Lucía Sanjurjo

Iris A Schulkens

Pauline Touarin

Roy Heusschen

Ed Aanhane

Kitty C M Castricum

Tanja D De Gruijl

Ulf J Nilsson

Hakon Leffler

Arjan W Griffioen

Latifa Elantak

Rory R Koenen

Victor L J L Thijssen
email: v.thijssen@amsterdamumc.nl

PhD Victor Ljl Thijssen

Chemokines modulate glycan binding and the immunoregulatory activity of galectins

Introduction

The human glycome is a complex and dynamic collection of cellular glycans and glycoconjugates that are involved in a plethora of cellular functions. Alterations in the glycan composition have been linked to different pathologies including genetic disorders, immune diseases and cancer [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF] . In vertebrates, the information that is encoded by the glycome is deciphered by specific glycan binding proteins, called lectins. Interestingly, the complex repertoire of the glycome greatly exceeds the number of lectins [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF] . This suggests that specific mechanisms exist to regulate and fine-tune lectin binding affinity in order to cover the broad spectrum of the glycome.

Recently, it was shown that galectin-3, a member of the galectin protein family, can form heterodimers with the chemokine CXCL12. While galectins are known to exert their function by binding to glycoconjugates [START_REF] Thijssen | Galectin expression in cancer diagnosis and prognosis: A systematic review[END_REF] , the interaction was found to be glycan-independent and to hamper CXCL12 activity [START_REF] Eckardt | Chemokines and galectins form heterodimers to modulate inflammation[END_REF] . Apart from galectin-3/CXCL12, the authors found interactions of other chemokines with either galectin-3 or galectin-1 [START_REF] Eckardt | Chemokines and galectins form heterodimers to modulate inflammation[END_REF] . These observations support our previous finding that chemokine-like peptides can directly bind to different members of the galectin family [START_REF] Thijssen | Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy[END_REF][START_REF] Salomonsson | The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins[END_REF] . We described that the heterodimerization induces changes in the galectin protein structure which affects the glycan-binding affinity and specificity of galectin-1 [START_REF] Thijssen | Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy[END_REF][START_REF] Salomonsson | The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins[END_REF] . More recently, modulation of glycanbinding activity was also observed in response to the interaction of galectin-1 with a specific fragment of the pre-B-cell receptor [START_REF] Elantak | Structural basis for galectin-1-dependent pre-B cell receptor (pre-BCR) activation[END_REF][START_REF] Bonzi | Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions[END_REF] . Collectively, these findings suggest that heterodimerization of galectins with other proteins could represent a mechanism to regulate galectin-glycan interactions. Such a mechanism would extend the functionality of this glycome-decoding protein family. However, whether chemokines could represent a protein family that regulates galectin function is not yet fully understood.

In the present study, we present evidence that the galectin-chemokine heterodimerization can indeed serve as a mechanism to modulate galectin glycan-binding and function. We found specific interactions between different chemokines and two immunomodulatory galectins i.e., galectin-1 and galectin-9. Focusing on the interaction between platelet factor 4 (PF4/CXCL4) and galectin-1, we show that the interaction modifies the glycan-binding properties of galectin-1 by inducing structural changes within its carbohydrate binding site. Moreover, we provide evidence that this interaction can modulate the immunomodulatory activity of galectin-1 as well as of galectin-9 on specific T cell subsets. Our findings provide evidence of galectin-chemokine cross-talk which can serve as a regulatory mechanism to guide and fine-tune glycoconjugate-galectin interactions leading to specific cellular responses.

Results

Several specific and glycan-independent galectin-cytokine heterodimers exist.

The current study aimed to explore whether galectin-chemokine interactions could serve as a mechanism to modulate galectin glycan-binding and function. First, we performed an interaction screen (Supplementary figure 1) and observed several specific interactions of galectin-1 and galectin-9 with different chemokines and cytokines. Based on our recent work showing complementary platelet responses induced by galectin-1 and platelet factor 4 (PF4 or CXCL4), we focused our investigation on this galectin-chemokine pair [START_REF] Dickhout | Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro[END_REF] . Spot blot analyses showed that CXCL4 binds galectin-1 to a similar extent as the positive control peptide, i.e., anginex 4 (Figure 1a). To ensure that the interaction was not caused by changes in cytokine structure due to immobilization on the nitrocellulose filter, soluble galectin-1 with either CXCL4 or anginex were incubated in the presence of the crosslinking agent DSS. Subsequent gel-shift analyses confirmed the expected shift in protein size of galectin-1 monomers (±14 kD) corresponding to the size of either ±8 kD (CXCL4) or ±4 kD (anginex) (Figure 1b). The intensity of the shifted band was weak suggesting interference of the bound chemokine with antibody binding to galectin-1. Of note, crosslinking with CXCL4 also revealed a band just above the galectin-1 dimer which could either represent a single galectin-1 monomer bound to two CXCL4 molecules or a galectin-1 dimer bound to a single CXCL4 molecule. To confirm that the interaction involved CXCL4 and was also not caused by structural abnormalities due to the crosslinking agent, excess heparin was added prior to crosslinking. Heparin, a known high affinity ligand of CXCL4, serves as a CXCL4 scavenger and indeed completely neutralized the galectin-1/CXCL4 interaction (Figure 1c). Heparin did not cause a shift in galectin-1 size suggesting no interaction of both molecules. At the same time, the addition of excess lactose, a carbohydrate ligand of galectin-1, did not prevent the interaction (Figure 1d). The latter indicated that CXCL4 does not directly compete for glycan binding in the carbohydrate recognition domain of galectin-1. Furthermore, the interaction was dose-dependent and extended beyond the galectin-1/CXCL4 pair (Figure 1e + Supplementary Figure 1).

To characterize the binding kinetics of the galectin-1/CXCL4 heterodimer formation, surface plasmon resonance analysis was performed. Similar as previously described for the galectin-1:anginex interaction 4 , the galectin-1:CXCL4 interaction showed a 1:2 stoichiometry (single association followed by biphasic dissociation) with binding affinities in the high nanomolar and low micromolar range (Supplementary Figure 2 + Table 1). This also suggests that the high molecular weight band observed in the gel-shift assay most likely represents a galectin-1 monomer bound to a CXCL4 dimer. Collectively, all these data provide clear evidence of a direct interaction between galectin-1 and CXCL4.

Structural characterization reveals that CXCL4 binds outside the galectin-1 carbohydrate binding site.

To further characterize the galectin-1/CXCL4 interaction, chemical shift perturbation (CSP) mapping was performed using [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] N-labeled galectin-1 with or without CXCL4 protein. [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF] H- [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] N HSQC spectra (Figure 2a) show that in the presence of CXCL4, several galectin-1 resonances experience CSPs (chemical shift deviations and peak intensity decrease), indicative of complex formation (Figure 2b + Supplementary figure 3). Remarkably, perturbed resonances localize at two different sites on galectin-1 structure: (i) a surface at the edge of the galectin-1 ß-sandwich fold (opposite to the dimer interface) (Figure 2c), and (ii) a surface at the carbohydrate binding site (CBS) (Figure 2c). The first surface is defined by perturbed resonances belonging mainly to the ß6, ß8 and ß9 strands. Within the CBS, residues from strands ß3 to ß6 are perturbed upon CXCL4 interaction impacting not only the CBS core known to interact with the galactose unit but also the surrounding areas. However, our cross-linking data show that CXCL4 interaction does not compete with glycan binding activity of galectin-1, indicating that CXCL4 docks onto the perturbed surface of galectin-1 at the edge of its ßsheets and that this binding induces changes inside the CBS. To further investigate the structural basis of this interaction we performed NMR data-driven docking to generate a structural model of the galectin-1/CXCL4 complex. Residues from galectin-1 experiencing CSPs upon CXCL4 interaction and localized at the edge of the ß-sandwich fold were defined as ambiguous restraints in the docking calculations. On the CXCL4 side, residues from the ß-sheet, homologous to the anginex peptide sequence, were also considered as ambiguous restraints. In the structural model showing the best HADDOCK score, the CXCL4 ß-sheet docks onto the galectin-1 surface involving mainly ß6 and ß9 strands (Figure 2d). In addition, the CXCL4 C-terminal helix establishes contacts with strands ß8 and ß9 strands. To further validate the proposed structural model of the galectin-1/CXCL4 complex formation, the CXCL4 ß-sheet region (CXCL4 ; Supplementary figure 4a+b) was produced after which the interaction with [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] N-labeled galectin-1 was tested. The perturbations observed on galectin-1 spectra corresponded to residues from the CBS and from the ß6 and ß9 strands (Supplementary Figure 4c). When compared to CSPs induced by full-length CXCL4, the same regions of galectin-1 were affected with CXCL4 except for a few residues belonging to the ß8 and ß9 strands, which belong to the galectin-1 residues that interact with the CXCL4 C-terminal helix that is absent from CXCL4 (Supplementary Figure 4d+e). Therefore, these data confirm the involvement of both the CXCL4 ß-sheet and its C-terminal helix in the interaction with galectin-1.

Binding of CXCL4 to galectin-1 affects the glycan-binding affinity and specificity of galectin-1.

