Dr Luz Zavala-Sanchez

Dr Ibrahim Khalil

Dr Laetitia Oliviero

Dr Jean-François F Paul
email: jean-francois.paul@univ-lille1.fr

Dr Françoise Maugé
email: francoise.mauge@ensicaen.fr

Structure and quantification of edge sites of WS 2 /Al 2 O 3 catalysts coupling IR/CO spectroscopy and DFT calculations

Keywords: Hydrotreating catalyst, Tungsten disulfide (WS 2 ), CO adsorption, IR spectroscopy, density functional theory, morphology

IR spectroscopy of CO adsorption (IR/CO) and Density Functional Theory (DFT) calculations on sulfide W/Al 2 O 3 catalyst were investigated for an insight into the sulfide edge sites. Parallel between experimental and theoretical results allow to assign the ν(CO) bands at 2121 and 2066 cm -1 to CO adsorption on M-and S-edge sites of WS 2 phase, respectively.

Through a careful analysis of CO spectra on well-selected W catalysts, the molar attenuation coefficients for CO adsorbed on the two exposed edges could be determined. This allowed the morphology of WS 2 slabs to be calculated. This work shows that increasing the W loading (6-20 wt% W) changes the slab morphology from an almost pure triangle exhibiting mostly M-edge to a truncated triangle.

On W as well as on Mo sulfide catalysts, IR spectroscopy and DFT calculation show that CO adsorption allows to account for M-and S-edge sites, and thus of the morphology of sulfide slabs. It was shown that increasing the W loading shifts the slab morphology from a triangle exposing predominantly M-edge towards a more hexagonal shape. For close to monolayer coverage of alumina, Mo and W sulfide slabs present similar shape i.e. truncated triangle. However, edge sites of WS 2 slabs clearly exhibit greater stability under H 2 atmosphere than Mo sulfided ones.

* Values corresponding to a metal close to monolayer (at the oxidic state) on the alumina surface. Sulfidation conditions: Mo/Al2O3 Ts = 423 K, 2h : W/Al2O3 Ts = 473 K, 2h.

INTRODUCTION

Hydrotreating (HDT) of petroleum fractions is one of the central refining processes to produce clean fuels that meet stringent current ecological requirements. The present challenge is to meet the current requirements for ultra-low sulfur content in fuels by extracting from heavier oil deposits. Hydrodesulfurization (HDS) is a key process in refineries where transition metal disulfides (TMS) supported on gamma alumina (γ-Al 2 O 3 ) are used for HDT processes. Among the different metals, Mo or W disulfides with Co or Ni metals as promoters have shown the most promising results in this field. The CoMo and NiMo/Al 2 O 3 catalysts have been extensively studied, meanwhile NiW catalysts have remained less investigated. [1] The importance of NiW/Al 2 O 3 sulfide catalysts is given by their excellent performance in hydrogenation reactions for deep HDS, providing a high potential for the conversion of alkyl-dibenzothiophenes (alkyl-DBTs). [2,3] HDS catalysts have been the subject of numerous characterization studies, [4] and presently there is a general agreement that the sulfide phase MoS 2 (WS 2 ) presents a lamellar structure, formed by layers of MS 2 (M=W, Mo). The 2D morphology of these layers, their average stacking (~2 to 4) and their average size (~3 to 6 nm) depends on the sulfidation conditions, the metal loading among other parameters. Many correlations between the structure and the activity of these systems link the catalytic reaction to the sites along the edges of the individual layers MS 2 . At this location, the promoter atoms are also found in the so-called CoMoS type structures (NiMoS, CoWS and NiWS). [START_REF] Topsøe | Catalysis Science and Technology[END_REF] The MS 2 slabs expose principally two types of edges: the metal terminated edge (M-edge) and the sulfur terminated edge (S-edge). Studies also indicates that M-edge and Sedge sites have different intrinsic activities in HDS reactions, S-edge being more active and more favorable for the promotion and the formation of the promoted phase. [START_REF] Okamoto | [END_REF] An informative way to characterize sulfide sites on these catalysts is the low temperature CO adsorption followed by IR spectroscopy (IR/CO). In addition to the detection of the sulfide sites, this characterization offers the advantage of quantifying their concentration by means of the molar attenuation coefficient, and thus gives access to sulfide phase dispersion and intrinsic activity of the sulfide sites (ref). [7] It should be mentioned that several ν(CO) bands characterized the sulfide sites, even on unpromoted catalysts. Unequivocal attribution of the CO adsorption bands to sites of specific sulfide structures cannot be achieved by IR spectroscopy alone. The continuous development of modeling techniques as the Density Functional Theory (DFT) allows defining realistic surfaces models. The modeling of the stable sulfide surfaces according to the sulfide atmosphere is a powerful approach to obtain a description of the various CO adsorption sites. [8,9] Hence for MoS 2 , Travert et al. demonstrated that the coupling of techniques as IR/CO and DFT permitted to attribute the main ν(CO) bands to M-edge sites and to S-edge sites as well as to show that these sulfide sites can be modified by post-treatments with hydrogen. [10,11] Further, determination of the molar attenuation coefficients of ν(CO) band characteristics of the M-edge and S-edge sites allowed quantification of these two edges and thus assessment of the morphology of MoS 2 slabs. [12] It was thus possible to demonstrate that the MoS 2 morphology (expressed by S edge /M edge ratio) varies with the preparation parameters (metal loading, chelating agent addition, support nature) [7,[13][14][15] and the sulfidation conditions. [13,16] Recent observations of the sulfide catalysts by STEM HAADF confirmed the morphology of the MoS 2 slabs deduced from the spectroscopic analysis [17] . These knowledge allows a controlled design of the MoS 2 -based slabs from a slightly truncated to heavily truncated triangle sulfided slab morphology.

In comparison with Mo catalysts, W based ones are less documented in literature. [18] Although a considerable amount of research has been made on their activity, selectivity and in general catalytic properties of the W catalysts, an accurate description of the active sites is still lacking. [13,19,20] So far the available CO spectra representing W-based catalyst are rather limited, and no reports were established on the assignment of their specific IR/CO bands, neither on the quantification of these edge sites. Hence, this paper presents an insight into the sulfided W/Al 2 O 3 system characterized through IR spectroscopy and DFT calculation. We first perform the IR/CO adsorption experiments over W/Al 2 O 3 catalyst, later the modeling of the WS 2 (1 0 0) stable surfaces and the calculation of the stretching frequencies of CO adsorbed on these surfaces. The parallel between experimental and theoretical results allows assigning the bands at 2121 and ~2066 cm -1 to CO adsorbed on M-and S-edge sites, respectively. In a further step, the molar attenuation coefficient of these bands was determined that allows accounting for the effect of W loading on the WS 2 slab morphology.

EXPERIMENTAL SECTION

Catalyst preparation.

The studied materials consisted in a series of non-promoted W/Al 2 O 3 catalyst prepared by a one-step pore volume impregnation method with variable W loading (6, 9, 12, 16 and 20 W wt. %), 20 W wt. % corresponding to 3. g -1 and pore volume of 0.82 ml•g -1 , precalcined in air flow at 723 K for 4 h. After impregnation, the materials were left for maturation at room temperature for 2 h and subsequently the complete catalyst series was thermally treated at 383 K for 16 h inside a rotatory furnace under air 100 ml·min -1 flow and a heating rate of 3 K·min -1 .

Hereinafter, the W/Al 2 O 3 catalysts are denoted as X_W/Al 2 O 3 where X refers to the W wt. % loading in the catalysts.

Low temperature CO adsorption followed by IR spectroscopy characterization (IR/CO).

The WS 2 edge sites of these catalysts were characterized by low-temperature (~100 K) CO adsorption followed by IR spectroscopy (IR/CO). CO adsorption was performed at low temperature to avoid any reaction with the catalysts surface and to strengthen the interaction with the weakest adsorption sites of the catalyst surface. The oxide catalyst was grounded and pressed into self-supported wafer (ca. 10 mg, precisely weighted, for a 2.01 cm 2 disc) and placed into a quartz IR cell with CaF 2 windows. After in situ sulfidation under 10% H 2 S/H 2 with a flow rate of 30 ml·min -1 for 2 h at 673 K (sulfidation starting from room temperature with a heating rate of 3 K.min -1 ), the cell was flushed with Ar for 10 min and evacuated down to P<1·10 -4 Pa maintaining the 673 K temperature during 1 h. Finally, the sample was cooled down to room temperature and further down to 100 K under vacuum. CO adsorption experiments were performed introducing small calibrated CO doses and then an equilibrium pressure of 133 Pa. Note that it was ensured that this protocol of sulfidation yields to catalysts with a WS 2 amount that is comprised between 92 and 100% whatever the W loading.

After the sulfidation following the previous procedure, pure hydrogen post-treatment was performed on the 20_W/Al 2 O 3 catalyst. In such a case, the sulfide catalyst was in situ reduced under pure H 2 with a flow rate of 50 ml•min -1 at 673 K for 2 h. After the aforementioned treatment, the sample was flushed under Ar flow for 10 minutes and the temperature was cooled down under high vacuum (P = 9.7 ˟ 10 -5 Pa residual pressure) to room temperature and then to 100 K to perform CO adsorption.

FTIR spectra were recorded with 256 scans and a resolution of 4 cm -1 using Nicolet Nexus FTIR spectrometer equipped with a MCT detector (FTIR= Fourier Transform Infrared; MCT=Mercury-Cadmium-Tellurium). All spectra were normalized to a disc of 5 mg·cm -2 . The bands of the adsorbed CO species were obtained by subtracting the spectra recorded after CO adsorption minus before. All experiments were duplicated, with a maximum error of 5%. The band areas of the different ν(CO) components that characterize the WS 2 can be obtained by careful spectrum decomposition. In this work, the decomposition was done with Peakfit V4.12 software using the "Autofit peak III decomposition Method". Before the decomposition, second derivative of the spectra was performed to determine the number of bands. For the decomposition, the center and full width at half height (FWHH) of each band are allowed to vary in a small range (± 3 cm -1 ).

Computational techniques.

Method description: computational parameters.

The density functional theory (DFT) calculations were carried out with the Vienna Ab initio Simulation Package (VASP) software. [21,22] The energy is calculated using the nonlocal functional generalized gradient corrections (GGA) approximation as parameterized by Perdew et al. [23,24] The wave function is expanded in a plane wave basis set, and the electron-ion interactions are defined using the Projector Augmented Plane Wave (PAW) method. [25] In order to compute reliable adsorption energies, Van der Waals forces were considered in the calculations. The Perdew-Burke-Ernzerhof (PBE) functional was employed with 0.1 eV Gaussian smearing. [26] It has been shown that PBE gives good results in ionic systems if the Van der Waals interactions are considered. [27] The solution of the Kohn-Sham equation was improved self-consistently until a difference lower than 10 -5 eV was obtained between successive iterations. The atomic positions were optimized until the forces become less than 0.03 eV·Å -1 . Test calculations were made by varying cut-off energy from 300 to 600 eV and using the Kpoints mesh from 3 × 1 × 1 to 5 × 3 × 1 following Monkhorst-Pack [28] schemes to define calculation parameters. From these evaluations, all the bulk and surface calculations were performed with a 550 eV cut-off energy, using a 3 × 3 × 1 K-point mesh for all surface calculations. Using these parameters, the accuracy of the DFT calculation is expected to be better than 0.1 eV for the calculated adsorption energy.

Surface model description.

