an integrative parameter that reflects the contribution to photosynthesis of the density of reaction centers and both the light and the dark reactions [START_REF] Poiroux-Gonord | Metabolism in orange fruits is driven by photooxidative stress in the leaves[END_REF]. All of the parameters were calculated from the measured fluorescence of leaves under saturating pulsed light (1 s at 3500 μmol m -2 s -1 ) after 20 min adaptation to the dark.

Total polyphenol and flavonoid contents

Harvested leaves were air dried on absorbent paper at room temperature for 3 weeks. The samples were prepared by maceration at room temperature for 96 h in 20 ml of 50% ethanol (v/v) (Carlo Erba, Italy). This step was followed by ultrasonic extraction for 30 min. The samples were then filtered into a 20 ml volumetric flask and adjusted to volume with the same solvent. The total polyphenol content was determined according to paragraph 2.8.14 of the current European Pharmacopoeia (Ph. Eur, 2017): the absorbance was measured at 760 nm (Shimadzu 1650pc, Japan), and the results are expressed as pyrogallol (Riedel-de-Haën, Germany) equivalents in percent (g/100 g of dried plant sample). The total flavonoid content was determined according to the aluminum chloride colorimetric method of monograph number 2386 (safflower flower) from the current European Pharmacopoeia. The absorbance was measured at 396 nm (Shimadzu 1650pc), and the results are expressed as the percentage of total flavonoids relative to luteoline (C 15 H 10 O 6 ; Mr 286.2).

High-performance liquid chromatography (HPLC) analyses

The extraction was performed by mixing 10 g of dried leaves with 100 ml of CH 2 Cl 2 (Carlo Erba, Italy) and introducing the mixture into a glass column in order to extract compounds by percolation with dichloromethane. After 18 h of maceration, 100 ml of dichloromethane extract was collected and evaporated to dryness. Next, 10 mg of dried extract were dissolved in 5 ml of methanol (Carlo Erba) and centrifuged. Then, 4 ml of the supernatant was brought to a final volume of 10 ml with distilled water. The solution was filtered through a 0.45-μm membrane. The analyses were performed using an Agilent 1200 series apparatus (G1379A degasser, G1313A autosampler, G1311A quaternary pump, G1316A column thermostat and G1315 B diode array detector (DAD)) (Agilent, Germany) with a Luna C18 adsorbent (3 μm, 150 mm × 4.6 mm) (Phenomenex, USA) and a SecurityGuard C18 column (3 mm ID × 4 mm cartridge) (Phenomenex). Instrument control, data acquisition and calculation were performed with ChemStation B.02.01. (Agilent). The mobile phase consisted of 52% MeOH (Carlo Erba) and 48% water (Millipore, Germany), and the pH of the mobile phase was 5.5. The flow rate was 1.0 ml/min. The detector was operated at 210 nm (sesquiterpene lactones absorption wavelength), and peaks were identified according to [START_REF] Garayev | New sesquiterpene acid and inositol derivatives from Inula montana L[END_REF]. The injection volume was 20 μl.

Statistical analysis

The principal component analysis (PCA) and non-parametric test were performed using R (R Foundation, Austria). For multiple comparisons, the post hoc Kruskal-Wallis-Dunn test with the Bonferroni adjustment method was used. The R libraries used were factomineR, PMCMR, and multcompView. The data are displayed as the means ± standard error of the mean and were considered significant at p < 0.05.

Results

Pedoclimatic characterization of I. montana habitats

Among the three Luberon Park sites assessed, Murs and Bonnieux showed a similar climatic pattern in terms of temperature and precipitation (Fig. 1). In addition, the 20-year-data suggested that both of these I. montana habitats experienced a drought period centered on July (1-2 months long). The Apt habitat, which is 500-600 m higher than the two other sites (Supplemental file), showed a lower mean temperature and higher precipitation. In addition, Apt notably displayed the absence of a drought period, according to the averaged data (Fig. 1), but it showed drier air throughout the year (Table 1).

The 3-year (2013-2015) satellite-based measurement of the global solar irradiation on the horizontal plane at ground level (GHI) (Fig. 2) showed a strong increase in irradiance from January to June, a stable amount of radiation from June to July and then a strong decrease until December. When investigating the irradiance in detail for the 3 I. montana populations, no difference was observed (GHI). However, when irradiance was estimated under clear sky, namely by virtually removing the clouds (Clear-Sky GHI), it appeared that the Apt site received ≈3% higher solar irradiation from May to July. Taken together, these results indicate that the cloudier Apt weather compensates on average for the higher solar irradiation at this altitude.

Considering the physical characteristics of the topsoil, Apt appeared clayey and loamy, whereas Murs and Bonnieux were much richer (6-12 times) in sand (Table 1). Concerning the chemical characteristics, the analysis of the topsoil aqueous extracts showed that all three growing sites appeared equally poor (Table 1). The Apt topsoil showed slightly lower levels of NH 4 , NO 3 , K, Ca, Mn and Zn than either of the other sites and also showed the lowest pH.

