Nicholas Kyriakides
email: nicholas.kyriakides@cut.ac.cy

Christis Z Chrysostomou
email: c.chrysostomou@cut.ac.cy

Panagiotis Kotronis
email: panagiotis.kotronis@ec-nantes.fr

Elpida Georgiou
email: es.georgiou@edu.cut.ac.cy

Panayiotis Roussis
email: roussis@ucy.ac.cy

Numerical simulation of the experimental results of a RC frame retrofitted with RC Infill walls

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

The most effective and economic method for retrofitting multi-storey RC buildings is the construction of new walls, especially those with pilotis (soft-storey). Their structural and economic effectiveness increases when selected bays of an existing RC frame are fully infilled. Most of the experimental research work performed in the last decades covers sufficiently the other frequently used types of retrofitting -in particular the use of fibre-reinforced polymers (FRP) and the concrete jackets. Some of these types of retrofitting for RC buildings with openings are discussed in Hemant (2009). Some more recent work (Antonopoulos T.A and Anagnostopoulos S.A. 2012) examines numerically the effectiveness of the use of diagonal steel braces to provide stiffness and ductility in cases of open ground stories. It is shown that the application of these braces improves the demand to capacity ratios for shear and bending in all floors of the building.

Alternatively, Misir (2014) proposes the use of locked brick infill walls, which have the potential to decrease the soft-story/weak-story formation in comparison to standard bricks due to their shear sliding mechanism and decreased upper-story/first-story stiffness, even in buildings that have non-infilled first stories. The use of these sliding brick walls increases the capacity of the building since it removes weaknesses due to openings but cannot provide significant stiffness and strength to the building to withstand severe earthquakes.

The use of RC Infill walls for retrofitting RC buildings with openings is a relatively new retrofit method, which can be used to increase the strength, stiffness and ductility of the building. There is no adequate experimental research work on the use of RC infill walls and most research has mainly targeted on what is feasible: testing of one-to two-storey specimens due to the practical difficulties of testing large specimens with high resistance. So, data is lacking for taller full-scale specimens that reflect real applications. Regarding to code provisions, Eurocode 8 -Part 3 fully covers retrofitting with FRP or concrete jackets, while it does not refer at all to new walls created by infilling frame bays. Other guidelines, like [START_REF] Kanepe | Code for Intervention in Reinforced Concrete Buildings, Earthquake Planning and Protection Organization (OASP)[END_REF] in Greece, refer to the design of such walls but only in terms of forces, without providing tools for calculation of their characteristic deformations (at yield and failure) and stiffness, unless the infill wall can be considered integral with the bounding frame. The inadequacy of design codes in this respect is due to our poor knowledge of the behaviour of walls created by infilling with RC a bay of an existing frame.

In order to start filling the gap of knowledge regarding infilling of existing RC frames with RC walls, the effectiveness of seismic retrofitting of multi-storey multi-bay RC-frame buildings by converting selected bays into new walls through infilling with RC was studied experimentally at the Elsa Laboratory of Structural Assessment (ELSA) facility at JRC, in Ispra. The research was ¡¢£¤ ¥¦£ §¤¨©£¥ £ ¡¡££¤¡ ££¤¦ ¡!¤¥¤ ¥ ¤£ !¨¤ ¤¨ §£¡ "¡£¤£# (SERIES), financed by the Seventh Framework Programme of the European Commission.

The scope of this paper is the numerical simulation of the tested frame and the comparison of the results with the experimental ones, using numerical models of different modelling sophistication. In the first part, a brief description of the design of the bare-frame specimen and the RC Infill walls is presented along with details of the testing campaign and some of the main results obtained from the experiment. Details can be found in Chrysostomou et al. (2013[START_REF] Chrysostomou | Seismic Evaluation and Rehabilitation of Structures, Chapter 17: RC Infilling of existing RC structures for seismic retrofitting[END_REF] and [START_REF] Poljansek | Seismic Retrofitting of RC Frames with RC Infilling (SERFIN Project)[END_REF]. In the second part, which is the main focus of the paper, the modelling and the results a 2D finite element micro-model, simulating the behaviour of one of the two planar frames tested, and a macro-model using fibre elements for the frame members and shell elements for the walls, simulating both planar frames and the connecting beams and slab, are presented. Finally, the results of the numerical simulation of the bare frame are also provided in order to emphasise the effectiveness of this retrofitting method and conclusions are drawn.

Description of the specimen and test campaign

The specimen was designed based on a four-storey prototype building structure consisting of four three-bay frames spaced at 6 meters, with RC infilling of the exterior frames only. The specimen was designed at full-scale to represent the two exterior frames of the prototype structure, ). The purpose of the experiment was to perform uni-directional pseudo-dynamic testing on these two parallel planar frames, which were infilled with RC infills reinforced with different amounts of reinforcement and connected to the frames with different connection details, as explained in the following paragraphs. These frames were named North and South as it is defined in Fig. 3. Hence, the direction towards the reaction wall (backside of Fig. 3) is East and the one in the opposite direction (front-side of Fig. 3) is West. The centre-line length dimension of the specimen was 8.5 m (central bay 2.5 m and the two exterior bays 3.0 m), the storey height 3.0 m, and the total height of the specimen (excluding the foundation) 12.0 m (Fig. 1(a)).

The dimensions of the columns were 0.25 m by 0.40 m with the long dimension along the plane of the three-bay frames (that was also the direction of uni-directional loading), while those of the beams 0.25 m by 0.50 m.

The proposed structure represents typical construction of the late 70 s and beginning of the 80 s in Cyprus. At that time, there were no provisions for earthquake loading, so the structures were designed for gravity loads only. There was no specific design standard and the authorities were accepting any standard that was acceptable to other countries such as CP110 [START_REF] Bsi | CP110-The structural use of concrete, Part 1, The Council for Codes of Practice[END_REF]BS8110 (1983), DIN, Greek Code, US code etc. For the mock-up design, it was decided to use the provisions of BS8110, which were very close to those of CP110 with minor differences.

The material properties used in the mock-up were constrained by the availability of materials in the Italian and European market. It was finally decided to use concrete C20/25 for both the frame and the walls, of unit weight 25 kN/m³ and modulus of elasticity, E=30,000 MPa. The yield strength of the ribbed bar reinforcing steel was fyk=400 MPa for both bending and shear reinforcement of the frame members and the slab, while for the RC infill and the dowels to be used for connecting the wall to the bounding frame members the yield strength was specified to fyk=450 MPa. The 400 MPa characteristic yield strength steel represents the one used in Cyprus construction practice in the 1970 s and 80 s, while the 450 MPa was the closest available in the Italian market to substitute for the 500 MPa steel that would have been used in the walls for retrofitting such a structure.

The self-weight was calculated using the unit weight of concrete specified above. The imposed dead load including the load of masonry infill walls was 3 kN/m 2 , and the live load was 1.5 kN/m². These loads were combined using partial factors of safety of 1.4 for self-weight and imposed deadload, and 1.6 for the live load. The material partial factors used were 1.5 for concrete and 1.15 for steel.

The reinforcement details of the beams, the columns and the slabs are given in Chrysostomou et al. (2013[START_REF] Chrysostomou | Seismic Evaluation and Rehabilitation of Structures, Chapter 17: RC Infilling of existing RC structures for seismic retrofitting[END_REF], [START_REF] Poljansek | Seismic Retrofitting of RC Frames with RC Infilling (SERFIN Project)[END_REF]. Herein, it is important to emphasise the fact that a compression lap was used for columns, in line with a design for gravity loads only, and was expected to fail when subjected to tension. Connection details at the joints, laps and between the infill-walls and the bounding frame are given in Fig. 2.

The walls in the two frames, which had a thickness of 0.25 m equal to the width of the beams and columns of the bounding frame, were reinforced with different amounts of reinforcement, with the north one being the stronger of the two, in order to facilitate the study of the effect of as many parameters as possible.

Two parameters were examined: a) the amount of web reinforcement in the walls and b) the connection detail between the wall and the bounding frame. Regarding the connection detail, two distinct connection details were used. In the first detail, the web bas were connected to the surrounding frame through lap splicing with the same diameter starter bars epoxy grouted into the fame members. Short dowels were then used in order to transfer the shear at the interface between the wall and the frame member. This detail was used to connect the wall at the bottom beam and right column at the 1 st and 2 nd floors of the North frame (Fig. 2(c), starter bars are shown here only for the bottom beam), while for the South frame it was used to connect the wall at the bottom beam of the 1 st and 2 nd floors, and the west and east columns of the 1 st and 2 nd floors, respectively.

In the second detail, longer dowels were used to act both as dowels and as starter bars of the web reinforcement, which is placed at the two faces of the wall. Thus, these dowels are considered as lap-spliced with the nearest -smaller diameter -web bars. However, in this case, the clear distance between the dowel and the nearest web bar, violates the maximum clear distance of 50 ¡¢ £¤ ¥¦ §¨¦¦© ¦ ¥¢ ¦¦ © ¢¡¡¦ ! (CEN 2004). This detail was used to connect the wall at the top beam and west column at the 1 st and 2 nd floors of the North frame (Fig. 2(c)), while for the South frame it was used to connect the wall at the top beam of the 1 st and 2 nd floors, and the east and west columns of the 1 st and 2 nd floors, respectively. In the 3 rd floor of both the North and South frames, only the second detail was used, while for the 4 th floor only two dowels per wall interface were used to provide safety against falling of the wall out of plane. The completed wall reinforcement (including web, starter bars and dowels) for the 1 st floor of the North wall is shown in Fig. 2(d). In all cases, the dowels were positioned along the centreline of the elements (i.e., at 0.125 m from the face of the wall). Detailed guidelines for the design of such walls can be found in Fardis (2013). Since the lapping of the column reinforcement could take only compression, then it was obvious that there would be lap splice failure at the ground floor columns next to the RC Infill wall, which could be detrimental to the whole experiment. Therefore, in order to safeguard against this type of failure and allow the experiment to be performed without any premature failure, it was decided to reinforce the edges of the wall at the 1 st floor with three-sided CFRP for the height of 0.60 m (lapping stopped at 0.55 m) from the base of the column. Based on the performance of the ground floor RC Infill wall during the experiments, this additional retrofit of the lap length region is crucial and constitutes part of the proposed retrofit strategy.

