Pascale Paul
email: pascale.paul@univ-amu.fr

PhD Christophe Picard

PhD Emmanuelle Sampol

PhD Luc Lyonnet

Julie Di Cristofaro

PhD Louise Paul-Delvaux

MSc Guillaume Lano

Corinne Nicolino Brunet

MD Eleonore Ravis

Frederic Collart

Julie Di Cristofaro

MSc Corinne Nicolino Brunet

PhD Francoise Dignat George

PhD Bertrand Dussol

PhD Florence Sabatier

PhD Annick Mouly Bandini

Genetic and Functional Profiling of CD16-Dependent Natural Killer Activation Identifies Patients at Higher Risk of Cardiac Allograft Vasculopathy

Keywords: Cardiac allograft vasculopathy, Natural killer cell cytotoxicity, Fc-gamma receptor polymorphism, Heart transplantation, Donor-specific anti-HLA antibodies mass index CAV: cardiac allograft vasculopathy CKD: chronic kidney disease CMV: cytomegalovirus CNI: calcineurin inhibitor DSA: donor-specific antibodies eGFR: estimated glomerular filtration rate FCGR: Fc gamma receptor HLA: human leucocyte antigen HT: heart transplantation HTR: heart transplant recipient MFI: mean fluorescence intensity MMF: mycophenolate mofetil NK: natural killer NK-CHAT: NK cell-Cellular Humoral Activation Test PBMC: peripheral blood mononucleated cells SNP: single-nucleotide polymorphism

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

Conclusions

This study unravels a prominent role for the CD16-dependent NK cell activation pathway in the complex array of factors that favor progression of transplant arteriosclerosis. It highlights the clinical potential of a non-invasive evaluation of the FCGR3A/CD16 in the early stratification of CAV risk. Recognizing CD16 as a major checkpoint controlling immune surveillance may promote the design of individualized NK cell-targeted therapies to limit vascular damage in highly responsive sensitized patients.

Clinical Perspective

What Is New?

• We used a non invasive NK-Cellular humoral activation test (NK-CHAT) to evaluate the association between genetic and phenotypic markers of the CD16-dependent NK cell activation pathway and development of Cardiac Allograft Vasculopathy (CAV).

• We report that the FCRGR3A-VV polymorphic variant, encoding the highly responsive CD16

Fc-receptor, can be identified as an independent baseline predictor of CAV and can be useful for stratifying patients at higher risk of rejection.

• This study identifies a potential immune mechanism underlying the inter-individual variability of the recipient's complement-independent humoral response that relies on its FCGR3A genotype and CD16-dependent NK cell activation profile.

What Are the Clinical Implications?

 The non-invasive profiling of FCRG3A polymorphism and CD16-dependent NK cell responses may warrant personalized monitoring of heart transplant outcomes.

 NK-CHAT appraisal of risk could potentially limit the recommendation of invasive biopsies to sensitized patients with high humoral responsiveness.

 Indexing of patient-to-patient variability regarding NK cell responses may be of value in defining potency markers that anticipate the response to rituximab or IV-Ig treatment of AMR.

 Recognizing CD16 as a major checkpoint controlling immune surveillance may promote the possibility of using NK cell-targeted therapies to limit vascular damage in patients identified being at high humoral risk.

Introduction

Heart transplantation (HT) is a life-saving treatment for patients with advanced heart failure.

Predicting the risk of a given patient developing coronary artery disease in the transplanted heart, also known as cardiac allograft vasculopathy (CAV), remains a major issue in the prevention of allograft loss and long-term mortality after heart transplantation [START_REF] Patel | Diagnosis of cardiac allograft vasculopathy: Challenges and opportunities[END_REF][START_REF] Chih | Allograft Vasculopathy: The Achilles' Heel of Heart Transplantation[END_REF][START_REF] Kobashigawa | The Search for a Gold Standard to Detect Rejection in Heart Transplant Patients: Are We There Yet?[END_REF] . In 2016, the International Society for Heart and Lung Transplantation (ISHLT) updated the guidelines and risk factors for development of CAV [START_REF] Mehra | The 2016 International Society for Heart Lung Transplantation listing criteria for heart transplantation: A 10-year update[END_REF] .

