Mohand-Tayeb T Boudjiet

Johan Bertrand

Fabrice Mathieu

Liviu Nicu

Laurent Mazenq

Thierry Leichle

Stephen M Heinrich

Claude Pellet

Isabelle Dufour

T Leïchlé

Geometry optimization of uncoated silicon microcantilever-based gas density sensors

Keywords: Density sensor, hydrogen sensor, microcantilever, geometry optimization, sensitivity optimization, Euler-Bernoulli beam theory, hydrodynamic function

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

In recent years microcantilever-based chemical, biological and physical sensors have attracted the interest of numerous researchers due to their high surface-to-volume ratio and their high performance in both gas and liquid phases [START_REF] Vančura | Liquid-phase chemical and biochemical detection using fully integrated magnetically actuated complementary metal oxide semiconductor resonant cantilever sensor systems[END_REF][START_REF] Goeders | Microcantilevers: Sensing chemical interaction via mechanical motion[END_REF][START_REF] Boisen | Cantilever-like micromechanical sensors[END_REF][START_REF] Johnson | Biosensing using dynamic-mode cantilever sensors: a review[END_REF][START_REF] Li | Integrated microcantilevers for high-resolution sensing and probing[END_REF][START_REF] Zhu | Microcantilever sensors in biological and chemical detections[END_REF]. For chemical and biochemical sensing applications, the microcantilevers are usually coated with a sensitive layer whose purpose is to selectively sorb the analyte of interest, resulting in either a static deflection (bilayer effect) in the static mode or a shift in the resonant frequency (mass effect) in the dynamic flexural mode. Furthermore, it has been demonstrated that uncoated micro-or millimeter size cantilevers operated in the dynamic flexural mode exhibit good sensitivities to gas mass density [START_REF] Rosario | Piezoelectric excited millimeter sized cantilever sensors for measuring gas density changes[END_REF][START_REF] Tétin | Modeling and performance of uncoated microcantilever-based chemical sensors[END_REF], liquid mass density [START_REF] Zhao | In-situ measurement of fluid density rapidly using a vibrating piezoresistive microcantilever sensor without resonance occurring[END_REF] and/or viscosity [START_REF] Lemaire | High-frequency viscoelastic measurements of fluids based on microcantilever sensing: New modeling and experimental issues[END_REF][START_REF] Bircher | Real-time viscosity and mass density sensors requiring microliter sample volume based on nanomechanical resonators[END_REF].

With a view towards chemical detection in gas media, the variation of the gas density can reflect the variation of a chemical species concentration in a gas mixture [START_REF] Tétin | Modeling and performance of uncoated microcantilever-based chemical sensors[END_REF][START_REF] Boudjiet | Preliminary results of the feasibility of hydrogen detection by the use of uncoated silicon microcantilever-based sensors[END_REF][START_REF] Kramer | High-precision density sensor for concentration monitoring of binary gas mixtures[END_REF][START_REF] Sparks | A MEMS-based low pressure, light gas density and binary concentration sensor[END_REF]. The operating principle of an uncoated silicon microcantilever (USMC) used as a density or chemical sensor is based on the influence of the mass of the fluid moved by the vibrating cantilever on the resonant frequency. In fact, when the surrounding fluid mass density increases (decreases), the equivalent effective mass of the cantilever increases (decreases), thereby causing the resonant frequency to decrease (increase) [START_REF] Boudjiet | Preliminary results of the feasibility of hydrogen detection by the use of uncoated silicon microcantilever-based sensors[END_REF].

The absence of a coating eliminates or significantly reduces several problems associated with microcantileverbased sensors such as long-time response, drift and aging effects. However, uncoated microcantilevers are nonselective and offer very low sensitivities, making it quite challenging to detect small concentration changes (small density changes). This last point serves as the motivation to increase the sensor sensitivity through geometry optimization.

In the literature geometry optimization has already been reported for other particular cases of chemical detectors. For example, in the static bending mode, A. Loui et al. [START_REF] Loui | The effect of piezoresistive microcantilever geometry on cantilever sensitivity during surface stress chemical sensing[END_REF] have studied the influence of the length-to-width aspect ratio on the sensitivity of rectangular cantilevers due to both surface stress and an end-force loading. They have found that structures with a low aspect ratio are better for surface-stress applications and structures with high aspect ratio are optimal for point-loading scenarios. In the case of dynamic mode operation, the cantilever mass sensitivity is proportional to its resonant frequency. The resonant frequency is proportional to the square root of the stiffness and inversely proportional to the square root of the effective mass. Therefore, the majority of the studies conducted in order to improve the mass sensitivity of cantilevers are focusing on increasing the stiffness (k) and/or decreasing the effective mass (m eff ) using different methods. H. Hocheng et al. [START_REF] Hocheng | Shape effects of micromechanical cantilever sensor[END_REF] have demonstrated using different microcantilever shapes that the higher the structural stiffness is, the better the sensitivity is. Similarly, S. Subramanian et al. [START_REF] Subramanian | Improved V-shaped microcantilever width profile for sensing applications[END_REF] suggested the use of a nonlinear width profile for V-shaped microcantilevers in order to increase the structural stiffness and subsequently the mass sensitivity. For biosensing applications and in order to improve the overall (static-mode and dynamic-mode) sensitivity of a microcantilever, as measured by the product of static deflection and resonant frequency, M.Z. Ansari et al. [START_REF] Ansari | Deflection, frequency, and stress characteristics of rectangular, triangular, and step profile microcantilevers for biosensors[END_REF] proposed using a non-uniform cantilever cross-section (giving increased k and decreased m eff ) and reducing the fixed-end area (increasing the static deflection). The authors suggested triangular or step cross-section profiles instead the conventional rectangular one. Another solution, proposed by M. Narducci et al. [START_REF] Narducci | Sensitivity improvement of a microcantilever based mass sensor[END_REF], consisted of reducing the microcantilever size (increasing k and reducing m eff ) and/or using higher-order modes. In the case of end-mass loading, S. Morshed et al. [START_REF] Morshed | Enhancing the sensitivity of microcantilever-based sensors via geometry modification[END_REF] have demonstrated via simulation studies that structures with high aspect ratio (length-to-width) are more sensitive to local end-mass variation; furthermore, they have suggested the use of a triangular microcantilever shape to enhance the stiffness and minimize the effective mass at the freeend of the structure. Furthermore, to enhance the capabilities of microcantilevers in liquid media, L.A. Beardslee et al. [START_REF] Beardslee | Geometrical considerations for the design of liquid-phase biochemical sensors using a cantilever's fundamental in-plane mode[END_REF] have studied the influence of the beam geometry on both the quality factor and the resonant frequency in a liquid medium (water) in order to limit the viscous damping effect, thus improving the detection limit of chemical sensing. The authors reported that the use of the in-plane bending mode reduces the damping and the mass loading due to the surrounding fluid, and that beams that are wide, thin and short and operated in the inplane mode are more suitable for liquid-phase chemical detection.

As reported above, the resonant frequency is a key parameter in determining the cantilever mass sensitivity and all researches are focusing on enhancing this parameter. The microcantilever operating in fluidic (gas or liquid) environments interacts with the surrounding fluid which causes a distributed mass depending on the fluid properties, frequency and cantilever width [START_REF] Sader | Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope[END_REF]. Thus, although the resonant frequency is an important parameter to mass density sensing, the structure's geometry and dimensions play an important role in the mass density sensitivity of microstructures. In the present work we study the effect of microcantilever shape (rectangular, Uand T-shaped microstructures) and geometrical dimensions on the gas mass density sensitivity (i.e., the ratio of resonant frequency variation to the density variation). To perform this study several uncoated silicon microcantilever shapes with different dimensions have been designed and fabricated. The structures have been tested at room conditions using different concentrations (0.2-2.0%) of hydrogen (H 2 ) in nitrogen (N 2 ). The density changes have been measured by monitoring the eigenfrequency (natural frequency) variation using the efficient phase linearization method [START_REF] Boudjiet | New characterization methods for monitoring small resonant frequency variation: Experimental tests in the case of hydrogen detection with uncoated silicon microcantilever-based sensors[END_REF].

Modeling

The Euler-Bernoulli equation taking into account the hydrodynamic force acting on the uncoated microcantilever is commonly used to model the behavior of resonating microcantilevers in fluid media when the influence of the beam's shearing deformation and rotational inertia can be neglected [START_REF] Tétin | Modeling and performance of uncoated microcantilever-based chemical sensors[END_REF][START_REF] Boudjiet | Preliminary results of the feasibility of hydrogen detection by the use of uncoated silicon microcantilever-based sensors[END_REF]. Figure 1 displays the out-of-plane cantilever flexural mode (w is the free-end transverse deflection) and the geometric parameters: length (L), width (b) and thickness (h). In this work, the Euler-Bernoulli model in a fluid medium is used to characterize the changes in structural behavior due to the gas density variation. This model is valid only when [START_REF] Sader | Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope[END_REF] • The beam has a uniform cross-section (geometry and materials) along the structure.