Within the testing campaign two PsD tests and one cyclic test were run. The accelerogram was scaled to a maximum acceleration of 0.10 g and 0.25 g for the first and second PsD tests, respectively. For the final cyclic test, a history of displacements was imposed at the fourth floor, while maintaining a triangular distribution of loads along the height of the North frame and zero rotation at each of the four floors (unidirectional test). The aim of this final test was to explore the final capacity of the specimen up to a 20 % drop of peak strength of the structure.

In a PsD test, it is assumed that the response of a structure can be determined by a discrete model with a limited number of degrees of freedom (DoF). In this test campaign, four DoFs were selected: the horizontal displacements of each storey with the assumption that all the mass is concentrated at the selected DoFs (i.e., the floor slabs). Horizontal displacements of the controlled DoFs were solved for a prototype time step using the explicit Newmark time integration method. These displacements were then computed and applied on the structure by horizontal actuators at each storey with a laboratory time step of 0.002 s corresponding to the sampling of the controllers. The forces measured by the load cells in the actuators, following the application of the controlled displacements, represent the restoring forces that are fed back to the computer and that are used in

¡¢ £¢¤ ¥¦¢ § ¢¨© ¡¢ ¥©£ ¢ § ©¥£ ©¢ § ¢ ¡ § © ¥£¢ ©¦ ¡¢ §¨¢¥¦¢£! §
response and reflect its state of damage. Since the inertial and viscous damping forces are modelled in the computer, the test does not have to run in real time scale. Hysteretic damping is automatically accounted for through inelastic deformation and damage progression of the test structure; consequently no viscous damping matrix was used (Molina et al. 2011). During the PsD test campaign the equation of motion was solved for restoring forces coming from the North frame only (calculated from static equilibrium of the load cell force measurements at each floor) and multiplied by a factor of two (the South frame is considered equal to the North frame in the numerical model). Equal displacements were applied to the two frames, in order to maintain zero rotation along the horizontal plane of the floor.

Experimental results and short discussion

Displacement transducers were installed to measure local displacements in critical areas. In particular, transducers were placed to monitor slip and crack opening between all walls and their bounding beams and columns, the displacements between the ground floor walls and the foundation beams, and the shear deformations of the two ground floor walls. Displacement transducers were also installed to measure at all storeys the vertical elongation of the bounding columns.

Inclinometers were used to measure the rotation of beams and columns at the first floor. They were placed at the centre joints and on beams and columns 30 cm away from the joints. Inclinometers were also placed at selected columns 30 cm above the foundation beam.

Heidenhain linear encoders were installed on two reference frames to measure the horizontal displacement of the two frames at each of the four floors in the direction of testing.

For the low-level acceleration, the structure behaved very well. There were no visible cracks on either the columns or the walls. Some hairline cracks that appeared on the surface of the wall closed down when the experiment was finished. The maximum top storey displacement was 24 mm and the displacement in the opposite direction was 26 mm. These displacements were the same for both the north and south frames, since the forces in the south frame were imposed in such a way so as to keep the displacements of the two frames equal, and hence avoid any torsional effects on the specimen. It was considered that both walls have reached their cracking moment, and this was the purpose of this experiment.

The 0.25 g test was performed the day after the first test, and it was designed to bring the specimen at its ultimate capacity. The maximum top storey displacement was 109 mm and the displacement in the opposite direction was -93 mm. Some difference was observed in the base shear between the two frames. The maximum base shear in the positive direction (specimen was moving towards the East, i.e., towards the reaction wall) was 1074 kN for the south frame and 1036 kN for the north, which are about the same, while a negative base shear of -843 kN was recorded for the south frame and -1011 kN for the north one in the negative direction (specimen was moving towards the West, i.e., away from the reaction wall). This was an indication that the south frame has suffered some damage and it could not take further load. The only visual indication of this was a crack that opened in the ground beam at the base of the wall and the lapsplice failure due to tensile forces of the outer column on the east side of the south frame. It should be noted that the presence of the CFRP on the bounding columns of the wall, have prevented a Regarding general behaviour of the specimen, it can be said that it withstood the loading imposed on it very well. There were no visible diagonal cracks on the walls, which behaved in a flexural mode. In nearly all the columns, a horizontal crack appeared at a height of 0.55 m where the lap-splice stopped, and in some cases, there was spalling of the concrete cover. Some vertical cracks appeared at the connection between the beams and the columns, but there was no severe damage, despite the fact that there were no ductile connections in the structure. In general, the stronger north frame had an overall better behaviour compared to the south one, but the differences were minor.

¡¢£¤¤¥¦ ¡¢ §¨© ne£¤ ¥¤¡ §© § £ ¡¤£©©¢ §-history was imposed at the top storey (92, - 92, 89, -125, 37, 0 mm) and a triangular distribution of forces was imposed. The objective of the test was to obtain a 20% reduction of the peak strength of the infill, in order to establish the strength envelope of the specimen. The base shear versus the top storey displacement is shown in Fig. 4. As it can be observed in the first cycle, the structure could reach 92 mm in both directions. In the second cycle, the attempt was to reach 125 mm, but in the positive direction only 89 mm was possible to be reached, while in the negative direction 125 mm was reached. This though had as a result a sudden drop of the strength of the south frame from 838 kN at -110 mm to 553 kN at a displacement of -125 mm. This amounts to a drop in strength of 34%. After that, the displacement was reduced to 37 mm in the positive direction and from there to zero. Detailed presentation of the experimental model and testing campaign, as well as discussion of the results are given in Chrysostomou et al. (2013[START_REF] Chrysostomou | Seismic Evaluation and Rehabilitation of Structures, Chapter 17: RC Infilling of existing RC structures for seismic retrofitting[END_REF], [START_REF] Poljansek | Seismic Retrofitting of RC Frames with RC Infilling (SERFIN Project)[END_REF].

2D Finite element model

As already mentioned, during the experiments equal displacements were applied to the two frames in order to maintain a zero rotation along the horizontal plane of the floor. The loading being unidirectional, a 2D finite element model was first chosen. All the calculations presented in §¨¡ © §¡ ¢ ¨£!© "©©¢ © © "© © §¨© ©#©¡©¢ § $"¤¡¢ £¤¤£ §¡ ¢%. Only one of the two frames was considered of width 0.25 m, with 75000 kg per floor and a total weight of 1211 kN at its base. The finite element code Cast3M developed at CEA in France was used for the [START_REF] Millard | Castem 2000 User Manual[END_REF]. Concrete was modelled using classical quadrilateral finite elements (Fig. 5(a)) and horizontal and vertical reinforcement bars with truss elements (Fig. 5(b)). Perfect bonding was assumed between concrete and steel, the existing structure and the RC infilling. Table ' ()012345 674 3256)289620@ 0A 674 )42@A0)B4C4@6 8D)5 2@ 674 A2@264 4E4C4@6 C034EF G74 HIPQ5 R4)4 not considered.

Results of the modal analysis are provided in Fig. 6. The first mode, corresponding to in-plane flexure and dominates the behaviour of the structure.

Advanced constitutive laws, suitable for cyclic loading were used for the materials. Concrete behaviour was reproduced using a damage mechanics law that takes into account the difference in tension and compression and the unilateral effect (opening and closing of cracks, Faria et al. 1998). Steel was modelled using a classical plasticity law able to reproduce the Bauschinger effect (cinematic hardening, Menegoto M., and Pinto P., 1973). The material parameters are shown in Table 2. Nonlinear calculations were performed considering a very low level of viscous Rayleigh damping (0.25%), so as to be compatible with the PsD testing (see section 2). The implicit Newmark numerical algorithm was adopted for the time integration of the equation of motion and the classical Newton-Raphson algorithm for the nonlinear material behaviour. Results are presented hereafter for 0.25 g (and for an earthquake signal of about 12 sec). The advantage when using continuum mechanics constitutive laws is that we have access to local results in terms of damage indices, strains in the reinforcement bars etc. Fig. 10 shows the damage index due to tension (a) and to compression (b) for a loading of 0.25 g. This damage index varies between zero and one (from undamaged to completely damaged cross-section). Therefore, a zero damage index corresponds to no tension or compression, whereas when the variable reaches the value of one it corresponds to the attainment of the tension or compression capacity of the members. It is clear from Fig. 10 that the structure has exhibited good behaviour in terms of the strong column-weak beam mechanism. However, the existence of damage due to compression at the bottom (Fig. 10(b)) indicates that this level of loading is important and that for a higher level of PGA the structure maybe at risk due to possible compression failure of the columns bounding the wall.

Simple numerical model

The analysis of the building was also conducted using a simple numerical model based on line and shell elements. The purpose of this model was to simulate the response of the specimen as it was tested in the lab, unlike the 2D micro-model presented in the previous section by which only one of the two frames was modelled, observe the correlation of the numerical to the experimental results, and examine the effect of modelling in increasing the accuracy of simulation. The simplicity in modelling results in computational-time reduction and thus provides the opportunity to study the 3D behaviour of the building. It should be repeated here that the PsD test was unidirectional and care was taken so no rotation was introduced in the two frames. Therefore, by 3D behaviour it is meant the contribution of the connecting slabs and beams to the response of the two planar frames. The numerical model was created in SAP2000 (2010), which is widely used both for commercial and academic purposes (unlike the sophisticated software needed for micromodelling), offers a friendly graphical interface, and includes non-linear models both for line and shell elements. A view of the model is shown in Fig. 11.

The modelling of the building was assembled using line fibre elements for the columns, which are expected to behave nonlinearly as it was observed during the experiment. The column crosssection was discretised in eight concrete fibres and four steel ones as shown in Fig. 12. The fibre element properties were distributed along the length of the column elements.

