Bruna Lima Correa

Nadia El Harane

Hocine Ingrid Gomez

Rachid Hocine

José Vilar

Manon Desgres

Valérie Bellamy

Kamaleswaran Keirththana

Chloé Guillas

Maria Perotto

Nadia El Harane1

Ingrid Gomez1

Hocine Rachid Hocine2

José Vilar1

Manon Desgres1

Valérie Bellamy1

Keirththana Kamaleswaran

Chloé Guillas1

Maria Perotto1

Laetitia Pidial1

Paul Alayrac1

Thi Tran1

Sisareuth Tan3

Thomas Hamada1

Dominique Charron5

Alain Brisson3

Nisa K Renault4

Reem Al-Daccak5

Philippe Menasché1
email: philippe.menasche@aphp.fr

Jean-Sébastien Silvestre1
email: jeansebastien.silvestre@inserm.fr

Extracellular vesicles from human cardiovascular progenitors trigger a reparative immune response in infarcted hearts

Keywords: Acellular therapies, Heart Failure, Extracellular vesicles, Immunomodulatory effects

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite therapeutic improvements, chronic heart failure still carries a poor prognosis with a 5-year mortality averaging 50% [START_REF] Bui | Epidemiology and risk profile of heart failure[END_REF] . When drug-based regimens are no longer effective, heart transplantation or mechanical assist devices may be indicated, but their invasiveness and limitations call for alternate options, include cell therapy. Even though, so far, cell therapy trials have largely failed to significantly improve patient outcomes, they have provided some insights into the mechanism of action of transplanted cells which appears to be predominantly mediated by paracrine signaling [START_REF] Gnecchi | Paracrine mechanisms in adult stem cell signaling and therapy[END_REF] . This concept is largely based on the observations that the secretome, including extracellular vesicles (EV), is as effective as the parent therapeutic cells in the treatment of experimental cardiomyopathies [START_REF] Gallet | Exosomes secreted by cardiospherederived cells reduce scarring, attenuate adverse remodelling, and improve function in acute and chronic porcine myocardial infarction[END_REF][START_REF] Lai | Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury[END_REF] . We have previously shown, in a mouse model of chronic heart failure, that EV released by embryonic stem cell derived cardiovascular progenitor cells (CPC) [START_REF] Kervadec | Cardiovascular progenitorderived extracellular vesicles recapitulate the beneficial effects of their parent cells in the treatment of chronic heart failure[END_REF] and EV released by induced pluripotent stem cells (iPSC) derived CPC [START_REF] El Harane | Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors[END_REF] were therapeutically effective. Others have shown this to be the case for the secretome of other cell types, primarily mesenchymal stem cells [START_REF] Timmers | Human mesenchymal stem cell-conditioned medium improves cardiac function following myocardial infarction[END_REF][START_REF] Gnecchi | Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement[END_REF] and cardiac stem cells [START_REF] Ibrahim | Exosomes as critical agents of cardiac regeneration triggered by cell therapy[END_REF][START_REF] Barile | Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction[END_REF] .

Extracellular vesicles are membrane-bound particles ranging in size from less than 100 nm to 1 µm and found in all biological fluids and conditioned media [START_REF] Zhang | Exosomes: biogenesis, biologic function and clinical potential[END_REF] . They are composed of a bi-lipid membrane, similar to the cell membrane, which encloses an aqueous, nucleus-free compartment, harboring mRNAs, microRNAs, proteins, lipids, and other bioactive molecules [START_REF] Yuan | Transfer of microRNAs by embryonic stem cell microvesicles[END_REF][START_REF] Vrijsen | Cardiomyocyte progenitor cell-derived exosomes stimulate migration of endothelial cells[END_REF][START_REF] Sahoo | Exosomes from human CD34(+) stem cells mediate their proangiogenic paracrine activity[END_REF][START_REF] Chen | Paracrine factors from adipose-mesenchymal stem cells enhance metastatic capacity through Wnt signaling pathway in a colon cancer cell co-culture model[END_REF][START_REF] Cosme | The cardiovascular exosome: current perspectives and potential[END_REF] . Although a consensus has not yet been reached on specific markers of EV subtypes, EV can be characterized by the expression of proteins like the tetraspanins (CD9, CD63 and CD81) and integrins while additional features of their profile depend on the phenotype of the parent cell [START_REF] Pols | Trafficking and function of the tetraspanin CD63[END_REF][START_REF] Colombo | Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles[END_REF][START_REF] Henne | The ESCRT pathway[END_REF][START_REF] Théry | Membrane vesicles as conveyors of immune responses[END_REF] . Furthermore, while the molecular mechanisms underpinning the cardio-protective effects of EV are still under investigation, the therapeutic potential of EV likely relies on pleiotropic effects including angiogenesis, modulation of the inflammatory environment and, possibly, activation of cardiac intrinsic regenerative pathways [START_REF] Kervadec | Cardiovascular progenitorderived extracellular vesicles recapitulate the beneficial effects of their parent cells in the treatment of chronic heart failure[END_REF][START_REF] El Harane | Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors[END_REF][START_REF] Lauden | Allogenicity of human cardiac stem/progenitor cells orchestrated by programmed death ligand 1[END_REF][START_REF] Fernandes | Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair[END_REF][START_REF] Hocine | Extracellular Vesicles Released by Allogeneic Human Cardiac Stem/Progenitor Cells as Part of their Therapeutic Benefit[END_REF][START_REF] De Couto | Exosomal MicroRNA Transfer Into Macrophages Mediates Cellular Postconditioning[END_REF] .

Yet, to the best of our knowledge, most of the studies investigating the cardio-reparative effects of human EV have been conducted in immune-deficient experimental animal models. Although these studies provide valuable mechanistic insights, a more clinically relevant assessment of the immune effects of EV, which is a prerequisite for their potential clinical use, requires their exposure to human cells and the use of immune-competent animal models. In the present study, we used tailored allogeneic in vitro human cell assays and immunocompetent mouse models of myocardial infarction (MI) to investigate the immune effects of EV derived from human CPC.

Material and Methods

Detailed Material and Methods are given in the Supplemental Material.

Ethics section

All procedures were approved by the Institutional Ethics Committee from the Université de Paris-Descartes (project #04073.01) and comply with European legislation (European Commission Directive 2010/63/EU) on animal care. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH guidelines).

All animals received anesthesia by isoflurane (2.5L/min isoflurane at 1.5L/min of O2) and were euthanized by dolethal injection at 140mg/kg. For in vitro tests with human cells, blood donors signed an informed consent and all methods and experimental protocols were approved by the institutional Ethics Committee (Hôpital St. Louis, Paris, France) and conducted in accordance with guidelines and regulation outlined in the Declaration of Helsinki.

Cell culture, EV preparation, quantification and characterization a) Cell Culture and cell characterization

Cardiovascular progenitor cells differentiated from human iPSC (iCell® Cardiac Progenitor Cells, Cellular Dynamics International, Madison, WI) were cultured according to the manufacturer's directions (for a detailed protocol, see Supplementary Material). After 4 days of culture, cells were harvested and immune-phenotyped with antibodies against relevant immune molecules using fluorescence-activated cell sorting (FACS) (CANTO-II Flow Cytometer, BD Bioscience, DIVA and FlowJo software).

For human cell in vitro experiments, human peripheral blood mononuclear cells (PBMC) were prepared from blood samples from healthy donors using Ficoll gradient (Eurobio) centrifugation and cryopreserved for later use.

b) EV isolation

Conditioned media were collected, pre-cleared by centrifugation for 6 minutes at 1,200g and EV were pelleted by ultracentrifugation (rotor 70Ti-Beckman) for 16 hours at 100,000g.

c) EV quantification and characterization

Extracellular vesicles were quantified by Nanoparticle Tracking Analysis (NanoSight LM-14, Malvern) and characterized by FACS [START_REF] Davidson | Exosomes and cardioprotection -A critical analysis[END_REF] after coupling with latex beads (Life Technologies, A37304) using a MACSplex kit (MACSPlex Exosome Kit human, Miltenyi Biotech) as follows to phenotype their membrane ligands.

In a second time, EV (25 μg of protein) were mixed with 4-μm Aldehyde/Sulfate Latex Beads at room temperature (RT); then suspended in Phosphate-Buffered Saline (PBS) and left at RT for another 2h. The coupling reaction was terminated by 100 mM glycine, and the mixture was centrifuged at 1,500 x g. The beads were then stained with conjugated antibodies for the relevant markers and analyzed by FACS.

Ultimately immuno-gold labeling and cryo-transmission electron microscopy (cryo-TEM) were also performed on EV. Gold nanoparticles (NP) were conjugated with an anti-CD81 monoclonal antibody (mAb) following procedures previously described [START_REF] Arraud | Extracellular vesicles from blood plasma: determination of their morphology, size, phenotype and concentration[END_REF] . Samples of EV were used either pure or diluted 10-fold with a HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)buffered saline containing 150 mM NaCl and 10 mM HEPES, pH 7.4. Samples were then labeled for 1 h with 10-nm anti-CD81-gold-NP at 1-3 × 10 15 gold-NP/L, at RT. Immuno-gold labeled samples were processed for cryo-TEM as follows. A 4-μL aliquot was deposited on an electron microscopy (EM) grid coated with a perforated carbon film. After draining the excess liquid with a filter paper, grids were quickly plunged into liquid ethane using a Leica EMCPC cryo-chamber, transferred to cryo-boxes and stored under liquid nitrogen until TEM observation. For cryo-TEM observation, grids were mounted onto a Gatan 626 cryoholder and transferred to a Tecnai F20 microscope (FEI) operated at 200 kV. Images were recorded with a USC1000-SSCCD camera (Gatan). These experiments were performed to best match the minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines [START_REF] Théry | Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines[END_REF] .