Diagnosis of CAV often relies on asymptomatic patients receiving a coronary angiography on a regular basis, which is an invasive procedure that is often performed at stages when lesions have already progressed and remain poorly accessible to treatment. The current challenge is to identify non-invasive biomarkers that allow for the early stratification of patients at risk [START_REF] Chih | Allograft Vasculopathy: The Achilles' Heel of Heart Transplantation[END_REF][START_REF] Crespo-Leiro | Clinical usefulness of gene-expression profile to rule out acute rejection after heart transplantation: CARGO II[END_REF][START_REF] Starling | Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study[END_REF][START_REF] Kransdorf | Biomarkers for Cardiac Allograft Vasculopathy: Still Searching After All These Years[END_REF][START_REF] Jasseron | Prediction of Waitlist Mortality in Adult Heart Transplant Candidates: The Candidate Risk Score[END_REF] to tailor individualized therapeutic approaches preventing heart transplant failure [START_REF] Starling | Multicenter Analysis of Immune Biomarkers and Heart Transplant Outcomes: Results of the Clinical Trials in Organ Transplantation-05 Study[END_REF][START_REF] Furiasse | Immunosuppression and adult heart transplantation: emerging therapies and opportunities[END_REF] .

CAV is the result of cumulative endothelial aggression resulting from activation of alloreactive cells. The humoral component is a major determinant of allograft injury after organ transplantation [START_REF] Burke | Late Cardiac Allograft Failure, Cardiac Allograft Vasculopathy, and Antibody-Mediated Rejection: Untangling Some Knots?[END_REF][START_REF] Loupy | Late Failing Heart Allografts: Pathology of Cardiac Allograft Vasculopathy and Association With Antibody-Mediated Rejection[END_REF][START_REF] Colvin | Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: a scientific statement from the American Heart Association[END_REF][START_REF] Hammond | ISHLT pathology antibody mediated rejection score correlates with increased risk of cardiovascular mortality: A retrospective validation analysis[END_REF] . The inflammatory signals resulting from stimulation of activating Fc-receptors (Fc-R) for C-reactive protein (CRP) serum amyloid P component (SAP) and IgG expressed on endothelial cells, vascular smooth muscle cells, monocytes/macrophages are identified as contributors and biomarkers of coronary heart disease pathogenesis [START_REF] Tanigaki | Fcgamma receptors and ligands and cardiovascular disease[END_REF] . Fc-R polymorphisms have been reported as individual predisposition factors contributing to vascular disease [START_REF] Tanigaki | Fcgamma receptors and ligands and cardiovascular disease[END_REF] , notably when development of cardiovascular disease associate with auto or alloimmune disorders and cannot be explained by traditional cardiovascular risk factors.

Donor-specific anti-human leukocyte antigen (HLA) circulating antibodies (DSA) with C1q binding activity have been associated with worsened prognosis after HT, but these markers lack the specificity to predict graft outcome in a given patient [START_REF] Coutance | Late antibody-mediated rejection after heart transplantation: Mortality, graft function, and fulminant cardiac allograft vasculopathy[END_REF] . In addition to complementdependent mechanisms, the Fc segment of IgG can stimulate the CD16-dependent cytotoxicity and inflammatory function of immune natural killer (NK) cells [START_REF] Legris | Humoral immunity after kidney transplantation: impact of two randomized immunosuppressive protocols[END_REF][START_REF] Resch | The Role of Natural Killer Cells in Humoral Rejection[END_REF] , whose critical role has been identified in antibody-driven allograft vasculopathy using animal models 18, 19 . Recent reports have highlighted the clinical value of tissue-based gene profiling of NKcell transcripts within the heart allograft in the classification of antibody-mediated rejection (AMR) [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF] . These approaches have identified a role for the CD16/FcR activation pathway in promoting antibody-mediated vascular damage of heart allograft [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF] . A single-nucleotide substitution (rs396991) in the FCGR3A gene leads to an allelic variation of amino acid 158 (phenylalanine-F or valine-V) in the IgG binding domain of the CD16 receptor that impacts the antibody-dependent cell cytotoxic (ADCC) function of NK cells. The FCGR3A-VV polymorphic variant binds with higher affinity to human IgG1 and IgG3 than does the FCGR3A-FF variant. The host FCGR3A genotype has been identified as a major determinant of the clinical response to therapeutic IgG such as rituximab [START_REF] Bowles | Anti-CD20 monoclonal antibody with enhanced affinity for CD16 activates NK cells at lower concentrations and more effectively than rituximab[END_REF] . In addition, the FCGR3A polymorphisms have been reported to contribute to infection susceptibility [START_REF] Li | Human FcR polymorphism and disease[END_REF][START_REF] Moraru | NK Cell and Ig Interplay in Defense against Herpes Simplex Virus Type 1: Epistatic Interaction of CD16A and IgG1 Allotypes of Variable Affinities Modulates Antibody-Dependent Cellular Cytotoxicity and Susceptibility to Clinical Reactivation[END_REF][START_REF] Das | Fcgamma receptor 3A polymorphism predicts the incidence of urinary tract infection in kidneytransplant recipients[END_REF] and coronary artery diseases [START_REF] Gavasso | Fcgamma receptor IIIA polymorphism as a risk-factor for coronary artery disease[END_REF][START_REF] Huang | The significant increase of FcgammaRIIIA (CD16), a sensitive marker, in patients with coronary heart disease[END_REF] and to predispose patients to severe complications after organ transplantation [START_REF] Castro-Dopico | Fcgamma Receptors in Solid Organ Transplantation[END_REF][START_REF] Thomas | The perfect storm: HLA antibodies, complement, FcgammaRs, and endothelium in transplant rejection[END_REF][START_REF] Shimizu | Fc-Gamma Receptor Polymorphisms Predispose Patients to Infectious Complications After Liver Transplantation[END_REF][START_REF] Ruttens | Genetic variation in immunoglobulin G receptor affects survival after lung transplantation[END_REF][START_REF] Venner | The molecular landscape of antibody-mediated kidney transplant rejection: evidence for NK involvement through CD16a Fc receptors[END_REF] . In the current study, we investigated whether CD16-dependent NK cell activation profiles and FCGR3A polymorphism may be associated with the development of CAV.