The Takeda (1970) model was used to describe the nonlinear properties of concrete including cracking. The Mander (1984) unconfined concrete model was used to describe the monotonic stress-strain relationship. In the case of the steel reinforcement, kinematic hysteretic type was assumed. The monotonic laws for both materials are shown in Fig. 13. Both longitudinal and crossbeams were modelled using elastic properties since they did not show nonlinear behaviour during the experiments. In order to account for the reduction in stiffness due to cracking at the 0.25 g test, a 10% reduced elastic stiffness was used for the longitudinal beams. The slabs were modelled with linear shell elements.

RC infill walls in the middle bay of the building were modelled using a nonlinear fibre shell element. The discretization of the wall cross-section was created automatically and accounting for the in-plane properties of the wall. The steel mesh reinforcement of each wall was modelled based on the corresponding detail as described in Chrysostomou (2014) in order to account for the strength reduction in higher floors. The discretization for the first floor wall is shown in Fig. 14. The same material constitutive laws were applied as for the column line elements. A single shell element was used to model the wall in each floor. The walls were assumed to be rigidly connected to the four corners of the bounding frame elements in each floor therefore no relative movement between the walls and the bounding frame was allowed, which is one of the shortcomings of the model. Based on the correlation of the results, it will be concluded whether such a movement took place or if the connection of the wall to the bounding frame using closely spaced dowels was close to rigid.

The full mass of the original building (600 Tonnes) was applied on the model as lumped masses on all the nodes. The floor loading included floor finishes and distributed masonry walls. The numerical period at the beginning of the 0.25 g test was equal to 0.48 sec., which was very close to the experimentally recorded one at the beginning of the test that was approximately equal to 0.50 sec. The evolution of the first mode frequency during the 0.25 g test is shown in Fig. 15.

Similar to the 2D finite element model, a 0.25% viscous damping was used in the model to account for the limited viscous damping activated in the structural elements during the slow process of the pseudo-dynamic testing. The hysteretic damping introduced by the nonlinear behaviour of the elements was modelled by the nonlinear hysteretic models used for concrete and steel.

The comparison of experimental to the numerical history of top storey displacements is shown in Fig. 16. It can be easily concluded that the numerical model captures with high accuracy the behaviour of the tested building. Both the evolution in period and strength during the experiment are very well simulated in the model and so is the nonlinear behaviour of the building.

To examine the correlation of the base shear force, energy dissipation and stiffness between the experimental and numerical model, the base shear versus top displacement hysteretic curves were plotted as shown in Fig. 17.

From the comparison in Fig. 18, it appears that the base shear in the numerical model simulates closely the experimental one. In addition, the stiffness of the numerical model defined by the slope of the hysteretic loop is close to the experimental one. Regarding the energy dissipation, based on the width of the hysteretic loops it appears that the numerical model absorbs slightly higher levels of energy (probably more damage).

#" H#"H H#H$ H#H% H#H& H#H H#HH H#H H#H& H#H% H#H$ H#"H H#" '()012345 678012 )09B45 f C D G X G C Y f `Y a G b k c d eg8 d2p85430)09B q)r
Fig. 17 Comparison of base shear vs. top storey displacement history for the 0.25 g test

Bare frame

In order to assess the seismic capacity of the original bare frame, and appreciate the efficiency of the proposed retrofit technique in enhancing its seismic capacity, the numerical analysis was repeated after removing the RC infill walls from all the floors in the simple numerical model. The good correlation of the global results of this model to the corresponding finite element ones allowed us to avoid using complicated and time-consuming finite element analysis for the bare frame. The capacity of the bare frame was determined by performing a number of non-linear timehistory analyses. In each one of those analyses the selected acceleration record was scaled to increasing PGA levels and the response of the column ends was monitored. A concentrated plasticity (plastic hinge) model was used at both ends of each column to model their nonlinear moment-curvature hysteretic behaviour. A fibre section was used to compute the momentcurvature relationship through cross-section analysis. The discretisation of the cross-section in fibres is shown in Fig. 12. Strength and stiffness degradation were not included in the behaviour.

Since strength degradation was not accounted for, the ultimate capacity of the frame was linked to the attainment of the yield moment in columns and thus the propagation of damage to columns and joints was recorded through the analyses. The number of plastic hinges defined the amount of damage to the building. Therefore, the attainment of the plastic capacity at the ends of all groundfloor columns was regarded as evidence of reaching the capacity of the building. Using this procedure and by examining the hysteretic behaviour of the ground floor columns, it was observed that at an acceleration of 0.05 g, yielding took place in most of the ground floor columns, which can be used to signal the attainment of the capacity of the frame. The hinge results for two of the corner columns are shown in Fig. 18, whereas the corresponding results for inside columns are shown in Fig. 19.

It is obvious from Figs. 18 and 19 that the yield moment was marginally reached and that hysteretic behaviour with some energy dissipation at the hinges took place. An attempt to increase the PGA of the record to 0.06g was conducted that showed significant plastic rotations in most of the ground floor columns. It is believed that since the building was not designed for seismic loading, these plastic rotations cannot be accommodated by the design and detailing of the crosssection. This analysis demonstrates the efficiency of this retrofitting method, since by introducing the RC infill walls in the central span of the three-span bare frame, which was designed for gravity loads only, there was a five-fold increase in its capacity to withstand horizontal loads (from 0.05 g to 0.25 g). Therefore, this method can be considered as a viable methodology for the seismic retrofitting of existing structures designed for gravity loads only.

Conclusions

The purpose of this paper was to examine the capabilities of a 2D finite element micro-model and a simple fibre macro-model to simulate the experimental nonlinear behaviour of a 4-storey RC building strengthened with RC infill walls. The numerical results from the two models were compared in the paper with the experimental results from a full-scale pseudo-dynamic test on a retrofitted RC building. The comparison was conducted using the floor displacement and baseshear time-histories. The elastic characteristics of the building were also well simulated as it is shown from the comparison of the fundamental frequency values. Both models managed to simulate the experimental behaviour of the tested retrofitted building with very good levels of accuracy in all the response parameters mentioned above. Since no relative movement was allowed in the interface between the RC Infill walls and the bounding frame in the numerical models, it was concluded that using the specific connection details leads to monolithic behaviour. The procedure followed for the design of the connections is described in Fardis (2013). It should be stressed out though that if less dowels were used at the connection interface, relative movement might have taken place, which would require modelling of the interface between the infill and the bounding frame in order to capture this behaviour, hence more complicated models would have been required. A comparison was also made between the capacity of the bare and the infilled frame using the macro-model and it was concluded that the latter one managed to withstand an earthquake with a PGA, which was five times larger than the one that the bare frame could resist. This shows that this retrofitting method is promising and further refinements will be made in the models in order to test various connection details and earthquake loads that will make it possible to propose a design methodology for such structures.

  ¡¢£¤¥¦ §¨©¥¢¡¨ §¥ £ ££¤¥¢£ §¨¤£©¡¨© § ¤ §¢£ ¤£¤¥£ ¥ !¥¨¨ §¨¨© Nicholas Kyriakides "# , Christis Z. Chrysostomou 1a , Panagiotis Kotronis 2b , Elpida Georgiou 1c and Panayiotis Roussis 4d $ %&'()0 123 45(03 6 & 7895@A27B 7C&D E5'F(6G526 78%3 43 BH2C3 255(3 2C F2I P57GF63 @0D QRSRT7U VWXY`DXaWX b5G5070D%&'()0R95BWWXVc```XVdWW e H@7B 5%526(FB 5I5fF2650 g 123 45(03 6& 78 %&'()0 hipqrstqu vwx xyyxxx y x xy y -yx x xxx x ww y w x xdxx y-x y-x x xx wwx dx-efghxwivwxyxxxxx xx y yyxx x x xxx wx wx yxi j x x wx xdxx xdxk x x xx yx yyx wx xx y xi vwx ddx y w ddx x wx dx y wxx x x wx xdxx x xw y wx xx xwxx w y x wx xyyxxxi vwx d xxx wx dk x y dxl xxk y wx xwxx yx wx x ywxx yxkwwxxkww yx-y xxi mnopqrrps xy y t y t x xt yx xxx t yx xk dx-xdxx x uv wxyz{|}~y{x

  Dimensions of the full-scale specimen without the RC infill (b) Layout of the full-scale specimen without the RC infill Fig. 1 Dimensions and layout of the full-scale specimen without the RC infill spaced at 6 m and linked by 0.15 m thick RC slab and four 0.25 m by 0.50 m beams perpendicular to the plane of the two three-bay frames (Fig. 1(b)

Fig. 3

 3 Fig. 3 Elevation of the specimen in the lab. The wall shown on the right is the South wall (frame) and the one on the left is the North wall (frame)

Fig. 4

 4 Fig. 4 Base shear versus top storey displacement for the south and north wall for the cyclic test

  mesh Fig. 5 2D finite element discretisation Table 1 2D finite element model: distribution of reinforcement bars Beam (long.) ¡¢£ ¤¥¦ §¨ ¡¢£ ¤© § Beam (transv.) ¡£ Column (transv.) £ ¡£ ¤ §¨£ ¡£ ¤ !"# § Column (transv.) ¡£ Wall (long.) ¡¢££ ¤£ $% & § Wall (transv.) ¡¢££ ¤£ $% & § (a) f 1 =2.85 Hz (b) f 2 =11.35 Hz (c) f 3 =13.6 Hz (d) f 1 =15.3 Hz Fig. 6 2D finite element model: the first four eigenmodes and the corresponding eigenfrequencies calculations

  storey Fig. 7 2D finite element model (0.25 g): displacements time history, experimental vs. numerical results

Fig. 7

 7 compares the numerical and the experimental results considering the displacements time ! "#$% &'$() $0#% #( %1 $ # maximum values and frequency content especially for the upper floors. Figs. 8 and 9 show the performance of the model in terms of base shear-top displacement and storey shear-interstorey drift at each floor. Results are particularly good for the upper floors. It can be noticed, however, that the model does not reproduce accurately the interstorey drift at the base.