In the present work we have used a large supercell (12.72 x12.50 x30 Å 3 ) that allows a good description of the WS 2 surface dimensions (see Figure 1). This supercell model containing four rows in the x-direction, two layers along the y-direction, and five in the zdirection is appropriate for describing the structural and adsorption properties of the (1 0 0) WS 2 surface. A vacuum layer of 15 Å is located above the WS 2 slab in the z direction in order to avoid interactions between slabs. The three upper W rows and the adsorbed molecules were allowed to relax during the geometry optimization, while the two lower ones were kept fixed at the bulk geometry. The CO vibration frequencies were calculated by numerical differentiation of the force matrix. The CO stretching vibrational mode being very weakly coupled with the vibration of the surface, only the part of the matrix corresponding to the atoms closest to CO was considered in the diagonalization process of the Hessian matrix. In order to determine the sulfur coverage of the stable edges, we performed a systematic study varying the sulfur coverage of both edge between 0 and 8, ie starting for the pure W termination to a fully sulfur satured surfaces. We have taken into account the effect of the temperature and pressure by calculation the thermodynamic correction using the classical statistical thermodynamic formalism. [29] At 673 K, the main

Determination of molar attenuation coefficients for CO adsorbed on M-edge and S-edge of site WS 2 catalysts.

According to the adapted Beer-Lambert law (Eq. 1) to solid materials, the concentration of sites (n, μmol·g -1 ) on which CO is adsorbed can be calculated using the associated CO molar attenuation coefficient (Ɛ, cm·μmol -1 ) and the area of ν(CO) band (A, cm -1 ) normalized to the surface (S, cm 2 ) and to the mass (m, g) of the catalyst wafer measured after sulfidation:

(1)

The typical CO adsorption experiments on WS 2 /Al 2 O 3 are performed by adding small CO doses, starting from 9.0x10 -3 μmol up to 8.64 μmol (cumulative doses) . The Ɛ values can be deduced from the slope of the linear relationship between the area of ν(CO) band (A CO ) and the amount of CO introduced onto the sample (n CO ) considering that all the introduced doses are adsorbed on the pellet (no residual pressure could be detected in the IR cell). However, the determination of the amount of CO adsorbed on a given site can be very delicate when the CO introduced interacts with several adsorbing sites.

In the present case, we had to determine the molar attenuation coefficient of two ν(CO) bands corresponding to two adsorption sites. It has firstly required the selection of samples for which analysis of the spectra from the very first doses of CO shows adsorption on only one type of site (Type 1). Thus, the molar attenuation coefficient (Ɛ 1 ) could be determined using eq. ( 1).

Then for Type 2 site, the methodology had to be different since none of the spectra whatever the samples show only CO adsorption on this site. Thus, for spectra that present two contributions, the Beer Lambert law can be written as:

(2)

The molar attenuation coefficient of CO adsorbed on the second contribution (Ɛ 2 ) can be determined as follows: after the determination of the area of the two contributions (A CO1 , A CO2 ), the number of CO adsorbed on the site 1 (n CO1 ) can be calculated from the previously determined Ɛ 1 value. Then, the number of CO adsorbed on the site 2 (n CO1) is determined by subtracting from the total amount of CO introduced n CO , the amount of CO adsorbed on site 1, n CO1 . Further, the Ɛ CO2 value can be calculated from eq. ( 2).

The ν(CO) band area of the components of CO adsorbed on WS 2 massif was obtained by careful spectrum decomposition using parameters described previously in Section 2.2.

RESULTS AND DISCUSSION

Characterization by low-temperature IR spectroscopy of CO.

IR/CO of sulfided WS 2 /Al 2 O 3 catalyst.

The IR spectra of CO adsorbed on sulfided 20_W/Al 2 O 3 catalyst are presented in Figure 2. Four main bands at 2185, 2160, 2121, and 2066 cm -1 were detected. The intensity of these bands increases with the amount of CO introduced. The higher wavenumber bands, namely 2185 and 2160 cm -1 , are attributed to coordination of CO on Lewis acid sites (Al 3+ ) of the alumina support and CO on hydrogen bonding with the support hydroxyl groups (OH), respectively. [12,30,31] Whereas the bands located at 2121 cm -1 and 2066 cm -1 are attributed to CO adsorbed on edge sites of the WS 2 slabs. At higher CO uptake appears a shoulder at 2141 cm -1 , corresponding to physisorbed CO. The two bands at 2121 cm -1 and 2066 cm -1 were observed since the very first introduced CO doses and were attributed to CO in interaction with coordinatively unsaturated W sites (CUS) of low oxidation state. [32] Early work of Duchet [30] on W/Al 2 O 3 catalysts already reported the presence of two bands at 2110 cm -1 and 2060 cm -1 on sulfided phase, called W 1 and W 2 , respectively. On the other hand, Crepeau in his work on WS 2 /ASA, [START_REF] Crepéau | Caracterisation de l'acidite de Catalyseurs d'hydrocraquage Amorphes Sulfures[END_REF] reported the presence of W 1 and W 2 bands at 2127 cm -1 and 2072 cm -1 . The difference in the wavenumber between that reported by Duchet on WS 2 /Al 2 O 3 and by Crepeau on WS 2 /ASA shows that the bands characteristic of the WS 2 phase are also sensitive to the nature of the support.

The inset Figure 3-A that reports the spectra normalized to the most intense band, reveals that the band width located at 2121 cm -1 (W 1 ) remains without alteration whatever the amount of CO introduced meanwhile the band at ~ 2066 cm -1 shifts and widens. A closer inspection to the spectra at intermediate CO doses was carried out with the help of the second derivative function (Figure 3-A). This analysis points out that at least three components (2075, 2066 and 2051 cm -1 ) can be distinguished in the W 2 band whereas the W 1 band only comprised one component at 2121 cm -1 . This analysis that specifies the maximum of the ν(CO) bands allows to carry out a proper decomposition of the CO spectra. order to fit properly the v(CO) bands on WS 2 phase. One peak was used to fit the ν(CO) band on W 1 -edge, which is located at 2121 cm -1 . Three peaks were employed to fit the ν(CO) band on W 2 -edge, which are located at 2075, 2066 and 2051 cm -1 . This confirms that W 1 band only comprised one single component whereas W 2 band comprised several components whose ratios change with CO doses. As hydrogen is one of the reactants during the HDS process, its reactivity with HDS catalysts is of great importance. [7,[START_REF] Travert | [END_REF] Molecular hydrogen can be adsorbed dissociatively on WS 2 edges, leading to the creation of sulfur vacancies by removing sulfur in the form of H 2 S and resulting in the WS 2 edges with lower sulfur coverage. The sulfur coverage at the edges of TMS nanoparticles depends on the H 2 S/H 2 ratio. Thus, the ease for the creation of sulfur vacancies on the edge sites of the TMS catalyst can be evaluated by comparing the CO uptake before and after the H 2 treatment. [START_REF] Cristol | [END_REF] In order to investigate the effect of reductive treatment, the sulfided 20_W/Al 2 O 3 catalyst was treated under pure H 2 flow for 2 h at 673 K at 1 atm pressure before being characterized by IR/CO.

Figure 4 shows that H 2 post-treatment leads to the increase of intensity of the 2160 and 2186 cm -1 bands ascribed to Al 3+ vacancies and hydroxyl groups of the support: this increase could be explained by evacuation of some adsorbed H 2 S during the heating process. As observed in Figure 4, the H 2 post-treatment also leads to some increase of the ν(CO) intensity in the region corresponding to W 2 -edge (2000-2100 cm -1 ), while the v(CO/W 1 -edge) remained practically unchanged. These results indicate that vacancies creation occurs more easily on the W 2 -edge and only limited amount of S atoms can be removed from the W 1 edge by H 2 treatment. An inspection in more detail of the three components associated with the W 2 -edge reveal that they all increase with a same proportion (Inset Figure 4). Nevertheless, it should be underlined that intensity increases of the W 2 bands are small, suggesting that the edges of the sulfided WS 2 slabs are rather stable upon H 2 -treatment. In sulfidation conditions, the most stable surface corresponds to [4 -7] (Figure 6-A).

The coordination of both the W atoms of the M-edge is six (VI) while on the sulfur edge, Considering only coordination numbers, we can suppose that CO adsorption on the Sedge will be favored in a reductive environment. Here, the coordination of the W atoms of the S-edge decreases from V-VI to IV and the number of CUS increases when increasing the H 2 /H 2 S ratio, while no change was observed in the geometry of the Medge.

Calculation of CO adsorption

The treatments to which the catalysts were subjected before the adsorption of CO were carried out to correspond to two of the three stable surfaces of the WS 2 edges. The stable zone corresponding to the sulfidation procedure under pure H 2 S was not considered in our case since it is less frequently used at industrial level. All computed adsorption energies and vibrational frequencies of CO on [4 -7] and [4 -4] surfaces for both edges are reported in Table I.

S-edge Surface [4 -4] M-edge S-edge M-edge Surface [4 -7]

A) B) On the M-edge either in sulfiding or in reducing conditions [4x], CO adsorption can take place with an adsorption energy ranging between -0.63 to -0.70 eV, depending on the exact geometry of the M-edge. The geometry of the W surface is subject to Peierls distortion, but this does not lead to any effect on the ν(CO) frequency that is 2025 cm -1 . In sulfiding condition, the stable surface of the S-edge is [x -7], a top CO adsorption on W 5c is also possible with adsorption energy of -0.40 eV. In this configuration, the CO molecule is almost perpendicular to the surface and the ν(CO) wavenumber is now calculated at 1957 cm -1 . In conditions corresponding to high hydrogen amount, the stable surface is the [x -4]. The 3 S atoms are removed, creating 4 CUS sites (W 4c atoms) on the S-edge. The S atoms on the S-edge form a zigzag chain that is flexible.

The sulfur atom can move from one side of the plan defined by the W atoms to the other side easily. On the most stable geometry, the adsorption energy is -1.04 eV and the associated wavenumber is 1959 cm -1 . The displacement of a sulfur atom form one side of the W plane to the other one reduced the stability of the S-edge, the adsorption energy is then -0.83 eV. In this configuration, the CO stretching wavenumber is computed at 1949 cm -1 . Thus there is a difference around 70 cm -1 between CO adsorbed on the S-edge and on the M-edge. Due to the multiple configurations of the S atoms on the S-edge, a broadening of the ν(CO) band on S-edge is expected.

A representation of the most stable structure is given in Figure 8. The CO molecule is adsorbed on the S-edge perpendicular to the surface (Figure 8-A).

CO adsorption: Experiment vs Calculation

Experimental spectra present two main ν(CO) bands (W 1 , W 2 ) separated by ~ 55 cm -1 .

Their intensity ratio varies significantly with the conditions of treatment and preparation (Figure 4 and Figure 9) indicating that they are characteristics of two distinct sites of the sulfided slabs. In parallel, DFT calculations show that CO adsorption on M-edge and on S-edge of WS 2 surface gives rise to ν(CO) bands separated by ~ 70 cm -1 . This allows us to propose assigning the high wavenumber band (W 1 , 2121 cm -1 ) to CO adsorbed on M-edge and the low wavenumber band (W 2 , 2066 cm -1 ) to CO adsorbed on S-edge. It should be mentioned that, as for DFT calculation, the differences of wavenumber are more reliable than the value of wavenumbers since no scaling factor nor anharmonic correction have been taken into account.

Other interesting parallels between experiment and calculation on W system appears. First, the experimental spectra (Figure 3 A and B) point out that the W 1 band at 2121 cm -1 correspond to one single species and DFT calculation reports that there is one single geometry for CO adsorption on M-edge. Second, experiments show that the W 2 component comprised several bands (2075, 2066, 2051 cm -1 ) and calculation shows for the CO adsorption on the S-edge that numerous geometries and orientations of the S atoms are stable, giving rise to more than one type of possible adsorption of CO at this edge. Moreover, despite the existence of different types of geometry, the variation of the calculated wavenumber from one band to another is relatively small (10 cm -1 ).

Another feature to mention is that reductive treatment leads to almost no change of intensity of W 1 bands, while W 2 band increases slightly (Figure 4). This is in good agreement with trends reported by calculation that indicates no change in W coordination on M-edge and some changes in the W coordination on S-edge between sulfiding and reductive conditions (Table I) and also that the number of CUS site on the S-edge will increase in reducing conditions, while this number is constant on the Medge.