On the basis of a previous study [START_REF] El Harane | Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors[END_REF] in which the cardioprotective effects of 1.4x10 [START_REF] El Harane | Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors[END_REF] CPC could be recapitulated by delivering the amount of EV they had secreted in 48 hours, we selected in the present study the same EV dose which equates here to 1x10 10 particles (100 µg of protein) concentrated in a 50-60µL-volume.

Allogeneic immune responses and immunomodulation assays

Tailored mixed lymphocyte reactions (MLR) were performed using PBMC from different donors.

Briefly, responding PBMC (1x10 5 cells) were labeled with 2.5 µM carboxyfluorescein succinimidyl ester (CFSE, Thermofisher Scientific) and then co-cultured in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% Fetal Bovine Serum (FBS, Gibco Life Technologies) in U-bottom 96-well plates (VWR) with human leukocyte antigen mismatched (HLA) mitomycin-C-treated (Sigma) stimulatory PBMC (1x10 5 cells), mitomycin-C-treated CPC (at CPC:PBMC ratios of 1:5 and 1:10), or increasing doses of EV (EV1: 5.5x10 8 , EV2: 5.5x10 [START_REF] Ibrahim | Exosomes as critical agents of cardiac regeneration triggered by cell therapy[END_REF] or EV3: 1.7x10 10 particles) or PBS as a control. After 6 days of co-culture, cells were harvested and stained with anti-CD3-PE/Cy7, anti-CD4-APC, anti-CD8-APC-H7 antibodies and 7AAD (BD Biosciences). Proliferation and cell death of T-cell subsets were monitored by FACS.

In assays determining the immunomodulatory effects of CPC and EV-CPC, HLA-mismatched CFSE-labeled PBMC (1x10 5 cells) were stimulated with 1 μg/mL phytohemagglutinin (Sigma-Aldrich) in the presence or absence of mitomycin-C-treated CPC (at CPC: PBMC ratios of 1:5 and 1:10) or with increasing concentrations of EV (EV1: 5.5x10 8 , EV2:5.5x10 [START_REF] Ibrahim | Exosomes as critical agents of cardiac regeneration triggered by cell therapy[END_REF] or EV3: 1.7x10 10 particles) for 5 days. At the end of the co-culture, the proliferation and death rate of the T cells were monitored by FACS.

Natural killer cell degranulation

Natural Killer (NK) cells were isolated from PBMC by negative selection using immune-magnetic cell sorting (Miltenyi Biotech, Germany) according to the manufacturer's instructions.

Experiments were conducted with NK cells primed overnight with recombinant human interleukine (Il), IL-15 (50 ng/mL) (Sigma) in RPMI medium supplemented with 10% FBS to ensure their proper expression of NK cell activating receptors and functionality. Cytokine-activated NK cells were cultured alone or in the presence of K562 cells used as positive controls (ATCC, USA), CPC (ratio 1:1) or increasing doses of EV (5.5x10 8 ; 5.5x10 9 or 1.7x10 10 particles) and labelled with an anti-CD107a-PE antibody (BD Bioscience). After 18 hours, cells were harvested, washed and stained with CD3-APC-H75R (BD Bioscience) and CD56-APC specific antibodies (BD Bioscience), and the expression of CD107a was analyzed on CD3 -CD56 + NK cells by FACS.

Macrophage polarization assay

PBMC from healthy donors were suspended at a concentration of 100 million cells per mL in Monocyte Attachment Medium (Promocell) and seeded at a density of 1 million viable cells/cm 2 with a monocyte content ≥25% and 1.5 million viable cells/cm 2 for a monocyte content of <25% (4 million cells per P24-well). At day 0, monocytes were incubated for 90 min at 5% CO2 and 37°C to allow adherence. Non-adherent cells were discarded by vigorously swirling the culture and aspirating the supernatant. Adherent cells were then washed and the appropriate amount of complete M0, M1, M2 Macrophage Generation Medium (Promocell) was added to the cells for 6 days. Under these conditions, monocytes differentiate into M1-like or M2-like polarized macrophages. After 6 days, the culture medium was replaced with fresh complete M0, M1, M2 Macrophage Generation Medium DXF with or without EV (5.5x10 9 particles per well).

Macrophages were then incubated for another 2 days at 37°C and 5% CO2. At day 8, the culture medium was supplemented with IFN-γ (50 ng/mL) and LPS (10 ng/mL) for M1 macrophage polarization, with 20 ng/mL IL-4 and 20ng/mL IL-13 for M2 macrophage polarization (all from Merck) or with macrophage colony-stimulating factor (MCSF) medium for M0 macrophages. Each of these conditions was exposed or not to EV (5.5x10 9 particles per well). The next day (D9), cells were detached with the Macrophage Detachment Solution DXF (Promocell) for 45 min at RT. Monocytes and macrophages were analyzed at day 9 of culture, after polarization and exposure to EV, as described above. They were suspended in MACSBuffer (Milteny), centrifuged for 10 min at 500 x g, counted and labelled at 200,000 cells per condition with anti-CD64 PE, anti-CD68 PE-Cy7, anti-CD80 PE-Cy5, anti-CD86 APC or BV605, anti-CD163 BV421, anti-CD206 BV711 and anti-CD14 FITC ((Fluorescein isothiocyanate) antibodies (all from BD Biosciences). Monocytes and macrophages were categorized by the following markers: Monocytes:CD14+; Proinflammatory monocytes: CD14+, CD86++; Macrophages: CD64+, CD68+; M1: CD64+, CD68+, CD80+, CD86+; M2a: CD64+, CD68+, CD206+; M2b: CD64+, CD 68+, CD163+. All antibodies were used at a dilution of 1:100. After incubation, cells were washed and suspended in 100 µL of MACS Buffer for FACS analysis. Events were acquired on a Fortessa X20 flow cytometer (BD Biosciences); results were analyzed with FlowJo software (FlowJo V10.4.2, FlowJo LLC) and are given as a percentage of the whole population. Experiments were performed in technical triplicates for each donor.

Bead-based multiplex immunoassay

A bead-based multiplex immunoassay (Bio-Rad) was used to measure the levels of multiple cytokines [basic Fibroblast Growth Factor, Eotaxin, G-CSF, GM-CSF, IFN-γ, IL-1β, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1 (MCAF), MIP-1α, MIP-1β, PDGF-BB, RANTES, Tumor Necrosis factor-alpha (TNF-α), and VEGF] from supernatants of macrophage cultures at day 9. Concentration were calculated using a standard curve (5 PL regression, with Bio-Plex Manager™ software).

Immunoglobulin assay following EV treatment

To assess whether EV-CPC could induce antibody production, mice were subcutaneously injected at day 1, 13 and 20 with either EV (n=5, at the dose of 1x10 10 particles per injection) or PBS (n=5) according to a classical immunization protocol [START_REF] Gunasekaran | Circulating Exosomes with Distinct Properties during Chronic Lung Allograft Rejection[END_REF] . Blood samples were collected at days 10, 20 and 30 post injection. Ig isotypes in plasma were qualitatively detected using the mouse immunoglobulin isotyping ELISA kit (ThermoFisher Scientific).

Enzyme-linked immunospot

The enzyme-linked immunospot (ELISPOT) is a sensitive technique for the detection of cytokineproducing cells at the single cell level. The IFN-γ ELISPOT (Diacole) method was used for measuring the antigen-specific response to EV-CPC elicited by NK cells, T cells, antigen-specific Th1 CD4 + and CD8 + effector T cells. Primed splenocytes of each vaccinated mouse were isolated, plated (at a seeding density of 1.5 million cells/cm 2 in 96-well plates), and re-exposed to either EV (2x10 9 particles) or PBS in vitro for 21h; the positive control was a mixed solution of phorbol 12myristate 13-acetate (PMA, 100ng/mL) and ionomycine (2ug/mL; both from Sigma) that triggers unspecific IFN-γ production. When spots indicating IFN-γ production appeared, they were counted using a Cellular Technology Ltd system (USA) and analyzed using ImmunoSpot 5.0.3 software.

Results are reported as the mean of triplicate measurements of the number of IFN-γ spot-forming cells (SFCs) per 5x10 5 plated cells.

Mouse model of myocardial infarction (MI) and administration of EV-CPC

Myocardial infarction was induced in C57BL/6JRj male mice (Janvier Laboratories) by permanent ligation of the left anterior descending coronary artery (LAD). In a first series of chronic heart failure, mice received 3 echo-guided, trans-cutaneous injections of 10-20 µL each, in peri-infarcted myocardium, 3 weeks after MI. The experimental groups were as follows: PBS, CPC (1.4 million cells), EV-CPC (secreted by 1.4 million CPC in 48 hours, which was 1x10 10 particles or 100 ug of protein) or lipopolysaccharide (LPS dose of 22.5 µg, used as a positive control of inflammation).

Six additional mice were also infarcted but did not receive any injection (controls). Except for the LPS injection group, which had a 33% post-treatment mortality rate, all treated animals survived and were sacrificed 3 days after injection.