Interestingly, while interacting at the edge of the galectin-1 ß-sheet, CXCL4 induced chemical shift perturbations within the galectin-1 CBS. Previously, we have shown that the direct proteinprotein interactions can modulate the glycan binding affinity of galectin-1 [START_REF] Salomonsson | The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins[END_REF][START_REF] Bonzi | Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions[END_REF] . To explore whether binding of CXCL4 could also affect the carbohydrate binding affinity of galectin-1 we performed fluorescent anisotropy using a fluorescein-tagged high affinity tdga-probe [START_REF] Salomonsson | Monovalent interactions of galectin-1[END_REF] . Upon addition of 8 µM CXCL4, the baseline signal slightly increased, indicating only minimal interaction between CXCL4 and the probe. Subsequent titration of increasing concentrations of galectin-1 showed that the binding affinity of galectin-1 for this probe increased almost 10-fold to a K d of 0.09±0.01 as compared to galectin-1 alone (K d of 0.88±0.05 µM) (Figure 3a). In line with previous results 5 , the A max (anisotropy of the galectin-probe complex) was also increased in the presence of CXCL4, which indicates a reduced mobility of the fluorescein tag. Similar as in the gel-shift experiments, the increased binding was neutralized by the addition of heparin (Figure 3a). To further explore the modulation of glycan binding by CXCL4, comparable experiments were performed with a low affinity probe (lacto-Ntetraose; LNT-probe). While in the absence of CXCL4 no binding was observed due to very low A max , the addition of CXCL4 induced a measurable anisotropy and binding of the LNT-probe to galectin-1 with a K d in the range of the high-affinity tdga-probe, i.e., 0.48±0.18 µM (Figure 3b). At high galectin concentrations (>1.25 µM) the anisotropy levels decreased again, due to competition of the free galectin for the probe. The altered carbohydrate binding affinity was further confirmed by a competition experiment in which increasing concentrations of an unlabeled inhibitor of the galectin-1-probe interaction, i.e., the glycoprotein asialofetuin (ASF), was added to a fixed concentration of galectin-1 (the EC 50 of the corresponding slope in the absence of ASF). In the presence of CXCL4, the inhibitory potency of ASF was more than 600-fold increased (Figure 3c). Of note, the effects were galectin-1/CXCL4 specific as CXCL4 did not affect glycan binding of e.g. galectin-3 nor did CCL5 and IL-8 affect glycan binding affinity of galectin-1 (Supplementary figure 5). Altogether, these results show that heterodimerization with CXCL4 can alter the affinity and specificity of galectin-1 for glycan binding.

Galectin-chemokine interactions can modify the pro-apoptotic activity of galectins in immune cells.

To explore whether galectin-1/CXCL4 interactions represent an endogenous mechanism to control the function of galectin-1, we analyzed the effect of the heterodimer formation on immune cells. We focused on T cell apoptosis since galectins are known to induce apoptosis in activated T cells [START_REF] Toscano | Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death[END_REF][START_REF] Bi | Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways[END_REF][START_REF] Perillo | Apoptosis of T cells mediated by galectin-1[END_REF] . Indeed, in line with previous findings, we did see time-and concentration-dependent induction of T cell (Jurkat) apoptosis by galectin-1 (Supplementary figure 6a). Interestingly, apoptosis induction by galectin-1 was significantly increased by CXCL4 and not by CCL5 or CXCL8 (Figure 4a). The latter two were included since spot blot or gel shift assays indicated an interaction with galectin-1 as well (Figure 1e and Supplementary figure 1). The effects of CXCL4 were dose-dependent (Figure 4b) and could be blocked by the addition of either lactose, which competes for galectin-glycan binding, or heparin which scavenges CXCL4 (Figure 4c). CCL5 (RANTES) showed an interaction with both galectin-1 and galectin-9 (Supplementary figure 1), but did not affect galectin-1 function.

Therefore, we explored whether CCL5 could affect galectin-9-induced T cell apoptosis. Similar as for galectin-1, treatment with galectin-9 alone resulted in a time-and concentration-dependent induction of Jurkat apoptosis (Supplementary figure 6b). Interestingly, the induction of apoptosis by galectin-9 was specifically inhibited by CCL5 and not by CXCL4 or IL-8 (Figure 4d). Again, the effect was dose-dependent (Figure 4e) and could be neutralized by lactose (Figure 4f). These results suggest that heterodimerization with specific chemokines can have opposite effects on T-cell apoptosis induction by specific galectins.

Regulation of apoptotic activity by galectin-chemokine heterodimers depends on immune cell type and activation status.

To further extend these findings, we next analyzed the effects of specific galectin-chemokine heterodimers on peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. Firstly, we evaluated the effect of galectin-chemokine heterodimers on apoptosis in resting and activated PBMCs, the latter being more susceptible to galectin-induced apoptosis [START_REF] Perillo | Apoptosis of T cells mediated by galectin-1[END_REF] . Cell activation was induced by PHA-L lectin treatment and confirmed by CD25 surface staining (Supplementary figure 7). In resting PBMCs, neither CXCL4 nor CCL5 affected the induction of apoptosis by galectin-1 and galectin-9, respectively (Figure 5a). However, in activated PBMCs (1 µg PHA-L/mL), the pro-apoptotic effect of galectin-1 was significantly induced by CXCL4 while the pro-apoptotic effect of galectin-9 was significantly inhibited by CCL5 (Figure 5b). These observations corroborated our findings in Jurkat cells. Of note, in the absence of galectins, neither CXCL4 nor CCL5 induced apoptosis in resting or activated PBMCs (Supplementary figure 8).

To determine whether the effects could be linked to specific immune cell subsets, analyses were performed on different immune cell subpopulations, i.e., CD3 + T cells, CD3 + /CD8 + cytotoxic T cells, CD3 + /CD4 + T helper cells, and CD14 + monocytic cells (For gating strategy see Supplementary figure 9). Regarding the galectin-1/CXCL4 heterodimer, we observed a significant increment in the percentage of apoptotic cells in CD3 + cells (Figure 5c) upon the addition of CXCL4 to galectin-1.

Within the CD3 + population, the increase of galectin-1-mediated apoptosis by CXCL4 could be mainly attributed to the CD8 + cells while no effect was observed in CD4 + cells (Figure 5d). This suggests that within the analyzed T cell subsets, CD3 + /CD8 + cells are particularly susceptible to galectin-1/CXCL4induced apoptosis. The modulation of galectin-9 by CCL5 appears to display a different specificity regarding the modulation of immune cell survival. While CCL5 reduced the galectin-9-induced percentage of apoptotic CD3 + cells (Figure 5e), this was only significant for CD3 + /CD4 + cells and not for CD3 + /CD8 + cells (Figure 5f). Comparable results were observed in CD14+ cells in which CXCL4 increased the effect of galectin-1 and CCL5 reduced the effect of galectin-9 (Supplementary figure 10). Altogether, these results suggest that galectin-chemokine heterodimers can alter the apoptotic activity of specific galectins on specific immune cell subpopulations.

Discussion

In this study, we present evidence that specific galectins and chemokines can form heterodimers that affect the function of galectins through modifications of their glycan binding activity. In particular, we show that galectin-1/CXCL4 and galectin-9/CCL5 heterodimers can exert opposing immunoregulatory activity with regard to immune cell apoptosis. This reveals a cross-talk mechanism by which galectins and chemokines can jointly control the immune response.

Both galectins and chemokines are well established (immuno)regulatory protein families that exert a plethora of biological functions, including regulation of (immune) cell recruitment, growth, differentiation, and survival [START_REF] Nagarsheth | Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy[END_REF][START_REF] Rabinovich | Regulatory circuits mediated by lectin-glycan interactions in autoimmunity and cancer[END_REF][START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] . Thus far, both galectins and chemokines have been considered as distinct immunomodulatory protein families. However, there is increasing evidence that suggests a link between both. For example, it has been recently shown in two studies that galectin-3 can directly interact with interferon-gamma [START_REF] Gordon-Alonso | Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration[END_REF] and CXCL12 3 , thereby affecting their activity. While these findings show that galectin heterodimers can govern cytokine/chemokine function, our results show that the interaction can also regulate the function of galectin-1 and galectin-9. This adds an additional level to the immunomodulatory activity of these two galectins. As shown in the current study, a potential mechanism underlying the observed effects might involve modulation of the galectin glycan-binding affinity and specificity by inducing structural changes in the carbohydrate binding site. Previously, it has been shown that introducing mutations distal from the carbohydrate binding site can also result in small modifications in the structure of galectin-1 that affect glycan binding affinity [START_REF] López-Lucendo | Growth-regulatory human galectin-1: crystallographic characterisation of the structural changes induced by single-site mutations and their impact on the thermodynamics of ligand binding[END_REF] . In addition, we and others have previously reported on a close relationship between the galectin CRD structure and ligand binding [START_REF] Salomonsson | Monovalent interactions of galectin-1[END_REF][START_REF] Nesmelova | Lactose binding to galectin-1 modulates structural dynamics, increases conformational entropy, and occurs with apparent negative cooperativity[END_REF][START_REF] Chien | Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange[END_REF][START_REF] Romero | Impact of human galectin-1 binding to saccharide ligands on dimer dissociation kinetics and structure[END_REF] . In line with this, we have previously described that non-endogenous chemokine-like peptides can directly bind to galectins and affect glycan binding affinity [START_REF] Salomonsson | The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins[END_REF] . More recently, we provided the first evidence that galectin-1 directly interacts with an endogenous protein, i.e., the pre-B cell receptor. This interaction also induces structural changes in galectin-1 which modulates glycan binding affinity, particularly with regard to LacNAc containing epitopes [START_REF] Elantak | Structural basis for galectin-1-dependent pre-B cell receptor (pre-BCR) activation[END_REF][START_REF] Bonzi | Pre-B cell receptor binding to galectin-1 modifies galectin-1/carbohydrate affinity to modulate specific galectin-1/glycan lattice interactions[END_REF] .