The agreement between experimental and calculated features supports the assignment of the band at 2121 cm -1 to CO adsorption on M-edge (CO M-edge ) and that at ~2066 cm -1 to CO adsorption on S-edge (CO S-edge ).

Influence of W loading on W-and S-edge bands.

A series of W/Al 2 O 3 catalysts, in which the W coverage was increased from 0.85 to 3.3 atoms·nm -2 (i.e. from 6 to 20 wt% W), has been analyzed by IR/CO from small CO doses up to 133 Pa at equilibrium. As a general manner, bands similar as those reported on W_20/Al 2 O 3 catalyst (Figure 2) are observed although with intensity and ratio that depends on the W amount and on the alumina coverage Figure 9. At the lowest metal loading (6 wt. %) and under 133 Pa of CO, ν(CO) bands in the 2130-2000 cm -1 region are almost undetectable (Figure 9). This is partly due to the high intensity of the ν(CO) bands associated with the support, which overshadow the signal corresponding to the sulfided sites. As the W concentration increases, the ν(CO) bands associated to WS 2 sites gain intensity and are better defined (Figure 9 see insert). In addition to the well-visible band CO M-edge band, centered at 2121 cm -1 , the CO S-edge band begins to appear at 2066 cm -1 . The intensity of CO S-edge band increases with the W loading in the catalyst. On the other hand, the intensity of the ν(CO) bands associated to the support (Al 3+ Lewis acid sites and OH groups of Al 2 O 3 ) decrease as a result of a more covered surface of the alumina support by WS 2 , which agrees with the increase of the W loading. 133 Pa CO at equilibrium, Insert: at sulfide site saturation, both at 100 K). The WS 2 /Al 2 O 3 catalysts were obtained by sulfidation at 673 K and 0.1 MPa with 10% H 2 S/H 2 .

Determination of molar attenuation coefficients for W-and S-edge bands

In previous works of our group on MoS 2 -based catalysts, [START_REF] Chen | [END_REF] distinct molar attenuation coefficient values were obtained for CO adsorbed on either M-edge (Ɛ CO /M-edge) or Sedge (Ɛ CO /S-edge) (Table II). These values allowed the quantification of MoS 2 sites of each edges and thus calculation of the S-/M-edge ratio that defines the morphology of the MoS 2 slabs. [14] Recent work on sulfided Mo/Al 2 O 3 catalysts prepared with and without citric acid showed a good agreement between sulfided slab shape determined from IR/CO, and that observed on transmission electron microscope by STEM-HAADF. [17] This gave a proof by image of the morphological changes of the catalyst according to the preparation modes, and also underlined that IR/CO method allows to reach a representative average picture of the sulfided slab shapes. Thus, the In the literature, no Ɛ CO values for M-edge and S-edge are reported for any of the species formed in the WS 2 or NiWS catalysts. A close inspection of the spectra obtained for the first doses of CO introduced shows that, at low W loading, only one ν(CO) band characteristic of M-edge sites appears (Figure 10 A-C). However, spectra obtained on 6_W/Al 2 O 3 present signals associated to CO adsorbed on alumina Lewis acid sites from the first CO doses. This is in agreement with the very limited support coverage by W phase (0.85 atoms·nm -2 ), but this prevents using the spectra obtained on the catalyst with the lowest W amount to calculate Ɛ CO/M-edge sites. However, when the W amount is somehow greater as for 9_W/Al to CO interaction with the M-edge site (2121 cm -1 ) and the S-edge site (2072 cm -1 ). With the molar attenuation coefficient of CO adsorbed on M-edge (Ɛ CO/M-edge ) already determined, the amount of CO adsorbed specifically on M-edge sites can be calculated from the band area of these sites and using the methodology described in section 3.5.

Hence, a relationship between the variation of the band area of CO adsorbed on S-edge and the number of CO adsorbed on S-edge sites (calculated from the total number of CO introduced in the cell minus the number of CO adsorbed on M-edge sites) was used to calculate the attenuation coefficient of CO adsorbed on S-edge (Ɛ CO/S-edge ). The slope of this relationship is the Ɛ CO/S-edge .

(3) Note that a CO band specific of Lewis acid sites appears from the third CO doses, but the signal being low, it has been neglected in the Ɛ CO/M-edge calculations. Figure 11 shows that close Ɛ CO/S-edge values are obtained for these two catalysts i.e. 22.9 ± 6 cm·μmol -1 for 16_W/Al 2 O 3 and 23.4 ± 6 cm·μmol -1 for 20_W/Al 2 O 3 . Thus, the average molar attenuation coefficients ε CO/M-edge and ε CO/S-edge for sulfided W/Al 2 O 3 catalysts are 11.9 ± 2 cm·μmol -1 and 23.1 ± 6 cm·μmol -1 respectively. Note that in all the calculations (Figure 11

The influence of W loading on WS 2 slab morphology.

As observed in Figure 9, the increase of metal loading has an effect not only on the intensity of the bands associated to the M-and S-edges, but also on their ratio. From the molar attenuation coefficients for both edge sites, the S-edge/M-edge ratio can be calculated for the various W catalysts (Figure 12). The value of the ratio undergoes a steady increase along with the W loading and shows that the morphology of the WS 2 slabs changes from slightly truncated triangles exposing mainly the M-edge (S-edge/Medge = 0.17) to a more heavily truncated triangle exposing more S-edge (S-edge/M- catalysts. [13] In Mo system, this phenomenon was associated to a change in the sulfide phase -support interactions with metal loading. This, since the MS 2 phase interacts with Al 2 O 3 support by the M-O-Al linkages that are located at the edges of the slabs instead of at the core slab sites. [13,37] Similarly, the concentration of the W-O-Al linkages will decreases with increasing WS 2 size. It is observed that the WS 2 slabs on WS 2 /Al 2 O 3 catalysts exposed more S-edge with increasing the W loading. All these results suggest that the WS 2 -Al 2 O 3 interactions decrease with increasing W loading. For sulfide W as well as for sulfide Mo catalysts, CO adsorbed on sulfide slabs gives rise to 2 IR bands separated by about 50 cm -1 that allows distinguishing M-edge (CO M- edge ) and S-edge (CO S-edge ). On both systems, the frequencies of CO M-edge appear at greater frequency than the CO S-edge . This is in agreement with the calculated Mo and W coordination that is greater on M-edge (M 6C ) than on S-edge (M 4C ) (Table 1, Ref. [10] ). In agreement with their higher frequencies, molar attenuation coefficient (Ɛ) for CO M-edge is greater than for CO S-edge . From these values, the morphology of the TMS slabs could be calculated. For alumina supported catalysts with metal amount corresponding to monolayer coverage, sulfided slabs with very close morphology i.e. highly truncated triangle (S-/M-ratio = 0.30-0.32) are formed on alumina supported Mo and W catalysts.

Comparison between W and Mo sulfide catalysts.

As summarized in

In both systems, an accurate analysis shows that CO adsorbed on S-edge is characterized by a broad band consisting of several ν(CO) components. The three bands (2065, 2045 and 2020 cm -1 ) characterizing CO adsorption on S-edge of Mo/Al 2 O 3 have been associated to different CO coverages. [10,11,[START_REF] Chen | [END_REF]38] Whereas the three components (2075, 2066 and 2051 cm -1 ) detected on S-edge of W/Al 2 O 3 have been associated to various orientations of the edge sulfur atoms giving rise to more than one ν(CO) band. It must be noted that the relatively small model used to describe the MoS 2 S edge, does not allows various geometries. By contrast, M-edge sites present a more homogeneous geometry. For W catalyst, only one component is detected whatever the H 2 S/H 2 ratio whereas for Mo/Al 2 O 3 system, a band is detected at 2110 cm -1 after sulfidation while a second one develops under reductive atmosphere (2098 cm -1 ). [10,[START_REF] Chen | [END_REF] A noticeable difference between the Mo and W systems is their difference of stability toward reductive treatment. When submitted to H 2 -post-treatment, the stability of the edge sites of sulfide W catalyst is remarkable (only S-edge site amount increases by 16%). By contrast, on Mo system, the CO uptake increases of about 55% both on Mand S-edges. This great sulfur depletion of MoS 2 edge is in agreement with the appearance of a new band (2098 cm -1 ) after reductive treatment whereas no frequency shift are observed on W catalysts.

Hence, sulfided slabs of Mo and W alumina supported catalysts present similar morphologies but different stability regarding H 2 treatment. to CO adsorbed on the M-edge and S-edge sites respectively. The molar attenuation coefficients for CO adsorbed at the M-edge and the S-edge were determined. This allowed the morphology of the WS 2 slabs to be calculated.

Figure 1 .

 1 Figure 1. Representation of the supercell used for the calculations, showing the axes. On the left, cell showing the cut WS 2 (1 0 0) surface. Dark circles: W; light circles: S

  correction (c.a. 95%) is due to gas phase molecules (H 2 and H 2 S) entropy differences. The surfaces are labeled according to the number of sulfur atoms on the two edges: this is [S atoms M-edge -S atoms S-edge ]. The stability of the surface have been computed using the[4-4] one as reference.

Figure 2 .

 2 Figure 2. IR spectra of increasing CO doses (up to 133 Pa at equilibrium) adsorbed at 100 K on 20_W/Al 2 O 3 catalyst sulfided with 10% H 2 S/H 2 at 673 K and 0.1 MPa.

Figure 3 .

 3 Figure 3. (A) IR spectra of intermediate CO doses adsorbed at 100 K on 20_W/Al 2 O 3 catalyst sulfided at 673 K, with their second derivative ; (B) decomposition of the IR spectrum of CO adsorption (P CO =133 Pa at equilibrium).

Figure 4 .

 4 Figure 4. Effect of H 2 treatment at 673 K on CO adsorption (100 K, 133 Pa at equilibrium) on sulfided 20_W/Al 2 O 3 catalyst. IR/CO spectrum obtained after sulfidation (continuous line) and after 2 h of H 2 treatment at 673 K (dashed line); Inset: Effect of the H 2 treatment on the area of the IR/CO bands of the three components of W 2 sites.

1 .

 1 Sulfur coverage and surfaces to study.By observing the crystallographic structure of the WS 2 slabs, two types of edges are considered, namely, the metallic edge (M-edge) and the sulfur edge (S-edge). It has been shown that the number of sulfur atoms present at the edges of the slabs depends 2000 on the H 2 /H 2 S molar ratio. Each surface is designated by the number of sulfur atoms exposed on each of the two edges: this is [S atoms M-edge -S atoms S-edge ]. The stability of the surface, compared to the[4-4] one, at 673K, as a function the partial pressure P(H 2 )/P(H 2 S) logarithm have been computed (Figure5).

Figure 5 .

 5 Figure 5. Free formation enthalpy (in eV) of the edges starting from the [4-4] surface at 673 K, as a function of .

  the coordination is five (V) for two W atoms and six (VI) for two W ones. Under this configuration, each W atom in the M-edge is bridged to 2 S atoms in the plane above the surface of the W atoms, while two W atoms in the S-edge are bridged to 4 S atoms in the plane above the surface of the W atoms and the two other ones bridges to only 3 S atoms. The second stable surface under reductive conditions is the[4 -4] (Figure6-B). The coordination numbers of W atoms are 6 on the M-edge (W 6c ) and 4 (W 4c ) on the S-edge. The terminal S atoms are in bridging position between two W atoms on both edges, the W-S distances are similar to the distances in the crystal (2.40 Å).

Figure 6 .

 6 Figure 6. Depiction of the thermodynamic WS 2 stable surfaces according to the H 2 S/H 2 partial pressures: (A) surface [4-7], stable surface when H 2 /H 2 S < 500; (B) surface [4 -4], stable surface when H 2 /H 2 S>500.