Methods

Patient population and CAV grading

We performed a prospective single-centre observational study including 103 HT recipients

Cells, plasma and sera

Cell and sera samples of HTRs and healthy donors were collected from AP-HM and from EFS respectively. Peripheral blood mononuclear cells (PBMCs) were isolated from HTR and healthy donor blood samples using Ficoll gradients.

Anti-HLA antibody detection

Sera from all patients were tested for anti-HLA antibodies using multianalyte bead assays performed on the Luminex-based LABScan™ 200 flow analyzer. The antibody specificity and binding levels (mean fluorescence intensity: MFI), were determined by the LABScreen® Single Antigen bead assay (One Lambda, Thermo Fisher Scientific, California, USA) and analyzed with the HLA Fusion software. The binding reactivity was considered saturated with an MFI >15000, positive with a MFI>500 to 10000 or negative with an MFI MFI<500. The HLA antibody specificities were analyzed in reference to the HLA typing of donors to identify DSA.

C1q detection

The C1qScreen™ assay was used to test the capacity of DSA to bind C1q (One Lambda, Part of ThermoFisher Scientific). The C1q complement component bound by the antigen-antibody complex was detected with an R-PE-labeled anti-C1q antibody. Fluorescence intensity was measured using a Luminex-based LABScan™ 200 flow analyzer. MFI>1000 for C1qScreen™ assay was considered positive.

FCGR3A genotyping

The detection of FCGR3A (F158V/rs396991) polymorphisms was performed by using the SNAPSHOT technique. Data were analyzed using the GeneMapper 4.0 and GeneRate computer tool package (http://geneva.unige.ch/generate). For each of the different alleles obtained, both strands of the PCR products were sequenced using the BigDye Terminator v1.1

Sequencing Kit (Applied Biosystems) and analyzed on an automated ABI PRISM 3130 XL genetic analyzer.

Phenotypic analysis of peripheral lymphocyte cell subset

Multiparameter flow cytometry analysis of NK cells was conducted on PBMCs isolated with Ficoll-Paque (Eurobio, Les Ullis, France). Antibodies were obtained from Beckman Coulter:

CD3-ECD (IgG1, UCHT1); CD56-PC7 (IgG1, N901); CD16-AA750 (IgG1, Clone 3G8). The cells were labeled for 15 min at room temperature, and the NK cells were defined as the percentage of CD3-CD56+ NK cells expressing the CD16 receptor. The MFI of CD16 staining was further evaluated within the CD16+CD3-CD56+ NK cell subset (Supplemental figure 2). At least 3000 events were analyzed in the NK lymphocyte gate using the Navios cytometer (Beckman Coulter).

Phenotypic analysis of antibody-dependent NK cell activation

The NK-CHAT analysis was performed as previously detailed [START_REF] Legris | Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies[END_REF] . Briefly, effector PBMCs and 500,000 target cells (CD20 + B-EBV-transformed or K562 cell lines) were incubated with 10% SVF for 3 h at 37°C using a 1:1 effector/target ratio in the presence of Golgi Stop and

Statistical analysis

Statistical analysis was performed using Graph Pad Prism 5 software (GraphPad Software, La Jolla, CA) and IBM SPSS Statistics for Windows, Version 20.0 (Armonk, NY: IBM Corp).

