Aline Migné
email: migne@sb-roscoff.fr

Gaspard Delebecq
email: gasp.delebecq@hotmail.fr

Dominique Davoult
email: davoult@sb-roscoff.fr

Nicolas Spilmont
email: nicolas.spilmont@univ-lille1.fr

Dominique Menu
email: dominique.menu@univ-lille1.fr

François Gévaert
email: francois.gevaert@univ-lille1.fr

Photosynthetic activity and productivity of intertidal macroalgae: in situ measurements, from thallus to community scale

Keywords: Primary production, Rocky shores, Fucoids, Kelps

est destinée au dépôt

Introduction

Rocky shores are frequently dominated by dense populations of macroalgae that exhibit distinct patterns of vertical zonation. On northern hemisphere temperate coasts, this pattern typically consists of the succession of fucoid algae from the high to the low mid shore and of laminarians (or kelps) in the lower shore. The productivity of such seaweed populations has long been shown to be important, ranging from 1 to several kg C m -2 y -1 [START_REF] Mann | Seaweeds: their productivity and strategy for growth[END_REF]. However, these estimations were rather raw and still need to be specified. In particular, the ecological performance of macroalgae has to be measured in situ and at the multi-species community scale to take into account not only the dominating species but all the species as well as their interactions in relevant conditions. Intertidal macroalgae experience two very different environments for photosynthesis, aerial and aquatic. The capacity of species from different shore levels to maintain photosynthesis during emersion periods has long been investigated based on measurements performed on isolated thalli under controlled laboratory conditions (e.g. [START_REF] Johnson | Comparative photosynthetic capacities of intertidal algae under exposed and submerged conditions[END_REF][START_REF] Quadir | Effects of emergence and submergence on the photosynthesis and respiration of marine macrophytes[END_REF][START_REF] Dring | Photosynthesis of intertidal brown algae during and after periods of emersion: a renewed search for physiological causes of zonation[END_REF]. Since then, differences between some shallow-water macroalgae species have been shown to be smaller at the communities scale than experiments on single thallus pieces suggested (Binzer and Middelboe, 2005) and an increasing productivity of fucoid assemblages down a shore-height gradient has been shown [START_REF] Tait | Dynamics of productivity in naturally structured macroalgal assemblages: importance of canopy structure on light-use efficiency[END_REF]. These community-scale measurements were limited, however, to immerged conditions and trends might be different under peculiar conditions of the emersion periods. The present study compares the photosynthetic activity and productivity of thalli of the dominant canopy macroalgae (i.e. the zone-forming species typical of the northern hemisphere temperate shores) and the productivity of the communities, based on in situ measurements performed either during emersion or immersion periods. First, the photosynthetic activity of adult thalli was examined using pulse-amplitude modulated (PAM) fluorescence. This technique, which relies on the variable fluorescence of chlorophyll a, is not a direct measurement of photosynthesis but allows an examination of the dynamic properties of photosynthesis in response to environmental changes [START_REF] Enriquez | The use of the fluorescence signal in studies of seagrasses and macroalgae[END_REF] during the whole tidal cycle (emersion and immersion).

Second, the productivity of single individual, or several individuals of a single species, was assessed using a closed chamber system. This monospecific approach aimed at understanding the productivity dynamic of each species in response to the environment variation (aerial versus aquatic). Third, the productivity of multi-species communities was assessed using closed chamber systems under emersion. This assemblage-based approach integrated the resource partitioning among canopyforming macroalgae and their sub-canopy assemblages. The aim of the study was to test two hypotheses: (1) the in air versus under water ratio of photosynthetic performance decreases down a shore-height gradient; (2) the variation in photosynthetic performance among dominant canopy species of the shore is reduced when the measurements are scaled up from thallus to community.

Materials and Methods

Target species and study sites

The target species were the three fucoids Pelvetia canaliculata (the uppermost zone-forming species), Fucus spiralis (the immediately contiguous zone-forming species) and Fucus serratus (the lowermost zone-forming fucoid) and the kelp Laminaria digitata (the lowermost zone-forming species). Two seasons (spring and summer) and two sites, differing for the timing of the tide and for turbidity, were investigated. Measurements were performed at the rocky shore of Wimereux (50°44'48" N -1°35'54" E, eastern English Channel, France) during spring tides in September 2009 (summer) on the four seaweed communities (P. canaliculata, F. spiralis, F. serratus and L. digitata) and in April 2010 (spring) on three seaweed communities (P. canaliculata, F. spiralis and L. digitata) and at the rocky shore of Roscoff (48°43'53" N -3°59'16" W, western English Channel, France) during spring tides in August 2010 (summer) and in May 2011 (spring) on the uppermost and the lowermost seaweed communities (P. canaliculata and L. digitata). At Wimereux, water turbidity is high and low tides of spring tide occur in the early morning (low tides occurred between 7:30am and 10:00am, UT+2h, for summer measurements and between 7:00am and 8:30am, UT+2h, for spring measurements). At Roscoff, water turbidity is low and low tides of spring tide occur in the early afternoon (low tides occurred between 2:00pm and 3:30pm, UT + 2h, for summer measurements and between 1:30pm and 2:30pm, UT + 2h, for spring measurements).

Light and temperature

Photosynthetically available radiation (PAR in μmol photons m -2 s -1 ) was measured in air and underwater and recorded every minute using a WinCQ flat sensor (Alec Electronics). Temperature was measured in air and underwater and recorded every minute using a MDS MkV-T sensor (Alec Electronics). Average temperature during the periods of selected measurements was 18°C underwater and 20°C in the air in summer and 11°C underwater and 14°C in the air in spring, at Wimereux. It was 17°C underwater and 27°C in the air in summer and 14°C underwater and 22°C in the air in spring, at Roscoff.

Fluorescence

In vivo chlorophyll fluorescence properties were measured in situ using a submersible PAM fluorometer (Diving PAM, Walz). The fluorescence signal was always taken from the middle of the frond in the same place for three haphazardly-selected and marked individuals of the target species.

The effective quantum yield of photosystem II (Φ PSII ) was measured under ambient light at different stages of the tidal cycle. The fiber optic was mounted in a home-made transparent Plexiglas holder applied to one side of the thallus in such a way that the distance between the fiber optic and the algal tissue was constant and standard. The fiber optic forms a 60° angle with the sample, avoiding shading or darkening. Φ PSII was calculated as (F m '-F t ) /F m ' [START_REF] Genty | The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence[END_REF], where F m ' is the maximal fluorescence level measured during a single saturating light pulse (0.8 s) for light-adapted samples, and F t is the fluorescence steady-state level immediately prior to the flash. Then, the electron transport rate (ETR in mol electrons m -2 s -1 , here after referred to as μmol e -m -2 s -1 ) was estimated using the following equation:

ETR = Φ PSII × PAR × 0.5 x A (1)
where PAR is the photosynthetically available radiation (in μmol photons m -2 s -1 ) , 0.5 is a correction factor based on the assumption that the incident photons are absorbed equally by the pigments of the two photosystems and A is the absorption coefficient determined in the laboratory using an integrating sphere (ISR-240A, Shimadzu). The absorption coefficient varied between 0.93 and 0.97 according to the species and season.

Carbon fluxes

Productivity of single or several individuals (depending on the size of the species) was measured using a benthic chamber placed on the shore at the target species level. The system was made of a transparent closed Perspex dome tightly sealed on the polyvinylchloride (PVC) base of the chamber and enclosed a volume of 35.3 L. During immersion periods, an electronic management system controlled three external pumps; two pumps ensured the rapid and constant homogenization of the media while the third one ensured the renewal of the media by flushing ambient seawater between two consecutive incubations [START_REF] Gévaert | A fully automated system for measurements of photosynthetic oxygen exchange under immersed conditions: an example of its use in Laminaria digitata (Heterokontophyta: Phaeophyceae)[END_REF]. pH was measured with a WTW sentix 41 probe (Multi 350i, WTW) and monitored every minute during 10 min incubations. Seawater was collected from inside the benthic chamber using a 100 ml syringe, then passed through cellulose acetate membrane filters (0.8 μm) and spiked with HgCl 2 pending potentiometric laboratory determination of total alkalinity [START_REF] Millero | Titration alkalinity of seawater[END_REF] on 3 subsamples of 20 ml. The dissolved inorganic carbon (DIC) concentration of seawater was calculated from the pH, total alkalinity (TA), temperature and salinity according to [START_REF] Strickland | A practical handbook of seawater analysis[END_REF] and using the formula given in [START_REF] Oviatt | A comparison of system (O 2 and CO 2 ) and C-14 measurements of metabolism in estuarine mesocosms[END_REF]. DIC flux (µmol h -1 ) was calculated as the difference between the final and the initial concentrations after the linearity of the pH (corrected from temperature) change has been checked. During emersion, inorganic carbon fluxes were measured in the benthic chamber using closed air circuit for CO 2 analysis [START_REF] Migné | A closed-chamber CO 2 -flux method for estimating intertidal primary production and respiration under emersed conditions[END_REF]. Changes in air CO 2 concentration (ppm) were measured in the chamber with a CO 2 infrared gas analyser (LiCor Li-6251) and recorded with a data logger (LiCor Li-1400) at 15 s intervals during incubations of about 10 minutes duration. CO 2 fluxes (μmol h -1 ) were calculated from the slope of CO 2 concentration against time assuming a molar volume of 22.4 L at standard temperature and pressure. The benthic chamber was opened between two consecutive incubations to renew the ambient air, and algae were weighted to assess their water loss during the emersion period. At the end of the series of measurements, algae were rehydrated to assess their fresh weight (FW) and then dried for 48 h at 60°C to assess their dry weight (DW). Fluxes were expressed as μmol C g DW -1 h -1 . The relative tissue water content at time t was calculated as the percent of total water content: [(W t -DW)/(FW-DW)] x 100 (where W t represents the weight measured at time t).