Figure 7

 7 Figure 7 represents the optimized geometry of the structure of the M-edge where

Figure 7 .

 7 Figure 7. Optimized configuration for one CO molecule adsorbed on the M-edge [4-x] of WS 2 surface. A) Front view of the optimized distances between the W atom and the CO molecule. B) Side view of the M-edge showing the W coordination.

Figure 8 .

 8 Figure 8. Optimized configuration for one CO molecule adsorbed on the S-edge [x-4] of WS 2 surface. A) Front view of the optimized distances between the W atom and the CO molecule. B) Side view of the S-edge showing the W coordination.

Figure 9

 9 compares the spectra obtained at 113 Pa CO at equilibrium of the different W-catalysts, the inset shows the IR spectra obtained at sulfide site saturation.

Figure 9 .

 9 Figure 9. IR spectra of CO adsorption on WS 2 /Al 2 O 3 prepared with different W loading (Main:

  determination of the morphology of the WS 2 particles requires the measurement of the molar attenuation coefficients of the ν(CO) bands characteristics of M-edge and S-edge.

  2 O 3 and 12_W/Al 2 O 3 , the relationship between the variation of the band area of CO adsorbed on M-edge and the number of CO introduced can be used to calculate the Ɛ CO/M-edge. As shown on Figure 11, close Ɛ CO/M-edge values were obtained for these two catalysts i.e. 11.4 ± 2 cm·μmol -1 for 9_W/Al 2 O 3 and 12.2 ± 2 cm·μmol -1 for 12_W/Al 2 O 3 . At higher W loading (Figure 10 D-E) i.e. for 16_W/Al 2 O 3 and 20_W/Al 2 O 3 , two ν (CO) bands are detected even for low CO doses. They correspond

Figure 10 .

 10 Figure 10. IR spectra of the first CO doses adsorbed (T = 100 K) on sulfided W/Al 2 O 3 at different metal loadings. Intermediate W concentrations results on CO adsorption only on the metallic edge. (first doses: 0.001, 0.002, 0.003, and 0.004 μmol CO·mg -1 sulfided catalyst).

Figure 11 .

 11 Figure 11. Determination of the molar attenuation coefficients (Ɛ) of CO adsorbed on WS 2 . The spectra for Ɛ determination are reported in Figure 9. The Ɛ values are the slope of the linear relationship between the area of v(CO) band and the amount of CO introduced onto a cm 2 of the sample.

Figure 12 .

 12 Figure 12. The ratios of S-edge/M-edge and corresponding morphologies of WS 2 slabs as function of W loading of WS 2 /Al 2 O 3 catalysts.

  In the present work, carbon monoxide adsorption on alumina-supported sulfided W catalysts was studied by means of IR spectroscopy pairing with DFT calculations. The parallel between experimental and calculated data allowed us to attribute, for the first time, the two ν(CO) bands observed at 2121 cm -1 and about 2066 cm -1 to CO adsorbed on the M-edge and S-edge sites of the WS 2 slabs, respectively.The integrated molar attenuation coefficients of CO associated with the M-and Sedge sites were determined, allowing determination of the WS 2 morphology. Increasing the W loading (3-20 wt% W) allows going from an almost pure triangle exhibiting mostly M-edge to a truncated triangle.Parallel between experiments and DFT calculation of CO adsorption on W and Mosulfide catalysts provides evidence for many similarities between the two systems. On both TMS, CO adsorption give rise to two main ν (CO) bands separated by about 50 cm - 1 that allowed to discriminate M-edge and S-edge sites. For M loading close to monolayer coverage of alumina (in oxidic form), Mo and W sulfide slab present a similar shape i.e. truncated triangle. However, edge sites of WS 2 slabs clearly exhibit greater stability under H 2 atmosphere than Mo sulfided ones.325-343.[38] C. Dujardin, M. A. Lélias, J. Van Gestel, A. Travert, J. C. Duchet, F. Maugé, 2007.Entry for the Table of ContentsText for Table of Contents:CO adsorption on sulfided W/Al 2 O 3 catalysts was studied by IR spectroscopy pairing with DFT calculations. Parallel between experimental and calculated data allowed to attribute, for the first time, the two ν(CO) bands observed at 2121 cm -1 and ~2066 cm-1

  3 atoms•nm -2 , e.g. a submonolayer of W atoms (oxidic form). The catalysts were impregnated with an aqueous solution of appropriate amounts of hydrated ammonium metatungstate ((NH 4 ) 6 H 2 W 12 O 40 •H 2 O SIGMA-ALDRICH) on commercial γ-Al 2 O 3 support (SASOL extrudates, specific surface area of 252 m

  2

Table I .

 I Calculated parameters of CO adsorbed on M-edge (W) and S-edge (S) of WS 2 .

	Edge	Conditions	Surface	W coordination	E ads (eV)	v(CO) (cm -1 )	D W-C (Å)	D C-O (Å)
	M-	sulfidation	[4-x]	6	-0.63 to -0.70	2025	2.11	1.15
	M-	reductive	[4-x]	6	-0.63 to -0.70	2025	2.11	1.15
	S-	sulfidation	[x-7]	5	-0.40	1957	2.09	1.16
	S-	reductive	[x-4]	4	-0.83 ; 1.04	1949 ; 1959 2.09	1.16

Al 2 O 3 11.4 0.966 12_W/Al 2 O 3 12.2 0.989 S-edge 16_W/Al 2 O 3 22.9 0.980 20_W/Al 2 O 3 23.4 0.962 CO amount on M-edge or S-edge (μmol·cm -2 ) edge

  = 0.32). Similar effect of metal loading has been earlier reported for MoS 2 /Al 2 O 3

		A(CO)=nCO·ƐCO
		sample	(Ɛ)	R 2
	M-edge	9_W/

Table II

 II

, many similarities appear for CO adsorption on W and Mo sulfide catalysts.

Table II .

 II Comparison of experimental ν(CO) frequencies and molar attenuation coefficients of CO on M-and S-edge, as well as MS 2 slab morphology for (9 %) Mo/Al 2 O 3 and (20 %) W/Al 2 O 3 catalysts sulfided under H 2 S/H 2 * or post -treated under H 2 at 473K atmosphere.

					H 2 post-
			Sulfidation	treatment vs
					sulfidation
	Catalyst	Edge type	ν(CO) frequency (cm -1 )	Epsilon, Ɛ CO (cm·μmol -1 )	CO band area enhancement

Acknowledgements

L. Zavala-Sanchez acknowledges the Mexican National Council for Science and Technology (CONACYT) for the Ph. D. grant. The authors acknowledge LABEX EMC3 from ANR for the financial support to MIMOSA project. DFT calculations have been performed on the Lille University Calculation center partially funded by Feder.


Dalil Hannani

Clara Locher

Takahiro Yamazaki

Véronique Colin-Minard

Marie Vetizou

Laetitia Aymeric

Sophie Viaud

Daniel Sanchez

Mark J Smyth

Pierre Bruhns

Guido Kroemer

Laurence Zitvogel
email: zitvogel@igr.fr

Pr Laurence Zitvogel

Contribution of humoral immune responses to the antitumor effects mediated by anthracyclines

Keywords: antibodies, immunogenic cell death, chemotherapy, humoral immunity, FcR, cancer Antibody-dependent cell cytotoxicity ATP: Adenosine triphosphate CRT: Calreticulin CTL: Cytotoxic T Lymphocyte DC: Dendritic cell ER: Endoplasmic Reticulum FcR: Fc Receptor HMGB1: High mobility Group Box-1 ICD: Immunogenic Cell

Abstract

Immunogenic cell death (ICD) induced by cytotoxic compounds contributes to the success of selected chemotherapies by eliciting a protective anticancer immune response, which is mediated by CD4 + and CD8 + T cells producing interferon- (IFN). In many instances, cancer progression is associated with high titers of tumor-specific antibodies, which become detectable in the serum but whose functional relevance is elusive. Here we explored the role of humoral immune responses in the anticancer efficacy of anthracyclines.

Chemotherapy reduced the number of tumor-infiltrating B cells and failed to promote humoral responses against immunodominant tumor antigens. While anthracycline-based anticancer chemotherapies failed in T cell-deficient mice, they successfully reduced the growth of cancers developing in mice lacking B lymphocytes (due to the injection of a B celldepleting anti-CD20 antibody), immunoglobulins or immunoglobulin receptors (FcR) due to genetic manipulations. These results suggest that the humoral arm of antitumor immunity is dispensable for the immune-dependent therapeutic effect of anthracyclines against mouse sarcoma. In addition, we show here that the titers of IgA and IgG antibodies directed against an autoantigen appearing at the cell surface of tumor cells postchemotherapy (calreticulin) did not significantly increase in patients treated with anthracyclines and that anti-calreticulin antibodies had no prognostic or predictive significance. Collectively, our data indicate that humoral anticancer immune responses differ from cellular responses in thus far that they do not contribute to the success of anthracycline-mediated anticancer therapies in human breast cancers and mouse sarcomas.

Introduction

Cancer often results from a chronic inflammatory process in which multiple hematopoietic and/or immune components participate. [START_REF] Fridman | Prognostic and predictive impact of intra-and peritumoral immune infiltrates[END_REF][START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF] The question arises as to which immune cell subset directly or indirectly promote malignancy, and whether these chronic infiltrates are generic across multiple solid tumor types or instead reflect tissue-selective, or oncogenic pathway-selective, profiles. If the contribution of leukocyte populations to oncogenesis or tumor progression was generic, therapeutics aimed at quelling the protumorigenic activities of such subsets could be proposed. Hence, it is of utmost importance to investigate how the interplay between inflammatory cells is orchestrated and modulated during anticancer therapies. Dendritic cells (DCs) are bone marrow-derived cells that are present in all tissues [START_REF] Steinman | Taking dendritic cells into medicine[END_REF] where they scan the environment and take up foreign antigens (including tumor antigens) before migrating to lymphoid organs to trigger adaptive immune responses. [START_REF] Steinman | Taking dendritic cells into medicine[END_REF] Upon recognition of a foreign antigen, T and B lymphocytes undergo clonal expansion and mount antigenspecific "cognate" responses. Once the adaptive immune response has ensued, the source of danger or damage is eliminated, inflammation declines and tissue homeostasis is restored.

In tumors, these well-orchestrated series of events fail to resolve and lead to chronic inflammation. Activated leukocytes (mostly represented by myeloid cells) supply direct and indirect mitogenic growth mediators and proteolytic enzymes that stimulate expansion and dissemination of both neoplastic and stromal cells [START_REF] Balkwill | Smoldering and polarized inflammation in the initiation and promotion of malignant disease[END_REF] . Moreover, the chronic presence of paracrine and juxtacrine mitogenic and tissue-remodeling molecules fuels programs that impair anti-tumor cytotoxic T lymphocyte (CTL) or NK/NKT cell-mediated killing of "damaged" (cancer) cells (reviewed in [START_REF] Hanahan | Accessories to the crime: functions of cells recruited to the tumor microenvironment[END_REF] ).