Quantitative variables were summarized by means and standard deviation (±SD) or medians and interquartile ranges. Continuous variables were compared using Student's t-test or non-parametric Mann-Whitney test. One-way analysis of variance (ANOVA) was used to compare more than two groups. The association between continuous variables was analyzed using Spearman's or Pearson's rank correlation analysis. Categorical variables were reported as counts or percentages and compared using a chi-square or Fisher's exact test. A p value<0.05 was considered to represent a statistically significant difference. Time of first CAV diagnosis was compared across the FCGR3A genotypes with the use of Kaplan-Meier estimation combined with the log-rank test. Association between CAV and clinical, demographic and immunological variables were assessed by using a univariate analysis. Variables identified as significantly associated with CAV (p value <0.05, Table 1) allowed for the construction of logistic regression multivariate models enabling the calculation of adjusted tests characterizing the risk factors associated with CAV.

Results

Clinical characteristics of HTRs

The characteristics of the study cohort are shown in Table 1. transplant. Donor age and recipient body mass index (BMI) were significantly higher in the CAV + group. The rates of CAV tended to be higher when a female recipient received a heart from a male donor (n=13 CAV + out of 20 patients, p=0.161) or when the patient had signs of hypertension before transplant (p=0.071). Renal dysfunction at the time of enrolment (eGFR <60 ml/min/1.73 m 2 ) was significantly associated with CAV (Table 1).

Maintenance immunosuppressant mainly consisted of a tritherapy regimen combining calcineurin inhibitors (CNIs), MMF and steroids (group A, n=68) that associated with lower CAV incidence ( 1). Post-transplant CMV infectious rates were higher in CAV+ HTR (14 CAV+ out of 18 CMV + HTR). The occurrence of CAV was also significantly higher when the recipient was not immunized against Epstein-Barr virus (EBV) prior to the graft (16 CAV+ out of 21 EBV -patients at transplant). Circulating anti HLA-antibodies were detected in 30 HTRs (29%). Circulating DSAs were detectable in 22% of HTRs. Among the DSA, 14 were further identified as C1q-binding DSA, mostly associating DSA with MFI>10000 and HLA Class II recognition (online Supplemental Table 1). No significant association was noted between the presence of circulating DSA or C1qbinding DSA or a history of cellular rejection and the occurrence of CAV.

Quantitative analysis of lymphocyte cell subset counts

The distribution (% and cell numbers) of peripheral blood CD3 -CD56 + NK, CD19 + and CD4+/CD8+ T lymphocyte subsets was analyzed in HTRs. CD3 + CD8 + and NK peripheral blood cell counts were preserved in HTRs and exhibited values that were comparable to the reference values observed in a cohort of healthy controls. In contrast, B and CD4 + T lymphocyte cell counts were significantly lower in HTRs than in healthy controls. No significant difference in the distribution of lymphocyte cell subsets was observed between the CAV + or CAV -groups (online Supplemental Figure 1).

NK cell cytotoxic activity is preserved in HTRs

Flow-cytometry analysis of the membrane expression of the cytotoxic granule CD107a/Lamp1 NK cell marker revealed that constitutive NK cell activation (% CD107a/Lamp1-positive NK cells in the absence of target stimulation) and K562-driven natural cytotoxicity of NK cells were comparable in the HTR and controls groups (Figure 1A). Quantification of the antibody-dependent cytotoxic activity of peripheral NK cells towards autologous B-lymphocytes challenged with rituximab (NK-ADCC) revealed a high inter-individual heterogeneity among HTRs (Figure 1A). The NK-ADCC intensity was correlated with the number of autologous circulating B-lymphocytes expressing the CD20 target antigen (Figure 1B). Flow-cytometry analysis of CD16 expression on CD3-CD56+ NK cells gated within PBMCs in a standardized NK-CHAT assay [START_REF] Legris | Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies[END_REF]

The FCGR3A polymorphism is associated with the level of CD16-dependent NK cell activation

The CD16DRI was also shown to be significantly associated with the constitutive CD16 expression on circulating NK cells (CD16MFI, Figure 2A).