In an additional series of acute myocardial infarction, mice received similar transcutaneous echoguided injections in 3 peri-infarcted areas of either PBS (vehicle control) or EV-CPC (secreted by 1.4x10 [START_REF] El Harane | Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors[END_REF] CPC which was 1x10 [START_REF] Barile | Extracellular vesicles from human cardiac progenitor cells inhibit cardiomyocyte apoptosis and improve cardiac function after myocardial infarction[END_REF] particles or 100 µg of proteins) 2 days after the infarct. Efforts were made to reproducibly control the degree of cardiac injury in each series of experiments by carefully standardizing the coronary artery ligation site 2-3 mm below the atria all the samples respected this histomorphometric inclusion criteria.

Tissue digestion and flow cytometry

Tissues (heart, spleen, femur and blood) were collected 3 days after EV-CPC injections in both acute and chronic models and kept on ice during all procedures (except for blood). Hearts were rinsed in PBS to remove blood clots. The left ventricle was isolated, minced with fine scissors and gently passed through the Bel-Art™ Scienceware™ 12-Well Tissue Disaggregator (Bel-Art Products). Cells from heart, spleen and bone marrow were then collected in PBS, filtered through a 40-μM nylon mesh (Falcon) and washed. Red blood cells were removed by using the Red Blood Cell Lysing Buffer (Sigma). Cells harvested from the heart were immuno-stained at 4°C for 30 min. Total cardiac immune cells were gated after staining with an Alexa Fluor 700-conjugated anti-CD45 antibody (BD Biosciences) and the following antibodies were used: PE/Cy7-conjugated anti-CD11b (BD Pharmingen), PA-conjugated anti-Ly6G (BD Pharmingen), BV421-conjugated anti-CD64 (Biolegend), APC-conjugated anti-F4/80 (Bio-Rad), FITC-conjugated anti-Ly6C (BD Pharmingen), Percp5.5-conjugated MHC class II (Biolegend), FITC-conjugated anti-CD45R/B220 (eBioscience) and BV421-conjugated anti-CD3 (BD Pharmingen). All antibodies were used at a dilution of 1:100. After incubation, cells were washed and suspended in 100 µL of PBS for FACS analysis. The total number of cells was normalized to heart weight. Events were acquired on an LSR II flow cytometer (BD Biosciences). Monocytes were categorized as Ly6C high monocytes (CD45+CD11b + F4/80 -Ly6G -Ly6C high ), and Ly6C low monocytes (CD45+CD11b + F4/80 -Ly6G - Ly6C low ). Neutrophils were categorized as CD45+CD11b + Ly6G ++ F4/80 -Ly6C med , and macrophages, as CD45+CD11b + Ly6G -F4/80 + 29 .

Bio-Plex ProTM

The bead-based multiplex immunoassay (Bio-Rad) was used to measure the levels of pro-and antiinflammatory mediators from mouse plasma (chronic heart failure model) or cardiac tissue homogenates (chronic heart failure and acute myocardial infarction models), 3 days after EV-CPC injection. A 21-plex immunoassay panel (IL-1 α and β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, IL-13, IL-17A, eotaxin, Granulocyte-colony stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), IFN-γ, KC, Monocyte chemoattractant protein (MCP-1 or CCL-2), MIP-1α, RANTES and TNF-α) was used according to the manufacturer's directions and analysed on a Bio-Plex® 200 (Bio-Rad). Analyte concentration was calculated using a standard curve (5 PL regression, with Bio-Plex Manager™ software).

Statistical Analyses

For all analyses, the Wilcoxon signed rank test was used and a p-value <0.05 was considered statistically significant, Mann-Whitney test was also used to compare two groups. Analyses were conducted using GraphPad InStat3 (8.0.1).

Results

EV-CPC and their parental cells display an immune phenotype compatible with allogeneic applications

The iPSC-derived CPC express SSEA-1/CD15, a marker of loss of pluripotency, in line with their cardiac progenitor identity. Immunologically, they express low levels of HLA-I and costimulatory/regulatory CD274 [PDL1], CD275[ICOS-l] and CD86 molecules, low levels of CD80 and of the ligands of the NK cell activating receptor NKG2D, ULBP-1, ULBP 2/5/6 and MIC-A/B and they did not express HLA-II (Figure 1a). The EV-CPC also express CD15 of their parent cells. Immunologically, they do not express HLA-I, HLA-II or costimulatory CD86 molecules. They display low levels of CD80, PD-L1 and ICOS-1, and only modest levels of the ligands of NKG2D (Figure 1b).

To further characterize EV, we used the MACSplex kit to more extensively determine their surface marker profile. We detected CD9, CD29, CD49e, CD63, CD81 and CD326 (Epithelial Cell Adhesion Molecule-EPCAM), which are known to be abundantly expressed by EV, (Figure 1c and Supplemental Figure 1). The EV-CPC also expressed markers of an early differentiation stage including SSEA-4, CD133, CD105 and ROR1 and lacked the expression of markers not expected to be present on EV derived from CPC such as CD3, CD4 and CD14 (Supplemental Figure 1). The EV identity was further confirmed by the expression of CD81, as assessed by cryo-immuno gold electron microscopy (Figure 1d). EV consisted mostly of spherical vesicles ranging in size from 50 to 500 nm (Figure 1d-lower panel) and 40% of them were found to express the tetraspanin CD81.

EV-CPC neither induce T cell activation nor modulate their on-going proliferation

The capacity of both CPC and their secreted EV to activate T cells in allogeneic settings was then evaluated. Co-culture of PBMC with allogeneic mitomycin-C-treated PBMC significantly activated both CD4 + and CD8 + cells, increasing the percentage of proliferating cells from 4% to 40%. In contrast, neither CD4 + nor CD8 + cells responded to allogeneic mitomycin-c-treated CPC (Figure 2a). On the same note, PBMC cultured with EV-CPC maintained their basal proliferation rate and were not affected by EV (Figure 2a). The possibility of PBMC death by apoptosis as a potential cause of their non-proliferation was excluded by aminoactinomycine D7-AAD labeling (data not shown). Thus, neither CPC nor their secreted EV trigger any direct or indirect in vitro allogeneic T cell proliferation.

We then investigated the immunomodulatory capacity of EV-CPC in comparison to their parent cells. We evaluated PHA-induced T cell proliferation as an on-going T cell immune response model in the presence or absence of CPC or their secreted EV. The presence of CPC decreased both PHA-induced CD4 + and CD8 + T cell proliferation by approximately 33%, in a ratio-dependent manner (Figure 2b). In contrast, the presence of EV did not modulate PHA-induced CD4 + or CD8 + T cell proliferation (Figure 2b), suggesting that they neither inhibit nor promote a T cell-mediated on-going immune response.

EV-CPC do not induce NK degranulation

Similar to their parent cells, EV-CPC express ligands of the NKG2D receptor, albeit at a lower level. Therefore, we evaluated their capacity to trigger NK cell cytotoxicity in allogeneic settings by assessing their ability to induce degranulation of IL-15-primed NK cells. Compared to the classical NK cell target, (HLA-I-negative K562 cells), CPC modestly activated allogeneic NK cells. The percentage of NK cells specifically expressing the degranulation marker CD107a was approximately 26% in the presence of CPC compared to 45% in the presence of K562 cells. In contrast, the expression of CD107a on IL-15-primed NK cells was not affected by the presence of EV-CPC, even when a high dose of 1.7x10 10 particles was used (Figure 3). Thus, in line with their expression of ligands of NK cell activating receptors, only CPC, but not their secreted EV, are able to modestly activate NK cells.

EV-CPC do not induce humoral nor cellular response in vivo

To validate our in vitro findings, we then investigated the immune behavior of EV-CPC in an immunocompetent mouse model subjected to an immunization protocol. Mice were subcutaneously injected 3 times (at day 1, 10, 20) with either EV-CPC (n=5) or PBS (n=5) and the humoral or cellular responses of injected mice against EV-CPC were determined. No statistically significant differences in immunoglobulin production were observed between controls and EV-CPC-treated mice at any time point (Figure 4a). To evaluate the cellular immune response, splenocytes from treated and untreated mice were re-challenged in vitro with EV-CPC or PBS and the production of IFN- was evaluated as a marker of effector T (CD4 + and CD8 + ) and NK cell activation. Neither the splenocytes from EV-CPC-treated mice nor those from untreated mice produced detectable levels of IFN-γ upon their re-exposure to EV-CPC (Figure 4b). Together, these data suggest that injecting immunocompetent recipients with EV-CPC is unlikely to trigger a pronounced humoral or cellular immune response.

Immuno-inflammatory effects of EV-CPC in an immunocompetent mouse model of chronic heart failure

We then investigated the immune response to EV-CPC in immunocompetent mice in which a MI had been created 3 weeks earlier. Inflammatory cells were harvested 3 days after treatment with PBS, CPC or EV-CPC. Lipopolysaccharide administration was used as a positive control to stimulate inflammation in the targeted cardiac tissue. As expected, the injection of LPS in the myocardium altered the profile of myeloid cell lines (Figure 5a), thereby confirming the capacity for these mice to mount an appropriate response to this endotoxin [START_REF] Prabhu | The Biological Basis for Cardiac Repair After Myocardial Infarction: From Inflammation to Fibrosis[END_REF] . In infarcted hearts which were left untreated (controls), the overall number of inflammatory cells was low, consistent with a lower level of inflammation in chronic heart failure compared to acute MI [START_REF] Cochain | The chemokine decoy receptor D6 prevents excessive inflammation and adverse ventricular remodeling after myocardial infarction[END_REF] . Furthermore, PBS injection did not lead to significant changes in the immune profile of heart tissue when compared to untreated animals, thereby ruling out a significant immune response to the needle poke or introduction of fluid. Likewise, injection of EV-CPC or their parental cells did not induce any significant alteration in the numbers of adaptive cells (B and T), innate macrophages or neutrophils, as compared to controls. In contrast, they significantly decreased the number of inflammatory Ly6C high monocytes without any pronounced effect on Ly6C low monocytes.