Our current findings extend these observations and suggest that the observed galectin-chemokine heterodimerization could represent a mechanism that allows the modification of the glycan-binding affinity and even specificity of galectin-1 and galectin-9. At the same time, the exact mechanism underlying the glycan-binding effects of the galectin-cytokine interactions reported here is still unknown and might also involve, e.g., allosteric effects, as shown for other peptides/molecules [START_REF] Dings | Antitumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding[END_REF][START_REF] Dings | Structure-Based Optimization of Angiostatic Agent 6DBF7, an Allosteric Antagonist of Galectin-1[END_REF][START_REF] Ermakova | Lactose binding to human galectin-7 (p53-induced gene 1) induces longrange effects through the protein resulting in increased dimer stability and evidence for positive cooperativity[END_REF][START_REF] Miller | Targeting the CRD F-face of Human Galectin-3 and Allosterically Modulating Glycan Binding by Angiostatic PTX008 and a Structurally Optimized Derivative[END_REF] .

Thus, while the exact reach and mechanisms of galectin-chemokine interactions remain to be explored, it is tempting to speculate that such interactions might be involved in adapting the glycanbinding repertoire of galectins. This is supported by our current results showing heterodimer formation of different chemokines with galectin-1 and/or galectin-9 as well as by a recent study by Eckardt et al. that identified several specific chemokine interactions with galectin-1 and/or galectin-3 [START_REF] Eckardt | Chemokines and galectins form heterodimers to modulate inflammation[END_REF] . Of note, while some interactions were confirmed by both Eckhardt et al. and us, e.g., gal-1/CCL5, others were not, including gal-1/CXCL4. This is most likely related to differences in experimental setup and these findings warrant further studies to unravel the specific features that govern galectinchemokine heterodimerization and to explore the functional consequences of these heterodimeric complexes.

Regarding the functional implications, we focused here on T cell apoptosis, instigated by the established role of galectin-1 and galectin-9 in this process [START_REF] Toscano | Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death[END_REF][START_REF] Bi | Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways[END_REF][START_REF] Perillo | Apoptosis of T cells mediated by galectin-1[END_REF] . For example, these two galectins, as well as others, can impair T cell function by inducing T cell apoptosis and skewing T cell differentiation towards a suppressive phenotype [START_REF] Toscano | Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death[END_REF][START_REF] Bi | Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways[END_REF][START_REF] Stillman | Galectin-3 and galectin-1 bind distinct cell surface glycoprotein receptors to induce T cell death[END_REF][START_REF] Wada | Developmental regulation, expression, and apoptotic potential of galectin-9, a beta-galactoside binding lectin[END_REF] . Our results confirm the previously described pro-apoptotic activity of galectin-1 and galectin-9 on T cells [START_REF] Toscano | Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death[END_REF][START_REF] Bi | Structural features of galectin-9 and galectin-1 that determine distinct T cell death pathways[END_REF] . In addition, our current data show that this pro-apoptotic activity can be either increased or inhibited by specific galectinchemokine heterodimers, i.e., gal-1/CXCL4 or gal-9/CCL5, respectively. The mechanism underlying this regulation should be further explored but it most likely involves an altered glycan-binding affinity of the specific galectin-chemokine heterodimers. For example, several T cell glycoproteins, e.g., CD3, CD7, CD43, CD45, have been linked to galectin-1-mediated T cell apoptosis [START_REF] Perillo | Apoptosis of T cells mediated by galectin-1[END_REF][START_REF] Pace | CD7 delivers a pro-apoptotic signal during galectin-1-induced T cell death[END_REF][START_REF] Pace | Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1[END_REF] . In addition, it has been shown that the presence of specific glycan repertoires controls the ability of galectin-1 to induce apoptosis in specific T helper cells [START_REF] Toscano | Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death[END_REF] . Thus, while speculative, it can be hypothesized that (changes in) the immune cell glycan signature together with altered glycan-binding affinity of galectin-chemokine heterodimers will determine whether or not certain immune cell subset will undergo apoptosis. The observation that the effects were more potent in Jurkat cells as compared to primary PBMCs could reflect the diverse glycosylation of different immune cell subsets in the latter.

In line with this, the regulatory effects in PBMCs were only observed after PHA activation, suggesting a role for altered glycosylation patterns in activated vs. non-activated cells. Thus, future studies should thus aim at deciphering which immune cell subsets are particularly sensitive to specific galectin-chemokine heterodimers and whether this is linked to the cell surface glycan signatures and activation status.

Apart from further exploring the role of galectin-chemokine interactions in the regulation of immune cell apoptosis, our findings raise additional questions that should be addressed. For example, it is currently unknown to what extent specific galectin-chemokine heterodimerization affects other processes of the immune response, e.g., cell growth or cell differentiation/maturation. It has been shown that both galectin-1 and CXCL4 are involved in Th17 differentiation [START_REF] Cedeno-Laurent | Galectin-1 triggers an immunoregulatory signature in Th cells functionally defined by IL-10 expression[END_REF][START_REF] De La Fuente | The leukocyte activation receptor CD69 controls T cell differentiation through its interaction with galectin-1[END_REF][START_REF] Affandi | CXCL4 is a novel inducer of human Th17 cells and correlates with IL-17 and IL-22 in psoriatic arthritis[END_REF] . Whether the galectin-1/CXCL4 heterodimers stimulate or hamper this effect is unknown. In addition, next to immune cells, both galectin-1 and CXCL4 have been shown to regulate other cell types as well, e.g., endothelial cells during angiogenesis [START_REF] Thijssen | Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy[END_REF][START_REF] Croci | Glycosylation-Dependent Lectin-Receptor Interactions Preserve Angiogenesis in Anti-VEGF Refractory Tumors[END_REF][START_REF] Maione | Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides[END_REF][START_REF] Sharpe | Growth inhibition of murine melanoma and human colon carcinoma by recombinant human platelet factor 4[END_REF] and platelets during hemostasis [START_REF] Pacienza | The immunoregulatory glycan-binding protein galectin-1 triggers human platelet activation[END_REF][START_REF] Eslin | Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin[END_REF] . Regarding the latter, we have recently shown that galectin-1 and CXCL4 colocalize on the platelet surface [START_REF] Dickhout | Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro[END_REF] . Since coincubation did not affect binding of galectin-1 to platelets and the observed effects on platelet function appeared to be additive rather than synergistic, it appears that this specific heterodimer does not play a major effect in platelet homeostasis. Whether this is true for other cell types and other heterodimers remains to be studied. However, given our current observations, it can be assumed that the effects of certain galectin-chemokine heterodimerization extend beyond the immune cell compartment.

Our finding that galectin-chemokine interactions modulate glycan binding affinity and specificity could also have implications for the efficacy of glycan-based small molecule inhibitors that target galectins [START_REF] Hirani | Target-inhibition of Galectin-3 by Inhaled TD139 in Patients with Idiopathic Pulmonary Fibrosis[END_REF] and the growing arsenal of drugs that unleash the immune system against tumors [START_REF] Berraondo | Cytokines in clinical cancer immunotherapy[END_REF][START_REF] Mahoney | Combination cancer immunotherapy and new immunomodulatory targets[END_REF] . It is essential to unravel the mechanisms by which the local tumor microenvironment (TME) controls the immune response and how these mechanisms interact [START_REF] Toscano | Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death[END_REF][START_REF] Munn | Immune suppressive mechanisms in the tumor microenvironment[END_REF] . Especially since tumor-controlled immunomodulation frequently involves impaired recruitment and/or function of immune cells due to the presence or absence of soluble factors like galectins and chemokines [START_REF] Sharma | Adaptive, and Acquired Resistance to Cancer Immunotherapy[END_REF][START_REF] Crome | A distinct innate lymphoid cell population regulates tumor-associated T cells[END_REF][START_REF] De Henau | Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells[END_REF] . In that regard, the local balance between galectins, chemokines and their heterodimers could underlie the opposing effects seen in different experimental settings and biological contexts, e.g., the pro-or antiapoptotic activity of CXCL4 [START_REF] Deng | Non-platelet-derived CXCL4 differentially regulates cytotoxic and regulatory T cells through CXCR3 to suppress the immune response to colon cancer[END_REF][START_REF] Gao | Activation of p38-MAPK by CXCL4/CXCR3 axis contributes to p53-dependent intestinal apoptosis initiated by 5-fluorouracil[END_REF][START_REF] Liang | Platelet factor 4 induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in multiple myeloma[END_REF][START_REF] Kasper | CXCL4-induced monocyte survival, cytokine expression, and oxygen radical formation is regulated by sphingosine kinase 1[END_REF][START_REF] Kasper | Platelet factor 4 (CXC chemokine ligand 4) differentially regulates respiratory burst, survival, and cytokine expression of human monocytes by using distinct signaling pathways[END_REF] . This also links to the recent findings that galectins can serve as scavenger molecules for cytokines/chemokines, thereby hampering immune cell recruitment [START_REF] Eckardt | Chemokines and galectins form heterodimers to modulate inflammation[END_REF][START_REF] Gordon-Alonso | Galectin-3 captures interferon-gamma in the tumor matrix reducing chemokine gradient production and T-cell tumor infiltration[END_REF] . An additional intriguing but unexplored concept is whether galectins not only scavenge but also serve as chaperones for cytokines and chemokines by directing them, in a cytokine/chemokine-specific manner, to sites displaying a specific glycosylation pattern, e.g., glycoprotein cell-surface receptors. Further insight in the reciprocal relationship between galectins and cytokines/chemokines will therefore likely benefit the development of more effective treatment strategies.