Impact of the geographic location and seasonal progress on I. montana morphology and physiology

Until autumn, I. montana plants from the Apt population showed less leaf blade surface area than leaves from the other sites (Fig. 3A). All three habitats displayed an intensive early-spring growth period, but during late spring, the leaf surface area was 45% lower at the Apt site than at the other sites. However, at Apt, the leaf area was quite stable after this time and for the remaining period, but the leaf surface decreased progressively from late spring to autumn at the two lower-altitude sites (Murs and Bonnieux). There was no significant variation in the number of leaves during the season, with the exception of a slight difference during summer between Bonnieux and the two other habitats (Fig. 3B).

In addition, the geographic location of I. montana habitats seemed to influence both the length of the flowering stem and the number of glandular trichomes per leaf (Table 2). It appeared that I. montana plants from the Apt habitat showed a significantly shortened floral stem (≈-12%) and fewer leaf glandular trichomes (≈-23%) in comparison with the other sites (Murs and Bonnieux).

Dry and fresh weights increased from spring to summer and then decreased until autumn (Fig. 4A,B). When comparing the three I. montana habitats in terms of plant dry weight, they showed no difference during the overall season (Fig. 4A). Similarly, all plants showed essentially the same water content until late spring (Fig. 4C). Then, Apt plants displayed a water content of over 70% during the remaining seasons, while plants from the two low-elevation locations stayed below 65% (Bonnieux showed the lowest value in the summer: 55%).

Both indicators from chlorophyll a fluorescence measurements (the maximum quantum yield of primary photosystem II photochemistry and the performance index) showed slight decreases during the summer regardless of the geographic location of the plants (Fig. 5). The overall values then increased until autumn, but they did not return to their initial levels. In addition, I. montana plants from the Apt population showed higher Fv/Fm and PI values during the whole growing period than did plants in the two low-altitude habitats.

Phytochemical contents of I. montana according to geographic location

The amounts of total polyphenols and their flavonoid subclass during the overall period did not differ among the three habitats, with the exception of a lower level of polyphenols during late-spring for the plants in Bonnieux (Fig. 6A). For the three habitats, the total polyphenol level was 49% lower in autumn than in the early spring. The total flavonoids (Fig. 6B) showed an average increase of 56% from early spring to summer (higher level) but then decreased drastically thereafter (-68%).

We conducted high-performance liquid chromatography analysis on the leaves of the I. montana plants. The chromatograms (Fig. 7) showed 10 major peaks, in which we recently identified (by the external standard method) 5 sesquiterpene lactones [START_REF] Garayev | New sesquiterpene acid and inositol derivatives from Inula montana L[END_REF] respectively artemorin (p1), 9B-hydroxycostunolide (p2), reynosin (p3), santamarine (p5) and costunolide (p10). Other peaks were determined to be a mix of two flavonoids (Chrysosplenol C and 6-hydroxykaempferol 3,7-dimethyl ether; p4) and four inositol derivatives (myoinositol-1,5- diangelate-4,6-diacetate, myoinositol-1,6-diangelate-4,5-diacetate, myoinositol-1-angelate-4,5-diacetate-6-(2-methylbutyrate), myoinositol-1-angelate-4,5-diacetate-6-isovalerate; p6 to p9). The cross-location and cross-time relative quantification of the 5 sesquiterpene lactones (Table 3) suggested that I. montana plants from the low-altitude Murs and Bonnieux populations contained approximately three times more phytochemicals than plants from Apt. The data also showed that p1, p3 and p5 tended to accumulate throughout the seasons, unlike p2 and p10, which decreased. Lastly, p10 appeared to be the most abundant compound (roughly 50% more than the other lactones) regardless of the location or season.

Discussion

4.1. Inula montana morphology montana plants exhibited shorter floral stem length and reduced leaf surface at high altitude (Apt; Table 2 and Fig. 3A). This is consistent with the tendency of many plants to shorten their organs during winter (Åström et al., 2015) or at high elevation due to low temperatures and strong wind speeds, as shown previously in three Asteraceae species [START_REF] Yuliani | The relationship between habitat altitude, enviromental factors and morphological characteristics of Pluchea indica, Ageratum conyzoides and Elephantopus scaber[END_REF]. This behavior allows for limiting dehydration and ameliorate the photosynthetic conditions by setting plant organs closer to the warmer soil surface [START_REF] Cabrera | Effects of temperature on photosynthesis of two morphologically contrasting plant species along an altitudinal gradient in the tropical high Andes[END_REF]. The seasonal modification of the leaf morphology has also been shown to optimize photosynthetic capacity (Åström et al., 2015). The slightly lower nutrient availability at Apt (Table 1) may also contribute to the smaller organ sizes. In addition, the leaf surface of I. montana remained stable during the hot period at Apt, whereas it decreased at low altitude (Fig. 3B). This result is correlated with both the higher temperature and the drought period present at the low-elevation sites. Taken together, the data suggest that the plant morphological response is clearly adapted to both the climate and the location.