We then investigated whether such inter-individual NK-ADCC variation may relate to polymorphic variations in the FCGR3A gene encoding CD16. We showed that the CD16DRI was associated with the FCGR3A genotype of HTRs and was higher in homozygous FCGR3A-VV patients than in HTRs bearing the FCGR3A-VF or FCGR3A-FF genotype (Figure 2B). Similarly, we confirmed that the basal level of CD16 expression (CD16MFI) in circulating NK cells was significantly higher in recipients bearing the homozygous FCGR3A-VV genotype (Figure 2C).

The FCGR3A-VV genotype is an independent predictor of CAV

The frequency of the FCGR3A-VV genotype was significantly higher in the CAV + group (Odds ratio: 3.9, 95%, p=0.0317, CI: 1.18 to 12.99, positive predictive value=0.92, negative predictive value=0.25) than in the CAV-free or healthy control groups (p=0.03, Figure 3A). A Kaplan-Meir analysis adjusting for the time post-transplant at first CAV diagnosis confirmed that the median CAV-free survival was 5 years in FCGR3A-VV HTRs, while individuals who bear the FF/VF genotype had a median CAV-free survival of 9 years post-HT (Figure 3B).

The FCGR3A-VV genotype was also associated with a higher incidence of post-transplant viral infections (p=0.005, Odds ratio: CMV infection in absence of CAV exhibited this FCGR3A-VV susceptibility genotype (Online Supplemental Table 3).

We further challenged the independent value of these markers of the CD16 NK cell activation pathway as susceptibility markers for CAV using regression multivariate analysis models introducing confounding factors identified in the univariate analysis (Table 1). We identified an elevated risk of developing CAV in FCGR3A-VV ( donor to female recipient status (Table 3). High constitutive NK cell surface expression of CD16 (online supplemental Table 4) and HTR NK-ADCC responsiveness, as evaluated by the CD16DRI (online supplemental table 5) were also correlated with the angiographic CAV diagnosis at the time of enrolment.

Discussion

As CAV is a major cause of late mortality after HT, there is an urge to identify early noninvasive markers that reflect immune mechanisms contributing to its onset. Non-invasive early stratification of patients at risk is also a major challenge that favors the personalized indication of treatment. The potential contribution of genetic variation conditioning inflammatory processes resulting from CRP ligation to Fc-R has been evoked as a mechanism promoting vascular injury and predisposition to cardiovascular disease. Targeted antiinflammatory therapies have been proposed as a novel approach to reduce cardiovascular risk [START_REF] Passacquale | The role of inflammatory biomarkers in developing targeted cardiovascular therapies: lessons from the cardiovascular inflammation reduction trials[END_REF] . Our study is the first establish a link between the genetic variation of the FCGR3A/ CD16 activating Fc-R and the predisposition for heart vasculopathy. Recent reports have identified the Fc-Receptor CD16 as a major checkpoint controlling inflammatory and vascular cytotoxicity resulting from immune alloreactivity. A recent report [START_REF] Loupy | Gene Expression Profiling for the Identification and Classification of Antibody-Mediated Heart Rejection[END_REF] We show that despite immunosuppression, the number and cytotoxic function of circulating immune NK cells are maintained in HTRs and exhibit high patient-to-patient variability.

Although preservation of this immune potential may be beneficial for thwarting the neoplastic and infectious complications that often arise in immunosuppressed HTRs, it may cause adverse effects of auto or alloantibodies towards graft endothelium in immunized patients bearing the high-affinity FCGR3A-VV allele. The phenotypic NK-CHAT evaluation of peripheral blood NK cell activation in a relevant cohort of HTRs showed that patients developing post-transplant CAV are predominantly those that exhibit the highest CD16 expression and CD16-dependent NK cell activation potential in response to antibodies. We show that the intensity of the NK cell response to rituximab is dependent not only on the number of HTR peripheral B cells expressing the CD20 antigen but also on the intrinsic level of CD16 expression of the recipient, which is in part conditioned by the FCGR3A-VV genotype. These data provide a mechanistic link between the CD16 signaling pathway and the highly variable capacity of a given recipient to trigger immune activation in response to a viral or humoral challenge, both identified as risk factors of CAV.

As previously reported, history or persistent chronic kidney disease (CKD, eGFR <60), evaluated in transplant recipients before heart transplant and at time of enrolment in the study, was identified as an independent risk factor for CAV. The pathological crosstalk between heart and kidney organ dysfunction, also referred to as the cardio-renal syndrome is now widely recognized. It is admitted that various factors participate to alteration of renal function in HTR, both before and after heart transplant. While multivariable analysis retained a kidney dysfunction before graft, recipient age and % LVEF as independent risk factors associating with CKD (eGFR <60), no significant association could be observed between renal dysfunction and calcineurin inhibitors dosage or CD16-dependent NK cell activation.