A limitation of the current study is that we only explored the effects of specific galectinchemokine combinations in vitro. To better understand the contribution of these interactions in different biological processes, in vivo experiments should be performed. However, to our opinion, this first requires further insight in the complex interactions between galectins and cytokines that might occur in vitro. For example, it has been suggested that heterodimers can also form between different members of the galectin family [START_REF] Miller | Adhesion/growth-regulatory galectins tested in combination: evidence for formation of hybrids as heterodimers[END_REF] or the chemokine family [START_REF] Von Hundelshausen | Heterophilic interactions of platelet factor 4 and RANTES promote monocyte arrest on endothelium[END_REF][START_REF] Koenen | Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice[END_REF] . This suggests the existence of a complex regulatory network that links the local expression levels of galectins and chemokines to the complex glycome in order to regulate cellular functions in vivo. The width of such a network is still unknown and while the present in vitro study adds to the identification of this complex network, a full understanding of the extent of specific galectin-chemokine interactions and how they contribute to in vivo biology requires further research.

In summary, we present evidence that galectins and chemokines can directly interact and that this heterodimerization can affect the glycan-binding properties and immunoregulatory activity of galectins. These findings suggest the existence of a reciprocal mechanism to fine-tune the activity of galectins by chemokines. A better understanding of this mechanism will provide insight in the complex process of immunomodulation and will help the development of future (immuno)therapeutic strategies.

Materials and Methods

Materials

Recombinant galectin-1 and galectin-9 were in-house produced or purchased from R&D systems. For fluorescent anisotropy and apoptosis assays, the oxidation resistant mutant galectin-1 [START_REF] Hirabayashi | Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin[END_REF] , i.e., galectin-1 C3S (in-house produced) was used.

The synthetic high-affinity tdga-probe (0.1 µM, 3'-[4-(fluorescein-5-amidomethyl)-1H-1,2,3-triazol-1yl]-3'-deoxy-β-D-galactopyranosyl 3-(3,5-dimethoxybenzamido)-3-deoxy-1-thio-β-Dgalactopyranoside) [START_REF] Salomonsson | Monovalent interactions of galectin-1[END_REF] as well as the low affinity probe (lacto-N-tetraose; LNT-probe) [START_REF] Carlsson | Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface[END_REF] were in-house produced.

Chemokines CXCL4 and CCL5 were either in-house produced [START_REF] Koenen | Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice[END_REF] or purchased from R&D systems. All other chemokines and cytokines were purchased from R&D systems. The beta-sheet region, CXCL4 22- 54 , was chemically synthesized and purchased from Schafer-N. Anginex was a kind gift from prof. K.

Mayo (University of Minnesota, Minneapolis, USA). Antibodies were purchased from BD Biosciences unless indicated otherwise.

Primary cells and cell lines

The human leukemia cell line Jurkat (originally purchased from and STR authenticated by ATCC) was maintained in RPMI 1640, 2 mM glutamine medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). Cells were cultured in a humidified incubator at 37°C and 5% CO2.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors (Purchased from Sanquin, no approval from ethical committee required) by density gradient centrifugation using Lymphoprep (Nycomed Pharma). Recovered cells were washed twice in sterile PBS with 0.1% bovine serum albumin and resuspended in RPMI-1640 2 mM glutamine (Gibco), containing 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin (Invitrogen). When indicated, PBMCs were treated with 1 µg/mL phytohemagglutinin-L (PHA-L, Invitrogen) for 24 hours prior to stimulation with galectins/cytokines. All studies involving human samples were conducted following the Declaration of Helsinki principles and current legislation on the confidentiality of personal data.

Spot blot

Spot blot analysis was performed with 10 µL drops containing 3 µg of the specific proteins that were spotted onto a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 hour, washed with PBS/0.1% Tween, and incubated with 1 µg/ml recombinant galectin-1 or galectin-9 in PBS/0.1% Tween for 1 hour. As control, the incubation step with recombinant galectin-1 was omitted. After washing with PBS/0.1% Tween, the membrane was incubated with rabbit anti-galectin-1 antibody (1:1000, Peprotech) or goat anti-galectin-9 antibody (1:250, R&D Systems) for 1 hour. Following three washing steps, the appropriate IRDye-labeled secondary antibody (1:10000, LI-COR Biosciences) was applied for 1 hour. Finally, the membrane was washed twice with PBS/0.1% Tween and rinsed in PBS. Images were obtained by scanning the membrane with the Odyssey infrared imaging system (LI-COR Biosciences).

Gel-shift analysis

For gel-shift analysis, 2 µg of the specific proteins were combined in a total volume of 10 µL in PBS and incubated together with 2 µL DSS (Disuccinimidyl suberate, Thermo Scientific) for 2 hours on ice to fix existing protein interactions. Where indicated, 25 mg/mL heparin or 5 mM lactose (Sigma Aldrich) was added. The reaction was quenched with 1 µL Tris buffer pH 7.5 for 15 minutes at room temperature. Subsequently, the protein mixture was suspended in Laemmli sample buffer (Biorad) supplemented with 1:20 β-mercapto-ethanol, boiled for 5 minutes and subjected to SDS-PAGE followed by Western immunoblotting using anti-galectin-1 antibody (1:1000, Peprotech). Following three washing steps, the appropriate IRDye-labeled secondary antibody (1:10000, LI-COR Biosciences) was applied for 1 hour. Finally, the membrane was washed twice with PBS/0.1% Tween and rinsed in PBS. Images were obtained by scanning the membrane with the Odyssey infrared imaging system (LI-COR Biosciences).

Surface plasmon resonance

Binding kinetics between galectin-1 and CXCL4 were studied using the BIAcore 1000 biosensor system (BiaCore) as described previously [START_REF] Thijssen | Galectin-1 is essential in tumor angiogenesis and is a target for antiangiogenesis therapy[END_REF] . In short, galectin-1 was covalently attached to CM5 Sensor Chips (BiaCore) using amine-coupling chemistry according to the manufacturer's instructions. Next, binding measurements were performed at 25°C using HBS-EP buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.005 % surfactant P20 adjusted to pH 7.4. Interactions between galectin-1 and CXCL4 were analyzed by injection of different CXCL4 concentrations (20 µL at a flow rate of 30 µL/minute). Flow cells were regenerated by injection of 20 µL regeneration buffer (10 mM glycine-HCl, pH 2.0). Association-rate (K a ) and dissociation-rate (K d ) constants were obtained by analysis of the sensorgrams using the Biaevaluation software, version 3.2. All measurements were performed at least in duplicate at all concentrations and the experiment was performed in duplo.

Fluorescence anisotropy

Fluorescence anisotropy analysis was performed as described previously [START_REF] Salomonsson | The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins[END_REF][START_REF] Sörme | Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions[END_REF] . For these experiments, the oxidation-resistant mutant galectin-1, i.e., galectin-1 C3S, was used to avoid the necessity to add β-mercaptoethanol, which may disturb interacting glycoprotein ligands [START_REF] Hirabayashi | Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin[END_REF][START_REF] Nishi | Functional and structural bases of a cysteine-less mutant as a long-lasting substitute for galectin-1[END_REF] . In brief, 40 µL reaction mixture containing different concentrations of galectin-1 C3S and 0.1 µM of fluorescein labeled saccharide probes either with or without CXCL4 at the indicated concentrations was incubated under slow rotary shaking for 5 minutes in black 96-well half-area polystyrene microplates. All dilutions were prepared in HEPES buffer with pH 7.4 and in the absence of salt to prevent precipitation of CXCL4. Fluorescence anisotropy of fluorescein-tagged saccharide probes was measured with excitation at 485 nm and emission at 520 nm using a using a PheraStarFS plate reader with software PHERAstar Mars version 2.10 R3 (BMG). Binding curves were generated by plotting anisotropy against increasing concentrations of galectin-1. Anisotropy was measured without galectin-1 (A 0 representing free probe), at experimental conditions (A) and at saturating concentrations of galectin-1 (A max ). The experiments were performed at least in duplicate. For the inhibition assay, increasing concentrations of the glycoprotein asialofetuin (ASF), an inhibitor of the galectin-1-probe interaction, were added to fixed low concentrations of galectin-1C3S (0.1 µM) and tdga-probe in the presence or absence of 8 µM CXCL4. Data plotting, non-linear regression analysis and curve construction was performed with Prism 9 software (Graphpad).

Nuclear magnetic resonance

For NMR experiments, galectin-1 has been produced as [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] N-labeled protein as previously described [START_REF] Elantak | Structural basis for galectin-1-dependent pre-B cell receptor (pre-BCR) activation[END_REF] and prepared in 20 mM KPO 4 pH 5.6 buffer with 8% D 2 O. All NMR spectra were acquired at 30°C on a Bruker AVANCEIII spectrometer operating at 600 MHz and equipped with cold probe. NMR data were processed with TopSpin and analyzed with Ccpnmr Analysis [START_REF] Vranken | The CCPN data model for NMR spectroscopy: development of a software pipeline[END_REF] . For interaction experiments with CXCL4, 1 H-15 N-HSQC spectra have been recorded on [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] N-labeled galectin-1 (40 µM) in the absence and in the presence 80 µM of CXCL4. The same experiments have been performed with [START_REF] Zlotnik | The chemokine superfamily revisited[END_REF] N-labeled galectin-1 (350 µM) in the presence of CXCL4 (700 µM). The following formula was used for extracting chemical shift perturbations (CSPs), which takes into consideration the [START_REF] Cummings | The repertoire of glycan determinants in the human glycome[END_REF]

NMR-Data Driven Docking

Models of galectin-1 in complex with CXCL4 were built with the version HADDOCK2.4 of "The HADDOCK web server for data-driven biomolecular docking" [START_REF] Van Zundert | The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes[END_REF] . As starting structures, we used the structures of galectin-1 (PDB ID 2KM2) and CXCL4 (PDB ID 1F9Q). The galectin-1 and CXCL4 structures were then docked within HADDOCK. Residues of galectin-1 that showed CSPs above the threshold were considered as active Ambiguous Restraints (AIR). On the CXCL4 side, the beta-sheet followed by FACS analysis with gating for specific subpopulations, i.e., CD3 + , CD3 + /CD4 + , CD3 + /CD8 + .