Productivity was assessed at the community scale during emersion using benthic chambers as described in [START_REF] Crowe | Large-scale variation in combined impacts of canopy loss and disturbance on community structure and ecosystem functioning[END_REF]. Three chambers were haphazardly-positioned (approximately 2 m apart) to account for spatial variability in each zone. Each chamber (of a total volume of 18.9 L) was made of a transparent Perspex dome with a 0.3 x 0.3 m transparent Perspex base air-tightly sealed with neutral silicon to the substratum. Changes in CO 2 mol fraction in the air were measured by an infrared CO 2 gas analyser (LiCor Li-800). CO 2 fluxes were estimated following the same procedure as described for single species measurements. The benthic chambers were opened between two consecutive incubations to renew the ambient air. In the Fucoid zones, algae were collected at the end of the series of measurements, and the zone-forming algae were dried for 48 h at 60°C and weighted. In the Laminaria digitata zone, there was no individual of the zone forming species inside the chambers. Incubations were performed either in ambient light conditions to assess the net productivity (NP) or in dark (after the chamber has been covered with an opaque tarp) in order to assess the respiration (R) rate. The gross productivity (GP) was then calculated by correcting NP by R.

Data analyses

To compare the photosynthetic activity and the productivity of the different species measured at the different scales at the two seasons and sites, the highest electron transport rates (ETR) and the highest carbon fluxes obtained at each occasion under emersion or immersion either in ambient light (mean PAR values given in Table 1) or in darkness were selected. Care was taken to use measurements performed in ambient light at saturating irradiance levels. A threshold of 300 mol photons m -2 s -1 was chosen based on the average value of 291 mol photons m -2 s -1 given for the onset of light saturation of coastal macroalgal communities by [START_REF] Middelboe | Highly predictable photosynthetic production in natural macroalgal communities from incoming and absorbed light[END_REF]. For under emersion measurements, care was also taken to use measures obtained during the early stages of emersion on still hydrated algae (relative tissue water content between 72 and 97 % for individual scale measurements). Some measurements could not be achieved under immersion due to the difficulty of working while diving in the backwash of the intertidal zone and some data are thus missing. The effect of site and season on the results obtained for each target species was tested when no data were missing using the Scheirer-Ray-Hare H-test (non-parametric 2-way ANOVA).

Results

Photosynthetic activity and productivity under emersion

Mean ETR was greatly higher for P. canaliculata than for L. digitata at each site and season (between 9 and 16 times higher, Fig 1 .A). There was no significant difference according to the season or site for P. canaliculata (Table 2). For L. digitata, mean ETR was significantly higher in spring than summer but there was no significant difference according to the site (Table 2).

The gross productivity rate was also higher for P. canaliculata than for L. digitata at each site and season (more than 2 times higher, Fig 2 .C). For L. digitata, respiration rates were sometimes higher than gross productivity leading to positive carbon fluxes in the benthic chamber under light at Wimereux in summer and in both seasons at Roscoff (Fig 2 .A).

The mean gross productivity rate measured at the community scale was higher for the P. canaliculata zone than the L. digitata zone at Wimereux at the two seasons (between 3 and 4 times higher) while the order of magnitude was the same for the two zones at Roscoff at the two seasons (Fig 3 .C). At this scale, carbon fluxes in the benthic chambers under light were always negative, i.e. gross productivity rates were higher than respiration rates. Mean gross productivity rates measured on the L. digitata zone were significantly higher at Roscoff than at Wimereux but there were no significant differences according to the season (Table 2). There were no significant differences according to the season or site for P. canaliculata (Table 2). The mean biomass of P. canaliculata (Table 3) was significantly lower at Wimereux than Roscoff but there were no significant differences between seasons (Table 2).

Photosynthetic activity and productivity under immersion

The mean ETR measured during immersion was greater for L. digitata than for P. canaliculata at Roscoff in summer, the single occasion when ETR was measured under water for both species (Fig

1.B).

At the individual scale, measurements under dark could be achieved only in three experiments (Fig 2.

Under emersion versus under immersion photosynthetic activity and productivity

The electron transport rate was higher in air than underwater for P. canaliculata and F. spiralis (the ratio reaching 3.72) but lower for F. serratus and L. digitata (the ratio reaching 0.14). For P.

canaliculata, the higher ETR in the air was explained by the greater photon flux density in the air than underwater at Wimereux in spring (Table 1, while the effective quantum yield of photosystem II was 0.5 and 0.7 in air and water respectively), but was also due to a greater Φ PSII in the air at Roscoff in summer (0.6 versus 0.4). For F. spiralis, it was mainly due to a greater Φ PSII in the air (0.5 versus 0.2).

Particularly low values of Φ PSII (< 0.1) were measured for L. digitata under emersion at Roscoff while they were between 0.2 and 0.7 in the other cases. The ratio of individual scale net productivity in air versus water was lower than 1 at each occasion (the maximum was 0.95 for F. spiralis) and was even negative for L. digitata at Roscoff (due to respiration rates higher than gross productivity under emersion). The ratio of gross productivity in air versus water was also lower than 1 in the only three occasions it could be calculated.

Discussion

The present study of the photosynthetic performance of intertidal macroalgae based on in situ measurements emphasizes on a major result: the trends observed under emersion according to the position of the algae along the shore vary with the scale of measurements. The uppermost species

Pelvetia canaliculata had higher photosynthetic activity and productivity under emersion than the lowermost species Laminaria digitata, while the few available data showed no trend between the zone-forming species under immersion. Futhermore, the photosynthetic activity measured at the thallus scale was higher in air than underwater for the uppermost species and lower in air than underwater for the lowermost species. Differences between zone-forming species were, however, greatly reduced when measurements were scaled up from the electron transport rate of thallus to the gross productivity of individuals and communities.

Photosynthetic performance according to the shore-level of the algae

In the present study, carbon flux measured at the individual scale, both under immersion and emersion, in ambient light conditions (with PAR > 300 mol photons m -2 s -1 ) suggested that photosynthetic performance was higher underwater whatever the species was, for both sites and seasons and even if the emerged individuals were not significantly dehydrated (relative tissue water content > 72%). Furthermore, a net negative productivity (respiration higher than gross productivity) was observed for L. digitata under emersion. However, the difference between underwater and aerial performance was lower for upper species, with a NP em /NP im ratio as high as 0.95 for Fucus spiralis. At the thallus scale, the electron transport rate also measured both under immersion and emersion in ambient light conditions was higher in air for P. canaliculata and F. spiralis but higher underwater for Fucus serratus and L. digitata. Moreover, at that scale of measurement, the upper species on the shore had by far the highest photosynthetic activity under emersion and the lower species had the lowest. This obvious trend was observed independently of the season or site and could not be only explained by better light conditions during measurements for upper species.