Strength of the study is to provide evidence that the identification of CD16 profile of the recipient as a risk factor associated to development of CAV is in part independent of other risk factors that associate to the cardio renal syndrome such as donor/recipient age, uremic or infectious status and immunosuppressive regimen.

Strength of the study is to provide evidence that the identification of CD16 profile of the recipient as a risk factor associated to development of CAV is in part independent of other risk factors that associate to the cardio renal syndrome such as donor/recipient age, uremic or infectious status and immunosuppressive regimen.

Genotyping of the FCGR3A polymorphic variant would be easy to implement in the pretransplant setting; therefore, we challenge the view that this susceptibility marker may represent an independent predictor with potential value in the identification of patients at higher risk of developing CAV. Although previous studies have reported associations between FCGR polymorphisms and adverse infectious events or accelerated atherosclerosis after organ transplant [START_REF] Castro-Dopico | Fcgamma Receptors in Solid Organ Transplantation[END_REF][START_REF] Shimizu | Fc-Gamma Receptor Polymorphisms Predispose Patients to Infectious Complications After Liver Transplantation[END_REF][START_REF] Ruttens | Genetic variation in immunoglobulin G receptor affects survival after lung transplantation[END_REF] , the link between the FCGR3A SNP and infection has yet to be elucidated in the context of HT. A recent study reported that patients with a combined FCGR2A-131H/H and FCGR3A-FF (low IgG binding affinity) genotype were more susceptible to blood-borne infections and exhibited increased mortality after liver transplant.

Previous studies have identified CMV infection as a risk factor associated with the development of allograft vascular lesions [START_REF] Delgado | Influence of cytomegalovirus infection in the development of cardiac allograft vasculopathy after heart transplantation[END_REF][START_REF] Sarmiento | Evaluation of humoral immunity profiles to identify heart recipients at risk for development of severe infections: A multicenter prospective study[END_REF] . In the present cohort, the FCGR3A-VV genotype was identified as a risk factor associated with viral infections. We provide evidence that the FCGR3A-VV genotype is associated with an increased susceptibility to CAV independent of the pre-transplant recipient and donor CMV combination, post-transplant infectious status, or immunosuppressive regimen. This suggests that, upon CMV infectious challenge, FCGR3A-VV HTRs may develop more potent humoral inflammatory responses that favor the development of allograft vasculopathy.

Interestingly, the association of CAV with the genetic and functional biomarkers of the CD16 activation pathway was independent of the DSA C1q binding activity. Our study thus reveals a potential role for the FCGR3A-VV SNP as a susceptibility factor that controls the chronic cytotoxicity of antibodies independent of the complement pathway. Although this association remains unclear, enhanced MFI and C1q binding activity of DSA and skin cancer rates were associated with the FCGR3A-FF genotype, thus suggesting that the threshold of innate immune NK cell immunosurveillance may be lowered in these HTRs. Interestingly, among the 8 patients who exhibited the highest levels of circulating DSA (MFI>15000), all 4 patients who remained CAV-free at the time of enrollment had the FCGR3A-FF low-responsive genotype. As shown in the kidney transplant setting (28), we suggest that such non-invasive NK-CHAT evaluation of CD16-mediated reactivity may reveal features of underlying humoral mechanisms involving NK cell chronic activation towards the donor heart that may not be fully appreciated from a conventional Luminex assessment of HLA circulating antibodies. We expect that the non-invasive CD16DRI evaluation of recipient NK cell reactivity towards donor cells could reveal the pathogenic activity of anti-endothelial antibodies that remains undetectable with conventional assays but exhibits a high potential to promote vasculopathy in high responders with the FCGR3A-VV genotype.

Our work has several limitations. First, the FCGR3A profiles were only analyzed in 103

HTRs, and the ability of the NK-CHAT to predict CAV needs to be further demonstrated in larger prospective and multicentric cohorts of HTRs with defined pre-and post-transplant time points. Nevertheless, given the multiple risk factors evaluated, none of the other Fc-R polymorphisms evaluated proved to be significantly associated to CAV in the present cohort.

Considering this primary analysis, we also need to challenge the predictive value of these CD16-dependent NK cell activation indexes evaluated at time of transplant with more stringent end-points, such as graft failure, cancer and viral immune surveillance, AMR and patient mortality. FcγRs are also expressed on neutrophils and monocyte/ macrophage subsets; another limitation of our study is that we only focused on the implication of CD16 in NK cell immune cytotoxic activation.