Inula montana displays two different trichome types on its leaves: hairy and glandular. Trichomes are well described as being plastic and efficient plant weapons against herbivory, notably through their high contents of protective secondary metabolites. Insect feeding can modify both the density and the content of trichomes [START_REF] Tian | Role of trichomes in defense against herbivores: comparison of herbivore response to woolly and hairless trichome mutants in tomato (Solanum lycopersicum)[END_REF]. Abiotic factors also strongly influence plant hairs; for example dry conditions, high temperatures or high irradiation can increase the number of trichomes per unit leaf area [START_REF] Pérez-Estrada | Variation in leaf trichomes of Wigandia urens: environmental factors and physiological consequences[END_REF]. Conversely, trichome density decreases in the shade or in well-irrigated plants. In this context, water availability in the plant environment is an integrative factor [START_REF] Picotte | ), respectively. A: Variable factor map. The bold lines and squares show sesquiterpene lactones; B: Individual factor map with confidence ellipses (95%) around the descriptive variables. temporal variation in moisture availability: consequences for water use efficiency and plant performance[END_REF]. Our data are consistent with this model, since plants from the Apt habitat (showing the highest altitude and precipitation but the lowest temperatures) displayed fewer glandular trichomes on their leaves than either of the other growing sites that suffered from drought periods (Table 2). These results also indicate that I. montana undergoes a stronger or at least a different type of stress at low altitude.

Inula montana physiology

It appeared that I. montana biomass increased from early spring to summer but then decreased, consistent with the hemicryptophytic strategy of this plant (dry weight, Fig. 4A). The location of the I. montana habitats had no effect on the dry weight but significantly influenced the plant water content, which markedly decreased during the summer at low elevation (Murs and Bonnieux; Fig. 4C). This is consistent with the expectation that low-elevation regions in the Mediterranean area would be hotter and drier than high-altitude regions, leading to more stressful conditions for plants [START_REF] Kofidis | Combined effects of altitude and season on leaf characteristics of Clinopodium vulgare L. (Labiatae)[END_REF][START_REF] Wolfe | Adaptation to spring heat and drought in northeastern Spanish Arabidopsis thaliana[END_REF]. The absence of an effect on the dry weight of I. montana here illustrates the strong variability of this xerophilous plant, namely its capacity to grow in various habitats and its ability to resist drought. Chlorophyll a fluorescence has been described as an accurate indicator of the plant response to environmental fluctuations and biotic stress [START_REF] Murchie | Chlorophyll fluorescence analysis: a guide to good practice and understanding some new applications[END_REF][START_REF] Guidi | Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas[END_REF] and has gained interest in ecophysiological studies [START_REF] Åström | Morphological characteristics and photosynthetic capacity of Fragaria vesca L. winter and summer leaves[END_REF][START_REF] Perera-Castro | Light response in alpine species: different patterns of physiological plasticity[END_REF]. The maximum photochemical quantum yield of PSII (Fv/Fm) and the performance index (PI) reflect photooxidative stress and plant fitness [START_REF] Strasser | The fluorescence transient as a tool to characterize and screen photosynthetic samples[END_REF]. Fv/Fm values usually vary from 0.75 to 0.85 for non-stressed plants. Any decrease indicates a stressful situation, reducing the photosynthetic potential [START_REF] Maxwell | Chlorophyll fluorescence -a practical guide[END_REF]. In the Mediterranean climate, plant photoinhibition frequently occurs [START_REF] Guidi | Non-invasive tools to estimate stress-induced changes in photosynthetic performance in plants inhabiting Mediterranean areas[END_REF]. Below a certain limit of solar radiation, this protective mechanism allows the dissipation of excessive photosynthetic energy as heat [START_REF] Dos Santos | Seasonal variations of photosynthesis gas exchange, quantum efficiency of photosystem II and biochemical responses of Jatropha curcas L. grown in semi-humid and semi-arid areas subject to water stress[END_REF]. Here, both of the indicators (Fv/Fm and PI) displayed lower values at the lowelevation sites (Murs and Bonnieux; Fig. 5), confirming that I. montana was subjected to greater stress there. These results are in agreement with the observed drought periods at those sites and reflect the adaptive response of the plants to avoid photodamage under high temperature and drought stress in order to preserve their photosynthetic apparatus [START_REF] Poiroux-Gonord | Metabolism in orange fruits is driven by photooxidative stress in the leaves[END_REF]. It is not possible to easily correlate these results to the solar radiation because no difference was observed among the 3 habitats, as described above (Fig. 2). However, a similar study that focused on the combined effects of altitude and season on Clinopodium vulgare highlighted a decrease in Fv/Fm values in lowland populations at the beginning of a drought period [START_REF] Kofidis | Combined effects of altitude and season on leaf characteristics of Clinopodium vulgare L. (Labiatae)[END_REF].