• The cross-sectional dimensions are negligible compared to the length of the structure: h << b << L.

• The deflection is negligible compared to the structural dimensions: w << h.

The solution of the differential equation governing the cantilever's motion in the presence of a surrounding fluid gives [START_REF] Sader | Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope[END_REF][START_REF] Maali | Hydrodynamics of oscillating atomic force microscopy cantilevers in viscous fluids[END_REF]:

00 0 00 8 ff ,vac f d df df b a ff h      (1)
with

0 21 2 n ,vac ²h E f L²    (2)
being the natural frequency in vacuum and f 0 the in-fluid natural frequency. Symbols E and  denote the Young's modulus and density of silicon,  f is the fluid density,  n is a coefficient depending on the eigenfrequency mode [START_REF] Blevins | Formulas for natural frequency and mode shape[END_REF] ( 1 =1.875,  2 =4.695,  3 =7.854 …), and a 0 =1.0553. The latter parameter is associated with an approximation introduced by A. Maali et al. [START_REF] Maali | Hydrodynamics of oscillating atomic force microscopy cantilevers in viscous fluids[END_REF].

The absolute density sensitivity of resonating sensors is defined as the ratio between the resonant frequency variation and the density variation. Based on Eqs. (1)-( 2), the absolute sensitivity can be written as 00 [START_REF] Hocheng | Shape effects of micromechanical cantilever sensor[END_REF] 12

n a f fa ² bE S L²       (3)
According to Eq.( 3), it can be observed that the sensitivity of a rectangular beam, based on the Euler-Bernoulli assumption, depends only on two geometric parameters --width (b) and length (L) --and does not depend on the thickness (h). This result shows that changes in sensitivity need not be accompanied by changes in the resonant frequency. For example, increasing the width increases the sensitivity but doesn't affect the resonant frequency.

It is expected from this equation that shorter and wider cantilevers will yield higher values of absolute sensitivity S a . Of course, the range of applicability of Eq. ( 3) is limited to that of the Euler-Bernoulli theory on which it is based. Thus, an extreme reduction in length L would need to be accompanied by a corresponding reduction in thickness h in order to maintain the validity of Eq. (3); otherwise, the effects shear deformation and rotational inertia, neglected in elementary beam theory, will become important. Such a reduction in h will also serve a more practical purpose: the use of shorter and wider cantilevers, which by Eq. (3) will improve the absolute sensitivity, will eventually become too stiff to render a measurable signal; reducing h will help to delay the onset of this practical limit to the applicability of Eq. (3).

Experiments

In order to experimentally study the optimization of the sensor sensitivity, several microcantilevers with different geometries (rectangular-, U-and T-shaped microstructures) and dimensions (L, b and h) have been fabricated (Fig. 2b and Table 1) with electromagnetic actuation and piezoresistive read-out (Fig. 2a).

Actuation and read-out systems

To actuate the cantilevers (Fig. 2a), an AC current is passed through the conductive loop wire placed along the cantilever periphery. In the presence of a magnetic field collinear to the longitudinal axis of the beam, an AC Lorenz force is created at the microcantilever free-end and induces out-of-plane vibrations (Fig. 1).

Semiconductor strain gauges which are boron-doped piezoresistors have been fabricated during the process in order to read-out the vibrations. They are arranged in a half Wheatstone bridge configuration: a first gauge is located at the clamped-end of the beam where the strain is maximum and the second one is on the rigid substrate.

Microcantilever fabrication

The main steps of the fabrication process are as follows. The starting substrate was a 100 mm-diameter, <100>, N-type Silicon-On-Insulator (SOI) wafer, with a 1 μm-thick buried oxide and a 5 μm-thick (or 10 µm-thick) top silicon layer (resistivity of 4-6 Ω.cm). The use of the SOI wafer enabled the precise control of the cantilever thickness, ensuring the consistency of their mechanical properties. The first step consisted of creating the piezoresistor in the bulk silicon. In order to optimize the piezoresistor sensitivity, the cantilevers were patterned along crystal axes for which the longitudinal piezoresistive coefficient is maximum, i.e., along the <110> direction in the case of a p-silicon piezoresistor. The fabrication method relied on the implantation of germanium (Ge) and boron fluorine (BF 3 ) in order to obtain an ultrathin piezoresistor [START_REF] Bergaud | Formation of implanted piezoresistors under 100-nm thick for nanoelectromechanical systems[END_REF]. The localization of the piezoresistive layer at the anchored edge of the cantilevers was achieved by using silicon dioxide as a masking layer. For that purpose, 300 nm of silicon dioxide was thermally grown and patterned with a photolithographic step. Germanium was implanted with an energy of 60 keV and a dose of 5*10 14 ions/cm 2 through a 6-nm silicon dioxide layer to create a preamorphized layer. This layer avoided channeling effects during the boron implantation and led to a very thin doped region. Boron fluorine was then implanted with an energy of 15 keV and a dose of 1*10 16 ions/cm 2 . Owing to the heavier mass of the BF 3 molecules relative to boron, the use of BF 3 resulted in a thickness reduction of the p + -doped region. The implantation process was followed by rapid thermal annealing at 1000°C for 15 s to minimize boron diffusion during the recrystallization of the amorphized layer and the electrical activation. This was followed by conventional annealing at 850°C during 20 min. The next step consisted of the deposition of 200 nm of Plasma Enhanced Chemical Vapor Deposition (PECVD) silicon dioxide on the entire SOI wafer before the sputtering of Aluminum (Al) (500 nm) for the electrode used for electromagnetic actuation. The oxide prevented shortcircuiting between the piezoresistors and the actuation electrodes. Lift-off of the Al film was achieved by using an AZ nLOF negative photoresist to define the electrodes. A passivation silicon oxide film (200 nm thick) was then deposited by PECVD. Contact pads were opened by dry etching of PECVD-deposited oxide. To finish, the microcantilever shapes were defined by a front Reactive Ion Etching of silicon, followed by vertical sidewalls etching on the backside of the SOI wafer using the Deep Reactive Ion Etching technique to release the structures. The 1 μm-thick SiO 2 acted as an etch stop layer for the dry silicon etching. This layer was then removed by Reactive Ion Etching. The resulting microstructural shapes, dimensions and surface areas are reported in Table 1.

Experimental setup

With the aim of comparing the different microcantilever geometries and dimensions in terms of sensitivity to density variation, measurements of various H 2 in N 2 concentrations (0.2, 0.6, 1 and 2%) have been performed using a gas line with a flow of 100 ml/min. The hermetic cell containing the tested microcantilever had a volume of 500 µl. A gain/phase analyzer (HP4194A) controlled with a LabVIEW program was used to acquire the phase spectrum every nine seconds (1 acquisition/9s). In order to measure the natural frequency variation (f 0 ), a linearization of the phase spectrum around the resonance was used to extract the natural frequency (f 0 ) as detailed in [START_REF] Boudjiet | New characterization methods for monitoring small resonant frequency variation: Experimental tests in the case of hydrogen detection with uncoated silicon microcantilever-based sensors[END_REF].

The study of both the geometry and the size influence on the sensor sensitivity was performed with tests using the first resonant mode of each structure. The first step of the study consisted of determining the structural shape influence (rectangular-, U-or T-shaped) by comparing the sensitivities of the structures A2_5μ, T1_5μ, T2_5μ, U1_5μ and U2_5μ , all having the same total length (L), total width (b) and thickness (5μm). The second step involved an investigation of the influence of the dimensions (L, b and h) on the sensitivity for the best sensitive shape (geometry) revealed by the results of the first step. An example of the measurement performed for each microstructure of Table 1 is presented in Figure 3. The optimization was performed by consideration of the absolute sensitivity defined in Eq. (3). Other sensor characteristics and performance metrics were also determined using the hydrogen detection measurements:  The sensor noise (Noise) has been estimated by the standard deviation on the stabilized natural frequency (f 0 ≈0)  The signal-to-noise ratio (SNR) and the eigenfrequency variation (f 0 ) have been calculated at 1% of H 2 in N 2 .  The limit of detection (LOD) has been estimated: it corresponds to a signal-to-noise ratio equal to 3.  The absolute sensitivity is the slope of the fitted line of the experimental measurements.

Results and discussion

Experiment set #1: influence of the shape

This first experiment set was used for the first step of the sensitivity study consisting of the examination of the shape influence (rectangular-, T-or U-shaped structure) on the sensitivity. Figures 4a and4b show the experimental measurements (markers) and the fitting lines of the first-mode natural frequency variation as a function of H 2 in N 2 concentration and density variation, respectively. The A2_5μ structure has the highest sensitivity (slope) among all of the other structures considered, namely U1_5μ, U2_5μ, T1_5μ and T2_5μ. The performance metrics are presented in Fig. 4c and the numerical results are reported in Table 3.