In addition to myeloid cells, lymphocytes play an important role in chronic inflammation. B cells display variable pro-and anti-tumor bioactivities deriving from their functional plasticity and phenotypic heterogeneity. As the sole producers of immunoglobulins (Ig), B cells are critical initiators and modulators of humoral immunity, tailoring specific responses to infections. In addition, B lymphocyte-derived paracrine factors are causative and/or potentiating mechanisms of disease, as shown in autoimmunity. [START_REF] Pillai | B cells and autoimmunity[END_REF] Moreover, there is accumulating evidence that B cell function is relevant to carcinogenesis. Cancer patients often develop specific antibody responses against tumor antigens, 6 which are mostly correlated with poor survival. [START_REF] Tanchot | Tumor-Infiltrating Regulatory T Cells: Phenotype, Role, Mechanism of Expansion In Situ and Clinical Significance[END_REF] Progression of human papillomavirus type 16 E6/E7-driven squamous carcinogenesis in transgenic mice is significantly diminished in the absence of B cells. [START_REF] Andreu | FcRgamma activation regulates inflammation-associated squamous carcinogenesis[END_REF][START_REF] De Visser | De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent[END_REF] In this preclinical tumor model, promotion of the early steps of carcinogenesis is achieved through deposition of IgG autoantibody in neoplastic parenchyma and immune complex/FcγR ligation of mast cells and macrophages, eventually fostering pro-angiogenic and immunosuppressive gene expression programs. [START_REF] Andreu | FcRgamma activation regulates inflammation-associated squamous carcinogenesis[END_REF][START_REF] De Visser | De novo carcinogenesis promoted by chronic inflammation is B lymphocyte dependent[END_REF] Similarly, skin chemical carcinogenesis by two-stage application of the DNA damaging agent DMBA and the tumor promoter TPA occurs more rapidly in B cell -proficient mice than in B cell-deficient mice. [START_REF] Schioppa | B regulatory cells and the tumor-promoting actions of TNF-alpha during squamous carcinogenesis[END_REF] As a possibility, B cells may modulate myeloid cell phenotypes through the secretion of IL-10 and TNFα, thereby accelerating tumor progression. Finally, B cell-derived lymphotoxin β is responsible for promoting castration resistance in mice with transgene-induced prostate cancer, likely by stimulating IKKα and STAT3 activity in malignant cells. [START_REF] Ammirante | B-cell-derived lymphotoxin promotes castration-resistant prostate cancer[END_REF] Whereas these studies revealed protumorigenic potential for B cells, others reported no specific functional significance for B cells during mammary carcinogenesis. [START_REF] Denardo | CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages[END_REF] The efficacy of anticancer therapies is determined by tumor cell-intrinsic as well as by cellextrinsic, in part immune factors. Thus, therapeutic settings that induce tumor antigenspecific Th1/Tc1 responses (i.e. immune responses that involve specific T cell subsets producing IFN) are particularly efficient in eliminating residual tumor cells. The way cancer cells die when exposed to selected chemotherapeutics (such as anthracyclines and oxaliplatin) die, ultimately dictates the immunogenicity of cell death. "Immunogenic cell death"(ICD) can be defined as a process in which cell death is preceded by a preapoptotic endoplasmic reticulum stress response and autophagic program, facilitating the recruitment, activation and antigen-presenting function of DCs. [START_REF] Kroemer | Immunogenic Cell Death in Cancer Therapy[END_REF] Indeed, ICD is accompanied by exposure or secretion of cell death-associated molecular patterns such as calreticulin (for the engulfment of dead bodies by DC), HMGB1 (for the TLR4/Myd88dependent processing of the phagocytic cargo by DC) and ATP (for DC recruitment and the Nlrp3-dependent IL-1 release culminating in Tc1 polarization). [START_REF] Kroemer | Immunogenic Cell Death in Cancer Therapy[END_REF] Accordingly, after exposure to neoadjuvant chemotherapy, approximately one third of stage 2/3 breast cancers appeared highly infiltrated by cytotoxic T cells with a concomitant reduction of B lymphocytes and Th2 cells. [START_REF] Ruffell | Leukocyte composition of human breast cancer[END_REF] Moreover, the relative proportions of macrophages and CD8 + T cells predict pathological responses to neoadjuvant anthracycline-based therapy and overall survival in locally advanced breast cancer. [START_REF] Denardo | Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy[END_REF] Since conventional chemotherapies can elicit tumor-specific T cell immunity, which then contribute to durably and negatively inflecting the tumor growth curve, we addressed the question whether anticancer therapies would also stimulate B cell-or FcR-dependent immune responses. Using a variety of complementary experimental settings, we came to the conclusion that humoral immune responses are not specifically induced by chemotherapy and that they do not significantly contribute to tumor control.

Results

Intratumoral accumulation of antigen-specific T cells but depletion of B cells after chemotherapy.

To evaluate possible anticancer immune responses mediated by B cells, we determined the absolute numbers of CD19 + B220 + B lymphocytes in the CD45 + leukocytic fraction from tumors established subcutaneously for 18 days and harvested at day 8 postchemotherapy with anthracyclines, following previously established experimental settings.

16, 17 MCA205-OVA, a methylcholanthrene-induced sarcoma cell line genetically modified to express the candidate tumor antigen OVA, was injected under the skin of histocompatible C57Bl/6 mice, and the developing cancers were treated by intratumoral injections of anthracyclines (doxorubicin). CD45.2 OVA (SIINFEKL)-specific H2-K b -restricted TCR transgenic CD8 + T cells (CTL) that were adoptively transferred into CD45.1 congenic mice on the first day post-chemotherapy proliferated and accumulated in tumor beds (Fig. 1A-B).

This expansion of OVA-specific T cells reflects a general increase in the frequency of Th1 and Tc1 cells post-chemotherapy. [START_REF] Ma | Contribution of IL-17-producing gamma delta T cells to the efficacy of anticancer chemotherapy[END_REF][START_REF] Ma | Anticancer chemotherapy-induced intratumoral recruitment and differentiation of antigen-presenting cells[END_REF] In sharp contrast, doxorubicin depleted intratumoral B cells by more than 70% (Fig. 1C). Moreover, a single systemic injection of cyclophosphamide reduced the number of splenic B cells, while it increased the frequency of CTL in the spleen (Suppl. Fig. 1A-C). Thus conventional chemotherapeutic regimens that stimulate local or systemic T cell responses, can reduce the number of B cells.

Contribution of cytotoxic T cells but not of B lymphocytes to the therapeutic effect of anthracyclines.

To further assess the role of B cells, we compared the antitumor effects of doxorubicin on MCA205 cancers established in C57Bl6 WT versus MT mice, which lack immunoglobulins and B lymphocytes. [START_REF] Angeli | B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization[END_REF] In MT mice, anthracyclines successfully reduced tumor growth, and this effect was undistinguishable from that observed in WT control mice (Fig. 2A). Next, we took advantage of an anti-CD20 antibody, [START_REF] Horikawa | Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice[END_REF] which was applied at different time points pre/post-tumor inoculation and pre/post-doxorubicin (Fig. 3A) to deplete CD19 + CD20 + B lymphocytes (Fig. 3B, not shown). B cell depletion did not affect the natural expansion of MCA205 cells (which lack CD20 expression, not shown) in vivo and failed to compromise the efficacy of chemotherapy against this MCA205 sarcomas (Fig. 3C-E). In contrast, depletion of CTLs using anti-CD8 antibodies completely abrogated the antitumor effects of anthracyclines (Fig. 4A-B), confirming their contribution to the efficacy of chemotherapy. [START_REF] Kroemer | Immunogenic Cell Death in Cancer Therapy[END_REF] Hence, genetic deficiency or antibody-induced depletion of B lymphocytes did not interfere with the therapeutic activity of anthracyclines.

No contribution of immunoglobulins and Fc receptos to the efficacy of anticancer chemotherapy.

To investigate to which extent immunoglobulins (Ig) might modulate the therapeutic efficacy of chemotherapy, we analyzed the titers of Ig directed against the immunodominant tumor antigen OVA in our model system. OVA-specific IgG/M/A antibody titers (monitored at day 15 and 21 post-therapy) were not boosted by chemotherapy (not shown and Fig. 2B). It is known that leukocyte FcRγ is necessary for squamous carcinogenesis, [START_REF] Andreu | FcRgamma activation regulates inflammation-associated squamous carcinogenesis[END_REF] as well as for short and long-term effects (involving memory T cell responses) in lymphoma bearing mice treated with antibody-dependent cell cytotoxicity (ADCC)-mediating Ab. [START_REF] Minard-Colin | Lymphoma depletion during CD20 immunotherapy in mice is mediated by macrophage FcgammaRI, FcgammaRIII, and FcgammaRIV[END_REF] Therefore, we assessed the putative contribution of FcR to the efficacy of chemotherapy against MCA205-OVA sarcoma. FcγRs are broadly expressed on immune cells and encompass both activating, e.g., FcγRI, III, and IV (including the FcRγ subunit), and inhibitory, e.g., FcγRIIB, subtype complexes. [START_REF] Bruhns | Properties of mouse and human IgG receptors and their contribution to disease models[END_REF] MCA205-OVA was inoculated into mice genetically manipulated to lack specific FcRγ subtypes, namely FcRIII, [START_REF] Hazenbos | Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice[END_REF] FcRI, [START_REF] Ioan-Facsinay | FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection[END_REF] FcRIV, [START_REF] Nimmerjahn | FcgammaRIV deletion reveals its central role for IgG2a and IgG2b activity in vivo[END_REF] as well as into « FcRIV-only » mice (which lack FcRI/IIB/III/FcRI/II genes) [START_REF] Mancardi | FcgammaRIV is a mouse IgE receptor that resembles macrophage FcepsilonRI in humans and promotes IgEinduced lung inflammation[END_REF] or « FcRIIB only » mice (which lack the FcRI/IIIA/IV genes). [START_REF] Takai | FcR gamma chain deletion results in pleiotrophic effector cell defects[END_REF] Thus anthracyclines similarly reduced tumor growth in these FcRγ-deficient mice (Fig. 5A-F) as it did in WT C57Bl6 controls (see above, Fig. 2A). Of note, FcR does not bind immunoglobulins but is a subunit that mediates signal transduction by other receptors relevant to myeloid cell function including the macrophage-inducible C-type lectin (Mincle), [START_REF] Yamasaki | Mincle is an ITAMcoupled activating receptor that senses damaged cells[END_REF] which plays a cardinal role in antimycobacterial responses. [START_REF] Behler | Role of Mincle in alveolar macrophage-dependent innate immunity against mycobacterial infections in mice[END_REF] In accord with the observation that FcRIII did not affect anthracycline responses (Fig. 5), the efficacy of anthracyclines was not compromised in Mincle knockout mice either (Suppl. Fig. 2).

Altogether, humoral immune responses directed against immunodominant tumor antigens as well as FcR are dispensable for the chemotherapeutic activity of anthracyclines.

Humoral immune responses against ICD-related auto-antigens in patients.

The chaperone calreticulin (CRT) is sequestered in the endoplasmic reticulum (ER) in unstressed cells but relocates to the plasma membrane upon induction of ER stress by anthracyclines [START_REF] Kroemer | Immunogenic Cell Death in Cancer Therapy[END_REF] or hypericin-based photodynamic therapy. [START_REF] Galluzzi | Enlightening the impact of immunogenic cell death in photodynamic cancer therapy[END_REF] Its immunohistochemical detection in paraffin-embedded primary breast cancer tissues has been correlated with serum levels of anti-CRT antibodies. [START_REF] Milovanovic | Overexpression of calreticulin in malignant and benign breast tumors: relationship with humoral immunity[END_REF] We analyzed the titers of IgA and IgG antibodies in 75 healthy controls and 63 breast cancer patients (Table 1, Fig. 6A) at diagnosis, during and after systemic chemotherapy (Fig. 6B). Both anti-CRT IgA and IgG serum levels were slightly higher in cancer patients than in control individuals (Fig. 6A). However, chemotherapy failed to significantly increase the humoral immune response against CRT (Fig. 6B). Paired serum specimens were available in 4 cases, revealing no specific increase post-therapy (Suppl. Fig. 3). The seroconversion (defined as antibody titers reaching levels above the detection threshold of the ELISA test [START_REF] Milovanovic | Overexpression of calreticulin in malignant and benign breast tumors: relationship with humoral immunity[END_REF] ) of patients either at diagnosis or after neoadjuvant chemotherapy failed to correlate with pathological complete responses (Fig. 6C) or metastases-free survival (Fig. 6D). We conclude that in human breast cancer, humoral immune responses directed against CRT lack clinical significance.