Secondary metabolites

Plant secondary metabolites are well known for accumulating in response to environmental conditions that induce oxidative stress. Many studies have proposed that polyphenols might play a protective anti-oxidative role in plants [START_REF] Bartwal | Role of secondary metabolites and brassinosteroids in plant defense against environmental stresses[END_REF][START_REF] Bautista | Environmentally induced changes in antioxidant phenolic compounds levels in wild plants[END_REF]. Consequently, phenolics and other secondary metabolites usually accumulate under drought stress, salt stress [START_REF] Adnan | Desmostachya bipinnata manages photosynthesis and oxidative stress at moderate salinity[END_REF], high or low temperatures and at high altitude; this is exacerbated in Mediterranean plants [START_REF] Kofidis | Combined effects of altitude and season on leaf characteristics of Clinopodium vulgare L. (Labiatae)[END_REF][START_REF] Scognamiglio | Chemical composition and seasonality of aromatic Mediterranean plant species by NMR-Based Metabolomics[END_REF]. Phenology and plant development also strongly influence the concentrations of phenolic compounds [START_REF] Radušienė | Effect of external and internal factors on secondary metabolites accumulation in St. John's Worth[END_REF]. Our data showed that the I. montana leaf total polyphenol and flavonoid contents both varied over the season and reached their maximum value during latespring and summer (Fig. 6). This physiological behavior follows the seasonal solar radiation profile (Fig. 2) and is consistent with the welldescribed photoprotective role of polyphenols [START_REF] Agati | Multiple functional roles of flavonoids in photoprotection[END_REF]. It has long been known that the quantity of solar radiation increases with altitude [START_REF] Spitaler | Altitudinal variation of secondary metabolite profiles in flowering heads of Arnica montana cv[END_REF]. Accordingly, we expected a higher content of phenolics at high elevation due to the higher irradiance, including UV. However, the cloudier weather at the Apt site appeared to compensate for the theoretical 3% difference in sunshine between that site and the 2 other I. montana habitats (Fig. 2). As such, our results cannot explain the low polyphenol content observed in latespring at Bonnieux. Either way, it appears that the stress perceived by I. montana plants at low altitude is not due to the simple variation in solar radiation but rather to a significant susceptibility to drought stress and/ or high temperatures.

Sesquiterpenes are an important group of organic compounds released by plants and are characteristic of the family Asteraceae. Most of them are volatile molecules used as hormones and for functions such as communication and defense against herbivory [START_REF] Rodriguez-Saona | The role of volatiles in plant-plant interactions[END_REF][START_REF] Chadwick | Sesquiterpenoids lactones: benefits to plants and people[END_REF]. In this work we have identified 5 sesquiterpene lactones that tend to accumulate in higher amounts in lowelevation habitats (Table 3). These compounds also showed quantities that were positively or negatively correlated with the seasonal progression. Sesquiterpene lactones are well described to follow a seasonal pattern and to accumulate in response to biotic and abiotic stresses [START_REF] Chadwick | Sesquiterpenoids lactones: benefits to plants and people[END_REF][START_REF] Sampaio | Effect of the environment on the secondary metabolic profile of Tithonia diversifolia: a model for environmental metabolomics of plants[END_REF]. Since these compounds play essential roles in the plant defense response, their accumulation under abiotic stress is consistent with the carbon balance theory, which states that the investment in plant defense increases in response to a growth limitation [START_REF] Mooney | Response of Plants to Multiple Stresses[END_REF]. However, in Arnica montana, no positive correlation between the production of these molecules and altitude was found [START_REF] Spitaler | Altitudinal variation of secondary metabolite profiles in flowering heads of Arnica montana cv[END_REF]. In addition, plant terpenoid release has been reported to be modulated by temperature, drought and UV radiation [START_REF] Loreto | Abiotic stresses and induced BVOCs[END_REF].

The topsoil at the low altitude sites (Murs and Bonnieux) was significantly richer in sand than that at Apt (Table 1). This confers a high draining capacity to the low-elevation sites that would inevitably increase the water deficiency and contribute to the drought stress perceived by the plants. Last, our data from aqueous extracts highlighted a slightly lower topsoil nutrient content at the Apt site (Table 1). Although the literature on this topic is scarce, and no information is available concerning N and K, some soil nutrients (namely P, Cu and Ca) can influence the plant sesquiterpene lactone content [START_REF] Foster | Influence of cultivation site on sesquiterpene lactone composition of forage chicory (Cichorium intybus L.)[END_REF][START_REF] Sampaio | Effect of the environment on the secondary metabolic profile of Tithonia diversifolia: a model for environmental metabolomics of plants[END_REF]. More broadly, deficiencies in nitrogen have been described to induce the accumulation of plant phenylpropanoids [START_REF] Ramakrishna | Influence of abiotic stress signals on secondary metabolites in plants[END_REF]. Apt also showed the lowest Ca content and pH. Although the values were only slightly higher at the two other sites, they may globally contribute to decreasing the availability of topsoil cations. We cannot exclude the possibility that this would also contribute to the stress on the plants at these locations, but it is not easy to make a connection with the plant phytochemical production.

Conclusion

The morpho-physiological characteristics of I. montana showed that the plant undergoes higher stress at its lower-altitude growing sites (Murs and Bonnieux). Four plant and environmental variables (chlorophyll fluorescence, plant water content, climate and topsoil draining capacity) specially converged to highlight the site water availability as the primary source of stress. In addition, the sesquiterpene lactone production by I. montana was higher at these low-elevation stress-inducing habitats.

The overall data are summarized in the principal component analysis (Fig. 8). The I. montana growing location (dimension 1) and the seasons (dimension 2) encompass more than 76% of the total variability, and the location itself exceeds 50%. The map confirms that plant stress (expressed as water content or Fv/Fm) and the subsequent release of sesquiterpene lactones (including 4 of the 5 compounds) are correlated to the integrative altitude parameter. The individual factor map (B) clearly discriminates the I. montana growing locations from the seasons and highlights the interaction of these two factors.