Graphs show percentage of Annexin A5+ cells within the indicated populations. N≥6.

2 +(Δδ N 2 /

 22 H and 15 N chemical shift range of NMR signals: CSP (ppm)= =*Δδ HN 25)] 1/2 where Δδ HN and Δδ N are, respectively, the proton and nitrogen chemical shift variations of each residue.

Figure 1 .a

 1 Figure 1. Specific protein-protein interactions occur between galectin-1 and different chemokines.

Figure 2 .ad

 2 Figure 2. Structural characterization of the galectin-1/CXCL4 complex. a Overlay of 1 H-15 N HSQC spectra of 15 N-labeled galectin-1 free (black trace) and bound to CXCL4

Figure 3 . 1 .a

 31 Figure 3. Interactions between galectin-1 and CXCL4 affect glycan-binding affinity and specificity of

Figure 4 .a

 4 Figure 4. Specific galectin-chemokine interactions modify the pro-apoptotic function of galectins on

Figure 5 .a

 5 Figure 5. Specific galectin-chemokine interactions modify the pro-apoptotic function of galectins on

Table 1 .

 1 Rate and affinity constants for galectin-1 bindingThe association rate constant (k a ) was obtained by plotting the k obs as a function of analyte concentration. K 1 is defined as k d1 /k a and K 2 is defined as k d2 /k a . BSA = bovine serum albumin. -= No interaction detected.

	Analyte k a (Ms-1)	k d1 (s-1)	k d2 (s-1)	K 1	K 2
	Anginex 6.5x10 3 (±1.7)	4.2x10 -2 (±0.4) 5.9x10 -4 (±1.9) 6.4±1.7 μM 90.0±6.7 nM
	CXCL4	1.02x10 4 (±0.05) 9.2x10 -2 (±1.2) 5.0x10 -3 (±0.5) 9.0±0.8 μM 483±28 nM
	BSA	-	-	-	-	-

Acknowledgements

The authors thank prof. Kevin H Mayo (University of Minnesota) for providing anginex and Anita Stam for excellent technical assistance. Part of this research was supported by a grant of the Dutch Cancer Society (KWF11040 to VLT).

Data availability

The data that support the findings of this study are available in the supplementary material of this article (Supplementary data). This includes the original Western blot scans from Figure 1b-e and the data presented in Figures 345.

residues homologous to the anginex [START_REF] Griffioen | Anginex, a designed peptide that inhibits angiogenesis[END_REF] sequence were considered as AIR. Calculations were performed with 2,000 structures during the HADDOCK rigid body energy minimization, 400 structures during the refinement, and 200 structures during the refinement in explicit water.

Apoptosis assays

For analysis of apoptosis, either 1x10 [START_REF] Salomonsson | The anti-angiogenic peptide anginex greatly enhances galectin-1 binding affinity for glycoproteins[END_REF] Jurkat cells or PBMCs per well were plated in 24-well plates and incubated for the indicated time and concentration with galectin-1 or galectin-9 alone or in combination with the indicated concentration of chemokines.

Following treatment, Jurkat cells were washed twice in ice-cold PBS and stained with (FITC)conjugated annexin A5 (Immunotools) and propidium iodide, following the manufacturer´s instructions. Cells were subsequently analyzed using a FACSCalibur instrument (BD Biosciences).

When indicated, 0.1 M lactose (Sigma-Aldrich) or 0.1 M heparin (Leo Pharma) were added to the cell culture just prior to galectin/chemokine treatment. Experiments were performed at least in triplicate.

Following PBMC treatment, PBMCs were blocked during 20 minutes with 1% BSA/PBS at 4°C, washed in ice-cold PBS and incubated during 30 minutes at 4°C with a combination of fluorescently conjugated monoclonal antibodies against CD3, CD8, CD4 and CD14: Anti CD3-APC (BD Clone SK7), anti-CD8-V500 (BD Clone SK1), anti-CD4-PerCP Cy5.5 (BD Clone SK73), anti-CD14-PE BD Clone MφP9). Subsequently, cells were washed twice with 0.1% BSA/PBS and stained with (FITC)-conjugated annexin A5 (Immunotools) following the manufacturer´s instructions. Data acquisition was performed using a FACSCanto II instrument (BD Biosciences). Experiments were performed using PBMCs from at least six different donors. All data were analyzed using Kaluza analysis software (Beckman Coulter).

Statistics and reproducibility

Of all experiments, the individual data are presented together with the mean of at least three independent experiments unless indicated otherwise. To calculate statistically significant differences of the Jurkat and PBMC apoptosis data, the paired student's t-test and the one-way ANOVA with Tukey's multiple comparison tests were used. Statistical computations were performed using Graphpad Prism software (v8 or v9) and p-values < 0.05 were considered as statistically significant.

Competing interests

The authors declare the following competing interests: H.L. and U.J.N are shareholders in Galecto Biotech AB, a company developing galectin inhibitors. The remaining authors declare no no competing interests.

Author contributions

LS, IS, PT, RH, EA, KC, VT conducted experiments and generated data. LS, IS, PT, RH, HK, AWG, LE, RK, VT analyzed and interpreted data. TdG, UN, HK, AWG, LE, RK, VT conceived, designed and/or supervised experiments. LS, IS, VT wrote the main manuscript. All authors approved the final manuscript.


Jim Tesson

Lucilla Benedetti

Vincent Godard

Catherine Novaes

Jules Fleury

Aster Team

Slip rate determined from cosmogenic nuclides on normal-fault facets

INTRODUCTION

Facets are characteristic landforms associated with the activity of normal faults [START_REF] Wallace | Geometry and rates of change of fault-generated range fronts, northcentral Nevada[END_REF][START_REF] Armijo | Quaternary extension in southern Tibet: Field observations and tectonic implications[END_REF]). But their potential as tectonic markers and the possibility of retrieving quantitative information on long-term slip rates from their morphological attributes and rates of evolution have seldom been considered (Petit et al. 2009, Tucker et al.

1-THEORETICAL 36 CL BUILD UP ON A TRIANGULAR FACET

Faceted spurs are near planar triangular or trapezoidal surfaces produced by the erosion of normal fault scarps that are continuously rejuvenated at their base by sustained slip [START_REF] Wallace | Geometry and rates of change of fault-generated range fronts, northcentral Nevada[END_REF]). Here we focus on normal fault footwalls formed in resistant bedrock and undergoing a weathering-limited evolution [START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF]. Morphological evolution models [START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF][START_REF] Strak | Interaction between normal fault slip and erosion on relief evolution: Insights from experimental modelling[END_REF], Tucker et al. 2020) suggest facet morphology reflects an equilibrium between rock uplift from fault slip and erosion.

At the base of the facets, well-preserved bedrock fault scarps representing the cumulative displacement on the fault since the last glacial maximum can be observed [START_REF] Wallace | Geometry and rates of change of fault-generated range fronts, northcentral Nevada[END_REF], Armijo et al. 1992). This has been confirmed in the Mediterranean by exposure-age dating in Greece, Italy, Israel, Turkey (e.g. [START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF][START_REF] Cowie | Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults[END_REF]. Post-glacial changes in climatic conditions result in a decrease in hillslope erosion rates and preservation of those bedrock fault scarps (Armijo et al. 1998[START_REF] Allen | Rapid environmental changes in southern Europe during the last glacial period[END_REF]. The timing of this shift is not accurately dated and may spatially vary. In the Apennines, it is estimated to be 12-21 ka [START_REF] Giraudi | Middle Pleistocene glaciations in the Apennines, Italy: new chronological data and preservation of the glacial record[END_REF].

Based on these previous observations we use a two-stage kinematic model to predict 36 Cl buildup in material currently exposed along the facet surface. First, under glacial conditions, we assume topographic steady state, such that rock uplift of the footwall is equal to the denudation rate of the facet surface. Rock sample are progressively exhumed parallel to the slip vector at a rate set by the fault slip-rate (Fig 2). Second, in post glacial conditions, we assume denudation of the facet to be negligible, such that rock samples are continuously exposed at the surface, with no significant regolith cover [START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF].

Our approach relies on the interpretation of measured 36 Cl concentration at the surface in terms of this exhumation and exposure history of the facet. Based on the conceptual evolution described above, we parametrize this evolution using the time at which the sample starts moving towards the surface (T exhum ), the rate at which the sample moves parallel to fault motion prior to post-glacial conditions (SR), and the duration of post-glacial exposure assumed without erosion (T pg ). We combine this simple model with equations describing cosmogenic nuclides buildup to predict 36 Cl concentrations at the surface of the facet for any combination of the 3 parameters (T exhum , T pg , SR) defining the prescribed history (Suppl.).