EV-CPC and CPC injection did not affect the number of circulating (blood) adaptive immune cells, neutrophils or Ly6C low monocytes (Supplementary Figure 2a). However, as compared to controls, and in contrast to EV, CPC injection reduced the number Ly6C high circulating monocytes. No significant difference between groups were observed in the immune profile of bone marrow (Supplemental Figure 3 a) or spleen cells (Supplemental Figure 3b), with the exception of a drastic drop of myeloid and lymphoid bone marrow cells in the LPS group [START_REF] Chandra | Endotoxemia down-regulates bone marrow lymphopoiesis but stimulates myelopoiesis: the effect of G6PD deficiency[END_REF] .

The expression profile of blood cytokine levels was in accordance. Infarcted mice treated with EV-CPC or parental cells displayed decreased levels of the pro-inflammatory cytokines IL-1α, IL-1, TNF, and IFN-γ compared to the PBS control group (Figure 5b), while other cytokines were not affected (Supplemental Figure 2b).

Collectively, these data suggest that EV-CPC, similar to their parental cells, do not trigger any pronounced deleterious immune response in a chronic MI-induced left ventricle (LV) dysfunction model. They might rather downregulate pro-inflammatory compounds in cardiac tissue.

Immuno-inflammatory effects of EV-CPC in an immunocompetent mouse model of acute myocardial infarction

As the number of inflammatory cells in the infarcted heart was low 3 weeks post-infarct, the possibility was raised that the putative immunomodulatory potential of EV-CPC could have been missed due to the dampened inflammatory reaction at that stage of disease. Therefore, we assessed the immune-inflammatory effects of EV-CPC in an experimental model of acute MI which is associated with a large increase of immune/inflammatory cells in the cardiac tissue. In this model, mice were injected with EV-CPC or PBS 2 days after the infarct, and, as in the chronic model, cardiac inflammatory cells were harvested 3 days after treatment. Compared to control PBSinjected mice, EV-CPC treatment resulted in a significant decrease in neutrophils (from 140 to 50 cells per mg of tissue, p=0.02) and pro-inflammatory Ly6C high monocytes (from 160 to 70 cells per mg of tissue, p=0.01), but did not induce any significant differences in macrophages or adaptive T or B cell populations (Figure 6a). Cytokine profiling in cardiac tissue was in-line with these results, as EV-CPC induced a significant decrease in pro-inflammatory interleukins, IL-1, IL-2, and IL-6, and a significant increase in the anti-inflammatory cytokine, IL-10. No noticeable differences were observed for other cytokines between the two groups (data not shown). EV-CPC treatment did not induce any significant alteration of tissue-resident cells in the bone marrow or the spleen (Supplemental Figure 4).

Collectively, our findings in the acute MI model support those observed in the chronic heart failure model and emphasize the capacity of EV-CPC to modulate immune/inflammatory responses towards a reparative profile.

EV-CPC shape macrophage polarization state

As our in vivo data had suggested that EV-CPC could act as modulators of monocyte/macrophage populations towards an anti-inflammatory phenotype, we assessed whether these effects were relevant to human cells. CD14+ monocytes were isolated from human PBMC and differentiated to M0-(M-CSF), M1-(LPS and IFN), or M2-macrophages (IL-4 and IL-13). Pro-inflammatory M1 macrophages were characterized as CD64+, CD68+, CD80+, CD86+ cells; and reparative macrophages as M2a (CD64+, CD68+, CD206+) or M2b (CD64+, CD68+, CD163+) (Supplementary Figure 5). Exposure of M0 to EV-CPC decreased the percentage of proinflammatory monocytes (CD14 + CD86 + ) by 2.20-fold (data not shown), and that of proinflammatory macrophages (CD64+ CD68+ CD86+ CD80+) by 2.26-fold. In this experimental setting, the percentage of anti-inflammatory macrophages M2a (CD64+ CD68+ CD206+) was increased by 1.66-fold, (Figure 7a). Exposure of M1 macrophages to EV-CPC did not impact the percentage of CD64+ CD68+ CD86+ CD80+ cells but raised by 1.64-fold the percentage of M2a cells, suggesting that even under pro-inflammatory conditions, EV-CPC favors the occurrence of anti-inflammatory M2 macrophages. Exposure of M2 macrophages to EV-CPC did not significantly affect the percentage of M1, M2a or M2b cells (Figure 7a). Analysis of the culture supernatants confirmed the capacity of EV to modulate cytokine levels and, more specifically, to upregulate angiogenic and anti-inflammatory markers in the treated macrophages (Figure 7b).

Discussion

The principal findings of the present study are that EV derived from cardiovascular progenitor cells do not appear to be immunogenic and seem rather endowed with anti-inflammatory properties (Schematic representation).

The consistent observation of a discrepancy between the low engraftment rate of therapeutic cells and the maintenance of functional benefits beyond the disappearance of the grafted tissue has led the field to favor a paracrine mechanism of action whereby the transplanted cells release factors which stimulate endogenous repair pathways [START_REF] Théry | Membrane vesicles as conveyors of immune responses[END_REF][START_REF] De Couto | Exosomal MicroRNA Transfer Into Macrophages Mediates Cellular Postconditioning[END_REF][START_REF] Théry | Exosomes: composition, biogenesis and function[END_REF][START_REF] El Andaloussi | Extracellular vesicles: biology and emerging therapeutic opportunities[END_REF][START_REF] Camussi | Exosomes/microvesicles as a mechanism of cell-to-cell communication[END_REF][START_REF] Camussi | Exosome/microvesiclemediated epigenetic reprogramming of cells[END_REF] . This mechanistic hypothesis is strengthened by the finding that the effects of transplanted cells can be recapitulated by the administration of their secretome [START_REF] Kervadec | Cardiovascular progenitorderived extracellular vesicles recapitulate the beneficial effects of their parent cells in the treatment of chronic heart failure[END_REF][START_REF] Gnecchi | Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement[END_REF] . This equivalence has been demonstrated in various and multiple preclinical models targeting different diseases [START_REF] Lener | Applying extracellular vesicles based therapeutics in clinical trials -an ISEV position paper[END_REF] which lends further support for the therapeutic benefits of the cellular secretome. While this secretome is comprised of multiple biologically active components, one important class which has gained increasing interest is represented by EV [START_REF] Ibrahim | Exosomes as critical agents of cardiac regeneration triggered by cell therapy[END_REF][START_REF] Lener | Applying extracellular vesicles based therapeutics in clinical trials -an ISEV position paper[END_REF][START_REF] Xin | Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats[END_REF][START_REF] Kamata | Improvement of cardiac stem cell sheet therapy for chronic ischemic injury by adding endothelial progenitor cell transplantation: analysis of layer-specific regional cardiac function[END_REF][START_REF] Vlassov | Exosomes: current knowledge of their composition, biological functions, and diagnostic and therapeutic potentials[END_REF][START_REF] Menasché | Stem cell-derived exosomes and the failing heart: Small cause, big effect[END_REF] . All cell types secrete EV, but not all EVs are equivalent. For example, Barile et al [START_REF] Barile | Cardioprotection by cardiac progenitor cell-secreted exosomes: role of pregnancy-associated plasma protein-A[END_REF] have shown that, in comparison with EV from mesenchymal stem cells, those released by cardiac stem cells have a greater therapeutic efficacy in a ischemic rat occlusion-reperfusion model. The proteomic profile from the two cell types is actually different, possibly leading to a release of factors by cardiaccommitted cells which are better tailored to heart tissue repair. Therefore, and in-line with our previous studies, we have been committed to use EV secreted from cells phenotypically close to those of the target tissue. In this case, we use iPSC derived cardiovascular progenitors. These EV were primarily characterized by their size profile, surface markers and TEM characteristics which thus provided sound evidence for their identity.

While the composition of the EV cargo reflects that of their parental cells, it can be selectively enriched in, or deprived of, some molecules present in these cells [START_REF] Colombo | Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles[END_REF] . This is confirmed in the present study by the finding that, in contrast to CPC, the secreted EV express extremely low levels of most immune relevant molecules. Yet, they did express the ligands for NK cell activating receptors, albeit at a lower level compared to their parent cells. This expression pattern strongly suggests that EV-CPC may be more immunologically neutral towards lymphoid populations and innate lymphoid NK cells than their parent cells. This idea is supported by the fact that EV-CPC did not trigger allogeneic T cell activation either in vitro, as shown by the mixed lymphocyte reactions data. The EV-CPC are probably also unable to trigger neither T nor B cells response in vivo, since they were not able to affect the number of these adaptive cells upon their injection in immunocompetent infarcted mice. Further, EV-CPC did not trigger a humoral immune response, since their repeated delivery did not result in an increase in immunoglobulin production as compared to controls, thereby strengthening the idea that they may deviate the immune surveillance of the host. This makes them therapeutically attractive since, and in contrast to their parental allogeneic cells, their use in patients might not require immunosuppression. Of note, EV from other cell sources like antigen-presenting cells carry HLA-I and II antigens and can thus trigger T cell proliferation [START_REF] Robbins | Regulation of immune responses by extracellular vesicles[END_REF] . Conversely, EV from mesenchymal stem cells have been credited with immunosuppressive effects [START_REF] Börger | Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles and Their Potential as Novel Immunomodulatory Therapeutic Agents[END_REF] which we did not find to be the case for EV-CPC in our in vitro studies. Taken together, these data highlight the critical influence of the parental cell phenotype on the ultimate immune properties of their secreted EV.