Dissecting the manner in which molecules of interest fluctuate in plants (in response to biotic and abiotic stress) is of great interest scientifically and economically [START_REF] Pavarini | Exogenous influences on plant secondary metabolite levels[END_REF]. The present study shows that growing habitats that induce plant stress, particularly drought stress, can significantly enhance the production of sesquiterpene lactones by I. montana. Similar approaches have been conducted with A. montana [START_REF] Spitaler | Altitudinal variation of secondary metabolite profiles in flowering heads of Arnica montana cv[END_REF][START_REF] Perry | Sesquiterpene lactones in Arnica montana: helenalin and dihydrohelenalin chemotypes in Spain[END_REF][START_REF] Clauser | Differences in the chemical composition of Arnica montana flowers from wild populations of north Italy[END_REF] and have provided valuable information and cultivation guidelines that helped with its domestication [START_REF] Jurkiewicz | Optimization of culture conditions of Arnica montana L.: effects of mycorrhizal fungi and competing plants[END_REF][START_REF] Sugier | Propagation and introduction of Arnica montana L. into cultivation: a step to reduce the pressure on endangered and highvalued medicinal plant species[END_REF]. Appropriate cultivation techniques driven by the ecophysiological study of A. montana have succeeded in influencing its sesquiterpene lactone content for medicinal use [START_REF] Todorova | Developmental and environmental effects on sesquiterpene lactones in cultivated Arnica montana L[END_REF]. The manipulation of environmental stress has also been described to significantly promote the phytochemical (phenolic) content of lettuce [START_REF] Oh | Environmental stresses induce healthpromoting phytochemicals in lettuce[END_REF] and halophytes [START_REF] Slama | Water deficit stress applied only or combined with salinity affects physiological parameters and antioxidant capacity in Sesuvium portulacastrum[END_REF][START_REF] Adnan | Desmostachya bipinnata manages photosynthesis and oxidative stress at moderate salinity[END_REF]. Literature regarding I. montana is very sparse. The present results bode well for our ongoing field-work that aims to simulate and test environmental levers to augment the secondary metabolism and to develop innovative culture methods for I. montana.

Our data also illustrate the high morpho-physiological variability of this calcicolous plant. High-altitude habitat appears to primarly impact the morphology of the plant, while low-elevation sites mostly induce physiological responses to stress (chlorophyll fluorescence, phytochemicals synthesis). I. montana appears to grow well on south-facing sites possessing poor topsoil and low nutrient availability. It is also able to face high temperature and altitude gradients and to grow well on draining soil under a climate that induces drought stress. Table 3 Cross-location and cross-time relative quantification of the 5 sesquiterpene lactones found in Inula montana leaves. "-" indicates the absence of the molecule, and "+", "++", and "+++" indicate its relative increasing abundance.

Fig. 1 .

 1 Fig. 1. Annual climographs (20 years of averaged data) of the three Inula montana study sites. The black line represents the mean temperature, and the hatched area represents the mean precipitation per month (rainfall or snow). Drought periods are symbolized by an asterisk.

Fig. 2 .

 2 Fig. 2. Monthly means of satellite-based global solar irradiation as measured on the horizontal plane at ground level (GHI). The solid lines (GHI) represent the actual terrestrial solar radiation; the dashed lines (Clear-Sky GHI) estimate the irradiation under a cloudless sky. The data represent the means of 3 years of records (2013-2015) for the 3 Inula montana populations (spatial resolution was 3-8 km). The asterisks indicate significant differences between sites at p < 0.05.

Fig. 3 .Fig. 4 .

 34 Fig. 3. Mean leaf blade surface area (A) and number of leaves (B) of Inula montana plants according to the geographic location and seasonal progress. The data represent the mean values of 10 plants ± standard error. The lowercase letters represent significant differences at p < 0.05.

Fig. 6 .

 6 Fig. 6. Effect of the geographic location on Inula montana phytochemical contents. The data represent the contents of total polyphenols (A) and total flavonoids (B). The data represent the mean values of 10 plants ± standard error. The lowercase letters represent significant differences at p < 0.05.

Fig. 5 .

 5 Fig. 5. Effect of the geographic location on Inula montana photosystem II fluorescence. The data represent the mean Fv/Fm (A) and mean PI (B) values of 10 plants ± standard error. The lowercase letters represent significant differences at p < 0.05.

Fig. 7 .

 7 Fig. 7. HPLC chromatograms of Inula montana leaves harvested during the summer, according to the plant geographic location. S.l.: sesquiterpene lactone; Fl.: flavonoid; In.: inositol. Peaks were identified according to Garayev et al. (2017).

Table 1

 1 Pedoclimatic characterization of Inula montana habitats. Exp. Δ: expected theoretical range of element concentrations for a standard agricultural parcel.