2-36 CL CONCENTRATION PROFILES ON 4 FACETED SPURS IN CENTRAL

APENNINES

We focus on 4 facets formed in limestones located along 3 active faults in central Apennines (Italy). Since late Pliocene-early Pleistocene, active normal faults accommodate the SW-NE extension in the area [START_REF] Roberts | Spatial and temporal variations in growth rates along active normal fault systems: an example from The Lazio-Abruzzo Apennines, central Italy[END_REF]. The Velino-Magnola fault is a major structure of the range (~30 km long, Fig. 1) with facets up to 300-800 m high, where we sampled two sites MA1 and MA3 along the northern and central part of the fault, respectively. The Roccapreturo fault is shorter (10 km), with lower facets (100-150 m), and was sampled at site AR in the center of the fault. The Bazzano fault is a small (3 km long) antithetic fault, with several facets reaching 100-150 m high, sampled in its southern section at site BAZ (Suppl.). The surface of the 4 sampled facets displays regular slope 30-35° (Fig 3). The surface of the facet is characterized by an alternance of bedrock outcrop, small bedrock protrusions (< 1m high), and patches of soil and screes less than a few cm thick. The facets are generally bounded by entrenched non-perennial streams that flow perpendicular to the fault's strike, which suggests recent incision that is contemporaneous with fault development. The slopes of the colluvial wedge surface (α~25-30) and of the facets (γ~30-35) are similar across sites, the fault dips (β) from 40° at MA1, to 65° at BAZ (Suppl.).

The facets were sampled following an up-dip fault transect (Suppl.). We focused sampling on the lower part of the facet, where the surface appears less rugged, and near the facet center to avoid increased erosion by bounding gullies. Samples are 5-10 cm thick pieces of bedrock, taken on the top of protruding (> 0.5 m) bedrock surfaces, avoiding places that could have been covered by regolith. Each sample was chemically prepared for 36 Cl measurements (Suppl.). At MA3 and ARC, the 36 Cl concentrations are roughly constant all along the facet (Fig. 3), except at the base where they increase by about 30%. At MA1 and BAZ, facets are lower and there are fewer points of measurements, but we observe a similar pattern at MA1 with constant values at the top, a slight decrease and an abrupt increase at the base of facet.

There are only 3 data points at BAZ with decreasing concentrations toward the base. The 36Cl concentrations along all studied facets are thus roughly constant suggesting that the denudation rate on those features is approximately constant. This observation is consistent with a facet development model that assumes a constant denudation rate. Moreover while it has been usually assumed that erosion would vary with facet height (Tucker et al. 2020 and references there in), our results suggest that, on the facet we studied, this is not the case.

3-FAULT SLIP-RATE FROM 36 CL DATA INVERSION

To constrain the fault slip-rate we carried out a Bayesian inversion of the observed 36 Cl concentrations where the model parameters are the individual slip-rates of the faults (SR MA3 , SR MA1 , SR ARC , SR BAZ ), and the post-glacial period duration (T PG ), assumed to be the same for the 4 sites (T exhum is fixed a priori see suppl.). A complete description of the inversion procedure using the GW-MCMC algorithm [START_REF] Goodman | Ensemble samplers with affine invariance[END_REF], the corresponding parameters and the numerical code are provided in the Suppl. The inversion yielded mean slip-rates of 3.0 ± 1.2 mm/yr for MA3, 0.2 ± 0.02 mm/yr for MA1, 0.4 ± 0.3 mm/yr for ARC, and 0.06 ± 0.02 mm/yr for BAZ (Fig. 3 and4). The mean post-glacial duration, common to all sites, is 18.7 ± 2 ka.

The modeled 36 Cl concentrations recover the average observed values at the scale of the facet, but do not account for the variability in 36 Cl concentrations observed in the lower part of the transects (Fig. 3).

4-DISCUSSION

Local controls on 36 Cl concentration

In most cases, for the upper part, the inversion reproduces the mean 36 Cl concentration.

Overall, the discrepancy between observed and modeled concentrations is at most 0.5x10 5 at 36 Cl/g rock except for the points at the base of each site where observed concentrations are systematically higher by as much as 20% (except BAZ). The discrepancy for the upper part of the profile could be attributed to the stochastic dynamics of local processes such as temporary cover by a few cm of scree or abrupt removal of bedrock fragments that would lower the observed amount of 36 Cl with respect to the predictions of our model. A scree cover of 10 cm would yield a 10% decrease in 36 Cl concentration and could account for a large fraction of the observed intra-site variability (Suppl.). On the contrary, simulations for MA3 show that a scree cover of less than 5-10 cm yields an increase in [ 36 Cl]. This is due to a relatively large slip-rate (3 mm/yr), and high natural chlorine content of some samples (>50 ppm) that maximize 36 Cl production at depth 0 to 100 cm [START_REF] Schimmelpfennig | Sources of in-situ 36Cl in basaltic rocks. Implications for calibration of production rates[END_REF]. Another possibility that explains such a discrepancy could be that the facet slope might be locally steeper than the mean value used in the model. A difference of 10° could increase 36 Cl concentration by about 7% (Suppl.). At the base of the profile, where observed concentrations are systematically higher by as much as 20% (except BAZ), the observed systematic increase in 36 Cl concentration cannot be attributed to such short wavelength random variability in surface processes. This portion is at the intersection between the bedrock scarp and the facet slope, which is defined in the model by the altitude of the bedrock scarp top. There is a high uncertainty in the altitude of this point, usually affected by sliding scree that could explain the discrepancy. The shielding calculation might also underestimate the effect of secondary neutrons produced through colluvial material or bedrock [START_REF] Masarik | Production rates of cosmogenic nuclides in boulders[END_REF]Wieler 2003, Balco 2014). The colluvial density and the dip of the colluvial slope also affect the 36 Cl content at the base of the profile and could additionally account for this discrepancy (Suppl.).

Duration of exhumation versus post-glacial exposure

The value of 18.7±2 ka obtained for the post-glacial duration is consistent with the timing of the most important glacial retreat in the Apennines estimated at about 21 ka from lake deposits records and morainic deposit [START_REF] Giraudi | Middle Pleistocene glaciations in the Apennines, Italy: new chronological data and preservation of the glacial record[END_REF]. It also corroborates the exposure ages from the top of the bedrock scarp at 11-15 ka at similar sites around the Apennines [START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF][START_REF] Cowie | Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults[END_REF]. The amount of 36 Cl accumulated during the exhumation phase under glacial conditions is controlled primarily by fault slip rates and accounts for 9% of the total 36 Cl budget for MA3, 58% for MA1, 24% for ARC and 66% for BAZ. It means that at MA3, 95% of the measured 36 Cl has been accumulated over the last 20 ka, at the other sites this integration time is much longer, with durations of 185 ka, 47 ka and 210 ka at MA1, ARC and BAZ, respectively. Our approach thus allows determining slip-rates averaged over time scales that range from 20 to 210 ka. It is noteworthy that slower slip-rates are more tightly constrained because the 36 Cl buildup at depth outweigh the post-glacial 36 Cl production.

Slip-rate variability over time and relationships with facet morphology

Slip-rates derived from inversion for the four studied sites are between 3 and 0.06 mm/yr which is within the range of observations over the Holocene for similar faults in the Apennines [START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF][START_REF] Cowie | Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults[END_REF]. Sites MA3 and MA1 are less than 5 km apart and located on two segments of the Magnola fault (Suppl.). 36 Cl exposure dating of the bedrock scarp at each site yield Holocene-averaged slip rates of 1 to 1.5 mm/yr at MA3 and MA1 [START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF], which is comparable with the result obtained at MA3 (3±1.2 mm/yr over 20 ka). On the other hand, the slip-rate at MA1 (0.2±0.02 mm/yr over 185 ka) is much lower than the one determined over the last ≈15 ka by [START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF]. This discrepency may be related to the difference in time-scale, suggesting that the slip on the fault has considerably accelerated over the last ≈15ka. Slip-rate variability through time has already been reported for the few faults worldwide where multiple time-scales records are available (Perouse and Wernicke 2017, [START_REF] Friedrich | Comparison of geodetic and geologic data from the Wasatch region, Utah, and implications for the spectral character of Earth deformation at periods of 10 to 10 million years[END_REF]. Studies on normal fault growth have shown that slip-rate can be highly variable over the fault length and over time [START_REF] Manighetti | Evidence for selfsimilar, triangular slip distributions on earthquakes: Implications for earthquake and fault mechanics[END_REF]. Our results confirm those observations and provide data to link incremental earthquake slip events and fault growth. The Bazzano fault (site BAZ) is an antithetic fault of the Paganica fault that ruptured during the 2009 L'Aquila earthquake.

Several studies have allowed determining the Paganica slip-rate over a few thousand years to the Middle Pleistocene [START_REF] Pucci | Complexity of the 2009 L'Aquila earthquake causative fault system (Abruzzi Apennines, Italy) and effects on the Middle Aterno Quaternary basin arrangement[END_REF]. All converge toward a slip-rate of less than 0.4 mm/yr. The rate obtained for the antithetic Bazzano fault of 0.06±0.02 mm/yr over the last 210 ka would thus account for about 15% of the Paganica synthetic fault slip, which appears realistic [START_REF] Boncio | Coseismic ground deformation of the 6 April 2009 L'Aquila earthquake (central Italy, Mw6. 3)[END_REF]). The slip-rate at site AR of 0.4 ± 0.3 mm/yr over the last 47 ka is similar to the rate previously estimated from Falcucci et al. (2015) of 0.2-0.3 mm/yr over the last 1 ± 0.2 Ma. The slip-rates obtained for the four sites vary with the facet height, with the highest rate at MA3 where facet is 800 m-high [START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF]. This is in agreement with DePolo and Anderson (2000) who carried out a statistical analysis on normal faults located in the Basin and Range and observed a logarithmic relationship between the height of the facet and the vertical slip-rate of the fault (ranging from 0.001 to 2 mm/yr). Based on numerical modelling experiments, Petit et al. (2009) and [START_REF] Strak | Interaction between normal fault slip and erosion on relief evolution: Insights from experimental modelling[END_REF] also derived a linear relationship between facet height and throw rate in agreement with our results.