Fig. 8

 8 Fig. 8 2D finite element model (0.25 g): Base shear -top displacement, experimental vs. numerical results

Fig. 11

 11 Fig. 11 3D view of the numerical model in SAP2000

Fig. 14

 14 Fig. 14 Discretization of shell RC wall cross-section for the 1 st floor wall

  Fig. 16 Comparison of top storey displacement history for the 0.25 g test

  Fig. 18 Hinge results for corner columns (a) (b) Fig. 19 Hinge results for inside columns

Table 2

 2 2D finite element model: material parameters

	Concrete	Parameters	Steel	Parameters
	¡¢£¤¥¦ §¡¨¢©¢¦	20E+09 Pa	¡¢£¤¥¦ §¡¨ulus	200E+09 Pa
	Poisson coefficient	0.2	Poisson coefficient	0.3
	Tension stress limit	3.6 MPa	Stress limit	460 MPa
	Tension deformation limit	1.8E-04	Deformation limit	0.0023
	Compression stress limit	33.8 MPa	Stress ultimate limit	515 MPa
	Compression deformation limit	-3.0E-03	Deformation ultimate limit	~0.25

Acknowledgments

¡¢£ ¤¥¦ §¨© -!"#$%"% ¢£ &'()¨) '()er DESMI 2009-10 of the Research Promotion Foundation of Cyprus and by the Cyprus Government and the European Regional Development Fund.


Michele Mondini
email: michele.mondini@gustaveroussy.fr

Pierre-Louis Loyher

Pauline Hamon

Marine Gerbé De Thoré

Marie Laviron

Kevin Berthelot

Céline Clemenson

Benoît Salomon

Christophe Combadière

Eric Deutsch

Céline Clémenson

Benoit L Salomon

Alexandre Boissonnas
email: alexandre.boissonnas@upmc.fr

CCR2-Dependent Recruitment of Tregs and Monocytes Following Radiotherapy Is Associated with TNFα-Mediated Resistance

Keywords: radiotherapy, monocytes, regulatory T cells, chemokines, TNFα

INTRODUCTION

Radiation therapy (RT) is a widely employed anticancer treatment and serves as a mainstay for the management of head and neck squamous cell carcinoma (HNSCC) [START_REF] Caudell | The future of personalised radiotherapy for head and neck cancer[END_REF]. The effects of RT on the priming and effector phases of antitumor immunity makes it an appealing strategy to generate immunity against the patient's own tumor [START_REF] Demaria | Role of Local Radiation Therapy in Cancer Immunotherapy[END_REF] and an interesting combinational tool to improve several anticancer immunotherapies [START_REF] Vacchelli | Trial Watch: Immunotherapy plus radiation therapy for oncological indications[END_REF][START_REF] Hettich | Checkpoint Antibodies but not T Cell-Recruiting Diabodies Effectively Synergize with TIL-Inducing gamma-Irradiation[END_REF]. While radiation is known to enhance the infiltration of activated T cells in the tumor microenvironment (TME) [START_REF] Derer | Immune-modulating properties of ionizing radiation: rationale for the treatment of cancer by combination radiotherapy and immune checkpoint inhibitors[END_REF][START_REF] Frey | Hypofractionated Irradiation Has Immune Stimulatory Potential and Induces a Timely Restricted Infiltration of Immune Cells in Colon Cancer Tumors[END_REF], local immunosuppression in the TME is most frequently maintained or even amplified by RT. Regulatory T cells (Tregs) and tumor-associated macrophages (TAMs) represent the main protagonists shaping the immunosuppressive TME and promoting tumor growth, and experimental evidence in murine models has demonstrated that the absence of either of Tregs or TAMs mostly delay tumor progression [START_REF] Fontenot | Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[END_REF][START_REF] Ugel | Tumor-induced myeloid deviation: when myeloid-derived suppressor cells meet tumor-associated macrophages[END_REF]. Macrophage and Treg infiltration into tumors is associated with poor prognosis, therapeutic failure and tumor relapse in multiple solid cancers [START_REF] Waniczek | Tumor-Associated Macrophages and Regulatory T Cells Infiltration and the Clinical Outcome in Colorectal Cancer[END_REF]. The chemokine-chemokine receptor network plays key roles in shaping the TME into an immunosuppressive site by inducing the differential recruitment of regulatory and effector immune cells [START_REF] Franciszkiewicz | Role of chemokines and chemokine receptors in shaping the effector phase of the antitumor immune response[END_REF]. For instance, the CCL2/CCR2 axis represents a major marker of tumor development and has predictive value following cancer therapy in both human and mouse models [START_REF] Conti | CCL2 (monocyte chemoattractant protein-1) and cancer[END_REF]. Recent studies have demonstrated that RT-induced CCL2 production by the tumor contributes to therapeutic failure [START_REF] Connolly | Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes[END_REF][START_REF] Kalbasi | Tumor-Derived CCL2 Mediates Resistance to Radiotherapy in Pancreatic Ductal Adenocarcinoma[END_REF][START_REF] Liang | Host STING-dependent MDSC mobilization drives extrinsic radiation resistance[END_REF], but the mechanisms involved remain elusive. The CCR2-dependent recruitment of Ly6C high monocytes can substantially contribute to the TAM compartment [START_REF] Franklin | The cellular and molecular origin of tumor-associated macrophages[END_REF][START_REF] Qian | CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis[END_REF]. Recently, we showed that CCR2 mediates the recruitment of a subset of CCR2 + Tregs in different tumor models as well as in human oral squamous cell carcinoma (OSCC) [START_REF] Loyher | CCR2 Influences T Regulatory Cell Migration to Tumors and Serves as a Biomarker of Cyclophosphamide Sensitivity[END_REF]. We thus hypothesized that the CCL2/CCR2 axis might be implicated in the co-recruitment of TAMs and Tregs following RT and the constitution of TME immunosuppression. The action of Tregs on innate monocyte-derived cells in vivo has been poorly investigated thus far. Here, we showed that performing local RT on oral tumors in mice induced a CCR2-dependent accumulation of Tregs and TNFα-producing monocytes and macrophages in the tumor bed. Tregs interacted with monocytes/macrophages in irradiated tumors and, finally, TNFα inhibition reduced Treg activity and was associated with increased RT efficacy.

MATERIAL AND METHODS

Cells and animal models

Firefly luciferase-expressing TC1/Luc cells, generated by the HPV16 E6/E7 and c-H-ras retroviral transduction of lung epithelial cells of C57BL/6 origin, were kindly provided by T.C. Wu (Johns Hopkins Medical Institutions, Baltimore, MD, USA) in 2009. Expression of E6 and E7 genes was confirmed by RT-PCR, as well as MHC haplotype H2b by flow cytometry [START_REF] Mondini | Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer[END_REF]. The TdTomato TC1 cell line was generated by infection of TC1 cells with a tdTomato lentivirus (kindly provided by Dr. N. Bercovici and M. Lambert, Institut Cochin, Paris, France). Master stocks of the cells were prepared upon receipt and then used within 6 months after resuscitation. The cells were injected at submucosal sites on the right inner lips of mice to establish a head and neck tumor implantation model as previously described [START_REF] Mondini | Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer[END_REF]. The RAW 264.7 monocyte/macrophage cell line was obtained from ATCC (Manassas, VA, USA). C57BL/6 mice were purchased from Janvier CERT (Le Genest St. Isle, France) and housed at the Gustave Roussy animal facility (Plateforme d'Evaluation Preclinique, PFEP) and the Centre d'exploration fonctionelle, Pitié-Salpêtrière (Paris France). C57BL/6-Foxp3 tm1Mal mice (called Foxp3-EGFP mice herein) [START_REF] Wang | Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells[END_REF], Ccr2 -/-mice, and Csf1r-Gal4VP16/UAS-ECFP (MacBlue) [START_REF] Ovchinnikov | Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP doubletransgenic mice[END_REF] mice were intercrossed to generate Foxp3-EGFP x MacBlue, Foxp3-EGFP x MacBlue x Ccr2 -/- mice. These strains, Foxp3 tm3(DTR/GFP)Ayr (Foxp3-DTR) [START_REF] Kim | Regulatory T cells prevent catastrophic autoimmunity throughout the lifespan of mice[END_REF] and MMTV-PyMT-P2A-mCherry-P2A-OVA (PyMT-ChOVA) [START_REF] Engelhardt | Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells[END_REF], were bred at the Centre d'Exploration Fonctionelle, Pitié-Salpêtrière (Paris, France). All animal procedures were performed in accordance to protocols approved by the Ethical Committee CEEA 26 and CEEA 05 and validated by the "Service Protection et Santé Animales, Environnement".

Irradiation

Mice received single-beam local irradiation to the head and neck region 7 days after tumor inoculation using a 200 kV Varian X-ray irradiator. Selective irradiation of the tumor grafts was performed by the interposition of a 4-cm-thick lead shield on a schedule delivering 7.5Gy in a single fraction at a dose rate of 1.08Gy/min.

Antibodies and treatments

The anti-CD25 (InVivoMAb anti-mouse CD25, clone PC-61.5.3) and anti-TNFα (InVivoMAb antimouse TNFα, clone XT3.11) neutralizing antibodies as well as the corresponding isotype controls (InVivoMAb rat IgG1 isotype control, anti-horseradish peroxidase, clone HRPN) were purchased from BioXcell (West Lebanon, NH, USA). Anti-CD25 was administered at 20 mg/kg in PBS by intraperitoneal (IP) injection on days 4 and 6 post-RT. Anti-TNFα was administered at 25 mg/kg in PBS by IP injection on days 2 and 4 post-RT. To deplete Tregs in Foxp3-DTR mice, diphtheria toxin (DTx, Merck, USA) was solubilized in PBS and injected IP at 1 µg/mouse on days 2 and 3 post-RT. DTx treatment had no effects on tumor growth in WT mice (not shown).

In vivo imaging

To monitor tumor growth, bioluminescence imaging was performed using the Xenogen In Vivo Imaging System 50 (IVIS; Caliper Life Sciences, Hopkinton, MA, USA) as previously described [START_REF] Mondini | Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer[END_REF].