The fact that EV-CPC seem endowed with anti-inflammatory properties further supports the above notion. In vivo, we consistently observed that pro-inflammatory Ly6C high monocytes were decreased in heart tissue in both acute MI and chronic heart failure models. This anti-inflammatory effect was more pronounced at the acute stage under the form of a concomitant decrease in neutrophils. Further evidence includes a decrease in heart and plasma cytokine levels in both acute and chronic settings and the trend for human innate myeloid monocytes/macrophages exposed to EV-CPC to shift their polarity towards an anti-inflammatory phenotype. Yet, EV-CPC were unable to reverse the pro-inflammatory M1 pattern once established concerning in vitro experiments with human cells, in contrast to exosomes from adipose-derived mesenchymal stromal cells which can reverse the M1 conversion of microglial cells subjected to oxygen and glucose deprivation [START_REF] Jiang | Exosomes from MiR-30d-5p-ADSCs Reverse Acute Ischemic Stroke-Induced, Autophagy-Mediated Brain Injury by Promoting M2 Microglial/Macrophage Polarization[END_REF] .These divergent results might be due to differences in the content of the EV payload and/or the phenotype of the target cells.

Although the present experiments focused on the immune-inflammatory responses to EV and, as such, did not include functional assessments, it is noteworthy that in a previous study from our laboratory [START_REF] El Harane | Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors[END_REF] , EV from the same CPC source as in the present study were shown to improve post infarction contractile performance in a mouse model of chronic left ventricular dysfunction. The transcriptomic study of these EV had highlighted the strong expression of 16 microRNAs broadly conserved across species, among which at least half, i.e., miR-92a [START_REF] Lai | MicroRNA-92a negatively regulates Toll-like receptor (TLR)-triggered inflammatory response in macrophages by targeting MKK4 kinase[END_REF] , miR-24 [START_REF] Tahamtan | Anti-Inflammatory MicroRNAs and Their Potential for Inflammatory Diseases Treatment[END_REF] , miR-93-5p [START_REF] Liu | miR-93-5p-Containing Exosomes Treatment Attenuates Acute Myocardial Infarction-Induced Myocardial Damage[END_REF] , miR 20b-5p [START_REF] You | MiR-20b-5p relieves neuropathic pain by targeting Akt3 in a chronic constriction injury rat model[END_REF] , miR-107 [START_REF] Gao | microRNA-107 protects against inflammation and endoplasmic reticulum stress of vascular endothelial cells via KRT1dependent Notch signaling pathway in a mouse model of coronary atherosclerosis[END_REF] , miR 26a-5p 51 , miR-16-5p [START_REF] Lei | LncRNA SNHG1 alleviates IL-1β-induced osteoarthritis by inhibiting miR-16-5p-mediated p38 MAPK and NF-κB signaling pathways[END_REF] and miR-130b-3p [START_REF] Wang | MiR-130b attenuates vascular inflammation via negatively regulating tumor progression locus 2 (Tpl2) expression[END_REF] are to some extent involved in the regulation of inflammation. It is thus plausible that the effects of the EV cargo on the mitigation of immune-inflammatory responses demonstrated in the present study participate in the previously documented EV-associated preservation of post infarction heart function. Of note, these functional benefits were observed in an immune-deficient mouse model. However, recent work in our laboratory has reproduced this therapeutic effect in an immune-competent strain (unpublished data), suggesting that the possible link between the EV-related regulation of immuneinflammatory responses and the mitigation of post infarct heart injury is independent of the immune status of the recipient animals.

The finding of decreased circulating levels of IL-1α and IFN-γ in mice, following intramyocardial injection of EV three weeks earlier, suggests that these particles might induce a sustained systemic anti-inflammatory effect not strictly localized in the treated organ. It is thus conceivable that the opposite event may happen, i.e., that intravenously delivered EV, even though they are predominantly trapped in the lungs and spleen, may exert endocrine anti-inflammatory effects on remote organs, including the heart, as reported for mesenchymal stem cells [START_REF] Luger | Intravenously Delivered Mesenchymal Stem Cells: Systemic Anti-Inflammatory Effects Improve Left Ventricular Dysfunction in Acute Myocardial Infarction and Ischemic Cardiomyopathy[END_REF] . This could explain the therapeutic efficacy of intravenously injected EV in Duchenne [START_REF] Rogers | Disease-modifying bioactivity of intravenous cardiosphere-derived cells and exosomes in mdx mice[END_REF] and doxorubicin-induced cardiomyopathy [START_REF] Milano | Intravenous administration of cardiac progenitor cell-derived exosomes protects against doxorubicin/trastuzumab-induced cardiac toxicity[END_REF] which share in common a chronic inflammation of the myocardial tissue [START_REF] Mavrogeni | Myocardial inflammation in Duchenne Muscular Dystrophy as a precipitating factor for heart failure: a prospective study[END_REF][START_REF] Sun | Intravenous mesenchymal stem cell-derived exosomes ameliorate myocardial inflammation in the dilated cardiomyopathy[END_REF] . Should this hypothesis be validated, it would reinforce the clinical attractiveness of EV by opening the way to systemic delivery, making such a treatment simple, non-invasive, readily available and economically affordable.

Figure 1 :

 1 Figure 1: Surface marker profile of CPC and EV-CPC: a & b) Representative flow cytometry histograms for the fluorescence intensity of surface markers on CPC (a) and EV-CPC (b). The percentage of positive cells (%) and geometric mean fluorescence intensity (MFI) are indicated. mAb (monoclonal antibody). c) Main surface marker profile of EV-CPC, as identified by a cocktail of 39 fluorescently-labeled bead populations, each coated with a specific antibody binding the respective EV surface epitope. These data were derived from 4 independent preparations of EV. Data are presented as box-and-whisker plots, with the midline representing the median and the whiskers representing maximum and minimum values. d) The upper and middle panels present representative cryo-TEM images of CD81+ and CD81-EV embedded in a thin film of frozen buffer. The lipid bilayer is clearly resolved (arrow in the upper panel). The lower panel presents the size distribution of CD81+ and CD81-EVs, determined over a total of 350 EV. The median sizes are 175 nM and 265 nM for CD81+ and CD81-EV, respectively. CPC: iPS-derived cardiovascular progenitor cells; EV: Extracellular vesicles from iPS-derived cardiovascular progenitor cells.

Figure 2 :

 2 Figure 2: Effects of CPC and EV-CPC on allogeneic T-cell immune response. a) CFSE-labeled peripheral blood mononuclear cells (PBMC) were cultured alone (negative control), with human leukocyte antigen-mismatched mitomycin C-treated PBMC (Allo PBMC from 2 different donors, positive control), with mitomycin C-treated CPC at different CPC:PBMC ratios (CPC1: 1:10; CPC2: 1:5), or with EV-CPC at different doses (EV1: 5.5x10 8 ; EV2: 5.5x10 9 and EV3: 1.7x10 10 particles). Proliferation levels of CD4 + and CD8 + T cells were determined by loss of CFSE labelling. b) CFSE-labeled human leukocyte antigen-mismatched PBMC were activated with phytohemagglutinin ((PHA)-positive control), alone (negative control), in the presence of mitomycin C-treated CPC at different CPC:PBMC ratios (CPC1: 1:10; CPC2: 1:5), or with EV-CPC at different doses (EV1: 5.5x10 8 ; EV2: 5.5x10 9 and EV3: 1.7x10 10 particles). Proliferation levels of CD4 + and CD8 + T cells were determined by loss of CFSE labelling. The results are given as mean ± SD from 3 independent experiments performed in duplicate or triplicate. Wilcoxon signed rank test was used and a p-value <0.05 was considered statistically significant** p ≤ 0.01; *** p ≤ 0.001. Abbreviations as in Figure 1.

Figure 3 :

 3 Figure 3: Effects of CPC and EV-CPC on NK cell degranulation. a) Scatter plot of flow cytometry analysis showing the percentages of CD107a-positive cells in CD56+ IL-15-activated NK cells after overnight incubation with target cells: K562 (positive control), CPC or EV-CPC (EV1: 5.5x10 8 ; EV2: 5.5x10 9 and EV3: 1.7x10 10 particles) at a 1:1 effector:target cell ratio. b) Histogram presenting percentages of CD107a positive cells (mean ± SD) from 3 independent experiments performed in triplicate. The results are given as mean ± SD. Wilcoxon signed rank test was used, a p-value <0.05 was considered statistically significant. *** p ≤ 0.001. Abbreviations as in Figure 1.