CONCLUSIONS

Our study presents the first systematic CRN measurements across facets developed above normal faults. We observe strikingly consistent signals with near constant 36 Cl concentrations over the height of the studied facets, which suggests the facets are close to a morphological steady state with a constant denudation during their development. Accordingly, we performed inverse modeling of the concentration profiles and inferred slip rates over the last 20-200 ka, the results of which are in agreement with long-term rate independently determined on some of those faults over the last 1 Ma. The integration time span for 36 Cl accumulation over the facets is longer than the postglacial history of these landscapes, suggesting that such measurements could provide slip rates estimates over a time window that is usually undersampled in most tectonic geomorphology studies. [START_REF] Wallace | Geometry and rates of change of fault-generated range fronts, northcentral Nevada[END_REF][START_REF] Armijo | Quaternary extension in southern Tibet: Field observations and tectonic implications[END_REF]). But their potential as tectonic markers and the possibility of retrieving quantitative information on long-term slip rates from their morphological attributes and rates of evolution have seldom been considered (Petit et al. 2009[START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF]. This is an attractive prospect, as faceted spur development integrates cumulative displacements over a longer timeframe (100's of ka) than most other geomorphological markers and are ubiquitous in uplifting landscapes bounded by active normal faults.

FIGURES CAPTIONS

Quantifying tectonic deformation over Quaternary timescales usually relies on the analysis of passive morphological markers formed by various types of processes, such as fluvial or marine terraces, moraines and landslides. In many settings the temporal record of such markers spans only a few 10 ka, leaving the 100 ka timescale usually poorly documented [START_REF] Ryerson | Applications of morphochronology to the active tectonics of Tibet[END_REF], Gold et al. 2016), which is a major hindrance for our understanding of how deformation is accommodated. Triangular facets, on the other hand, are not passively deformed markers but result from the combination of sustained rock uplift of the normal fault footwall and its long-term erosion [START_REF] Burbank | Tectonic geomorphology[END_REF].

Here, we provide the first systematic investigation of the use of the 36 Cl cosmogenic nuclide to assess the rates of facet evolution. Our goal is to link cosmogenic nuclide buildup 2

). The inversion of data acquired from the 4 studied sites demonstrates that our approach provides accurate constraints on fault slip rates over time-spans ≤ 200 ka (Fig. 3 and4).

1-THEORETICAL 36 CL BUILD UP ON A TRIANGULAR FACET

Faceted spurs are near planar triangular or trapezoidal surfaces produced by the erosion of normal fault scarps that are continuously rejuvenated at their base by sustained slip [START_REF] Wallace | Geometry and rates of change of fault-generated range fronts, northcentral Nevada[END_REF]). Here we focus on normal fault footwalls formed in resistant bedrock and undergoing a weathering-limited evolution [START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF]. Morphological evolution models [START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF][START_REF] Strak | Interaction between normal fault slip and erosion on relief evolution: Insights from experimental modelling[END_REF], Tucker et al. 2020) suggest facet morphology reflects an equilibrium between rock uplift from fault slip and erosion.

At the base of the facets, well-preserved bedrock fault scarps representing the cumulative displacement on the fault since the last glacial maximum can be observed [START_REF] Wallace | Geometry and rates of change of fault-generated range fronts, northcentral Nevada[END_REF], Armijo et al. 1992). This has been confirmed in the Mediterranean by exposure-age dating in Greece, Italy, Israel, Turkey (e.g. [START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF][START_REF] Cowie | Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults[END_REF]. Post-glacial changes in climatic conditions result in a decrease in hillslope erosion rates and preservation of those bedrock fault scarps (Armijo et al. 1998[START_REF] Allen | Rapid environmental changes in southern Europe during the last glacial period[END_REF]. The timing of this shift is not accurately dated and may spatially vary. In the Apennines, it is estimated to be 12-21 ka [START_REF] Giraudi | Middle Pleistocene glaciations in the Apennines, Italy: new chronological data and preservation of the glacial record[END_REF].

Based on these previous observations we use a two-stage kinematic model to predict 36 Cl buildup in material currently exposed along the facet surface. First, under glacial conditions, we assume topographic steady state, such that rock uplift of the footwall is equal to the denudation rate of the facet surface. Rock sample are progressively exhumed parallel to the slip vector at a rate set by the fault slip-rate (Fig 2). Second, in post glacial conditions, we assume denudation of the facet to be negligible, such that rock samples are continuously exposed at the surface, with no significant regolith cover [START_REF] Tucker | Geomorphic significance of postglacial bedrock scarps on normal-fault footwalls[END_REF].

Our approach relies on the interpretation of measured 36 Cl concentration at the surface in terms of this exhumation and exposure history of the facet. Based on the conceptual evolution described above, we parametrize this evolution using the time at which the sample starts moving towards the surface (T exhum ), the rate at which the sample moves parallel to fault motion prior to post-glacial conditions (SR), and the duration of post-glacial exposure assumed without erosion (T pg ). We combine this simple model with equations describing cosmogenic nuclides buildup to predict 36 Cl concentrations at the surface of the facet for any combination of the 3 parameters (T exhum , T pg , SR) defining the prescribed history (Suppl.).

2-36 CL CONCENTRATION PROFILES ON 4 FACETED SPURS IN CENTRAL

APENNINES

We focus on 4 facets formed in limestones located along 3 active faults in central Apennines are similar across sites, the fault dips (β) from 40° at MA1, to 65° at BAZ (Suppl.).

The facets were sampled following an up-dip fault transect (Suppl.). We focused sampling on the lower part of the facet, where the surface appears less rugged, and near the facet center to avoid increased erosion by bounding gullies. Samples are 5-10 cm thick pieces of bedrock, taken on the top of protruding (> 0.5 m) bedrock surfaces, avoiding places that could have been covered by regolith. Each sample was chemically prepared for 36 Cl measurements (Suppl.). At MA3 and ARC, the 36 Cl concentrations are roughly constant all along the facet (Fig. 3), except at the base where they increase by about 30%. At MA1 and BAZ, facets are lower and there are fewer points of measurements, but we observe a similar pattern at MA1

with constant values at the top, a slight decrease and an abrupt increase at the base of facet.

There are only 3 data points at BAZ with decreasing concentrations toward the base. The 36Cl concentrations along all studied facets are thus roughly constant suggesting that the denudation rate on those features is approximately constant. This observation is consistent with a facet development model that assumes a constant denudation rate. Moreover while it has been usually assumed that erosion would vary with facet height (Tucker et al. 2020 and references there in), our results suggest that, on the facet we studied, this is not the case.

3-FAULT SLIP-RATE FROM 36 CL DATA INVERSION

To constrain the fault slip-rate we carried out a Bayesian inversion of the observed 36 Cl concentrations where the model parameters are the individual slip-rates of the faults (SR MA3 , SR MA1 , SR ARC , SR BAZ ), and the post-glacial period duration (T PG ), assumed to be the same for the 4 sites (T exhum is fixed a priori see suppl.). A complete description of the inversion procedure using the GW-MCMC algorithm (Goodman and Weare 2010), the corresponding parameters and the numerical code are provided in the Suppl. The inversion yielded mean slip-rates of 3.0 ± 1.2 mm/yr for MA3, 0.2 ± 0.02 mm/yr for MA1, 0.4 ± 0.3 mm/yr for ARC, and 0.06 ± 0.02 mm/yr for BAZ (Fig. 3 and4). The mean post-glacial duration, common to all sites, is 18.7 ± 2 ka.

The modeled 36 Cl concentrations recover the average observed values at the scale of the facet, but do not account for the variability in 36 Cl concentrations observed in the lower part of the transects (Fig. 3).

4-DISCUSSION

Local controls on 36 Cl concentration

In most cases, for the upper part, the inversion reproduces the mean 36 Cl concentration.

Overall, the discrepancy between observed and modeled concentrations is at most 0.5x10 5 at 36 Cl/g rock except for the points at the base of each site where observed concentrations are systematically higher by as much as 20% (except BAZ). The discrepancy for the upper part of the profile could be attributed to the stochastic dynamics of local processes such as temporary cover by a few cm of scree or abrupt removal of bedrock fragments that would lower the observed amount of 36 Cl with respect to the predictions of our model. A scree cover of 10 cm would yield a 10% decrease in 36 Cl concentration and could account for a large fraction of the observed intra-site variability (Suppl.). On the contrary, simulations for MA3 show that a scree cover of less than 5-10 cm yields an increase in [ 36 Cl]. This is due to a relatively large slip-rate (3 mm/yr), and high natural chlorine content of some samples (>50 ppm) that maximize 36 Cl production at depth 0 to 100 cm [START_REF] Schimmelpfennig | Sources of in-situ 36Cl in basaltic rocks. Implications for calibration of production rates[END_REF]. Another possibility that explains such a discrepancy could be that the facet slope might be locally steeper than the mean value used in the model. A difference of 10° could increase 36 Cl concentration by about 7% (Suppl.). At the base of the profile, where observed concentrations are systematically higher by as much as 20% (except BAZ), the observed systematic increase in 36 Cl concentration cannot be attributed to such short wavelength random variability in surface processes. This portion is at the intersection between the bedrock scarp and the facet slope, which is defined in the model by the altitude of the bedrock scarp top. There is a high uncertainty in the altitude of this point, usually affected by sliding scree that could explain the discrepancy. The shielding calculation might also underestimate the effect of secondary neutrons produced through colluvial material or bedrock [START_REF] Masarik | Production rates of cosmogenic nuclides in boulders[END_REF]Wieler 2003, Balco 2014). The colluvial density and the dip of the colluvial slope also affect the 36 Cl content at the base of the profile and could additionally account for this discrepancy (Suppl.).