This preliminary result allows us to conclude that for structures having the same total length (L), the same total width (b) and the same thickness (h), structures having more contact area (Tab.1) with the surrounding fluid have the best absolute sensitivity (S a ) to the density variations of the surrounding gas (Fig. 4b and Tab.3). This is a simplistic conclusion because structures having more contact area and keeping the same total length (L), total width (b) and total thickness (h), also have higher resonant frequency. According to these first experiments, we can conclude that the rectangular shape (A2_5µ in these experiments) is the best geometry in terms of sensitivity for gas density sensing applications. It can be seen in Figure 4c that the A2_5µ structure also has the best quality factor (Q) and the best signal-to-noise ratio (SNR) and, thus, the best limit of detection (LOD= 100 ppm or 0.11mg/l).

Experiment set #2: influence of the dimensions

The previous experiments have shown that, for the same overall dimensions, the rectangular structures are the best geometries in terms of sensitivity to the gas density changes. In this section we therefore study the effect of the geometric parameters (L, b and h) on the sensitivity of rectangular structures by analyzing the structural responses to the different concentrations of H 2 in N 2 .

Thickness influence (a) (b) (c)

The structures A2_5μ and A2_10μ have the same length and width (L, b) with thicknesses (h) of 5µm and 10µm, respectively. The detection curves of both structures are presented in Figs. 5a-b where it can be seen that thickness has no effect on the absolute sensitivity (S a ) (red triangle and green diamond markers). The fact that the absolute sensitivity is independent on the thickness is consistent with Eq. (3). Furthermore, doubling the thickness doubles the quality factor as can be seen in Table 4.

Length and width influence

Keeping in mind that the primary goal is to identify the best structure in terms of sensitivity to gas density, this part has three objectives:  Objective 1 consists of comparing the geometries A2_5μ and A3_5μ in terms of their absolute sensitivity (S a ). These structures have been selected because they have the same widths and thicknesses, but A3_5µ is two times longer than A2_5µ.  Objective 2 involves the sensitivity comparison between the geometries A1_5μ and A0_5μ having the same lengths and thicknesses, but A1_5µ is two times wider than A0_5µ.  Objective 3 is to compare sensitivities of the two square geometries A1_5μ and A2_5μ having the same thickness, but the second structure has length and width two times larger than the first one.

The structural dimensions are reported in Table 1. Figure 5 summarizes the detection results obtained with the different geometries and Table 4 reports the numerical characteristics and performance metrics of the different sensors. Performing in order the three comparisons mentioned above, the experimental results demonstrate the following:

 For a fixed width, the shorter beams have higher absolute sensitivities.  For a fixed length, the wider beams have higher absolute sensitivities.  When the width and length are both halved, the structure's sensitivity is increased by a factor of approximately two.

These points confirm the scale effects indicated by Eq. (3), which shows that the absolute sensitivity is proportional to the ratio of width to the square of the length (b/L 2 ). We note that all structures (experiment sets #1 and #2) detect 0.2% of H 2 in N 2 , corresponding to 2 mg/l as density variation. Furthermore, we announce a theoretical detection limit (LOD) of 0.01% (100 ppm) of H 2 in N 2 corresponding to 0.11 mg/l as density variation for the A2_5µ structure, which is 800 times smaller than the published value of R. Rosario et al. [START_REF] Rosario | Piezoelectric excited millimeter sized cantilever sensors for measuring gas density changes[END_REF]. We also note that this limit of detection can be improved considerably by increasing the actuation force.

Theory vs. experiment

In the literature there are no theoretical models that permit one to accurately predict the resonant frequency variation due to fluid density change for the case of complex geometries such as T-shaped, U-shaped and Vshaped cantilevers. The only existing model concerns parallelepiped-cantilever (rectangular) geometries that respect the Euler-Bernoulli conditions listed in Sect. 2. Therefore, only rectangular-shaped microcantilevers are considered for this comparison although their dimensions do not always respect the Euler-Bernoulli conditions.

In Fig. 6 comparisons are made between the model [Eqs. (1)-( 2)] and measurements of natural frequency (f 0 ) and natural frequency variation (f 0 ) at 1% of H 2 in N 2 .

We observe in Fig. 6a that the accuracy of the theoretical model in terms of f 0 estimation is satisfactory with the exception of the A0_5µ structure having a relative error of 25%. This error is most likely due to the structure etching defects [START_REF] Fadel-Taris | Influence of non-ideal clamping in microcantilever resonant frequency estimation[END_REF] which have substantially modified the length of the cantilever (reduction). The evidence is provided by the fact that the structure A3_5µ, having exactly the same shape (L=2b) as the A0_5µ structure but with a size four times larger (Table 1), shows good agreement between measurement and theory.

(c)

It can be observed in Fig. 6b that the relative deviation between model and measurement for the frequency shift estimation is about 50% for all structures except A3_5µ. These relatively high deviations are due to the fact that the microcantilevers don't respect the Euler-Bernoulli conditions; thus, the fluid-structure interaction model proposed by E. Sader et al. [START_REF] Sader | Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope[END_REF] is not expected to give a good approximation. The A3_5µ structure is two times longer than its width (L=2b) and therefore has a smaller relative deviation (31%) than the other cases since the model is expected to be more applicable for longer geometries. The A0_5µ structure has a larger error (46%) than A3_5µ (31%) for the same reason mentioned in the previous paragraph. In order to verify the effect of geometry in modeling accuracy, another measurement has been realized at the same room conditions (temperature =23°, pressure=1 atm) and gas conditions (H 2 -N 2 : 0.2-2%, gas flow = 100 ml/min) using another microcantilever (L x b x h= 2000 x 400 x 5µm 3 ) having a length five times greater than its width (L=5xb).

The frequency-shift results of this experiment are reported in Fig. 7 from which we see that the relative deviation between the model and the measurement for 1% of H 2 in N 2 is 3.12%. This result confirms that the Euler-Bernoulli conditions must be respected in order to achieve a good estimate using Sader's model [START_REF] Sader | Frequency response of cantilever beams immersed in viscous fluids with applications to the atomic force microscope[END_REF] (or any model based on elementary beam theory). We also remark that the error increases for lower concentrations (0.6% and 0.2% in Fig. 7). The reason is the relatively large frequency step between each measurement caused by the relatively large span measurement in this case (20Hz). In fact, the gain-phase analyzer (HP4194A) has a maximum of 400 measurement points; thus, configuring a span measurement of 20 Hz for the acquisition of the gain and phase spectra, a 50-mHz frequency step is achieved. However, using the phase linearization method [START_REF] Boudjiet | New characterization methods for monitoring small resonant frequency variation: Experimental tests in the case of hydrogen detection with uncoated silicon microcantilever-based sensors[END_REF], the resonant frequency can be estimated with a better accuracy than the frequency step.

Conclusions

We have demonstrated that uniform rectangular cantilevers are more suitable for density measurement than the other tested T-and U-shapes. Moreover, wide and short beams are more sensitive to the density variation, with the sensitivity of the rectangular beams being proportional to b/L 2 . Furthermore, the thickness doesn't affect the sensitivity of rectangular cantilevers to the mass density changes despite the fact that it affects the resonant frequency. However, the noise on the resonant frequency estimation depends on the thickness of the microcantilever. Thus, in order to select the most appropriate thickness for a given structure size (length and width) in view of limit-of-detection optimization, noise consideration has to be studied.

We have also modeled the fluid-structure interaction using the Euler-Bernoulli beam model combined with the hydrodynamic force. This modeling shows good agreement with measurements when the rectangular structures are narrow and sufficiently long (length 5 x width in our case). These results may help designers to optimize the cantilever geometry and dimensions in order to be more or less sensitive to the gas density variation depending on the application.

Moreover, due to the implementation of the phase linearization method [START_REF] Boudjiet | New characterization methods for monitoring small resonant frequency variation: Experimental tests in the case of hydrogen detection with uncoated silicon microcantilever-based sensors[END_REF] used to estimate the small natural frequency variation, detection of 2 mg/l of the gas mass density change has been achieved with all the microstructures and a limit of detection of 0.11 mg/l of the gas mass density variation (corresponding to a concentration of 100 ppm of H 2 in N 2 ) has been estimated, which is much smaller than the previous state-of-theart value of 88mg/l [START_REF] Rosario | Piezoelectric excited millimeter sized cantilever sensors for measuring gas density changes[END_REF].

Fig. 1 :

 1 Fig.1 : Schematic representation of both the microcantilever geometry and the transverse bending deflection (w). The beam dimensions are width (b), length (L) and thickness (h).

Fig. 2 :

 2 Fig.2 : (a) Uncoated silicon microcantilever design: (1) Printed Circuit Board, (2) adhesive, (3) silicon, (4) constant magnetic field, (5) metal, (6) AC current, (7) piezoresistor and (8) AC Lorentz force. (b) Different microcantilever geometries (rectangular-, U-and T-shaped) and geometric parameters: total length 'L', total width 'b', thickness 'h', leg length 'L leg ' and leg width 'b leg '.