			Murs	Bonnieux	Apt
	Climate
	Mean temperature (°C)	11.0	12.1	7.7
	Air moisture (%)		66.5	69.7	54.4
	Annual precipitation (mm)	774	702	928
	Topsoil
	Composition (%) & texture	Sandy loam	Sandy clayey	Clayey loam
				loam
	Organic matter (from aqueous	5.01	4.98	5.00
	extract)
	Clay		6.4	16.1	18.0
	Sand		37.3	20.4	3.1
	Silt		56.3	63.5	78.8
	Macro-and microelements (mg/kg from aqueous extract)	Exp. Δ
	pH	8.3	7.8	7.7
	NH 4	3.96	2.56	2.09	4.0-8.0
	NO 3	3.79	3.14	1.29	4.0-8.0
	K	5.4	5.4	2.7	40-80
	PO 4	0.2	0.2	0.2	15-25
	Mg	2.5	2.5	3.3	20-40
	Ca	96.2	71.8	68.7	100-200
	Fe	0.33	0.07	0.14	8.0-12.0
	Cu	0.01	0.01	0.01	0.30-0.50
	Mn	0.13	0.14	0.08	0.30-0.50
	Zn	0.09	0.08	0.07	0.30-0.50
	Bo	0.51	0.49	0.53	1.0-2.0

Acknowledgments

This work was supported by the French region Provence-Alpes-Côte d'Azur (project n°2013_13403), the Luberon Regional Natural Park and the TERSYS Research Federation of the University of Avignon. We thank Prof. Vincent Valles (Avignon University) for his advice on the statistics. We thank Didier Morisot, collections manager of the plant garden of the Faculty of Medicine of the University of Montpellier, for the I. montana identification.


Olivier Briant
email: olivier.briant@grenoble-inp.fr

Hadrien Cambazard
email: hadrien.cambazard@grenoble-inp.fr

Diego Cattaruzza
email: diego.cattaruzza@centralelille.fr

Nicolas Catusse
email: nicolas.catusse@grenoble-inp.fr

Anne-Laure Ladier
email: anne-laure.ladier@insa-lyon.fr

Maxime Ogier
email: maxime.ogier@centralelille.fr

A column generation based approach for the joint order batching and picker routing problem

Keywords: order batching, picker routing, column generation

Introduction

Picking is the process of retrieving products from the inventory and is often considered a very expensive operation in warehouse management. A set of pickers perform routes into the warehouse, pushing a trolley and collecting items to prepare customer orders. However, customer orders usually do not fit the capacity of a trolley. They are typically grouped into batches, or on the contrary divided into subsets, with the aim of collecting all the orders by minimizing the walked distance. This problem is known as the joint order batching and picker routing problem [START_REF] Cristiano | Optimally solving the joint order batching and picker routing problem[END_REF].

This work presents an exponential linear programming formulation where variables or columns are related to single picking routes in the warehouse. More precisely, a column refers to a route involving a set of picking operations and satisfying the side constraints required at the trolley level such as the mixing of orders or the capacity. Computing such a picking route is an intractable routing problem in general and, depending on the warehouse layout, can closely relate to the traveling salesman problem (TSP). The rational of our approach is however to consider that the picking problem alone, in real-life warehouses, is easy enough in practice to be solved exactly. We apply this approach on two different industrial benchmarks, based on different warehouse layouts.

Problem specification and industrial applications

The warehouse layout is modeled as a directed graph G = (V, A) with two types of vertices, locations and intersections. Locations contain one or more product references to be picked.

Two typical examples of warehouse layouts are used as benchmarks in the present work.

-A regular rectangular layout made of vertical aisles and horizontal cross-aisles. Such a layout has been used by numerous authors in the past [START_REF] De Koster | Design and control of warehouse order picking : A literature review[END_REF] to define the order picking problem. It is the setup of the Walmart benchmark. -An acyclic layout where pickers are not allowed to backtrack. It is another typical industrial setup where the flow is constrained in a single direction and an aisle must be entered and exited on the same side. It is the setup of the HappyChic benchmark. Each product reference p ∈ P is characterized by its location in the warehouse and its size V w p in each dimension w ∈ W. A product reference may have several dimensions such as weight and volume and we refer to the set of dimensions as W.

An order from a customer is defined as a set of order lines. An order line l ∈ L is related to an order o and defined as a pair (p l , Q l ) where p l ∈ P is a product reference and Q l is the number of items to pick. An order o ∈ O is a set of order lines L o ⊆ L. Moreover, an order can be split in at most M o boxes.

Order lines are collected by trolleys, each carrying a set of B boxes. A box has a capacity V w in dimension w ∈ W and an order line can be assigned into several boxes (the quantity Q l of an order line l can be split among several boxes). A box is therefore filled with partial order lines. A partial order line l is a pair (p l , Ql ) with Ql ≤ Q l . A box can only contain partial order lines from a single order.

A solution is a collection of routes R in the warehouse layout G. Each route r is travelled by a trolley which collects partial order lines into its boxes. The capacities of the boxes must be satisfied in each dimension w ∈ W. An order o ∈ O can not be assigned to more than M o boxes. Finally all order lines must be picked with the required number of items. The objective is to minimize the total distance to perform all the routes in R.

The two industrial cases addressed in the present work, from the Walmart and HappyChic, differ slightly. In particular, for the Walmart case, only one dimension is considered for a box, representing the maximum number of items in a box. Additionally, an order must be picked entirely by a single trolley.