Discussion

Our findings strongly suggest that humoral immunity directed against immunodominant tumor antigens or self-epitopes related to ER stress-associated chaperones (CRT) may not contribute to the chemotherapy-mediated control of tumor 31, 32 However, NY-ESO-1 seropositivity was only predictive of clinical benefit in cases of the concomitant elicitation of NY-ESO1-specific CD8 + T cell responses. [START_REF] Yuan | Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab[END_REF] Repeated vaccination with NY-ESO-1 protein in adjuvants (Montanide plus CpG ODN) induced an integrated Th1 CD4 + T cell and antibody response against NY-ESO-1 that was eventually completed by CD8 + T cell immunity. Vaccine-induced antibodies facilitated cross-presentation of NY-ESO-1 epitopes by dendritic cells to CD8 + T cells. [START_REF] Valmori | Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody/Th1 responses and CD8 T cells through cross-priming[END_REF] Similarly, therapy-induced antibodies directed against anti-MHC class I -like chain related protein A molecules (MICA) enhanced cross-presentation to T cells and immune-mediated antitumor effects. [START_REF] Jinushi | Therapy-induced antibodies to MHC class I chainrelated protein A antagonize immune suppression and stimulate antitumor cytotoxicity[END_REF] Antibody responses developed towards neovessels following immunization were instrumental to create ischemic tumor necrosis. [START_REF] Schoenfeld | Active immunotherapy induces antibody responses that target tumor angiogenesis[END_REF] Hence, GM-CSF-engineered tumor vaccines combined with anti-CTLA4 engendered a coordinated cellular and humoral immunity associated with clinically significant tumor regressions in advanced solid cancers. [START_REF] Hodi | Immunologic and clinical effects of antibody blockade of cytotoxic T lymphocyte-associated antigen 4 in previously vaccinated cancer patients[END_REF] In such regressing tumor burdens, T cell infiltrates were juxtaposed to antibody-producing B cells in long term responders. Humoral reactivity was reported against proangiogenic cytokines such as angiopoietin-1 and -2 blocking endothelial cell tube formation and/or macrophage inhibitory factor attenuating macrophage Tie-2 and MMP9 expression [START_REF] Schoenfeld | Active immunotherapy induces antibody responses that target tumor angiogenesis[END_REF] . Likewise, antibodies against protein disulfide isomerases (PDI, ERp5, [START_REF] Fonseca | Protein disulfide isomerases are antibody targets during immune-mediated tumor destruction[END_REF] ) or an accessory unit of the vacuolar H + -ATPase complex [START_REF] Hodi | ATP6S1 elicits potent humoral responses associated with immune-mediated tumor destruction[END_REF] appeared of clinical significance. These data are in line with other observations stating that the presence of tertiary lymphoid organogenesis (TLO), high endothelial venules (HEV) or germinal centers (GC) residing around tumor nests are associated with good prognosis in breast carcinoma, non-small cell lung cancer and melanoma. [START_REF] Erdag | Immunotype and immunohistologic characteristics of tumor-infiltrating immune cells are associated with clinical outcome in metastatic melanoma[END_REF][START_REF] Martinet | Tumor high endothelial venules (HEVs) predict lymphocyte infiltration and favorable prognosis in breast cancer[END_REF][START_REF] Martinet | High endothelial venules (HEVs) in human melanoma lesions: Major gateways for tumorinfiltrating lymphocytes[END_REF][START_REF] Dieu-Nosjean | Long-term survival for patients with non-small-cell lung cancer with intratumoral lymphoid structures[END_REF] Although some particular chemotherapy regimens elicit a therapy-relevant T cell immunity, they fail to ignite protective B cell responses, at least in mouse sarcomas and human breast cancers, which however have been investigated at relatively early time points. Further in-depth investigation should address antitumor serum reactivities in clinical cases where TLO or HEV or GC are observed post-chemotherapy.

We found that anti-CD20 antibodies did not affect the efficacy of anthracyclines. As a caveat, it should be noted that a subset of regulatory CD5 + CD1d high IL-10 producing B cells (B1 cells) fails to be depleted by anti-CD20 antibodies. [START_REF] Haas | B-1a and B-1b cells exhibit distinct developmental requirements and have unique functional roles in innate and adaptive immunity to S. pneumoniae[END_REF] Interestingly, secretion of autoantibodies is a function exclusive to the B1 subset and the majority of human cancer patients mount specific autoantibody responses against their tumors. [START_REF] Reuschenbach | A systematic review of humoral immune responses against tumor antigens[END_REF] Moreover, the population of CD20-resistant B cells has been reported to enhance A20 lymphoma growth in an IL-10-dependent manner. [START_REF] Horikawa | Regulatory B cell production of IL-10 inhibits lymphoma depletion during CD20 immunotherapy in mice[END_REF] Future studies must evaluate in which particular settings B1 cells may modulate the growth of human tumors. FcR (CD64 and CD16) have been shown to have a positive role in the treatment of mouse melanomas with specific antibodies. [START_REF] Albanesi | Cutting Edge: FcgammaRIII (CD16) and FcgammaRI (CD64) Are Responsible for Anti-Glycoprotein 75 Monoclonal Antibody TA99 Therapy for Experimental Metastatic B16 Melanoma[END_REF] These preclinical findings are in accordance with human data showing that single nucleotide polymorphisms (SNP) affecting the protein structure of FcRIIIA/CD16A determine the in vivo efficacy of monoclonal ADCC-mediating antibodies (such as Rituximab, Trastuzumab and Erbitux). [START_REF] Lejeune | Evidence for linkage disequilibrium between Fcgamma RIIIa-V158F and Fcgamma RIIa-H131R polymorphisms in white patients, and for an Fcgamma RIIIa-restricted influence on the response to therapeutic antibodies[END_REF] To our knowledge, the clinical significance of such SNPs has not been reported for anthracyclines or oxaliplatin, supporting the findings reported in this paper.

Antibodies against calreticulin (CRT), a Ca + -binding ER residing chaperone, represent a diagnostic tool for various infectious, autoimmune and cancer-associated disorders. [START_REF] Boehm | Systemic lupus erythematosus is associated with increased auto-antibody titers against calreticulin and grp94, but calreticulin is not the Ro/SS-A antigen[END_REF][START_REF] Aswad | Use of recombinant calreticulin and cercarial transformation fluid (CTF) in the serodiagnosis of Schistosoma mansoni[END_REF][START_REF] Watanabe | Anti-calreticulin antibodies in patients with inflammatory bowel disease[END_REF][START_REF] Winter | The assessment of hookworm calreticulin as a potential vaccine for necatoriasis[END_REF] Interestingly, anti-CRT IgA titers are more useful than anti-CRT IgG levels for detecting mammary, colorectal, hepatocellular and pancreatic carcinomas, alcoholic hepatitis, refractory coeliac disease, or primary biliary cirrhosis. [START_REF] Milovanovic | Overexpression of calreticulin in malignant and benign breast tumors: relationship with humoral immunity[END_REF][START_REF] Kreisel | High prevalence of antibodies to calreticulin of the IgA class in primary biliary cirrhosis: a possible role of gutderived bacterial antigens in its aetiology?[END_REF][START_REF] Pekarikova | Calreticulin is a B cell molecular target in some gastrointestinal malignancies[END_REF][START_REF] Sanchez | Anti-calreticulin immunoglobulin A (IgA) antibodies in refractory coeliac disease[END_REF] High concentrations of anti-CRT IgA antibodies were more frequently detected in lymph node-positive breast cancers than IgG antibodies but failed to correlate with CRT membrane expression [START_REF] Milovanovic | Overexpression of calreticulin in malignant and benign breast tumors: relationship with humoral immunity[END_REF] . The biological significance of anti-CRT antibodies is unclear. However, experimental immunization against CRT could induce liver focal necrosis and hepatitis. [START_REF] Abe | Breakthrough of immune self-tolerance to calreticulin induced by CpG-oligodeoxynucleotides as adjuvant[END_REF] Trypanosoma cruzi CRT is highly immunogenic and exhibits cross-reactivity with isogenic cardiomyocytes, thus causing a local autoimmune reaction relevant to Chagas disease. [START_REF] Ribeiro | Trypanosoma cruzi calreticulin: a possible role in Chagas' disease autoimmunity[END_REF] The observation that anti-CRT IgA or IgG serum levels did not significantly increase after anthracycline-based chemotherapy of breast cancer patients may reflect the inability of many tumors to translocate CRT to the cell surface [START_REF] Panaretakis | Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death[END_REF][START_REF] Tufi | Reduction of endoplasmic reticulum Ca2+ levels favors plasma membrane surface exposure of calreticulin[END_REF] , defects in other hallmarks of ICD such as absent expression of HMGB1 (Yamazaki T et al. same issue), or deficient autophagy [START_REF] Ladoire | Immunohistochemical detection of cytoplasmic LC3 puncta in human cancer specimens[END_REF] or the general irrelevance of humoral responses to clinical outcome. Future studies must correlate the therapy-induced raise in anti-CRT IgA antibodies or other immunological alterations, including changing frequencies in tumor antigen-specific T lymphocytes, with tumor cell-intrinsic, ICD-relevant parameters.

Material and Methods

Mouse strains. All mice were bred and maintained according to both the FELASA and the Animal Experimental Ethics Committee Guidelines (Val de Marne, France). WT SPF C57Bl/6J mice were obtained from Harlan. OT-1 C57Bl/6J mice and Ly5.1 congenic mice were obtained from Jackson Lab. C57Bl/6J FcγRI -/-mice [START_REF] Ioan-Facsinay | FcgammaRI (CD64) contributes substantially to severity of arthritis, hypersensitivity responses, and protection from bacterial infection[END_REF] , FcRI/IIIA/IV ('FcRIIB-only') [START_REF] Takai | FcR gamma chain deletion results in pleiotrophic effector cell defects[END_REF] , FcγRIII -/-mice, [START_REF] Hazenbos | Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice[END_REF] FcγRIV -/-mice, [START_REF] Nimmerjahn | FcgammaRIV deletion reveals its central role for IgG2a and IgG2b activity in vivo[END_REF] and FcγRI/IIB/III FcεRI/II (5KO, also known as "FcγRIV-only") mice [START_REF] Mancardi | FcgammaRIV is a mouse IgE receptor that resembles macrophage FcepsilonRI in humans and promotes IgEinduced lung inflammation[END_REF] have been reported previously and bred at the Institut Pasteur animal facility. C57Bl/6J µM KO mice were obtained from Centre d'Elevage d'Orléans (Dr Ryffel) and bred at the Institut Gustave Roussy animal facility.

Reagents and antibodies. Doxorubicin was purchased from Sigma Aldrich. Anti-mouse antibodies for CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD16/32 (2.4G2), CD19 (1D3), CD45.2 (104), were obtained from BioLegend or eBioscience. LIVE/DEAD fixable yellow stain fluorescence for viability staining was purchased from Invitrogen/Molecular Probes. Flow cytometry analyses have been performed on a Cyan (Beckman Coulter) flow cytometer with FloJo (Tree Star) software or on a FACSCanto II and Diva software (BD Bioscience).