Our study provides evidence of a role for NK immune cells as effectors of cardiac allograft dysfunction. As CD16 is a major component that controls complex IgG-mediated effector functions of immune cells in response to alloimmune, neoplastic and infectious challenge, we expect that such a genetic and phenotypic assessment of CD16/FCGR3A may provide biomarkers that reflect the complexity of immune mechanisms associated with heart allograft loss.

In addition, as shown in the kidney transplant setting [START_REF] Legris | Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies[END_REF] , the non invasive assay NK-CHAT monitoring of Fc-R and antibody dependent NK cell alloreactivity may be a valuable tool to identify patients at higher risk to develop CAV after heart transplantation. Such indexing of patient-to-patient variability regarding NK cell responses also suggests that NK-CHAT may also be of value in defining potency markers that anticipate response to therapy, notably in the rituximab or IV-Ig treatment of AMR.

Conclusion

Our study unravels the relevance of the CD16 axis in controlling the intensity of IgG-mediated NK cell cytotoxic activation in HTR. Our work suggests that individualized scoring of the recipient's intrinsic capacity to elicit CD16-dependent NK cell activation may provide an integrated view on the interplay between susceptibility SNPs, humoral innate cell alloreactivity and CMV in the promotion of coronary artery disease in the transplanted heart.

Such indexing of patient-to-patient variability regarding NK cell responses may be of value in defining non-invasive biomarkers that anticipate CAV risk and response to therapy.

Translated to a simplified test, such analysis of the FCGR3A polymorphism and NK-ADCC activation potential may warrant informative non-invasive monitoring of heart transplant outcomes both before transplant and after transplant. Such an appraisal of risk could potentially limit the recommendation of invasive biopsies to a subset of immunized patients with high ADCC responsiveness, whereas less stringent monitoring may be considered in patients with a lower ADCC reactive potential. This method may also provide novel tools to quantify the individual benefit/risk profile of the patient to adapt immunosuppressive therapies. Recognizing CD16 as a major checkpoint controlling immune surveillance in the setting of HT may promote the possibility of designing and testing novel individualized NK cell-targeted therapies to limit vascular damage in sensitized patients identified being at high humoral risk. Log-rank (Mantel-Cox) Test p=0,0491

(

  HTRs) undergoing a coronary angiography in the framework of their routine follow-up (performed on the first year after HT, every two years or in case of unexplained left ventricular dysfunction) in the cardiac surgical unit of Assistance Publique-Hôpitaux de Marseille (AP-HM) from 2011 to 2014. The study was approved by the Agence Française de Sécurité Sanitaire des Produits de Santé (June 2011, reference B101345-70) and the Comite de Protection des Personnes Sud-Méditerranée I (January 2011, reference 10-61) and supervised by AP-HM under ID numbers NCT01569334/2010-A01145-34). Written informed consent was obtained from all participants. The healthy donors' blood samples were anonymized according to the French Blood Centre regulation procedures. CAV diagnosis was defined by coronary angiography and graded according to the ISHLT recommendations. All patients received perioperative induction therapy with rabbit anti-thymocyte globulin (ATG) or basiliximab. Statin therapy was given postoperatively. Maintenance immunosuppressant therapy included calcineurin inhibitors (CNIs), such as cyclosporine or tacrolimus, mycophenolate mofetil (MMF) or everolimus and steroids. Blood samples were collected in a fasting state 12 hours after CNI administration.

CD107a-

  PC5 antibodies (Becton Dickinson). Cells were washed and labeled with CD3-ECD, CD16-PE and CD56-PC7 antibodies (Beckman Coulter) for 15 min at room temperature. Data acquisition was performed on a Navios cytometer. CD107a/Lamp1 expression patterns were analyzed in the CD3 -CD56 + NK subset gated within the PBMCs. Analysis of the K562induced NK cell expression of CD107a/Lamp1 cytotoxic granule exocytosis allowed for the evaluation of the natural cytotoxic activity of NK cells. NK-ADCC activity was evaluated after addition of 10/ml of rituximab to the PBMC-B cell target co-culture for 3h by measuring the % of CD107a/Lamp1-positive CD3 -CD56 + NK cells. The level of CD16 engagement was quantified as the ratio between the MFI of NK cell CD16 expression (CD16MFI) observed when PBMCs were incubated with CD20 + B cell targets in the presence or absence of rituximab. The level of rituximab-induced NK cell CD16 engagement was defined as the CD16 down-regulation index (CD16DRI).