For multiphoton imaging of Foxp3-EGFP x MacBlue mice, HNSCC tumors were carefully collected and immobilized in an imaging chamber perfused with oxygenated (95% O2 plus 5% CO2) RPMI medium containing 10% fetal calf serum (FCS). The local temperature was monitored and maintained at 37°C. The two-photon laser scanning microscopy setup used was previously described [START_REF] Ovchinnikov | Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP doubletransgenic mice[END_REF], and the excitation wavelength used was 870 nm. The system included a set of external nondescanned detectors in reflection with a combination of LP-600 nm followed by LP-462 nm and LP-500 nm dichroic mirrors to split the light and collect the second-harmonic generation (SHG) signal with a 417/60 nm emission filter, ECFP with a 480/40 nm emission filter and EGFP with a 525/50 nm emission filter.

Cell motility was measured every 30 s by 9 consecutive 5 μm z spacing stacks (total of 40 μm thickness) using a plan apochromat 20× (NA=1) water immersion objective. Fluorescent cells were monitored over time with 3-dimensional automatic tracking and manual correction with Imaris software (Bitplane, Zurich, Switzerland). The acquisition and analysis protocols for all experimental conditions to be compared were identical. Velocity and straightness were determined using Imaris. The track straightness corresponded to the ratio of the distance between the initial and final positions of each cell to the total distance covered by the same cell.

Histological analysis

Tumors were harvested, fixed in 4% formalin for 4 h, incubated in 30% sucrose-PBS overnight at 4°C, embedded in Tissue-Tek OCT compound (Sakura Finetek, Alphen aan den Rijn, Netherlands) and frozen at -80°C. Sectioning was completed on an HM550 cryostat (Thermo Fisher Scientific, Waltham, MA, USA) at -20°C; 5-μm-thick sections were collected on Superfrost® Plus Slides (Thermo Fisher Scientific, Waltham, MA) and stored at -20°C until use. The slides were counterstained and mounted with Vectashield Mounting Medium with DAPI (4,6-diamidino-2-phenylindole; Vector Laboratories). Images were acquired with a Zeiss Axio Z1 fluorescence microscope (Carl Zeiss, Germany).

Flow cytometry analysis

Phenotypic characterization was performed using the LSRFortessa instrument (Becton Dickinson) for murine cells. For analysis, FlowJo software (Tree Star Inc.) was used. TC1/Luc and spontaneous MMTV-PyMT oral tumors were digested in RPMI medium (Gibco, Invitrogen, Cergy Pontoise, France) supplemented with 1 mg/mL collagenase IV (Sigma) for 30 minutes at 37°C, entirely resuspended in PBS supplemented with 0.5% BSA/2 mmol/L EDTA (FACS buffer) and filtered using a 70-μm cell strainer (BD Biosciences). Blood was drawn, and the cells were lysed in RBC lysis buffer containing 0.15 mol/L NH4Cl, 0.01 mmol/L KHCO3, and 0.1 mmol/L EDTA. Surface staining was performed by incubating 50 μL of the cell suspension (one tenth of the total) with 1 μg/mL purified anti-CD16/32 (2.4G2; BD Biosciences) for 10 minutes at 4°C followed by incubation with an appropriate dilution of specific surface antibodies for an additional 20 minutes. Dead cells were excluded using LIVE/DEAD Fixable Stain (Life Technologies) according to the manufacturer's instructions. Forward and side scatter parameters were used for doublet exclusion. After incubation, the cell suspensions were washed once in FACS buffer. The CCL2-binding assay was performed as previously described [START_REF] Loyher | CCR2 Influences T Regulatory Cell Migration to Tumors and Serves as a Biomarker of Cyclophosphamide Sensitivity[END_REF]. Briefly, after surface staining, the cells were incubated in the dark for 45 minutes at 37°C in RPMI medium supplemented with GlutaMAX containing 25 nmol/L murine CCL2 AF647 (Almac Sciences) and then washed with FACS buffer. For INFγ and TNFα staining, cells were preincubated for 2 h with a cell activation cocktail containing Brefeldin A according to the manufacturer's instructions (BioLegend). After surface staining, the cells were fixed in 4% paraformaldehyde for 20 minutes, washed twice in Perm/Wash solution (BD Biosciences), incubated for 10 minutes with 1 μg/mL purified anti-CD16/32 in Perm/Wash at room temperature, and incubated for 30 minutes in Perm/Wash in the presence of anti-IFNγ or anti-TNFα. For intracellular staining, the Foxp3/Transcription Factor Staining Buffer Set, anti-Foxp3 and anti-CTLA4 were used according to the manufacturers' instructions. Samples were washed in FACS buffer before acquisition. Calculation of the absolute numbers of different cell populations was performed by adding a fixed number (10,000) of nonfluorescent 10-μm Polybead Carboxylate Microspheres (Polysciences) to each vial according to the following formula: number of cells = number of acquired cells × 10,000/number of acquired beads. The number of cells obtained for each sample was extrapolated to the whole organ. The panel of antibodies used comprised the following: anti-CD11b (clone M1/70), anti-Ly6C (clone AL-21), anti-Ly6G (clone 1A8), anti-NK1.1 (clone PK136), anti-I-Ab (clone AF6-120.1), anti-CD11c (clone HL3), anti-Siglec-F (clone E50-2440), anti-CD103 (clone M290), anti-CD45 (clone 30-F11), anti-CD4 (clone RM4-5), anti-CD3 (clone 145-2C11), anti-CD8 (clone 53-6.7), anti-CD25 (clone PC61), anti-CD64 (clone X54-5/7.1), anti-TNFα (clone MP6-XT22), anti-IFNγ (clone XMG1.2), anti-CTLA4 (clone UC10-4F10-11), anti-TGFβ (clone TW7-16B4), anti-Foxp3 (clone MF23; Pharmingen, BD Bioscience), anti-CD206 (clone C068C2; Biolegend) and anti-CCR2 (clone 475301; R&D systems, Minneapolis, MN, USA).

CCL2 ELISA

The levels of CCL2 in cell supernatants and mouse sera were evaluated by ELISA using Mouse Quantikine ELISA Kits from R&D Systems.

Quantitative RT-PCR

RNA was extracted from cells and tumor samples using the Qiagen RNeasy kit. The samples were treated with DNAse-I (Qiagen), and RNA was retrotranscribed using the Bio-Rad I-Script kit. qRT-PCR analysis of CCL2 was performed using SYBR Green Mix (Applied Biosystems, Foster City, CA, USA) and the GeneAmp 7900HT Sequence Detection System (Applied Biosystems) as previously described [START_REF] Henke | Targeted deletion of CCR2 impairs deep vein thombosis resolution in a mouse model[END_REF].

Generation of the CCL2-deleted TC1/Luc cell line

The genomic TC1/Luc cell line was edited using CRISPR-CAS9 technology to disrupt the Ccl2 gene. The Ccl2 mouse gene knockout via CRISPR kit (product number KN302782, Origene, Rockville, MD, USA) was used to knockout a region of the Ccl2 gene and knockin a functional cassette containing GFP and the puromycin-resistance gene. Briefly, TC1/Luc cells were cotransfected with donor template DNA and pCas-guide plasmids using Lipofectamine 2000 (Thermo Fisher Scientific, USA), sorted for GFP expression, cultured for 7 weeks and then selected for puromycin resistance. Individual cell colonies were isolated by limiting dilution. Supernatants of clones were screened for CCL2 levels by ELISA, and CCL2-negative clones were selected.

Tumor-macrophage co-cultures

The TC1/Luc and RAW264.7 cells were mock-treated or irradiated at 7.5Gy and then respectively seeded into the lower and upper chambers of 6.5 mm transwells with a 0.4 µm Pore Polycarbonate Membrane Insert (Corning, NY, USA). The number of cells plated was adjusted considering the proliferation rate and the response to RT to yield an approximately equivalent number of cells regardless of the radiation dose delivered and the growth time. After 48 h of incubation, cell media were collected and analyzed by ELISA, and the mRNAs were separately extracted from TC1/Luc and Raw264.7 cells and analyzed by RT-qPCR.

Cytokine/chemokine array

The cytokine and chemokine levels in tumor tissues were profiled using the Mouse Cytokine Array/Chemokine Array 31-Plex (ref. at Eve Technologies Corporation (Calgary, AB, Canada). Protein extracts from tumor tissues were prepared by homogenization in RIPA buffer (Sigma-Aldrich, USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland) using Biomasher disposable homogenizers (Nippi, Tokyo, Japan). Protein extracts were diluted at 4 μg/μl, and the multiplex immunoassay Mouse Cytokine Array/Chemokine Array 32-Plex (Milliplex, Merck Millipore, USA) was analyzed with the BioPlex 200 instrument (Bio-Rad, USA).

Statistical analyses

Statistical analyses were performed using Prism Version 7 (GraphPad, La Jolla, CA, USA). Survival data were analyzed using the Kaplan-Meier and log-rank tests for survival distribution. The bioluminescent signal (serving as a measurement of tumor size), immune infiltrate and tumor perfusion levels were analyzed using appropriate tests. Accordingly, multigroup analyses of variances were performed using one-way or two-way ANOVA followed by Sidak's or Tukey's multiple comparisons tests, respectively, or the Kruskal-Wallis test followed by Dunn's multiple comparisons test. For simple comparison analysis, unpaired Student's t-test was used to compare parametric distributions, and the Mann-Whitney test was used to compare nonparametric distributions. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; ns=not significant.

RESULTS

Radiotherapy induces the CCR2-dependent recruitment of monocytes into the tumor.