Transplantable tumor cell lines. MCA205 OVA fibrosarcoma cells (syngenic from C57Bl/6 mice) were cultured at 37 °C under 5% CO2 in RPMI 1640 containing 10% FCS, 2 mM Lglutamine, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate and MEM nonessential amino acids (Invitrogen). OVA expressing tumor cells have been selected by antibiotic selection (Hygromycin (50µg/ml, Invitrogen))

Chemotherapy treatment of established tumors in mice. Tumors were subcutaneously inoculated in the right flank by injecting 1x10 6 of MCA205-OVA cells. When tumor size reached 30-45 mm 2 , mice were treated with 2µM doxorubicin, injected intratumorally in 50 μl of PBS, or PBS alone as control. Tumor growths were monitored every 3-4 days using a caliper. In some experiments, MCA205 OVA tumor cell line has been subcutaneously inoculated to CD45.1 C57Bl/6 mice. Seven days later, mice have been treated or not by doxorubicin (i.t). One day post chemotherapy, 1x10 6 CD45.2 congenic naive OT-I T cells have been injected i.v. day 8 post-chemotherapy. After mouse sacrifice and tumor harvesting and enzymatic dissociation (see below), the amounts of tumor infiltrating OT-I or B cells have been assessed by flow cytometry.

Tumor infiltrating lymphocytes analyses. At indicated time points, tumors were harvested, cut into small pieces and digested in Liberase TM 25µg/ml(Roche) and DNase I 150 U/ml (Calbiochem) for 30 min at 37°C. Single-cell suspension was obtained by crushing the digested tissue with a syringe plunger and filtering through a 100 μm cell strainer. After washing, cells have been used for subsequent immunostaining.

In vivo cell depletion. At indicated time point, MCA 205 OVA tumor bearing mice received a single 250 μg of anti-CD20 (MB20-11)or IgG2a control i.v, as previously described [START_REF] Hamaguchi | Antibody isotype-specific engagement of Fcgamma receptors regulates B lymphocyte depletion during CD20 immunotherapy[END_REF] . B cell depletion was checked in spleen and tumors from antibody administrated mice. CD8+ T cells have been depleted by using anti-CD8 Ab (53-6.72, Bioxcell) or Rat IgG2a control (2A3, Bioxcell). The depletions have been started 1 day prior chemotherapy treatment and maintained during the whole experiment. Mice received 100 µg of Ab in 100µl of PBS, i.p, every 3 days. T cell depletion was checked in spleen and tumor from antibody administrated mice.

Mouse Anti-OVA Ig dosing. MCA 205 OVA-bearing mice have been treated or not with doxorubicin at day 7 post-tumor inoculation. Two to 3 weeks later, sera have been collected and the presence of OVA specific IgG, IgA and IgM has been determined by using mouse anti-ovalbumin Igs (total G+A+M) ELISA kit (alpha diagnostic), according to manufacturer's instructions.

Patients' cohort. Breast cancer patients who has signed an informed consent for investigations of research purposes were selected from a database of 591 patients who received preoperative anthracyclines-based chemotherapy regimen (plus or minus taxanes and herceptin for HER2 BC) at the Institut Gustave Roussy between 1987 and 2003 (Table 1). Pathologic complete response (pCR) were defined as the absence of any invasive cancer or isolated tumor cells only in the breast and lymph nodes after completion of chemotherapy. Serum was drawn for CA15.3 dosages and remaining material kept frozen for parallel investigations including anti-CRT IgG/A concentration assessments. Therefore, serum withdrawn at diagnosis prior to chemotherapy, during the 8 cycles of therapy and/or after completion of neoadjuvant treatment at follow up consultations was kept at -20°C until ELISA was performed.

Human Anti-CRT IgG dosing. Human anti-CRT IgG and IgA sera concentrations have been evaluated as previously described. [START_REF] Sanchez | Epitopes of calreticulin recognised by IgA autoantibodies from patients with hepatic and coeliac disease[END_REF] Antibody response against CRT has been assessed in a cohort of 63 breast cancer patients and 75 healthy volunteers. The cut-off values for Ig were as follows: detection threshold of 30 UI/ml for IgA and 100 for IgG).

Statistical analyses.

Results are expressed as mean ± SEM. All experiments were repeated at least twice, yielding similar results. Normal distributions were compared by unpaired, two-tailed Student's t tests. Statistical analyses were performed by means of the software Prism 5 (GraphPad, San Diego, CA, USA) and p values < 0.05 were considered as statistically significant.

  growth. Using three distinct model systems (B cell depletion by anti-CD20 antibodies, constitutive Ig deficiency in MT mice, lack of activatory or inhibitory FcR resulting from homologous recombination), we demonstrated that B cells or Ig-specific receptors are dispensable for anthracycline-mediated antitumor effects, contrasting with the essential role of T-lymphocyte-mediated immune responses observable in the same settings. Indeed, in these OVA-engineered or wild type mouse sarcoma models, anthracyclines did not readily mobilize the humoral arm of immunity. These data do not rule out the possibility that, in other circumstances of dosing or scheduling or other therapeutic regimen, anti-tumor B cell responses be triggered Humoral immune responses have been described in the context of various immunotherapy strategies. Ipilimumab, a fully human mAb directed against cytotoxic T-lymphocyte antigen-4 that improves overall survival in metastatic melanoma patients, slightly increased antibody levels against tumor antigens (NY-ESO-1, MART-1, SSX2, p53, MAGE-A4) and boosted humoral immune responses against tetanus, pneumococcal and influenza vaccines.

Figure 1 . 01 Figure 2 .

 1012 Figure 1. Ag specific T cells but not B cell accumulation in the tumor bed post chemotherapy. (A-B). T cell accumulation in tumors post-anthracyclines. Day 7 established MCA205 OVA implanted in CD45.1 C57Bl/6 mice were treated with doxorubicin (i.t). One day post chemotherapy, 1x10 6 CD45.2 congenic naive OT-I T cells were injected i.v. and their frequency among CD8/CD45.2 cells (A) and absolute number (B) were assessed in flow cytometry in TILs. N= 5 mice/group. A representative dot plot is shown (A) and the data from one representative experiment out of 3 are depicted (B). (C) B cell depletion post-Doxo. Absolute intratumoral B cell numbers (defined as CD19 + B220 + live CD45+ cells) were assessed 8 days post-chemotherapy in TILs using flow cytometry. N= 5 mice/group. A representative graph out of two independent experiments is shown. Student t test: ** p < 0.01

Figure 3 .

 3 Figure 3. Anti-CD20 Ab-mediated B cell depletion does not impair chemotherapy efficacy. (A). Experimental setting. B cells have been depleted using anti-CD20 antibody either before tumor inoculation (D-14), before chemotherapy (D-2) or 6 days after chemotherapy treatment (D+6). Mice received doxorubicin (i.t) at day 0 and the tumor growth was monitored every 3-4 days. B cell depletion was analyzed by flow cytometry in the spleen 1 week after starting anti-CD20 injections. (B) Dot plots show the depletion in the « D-14 » group as a representative example. (C-E) Tumor growth curves of control or doxorubicintreated MCA205 bearing mice treated with anti-CD20 or IgG control Ab. N= 5-7 mice /group. Student t test or Anova:*** p<0.001, ns: not significant.

Figure 4 .

 4 Figure 4. CD8 + T cells are indispensable for chemotherapy efficacy. (A). Experimental setting. CD8 + T cells have been depleted from day -1 before chemotherapy throughout the experiment using 100µg of anti-CD8 antibodies (53.6.72) or rat IgG2a control Ab (2A3) every 3 days. (B). Tumor growth curves of control or doxorubicin-treated MCA205-bearing mice treated with anti-CD8 or IgG controls are depicted in a typical experiment out of three. Student t test:*** p<0.001, ns: not significant.

Figure 5 .Figure 6 .

 56 Figure 5. Chemotherapy efficacy does not rely on antibody response. MCA205 -OVA was established in various C57BL/6 mouse backgrounds (WT (A) or FcR deficient as indicated i.e "FcRIV only" (FcRI/IIB/III FcRI/II KO) (B) FcR KO (FcRI/III/IV KO) (C), FcRI KO (D) FcRIII KO (E) and FcRIV KO (F)) and treated or not with doxorubicin at day 7 post-tumor inoculation. Tumor growth kinetics is indicated in one representative experiment out of two for each group of mice. N= 5-7 mice/group. Student t test: ***p<0.001, ns= not significant

  ). Diagnosis n=26; After chemotherapy n=36. Student t test: *p<0.05, ns= not significant.

	Table1: Characteristics of breast cancer patients
	Patients	N=63	%
	Age at diagnosis
	Mean	48
	Range	10
	Sex
	Female	63	100%
	Histology
	Ductal invasive	63	100%
	Grade
	1	3	5%
	2	23	37%
	3	35	56%
	ND	2	3%
	ER status
	Positive	29	46%
	Negative	34	54%
	HER2-status
	Positive	4	6%
	Negative	59	94%
	Chemotherapy
	Neoadjuvant	63	100%
	Adjuvant	16	25%
	Neoadjuvant therapy
	FEC + TXT	50	79%
	TXT + FEC	4	6%
	Others (with
	Epirubicine)	9	14%
	Adjuvant therapy
	Hormonotherapy	28	44%
	RX	41	65%
	RX/FUN	12	19%
	RX/TXT	4	6%
	Others	6	10%
	Abbreviations : FEC = 5-fluorouracil / epirubicin /
	cyclophosphamide ; TXT = taxotere ; FUN = 5-fluorouracil /
	navelbine

We thank T.F Tedder (Duke University) for anti-CD20 mAb and useful discussions. D.H is supported by l'Association pour la Recherche sur le Cancer (ARC). N.P is supported by la Ligue contre le cancer. L.Z is supported by Institut National du Cancer (INCa), la Ligue contre le cancer (équipe labellisée), Fondation pour la Recherche Médicale (FRM) and Fondation de France. G.K. is supported by the European Commission (ArtForce); Agence National de la Recherche (ANR); Ligue contre le Cancer (Equipe labellisée); Fondation pour la Recherche Médicale (FRM); SIRIC SOCRATES, Institut National du Cancer (INCa); LabEx Immuno-Oncologie; Fondation de France; Fondation Bettencourt-Schueller; AXA Chair for Longevity Research; Cancéropôle Ile-de-France and Paris Alliance of Cancer Research Institutes (PACRI)


Thorsten Schramm

Alfons Hester

Ivo Klinkert

Jean-Pierre Both

Ron M A Heeren

Alain Brunelle

Olivier Laprévote

Nicolas Desbenoit

PhD Andreas Roempp
email: andreas.roempp@anorg.chemie.uni-giessen.de

Justus Liebig

Olivier Laprevote

Marie-France Robbe

Markus Stoeckli

Bernhard Spengler

Andreas Römpp

imzML -A common data format for the flexible exchange and processing of

Keywords:

come   L'archive ouverte pluridisciplinaire

Introduction

In mass spectrometry imaging (MS Imaging) a sample of interest is scanned and the resulting ion signals are reconstructed into a pixel image. Several overviews of this method have been published recently [START_REF] Chughtai | Mass Spectrometric Imaging for Biomedical Tissue Analysis[END_REF][START_REF] Mcdonnell | Imaging mass spectrometry[END_REF].The application of MS imaging is rapidly growing with a constantly increasing number of different instrumental systems and software tools. This results in a need of exchangeability of MS imaging data between different instruments and data analysis software. As for many analytical methods, data processing has become an essential part of the workflow for MS imaging. MS Imaging data consist of several thousand spectra which frequently result in data files of several gigabytes. Mass spectra of one experiment are usually acquired with identical measurement parameters. Existing standards such as the DICOM standard [START_REF] Dicom | Digital imaging and communications in medicine -DICOM[END_REF] for in-vivo imaging data or the mzML standard [START_REF] Martens | mzML -a Community Standard for Mass Spectrometry Data[END_REF] established by HUPO-PSI [START_REF] Hermjakob | The HUPO proteomics standards initiative -Overcoming the fragmentation of proteomics data[END_REF] are not suitable to adequately represent an MS imaging experiment. Therefore a common data format dedicated to mass spectrometry imaging data was developed within the EU project COMPUTIS (www.computis.org). The data format is in part based on the HUPO-PSI format mzML (see details below) and is thus called imzML for 'imaging mzML'.