Figure 4 :

 4 Figure 4: Humoral and cellular responses of vaccinated mice with PBS or EV-CPC. a) Immunoglobulin isotype detection in plasma of vaccinated mice at days 10, 20, 30 and 37. b) Spot counts of interferon-gamma (IFN-γ) detection secreted by splenocytes of each vaccinated mouse (technical triplicates), n = 6 per group. PBS: Phosphate-Buffered saline; other abbreviations as in Figure 1.

Figure 5 :

 5 Figure 5: Inflammatory cell numbers in heart tissue and cytokine blood levels in the chronic heart failure model. a) Heart tissue levels of immune cells: Three weeks after MI, mice were untreated (n=8-13) or injected with PBS (n=6-8), EV (n=7), CPC (n=6) or LPS (n=4). Three days after injection, the left ventricle was removed and analysed by FACS for the assessment of immune cells. b) Circulating blood cytokine measurements, assessed by FACS using a Luminex® based-bead immuno assay. Untreated (n=3-5), PBS (n=4-6), EV (n=5-6), CPC (n=3-5) or LPS (n=3). The results are given as mean ± SD. Wilcoxon signed rank test was used, a p-value <0.05 was considered statistically significant. *p ≤ 0.05; ** p ≤ 0.01. Abbreviations as in Figure 4.

Figure 6 :

 6 Figure 6: Inflammatory cell numbers and cytokine levels in heart tissue in the acute myocardial infarction model.

Funding: This work was supported by the Institut National de la Santé et de la Recherche

Médicale; the Assistance Publique Hôpitaux de Paris; the Université Paris Descartes; the Agence Nationale de la Recherche (LabexRevive, ANR -10-LABX-73 to P.M.); the Fondation de France (FDF 00066471 to P.M. and FDF 00066471 J-S. S.), and the Fondation pour la Recherche Médicale (DEQ20160334910 to J-S. S. and P.M.).

Conflict of interests:

The authors declare no competing financial interests. a) Heart tissue levels of immune cells: Two days after MI, mice were injected with PBS (n=6) or EV (n=6). Three days after injection, the left ventricle was removed and analysed by FACS for the assessment of immune cells. b) Cardiac cytokine levels, as assessed by FACS using a Luminex® based-bead immuno-assay; PBS (n=5-6) or EV (n=6-7). The results are given as mean ± SD. Wilcoxon signed rank test was used, a p-value <0.05 was considered statistically significant. *p ≤ 0.05. Abbreviations as in Figure 4. Of note, EV harbor an unchallenged capacity to fine-tune the response of monocyte/macrophages towards resolution of inflammation bolstering their therapeutic potential.


Lulin Zhang

Ewelina Rupnik
email: ewelina.rupnik@ign.fr

Marc Pierrot-Deseilligny
email: marc.pierrot-deseilligny@ensg.eu

GUIDED FEATURE MATCHING FOR MULTI-EPOCH HISTORICAL IMAGE BLOCKS POSE ESTIMATION

Keywords: Feature matching, Analogue aerial images, Multi-epoch, Pose estimation

Historical aerial imagery plays an important role in providing unique information about evolution of our landscapes. It possesses many positive qualities such as high spatial resolution, stereoscopic configuration and short time interval. Self-calibration reamains a main bottleneck for achieving the intrinsic value of historical imagery, as it involves certain underdeveloped research points such as detecting inter-epoch tie-points. In this research, we present a novel algorithm to detecting inter-epoch tie-points in historical images which do not rely on any auxiliary data. Using SIFT-detected keypoints we perform matching across epochs by interchangeably estimating and imposing that points follow two mathematical models: at first a 2D spatial similarity, then a 3D spatial similarity. We import GCPs to quantitatively evaluate our results with Digital Elevation Models (DEM) of differences (abbreviated as DoD) in absolute reference frame, and compare the results of our method with other 2 methods that use either the traditional SIFT or few virtual GCPs. The experiments show that far more correct inter-epoch tie-points can be extracted with our guided technique. Qualitative and quantitative results are reported.

INTRODUCTION

Historical (i.e. analogue) aerial imagery plays an important role in providing unique information about evolution of our landscapes. The imagery had been acquired by many countries all over the world and can be traced back to the beginning of 20 th century [START_REF] Cowley | Historic aerial photographic archives for European archaeology[END_REF]. In many cases high spatial resolution stereoscopic acquisitions were performed [START_REF] Giordano | Archiving and geoprocessing of historical aerial images: current status in europe,official publication no 70[END_REF], allowing for 3D restitution of territories. Coupled with high temporal sampling, the imagery becomes also suitable for long-term environmental studies. As of now, many historical images collections have been digitized [START_REF] Giordano | Archiving and geoprocessing of historical aerial images: current status in europe,official publication no 70[END_REF]USGS, 2019;IGN, 2019) and a number of attempts have been made to process the images with the state-of-the-art automated methods developed in photogrammetry and computer vision community over the last twenty years [START_REF] Ayoub | Co-registration and correlation of aerial photographs for ground deformation measurements[END_REF][START_REF] Bakker | Archival photogrammetric analysis of river-floodplain systems using Structure from Motion (SfM) methods[END_REF][START_REF] Chen | Study of automatic image rectification and registration of scanned historical aerial photographs[END_REF][START_REF] Ellis | Measuring long-term ecological changes in densely populated landscapes using current and historical high resolution imagery[END_REF][START_REF] Feurer | Joining multi-epoch archival aerial images in a single SfM block allows 3-D change detection with almost exclusively image information[END_REF].

Apart from imagery itself, sometimes corresponding metadata is archived too. For instance, camera focal length and physical size of the images are commonly found, whereas flight plans, camera calibration certificates or orientations are rarely available. Because the images were scanned, and prior to scanning they may have been kept in unsuitable conditions, a selfcalibration is always necessary.

Historical images are self-calibrated in a bundle block adjustment (BBA) routine, using tie-points and Ground Control Points (GCPs) as input observations. When dealing with singleepoch datasets, tie-points are efficiently recovered with modern extraction algorithms, such as SIFT [START_REF] Lowe | Distinctive image features from scaleinvariant keypoints[END_REF], while GCPs originate from (i) field surveys [START_REF] Micheletti | Application of archival aerial photogrammetry to quantify climate forcing of alpine landscapes[END_REF][START_REF] Walstra | Time for change-quantifying landslide evolution using historical aerial photographs and modern photogrammetric methods[END_REF][START_REF] Cardenal | Use of historical flight for landslide monitoring[END_REF], (ii) existing orthophotos and DEM [START_REF] Nurminen | Automation aspects for the georeferencing of photogrammetric aerial image archives in forested scenes[END_REF][START_REF] Ellis | Measuring long-term ecological changes in densely populated landscapes using current and historical high resolution imagery[END_REF][START_REF] Fox | Unlocking the time capsule of historic aerial photography to measure changes in Antarctic Peninsula glaciers[END_REF], (iii) recent satellite images [START_REF] Ellis | Measuring long-term ecological changes in densely populated landscapes using current and historical high resolution imagery[END_REF][START_REF] Ford | Shoreline changes interpreted from multitemporal aerial photographs and high resolution satellite images: Wotje Atoll, Marshall Islands[END_REF]. In many scenarios none of the auxiliary data is at disposal. When processing multi-epoch image blocks, ideally a joint self-calibrating bundle adjustment is carried out, including inter-epoch image observations. However, extracting interepoch tie-points remains challenging because the scene changes over time, and images are often of low radiometric quality violating the brightness constancy rule that all modern extraction algorithms rely upon.

In this work we propose a new approach to detecting interepoch tie-points. Using SIFT-detected key-points we perform matching across epochs by interchangeably estimating and imposing that points follow two mathematical models: at first a 2D spatial similarity, then a 3D spatial similarity. We do not rely on any auxiliary data, making it a generic approach. We adopt GCPs only to quantitatively evaluate our results. In the following we briefly describe existing feature matching methods, as well as recent approaches to georeferencing of historical images. In Section 3 we introduce our methodology, and in Section 4 the experiments as well as results are given. The terms features and tie-points are considered synonymous and used interchangeably throughout this publication.

RELATED WORK

Feature Extraction

Feature extraction refers to finding discriminative structure in images such as corner, blob, edge and so on, followed by a matching step. According to whether machine learning techniques are applied, the image features can be categorized as hand-crafted or learned.

Hand-crafted Features

In the early stage, Moravec detects corner feature by measuring the sum-of-squareddifferences (SSD) by applying a small shift in a number of directions to the patch around a candidate feature [START_REF] Moravec | Obstacle avoidance and navigation in the real world by a seeing robot rover[END_REF]. Based on this, Harris computes an approximation to the second derivative of the SSD with respect to the shift [START_REF] Harris | A combined corner and edge detector[END_REF]. Since both Moravec and Harris are sensitive to changes in image scale, algorithms invariant to scale and affine transformations based on Harris are presented [START_REF] Mikolajczyk | Scale & affine invariant interest point detectors[END_REF]. Other than corner feature, SIFT (Scaleinvariant feature transform) [START_REF] Lowe | Distinctive image features from scaleinvariant keypoints[END_REF] detects blob feature in scale-space, which is an entire pipeline including detection and description. It uses a difference-of-Gaussian function to identify potential feature points that are invariant to scale and orientation. SIFT is a milestone among hand-crafted features, and comparable with machine learning alternatives. RootSIFT [START_REF] Arandjelović | Three things everyone should know to improve object retrieval[END_REF] uses a square root (Hellinger) kernel instead of the standard Euclidean distance to measure the similarity between SIFT descriptors, which leads to a dramatic performance boost. Similar to SIFT, SURF [START_REF] Bay | Surf: Speeded up robust features[END_REF] resorts to integral images and Haar filters to extract blob feature in a computationally efficient way. DAISY [START_REF] Tola | Daisy: An efficient dense descriptor applied to wide-baseline stereo[END_REF] is a local image descriptor, which uses convolutions of gradients in specific directions with several Gaussian filters to make it very efficient to extract dense descriptors. KAZE [START_REF] Alcantarilla | Kaze features[END_REF] is an algorithm that detects and describes multi-scale 2D feature in nonlinear scale spaces. AKAZE [START_REF] Alcantarilla | Fast explicit diffusion for accelerated features in nonlinear scale spaces[END_REF] is an accelerated version based on KAZE.