Some previous laboratory researches, made under controlled light conditions and based on either oxygen and carbon flux [START_REF] Johnson | Comparative photosynthetic capacities of intertidal algae under exposed and submerged conditions[END_REF][START_REF] Kawamitsu | Photosynthesis and carbon storage between tides in a brown alga, Fucus vesiculosus[END_REF] or fluorescence [START_REF] Skene | Key differences in photosynthetic characteristics of nine species of intertidal macroalgae are related to their position on the shore[END_REF] measurements on thalli, also suggested that the ability of intertidal algae for sustained photosynthesis in air is enhanced for upper shore species. [START_REF] Johnson | Comparative photosynthetic capacities of intertidal algae under exposed and submerged conditions[END_REF] even obtained a NP em /NP im ratio of 6.6 for the high-shore species Fucus distichus. This trend might be explained by the significant ecophysiological distinctions that had been made between algae living essentially completely submersed and those spending a part of tidal cycles in the emersed state. The upper shore algae had been shown to have the potential for rapid photosynthesis in air during the short time without desiccation [START_REF] Surif | Photosynthetic gas exchange under emersed conditions in eulittoral and normally submersed members of the Fucales and the Laminariales: interpretation in relation to C isotope ratio and N and water use efficiency[END_REF]). Thus, the less time is available for photosynthesis, as limited by desiccation, the greater is the photosynthetic rate during the time available. The "CO 2 concentrating mechanism", characteristic of photosynthesis in the eulittoral Fucaceae [START_REF] Surif | Exogenous inorganic carbon sources for photosynthesis in seawater by members of the Fucales and the Laminariales (Phaeophyta): ecological and taxonomic implications[END_REF], may be important in increasing the efficiency of emersed photosynthesis with limited capital of water available.

Some contrasting results have, however, been obtained in laboratory studies. For example, the carbon intake in the air was higher for the lower shore Saccharina latissima than the uppershore Pelvetia canaliculata [START_REF] Ni Longphuirt | Seasonal and species-specific response of five brown macroalgae to high atmospheric CO2[END_REF], the photosynthesis rates of intertidal Fucoids were always higher in water than in air [START_REF] Williams | High and dry: variation in net photosynthesis of the intertidal seaweed Fucus gardneri[END_REF] or either higher in water or in air according to the temperature [START_REF] Madsen | A comparison of air and water as environments for photosynthesis by the intertidal alga Fucus spiralis (Phaeophyta)[END_REF]) and the effective PSII quantum efficiency of Laminaria digitata increased after 3h of emersion [START_REF] Nitschke | Molecular iodine emission rates and photosynthetic performance of different thallus parts of Laminaria digitata (Phaeophyceae) during emersion[END_REF]. Furthermore, the effect of short-term emersion on chl a fluorescence responses to irradiance has been shown to be dependent on the protocol used for the acclimation of the algae to the different light levels [START_REF] Nitschke | Chlorophyll a fluorescence responses of temperate Phaeophyceae under submersion and emersion regimes: a comparison of rapid and steady-state light curves[END_REF] and the variation of the effective PSII quantum efficiency measured in situ has been shown to be dependent on the meteorological conditions [START_REF] Lamote | Photosynthetic responses of an intertidal alga to emersion: the interplay of intertidal height and meteorological conditions[END_REF]. Different sampling, keeping and/or measuring conditions could then have influenced both laboratory and field results, leading to various conclusions among studies.

Variation in the trends of photosynthetic performance according to the scale of measurements

The trend of photosynthetic activity under emersion according to the shore-level of the algae was obvious in the present study when photosynthetic activity was measured at the thallus scale as electron transport rate. When photosynthetic activity was measured as gross productivity at the individual scale, it was also typically higher for the uppermost than the lowermost species but the trend along the shore was less obvious (even if the lack of replication prevents clear comparisons to be performed). Finally, when photosynthetic activity was measured as gross productivity at the community scale, which is a more relevant ecological unit, the highest rates were measured on the intermediate mid-shore zones and, at one of the two sites, rates were higher for the lowermost zone than for the uppermost zone. Furthermore, considering the whole set of data the coefficient of variation of net productivity under emersion was 1.15 at the individual scale and 0.55 at the community scale.

The contrasting results using either fluorescence or carbon flux measurements can suggest that thalli are not relevant ecological units but also that the photosystem II activity measured as ETR is not always a relevant measure of the rate of photosynthesis. There is a general assumption that a linear relationship between ETR and gross photosynthesis exists, as long as environmental stresses do not impose restrictions on photosynthetic carbon fixation. The linearity between ETR and photosynthesis of macroalgae has been, however, only confirmed at low light intensities [START_REF] Enriquez | The use of the fluorescence signal in studies of seagrasses and macroalgae[END_REF]. ETR calculation is based on the assumption that light absorption is only due to photosynthetic pigments. There are, however, alternative electron sinks (e.g. the Mehler reaction, the photorespiration) which explain the deviation from linearity in the relationship between ETR and gross productivity (measured either as O 2 production or C uptake) observed at high irradiances in various primary producers (Beardall et al., 2009). This occurred as critical low values of Φ PSII were reached and the irradiance at which these values were reached depended on the previous light histories undergone by the algae (Beer and Axelsson, 2004). It is the irradiance relative to the algal growth conditions rather than the irradiance during the measurement that determines the threshold.

Beer and Axelsson ( 2004) measured critical values of Φ PSII (< 0.1) at lower irradiance for Laminaria saccharina than for Fucus serratus (250 versus 2000 mol photons m -2 s -1 ) while both species were acclimated to 400 mol photons m -2 s -1 for two weeks. In the present study, critical low values of Φ PSII were measured for Laminaria digitata under emersion at Roscoff. Such critical values were previously measured in situ underwater at ebb tide around midday on the Roscoff kelps L. saccharina and L. digitata. The response of Φ PSII to high irradiances was associated to an increase in the nonphotochemical quenching and was explained as a down-regulation of the photosystem II [START_REF] Gévaert | Laminaria saccharina photosynthesis measured in situ: photoinhibition and xanthophyll cycle during a tidal cycle[END_REF][START_REF] Delebecq | In situ photosynthetic performance of Laminaria digitata (Phaeophyceae) during spring tides in Northern Brittany[END_REF].

The contrasting results measuring carbon fluxes either at the individual or at the community scale do reflect the difference in use of resources by either isolated or grouped individuals of only one species and by mixed communities allowing species to interact and supplement each other. Although individual photosynthesis must influence the community response, individual metabolism was not expected to be closely related to community metabolism, because this represents a complex integrated response of many different species exposed to a wide range of local light conditions within the canopies [START_REF] Middelboe | Importance of canopy structure on photosynthesis in single-and multi-species assemblages of marine macroalgae[END_REF]Binzer and Middelboe, 2005;[START_REF] Middelboe | Highly predictable photosynthetic production in natural macroalgal communities from incoming and absorbed light[END_REF]. [START_REF] Tait | Dynamics of productivity in naturally structured macroalgal assemblages: importance of canopy structure on light-use efficiency[END_REF] showed that layering within macroalgal assemblages plays a major role in maintaining net photosynthesis throughout the natural range of irradiance and even enhances photosynthesis at high irradiance. [START_REF] Tait | Primary productivity of intertidal macroalgal assemblages: comparison of laboratory and in situ photorespirometry[END_REF] measured the underwater productivity of three fucoid assemblages. The rates, on a per area basis, showed a down shore-height gradient explained by the greater biomass and number of algal species of the lower-shore assemblage. In the present study, a down shore-height gradient was also shown when measurements were performed on the three fucoid assemblages (i.e. at Wimereux in summer). The highest rates at the community scale were measured on the Fucus serratus zone where the biomass of the dominant canopy species and the number of species were the highest (9 species were identified inside the chambers while it was 7 for the Pelvetia canaliculata and 8 for the Fucus spiralis zones). The zone of Laminaria digitata

  E), leading to only three available values of gross productivity (the highest being for P. canaliculata and the lowest for L. digitata, Fig 2.F). More experiments could be achieved under light, allowing the comparison of the net productivity rates. They were of the same order of magnitude for P. canaliculata than L. digitata at Roscoff in summer, the single occasion when NP was measured under water for both species (Fig 2.D).

Figure 1 :Figure 2 :

 12 Figure 1: Highest electron transport rate (ETR in mol e -m -2 s -1 , mean ± sd, n = 3) measured in

Figure 3 :

 3 Figure 3: Highest carbon fluxes measured under emersion in the benthic chambers at the community scale (in mmol C m -2 h -1 , mean ± sd, n = 3) in ambient light (a, PAR values given in table 1) or in dark

Table 2 :

 2 Significance of the effect of season and site on electron transport rates (ETR) and gross community productivity (GCP) measured under emersion and on dry weight inside benthic chambers (DW) of the zone-forming species (Pc: Pelvetia canaliculata, Ld: Laminaria digitata) as tested by Scheirer-Ray-Hare H-test.