A column generation based approach

In the industrial case of HappyChic, the picking takes place on an acyclic graph, thus boils down to an easy path problem. In Walmart's case, warehouse have the regular rectangular structure made of aisles and cross-aisles. In that case, dynamic programming algorithms can take advantage of that structure to efficiently solve the corresponding TSP when the warehouse contains up to eight cross-aisles, which is beyond most real-life warehouse's sizes [START_REF] Cambazard | Fixed-Parameter Algorithms for Rectilinear Steiner tree and Rectilinear Traveling Salesman Problem in the plane[END_REF]. We therefore assume that in both cases, an efficient oracle is available to provide optimal picking routes in the warehouse.

We show that such an oracle allows for a very effective exponential LP formulation of the joint order batching and picking problem. The pricing problem can be seen as a prize-collecting TSP with a capacity constraint and the pricing algorithm heavily relies on the picking oracle to generate cutting planes. A number of improvements are proposed to speed up the pricing. In particular, a procedure to strengthen the cutting planes is given when the distance function for the considered set of orders is submodular. For the industrial case of HappyChic, the graph is acyclic, so it is possible to propose a polynomial set of constraints to exactly calculate the distance, instead of generating cutting planes.

The proposed formulation is compared experimentally on Walmart's benchmark and proves to be very effective, improving many of the best known solutions and providing very strong lower bounds. Finally, this approach is also applied to the HappyChic case, demonstrating its generality and interest for this application's domain.


S Jacques

B Bonnetot

M.-P Berthet

H Vincent

BN interphase processed by LP-CVD from tris(dimethylamino)borane and characterized using SiC/SiC minicomposites

SiC/BN/SiC 1D minicomposites were produced by infiltration of a Hi-Nicalon (from Nippon Carbon, Japan) fiber tow in a Low Pressure Chemical Vapor Deposition reactor.

Tris(dimethylamino)borane was used as a halogenide-free precursor for the BN interphase processing. This precursor prevents fiber and CVD apparatus from chemical damage. FT-IR and XPS analyses have confirmed the boron nitride nature of the films. Minicomposite tensile tests with unload-reload cycles have shown that the minicomposite mechanical properties are good with a high interfacial shear stress. Transmission electron microscopy observation of the interphase reveals that it is made of an anisotropic turbostratic material.

Furthermore, the fiber/matrix debonding, which occurs during mechanical loading, is located within the BN interphase itself.

INTRODUCTION

In SiC/SiC type ceramic matrix composites, a good toughness can be achieved by adding between the fiber and the brittle matrix a thin film of a compliant material called "interphase" [START_REF] Evans | The physics and mechanics of fibre-reinforced brittle matrix composites[END_REF]. Anisotropic pyrolytic boron nitride obtained from BF3/NH3/H2 mixture can play such a role. However, its processing by LP-CVD (Low Pressure Chemical Vapor Deposition) from BF3 requires protecting the fiber from gaseous chemical attack [START_REF] Rebillat | Oxidation resistance of SiC/SiC minicomposites with a highly crystallised BN interphase[END_REF] [START_REF] Jacques | SiC/SiC minicomposites with structure-graded BN interphases[END_REF]. Furthermore, the CVD apparatus is quickly deteriorated by the aggressive halogenated gases and expensive maintenance is needed. On the other hand, some authors have reported the use of a halogenide-free precursor: B[N(CH3)2]3 (tris(dimethylamino)borane, TDMAB) for CVD semiconductor h-BN film processing [START_REF] Dumont | Deposition and characterization of BN/Si(0 0 1) using tris(dimethylamino)borane[END_REF].

The aim of the present contribution was to prepare within one-dimensional minicomposites a BN interphase from TDMAB and to characterize this interphase and the properties of these SiC/BN/SiC minicomposites.

EXPERIMENTAL

SiC/BN/SiC minicomposites were produced by infiltration of the BN interphase within a Hi-Nicalon (from Nippon Carbon, Japan) fiber tow by LP-CVD in a horizontal hot-wall reactor (inner diameter: 24 mm) at a temperature close to 1100°C during 90 seconds. TDMAB vapor was carried by hydrogen through a bubbler at 30°C (TDMAB is liquid at this temperature and the vapor pressure is 780 Pa). The H2 gas flow rate was 15 sccm. NH3 was added to the gaseous source with a flow rate of 100 sccm in order to enhance nitrogen source and favor amine group stripping from the precursor and carbon suppression in the coating. A BN film was also deposited with the same conditions on a Si wafer for Fourier transform infrared (FT-IR) spectroscopy (Nicolet spectrometer, Model MAGNA 550, USA) and X-ray photoelectron spectroscopy (XPS) analyses (SSI model 301 spectrometer). The SiC matrix was classically infiltrated in the fiber tow from CH3SiCl3/H2 precursor gases at 950°C in a second LP-CVD reactor. In both cases, the total gas pressure in the reactors was as low as 2 kPa in order to favor infiltration homogeneity within the fiber tows.