Learned Features

With the rise of machine learning, learned features have shown their feasibility in the image matching problem when enough ground truth data is available. FAST [START_REF] Rosten | Machine learning for highspeed corner detection[END_REF] uses decision tree to speed up the process of finding corner feature. LIFT (Learned Invariant Feature Transform) [START_REF] Yi | Lift: Learned invariant feature transform[END_REF] is a deep network architecture that implements a full pipeline including detection, orientation estimation and feature description. It is based on the previous work TILDE [START_REF] Verdie | Tilde: a temporally invariant learned detector[END_REF], the method of (Moo [START_REF] Yi | Lift: Learned invariant feature transform[END_REF] and DeepDesc [START_REF] Simo-Serra | Discriminative learning of deep convolutional feature point descriptors[END_REF]. SuperPoint [START_REF] Detone | Superpoint: Self-supervised interest point detection and description[END_REF]) is a self-supervised, fully-convolutional model that operates on full-sized images and jointly computes pixel-level feature point locations and associated descriptors in one forward pass. LF-Net [START_REF] Ono | Lf-net: learning local features from images[END_REF] is a deep architecture that embeds the entire feature extraction pipeline, and can be trained end-to-end with just a collection of images. Finally, D2-Net [START_REF] Dusmanu | D2-Net: A Trainable CNN for Joint Detection and Description of Local Features[END_REF]) is a single convolutional neural network that works as a feature detector and a dense feature descriptor simultaneously.

Even though the learned features demonstrate better performance when compared to hand-crafted features on certain benchmark, it does not necessarily imply a better performance in terms of subsequent processing steps. For example, in the context of Structure from Motion (SfM), finding additional correspondences for image pairs where SIFT already provides enough matches does not necessarily results in more accurate or complete reconstructions [START_REF] Schonberger | Comparative evaluation of hand-crafted and learned local features[END_REF].

Geo-referencing of Historical Images

Pose estimation describes the intrinsic and extrinsic parameters of an image and is classically solved with the SfM algorithms [START_REF] Snavely | Photo tourism: exploring photo collections in 3d[END_REF][START_REF] Pierrot-Deseilligny | Apero, an open source bundle adjusment software for automatic calibration and orientation of set of images[END_REF][START_REF] Schonberger | Structure-from-motion revisited[END_REF]. If the image pose should be known in a reference coordinate system, i.e. be georeferenced, a 7parameter transformation followed by a posteriori bundle block adjustment must be carried out.

Unlike in modern images where the image coordinate system overlaps with the planimetric camera coordinate system, in historical images the overlap is not maintained due to the scanning procedure. To account for this, an additional 2D transformation is estimated in the course of the processing [START_REF] Mcglone | Manual of photogrammetry[END_REF]. [START_REF] Giordano | Toward automatic georeferencing of archival aerial photogrammetric surveys[END_REF] demonstrates the importance of the selfcalibration of historical images and its impact on 3D accuracy. Poorly modelled intrinsic parameters result in the known domelike deformations that the authors eliminate with the help of automatic GCPs from exiting orthophotos.

Many cases reported in the literature calculate the image poses with SfM using features matched exclusively within the same epoch [START_REF] Nurminen | Automation aspects for the georeferencing of photogrammetric aerial image archives in forested scenes[END_REF][START_REF] Cardenal | Use of historical flight for landslide monitoring[END_REF][START_REF] Fox | Unlocking the time capsule of historic aerial photography to measure changes in Antarctic Peninsula glaciers[END_REF][START_REF] Walstra | Time for change-quantifying landslide evolution using historical aerial photographs and modern photogrammetric methods[END_REF]. In multi-epoch scenarios, the individual epochs should be defined in a common frame, be it the frame of a reference epoch or an absolute reference frame (i.e., a projection coordinate frame). Control points derived from recent orthophotos and DEM [START_REF] Nurminen | Automation aspects for the georeferencing of photogrammetric aerial image archives in forested scenes[END_REF][START_REF] Ellis | Measuring long-term ecological changes in densely populated landscapes using current and historical high resolution imagery[END_REF][START_REF] Fox | Unlocking the time capsule of historic aerial photography to measure changes in Antarctic Peninsula glaciers[END_REF] or GPS survey [START_REF] Micheletti | Application of archival aerial photogrammetry to quantify climate forcing of alpine landscapes[END_REF][START_REF] Walstra | Time for change-quantifying landslide evolution using historical aerial photographs and modern photogrammetric methods[END_REF][START_REF] Cardenal | Use of historical flight for landslide monitoring[END_REF] serve to transform the individual epochs to the common frame. Alternatively, a coarse flight plan may provide an approximate co-registration [START_REF] Giordano | Toward automatic georeferencing of archival aerial photogrammetric surveys[END_REF].

To increase relative accuracy between several epochs, [START_REF] Cardenal | Use of historical flight for landslide monitoring[END_REF][START_REF] Korpela | Geometrically accurate time series of archived aerial images and airborne lidar data in a forest environment[END_REF][START_REF] Micheletti | Application of archival aerial photogrammetry to quantify climate forcing of alpine landscapes[END_REF] manually insert inter-epoch tie-points. [START_REF] Giordano | Toward automatic georeferencing of archival aerial photogrammetric surveys[END_REF] extracts tie-points between analogue images and recent images based on the method of [START_REF] Aubry | Painting-to-3D model alignment via discriminative visual elements[END_REF]. [START_REF] Feurer | Joining multi-epoch archival aerial images in a single SfM block allows 3-D change detection with almost exclusively image information[END_REF] joins multi-epoch images in a single SfM block based on SIFT-like algorithm [START_REF] Lowe | Distinctive image features from scaleinvariant keypoints[END_REF][START_REF] Semyonov | Algorithms used in photoscan[END_REF] by making the assumption that a sufficient number of feature points remain invariant across each time period. Identifying permanent points over a long time-span has not reached a maturity and research in this domain remains insignificant. Most of the mentioned approaches rely on manual effort, auxiliary input or limiting hypothesis.

METHODOLOGY

Figure 1 exhibits the workflow of our algorithm proposed in this paper. It can be divided as two main parts: processing intraepoch, and processing inter-epoch. The images are resampled to the geometry of the fiducial marks prior to processing. For the sake of simplicity we only exhibit the processing flow of two epochs, however, it can be easily extended to more epochs. In the remaining of this section, both intra-epoch and inter-epoch processing will be explained in detail.

Intra-epoch Processing

Inter-epoch Processing

Using images of all epochs, we now extract inter-epoch tiepoints. We propose the following 3-step guided matching strategy to increase the robustness of the automated tie-points:

Get tentative inter-epoch tie-points: extracted with SIFT and matched based on mutual nearest neighbor (i.e., Euclidean distance) between respective descriptors. To increase computational efficiency and discard noise, features are extracted on images down-sampled with a factor of 1/6. No ratio test [START_REF] Lowe | Distinctive image features from scaleinvariant keypoints[END_REF] was applied since very few tie-points were otherwise retained. This obviously results in a higher number of false matches but as these are only our tentative matches, we accept that. When choosing which feature extraction algorithm to employ, we hesitated between SIFT as the state-of-the-art algorithm for hand-crafted features, and D2-Net -the state-ofthe-art in learned features. We chose SIFT based on a conducted experiment (see Section 4.3) where it outperformed D2-Net.

Get enhanced inter-epoch tie-points: out of the set of all tentative matches we now want to identify and eliminate the false matches. We introduce a 2D similarity transformation (cf., Equation 1) to describe the relation between feature points in a pair of inter-epoch images. This assumption is satisfactory for nadir images, which is our primary focus. The calculation is embedded in a RANSAC framework, and at each iteration the 2D similarity parameters (λ, θ, ∆x, ∆y) are computed from minimal points in inter-epoch images. All points falling within N pixels from the prediction are considered inliers. In our experiment, N was set to 5% of the image diagonal, and RANSAC iterations to 1000.

x y = λ • cosθ sinθ -sinθ cosθ • x y + ∆x ∆y . (1)
Get final inter-epoch tie-points: in the preceding step we got rid of a substantial number of outliers, however, we believe not all outliers could be identified. To obtain the final tie-points we introduce another filter, this time based on 3D similarity transformation:

P = λ • R • P + T (2)
Where the P are the 3D coordinates of a feature point in the reference frame of the intra-epoch reconstruction (e.g., epoch1) and P are its respective coordinates in epoch2. The 3D coordinates are retrieved from the individual depth maps computed within the intra-epoch processing step. We now carry out the estimation of the scale λ, the translation vector T, and the rotation matrix R in a RANSAC routine. We set the number of RANSAC iterations to 1000, and consider tie-points within 10m of its predicted position as inliers.