	Variable Zone-forming species	Factor	H	p-value
	ETR	Pc	Season	1.256	0.262
			Site	0.026	0.872
			Interaction	0.641	0.423
		Ld	Season	8.308	0.004
			Site	1.256	0.262
			Interaction	0.103	0.748
	GCP	Pc	Season	0.410	0.522
			Site	2.564	0.109
			Interaction	1.256	0.262
		Ld	Season	0.026	0.872
			Site	8.308	0.004
			Interaction	0.103	0.748
	DW	Pc	Season	0.923	0.337
			Site	7.410	0.006
			Interaction	0.103	0.748

Acknowledgements

This work was funded by the French program EC2CO/INSU. Thierry Cariou from the Station Biologique de Roscoff provided surface PAR data that was useful for intercalibration between our various measures. Thanks are due to Marie-Andrée Janquin, Renaud Michel and Lucie Camañez for assistance in the field. We would like also to thank the Service Mer & Observation from the Station Biologique de Roscoff for valuable support and particularly François Le Ven who managed to bring the wayfarer chamber back from the "Bigorneau langoureux" in the Batz Island. Finally, we acknowledge the improvements originated by the comments made by the editor and reviewers of this paper.

Literature cited

Beardall, J., Ihnken, S., Quigg, A., 2009. Gross and net primary production: closing the gap between concepts and measurements. Aquat. Microb. Ecol. 56, 113-122. Beer, S., Axelsson, L., 2004. Limitations in the use of PAM fluorometry for measuring photosynthetic rates of macroalgae at high irradiances. Eur. J. Phycol. 39, 1-7. Binzer, T., Middelboe, A.L., 2005. From thallus to communities: scale effects and photosynthetic performance in macroalgae communities. Mar. Ecol. Prog. Ser. 287,[65][66][67][68][69][70][71][72][73][74][75]

had also a great algal biomass (it was measured only once, at Roscoff in summer, and was 80.51 ± 26.62 g DW /0.09 m 2 ) and had a great species richness (9 and 22 species identified inside the chambers at Wimereux and Roscoff respectively) but lower community scale productivity under emersion, particularly at Wimereux. This certainly reflects the low efficiency in air of algae living essentially submersed. Further measurements should be performed at the community scale during immersion periods to test if the down shore-height gradient highlighted with fucoids is maintained when both fucoids and kelps are considered.

Abbreviations


Michelle Rosenzwajg

Guillaume Churlaud

Roberto Mallone

Adrien Six

Nicolas Dérian

Wahiba Chaara

Roberta Lorenzon

Alice Long

Jane H Buckner

Georgia Afonso

S Alice Long

Hang-Phuong Pham

Agnès Hartemann

Aixin Yu

Alberto Pugliese

Thomas R Malek

David Klatzmann
email: david.klatzmann@upmc.fr

Prof D Klatzmann

Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients

Keywords: Tolerance, Immunotherapy, Inflammation, Immunopathology, Pharmacokinetics

Introduction

Most self-reactive T lymphocytes are physically eliminated during thymic selection to avoid immune responses against self-antigens. Yet this process is imperfect, and potentially harmful self-specific effector T cells (Teffs) survive thymic deletion and populate the periphery. In the normal immune system, these autoreactive Teffs do not mediate autoimmunity as their expansion and function are inhibited by regulatory T cells (Tregs) [START_REF] Sakaguchi | FOXP3+ regulatory T cells in the human immune system[END_REF]. In most autoimmune diseases (AID), there is an imbalance between Teffs that attack normal tissues and Tregs that normally control them. Tregs are essential players in the control of all immune responses, including responses to self, tumors, and infectious agents [START_REF] Sakaguchi | FOXP3+ regulatory T cells in the human immune system[END_REF], and in the control of inflammatory disorders [START_REF] Ait-Oufella | Natural regulatory T cells control the development of atherosclerosis in mice[END_REF]. The discovery of Tregs revolutionized our understanding of AID pathophysiology and opened new avenues for the treatment of autoimmunity. Human beings and mice presenting a FOXP3 genetically-determined Treg defect develop multiple organspecific AID [START_REF] Campbell | Phenotypical and functional specialization of FOXP3+ regulatory T cells[END_REF][START_REF] Wing | Regulatory T cells exert checks and balances on self tolerance and autoimmunity[END_REF][START_REF] Bacchetta | Defective regulatory and effector T cell functions in patients with FOXP3 mutations[END_REF][START_REF] Bennett | The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3[END_REF][START_REF] Brunkow | Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse[END_REF][START_REF] Fontenot | Foxp3 programs the development and function of CD4+CD25+ regulatory T cells[END_REF]. Treg quantitative or qualitative defects have been described in common human AID and their mouse models [START_REF] Brusko | Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes[END_REF][START_REF] Lindley | Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes[END_REF][START_REF] Long | Defects in IL-2R signaling contribute to diminished maintenance of FOXP3 expression in CD4(+)CD25(+) regulatory T-cells of type 1 diabetic subjects[END_REF][START_REF] Buckner | Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+) regulatory T cells in human autoimmune diseases[END_REF][START_REF] Long | CD4+FOXP3+ T regulatory cells in human autoimmunity: more than a numbers game[END_REF]. Moreover, administration/restoration of Tregs improves symptoms in experimental animals and patients with AID [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] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Matsuoka | Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease[END_REF][START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Tang | CD4(+)Foxp3(+) regulatory T cell therapy in transplantation[END_REF][START_REF] Wood | Regulatory T cells in transplantation tolerance[END_REF]. Thus, while blocking Teffs with immunosuppressive drugs has long been the only therapeutic option for AID, activating/expanding Tregs is becoming a novel approach that may lead to improved outcomes with better safety [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] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Matsuoka | Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease[END_REF][START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Tang | CD4(+)Foxp3(+) regulatory T cell therapy in transplantation[END_REF][START_REF] Wood | Regulatory T cells in transplantation tolerance[END_REF].

Until recently, most Treg-based therapies have been based on the infusion of ex vivo expanded Tregs, as there were no drugs or molecules that could specifically expand/activate Tregs in vivo. We and others recently showed that IL-2, a molecule identified almost 30 years ago for its capacity to stimulate T cells in vitro, could be such a molecule [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] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Matsuoka | Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease[END_REF][START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF]. We performed the first clinical trial aimed at inducing Tregs with IL-2 in patients with an AID [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF], namely hepatitis C virus (HCV)-related vasculitis (NCT00574652) [START_REF] Cacoub | Extrahepatic manifestations of chronic hepatitis C. MULTIVIRC Group. Multidepartment Virus C[END_REF]. We treated these patients with low-dose (ld) IL-2 (1.5-3 million international units MIU/day; 52.5 MIU cumulative dose; Proleukin®). The treatment was very well tolerated. Importantly, we observed a major expansion of Tregs with subsequent clinical improvement in 8/10 patients [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF]. Our results showed for the first time that ld-IL-2 could be exploited as an immunoregulatory drug for the treatment of AID, acting via Treg expansion/activation.

Others have reported that low dose IL-2 is safe and beneficial for the treatment of alloimmunity, i.e. chronic graft versus host disease (GVHD) [START_REF] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Matsuoka | Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease[END_REF].