Fig. 3 :

 3 Fig. 3 : Example of detection curve obtained using A2_5µ structure with different concentrations of H 2 in N 2 with a gas flow of 100 ml/min at room conditions (Temperature≈23°C, pressure≈1 atm).

Fig. 4 :

 4 Fig. 4 : Results of the first experiments. (a) Natural frequency variation as function of H 2 in N 2 concentration (0, 0.2, 0.6, 1 and 2%). (b) Natural frequency variation as function of the density variation of the H 2 -N 2 gas mixture. (c) Performance of structures A2_5µ, U1_5µ, U2_5µ, T1_5µ and T2_5µ in terms of natural frequency (f 0 ), quality factor (Q), natural frequency variation (f 0 ) at 1% of H 2 in N 2 , noise (Noise) estimated by the calculation of the standard deviation, signal-to-noise ratio (SNR) calculated at 1% of H 2 in N 2 , absolute sensitivity (S a ) which is the slope of the linear characteristics and limit of detection (LOD) in terms of H 2 in N 2 concentration (%) and density variation (mg.l -1 ).

Fig. 5 :

 5 Fig. 5 : Results of the second set of experiments. (a) Natural frequency variation as function of H 2 in N 2 concentration (0, 0.2, 0.6, 1 and 2%). (b) Natural frequency variation as function of the density variation of the H 2 -N 2 gas mixture. (c) Performance of structures A1_5µ, A0_5µ, A2_10µ, A2_5µ and A3_5µ in terms of: natural frequency (f 0 ), quality factor (Q),natural frequency variation (f 0 ) at 1% of H 2 in N 2 , noise (Noise) estimated by the calculation of the standard deviation, signal-to-noise ratio (SNR) calculated at 1% of H 2 in N 2 , absolute sensitivity (S a ) which is the slope of the fitted straight lines and limits of detection (LOD) in terms of H 2 in N 2 concentration (%) and density variation (mg.l -1 ).

Fig. 6 :

 6 Fig. 6 : Comparison between model and measurements at 1% of H 2 in N 2 . (a) Natural frequency (f 0 ). (b) Natural frequency variation (f 0 ).

Fig. 7 :

 7 Fig. 7 : Measurement of concentration of H 2 in N 2 (0.2-2%) with gas flow of 100 ml/min using rectangular-shaped microcantilever (L x b x h= 2000 x 400 x 5µm 3 ). The triangle markers present experimental measurements, the blue line presents the theoretical modeling [Eq. (3)] and the red numerical values present the relative deviation between model and measurements.

Table 1 :

 1 Microcantilever dimensions and surface areas. Extensions "_5µ" and "_10µ" indicate the specimen thickness in µm.

			[mm]		[µm]		[mm²]
	Specimen L	b	h L Leg b Leg Surface
	U1_5µ	1	1	5 666 250	0.670
	U2_5µ	1	1	5 333 250	0.830
	T1_5µ	1	1	5 500 200	0.600
	T2_5µ	1	1	5 250 200	0.800
	A0_5µ	0.5 0.25 5	-	-	0.125
	A1_5µ	0.5 0.5 5	-	-	0.250
	A2_5µ	1	1	5	-	-	1.000
	A2_10µ	1	1	10	-	-	1.000
	A3_5µ	2	1	5	-	-	2.000
	Concentrations of
	H

A gas line

[START_REF] Boudjiet | New characterization methods for monitoring small resonant frequency variation: Experimental tests in the case of hydrogen detection with uncoated silicon microcantilever-based sensors[END_REF]

has been used to generate different concentrations of H 2 in N 2 and to control the gas mixture flow. The different gas densities ( H2-N2 ), gas density variations ( H2-N2 ) and relative gas density variations ( f / f ) of the gas fluid (H 2 -N 2 ) corresponding to different concentrations of H 2 in N 2 are reported in Table

2

.

Table. 2 : Densities ( H2-N2

), density variations ( H2-N2 ) and relative density variations ( f / f ) corresponding to different concentrations of H 2 /N 2 gas mixture. These values are calculated at room conditions (23°C and 1.01325 bar).

2 in N 2 (%)  H2-N2 (kg.m -3 )  H2-N2 (kg.m -3 or g/l)  f / f =  H2-N2 / N2 (%)

	100	0.0830	-1.0703	-93
	2	1.1314	-0.0214	-1.8
	1	1.1421	-0.0107	-0.93
	0.6	1.1464	-0.0064	-0.56
	0.2	1.1506	-0.0021	-0.19
	0	1.1533	0	0

Table . 3

 .

: Numerical values of performance metrics extracted from the experiments made with A2_5µ, U2_5µ, U1_5µ, T2_5µ and T1_5µ structures.

Specimen f 0 (Hz) Q f 0 (Hz) Noise (mHz) SNR S a (Hz.kg -1 .m 3 ) LOD (%) LOD (mg.l -1 ) A2_5µ

		7082 120 1.28	4.43	289	117	0.01	0.11
	U2_5µ	4580 94 0.80	4.1	195	73	0.015	0.16
	U1_5µ	4624 90 0.58	3.6	161	53	0.019	0.20
	T2_5µ	3480 92 0.56	3.80	147	53	0.02	0.22
	T1_5µ	3431 90 0.45	3	150	42	0.02	0.21

Table . 4

 . : Numerical performance values extracted from the experiments made with A0_5µ, A1_5µ, A2_5µ, A0_10µ and A3_5µ structures.

	Specimens	f 0 (Hz)	Q	f 0 (Hz)	Noise (mHz)	SNR	S a (Hz.kg -1 .m 3 )	LOD (%)	LOD (mg.l -1 )
	A0_5µ	41700 570 1.40 22.83	61	145	0.049	0.52
	A1_5µ	27099 250 2.50 12.60	198	228	0.015	0.16
	A2_5µ	7082 120 1.28	4.43	289	117	0.010	0.11
	A2_10µ	11260 240 1.38	9.55	145	123	0.021	0.22
	A3_5µ	1760	60 0.43	6.77	64	39	0.047	0.51

Acknowledgment

The authors gratefully acknowledge the French National Radioactive Waste Management Agency (http://www.andra.fr/) for their support of this research.

Biographies

Mohand-Tayeb Boudjiet received in 2012 the Master's degree in embedded electronics systems from University of Bordeaux in France. He is currently a Ph.D. student at IMS Laboratory working on microcantilevers for density measurement and chemical detection.

Johan


Louis Pérol

PhD Gaëlle H Martin

Sébastien Maury

PhD José L Cohen
email: jose.cohen@inserm.fr

PhD Eliane Piaggio
email: eliane.piaggio@yahoo.com

Potential limitations of IL-2 administration for the treatment of experimental acute graft-versus-host disease

Keywords: IL-2, bone marrow transplantation, graft-vs-host disease, regulatory T cell, immunotherapy BMT, Interleukin-2, GVHD, xeno-GVHD, regulatory T cell, immunotherapy

Low-dose IL-2 administration can control autoimmunity by specifically activating CD4+FoxP3+ regulatory T cells (Tregs). Here, we studied IL-2-based immunotherapy in experimental graft-versus-host disease (GVHD). IL-2 administration to donor mice induced a dose-dependent expansion of Tregs in the graft but was insufficient to control GVHD. IL-2 administration to allogeneic-grafted recipient mice activated Tconventional cells (Tcons) and did not prevent GVHD. This loss of IL-2 selectivity towards Tregs was explained by an IL-2-induced increase in the IL-2 receptor αchain expression on Tcons. Finally, in xeno-GVHD generated by human PBMCs transplanted into immunodeficient mice, low-dose IL-2 increased Treg frequencies but did neither control pro-inflammatory cytokine production by pathogenic Tcons, nor prevented GVHD. Furthermore, combination of low-dose IL-2 with rapamycin was ineffective in this model. Our results indicate that limitations on the use of IL-2 during acute GVHD are likely due to the massive activation of the allogeneic T cells unique to this setting.