Orthotopic inoculation of the TC1/Luc cell line was performed at a submucosal site of the inner lip in syngeneic wild-type (WT, Ccr2 +/+ ) and Ccr2 -/-mice to mimic an HPV-related oral cancer as described previously [START_REF] Mondini | Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer[END_REF]. Using the same model and irradiation treatment (Fig 1A ), we showed here that the lack of stromal CCR2 improved the survival rate after localized RT (Fig 1B ; median survival gain after RT for WT= 3 days, for Ccr2 -/-= 6.5 days), which was associated with a reduction in tumor burden (Fig S1A). RT induced a transient CCR2-dependent increase of classical Ly6C high monocytes (Ly6C high Mo) in the blood that was not observed after the local irradiation of tumor-free mice, indicating that the increase in blood Ly6C high Mo was dependent on irradiation of the tumor (Fig 1C). We next monitored the impact of RT on Ly6C high Mo and macrophage subsets within the tumor according to the gating strategy depicted in Fig S1B . The Ly6C high monocytosis induced by RT was associated with a massive and transient accumulation of Ly6C high Mo into the tumor, a higher CD64 expression and a subsequent accumulation of Ly6C low/-CD64 + macrophages, suggesting that the latter mainly arose from the differentiation of Ly6C high Mo between days 3 and 5 post-RT (Fig 1D). CCR2 deficiency resulted in a severe impairment of both monocyte and macrophage accumulation on day 5 post-RT (Fig 1E). Newly infiltrated macrophages in WT mice displayed a higher proportion of I-Ab + cells, while CD206 was expressed at lower levels in these mice than in nonirradiated mice (Fig 1F). This activation profile was severely impaired in Ccr2 -/-mice, with a lower expression of I-Ab and CD206 on the few infiltrated macrophages detected in these mice (Fig 1F). Histological analysis of MacBlue transgenic mice inoculated with tdTomato TC1 cells unveiled that the accumulation of ECFP + monocytes/macrophages mainly occurred at the tumor periphery after RT and was not observed in MacBlue x Ccr2 -/-mice (Fig S1C). Overall, our results suggest that local tumor irradiation induces a CCR2-dependent increase of monocytes in the blood that subsequently infiltrate the tumor and progressively differentiate into macrophages.

Radiotherapy induces the recruitment of CCR2 + Tregs.

We recently demonstrated that CCR2 represents an important homing receptor of Tregs toward the tumor and that a large fraction of Tregs are CCR2 + in human HNSCC [START_REF] Loyher | CCR2 Influences T Regulatory Cell Migration to Tumors and Serves as a Biomarker of Cyclophosphamide Sensitivity[END_REF]. In this orthotopic model of HNSCC, RT, together with an increased CCR2-dependent tumor infiltration of monocytes/macrophages, mediated a significant increase in CD4 + conventional T cells (Tconvs) and Tregs in RT vs untreated tumors at 5 days post-treatment (Fig 2A). In the TC1/Luc model, the accumulation of Tconvs and Tregs was severely reduced in Ccr2 -/-mice, in accordance with our previous study (Fig 2A) [START_REF] Loyher | CCR2 Influences T Regulatory Cell Migration to Tumors and Serves as a Biomarker of Cyclophosphamide Sensitivity[END_REF]. In agreement with the CCR2-mediated recruitment of Tregs to irradiated tumors, the proportion of the CCR2 + Treg subset was increased in WT mice following RT, while the percentage of CCR2 + Tconvs was not significantly changed (Fig 2B). To investigate the effect of Tregs following RT in this HNSCC tumor model, we selectively depleted Tregs using Foxp3-DTR mice. Diphtheria toxin (DTx) treatment was started on day 2 after irradiation to fully impair RT-induced Treg recruitment (Fig 2C). Treg depletion in irradiated tumor-bearing mice reduced the tumor burden compared to that in non-depleted control mice (Fig 2D), confirming that Tregs limit RT efficacy. RT-induced accumulation of macrophages and Tregs was also confirmed in a spontaneous model of HNSCC, using male PyMT-ChOva mice that develop salivary tumors, suggesting that this observation is not restricted to engrafted models (Fig 3) [START_REF] Engelhardt | Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells[END_REF].

Radiotherapy triggers myeloid-associated cytokine secretion.

To better understand the mechanisms underlying RT-mediated cell recruitment, we We next performed cytokine profiling on whole-tumor extracts and compared the levels of a set of cytokine and growth factors relevant to monocyte-derived cell recruitment and differentiation in nonirradiated control tumors and irradiated tumors at 5 days post-RT, when macrophages and Tregs were accumulated into irradiated tumors (Fig 4D). Among the cytokines and growth factors analyzed, CCL2 accumulation was confirmed at 5 days after RT. In addition, the IL3, GM-CSF, TNFα and IFNγ levels significantly increased after RT, while the levels of M-CSF were similar to those in nonirradiated tumors. We concluded that local RT induces the secretion of CCL2 by tumor cells favoring the recruitment of Ly6C high Mo into the tumor, associated with an increased production of myeloid-cell related cytokines and growth factors involved in survival and differentiation.

TNFα limits the efficacy of RT in a Treg-dependent manner.

We speculated that the co-recruitment of Tregs and monocytes following RT might favor the crosstalk between these two subsets. TNFα is known to have a direct effect on Treg function [START_REF] Romano | Expanded Regulatory T Cells Induce Alternatively Activated Monocytes With a Reduced Capacity to Expand T Helper-17 Cells[END_REF][START_REF] Salomon | Tumor Necrosis Factor alpha and Regulatory T Cells in Oncoimmunology[END_REF]

Tregs interact with monocytes/macrophages in irradiated tumors.

To further investigate the mechanism of Treg/monocyte crosstalk, we performed live imaging using intravital biphoton microscopy. Using MacBlue x Foxp3-EGFP double transgenic mice, we monitored the dynamic interactions of co-recruited monocytes/macrophages and Tregs within irradiated tumors 5 days after RT, at the time Tregs accumulated within the tumor.

DISCUSSION

It is well recognized that RT severely impacts the TME and tumor immune response [START_REF] Frey | Immunomodulation by ionizing radiation-impact for design of radio-immunotherapies and for treatment of inflammatory diseases[END_REF]. Radiation promotes the recruitment of inflammatory cells into the TME but can also attract immunosuppressive cells [START_REF] Weichselbaum | Radiotherapy and immunotherapy: a beneficial liaison?[END_REF]. Our data, obtained using an orthotopic model of HPVassociated oral cancer, indicates that a marked influx of Ly6C high Mo, driven by the CCL2/CCR2 axis, is observable 3 days after a single exposure to 7.5Gy of ionizing radiation. Our observation on Ly6C high Mo mobilization is in accordance with others made using subcutaneous models of pancreatic ( 13) colon and lung adenocarcinoma [START_REF] Liang | Host STING-dependent MDSC mobilization drives extrinsic radiation resistance[END_REF] and with intramuscular models of colon adenocarcinoma, glioblastoma and lung carcinoma [START_REF] Connolly | Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes[END_REF] with different RT regimens (15-20Gy), suggesting that the CCL2/CCR2-mediated recruitment of Ly6C high Mo can be a general mechanism that follows tumor exposure to ionizing radiation, at least at high doses (>7.5Gy). This sub-optimal RT regimen was chosen in order to monitor the added-value of the combined immunotherapy targeting CCR2 axis. Unpublished data (data not shown in Connolly et al. [START_REF] Connolly | Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes[END_REF]) indicate that anti-CCR2/CCR5 treatment is ineffective at doses of 5x5Gy but synergizes with RT at 1x15Gy, indirectly suggesting that the mechanism of CCR2-mediated immunosuppressive cell recruitment could be different at lower and/or fractionated doses.

Nevertheless, it remains to be clearly elucidated whether CCR2-mediated recruitment occurs at lower RT doses, and especially after multiple RT exposures, to more closely recapitulate fractionated RT regimes in the clinical setting.

The mechanism was shown to involve the induction of increased CCL2 expression by irradiated tumor cells based on the following: 1 -CCL2 was still detectable and increased after RT when TC1/Luc cells were engrafted into Ccl2 -/-mice; 2no upregulation of CCL2 was detectable in in the blood of mice engrafted with a Ccl2 -/-cell line (in line with Kalbasi et al. [START_REF] Kalbasi | Tumor-Derived CCL2 Mediates Resistance to Radiotherapy in Pancreatic Ductal Adenocarcinoma[END_REF]) or in the blood of tumor-free mice after RT; and 3 -CCL2 was upregulated in tumor cells in vitro after RT. CCL2 upregulation in irradiated tumor cells is likely enhanced by the release of soluble factors from TAMs, as suggested by the tumor-macrophage co-culture experiments. Such mediators could include IL-6 (29) and type I interferons [START_REF] Liang | Host STING-dependent MDSC mobilization drives extrinsic radiation resistance[END_REF]. Further experiments would be necessary to confirm whether primary tumor-monocytes as well as other stromal cells might enhance CCL2 upregulation after irradiation.

Our work clearly demonstrates that not only Ly6C high Mo but also Tregs recruitment to the tumor after RT involves the CCL2/CCR2 pathway. The intratumor Treg strongly accumulated 5 days post-RT while the Treg accumulation in irradiated Ccr2 -/-mice was severely reduced. We cannot exclude that the increase in Tregs was partly due to the local proliferation of intratumor Tregs after radiation, as this observation was recently described using different tumor models [START_REF] Muroyama | Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment[END_REF]. Nevertheless, the lack of increase in Tregs in irradiated tumors from Ccr2 - /-mice and the increased proportion of CCR2-expressing Tregs in WT mice after RT are supportive of a significant role of CCR2-mediated Treg recruitment in the tumor bed.