Based on these results, we evaluated ld-IL-2 in patients with type 1 diabetes (T1D), a condition resulting from the chronic autoimmune destruction of pancreatic beta-cells, eventually leading to insulin deficiency. There are several reasons for using IL-2 in T1D patients: (i) several susceptibility genes are directly connected to the IL-2 pathway: IL-2, CD25 (IL-2RA) [START_REF] Dendrou | Cell-specific protein phenotypes for the autoimmune locus IL2RA using a genotype-selectable human bioresource[END_REF][START_REF] Qu | Toward further mapping of the association between the IL2RA locus and type 1 diabetes[END_REF][START_REF] Vella | Localization of a type 1 diabetes locus in the IL2RA/CD25 region by use of tag single-nucleotide polymorphisms[END_REF], IL-2RB [START_REF] Todd | Etiology of type 1 diabetes[END_REF], and PTPN2 [START_REF] Todd | Robust associations of four new chromosome regions from genome-wide analyses of type 1 diabetes[END_REF][START_REF] Wang | Genome-wide association studies: theoretical and practical concerns[END_REF]; (ii) some studies reported Treg deficiency in peripheral blood [START_REF] Brusko | Functional defects and the influence of age on the frequency of CD4+ CD25+ T-cells in type 1 diabetes[END_REF][START_REF] Lindley | Defective suppressor function in CD4(+)CD25(+) T-cells from patients with type 1 diabetes[END_REF], and in pancreatic lymph nodes [START_REF] Ferraro | Expansion of Th17 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes[END_REF][START_REF] Bluestone | Genetics, pathogenesis and clinical interventions in type 1 diabetes[END_REF]; (iii) at the time of clinical diagnosis, there is significant residual beta-cell function in most patients, so that immunotherapy could curtail inflammation, promote immune tolerance, and in turn preserve beta-cell mass and function [START_REF] Battaglia | Immune intervention with T regulatory cells: past lessons and future perspectives for type 1 diabetes[END_REF]. In Non Obese Diabetic (NOD) mice, a model of spontaneous autoimmune diabetes with remarkable similarities to the human disease, IL-2 prevents T1D and we showed that a short course of IL-2 at diabetes onset led to disease reversal in one third of the mice [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] Tang | Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction[END_REF]. Finally, the use of immunosuppressants such as cyclosporine A (CsA), a calcineurin inhibitor that reduces T cell activation and expansion, provided proof of principle that newly-diagnosed T1D could be treated with immunotherapy [START_REF] Bougneres | Factors associated with early remission of type I diabetes in children treated with cyclosporine[END_REF][START_REF] Feutren | Cyclosporin increases the rate and length of remissions in insulin-dependent diabetes of recent onset. Results of a multicentre double-blind trial[END_REF][START_REF] Stiller | Effects of cyclosporine immunosuppression in insulin-dependent diabetes mellitus of recent onset[END_REF]. CsA demonstrated clinical efficacy in prolonging endogenous insulin production, but remission from autoimmunity was limited to the duration of the treatment and CsA toxicity precluded its clinical use. Other immunosuppressive drugs or immunomodulators have been tested in clinical trials in T1D, both as short therapy courses or chronic regimens, only in some cases resulting in temporary preservation of insulin secretion with better results in subsets of responder patients, as reported recently for therapies with anti-CD3, anti-CD20 and CTLA4-Ig [START_REF] Skyler | Immune intervention for type 1 diabetes, 2012-2013[END_REF][START_REF] Orban | Costimulation modulation with abatacept in patients with recent-onset type 1 diabetes: follow-up 1 year after cessation of treatment[END_REF][START_REF] Herold | Teplizumab (anti-CD3 mAb) treatment preserves C-peptide responses in patients with new-onset type 1 diabetes in a randomized controlled trial: metabolic and immunologic features at baseline identify a subgroup of responders[END_REF][START_REF] Hagopian | Teplizumab preserves C-peptide in recent-onset type 1 diabetes: two-year results from the randomized, placebo-controlled Protege trial[END_REF][START_REF] Orban | Reduction in CD4 Central Memory T-cell subset in Co-Stimulation Modulator Abatacept-Treated Patients with Recent-Onset Type 1 Diabetes is Associated with Slower C-Peptide Decline[END_REF].

The identification of a dose of IL-2 capable of safely tipping the Treg/Teff balance towards Tregs is of major importance. In our vasculitis trial, we showed that IL-2 at the dose of 1.5 MIU induced Tregs in all 10 patients and was well tolerated. However, the dose to be used in T1D was not predictable as (i) some T1D patients may have defects in the IL-2/IL-2R activation pathway [START_REF] Garg | Type 1 diabetesassociated IL2RA variation lowers IL-2 signaling and contributes to diminished CD4+CD25+ regulatory T cell function[END_REF] (ii) HCV vasculitis is an antibody-mediated disease, while in T1D beta-cell destruction is mediated by pathogenic Teffs that could respond to IL-2 and thus exacerbate disease. Hence, we designed a dose finding trial to define safety and immunological responses; we previously reported that a 5-day course of single IL-2 injections, given at 0.33, 1 or 3 MIU, were well tolerated and stimulated Tregs [START_REF] Hartemann | Lowdose Interleukin-2 induces regulatory T cells and is well tolerated in patients with type-1 diabetes: results of a phase I/II randomized, double-blind, placebo-controlled trial[END_REF].

Here, we report the results of detailed immunomonitoring of these patients, showing that ld-IL-2 induces a dose-dependent, regulatory milieu characterized by broad changes extending beyond the primary effect on Tregs. Thus, our study provides important information for further developing ld-IL2 therapies.

Materials and methods

Patient characteristics

Male and female patients aged from 18 to 55 years, with confirmed T1D were recruited at the Diabetology Department of the Pitié-Salpêtrière Hospital (Paris, France) and thereafter followed at the Clinical Investigation Centre-Paris Est. Written informed consent was obtained from all subjects before they were enrolled in the DF-IL2 trial (NCT01353833).

Detailed patients description, safety analyses and peak effects of the treatment on Tregs have been reported [START_REF] Hartemann | Lowdose Interleukin-2 induces regulatory T cells and is well tolerated in patients with type-1 diabetes: results of a phase I/II randomized, double-blind, placebo-controlled trial[END_REF]. This clinical trial was conducted according to Declaration of Helsinki principles. All human studies were approved by the appropriate institutional review boards.

Immunomonitoring

Blood samples were collected according to the planned protocol: absolute numbers of lymphocyte subsets and Tregs were monitored at each patient visit: day-1, before treatment; day 2-5, during IL-2 administration; day-6; 15, 22, and 60 end of the follow up.

Tregs phenotyping and cytokines assay were performed at baseline, day 6, 15, 22 and 60.

Because of the limited amount of blood collected, other phenotypic markers where only investigated at baseline and day 15. When possible, cells were frozen for other investigations.

Stat5 phosphorylation (pSTAT5) was examined on frozen cells from 2 controls and 3 patients per treated group. Beta-cell-specific Teff responses were studied in 1 control, 3 patients for the dose 0.33, 2 for the dose 1 and 4 for the dose 3.

Flow Cytometry and pStat5 analysis

Flow cytometry and basal pStat5 experiments were performed according to previously published methods [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Long | Low-dose antigen promotes induction of FOXP3 in human CD4+ T cells[END_REF]. Monoclonal antibodies (mAbs) used were CD3-APCa750, CD4-ECD, CD8-APCa700, CD16-FITC, CD19-ECD, CD45RO-FITC, CD56-PCy7 and CD152-PE from Beckman Coulter; CD25-PE, CD45RA-APC and CD45RA-PCy7 from BD Biosciences; CD127-FITC from eBioscience; CD25-APC and LAP-PE from R&D Systems and GITR-PE from Miltenyi.

For pSTAT5 activation experiments, peripheral blood mononuclear cells (PBMCs) were cultured (500,000/well) for 30 min at 37 o C and IL-2 was added for 15 min. Cells were then fixed with 1.6% paraformaldehyde, permeabilized with 100% methanol, stained for pSTAT5 and appropriate surface markers. Events were collected using a LSRFortessa flow cytometer (BD Biosciences) and data were analyzed using Diva software (BD Biosciences). To calculate EC50, after subtracting the value from the media control, binding data for each sample was normalized to 100% based on the maximal response and non-linear regression of these data was performed assuming a variable slope using Graph-Pad Prism 6.0.

Plasma cytokine measurement

Plasma samples were collected, aliquoted and stored at -80°C until analyzed. Quantitative determination of 26 cytokines/chemokines (GM-CSF, IFN-α2, IFN-γ, IL-1RA, IL1a, IL1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12p40, IL-12p70 , IL-13, IL-15, IL-17a, CXCL-10, CCL-2, CCL-3, CCL-4, CCL-2, TNF-α and IL-2RA) was performed using Human Milliplex HCYTOMAG-60 kits (Millipore) and TGF-beta using Human TGF-beta1 Platinium ELISA (eBioscience) in accordance with the manufacturer protocols.

Analysis of beta-cell antigen-specific T-cell responses

CD4 + and CD8 + T-cell responses were evaluated according to previously published assay [START_REF] Martinuzzi | acDCs enhance human antigen-specific T-cell responses[END_REF].

Anti-IL-2 antibodies assay

The presence of plasma IL-2-binding antibodies was studied by ELISA, as previously described [START_REF] Scharenberg | The development of anti-interleukin-2 (IL-2) antibodies in cancer patients treated with recombinant IL-2[END_REF]. Data are the average of two replicates. Positive controls are from ApoE -/-mice that were injected three times a week with 25,000 IU of Proleukin® for 4 months. Mouse studies were approved by the appropriate institutional review boards. IL-2 neutralizing activity of plasma was assayed in a proliferation assay with Kit 225 cells [START_REF] Hori | Establishment of an interleukin 2-dependent human T cell line from a patient with T cell chronic lymphocytic leukemia who is not infected with human T cell leukemia/lymphoma virus[END_REF]. 10,000 cells/well were cultured (37°C in 5% CO 2 ) in 100 µL of RPMI (Gibco) and 10% of BSA. Plasma were preincubated for 1 hour with IL-2 10 IU/ml at 37°C and then added at the start of the cultures at a concentration of 20%. Cell proliferation was assayed by counting cells at day 3.

Transcriptomic analysis

Transcriptomic analysis was performed using the SurePrint G3 Human GE v2 8x60K Microarray (Agilent Technologies, ID: 039494) according to the manufacturer's protocol and to previously published [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF]. Cyanine-3 labeled cRNAs were prepared using the One-Color Low Input Quick Amp Labeling kit (Agilent Technologies).