INTRODUCTION

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is the treatment of choice for many hematological malignancies and primary immunodeficiencies. The efficacy of such therapy is compromised by the presence of allogeneic donor mature T cells within the graft, which are responsible for a life threatening complication: graftversus-host disease (GVHD) [START_REF] Ferrara | Graft-versushost disease[END_REF]. It is now assumed that thymus-derived regulatory T cells (Treg) could modulate allogeneic immune responses as demonstrated in different experimental models (for recent review see [START_REF] Juvet | Harnessing regulatory T cells for clinical use in transplantation: the end of the beginning[END_REF]). Initially, we and others have demonstrated that adding high numbers of regulatory T cells (Tregs) to the graft could prevent GVHD [START_REF] Cohen | CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graftversus-host disease[END_REF][START_REF] Hoffmann | Donor-type CD4(+)CD25(+) regulatory T cells suppress lethal acute graft-versus-host disease after allogeneic bone marrow transplantation[END_REF][5]. In particular, Tregs that were rendered specific for recipient allo-antigens [START_REF] Trenado | Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia[END_REF][START_REF] Gaidot | Immune reconstitution is preserved in hematopoietic stem cell transplantation coadministered with regulatory T cells for GVHD prevention[END_REF] or exogenous nominal Ag through ex-vivo activation [8] prevented GVHD while retaining the ability of donor T cells to mediate graft-versustumor effects and promote immune reconstitution. However, despite the fact that the preparation of clinical grade Treg was strongly improved in the recent years, this remains a complex process, particularly for the generation of antigen specific Treg [START_REF] Landwehr-Kenzel | Novel GMP-compatible protocol employing an allogeneic B cell bank for clonal expansion of allospecific natural regulatory T cells[END_REF][START_REF] Putnam | Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation[END_REF]. Consequently, in vivo direct stimulation of Treg compartment could be a more accessible alternative for decreasing GVHD incidence.

Interleukin-2 (IL-2) was initially described for its capacity to stimulate T-cell proliferation in vitro [START_REF] Morgan | Selective in vitro growth of T lymphocytes from normal human bone marrows[END_REF][START_REF] Smith | Lymphocyte activating factor promotes T-cell growth factor production by cloned murine lymphoma cells[END_REF]. Consequently, IL-2 at high doses has been used clinically to stimulate anti-tumor or anti-viral immune responses in patients with metastatic melanoma, metastatic renal carcinoma and HIV, respectively [START_REF] Ahmadzadeh | IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients[END_REF][START_REF] Levy | Effects of interleukin-2 therapy combined with highly active antiretroviral therapy on immune restoration in HIV-1 infection: a randomized controlled trial[END_REF][START_REF] Rosenberg | Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2[END_REF][START_REF] Weiss | In vivo expansion of naive and activated CD4+CD25+FOXP3+ regulatory T cell populations in interleukin-2-treated HIV patients[END_REF][START_REF] Zhang | Lymphopenia and interleukin-2 therapy alter homeostasis of CD4+CD25+ regulatory T cells[END_REF].

Although these results have demonstrated a pleïotropic effect of IL-2 on immune cells, recent advances have shown that the principal non-redundant role of IL-2 is the maintenance of Treg homeostasis [START_REF] Malek | The biology of interleukin-2[END_REF]. Indeed, mice lacking functional IL-2 or IL-2 receptors develop severe autoimmunity in association with abnormal homeostasis of Tregs [START_REF] Sadlack | Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene[END_REF][START_REF] Suzuki | Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor beta[END_REF] establishing that IL-2 is essential for Treg development, proliferation and peripheral survival [START_REF] Papiernik | Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency[END_REF][START_REF] Scheffold | Regulation of CD4+CD25+ regulatory T cell activity: it takes (IL-)two to tango[END_REF]. In this line, we recently demonstrated that treatment of young non-obese diabetic (NOD) mice with low-dose IL-2 prevents [START_REF] Tang | Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[END_REF] and reverts [START_REF] Grinberg-Bleyer | IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[END_REF] type 1 diabetes (T1D) development. In this setting, IL-2 can revert established autoimmunity by acting with exquisite selectivity, expanding and activating Tregs only in the inflamed pancreas while leaving pathogenic conventional T cells (Tcons) unaffected. IL-2-based treatment was also envisaged for GVHD prophylaxis. During the 90's, pioneer work by the group of David Sachs [START_REF] Sykes | IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity[END_REF][START_REF] Sykes | In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment[END_REF][START_REF] Sykes | Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukemia effect of allogeneic T cells[END_REF] showed that 3 days of IL-2 administration could control experimental GVHD development. In contrast, Shin et al reported the lack of therapeutic effect of IL-2 on experimental GVHD [START_REF] Shin | Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells[END_REF]. In this last study, GVHD prophylaxis was only observed when IL-2 treatment was combined to rapamycin administration. It is thus to date difficult to conclude on the possibility to prevent GVHD by the sole IL-2 administration in experimental GVHD. In humans, a recent clinical trial aimed at evaluating the effect of ultra low dose (ULD) IL-2 for GVHD prophylaxis after allo-HCT. When IL-2 treatment was combined with a standard immunosuppressive regimen, an increased proportion of Treg was observed in approximately half of patients [START_REF] Kennedy-Nasser | Ultra Low-Dose IL-2 for GVHD prophylaxis after Allogeneic HSCT mediates Expansion of Regulatory T Cells Without Diminishing Antiviral and Anti-leukemic Activity. Clinical cancer research : an official journal of the American Association for Cancer Research[END_REF]. Additionally, Ito et al. [START_REF] Ito | Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers[END_REF] recently tested the possibility to increase the Treg content in twenty-one healthy volunteer donors by administrating ultra low dose (ULD) IL-2 treatment for 5 days. They observed that ULD IL-2 induced a significant increase in the frequency and the suppressive function of Treg that was maintained until day 7. However, by showing that healthy donor could also be treated by IL-2 with minimal adverse events, the authors clearly pave the way for new clinical application for IL-2 therapy to prevent GVHD after HSCT. To directly address this issue, we tested for the first time the possibility to impact experimental GVHD by administrating low-dose IL-2 in donor mice over 5 days (as performed in the Ito's study) in order to selectively induce Treg expansion/activation. We also compared this approach with a second prophylactic strategy consisting in the direct administration of IL-2 to grafted mice in a model of acute allogeneic GVHD and in a model of xenogeneic (xeno-) GVHD consisting in the administration of human PBMCs to immunodeficient mice.

RESULTS

Low-dose IL-2-treatment of donor mice specifically increases Treg proportions

We reasoned that administration of low-dose IL-2 to immunocompetent donor mice could expand Tregs in the graft as recently showed by Ito et al. [START_REF] Ito | Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers[END_REF] in humans, and consequently prevent GVHD. Thus, we first studied the capacity of different doses of IL-2 administered over 5 days to selectively induce Treg expansion/activation in donor B6 mice (Figure 1a). Indeed, in this context of "healthy" donors (immunocompetent, unmanipulated mice), IL-2 treatment induced a dose-dependent increase in the proportion of Tregs in peripheral lymph nodes (Figure 1b-c). These expanded Tregs showed an activated phenotype, as attested by their IL-2-induced increased expression of CD25 and Foxp3 levels (Figure 1b-c). The distribution, of naïve and memory Tregs, Tconv and CD8+ T cells, as defined by the expression of CD44 and CD62L, was not significantly modified by the treatment (Figure 1d).

Low-dose IL-2-treatment of donor mice does not prevent GVHD

We then evaluated the capacity of T cells collected from IL-2-treated donor mice to induce GVHD (Figure 2a). We observed that independently of the IL-2 pre-treatment, the transferred T cells induced similar GVHD, as attested by the similar pattern of body weight loss (Figure 2b). Survival of the grafted mice was not ameliorated when grafts were obtained from IL-2 treated donors, and even tended to accelerate GVHDlinked mortality at the highest dose (Figure 2c). These results show that despite an IL-2-induced Treg enrichment in the graft, GVHD incidence in the recipient mice was not affected.

IL-2 administration to grafted mice does not affect GVHD incidence

We previously demonstrated that Treg administration could reduce the activation and expansion of donor T cells in experimental allogeneic BM transplantation [START_REF] Cohen | CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graftversus-host disease[END_REF][START_REF] Trenado | Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia[END_REF]. We therefore investigated whether IL-2 administration to recipient mice, by boosting Tregs, could reduce acute GVHD. We used a parent into F1 strain combination, which mimics the uncommon and very aggressive clinical scenario of haplomismatch SCT because: (i) this combination enables evaluation of the sole donor versus host immune response since in fully allogeneic HSCT, the anti-donor immune response mediated by residual recipient immune cells could attenuate GVHD. (ii) Our aim was also to evaluate the impact of the inflammatory setting on the IL-2 effect. This model is the sole to allow GVHD in irradiated and non-irradiated recipient mice without modifying the genetic combination between donor and recipient mice. For this, recipient B6D2F1 mice were lethally irradiated and grafted with B6 bone marrow cells along with B6 CD3+ T cells and treated daily with different IL-2 doses for 10 days (Figure 3a). We did not observe any protective effects against GVHD at any of the tested doses, nor were there any effects on survival or the body weight profile of grafted mice (Figure 3b-c). Moreover, high IL-2 doses significantly accelerated death of the grafted mice compared to untreated mice (p <0.05).

To rule out a direct toxic effect of IL-2, recipient mice were grafted only with BM cells and were treated with IL-2. In this setting, IL-2 was not lethally toxic but induced a mild delay in body weight gain only at the highest dose (Supplemental Figure 1), which could contribute to GVHD-related morbidity in semi-allogeneic BMT.