The interphase thickness was about 150 nm and the fiber volume fraction was about 40 % (measured by weighing). The minicomposites were tensile tested at room temperature with unload-reload cycles using a machine (MTS Systems, Synergie 400, USA) equipped with a 2 kN load cell. The minicomposite ends were glued with an epoxy resin (Lam Plan, ref 607, France) in metallic tubes separated by 40 mm that were then gripped into the testing machine jaws. The crosshead speed was 0.05 mm/min. The strain was measured with an extensometer (MTS, model 634.11F54, USA) directly gripped on the minicomposite itself.

The extensometer gauge length was 25 mm. The total number of matrix cracks was verified by optical microscopy on polished longitudinal sections of the failed minicomposites after chemical etching (Murakami reactant) in order to reveal the matrix microcracks which were closed during unloading. The interfacial shear stress  was then estimated from the last hysteresis loop recorded before failure by following the method described in reference [START_REF] Lamon | Microcomposite test procedure for evaluating the interface properties of ceramic matrix composites[END_REF].

Thin longitudinal sections of minicomposites were studied by transmission electron microscopy (TEM: Topcon 002B, Japan) after tensile test using bright-field (BF), high resolution (HR) and selected area electron diffraction (SAED) techniques. The samples were embedded in a ceramic cement (CERAMABOND 503, Aremco Products Inc., USA) and mechanically thinned. The thin sheets (~60 µm in thickness) were then ion-milled (GATAN PIPS, USA) to electron transparency.

RESULTS AND DISCUSSION

Only two absorption bands are seen on the transmittance FT-IR spectra at 810 cm -1 and 1380 cm -1 typical of h-BN; OH bonds are not detected (Fig. 1).

At the film surface, the B/N atomic concentration ratio determined by XPS is close to one.

After ionic etching, the carbon content due to surface pollution decreases drastically; the nitrogen deficit is due to a preferential etching (Fig. 2). Both analyses confirm the BN nature of the films.

Figure 3 displays a typical force-strain curve for SiC/BN/SiC minicomposites. 588 matrix cracks were detected after failure along the 25 mm gauge length. The composites exhibit a non-brittle behavior: a non-linear domain evidencing matrix microcracking and fibre/matrix debonding follows the initial linear elastic region up to a high force at failure (170 N).

Therefore, (i) the BN interphase acts as a mechanical fuse and (ii) the Hi-Nicalon fibers were not damaged during the interphase BN processing from TDMAB. Furthermore, the calculated  is 230 MPa. This value corresponds to a good load transfer between the matrix and the fibers and is as high as the best values obtained with BN interphases processed from classical halogenated gases [3] [6].

TEM observation of minicomposite pieces after failure (Fig. 4) shows that the matrix cracks deflections are preferentially localized within the BN interphase. Figure 4.a exhibits a thin matrix microcrack with a small opening displacement that is stopped within the interphase before reaching the fiber. In figure 4.b, a larger matrix crack which has been widened by the ion-milling is observed. In that case, some BN material remains bonded on both the fiber and the matrix. Thus, neither the interface with the fiber as in reference [START_REF] Naslain | Boron nitride interphase in ceramic-matrix composites[END_REF] nor the interface with the matrix is a weak link. The role of mechanical fuse is played by the boron nitride interphase itself. This feature agrees with the good interfacial shear stress measured for these minicomposites and corresponds to a strong fiber bonding characterized by a high strength and a high toughness [START_REF] Droillard | Strong interface in CMCs, a condition for efficient multilayered interphases[END_REF].

In figure 4.c, a crack is observed within the interphase. A higher magnification in HR mode (Fig. 4.e) reveals that the orientation of the 002 BN planes seems to influence the crack path: the crack and the lattice fringes have the same curvature. Furthermore, the existence of two distinct BN 002 diffraction arcs in the SAED pattern (Fig. 4.d) is due to a preferential orientation parallel of the 002 planes to the fiber axis. This structural anisotropy promotes the mode II crack propagation observed in the interphase.

CONCLUSION

A BN interphase was processed by LP-CVD within SiC/SiC minicomposites from tris(dimethylamino)borane, a halogenide-free precursor. The structure of the BN material is anisotropic and allows deflecting the matrix cracks during mechanical damaging. This interphase is strongly bonded to the fiber and plays the role of a mechanical fuse. The good mechanical properties of the composites allow considering the TDMAB as a new alternative precursor to classical aggressive halogenated gases for LP-CVD boron nitride interphase processing.

Figure 1 :

 1 Figure 1: Transmittance FT-IR spectra of the BN film.

Figure 2 :

 2 Figure 2: XPS depth atomic concentration profiles for the BN film (sputter rate:  1 -4 nm/min).

Figure 3 :F

 3 Figure 3: Tensile force-strain curve with unload-reload cycles for the minicomposites (for clarity only a few hysteresis loops are represented).

Figure 4 :

 4 Figure 4: TEM observation of the SiC/BN/SiC minicomposite according to the BF mode (a), (b) and (c), the SAED technique (negative pattern of the Hi-Nicalon fiber and the interphase) (d) and the HR mode (e).

ACKNOWLEDGEMENT

The authors are grateful to G. Guimon from LPCM (University of Pau, France) for XPS analysis.