The slides were scanned using a G2565CA Scanner System (Agilent Technologies), and using a scan protocol with a resolution of 3 µm and a dynamic range of 20 bit. The resulting .tiff images were analyzed with the Feature Extraction Software v10.7.3.1 (Agilent Technologies) and using the GE2_107_Sep09 protocol. Original dataset was normalized using quantile method implemented in Limma's package. Probes with bad detection p-values (<0.01) in half of the samples of a biological group were discarded. Ranked gene lists were obtained using statistical score of paired eBayes implemented in Limma's package. Gene set enrichment analyses were performed using the Blood Transcription Modules [START_REF] Li | Molecular signatures of antibody responses derived from a systems biology study of five human vaccines[END_REF] added with a selection of molecular signatures from C2 database provided by GSEA software [START_REF] Mootha | PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[END_REF][START_REF] Subramanian | Gene set enrichment analysis: a knowledge-based approach for interpreting genomewide expression profiles[END_REF].

Network analyses were performed using Cytoscape 2.8.3 and the plugin Enrichment Map dedicated to gene set enrichment analyses.

All datasets can be accessed in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-2858.

Statistical analysis

Data were compared between patients groups using a two-way analysis of variance (ANOVA) for repeated measures using GraphPad Prism version 5.0. When the p value of the time by treatment interaction was significant (p<0.05), each IL-2 group of patients was compared to placebo group.

Detailed statistical results of two-way ANOVA for repeated measures are shown in the Supplementary table 1.

Principal Component Analyses were then performed on log10-transformed data expressed as fold change compared to day 1. Multivariate analyses were performed using R platform (www.r-project.org).

Results

Clinical trial design, patients and safety

Results presented here are from a randomized, double-blind, placebo-controlled, dosefinding trial. Patients received a daily sub-cutaneous injection of placebo or 0.33, 1 or 3 MIU of IL-2 for 5 consecutive days. They were sampled every day just before the IL-2 injection for the first 5 days, then at day 6, 15, 22 and 60 (Supplementary Fig 1). Twenty-four patients (6 per dose) were included in the per-protocol analysis. No serious adverse events were reported during the trial [START_REF] Hartemann | Lowdose Interleukin-2 induces regulatory T cells and is well tolerated in patients with type-1 diabetes: results of a phase I/II randomized, double-blind, placebo-controlled trial[END_REF].

Dose dependent effects on peripheral blood mononuclear cells

T, B and NK cells

There were no significant changes in CD4 + and CD8 + T cell, B cell and NK cell numbers in the two lower dose groups compared to placebo (Supplementary Fig 2). At the dose of 3 MIU/day, T, B and NK cell numbers were decreased approximately 2 fold during treatment, followed by an immediate return to baseline thereafter (Supplementary Fig 2). Regarding cell percentages (Supplementary Fig 3), a B cell reduction was observed at the two highest doses (p=0.03 at 1 MIU/day and 0.003 at 3 MIU/day) during treatment, followed by a return to baseline levels thereafter. No statistically significant variations of NK cell percentages were observed at the doses of 0.33 and 1 MIU/day while increase NK proportions were observed at the dose of 3 MIU/day (p=0.003). However, at this dose, individual variations of NK cell percentages remained in the range observed in the placebo group (see below). Finally, there was an increase in CD56bright regulatory NK cells [START_REF] Vivier | Innate or adaptive immunity? The example of natural killer cells[END_REF] at the dose of 1 MIU/day (p=0.015)

(Supplementary Fig 4).

CD4 and CD8 Tregs

Effects of treatment on the proportions of CD4 + Tregs are shown in Fig 1A . Comparisons between placebo and each of the three doses of IL-2 by repeated measures ANOVA showed that the day/treatment interaction was highly significant (p<0.0001). Tregs were expanded at all doses, and the duration of increase was dose-dependent. By day 15, Tregs returned to baseline at the dose of 0.33 MIU/day, but persisted elevated at the dose of 1 MIU/day; increased Treg levels were still detected at day 60 at the dose of 3 MIU/day. The increased percentage of CD4 + Tregs was associated with an increase in their absolute numbers (p<0.0001) (Fig1B), leading to an increase in the Tregs/Teff ratio (p<0.0001) (Fig 1C).

We previously reported that the very small population of CD8 + CD25 + Foxp3 + T cells, which are suppressive and expanded in colorectal cancer [START_REF] Chaput | Identification of CD8+CD25+Foxp3+ suppressive T cells in colorectal cancer tissue[END_REF], was expanded in the VASCU-IL-2 trial [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF]. Here, we show a dose-dependent expansion of these CD8 + Tregs (p=0.0002), remarkably high at the highest IL-2 dose (p< 0.0001) (Fig 1D).

Treg immunophenotyping

Expanded Tregs displayed a memory phenotype (p=0.0051) [START_REF] Miyara | Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor[END_REF] Overall, these data are consistent with stable, highly sensitive, and selective responses by Tregs to IL-2 in these T1D patients.

Dose dependent modulation of plasma cytokine profiles

A panel of 26 cytokines and chemokines were assayed to investigate whether IL-2 could influence the balance of Th1/Th2/Th17 cytokines during treatment. The entire cytokine profile was tested at day 6, according to dose and to the functional classification of cytokines.

Transcriptomic analysis

We compared the global transcriptome of PBMCs at baseline and day-6. For each IL-2 treatment dose (0.33, 1 and 3 MUI), Enrichment Map analysis was performed on significantly up-and down-regulated molecular signatures (q-value < 0.05). Network analyses of significantly enriched molecular signatures show a good consistency for all IL-2 doses (Fig 5). Whatever the IL-2 dose, there is a striking down regulation of B-cells related signatures, and an up regulation of molecular signatures related to cell cycle and transcription.

Chemokines associated signatures are found down regulated with the 2 lowest IL-2 doses, but up-regulated at the dose of 3 MIU/day, a dose at which NK cells signatures also became upregulated.

The molecular signature related to FOXP3 target genes [START_REF] Gavin | Foxp3-dependent programme of regulatory T-cell differentiation[END_REF] is non-significantly up-regulated at the lowest dose (q-val=0.2), and significantly enriched and up-regulated at the dose of 1 and 3 MIU/day.

Modulation of beta-cell-specific Teff responses

Anti-IL-2 antibodies are not induced by ld-IL-2

We did not observe induction of anti-IL-2 antibodies and neutralizing antibodies in the plasma, at any time during the follow-up (Fig 7). While a very small reactivity against IL-2 could be detected in some patients, (i) it was not increased by IL-2 administration (Fig 7A andB) and (ii) it did not neutralize the effect of IL-2 on the proliferation of an IL-2-dependent cell line (Fig 7C).

Discussion

On finding an optimal IL-2 dose for Treg specific expansion/activation

IL-2 has been initially described as a T cell growth factor [START_REF] Morgan | Selective in vitro growth of T lymphocytes from normal human bone marrows[END_REF] and developed for the treatment of conditions calling for the boosting of Teffs, i.e. cancer and infections [START_REF] Chang | Overview of interleukin-2 as an immunotherapeutic agent[END_REF][START_REF] Giedlin | The use of recombinant human interleukin-2 in treating infectious diseases[END_REF]. However, IL-2 is dispensable for the differentiation, survival and function of Teffs, as IL-2 -/- mice develop T-cell-mediated autoimmunity [START_REF] Sadlack | Generalized autoimmune disease in interleukin-2-deficient mice is triggered by an uncontrolled activation and proliferation of CD4+ T cells[END_REF]. In contrast, IL-2 is essential for the differentiation, survival and function of Tregs [START_REF] Zorn | IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo[END_REF][START_REF] Malek | Interleukin-2 receptor signaling: at the interface between tolerance and immunity[END_REF]. IL-2 also has pleiotropic functions and can activate Teffs and notably memory CD8 + Teffs, and using IL-2 for treating T-cell mediated AID by promoting Treg function carries the risk of stimulating Teffs and could potentially worsen disease. Thus, therapeutic use of IL-2 to control autoimmunity requires identifying doses that selectively promote Treg function without activating effector populations.