Finally, we evaluated the therapeutic capacity of delayed IL-2 administration. For this, we tested a curative IL-2 schedule beginning at day 10 or 15 after grafting, or alternatively, at detection of the first clinical signs of GVHD. As showed in Supplementary Figure 2, neither of these therapeutic approaches could control GVHD course.

IL-2 administration to grafted mice does not affect T cell reconstitution kinetics

To follow the effect of IL-2 administration on the fate of donor T cells [START_REF] Zeiser | Inhibition of CD4+CD25+ regulatory T-cell function by calcineurindependent interleukin-2 production[END_REF], we induced GVHD by infusing B6Luc+ LN T cells in lethally irradiated B6D2F1 recipient mice, treated or not with IL-2. As shown in Figure 4a, the same kinetics of donor T cell expansion measured by bioluminescence detection was observed independent of IL-2 treatment. Similarly, by flow cytometry analysis no significant differences were observed between PBS or IL-2-treated mice, neither in the frequency nor in the absolute number of Tregs or Tcons (Figure 4b-d), confirming that IL-2 treatment did not affect the kinetics of early T cell reconstitution.

During acute GVHD, IL-2 looses it selectivity for Tregs

We studied the effect of IL-2 treatment on the T cell activation status. As shown in Figure 5, IL-2 administration preferentially activated CD8+ T cells as attested by the IL-2-dose-dependent increase in the proportion of effector/memory CD8+ T cells (CD44high, CD62L-) at expenses of the naïve CD8+ T cells (CD44low, CD62L+) (Figure 5a-b). Also, at the higher IL-2 dose, an increase in the MFI of ICOS and GITR among the CD8+ Tcon population was observed (Figure 5c-d). For the CD4+ Tcons, high-dose IL-2 administration only induced a significant increase in the MFI of GITR (Figure 5d).

In experimental allogeneic BMT, we previously observed that donor T cells rapidly divide and transiently acquire CD25 expression after transfer into recipient mice [START_REF] Maury | Division rate and phenotypic differences discriminate alloreactive and nonalloreactive T cells transferred in lethally irradiated mice[END_REF].

Of note, this transient expression of the high affinity IL-2 receptor likely endows the activated T cells with the ability to respond to reduced doses of IL-2 [START_REF] Long | Cutting edge: Paracrine, but not autocrine, IL-2 signaling is sustained during early antiviral CD4 T cell response[END_REF][START_REF] Yu | A low interleukin-2 receptor signaling threshold supports the development and homeostasis of T regulatory cells[END_REF][START_REF] Kalia | Prolonged interleukin-2Ralpha expression on virus-specific CD8+ T cells favors terminal-effector differentiation in vivo[END_REF]. We thus evaluated the kinetics of CD25 expression on the different donor T cell subpopulations after in vivo IL-2 administration (Figure 6). In untreated grafted mice, donor CD4+ and CD8+ T cells showed a pick of CD25 expression by 3 days postgraft, which diminished by day 6. Notably, in the presence of exogenous IL-2, The percentage of both CD4+ and CD8+ CD25+ allogeneic Tcons increased in a dosedependent manner (Figure 6a-b), and maintained very high expression levels of CD25 throughout the treatment (Figure 6a andc). Importantly, in IL-2 treated mice, increased expression of CD25 represented not only a biomarker of response to IL-2, but also a biomarker of cell activation (Supplemental Figure 3). Indeed, the CD25+ fraction of CD4+ and CD8+ Tcons obtained from IL-2 treated mice was significantly enriched in effector memory cells (CD44high, CD62L-) and showed increased expression of ICOS and GITR, compared to the CD25-fraction (Supplemental Figure 3a-b). Collectively, these results demonstrate that IL-2 treatment of recipient mice increased and sustained CD25 expression on allogeneic Tcons, endowing them with the capacity to respond to reduced amounts of IL-2, and thus explaining the lost of low-dose IL-2 administration specificity for Tregs.

In the autoimmune setting, low-dose IL-2 administration halts the pathogenic immune response through targeted boosting of Tregs in inflamed tissue [START_REF] Tang | Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[END_REF][START_REF] Grinberg-Bleyer | IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[END_REF]. However, it could be argued that in allo-HCT, the lymphopenia associated with a cytokine storm due to conditioning [START_REF] Ferrara | Graft-versushost disease[END_REF] could be responsible for the absence of therapeutic effect of IL-2 during GVHD. To address this question, we turned to a mouse model of acute GVHD that allowed for controlling lymphopenia and the cytokine storm. We used a non-irradiated mouse model of GVHD [START_REF] Pickel | Suppressor T cells arising in mice undergoing a graft-vs-host response[END_REF] (Supplementary Figure 4). In this setting, the infusion of large numbers of parental T cells in F1 recipients resulted in 64% mortality at day 65 after transplantation and IL-2 administration did not significantly modify the course of GVHD (Supplementary Figure 4). Overall, these data suggest that neither inflammation induced by the radiation of the recipient, nor this conditioning-associated lymphopenia are responsible for the lack of protective effect of IL-2 during acute GVHD.

Low-dose IL-2 expands human Tregs in PBMC administered in immunodeficient mice

Low-dose IL-2 administration was recently described to reproducibly increase human Treg proportions in different immune-related pathologies, such as hepatitis C virusinduced vasculitis [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF], autoimmune diabetes [START_REF] Hartemann | Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial[END_REF] and the allogeneic setting of patients suffering from chronic GVHD [START_REF] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF] or for GVHD prophylaxis in children [START_REF] Kennedy-Nasser | Ultra Low-Dose IL-2 for GVHD Prophylaxis after Allogeneic Hematopoietic Stem Cell Transplantation Mediates Expansion of Regulatory T Cells without Diminishing Antiviral and Antileukemic Activity[END_REF].

We first wanted to test whether IL-2 administration could also increase Treg proportion in our experimental model of xenogeneic (xeno-) GVHD consisting in the administration of human PBMCs to immunodeficient mice. Thus, we administered 25,000 and 250,000 IU IL-2 (low-and high-IL-2 doses) and measured whether a similar effect on human Tregs was observed with PBMCs cells adoptively transferred into immunodeficient mice. At day 5, both IL-2 doses significantly increased Treg frequencies and proliferation (Figure 7a). At day 10, Treg proportions were increased in all conditions compared to day 5, but only low doses of IL-2 significantly increased Treg proportions compared with untreated mice (P=0.04).

Next, we studied the specificity of IL-2 for Tregs as compared to the effect on Tconvs.

Low-dose IL-2 treatment did not measurably affect CD4 or CD8 Tconv activation and proliferation as estimated by CD25 and Ki67 expression (Figure 7b). In marked contrast, although high-dose IL-2 administration increased Treg proportions, it also amplified the percentage of CD25 expressing CD4 Tconvs at day 10, as well as the percentage of Ki67 expressing CD4 and CD8 Tconvs at days 5 and 10. Thus, low dose IL-2 selectively expands Treg in vivo after adoptive transfer of PBMCs in immunodeficient mice.

Treg expansion by low-dose IL-2 does not inhibit in vivo Tconv activation and differentiation during xeno-GVHD

We next aimed to evaluate in vivo the biological effects of Treg expansion on human Tconv activation and differentiation in the context of GVHD. For this, we examined TNF and IFN production by Tconvs in the spleen and liver (a target organ of xeno-GVHD), as both cytokines represent pro-inflammatory mediators of acute GVHD [START_REF] Cooke | Tumor necrosis factor-alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease[END_REF][START_REF] Mowat | Antibodies to IFN-gamma prevent immunologically mediated intestinal damage in murine graft-versus-host reaction[END_REF]. While low-dose IL-2 administration did not modify Tconv cytokine production compared to untreated mice, high-dose IL-2 dramatically increased the percentage of TNF and INFproducing CD4 and CD8 Tconvs (Figure 7c).

Overall, using human cells, we observed for the first time that high-doses of IL-2 increased pathogenic Tconv activation, proliferation and cytokine production, whereas low-dose IL-2 increased only Tregs proportions, which nevertheless were insufficient to control the production of inflammatory cytokines by the activated Tconvs.

Low-dose IL-2 alone or combined to rapamycin does not prevent xeno-

GVHD

We then tested the clinical impact of Treg expansion on GVHD following several therapeutic schemes described in Figure 8a. As previously shown [START_REF] Hannon | Infusion of clinical-grade enriched regulatory T cells delays experimental xenogeneic graft-versus-host disease[END_REF][START_REF] Parmar | Third-party umbilical cord blood-derived regulatory T cells prevent xenogenic graft-versus-host disease[END_REF], adoptive transfer of human PBMCs induced lethal xeno-GVHD (median survival time, MST = 42.5 days). Administration of low-doses of IL-2 did not significantly modify the disease course (MST = 37.5 days; P = 0.0626 versus PBS-treated mice). Addition of rapamycin to low-dose IL-2 therapy did not ameliorate GVHD (Figure 8b).