The co-recruited monocytes/macrophages and Tregs displayed dynamic interactions within the irradiated tumor tissue, suggesting a direct cell-to-cell mediated crosstalk between these two subsets. Anti-CD25 treatment strongly reduced the contact duration between these subsets, showing that these interactions are not random but rather guided by a specific activation state of the different subsets. Recently, Romano et al. showed that expanded Tregs polarize in vitro monocytes toward alternatively activated macrophages [START_REF] Romano | Expanded Regulatory T Cells Induce Alternatively Activated Monocytes With a Reduced Capacity to Expand T Helper-17 Cells[END_REF]. In the tumor context, we observed that functional Tregs induce monocyte/macrophage activation by upregulating the expression of MHC class II molecules, suggesting that they might favor direct T cell receptor (TCR) engagement. In this system, anti-CD25 treatment did not improve the RT efficacy showing that reducing the duration of the interaction between Tregs and monocytes/macrophages is not sufficient to abrogate their immunosuppressive activity. Whether local CCL2 production favors physical interactions between the two subsets deserves further investigation. Among local cytokines produced following RT, TNFα was previously demonstrated to exert a radioprotective effect, which was proposed to occur via VEGF production by TAMs [START_REF] Meng | Blockade of tumor necrosis factor alpha signaling in tumor-associated macrophages as a radiosensitizing strategy[END_REF]. We further demonstrated that TNFα neutralization enhances tumor response to RT by reducing Treg frequency and TGFβ production. Considering the role of TGFβ in Treg-mediated immunosuppressive activity (32), the activation of this axis is strongly relevant to the radioprotective actions of Tregs. Among these protective actions, we observed that anti-TNFα increased the frequency of IFNγ-producing CD8+ T cells after RT. While CTLA4 expression was increased in Tregs following RT, its expression was not modified by anti-TNFα, suggesting that TNFα might regulate specific pathways of Treg activation. Several pieces of data have indicated that TNFα induces more specific signaling in Tregs via TNFR2 to maintain their suppressive activity [START_REF] Salomon | Tumor Necrosis Factor alpha and Regulatory T Cells in Oncoimmunology[END_REF][START_REF] Yang | Role of TNF-TNF Receptor 2 Signal in Regulatory T Cells and Its Therapeutic Implications[END_REF][START_REF] Zaragoza | Suppressive activity of human regulatory T cells is maintained in the presence of TNF[END_REF]. Deciphering the pathway of Treg activation following RT is crucial considering the potential of combined immunotherapies targeting Tregs with RT. Considering the cytotoxic effect of TNFα [START_REF] Larrick | Cytotoxic mechanism of tumor necrosis factor-alpha[END_REF], one could assume that a shortened anti-TNFα treatment might efficiently block Treg accumulation while avoiding inhibition of CD8 + T cell activity. In contrast to most cancers, paradoxical observations have been gathered on the role of Tregs in patients with HNSCC, suggesting that tumor Treg density correlate with increased survival [START_REF] Lim | CD4+CD25hiCD127low regulatory T cells are increased in oral squamous cell carcinoma patients[END_REF]. This discrepancy is poorly understood, and some observations suggest that Tregs suppress the induction of a chronic inflammatory state in the tissue [START_REF] Al-Qahtani | Tumour infiltrating CD25+ FoxP3+ regulatory T cells (Tregs) relate to tumour grade and stromal inflammation in oral squamous cell carcinoma[END_REF]. However, these hypotheses are still being debated, as other research showed no such correlation [START_REF] Song | Foxp3 overexpression in tumor cells predicts poor survival in oral squamous cell carcinoma[END_REF]. Our data clearly indicate that Tregs play a detrimental role in the response to RT, as their depletion increases RT efficacy. Thus, targeting Tregs could be an interesting therapeutic approach to enhance the RT index, as previously suggested (see Liu et al. (39) for a review).

The results of our study offer interesting perspectives. Our data provide strong evidence for the thus far poorly studied crosstalk between adaptive and innate immune system components and indicate that impacting the monocyte/Treg interaction after RT could increase the antitumor effect of RT. The extent of CCL2 and TNFα production was heterogenous between tumors following RT, possibly reflecting the differential response to radiation. It would thus be interesting to evaluate the prognostic value of these cytokines. As modulators of CCL2 and CCR2 are already being tested in clinical settings, translating the results from this study into the clinic is conceivable. An alternative (and potentially synergistic) approach could be to use anti-TNFα or anti-TGFβ therapeutics to overcome the immunosuppressive effects of Tregs and thus potentiate the effects of RT. Our data underline the relevance of using combined strategies to improve the biological efficacy of ionizing radiation-based therapies and limit the toxicities related to dose escalation. Indeed, deintensifying treatments to avoid late toxicity is a foremost goal and an active area of research. The use of RT in combination with CCL2/CCR2 and anti-TNFα modulators represents a promising approach to achieve such goals. (A) C57Bl/6 wild-type (WT, Ccr2 +/+ ) and Ccr2 KO (Ccr2 -/-) mice were injected with TC1/Luc cells in their inner lip and then locally irradiated at 7.5Gy. Blood and tumor samples were collected at different time points as depicted in the schematic diagram. (B) Kaplan-Meier survival curves for mock-or radiotherapy (RT)-treated mice. The log-rank test was performed (n=8-15 mice/group from 3 independent experiments). (C) Numbers of Ly6C high monocytes in the bloodstreams of tumor-bearing WT (Ccr2 +/+ ) and Ccr2 KO (Ccr2 -/-) mice and nontumorchallenged WT mice (Tumor-free) with or without RT treatment (n= more than 4 mice/group per time point from at least two independent experiments). (D) Left, representative Ly6C/CD64 dot plots of tumor-infiltrating monocytes/macrophages in nonirradiated (Ctrl) and irradiated (RT) mice at 3 and 6 days post-RT (the mean±SD percentages of cells in each gate are indicated, n=8 mice/group from two independent experiments). Right panels, numbers of tumor Ly6C high monocytes (Ly6C high Mo) and tumor-associated macrophages (TAMs) per mg. Two-way ANOVA with Tukey's multiple comparisons test was performed for RT vs nonirradiated mice at each time point (n=8-15 mice/group per time point from at least two independent experiments). (E) Absolute numbers of Ly6C high Mo and TAMs in WT and Ccr2 -/- mice per mg at 5 days post-RT. One-way ANOVA with Sidak's multiple comparisons test was performed for RT vs nonirradiated mice at each time point (n=9-10 mice/group from three independent experiments). (F) Representative Ly6C/I-Ab (left panels) and Ly6C/CD206 (right panels) dot plots of tumor-infiltrating monocytes/macrophages in nonirradiated (Ctrl) and irradiated (RT) mice at 5 days post-RT (the mean±SD percentages of cells in each gate are indicated, n=6-8 mice/group from two independent experiments). For all panels: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

  investigated the effect of RT on CCL2 production. Local irradiation of tumor-bearing mice induced a transient increase in the serum CCL2 concentration at 3 days post-treatment (Fig 4A), which directly correlated with the Ly6C high Mo release into the blood (Fig 1C). In the absence of a tumor, the serum CCL2 concentration was not increased after RT (Fig 4B), while CCL2 accumulation was still detectable after irradiation of TC1/Luc tumor-bearing Ccl2 -/-mice, indicating that the circulating CCL2 mainly originated from the tumor cells (Fig S2A). In contrast, no serum CCL2 accumulation was observed in either WT or Ccl2 -/-mice bearing CCL2 knockdown (Ccl2 -/-) TC1/Luc cells (Fig S2B). These results suggest that TC1/Luc tumor cells represent the main source of CCL2 production after RT rather than stromal cells. In the tumor, increased CCL2 transcription was detected by quantitative PCR 3 days after RT and was maintained on day 5 (Fig S2C). In vitro, when TC1/Luc tumor cells were co-cultured with the macrophage cell line RAW264.7, CCL2 secretion was increased and peaked when both TC1/Luc and RAW264.7 cells were exposed to ionizing radiation (Fig 4C), indicating that secreted factors from irradiated monocytes/macrophages further contribute to CCL2 upregulation at both the mRNA (Fig S2D) and secreted protein levels (Fig 4C).

  and was found to be upregulated in irradiated tumors (Fig 4D). In the tumor, Ly6C high Mo and TAMs represented important TNFα producers. The production of this cytokine was maintained in the newly infiltrated cells that accumulated 5 days after RT (Fig 5A). Anti-TNFα treatment after irradiation strongly improved the efficacy of RT (Fig 5B), while the effect of anti-TNFα was abrogated in Foxp3-DTR mice (Fig 5C), suggesting that the antitumor effect of anti-TNFα was mediated by Tregs. Anti-TNFα treatment reduced the Tregs frequency among CD4 + T cells following RT (Fig 5D) and significantly reduced TGFβ expression by Tregs (Fig 5E), while CTLA4 expression was not modified (Fig S3A). Conventional CD4 + and CD8 + T cell infiltration was not modulated by anti-TNFα (Fig S3B), but RT increased the proportion of IFNγproducing CD8 + T cells only when mice were treated with anti-TNFα (Fig 5F). Our results suggest that RT induces the CCR2-dependent accumulation of TNFα-producing myeloid cells that activate Tregs and dampen the CD8 + T cell response to ionizing radiation via TGFβ production.

  Clusters of monocytes/macrophages were present in close proximity to Treg cells (Fig 6A), and Tregs displayed multiple interactions with monocytes/macrophages (Fig 6A and Supplemental Video 1). Treatment with an anti-CD25 antibody is known to neutralize Treg activity without depleting the cell population (Fig S4A) (26). Anti-CD25 neutralized-Tregs displayed an increased mean velocity compared to that of IgG-treated controls (Fig 6B). While the rate of Treg interactions with monocytes/macrophages was not affected (Fig 6C), the duration of these interactions was significantly reduced in anti-CD25-treated mice likely explaining to the higher speediness of Tregs (Fig 6D). The very low number of Tregs in nonirradiated mice prevented us from analyzing their potential interactions with monocytes/macrophages. Anti-CD25 treatment was not sufficient to improve the RT effect (Fig S4B-C), but the altered pattern of interaction between Tregs and monocytes/macrophages upon CD25 inhibition was associated with an altered activation profile, as depicted by the reduced expression of major histocompatibility complex (MHC) class II molecules (I-Ab) in both tumor Ly6C high Mo and TAMs (Fig 6E-F), suggesting that Tregmonocyte/macrophage crosstalk occurs via direct cell-to-cell interactions.