Combined Processing

With the help of the 3D similarity transformation we can move our multi-epoch acquisitions to a common reference frame. Finally, we perform a BBA to refine all the camera poses and camera calibrations. The intra-and inter-epoch features are the observations in our BBA. To analyze the results in a metric scale, the BBA is followed by another spatial similarity transformation that brings the arbitrary reference frame tied to a selected acquisition to a global reference frame. In our experiments, we derived this transformation thanks to several manually identified points in a recent orthophoto and a digital surface model over the area. If precise poses for one of the epochs were known, we fixed their parameters during the BBA and skipped the final spatial similarity transformation.

EXPERIMENTS

Test Site and Data

The test site is a 420 km 2 rectangular area located in Pezenas in the Occitanie region in southern France. The area is mainly covered with vegetation, and several sparsely populated urban zones. We have at our disposal three sets of images acquired in June 1971, in June 1981and in 2015 (cf., Table 1). The 2015 images were acquired with the IGN's digital metric camera [START_REF] Souchon | The IGN CAMv2 System[END_REF]. The area exhibited changes in scene appearance due to land-use changes in the 44-year period. Our ground truth (GT) is a reference 0.25m

Evaluation Method

As presented in Figure 1, we perform the BBA in a relative coordinate system embedded in a selected epoch. Subsequently, for evaluation purposes we transform the relative results to a reference frame of our GT with the help of several manually measured GCPs. We evaluate the results by computing DSM differences (DoD), between the GT DSM and a DSM resulting from our computations in respective years (cf. Figure 5). We compare Our method to two other approaches:

-SIF Tintra-inter: intra-and inter-epoch tie-points are extracted and matched with SIFT;

-SIF TintraV GCPinter: intra-epoch tie-points are extracted with SIFT but no inter-epoch tie-points are present; the individual epochs are co-registered with the help of virtual GCPs (i.e., points triangulated in one epoch and used as GCP in another).

Result

Comparison between D2-Net and SIFT. To decide which feature extracting algorithm to use in our guided matching, we performed an experiment based on D2-Net and SIFT. D2-Net is employed with the off-the-shelf trained model provided by the authors, and we use their multiscale detection version to achieve better performance with scale changes. We always apply the ratio test for SIFT, and for D2-Net we test scenarios with and without the ratio test. We used an inter-epoch image pair from 1971 and 1981. The images have a scale difference of factor 1.8 and are rotated by 180 • with respect to each other. D2-Net failed at finding corresponding features. This is probably due to its sensitivity to severe rotation. Meanwhile, SIFT correctly identified several matching points as shown in Figure 2. To be able to compare the two feature extractors we removed the scale difference and rotated one image to the orientation of the second image. The matching results are presented in Figure 3. D2-Net detects and matches more points, however, their precision is inferior to that of SIFT (see Figure 3 bottom).

As mentioned in [START_REF] Dusmanu | D2-Net: A Trainable CNN for Joint Detection and Description of Local Features[END_REF], D2-Net is a detection based on higher-level information which inherently leads to more robust but less accurate keypoints. At the same time, D2-Net features are also detected at lower resolution of the CNN features (feature maps have a resolution of one fourth of the input resolution). SIFT was the preferred algorithm because even though it finds few points, they are much more reliable than D2-Net, and they are robust to image rotation.

Inter epoch matching and BBA. In the BBA, all poses corresponding to 1971 and 1981 were considered as free parameters. Since the total number of intra-epoch tie-points is by far larger than the inter-epoch tie-points (cf. Table 2), we set the relative weight to balance the effect. We adopted the Fraser model [START_REF] Fraser | Digital camera self-calibration[END_REF] to calibrate the cameras, and allowed imagedependent affine parameters. The remaining parameters were shared among all images. The poses of the 2015 acquisition were accurately known a-priori thereby treated as fixed during the combined BBA.

Comparison between three methods. 1971 and 2015). Figure 5 shows the computed DoDs and Table 3 reports the average and standard deviations computed on Z-coordinate difference. A dome artifact is present in DoDs corresponding to methods SIF TintraV GCPinter and SIF Tintra-inter. This kind of systematic error is known to originate from poorly modelled camera internal parameters [START_REF] Giordano | Toward automatic georeferencing of archival aerial photogrammetric surveys[END_REF]. In Ours, given the reasonable dense inter-epoch features and the calibrated 2015 acquisition, we are able to effectively remove this discrepancy. Even though our proposed method performs best of all, we still observe some systematic errors, e.g. the stripe-like pattern that coincides with the acquisition geometry, and the increasing differences towards the edge of the image block in 1971, where few interepoch tie-points were found.

CONCLUSION

A new approach to detecting inter-epoch tie-points in historical images has been proposed. Our validation datasets consisted of three epochs: 1971, 1981 and 2015. The proposed method outperformed the classical SIFT and D2-Net extraction algorithms in detecting many more and reliable points. This resulted in better pose estimation outcomes. The approach is generic as no auxiliary data is required, and it is independent of limiting initial assumptions. We also performed an experiment in which we show that (i) SIFT often provides less tie-points than D2-Net but they are more precise; (ii) D2-Net is sensitive to image rotation. Future work might concentrate on further improving robustness of the proposed method, in order to handle more challenging conditions, such as larger temporal gap. This could for example be done by automatically searching for a more suitable scale factor to downsample the input image to perform the tentative matching, or resorting to line features when necessary.

  We process each single-epoch data individually as follow: 1. Intra-epoch tie-points are effectively obtained by extracting and matching feature points based on SIFT algorithm (Lowe, 2004); 2. Interior and relative orientations are performed with standard SfM algorithm implemented in MicMac software (Pierrot-Deseilligny et al., 2015); 3. Individual depth maps are obtained with semi-global dense matching available in MicMac (Pierrot-Deseilligny, Paparoditis, 2006).

Figure 1 .

 1 Figure 1. Our processing workflow. The inputs are marked in yellow, the blue area corresponds to the inter-epoch processing, and the gray is the intra-epoch processing. The input images had been resampled to the geometry of the fiducial marks prior to the processing.

Figure 2 .

 2 Figure 2. SIFT matching result on an inter-epoch image pair with rotation and scale change.

  Figure 3. Matching result on inter-epoch image pair without scale change and rotation. Top: a global view, bottom: zoom over an area with the images transformed to a common geometry. Notice the offsets between the D2-Net corresponding points.

  Figure 4. Inter-epoch tie-points.

  Figure 5. (a) DoD between GT DSM and 2015 DSM, (b) Orthophoto of the corresponding area, (c) Overlap of block 1971, 1981 and 2015, (d-f) DoD between GT DSM and 1981 DSM with Ours, SIF Tintra-inter and SIF TintraV GCPinter, (g-i) DoD between GT DSM and 1971 DSM with Ours, SIF Tintra-inter and SIF TintraV GCPinter.

Table 1 .

 1 Details about 1971Details about , 1981Details about and 2015 acquisitions. The 2015 acquisition is obtained with two sets of camera. GSD is the ground sampling distance, F.o. and S.o. are forward and side overlap, H is the flying height, Nb is the number of images.

			resolution DSM gener-
	ated by the French Mapping Agency (IGN) in 2015.
		epoch 1971 epoch 1981	epoch 2015
	F [pix]	7589.06	7606.89	9967.5 9204.5
	Size [mm]	230,230	230,230	47,35	50,36
	GSD [m]	0.32	0.59	0.46	0.50
	F. o.	60%	60%	60%	60%
	S. o.	20%	20%	50%	50%
	H [m]	2400	4500	4600	4600
	Nb	57	27	308	74

  Figure 4 illustrates the inter-epoch tie-points extracted with Ours and SIF Tintra-inter between 1971 and 1981, 1971 and 2015 as well as 1981 and 2015. Our guided matching strategy detected far more points than the classical SIFT pipeline in SIF Tintra-inter (cf.Table 2), especially for the most challenging scenario (i.e. the one with the longest time gap between

Table 2 .

 2 Number of extracted tie-points with Ours (in bold), and with SIF Tintra-inter.

		epoch 1971 epoch 1981 epoch 2015
	epoch 1971	448165	160031	28759
		448165	9075		341
	epoch 1981	/	631290	61546
		/	631290	2031
	epoch 2015	/	/		18363596
		/	/		18363596
			µ [m] σ [m] |µ| [m]
	DoD2015		0.36	1.56	0.81
	DoD Ours 1981 DoD SIF T intra-inter 1981 DoD SIF T intra V GCP inter 1981 DoD Ours 1971 DoD SIF T intra-inter 1971 DoD SIF T intra V GCP inter 1971	1.10 -1.10 -1.53 0.45 -7.55 0.30	2.36 3.45 8.00 2.75 7.67 15.36	1.45 2.94 6.98 1.61 9.62 12.51

Table 3 .

 3 Average µ, standard deviation σ, and absolute average |µ| of the Z-coordinate in the 7 DoDs in Figure5.

ACKNOWLEDGEMENTS

This work was supported by ANR project DISRUPT (ANR-18-CE31-0012-0). We thank Denis Feurer and Fabrice Vinatier for their contribution to the interior calibration of the pezenas dataset. We would also like to thank Sebastien Giordano for the ground truth DSM and Benjamin Ferrand for the 2015 acquisition.