DISCUSSION

The few published studies of IL-2 administration during BMT are controversial. In this work, we observed that a wide range of low-dose IL-2 injections administered to grafted mice were inefficient for controlling GVHD. More precisely, 25,000 IU IL-2, which was the "low-dose IL-2" used to cure T1D [START_REF] Grinberg-Bleyer | IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[END_REF], and even lower doses (2,500, n=5; 10,000, not shown) did not induce any therapeutic effect in the GVHD setting, and higher IL-2 doses (100,000 and 250,000 IU) significantly accelerated GVHDrelated mortality. Like us, Shin et al reported the lack therapeutic effect of IL-2 alone administration in a different genetic combination [START_REF] Shin | Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells[END_REF]. In contrast, during the 90's, pioneer work by the group of David Sachs [START_REF] Sykes | IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity[END_REF][START_REF] Sykes | In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment[END_REF][START_REF] Sykes | Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukemia effect of allogeneic T cells[END_REF] showed that 3 days of IL-2 administration could control GVHD development. In their studies, they used a fully allogeneic GVHD model in which spleen cells were used as the source of allogeneic T cells. Moreover, their IL-2 had a different origin (Cetus corporation) than ours (Proleukin, Chiron), and the used doses were 2 doses/day over 5 days of either 10,000 or 50,000 Cetus units, corresponding to 60,000 or 300,000 IU. To understand the origin of the different therapeutic outcome, we also tested their experimental conditions in our model. However, the graft of spleen cells instead of lymph node cells on IL-2 treated mice did not improve GVHD incidence nor did the reduction of the treatment from 10 to 3 days (data not shown). These differences suggest the key factor underlying IL-2 therapeutic outcome is likely the type of GVHD (fully allogeneic, semi allogeneic, syngeneic) [START_REF] Sykes | IL-2 reduces graft-versus-host disease and preserves a graft-versus-leukemia effect by selectively inhibiting CD4+ T cell activity[END_REF][START_REF] Sykes | In vivo administration of interleukin 2 plus T cell-depleted syngeneic marrow prevents graft-versus-host disease mortality and permits alloengraftment[END_REF][START_REF] Sykes | Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukemia effect of allogeneic T cells[END_REF][START_REF] Shin | Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells[END_REF].

Indeed, contrary to T1D, which is an organ-specific slowly progressive chronic inflammatory disease; acute GVHD is a highly inflammatory systemic disease implying about 10% of the donor T cell repertoire [START_REF] Shlomchik | Graft-versus-host disease[END_REF]. This unique scenario, in which IL-2 administration increases and prolongs CD25 expression on these numerous activated allogeneic T cells, likely explains the opposite outcome of low-dose IL-2 administration in GVHD compared to T1D. Indeed, functional CD25 expression endows Tcons with the capacity to compete with Tregs for low-doses of IL-2, and the selective IL-2 action on Tregs is lost. Moreover, this scenario seems valid beyond the lymphopenia associated to the conditioning of the host, as suggested by the absence of protection from GVHD in IL-2-treated non-irradiated recipient mice. Notably, CD25 expression level is a sensitive biomarker of IL-2 effects in vivo, and follow-up of CD25 expression on Tcons upon IL-2 administration may contribute to appropriately assess the side effect of Tcon activation when implementing low-dose IL-2-based therapies.

An alternative approach is illustrated by the very recent work by Ito S. at al [START_REF] Ito | Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers[END_REF] in which ultra-low dose IL-2 administration to immunocompetent healthy volunteers induced the expansion of Tregs and prompted the authors to propose said strategy for SCT donors to prevent GVHD. Here, we tested this hypothesis for the first time in mouse GVHD, and observed that although grafts obtained from IL-2 treated donors were increased in Tregs, GVHD incidence was not modified compared to mice that received unmanipulated grafts. Absence of efficacy can be explained by our previous published observations showing that acute GVHD can only be controlled by the infusion of Tregs and Tcons at a 1:1 ratio [START_REF] Cohen | CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graftversus-host disease[END_REF]. Here, we observed that IL-2 administration to donor mice could not increase Tregs more than 1,3 folds. Similarly, ultra-low dose IL-2 administration to healthy volunteers [START_REF] Ito | Ultra-low Dose Interleukin-2 Promotes Immune-modulating Function of Regulatory T Cells and Natural Killer Cells in Healthy Volunteers[END_REF] induced an approximately 2-fold expansion of Tregs without activating Tcons. These IL-2 performances are still very far away from the theoretically desired more than 20-fold increase needed to approach a 1:1 Treg:Tcon ratio.

Unlike previous experimental models [START_REF] Sykes | Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukemia effect of allogeneic T cells[END_REF][START_REF] Shin | Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells[END_REF], the xeno-GVHD model used in this report uses for the first time of human cells thus reflecting probably more accurately IL-2 clinical effect on human cells and the disease process. Consequently, the results reported here are highly relevant and striking as they show that despite an increase in Tregs cells (mean 3.6% +/-0.57 at day 0 versus 5.60% +/-1.51 at day 5), which is comparable to what is observed in humans following administration of low doses of IL-2 in healthy donors (3.53% +/-1.17 at day 0 versus 5.68% +/-1.56 at day 4; [START_REF] Ito | Ultra-low dose interleukin-2 promotes immune-modulating function of regulatory T cells and natural killer cells in healthy volunteers[END_REF]) or in allogeneic stem cell transplant recipients (4.8% at day 0 versus 11.1% after one month of IL-2 treatment; [START_REF] Kennedy-Nasser | Ultra Low-Dose IL-2 for GVHD Prophylaxis after Allogeneic Hematopoietic Stem Cell Transplantation Mediates Expansion of Regulatory T Cells without Diminishing Antiviral and Antileukemic Activity[END_REF]), no clinical impact was observed on GVHD. Thus, the model seems to well reflect well the Treg increases that can be expected with this therapy, which makes the ultimate finding of lack of clinical response of high clinicaltranslational value. Of note, during xeno-GVHD, expanded Tregs were unable to control the production of inflammatory cytokines by the activated Tconvs, arguing again for a lack of efficacy associated to an insufficient increase of the Treg/Teff ratio (from 0.5:10 to 2:10 ratio, far from the theoretically optimal 1:1 ratio) (ref 1), as discussed above, and in accordance with our previous observations showing that Treg content is not a reliable predictive factor of GVHD [START_REF] Rosenzwajg | Regulatory T cell content in the bone marrow graft does not predict the occurrence of acute GVHD[END_REF].

The lack of therapeutic efficacy of low-dose IL-2 in the inflammatory context of acute GVHD does not invalidate the potential use of low-dose IL-2 as an immunosuppressive agent. On the contrary, two recent clinical trials -one in chronic GVHD refractory to glucocorticoid therapy [START_REF] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF] and another in a prophylactic regimen in pediatric patients [START_REF] Kennedy-Nasser | Ultra Low-Dose IL-2 for GVHD prophylaxis after Allogeneic HSCT mediates Expansion of Regulatory T Cells Without Diminishing Antiviral and Anti-leukemic Activity. Clinical cancer research : an official journal of the American Association for Cancer Research[END_REF] have provided the proof of concept that low doses of IL-2 can induce Treg expansion in vivo and that may provide therapeutic benefits. However, in these trials, IL-2 was concomitantly administered with conditioning/prophylaxis regimens, which included immunosuppressive drugs, such as rapamycin or glucocorticoids, making it difficult to weight IL-2 alone contribution to therapeutic outcome. Indeed, a recent publication by Negrin's group, showed that efficacy of IL-2 treatment to lower GVHD in mice is only achieved if IL-2 is combined with rapamycin [START_REF] Shin | Rapamycin and IL-2 reduce lethal acute graft-versus-host disease associated with increased expansion of donor type CD4+CD25+Foxp3+ regulatory T cells[END_REF]. This strategy combines the beneficial effect of IL-2 on Tregs with concomitant elimination of the pathogenic Tcons by rapamycin [START_REF] Battaglia | Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients[END_REF][START_REF] Battaglia | Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells[END_REF]. At odds with these results, we observed that during xeno-GVHD, addition of rapamycin to lowdose IL-2 administration was inefficient to control GVHD. One possible limitation in our model could be due to the lack of donor APC engrafting in the immunodeficient mice together with CD3 cells. Indeed, it has been recently shown that part of the effect of rapamycin was to induce an increase in IL-12 secretion by dendritic cells thus leading to INF-g-dependent apoptosis of alloreactive T cells [START_REF] Stenger | IL-12hi rapamycin-conditioned dendritic cells mediate IFN-gamma-dependent apoptosis of alloreactive CD4+ T cells in vitro and reduce lethal graft-versus-host disease[END_REF]. The capacity for murine recipient dendritic cells to produce IL-12 under rapamycin treatment and to induce such effect on human T cells is unknown.