The present study shows that the 0.3 to 3 MIU/day dose range is safe and effectively expanded/activated Tregs in T1D patients. The IL-2 dose-range of 0.3 to 1 MIU/day, given for 5 days effectively expands and activates Tregs without untoward effects on Teffs or NK cells. These results are in line with a recent study of low dose IL-2 (≈0.1 to 0.4 MIU/day for five days) in healthy adults [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]. At the dose of 3 MIU/day, the effect on Tregs is even more pronounced and importantly more durable; yet at this dose, we observed a trend toward NK cell expansion (Supplementary Fig 2 ), a trend for chemokine cytokine/chemokine increase (Fig 4), and more frequent mild to moderate side effects [START_REF] Hartemann | Lowdose Interleukin-2 induces regulatory T cells and is well tolerated in patients with type-1 diabetes: results of a phase I/II randomized, double-blind, placebo-controlled trial[END_REF].

Under the assumption that chronic diseases will require maintaining increased proportion of Tregs over time, repeated treatment will likely be necessary. The appropriate dosing should consider the patient population, the duration and frequency of the treatment, and the cumulative dosing effects in relation to time. The duration of Tregs expansion, which is clearly dose-dependent, is a major parameter to take in consideration for designing regimen of administration. Using results from this trial, we modeled that at a dose of 1 MIU/day, a 5-day induction course followed by one injection every two weeks would allow to maintain a modest increase (< 30%) of Tregs throughout the treatment period, a dosing that we currently use in a trial investigating ld-IL-2 in 11 different AIDs (NCT01988506).

On the effect of IL-2 on the immune environment

At the dose of 1 and 3 MIU/day, we observed a significant and unexpected decrease in B cell percentages, correlating with increases in Treg but not NK cells (Supplementary Fig 2).

Interpretation of this finding is not straightforward. A direct effect of IL-2 on mature B cells is unlikely because they do not express CD25 nor the signal transducing beta and gamma chains of the IL2 receptor [START_REF] Boyman | The role of interleukin-2 during homeostasis and activation of the immune system[END_REF]. The clinical significance of these findings remains to be clarified in future studies, particularly because a decrease in B cells could be advantageous for the treatment of B cell dependent AIDs. IL-2 induces a clear shift of the peripheral blood immune environment towards a regulatory milieu, as reflected not only by changes in cell proportions, but also by plasma proteomics. This shift is dose-dependent, as well illustrated by the unsupervised statistical analyses (Fig 4B). These results suggest a broad anti-inflammatory effect of ld-IL-2 [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF] and thus a broader therapeutic scope. IL-10 and TGF-beta, two cytokines involved in suppressive functions, are also increased by IL-2 therapy, more pronouncedly at 3 MIU/day. At the same dose, there is an increase in IFNgamma, which is usually classified as a Th1 cytokine. However, this is the only Th1 cytokine impacted and it was only modestly elevated, possibly coming from IFN-gamma secreting Tregs [START_REF] Oldenhove | Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection[END_REF]. IL-17 was moderately increased only at the 3 MIU/day dose, a dose at which IL-2, IL-10 and TGF-beta were markedly increased; thus, the overall balance still appears tipped towards a regulatory milieu at this dose. Consistent with this interpretation, beta-cell-specific Teff responses were reduced in 6/9 patients tested during treatment, importantly in 4/4 treated with the 3 MIU/day dose (Fig 6), with no reductions in the placebo-treated patient tested. Thus, altogether, there is a clear IL-2 dose-dependent tuning of the immune response towards regulation of effector responses, including disease-specific, autoreactive Teff responses.

At the dose of 3 MIU/day, there was a marked increase of IL-5 levels (Fig 4A ), a cytokine involved in eosinophil homeostasis. However, at the single and cumulative doses used, our available data show only a modest increase in eosinophil counts. This contrasts with the marked increase reported by others at doses of IL-2 that were higher and/or given over a longer time course [START_REF] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Long | Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs beta-cell function[END_REF]. These results are compatible with IL-2 triggering IL-5 production by stimulating ILC2 cells, which are CD25 + [START_REF] Walker | Innate lymphoid cells--how did we miss them?[END_REF]. However, ILC2 also produce IL-13, IL-9 and IL-4 [START_REF] Walker | Innate lymphoid cells--how did we miss them?[END_REF], which were not increased in patients' plasma. Of note, it has been reported that IL-5 promotes induction of antigen-specific Treg that suppress autoimmunity [START_REF] Tran | IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity[END_REF].

Importantly, at the 1 MIU/day dose, IL-5 was not induced, further pointing at this dose as safer choice for chronic administration.

On IL-2 and therapy and prevention of AIDs

AIDs often develop over a long period of time and are chronic, with a progressive aggravation. Early intervention with ld-IL2 could help reestablish a proper immune milieu and slow-down or even reverse the process. This is exemplified in T1D, for which at diagnosis most patients still maintain a functional mass of pancreatic β-cells and produce insulin upon stimulation. Halting beta-cell autoimmune destruction and preserving residual beta-cells soon after diagnosis could prevent further disease progression, thus improving clinical management and long-term outcomes for patients with T1D. In this perspective, our initial results showing the blunting of beta-cell-specific Teffs during ld-IL-2 are very encouraging. We caution that such analyses could be performed only in a subset of patients and thus further study and validation are needed, which we will pursue in our efficacy trial of ld-IL-2 in patients with T1D (NCT01862120).

We believe that there may be even greater potential for using ld-IL-2 to prevent AIDs.

Providing a boost to Tregs in patients at risk for AIDs, such as autoantibody-positive relatives of T1D patients, could help delay or prevent the onset of the full-blown disease. Moreover, boosting Tregs with ld-IL2 could also prevent or decrease the frequency and severity of relapses in relapsing-remitting diseases like multiple sclerosis (MS).

Finally, ld-IL-2 has now been used successfully in the setting of chronic and acute GVHD [START_REF] Koreth | Interleukin-2 and regulatory T cells in graft-versus-host disease[END_REF][START_REF] Kennedy-Nasser | Ultra lowdose 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] and several AIDs, such as HCV-vasculitis [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF], T1D [START_REF] Hartemann | Lowdose Interleukin-2 induces regulatory T cells and is well tolerated in patients with type-1 diabetes: results of a phase I/II randomized, double-blind, placebo-controlled trial[END_REF], alopecia areata [START_REF] Castela | Effects of Low-Dose Recombinant Interleukin 2 to Promote T-Regulatory Cells in Alopecia Areata[END_REF], and SLE [START_REF] Klatzmann | Immunoregulation Without Immunosuppression: The Promise of Low Dose IL-2[END_REF][START_REF] Spee-Mayer | Low-dose interleukin-2 therapy caused selective expansion of Tregs together with rapid reduction of disease activity in a patient with severe refractory SLE[END_REF][START_REF] Yu | Low-dose interleukin-2 in active systemic lupus erythematosus[END_REF]. A therapeutic benefit was observed in most patients treated in these trials. This changes the paradigm of a highly focused effect of ld-IL-2 on Tregs to one where IL-2 has a broader regulatory influence on the homeostasis of the immune system, and notably inflammation. As such, future studies should explore further the effects of IL-2 and define efficacious ld-IL-2 regimens that can benefit patients currently suffering from several forms of AID and inflammatory disorders. For each IL-2 treatment dose (0.33, 1 and 3 MIU), Enrichment Map analysis was performed on significantly up-and down-regulated molecular signatures, in red and green, respectively (q-value < 0.05). Clusters have been labelled according to their main biological feature. Size of nodes (circles) is proportional to the number of genes in the signature; width of bars linking nodes is related to the Jaccard coefficient between two signatures. Signatures without connection are not shown.

Dose Dose

  and phenotypic features characteristic of enhanced activation (Fig 2A). The expression of IL-2 receptor-alpha (CD25) was increased in Tregs (Fig 2B), but not in CD4 + CD45RA -T effector/memory (T EM ) cells (not shown), during IL-2 therapy, from day 2 or 3, for all doses (p<0.0001), and rapidly returned to baseline values thereafter. This was associated with increased expression of GITR (p=0.0058) and CTLA-4 (p<0.0001) (Fig 2C, 2D), whereas TGF-β latency-associated peptide (LAP) remained unchanged (data not shown).

3. 4 . 2 .

 42 Tregs show stable, selective, and highly sensitive responses to IL-An immediate consequence of IL-2 binding to the IL-2R is tyrosine phosphorylation of STAT5 (pSTAT5). Basal pSTAT5 levels in Tregs were transiently increased at the 3 MIU/day dose (p=0.0308) (Fig 2E), which may be related to the increased proportions of CD45RA -Tregs (Fig 2A). We also assessed the IL-2-dependent activation of pSTAT5 by CD4 + Treg and T EM cells by various amounts of IL-2, for patients' samples that were obtained before and 4-6 days after the initiation of IL-2 therapy. For each patient's sample examined, Tregs showed increased sensitivity to IL-2 when compared to T EM cells. These responses were largely comparable to cells from normal controls that did not receive IL-2 therapy (Fig 3A). This trend was similar regardless of the dose of IL-2 used to treat the patients. In addition, the responses during treatment were essentially identical to those detected before ld-IL-2 therapy. Thus, a short course of IL-2 therapy did not rapidly alter IL-2-dependent pSTAT5 activation. By calculating the EC50s of these responses, Tregs from T1D patients were approximately 15-20-fold more sensitive to IL-2 that T EM cells (Fig 3B). These analyses of frozen PBMCs resulted in IC50s in a similar range detected for fresh PBMCs, i.e 3-7 pM for Tregs and 30-35 pM for CD4 + T EM cells (A.Y, A.P. and T.R.M, unpublished data).