Figure 1 :

 1 Figure 1: Radiotherapy induces the CCR2-dependent recruitment of monocytes into the tumor.

Figure 2 :

 2 Figure 2: Radiotherapy induces the recruitment of CCR2 + Tregs.(A) Left panels, representative dot plots of regulatory T cells (Tregs) in nonirradiated (Ctrl) and irradiated (RT) wild-type (WT, Ccr2 +/+ ) mice at 5 days post-RT. Right panels, numbers of tumor Tregs and conventional CD4 + T cells (Tconvs) per mg in WT and Ccr2 -/-mice. One-way ANOVA with Sidak's multiple comparisons test was performed for RT vs nonirradiated WT and Ccr2 -/- mice (n=9-14 mice/group from three independent experiments). (B) Histograms showing the expression of CCR2 on the surface of Tregs (left panel) in WT and Ccr2 -/-mice and quantification of the percentages of CCR2 + Tregs and Tconvs in nonirradiated and irradiated (RT) WT mice (right panel). One-way ANOVA with Sidak's multiple comparisons test was performed for Tconvs and Tregs in RT vs nonirradiated mice (n=13-16 mice/group from three independent experiments). (C) Representative dot plots (left panels) and quantification of the percentages (right panel) of Tregs in irradiated tumors from WT and Foxp3-DTR mice treated with diphtheria toxin (DTx). Unpaired Student's t-test was performed (n=9-18 mice/group from at least two independent experiments). (D) Tumor growth in WT and Foxp3-DTR mice treated with DTx (black arrows) was monitored by bioluminescence in vivo imaging. Data for WT Ctrl mice (WT+DTx) at day 7 are not indicated since mice were euthanized before this time point. One-way ANOVA with Sidak's multiple comparisons test was performed (n=6-18 mice/group from at least two independent experiments). For all panels: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Figure 3 :

 3 Figure 3: Macrophages and Tregs accumulates in irradiated spontaneous oral tumors. Spontaneous oral tumors from MMTV-PyMT mice bearing fluorescent reporters for tumor cells (mCherry, red) and monocytes/macrophages (ECFP, light blue, the MacBlue reporter allows the specific visualization of tumor-associated monocytes/macrophages) were irradiated and analyzed 5 days later by flow cytometry (A) or confocal microscopy (B). Twoway ANOVA with Sidak's multiple comparisons test was performed (n=3-6 mice/group from two independent experiments).

Figure 4 :

 4 Figure 4: Radiotherapy triggers myeloid-associated cytokine secretion. The CCL2 levels in the sera of mice engrafted with TC1/Luc tumor cells (A) and nontumorchallenged wild-type mice (Tumor-free) (B) were evaluated by ELISA at different time points after 7.5Gy irradiation (RT). (C) TC1/Luc and Raw 264.7 cells were mock-treated (NI=nonirradiated) or irradiated at 7.5Gy (IR) and then co-cultured in transwell chambers for 48h; the cell supernatants were then collected for ELISA (-= no cells; data from two independent experiments performed in triplicate). (D) Cytokine profiling of whole-tumor protein extracts performed 5 days after the treatment of WT mice with 7.5Gy irradiation. One-way ANOVA with Sidak's multiple comparisons test (A and B) or two-way ANOVA with Tukey's multiple comparisons test (C) and the Mann-Whitney test (D) were performed. For A, B and D: n=4-20 mice/group per time point out of at least two independent experiments. For all panels: *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Figure 5 :

 5 Figure 5: TNFα limits the efficacy of RT in a Treg-dependent manner. (A) Representative histograms of flow cytometry analyses of intracellular staining to evaluate TNFα levels in tumor Ly6C high Mo and TAMs from nonirradiated (Ctrl) and irradiated (RT) WT mice at 5 days post-RT. The mean±SD of the percentage of TNFα + cells is indicated (n=6-10 mice from two independent experiments). Irradiated WT (B), Foxp3-DTR and DTx-treated (C) mice were injected with a control (isotype) or neutralizing anti-TNFα Ab (aTNFα, black arrows), and tumor growth was monitored by in vivo imaging. Two-way ANOVA with Tukey's multiple comparisons test was performed (n=9-10 mice/group per time point out of at least two independent experiments). (D) Absolute numbers per mg (left panel) and percentages (right panel) of CD4 + Tregs in tumors from WT mice treated with RT and/or anti-TNFα. (E) TGFβ expression in Tregs from tumors of WT mice treated with RT and/or anti-TNFα as quantified by flow cytometry. The mean±SD of the percentage of TGFβ + is indicated. (F) Percentage of CD8 + T cells expressing IFNγ as detected by flow cytometry in tumors from WT mice treated with RT and/or anti-TNFα. One-way ANOVA with Sidak's multiple comparisons test was performed for D, E and F (n=6-9 mice/group out of two independent experiments). For all panels: *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 6 :

 6 Figure 6: Tregs interact with monocytes/macrophages in irradiated tumors. Intravital biphoton microscopy analysis of explanted TC1/Luc tumors from MacBlue/Foxp3-EGFP mice was performed at 5 days after radiotherapy. (A) Time-lapse TPLSM image sequence from a representative field showing multiple interactions (red arrows) between Tregs (green) and monocytes (light blue). Scatter plots representing the quantification of Treg motility (B) and the rate (C) and duration (D) of Treg-monocyte/macrophage interactions in irradiated tumors from mice treated with anti-CD25 or control IgG (isotype) (data are pooled from 3-4 mice in each group). The expression of MHCII (I-Ab) on the surface of Ly6C high monocytes (E) and macrophages (F) was quantified by flow cytometry (n=12-17 mice/group out of three independent experiments). The Mann-Whitney test was performed for B, C, D, E and F. For all panels: ns: nonsignificant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

ACKNOWLEDGMENTS

The authors wish to thank the "NAC" animal facility for their assistance with mouse breeding and the Plateforme d'Évaluation Preclinique (PFEP) for providing animal care. CONTRIBUTIONS M.M., A.B. and E.D. initiated the project concept and supervised the project. A.B., M.M. and P.L.L. conceived and designed the experiments. M.M., P.L.L., P.H., M.G.D.T., M.L., K.B. and A.B. performed the experiments. C.Cl., B.S., C.Co. and E.D. provided suggestions and technical support. M.M., A.B. and P.H. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Financial support: P.L.L. was funded by the "Fondation ARC pour la Recherche sur le Cancer". P.H. was funded by the "Ligue contre le cancer". K.B. was funded by "Fondation pour la Recherche Médicale" and "Fondation ARC pour la Recherche sur le Cancer". This work was supported by funding from the European Community's Seventh Framework Programme (FP7/2007-2013) n°304810 -RAIDs (to A.B., E.D. and M.M.), Inserm, "Fondation ARC pour la recherche sur le cancer" (Projets Fondation ARC to M.M. and A.B.) and the "Ligue contre le cancer" (to A.B.).

Supplementary Figure S1: (A)

Tumor growth was monitored by bioluminescence imaging in C57Bl/6 wild-type (WT, Ccr2 +/+ ) or Ccr2 KO (Ccr2 -/-) mice locally irradiated or not at 7.5Gy (n=8-15 mice per group, two-way ANOVA with Sidak's multiple comparisons test was performed, **: p<0.01). (B) Flow cytometry dot plots show the gating strategy to discriminate tumor myeloid subsets. (C) Confocal analysis of tumor cryosections of tdTomato TC1 tumor-bearing Ccr2 +/+ x MacBlue and Ccr2 -/-x MacBlue mice showing the distribution of monocytes/macrophages (ECFP, light blue) in the tumor core and at the periphery. S2: (A) The CCL2 levels in the sera of Ccl2 KO (Ccl2 -/-) mice engrafted with TC1/Luc tumor cells were evaluated by ELISA 3 days after 7.5Gy irradiation. The Mann-Whitney test was performed (n=3-10 mice/group from two independent experiments). (B) Ccl2 -/-TC1/Luc cells generated by CRISPR-Cas9 were engrafted into Ccl2 +/+ or Ccl2 -/-mice. The CCL2 serum levels were analyzed by ELISA 3 days after RT. Two-way ANOVA with Sidak's multiple comparisons test was performed (n=3-10 mice/group from two independent experiments). (C) qPCR analysis of CCL2 mRNA in tumors from WT mice at different time points after radiotherapy. Two-way ANOVA with Sidak's multiple comparisons test was performed (n=3-4 mice/group). (D) TC1/Luc and Raw 264.7 cells were mock-treated (NI=nonirradiated) or irradiated at 7.5Gy (IR) and then co-cultured in transwell chambers for 48h; RNA was then extracted for qPCR analysis (-= no cells). The Kruskal-Wallis test with Dunn's multiple comparisons test was performed (data are from three replicates and three independent experiments). For all panels: *: p<0.05; **: p<0.01.

Supplementary Figure

Supplementary Figure S3: (A)

The expression of CTLA4 on the surface of Tregs in tumors from WT mice treated with RT and/or anti-TNFα was quantified by flow cytometry. (B) Absolute numbers of Tconvs CD4 + (left panel) and CD8 + cells (right panel) per mg in tumors from WT mice treated with RT and/or anti-TNFα. One-way ANOVA with Sidak's multiple comparisons test was performed for all panels (n=6-10 mice/group from two independent experiments). For all panels: *: p<0.05; **: p<0.01; ***: p<0.001. S4: (A) Quantification of the percentages of Tregs and their mean fluorescence intensities (MFIs) of CD25 expression in the tumors of irradiated wild-type mice treated with anti-CD25 or a control isotype. Unpaired Student's t-test was performed (n=11-18 mice/group from three independent experiments). *: p<0.05. (B) Tumor growth was monitored by bioluminescence imaging in C57Bl/6 wild-type mice locally irradiated or not at 7.5Gy (n=3-6 mice per group) and treated with anti-CD25 or control IgG (Ctrl). Two-way ANOVA with Sidak's multiple comparisons test was performed). (C) Kaplan-Meier survival curves for mock-or radiotherapy (RT)-treated mice. The logrank test was performed (n=3-6 mice/group) Supplementary movie 1: Intravital biphoton microscopy analysis of explanted TC1/Luc tumors from MacBlue/Foxp3-EGFP mice was performed at 5 days after radiotherapy. Time-lapse TPLSM video sequence from a representative field showing multiple interactions between Tregs and monocytes.

Supplementary Figure