In conclusion, the use of IL-2 as an immunosuppressive drug is an emerging concept, resulting in a previously unsought clinical use of this cytokine. IL-2 was initially used at high-doses in cancer to boost the anti-tumoral immune response [START_REF] Ahmadzadeh | IL-2 administration increases CD4+ CD25(hi) Foxp3+ regulatory T cells in cancer patients[END_REF][START_REF] Rosenberg | Treatment of 283 consecutive patients with metastatic melanoma or renal cell cancer using high-dose bolus interleukin 2[END_REF]; it is now used at low doses to slow down or even control autoimmune or chronic inflammation, probably by boosting Treg function [START_REF] Tang | Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[END_REF][START_REF] Grinberg-Bleyer | IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells[END_REF][START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Koulmanda | Modification of adverse inflammation is required to cure new-onset type 1 diabetic hosts[END_REF][START_REF] Webster | In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression[END_REF][START_REF] Wilson | Suppression of murine allergic airway disease by IL-2:anti-IL-2 monoclonal antibody-induced regulatory T cells[END_REF]. However, our results obtained in the particular setting of experimental acute GVHD indicate that IL-2 effects can be radically different depending on the underlying immune pathology.

Consequently, although our data documents the limitations associated to IL-2 treatment during GVHD; combination of IL-2 with other drugs that target activated conventional cells (rapamycin, MMF, etc) and definition of more appropriated settings such as patients receiving HSCT from matched related donors, may represent a valuable strategies to control GVHD.

MATERIALS AND METHODS: 1.4.1 Mice

Seven-to twelve-week-old C57BL/6 (B6) (H-2b), [B6 x DBA/2] F1 (B6D2F1, H-2bxd) mice were obtained from Janvier Laboratory (France). NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) and Luciferase-expressing (B6 luc+) mice were bred in our animal facility under specific pathogen-free conditions. Manipulations were performed according to European Union guidelines and with approval by The Ethics Committee in Animal Experiment Charles Darwin, France (Ce5/2012/021).

IL-2 treatment

Donor or recipient mice were treated with daily intraperitoneal (IP) injections of recombinant human IL-2 (Proleukin, Novartis) for 5 or 10 consecutive days as indicated. Rapamycin (Rapamune, Wyeth Laboratory) was administered per os (1.5 mg/kg).

Acute GVHD model

Acute GVHD was induced as has been described previously [START_REF] Cohen | CD4(+)CD25(+) immunoregulatory T Cells: new therapeutics for graftversus-host disease[END_REF]. Briefly, eight-to twelve-week-old B6D2F1 recipient mice were lethally irradiated with 11 Gy, given in 2 split doses of 5.5 Gy 5 hours apart, followed by an intravenous (IV) injection of 10x106 C57BL/6 donor bone marrow (BM) cells and 4x106 B6 lymph node (LN) CD3+ T cells to induce GVHD. These cells were collected from donor mice LN, and the percentage of CD3+ T cells was determined by flow cytometry. Grafted mice were monitored for weight loss and survival 3 times per week for 2 months. A body weight loss of more than 30% of initial weight resulted in euthanasia.

Non-irradiated Acute GVHD model

Briefly, non-irradiated eight-to twelve-week-old B6D2F1 recipient mice were injected IV with 20x10 6 B6 lymph node (LN) CD3 + T cells to induce GVHD. These cells were collected from donor mice LN, and the percentage of CD3+ T cells was determined by flow cytometry. Grafted mice were monitored for weight loss and survival 3 times per week for 80 days. A body weight loss of more than 30% of initial weight resulted in euthanasia.

In vivo bioluminescent imaging (BLI)

In vivo BLI was performed as previously described [START_REF] Duda | Methods for imaging cell fates in hematopoiesis[END_REF]. Mice received an IP injection of 30 µg/g of body weight of D-Luciferin (Caliper Life Sciences). Imaging of mice was done ten minutes later using an IVIS Spectrum imaging system (Caliper Life and analyzed with Living Image software (Caliper Life Sciences).

Xeno-GVHD model

Xeno-GVHD was induced as described [START_REF] Ito | Highly sensitive model for xenogenic GVHD using severe immunodeficient NOG mice[END_REF]. Ten-to fifteen-week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) recipient mice (Charles River) received an intravenous injection of PBMCs containing 12x10 6 human CD3+ obtained from healthy volunteers with their informed consent (EFS) and monitored for weight loss and survival for 2 months. Body weight loss higher than 30% of initial weight resulted in euthanasia.

Antibodies and flow cytometry

For cells of mouse origin: anti-CD3, anti-CD4, anti-CD8, anti-CD25, anti-CD44, anti-CD62L, anti-CD69, anti-GITR, anti-H-2Kb, anti-H-2Kd and anti-ICOS labeled with PE, APC, PerCP, FITC, PE-Cy7, AF700, V500 and biotin were purchased from BD (France). Anti-CD8 and anti-CD25 labeled with AF700, APC-H7, PE-Cy7, PerCP-Cy5.5,FITC and PerCP or Pacific Blue streptavidin were purchased from eBioscience (France). PE-or Pacific Blue-anti-Foxp3 staining was performed using the eBioscience kit and protocol. Events were acquired on an LSR II flow cytometer (BD Biosciences), and data were analyzed using FlowJo (Tree Star, USA).

For cells of human origin: Single cell suspensions of blood, spleens and livers were stained with anti-CD45-V500, anti-CD3-PE, anti-CD4-Alexa700 (all from BD Biosciences), anti-CD8ECD, anti-CD25-PE-Cy7 (both from Beckman Coulter) and anti-CD127-BV421 (BioLegend). For intracellular cytokine staining, cells were restimulated with PMA/Iono for 3h in the presence of brefeldin A (eBioscience) and stained with anti-CD45-APC-Cy7 (BD Biosciences), anti-CD3-PE-Cy5, anti-CD8-ECD (both from Beckman Coulter) anti-CD4-APC (Miltenyi Biotech). Anti-Foxp3-Alexa488, anti-IFN-PE-Cy7 (both from eBiosciences), anti-Ki67-Alexa647 and anti-TNFPE (both BD Biosciences) staining was then performed using the eBioscience Foxp3 staining kit. Events were acquired on a Fortessa flow cytometer (BD Biosciences) and data analyzed using FlowJo (Tree Star).

Statistical analysis

When comparing differences between groups, if sample distribution was normal (Shapiro-Wilk normality test) we applied a two-sided unpaired Student's t-test and if sample distribution was not normal we applied a two-sided non-parametric Mann-Whitney test. P < 0.05 was taken as statistical significance. Survival proportions were calculated using the Kaplan-Meyer method, and statistical significances were calculated using the Gehan-Breslow-Wilcoxon test. A p-value <.05 was considered significant. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, USA).

FIGURE LEGENDS

Figure 1 .

 1 Figure 1. Low-dose IL-2 administration in donor mice selectively expands Tregs. (a)

Figure 2 .

 2 Figure 2. Low-dose IL-2 administration in donor mice does allow GVHD prevention.

Figure 3 .

 3 Figure 3. Low-dose IL-2 administration in recipient mice does not affect GVHD

Figure 4 .

 4 Figure 4. IL-2 administration in recipient mice does not affect reconstitution of the T-

Figure 5 .

 5 Figure 5. IL-2 administration over-activates allogeneic donor T cells. Recipient

Figure 6 .

 6 Figure 6. IL-2 administration sustains CD25 expression on pathogenic donor T cells.

Figure 7 .

 7 Figure 7. Low doses IL-2 selectively expand human T reg cells during xenogeneic GVHD. Recipient NSG mice were grafted with 12.10 6 human PBMCs and then daily

Figure 8 .

 8 Figure 8. Treatment with low-doses of IL-2 alone or combined to rapamycin fails to

  Figure 1 b

  Figure 4

  Figure 5

CD4 1 .

 1 Figure 6

ACKNOWLEDGEMENTS

This work was supported by the "Agence de la Biomédecine". LP was supported by the French Government; GHM was supported by the French Government and by the "Ligue Nationale contre le cancer". The authors acknowledge Olivier Boyer (Rouen University, France) for providing proofreading of the manuscript and useful comments. We thank the animal care team members Christelle Enond, Flora Issert, François Bodin, Serban Morosan (all from the Centre d'Exploration Fonctionnelle, Université Pierre et Marie Curie) and Olivier Lebhar, Armelle Halle, Céline Daviaud, Isabelle Grandjean and Virgine Dangles-Marie (all from the Institut Curie, Paris).

CONFLICT OF INTEREST

E.P. is inventor of a patent application related to the use of low-dose IL2 owned by her public institutions. There is no other conflict of interest. 1.7 AUTHORS CONTRIBUTION GHM and LP performed the research, analyzed the data and wrote the paper; SM, JLC and EP designed the research, analyzed the data and wrote the paper. Graphs show cumulative data from 5 experiments (3 with PBS and IL-2 treatment only and 2 with PBS, IL-2, Rapa and IL-2/Rapa treatment). Errors bars are the S.E.M. of the mean value. * P < 0.05 as assessed by a Gehan-Breslow-Wilcoxon test.