2 (

 2 Dose dependent IL-2 levels could be detected in the plasma. A radar chart (Fig 4A) illustrates a clear trend for a dose-dependent increase of Treg-related cytokines, while the other cytokines are modestly affected at the 0.33 and 1 MIU/day doses. At 3 MIU/day, there are discrete and more pronounced cytokine increases, notably for CXCL10 and IL-5 whereas other cytokines tested remained unchanged (Fig 4A). Principal Component Analyses of these results illustrate this dose-dependent trend: the 0.33 MIU/day patients largely overlap with placebo-treated patients, while the 1 and 3 MIU/day patients show more distinct behaviors (Fig 4B). Repeated measures ANOVA for the day/treatment interaction were significant for IL-2, IL-2RA, IL-5, IL-10, IL-17, TNF-alpha, TGF-beta1, CCL22, and CXCL10 (p<0.0001 for IL-2, IL-2RA, IL-5, IL10, CXCL10 and TNF-alpha, p=0.0006 for TGF-beta1, p=0.0059 for IL-17 and p=0.0096 for CCL22) (Fig 4A and supplementary Table 1). The time-course changes for cytokines show a dose-dependent and rapid increase, with values already elevated at day Fig 4C). The increase of IL-17 was only observed at the 3 MIU/day dose (Fig 4C), but this was paralleled by an increase in the regulatory cytokine TGF-beta.

22 (

 22 Preproinsulin (PPI) and glutamic acid decarboxylase (GAD) are beta-cell autoantigens commonly targeted by pathogenic Teffs in T1D patients. We therefore investigated T-cell responses directed to GAD, proinsulin (PI), C-peptide and the PPI signal peptide during ld-IL-2 treatment in 10 patients for whom samples from different time points (day 1, 6, 15 and 22) were available. Compared to pre-treatment values, many treated patients showed a reduction or stabilization of IFN-gamma responses to most antigens between day 6 and Fig 6). Specifically, we observed consistent suppression of all Teff responses tested in 1/3, 1/2 and 4/4 patients treated with 0.33, 1 and 3 MIU of IL-2, respectively. The suppressive effect on these responses was maintained after cessation of IL-2 therapy at the higher 3 MIU/day dose (Fig 6), up to day 22.

  MR supervised immunomonitoring, participated to the study design and analyzed results; GC, RM, AS, ND, WC, RL, AL, JB, GA, HPP, AY, AP and TRM performed some experiments and analyzed results; AH was the principal clinical investigator of the study, participated to the study design and analyzed results; DK was the principal investigator of the study, conceived the study, participated to the study design, analyzed results and wrote the manuscript. All authors edited the manuscript. The DF-IL2 STUDY GROUP Principal Investigator: Prof D Klatzmann MD, PhD Principal Clinical Investigator: Prof A Hartemann MD Methodologist: G Bensimon MD Data management & Statistical Analysis: C A Payan MD Clinical Investigations Centre-Centre d'Investigation Clinique Paris-Est Hopital de La Pitié-Salpêtrière Paris France Prof C. Funck-Brentano MD, B. Charbit MD, N Nicolas MD, S Delroise MD Biological Investigation M Rosenzwajg MD, M Fontfrede PharmD Safety committee Pr P Bougnères MD, Pr C Boitard MD Scientific watch E Piaggio PhD Modeling and Interim Analysis Clinical Pharmacology, Angers Hospital, Angers, France (Prof B. Diquet PharmD, C. Abbara PharmD) Biometry, Curie Institute, Paris, France (Prof B Asselain MD, Dr Y de Rycke PhD) Regulatory affairs and Monitoring Clinical Research and Development Department, APHP, Paris, France (V. Millul PhD) Clinical Research Unit Pitié-Salpêtrière, APHP, Paris, France (N Dedise CRA, D Wenjie Gu CRT) Randomization Clinical Research Unit-EST GH St Antoine, APHP, Paris, France (A Rousseau PhD) Treatment Management Pharmacy, Pitié-Salpêtrière Hospital, APHP, Paris, France (MH Fievet PharmD, G. Churlaud PharmD, PhD) Clinicians at recruiting centres -Department of endocrinology-diabetology, AP-HP, Ambroise Paré Hospital, Boulogne, France Univ Versailles SQY, Versailles, France (M-L Raffin-Sanson, MD) -Department of diabetology, AP-HP, Hôtel-Dieu Hospital, Paris, France (D Dubois-Laforgue, MD) -Department of endocrinology-diabetology-nutrition, AP-HP, La Pitié-Salpêtrière Hospital, Paris, France (Prof A Hartemann, O Bourron MD, S Jacqueminet MD) -Department of Internal Medicine, AP-HP, Henri Mondor Hospital, Creteil, France (G Lagadec, MD) -Department of internal medicine, Institut Mutualiste Montsouris, Paris, France (C Deybach, MD) -Centre Hospitalier de Rambouillet, Department of internal medicine, France, (M Popelier MD)

Fig 1 : 2

 12 Fig 1: Dose-dependent increase of Treg in T1D patients treated by ld-IL-2Each curve represents changes in CD25 high CD127 -Foxp3 + among CD4 + T cells (percentages

Fig 2 : 2

 22 Fig 2: Dose-dependent Treg activation in T1D patients treated by ld-IL-2 Curves represent changes in Treg activation markers. Percentage of CD25 high CD127 - CD45RA -Tregs among CD4 + cells (A). CD25 MFI (B), percentage of GITR + (C), CTLA-4 + (D) and basal pSTAT5 expression (E) in CD4 + CD25 high CD127 -Foxp3 + Tregs. Data are shown

Fig 3 :

 3 Fig 3: Selective IL-2-dependent activation of STAT5 by Tregs does not vary after ld-IL-

Fig 4 : 2

 42 Fig 4: Dose-dependent changes in cytokines and chemokines in the plasma of T1D

Fig 5 :

 5 Fig 5: Clustering of significantly regulated molecular signatures from PBMCs.

Fig 7 :

 7 Fig 7: Anti-IL-2 antibodies study

Acknowledgments

We are indebted to Christophe Misse, director of the Département de la Recherche Clinique et du Développement (DRCD) of the AP-HP, for initiating the support for this study. We thank the patients and the members of the DF-IL2-study group (listed below), and more specially Gilbert Bensimon for the study design and Christine Payan for data management and statistical analyses. We also thank all the personnel of the Endocrinology department, the Clinical Investigation Center Paris-est (CIC-PE) and the Clinical Investigation Center for Biotherapies (CIC-BTi) for their participation and more especially Nathalie Fery and Cornélia Degbe. DK, MR and TRM are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. MR and DK are inventors on a patent application related to the therapeutic use of ld-IL-2, which belongs to their academic institutions and has been licensed to ILTOO pharma. MR, GC and DK hold shares in ILTOO pharma. HPP is an employee of ILTOO pharma. No other potential conflicts of interest relevant to this article were reported. This work was funded by the DRCD, the CIC-PE and CIC-BTi, the Inserm and French state funds managed by the ANR within the Investissements d'Avenir programme (ANR-11-IDEX-0004-02). RM was supported by the Aviesan/AstraZeneca program. JB and AL were supported by the Immune Tolerance Network (NIH Contract #N01 AI15416), an international clinical research consortium headquartered at the Benaroya Research Institute and supported by the National Institute of Allergy and Infectious Diseases and the Juvenile Diabetes Research Foundation. This work was also supported by the Diabetes Research Institute Foundation, Hollywood, FL, the Peacock Foundation, Inc., Miami, FL, and the Anton E. B. Schefer Foundation, which supported AY, AP and TRM. -Department of endocrinology-diabetology, AP-HP, Saint-Antoine Hospital , Paris, F-75012, France (N Bourcigaux, MD, E Laroche MD, S Christin-Maitre MD) -Department of endocrinology-diabetology-nutrition, AP-HP, Jean Verdier Hospital, Bondy, France (E Cosson MD, B Donadille, MD, B Lormeau, MD)

Online supplemental materials

Supplementary figures and table

Supplementary