This technical report provides an overview of the most important properties and features of the imzML format. Additional information and all technical details necessary to implement imzML is provided on the website www.imzml.org and in a recently published book chapter [START_REF] Römpp | Imaging mzML (imzML) -a common data format for imaging mass spectrometry[END_REF].

Methodology

MS imaging data in imzML is divided into two separate files (Figure 1). This structure, consisting of a small file (text or XML) and a larger (binary) file, has been shown to be efficient in previous data formats for MS imaging, for example in BioMap [START_REF] Biomap | [END_REF] and internal data formats at FOM Institute and Justus Liebig University. All metadata (e.g. instrumental parameters, sample details) are stored in an XML file. Mass spectral data is stored in a binary file to ensure efficient storage. Corresponding XML and binary files contain a universally unique identifier (UUID) [START_REF]RFC 4122 -A Universally Unique IDentifier (UUID) URN Namespace[END_REF] in order to link them unequivocally and to prevent loss of information.

The XML file (*.imzml) contains all relevant information about the MS imaging experiment.

In order to stay as close as possible to existing formats this metadata file is based on the mass spectrometry data standard mzML [START_REF] Martens | mzML -a Community Standard for Mass Spectrometry Data[END_REF] developed by HUPO-PSI [START_REF]The Human Proteome Organization: Proteomics Standards Initiative[END_REF]. Discussions with HUPO-PSI resulted in the strategy to maintain imzML as a separate format, but keep the XML structure consistent with mzML. Information is provided in the form of a 'controlled vocabulary' (CV) which is stored in an open biomedical ontology [START_REF] Smith | The OBO Foundry: coordinated evolution of ontologies to support biomedical data integration[END_REF]. This approach is used in order to unequivocally describe the parameters and to avoid redundancy in nomenclature.

The mzML controlled vocabulary [START_REF]Mass Spectrometry Standards Working Group -controlled vocabulary[END_REF] has been supplemented by parameters which are necessary for a comprehensive description of MS imaging experiments. These additional imzML CV parameters (including x/y position, scan direction/pattern, pixel size) are stored in the imagingMS.obo file [START_REF]MS Imaging controlled vocabulary[END_REF].

The imaging binary data file (*.ibd) contains the mass spectral data of the MS imaging measurement. In order to ensure efficient storage, two different binary modes are defined: continuous and processed. 'Continuous' means that an intensity value is stored for each m/z bin even if there is no measurable signal (resulting in an intensity of zero). This data structure is used for many MALDI-TOF mass spectrometers. As a result the m/z axis is identical for all mass spectra of one image. Therefore it is sufficient to store the m/z array only once in the binary file. This structure can reduce the file size significantly (up to a factor of 2). On the other hand mass spectra are often processed before they are stored e.g. for noise-reduction, peak-picking, deisotoping. This results in discontinuous and non-constant m/z arrays. In this case the m/z array has to be stored for each spectrum separately. This data structure was termed 'Processed'.

Implementation and Discussion

A number of software tools already support imzML. This allows for choosing from a (growing) number of options to display and process MS imaging data. Users will be no longer limited to proprietary software, but are able to use the processing software best suited for a specific question or application. Multiple software tools can also be combined in one workflow using imzML as demonstrated in Figure 2. In this example a whole body rat section was coated with α-Cyano-4-hydroxycinnamic acid (CHCA) and analysed with a QStar mass spectrometer (AB Sciex). Mass spectra were acquired for 344 x 87 pixels (500 µm pixel size) in the mass range m/z = 300-1000 u. Data was saved in the Analyze 7.5 data format and opened in 'BioMap' for initial inspection (Figure 2A). Subsequently the dataset was converted to imzML using the 'Toimzml' converter developed by the CEA-LIST institute.

The imzML file was opened in 'SpectViewer' (developed by CEA-LIST) and a wavelet denoising procedure was applied in order to improve image quality (Figure 2B). This particular processing function is not available in the other software tools. The processed data was saved in a new imzML file which was subjected to further analysis. This conversion tool has also been used in previous studies in combination with the Datacube Explorer for analyzing and displaying MS imaging data [START_REF] Li | Characterization of barley leaf tissue using direct and indirect desorption electrospray ionization imaging mass spectrometry[END_REF][START_REF] Thunig | Analysis of Secondary Plant Metabolites by Indirect Desorption Electrospray Ionization Imaging Mass Spectrometry[END_REF]. More information on sample details, image properties and conversion method are provided in the 'imzML gallery' on www.imzml.org. This page also includes additional examples of MS images generated with imzML.

The examples discussed above illustrate the functionalities and possibilities of the imzML data format. A number of additional software tools are currently adopted for usage with imzML. This includes 'MSImageView' the successor of BioMap which is probably the most widely used independent software to analyze MS imaging data. The first two commercial software packages supporting imzML have been released recently [START_REF]MALDIVision[END_REF][START_REF]Quantinetix MALDI Imaging Software[END_REF]. The concept of imzML is also actively supported by major vendors of instrumentation for MS imaging including Waters Corporation (Manchester, United Kingdom), Thermo Fisher Scientific (Bremen, Germany) and Bruker Daltonik (Bremen, Germany). Their data formats are integrated in the imzML workflow through export options in proprietary software suites (Waters) or by external converters being developed by the MS imaging community with technical support from the companies (Thermo, Bruker). An updated list of available tools and workflows can be found on the imzML website. imzML was chosen as the central platform for exchange of data in the European COST network 'Mass Spectrometry Imaging: New Tools for Healthcare Research' which includes more than 30 MS imaging groups [START_REF] Mcdonnell | Going forward: Increasing the accessibility of imaging mass spectrometry[END_REF].

The future development of imzML-based software will also be coordinated in this framework.

These activities ensure a wide distribution and active development of imzML as the data standard for mass spectrometry imaging data.

Conclusions

The data format imzML enables efficient and flexible exchange of MS imaging data. It allows for a flexible selection of data analysis tools. imzML is increasingly used in the MS imaging community. A number of software tools are available and many more are currently being adapted to imzML. A common data format is not a mere technical detail, but has significant impact on practical work: fully searchable mass spectrometry imaging data can be shared with collaborators in biological or clinical labs without being restricted to proprietary vendor software. imzML is thus an important step towards more efficient collaboration as well as more flexible and transparent data processing in mass spectrometry imaging. These are key requirements for MS imaging to finally be established as a routine method.

  The two files (XML and binary) are connected by offset values in the XML file that indicate the position of the corresponding data in the binary file. This allows for fast reading/access of large data sets. The combination of non-redundant metadata representation and binary data leads to efficient storage. The resulting datasets are comparable in size to the proprietary data and the separate metadata file allows flexible handling of large datasets. This is important since MS imaging experiments typically consist of several thousand spectra, and increased file size due to format conversion could lead to data sets which are very difficult to handle. imzML has been extensively discussed with academic and industrial users at various occasions in recent years. The current version 1.1.0 of imzML was released at the International Mass Spectrometry Conference (IMSC) in Bremen, Germany in September 2009. The flexible structure based on XML and the controlled vocabulary allows for compatibility with new instruments and methods in the future. Technical documentation including XML scheme, mapping file, controlled vocabulary (obo file) and example files are provided on www.imzml.org.

  The 'Datacube Explorer' developed by AMOLF allows for fast browsing of MS images and can thus be used to visually search for signals that show interesting spatial features. The denoised imzML data set was displayed in Datacube Explorer and spectra from two regions of interest (ROI) were extracted (Figure2C). The data analysis tool 'Mirion' developed by JLU can be used to semiautomatically generate MS images based on parameters such as pixel coverage or incremental mass. It can also be used to open multiple files as shown in Figure2D(original and denoised imzML data set), data from these files can be combined for example by RGB overlay (not shown). The combination of processing steps within the four programmes is only possible due to the standardised and open data format imzML. A detailed description of the different software packages would be beyond the scope of this technical note. The purpose of the example shown in Figure 2 is to demonstrate the possibility to open and process imzML files in different MS imaging software packages. Details about functionalities of SpectViewer, Datacube Explorer and Mirion will be provided in separate publications which are currently in preparation. The demonstrated workflow is only one example for the use of imzML for analysis of MSI data with different tools. Another application is the comparison of measurements from different instruments. Options for data processing (e.g. binning, normalization) and displaying (e.g. interpolation options, color schemes) vary strongly between different MS imaging software tools and can make a direct comparison of data difficult. This problem can be avoided, if all data sets are converted to imzML and the data is displayed with identical parameters in one software package. Examples of imzML data sets from different instrument platforms are shown in Figure 3. All images are displayed in the same software package (two masses in green and red, no interpolation). Data from the most commonly used matrixassisted desorption/ionization (MALDI) mass spectrometers (Thermo, Bruker, AB Sciex and Waters) are shown in Figures 3A-D. A data set originating from secondary ion mass spectrometry (SIMS) is shown in Figure 3E. The majority of MS imaging applications are based on MALDI and SIMS, but other ionization techniques are increasingly used. Data acquisition in desorption electrospray ionization (DESI) mode is based on one data file per line scan and conversion to imzML therefore differs from most MALDI experiments where usually a single file is acquired per image. The 'RAW to imzML' converter developed by Justus Liebig University was modified to convert multiple line scans into a unified imzML file. An example of a DESI imzML file generated with this converter is shown in Figure 3F.

Figure 1 :

 1 Figure 1: Scheme of imzML structure (adapted from www.imzml.org).

Figure 2 :

 2 Figure 2: Example of sequential processing of an MS imaging data set based on imzML. MS imaging data acquired on an AB Sciex QStar instrument was converted to imzML. The imzML file was opened and processed in different software packages for analysis of MS imaging data. A) BioMap: Selected ion image of m/z = 772 and averaged mass spectrum, B) SpectViewer: averaged mass spectrum and selected ion image of m/z = 772 for original data (left) and after application of wavelet denoising algorithm (right), C) Datacube Explorer: mass spectrum of full image and two regions of interest (ROI) which are indicated in selected ion image of m/z 772, D) Mirion: selected ion image of m/z = 772 from original (top) and denoised (bottom) data set.

Figure 3 :

 3 Figure 3: Examples of data sets from different instrument platforms that were converted to imzML.All images are displayed in the same software tool (Mirion) with identical settings (showing an overlay of two masses in red and green, no interpolation). Ionization type and mass spectrometer type are indicated for each measurement. A) mouse kidney, MALDI, LTQ Orbitrap (Thermo), data provided by Sabine Guenther (Justus Liebig University, Giessen, Germany), B) mouse brain (left half), MALDI, ultrafleXtreme (Bruker), data provided by Corinna Henkel (Bochum University, Germany), C) whole body rat section (head), MALDI, QStar (ABSciex), same data as shown in Figure2, D) whole bod yrat section, MALDI, Synapt G2 (Waters), data provided by Emmanuelle Claude (Waters, Manchester, UK), E) rabbit eye, SIMS, TOF-SIMS IV (IonTOF), data provided by Alain Brunelle (CNRS, Gif-sur-Yvette, France), F) tumor tissue, DESI, Exactive (Thermo), data provided by Zoltan Takats (Imperial College, London, UK). More details on these data sets can be found in the 'imzML gallery' on the www.imzml.org.

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 3 Figure 1 Click here to download high resolution image

Acknowledgements

First and foremost we would like to thank all the members of the HUPO Proteomics Standards Initiative (PSI) for developing the data format mzML which has been used as a model for imzML. We are especially thankful to Lennart Martens, Eric Deutsch, Randall Julian and Martin Eisenacher for many helpful discussions in the last years. We would like to thank Sabine Guenther, Corinna Henkel, Brendan Prideaux, Emmanuelle Claude, Zoltan Takats for providing MS imaging data sets. This work was supported by the European Union (Contract LSHG-CT-2005-518194 COMPUTIS). JLU acknowledges support by the Hessian Ministry of Science and Art (LOEWE focus Ambiprobe).