Duration of exhumation versus post-glacial exposure

The value of 18.7±2 ka obtained for the post-glacial duration is consistent with the timing of the most important glacial retreat in the Apennines estimated at about 21 ka from lake deposits records and morainic deposit [START_REF] Giraudi | Middle Pleistocene glaciations in the Apennines, Italy: new chronological data and preservation of the glacial record[END_REF]. It also corroborates the exposure ages from the top of the bedrock scarp at 11-15 ka at similar sites around the Apennines [START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF][START_REF] Cowie | Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults[END_REF]. The amount of 36 Cl accumulated during the exhumation phase under glacial conditions is controlled primarily by fault slip rates and accounts for 9% of the total 36 Cl budget for MA3, 58% for MA1, 24% for ARC and 66% for BAZ. It means that at MA3, 95% of the measured 36 Cl has been accumulated over the last 20 ka, at the other sites this integration time is much longer, with durations of 185 ka, 47 ka and 210 ka at MA1, ARC and BAZ, respectively. Our approach thus allows determining slip-rates averaged over time scales that range from 20 to 210 ka. It is noteworthy that slower slip-rates are more tightly constrained because the 36 Cl buildup at depth outweigh the post-glacial 36 Cl production.

Slip-rate variability over time and relationships with facet morphology

Slip-rates derived from inversion for the four studied sites are between 3 and 0.06 mm/yr which is within the range of observations over the Holocene for similar faults in the Apennines [START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF][START_REF] Cowie | Orogen-scale uplift in the central Italian Apennines drives episodic behaviour of earthquake faults[END_REF]. Sites MA3 and MA1 are less than 5 km apart and located on two segments of the Magnola fault (Suppl.). 36 Cl exposure dating of the bedrock scarp at each site yield Holocene-averaged slip rates of 1 to 1.5 mm/yr at MA3 and MA1 [START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF], which is comparable with the result obtained at MA3 (3±1.2 mm/yr over 20 ka). On the other hand, the slip-rate at MA1 (0.2±0.02 mm/yr over 185 ka) is much lower than the one determined over the last ≈15 ka by [START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF]. This

CONCLUSIONS

Our study presents the first systematic CRN measurements across facets developed above normal faults. We observe strikingly consistent signals with near constant 36 Cl concentrations over the height of the studied facets, which suggests the facets are close to a morphological steady state with a constant denudation during their development. Accordingly, we performed inverse modeling of the concentration profiles and inferred slip rates over the last 20-200 ka, the results of which are in agreement with long-term rate independently determined on some of those faults over the last 1 Ma. The integration time span for 36 Cl accumulation over the facets is longer than the postglacial history of these landscapes, suggesting that such measurements could provide slip rates estimates over a time window that is usually undersampled in most tectonic geomorphology studies.

FIGURES CAPTIONS

Fig. 1 :

 1 Fig.1 : A) Tectonic map of the Lazio-Abruzzo fault system[START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF][START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF] with the faults (red line) bounding the sampled facets (yellow arrows) and site names (sites names were kept as in[START_REF] Schlagenhauf | Using in situ Chlorine-36 cosmonuclide to recover past earthquake histories on limestone normal fault scarps: a reappraisal of methodology and interpretations[END_REF]. B) Photography of the F-MA1 facet with location of samples and Holocene bedrock fault scarp at the base of the facets.

Fig. 3 :Figure 4 INTRODUCTION

 34 Fig. 3 : Measured (black dot) and modeled 36 Cl concentrations obtained from Bayesian inversion (grey pdf) of samples collected on the 4 facets (MA3, MA1, AR, BAZ) in Central Italy, as a function of sample altitude. Reduced chi-squared values are MA3=6.4; MA1=8.2;AR=5.2; BAZ=19.6.

  on limestone faceted spurs and fault slip-rate by investigating 4 sites in the Central Apennines, Italy of different shape and height (Fig 1). Following on Tucker et al. (2011)'s assumptions linking facet slope, denudation rate and fault slip-rate, we compute the theoretical 36 Cl concentration buildup of a facet as a function of its evolution with time (Fig.

(

  Italy). Since late Pliocene-early Pleistocene, active normal faults accommodate the SW-NE extension in the area[START_REF] Roberts | Spatial and temporal variations in growth rates along active normal fault systems: an example from The Lazio-Abruzzo Apennines, central Italy[END_REF]. The Velino-Magnola fault is a major structure of the range (~30 km long, Fig.1) with facets up to 300-800 m high, where we sampled two sites MA1 and MA3 along the northern and central part of the fault, respectively. The Roccapreturo fault is shorter (10 km), with lower facets (100-150 m), and was sampled at site AR in the center of the fault. The Bazzano fault is a small (3 km long) antithetic fault, with several facets reaching 100-150 m high, sampled in its southern section at site BAZ (Suppl.). The surface of the 4 sampled facets displays regular slope 30-35° (Fig3). The surface of the facet is characterized by an alternance of bedrock outcrop, small bedrock protrusions (< 1m high), and patches of soil and screes less than a few cm thick. The facets are generally bounded by entrenched non-perennial streams that flow perpendicular to the fault's strike, which suggests recent incision that is contemporaneous with fault development. The slopes of the colluvial wedge surface (α~25-30) and of the facets (γ~30-35)

  discrepency may be related to the difference in time-scale, suggesting that the slip on the fault has considerably accelerated over the last ≈15ka. Slip-rate variability through time has already been reported for the few faults worldwide where multiple time-scales records are available (Perouse and Wernicke 2017,[START_REF] Friedrich | Comparison of geodetic and geologic data from the Wasatch region, Utah, and implications for the spectral character of Earth deformation at periods of 10 to 10 million years[END_REF]. Studies on normal fault growth have shown that slip-rate can be highly variable over the fault length and over time[START_REF] Manighetti | Evidence for selfsimilar, triangular slip distributions on earthquakes: Implications for earthquake and fault mechanics[END_REF]. Our results confirm those observations and provide data to link incremental earthquake slip events and fault growth. The Bazzano fault (site BAZ) is an antithetic fault of the Paganica fault that ruptured during the 2009 L'Aquila earthquake.Several studies have allowed determining the Paganica slip-rate over a few thousand years to the Middle Pleistocene[START_REF] Pucci | Complexity of the 2009 L'Aquila earthquake causative fault system (Abruzzi Apennines, Italy) and effects on the Middle Aterno Quaternary basin arrangement[END_REF]. All converge toward a slip-rate of less than 0.4 mm/yr. The rate obtained for the antithetic Bazzano fault of 0.06±0.02 mm/yr over the last 210 ka would thus account for about 15% of the Paganica synthetic fault slip, which appears realistic[START_REF] Boncio | Coseismic ground deformation of the 6 April 2009 L'Aquila earthquake (central Italy, Mw6. 3)[END_REF]). The slip-rate at site AR of 0.4 ± 0.3 mm/yr over the last 47 ka is similar to the rate previously estimated fromFalcucci et al. (2015) of 0.2-0.3 mm/yr over the last 1 ± 0.2 Ma. The slip-rates obtained for the four sites vary with the facet height, with the highest rate at MA3 where facet is 800 m-high[START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF]. This is in agreement with DePolo and[START_REF] Depolo | Estimating the slip rates of normal faults in the Great Basin, USA[END_REF] who carried out a statistical analysis on normal faults located in the Basin and Range and observed a logarithmic relationship between the height of the facet and the vertical slip-rate of the fault (ranging from 0.001 to 2 mm/yr). Based on numerical modelling experiments,Petit et al. (2009) and[START_REF] Strak | Interaction between normal fault slip and erosion on relief evolution: Insights from experimental modelling[END_REF] also derived a linear relationship between facet height and throw rate in agreement with our results.

Fig. 1 :

 1 Fig.1 : A) Tectonic map of the Lazio-Abruzzo fault system[START_REF] Schlagenhauf | Earthquake supercycles in Central Italy, inferred from 36Cl exposure dating[END_REF][START_REF] Benedetti | Earthquake synchrony and clustering on Fucino faults (Central Italy) as revealed from in situ 36Cl exposure dating[END_REF] with the faults (red line) bounding the sampled facets (yellow arrows) and site names (sites names were kept as in[START_REF] Schlagenhauf | Using in situ Chlorine-36 cosmonuclide to recover past earthquake histories on limestone normal fault scarps: a reappraisal of methodology and interpretations[END_REF]. B) Photography of the F-MA1 facet with location of samples and Holocene bedrock fault scarp at the base of the facets.

Fig. 3 :

 3 Fig. 3 : Measured (black dot) and modeled 36 Cl concentrations obtained from Bayesian inversion (grey pdf) of samples collected on the 4 facets (MA3, MA1, AR, BAZ) in Central Italy, as a function of sample altitude. Reduced chi-squared values are MA3=6.4; MA1=8.2;AR=5.2; BAZ=19.6.

G. Aumaître, D.L. Bourlès, K. Keddadouche

ACKNOWLEDGMENTS

This work has been partly funded by the LABEX OT MED. M. Rizza and B. Ourion are greatly acknowledged for their help and support in the field.

ACKNOWLEDGMENTS

This work has been partly funded by the LABEX OT MED. M. Rizza and B. Ourion are greatly acknowledged for their help and support in the field.