  Coronary angiograms were used to define a CAV-group of 51 HTRs without detectable angiographic lesions and a CAV+ group of 52 patients exhibiting mild (grade 1, n=35), moderate (grade 2, n=6) or severe (grade 3, n=11) CAV. Time post-transplant was highly associated with the occurrence of CAV and the median time at first CAV diagnosis was 3 years (25-75 percentile: 1-7.2). CAV was diagnosed in 8 of 29 HTRs (28%) examined 1-year post-transplant, 12 of 29 (41%) HTRs evaluated between 1 and 5 years post-transplant, 14 of 35 HTRs (40%) evaluated between 6 and 9 years post-transplant, and 32 of 45 HTRs (71%) evaluated after 10 years post-

  further allowed for the measurement of the level of Fc-dependent activation of HTR NK cells towards rituximabcoated B cell targets (online supplemental figure 2A). To normalize the variability of baseline CD16 expression in HTRs, the CD16DRI was calculated as the ratio between the NK cell CD16 staining (MFI) measured in response to B cells coated with control serum (baseline) and the NK cell CD16MFI measured in response to rituximab-coated cells (illustrated in online supplemental figure2B). The CD16DRI exhibited high inter-individual variability among HTRs (median CD16DRI: 8.6-fold rituximab-induced down-regulation, 25-75 percentile: 5.5-13.7) and was correlated with the cytotoxic function of HTR NK cells, as measured by CD107a/Lamp1 granule exocytosis in response to rituximab (Figure1C).

  illustrated the clinical potential of biopsy measurement of CD16 and NK cell transcripts to refine classification and diagnosis of antibody mediated heart transplant rejection. In this context, our study provides evidence that CD16 profiling identifies non-invasive biomarkers that reflect the intrinsic potential of a given recipient to mount deleterious cytotoxic immune responses towards heart vasculature. The strength of this study is to provide evidence that the FCGR3A-VV genetic variant encoding the CD16 receptor with high affinity for the Fc portion of antibodies is associated with an enhanced CD16-driven NK cell activation potential and behaves as a susceptibility factor identifying patients at higher risk to develop coronary artery disease of the transplanted heart. At the individual level, such a highly responsive NK cell profile associated with the VV genotype and CD16 expression levels is expected to shape the intensity of NK cell antibody-dependent cellular cytotoxic and inflammatory paracrine function through the release of CD107a/Lamp1 cytotoxic granules and inflammatory cytokines such as interferon (IFN)-γ.

Figure 1 (

 1 Figure 1 (A) Phenotypic analysis of NK cell degranulation: % of CD3-CD56+ NK cells expressing the

Figure 2 (

 2 Figure 2 (A) The level of rituximab-induced down-regulation of CD16 on NK cells (CD16DRI) correlated with the level of NK cell CD16 constitutive expression (CD16MFI) (B) Levels of CD16DRI across CAV + and CAV -HTR groups with the FCGR3A-FF, FCGR3A-VF or FCGR3A-VV genotypes. (C) Levels of

Figure 3 (

 3 Figure 3 (A) Distribution of the FCGR3A genotype in the CAV+ (n=52) and CAV-(n=51) HTR and

Figure 1 Figure 1 (

 11 Figure 1

Figure 2 Figure 2 (

 22 Figure 2

Figure 3 Figure 3 (

 33 Figure 3

Supplemental Figure 1 .Supplemental Figure 2 A

 12 Phenotypic analysis of CD3+, NK and B cell lymphocyte counts in heart transplant recipients (HTRs) and healthy individuals (CTL) . Illustration of the Flow Cytometry gating strategy. NK cells were defined within peripheral blood mononucleated cells (PBMCs) as a CD3 -CD56 + cell subset. CD3-CD56 dim (gate B) NK cells were identified as the major subset expressing CD16 while the CD56bright NK cell subset was CD16 negative. Variation of CD16 expression (MFI) resulting from antibody recognition was therefore further evaluated within HTR PBMC on the whole CD3-CD56+ NK cell subset without further discrimination of the CD56 bright and CD56 dim subsets.

Table 1 )

 1 , or everolimus, CNI and steroids (group B, n=15), or bi-or monotherapy (group C, steroid or MMF withdrawal n=18; CNI monotherapy n=2). Posttransplant infections (viral, fungal, bacterial or parasitic) were observed in 48 patients(30 of

52 CAV+ HTR). Post-transplant viral (15 CAV+ out of 21 HTR) and bacterial infections (

24

CAV+ out of 36 HTRs) were associated with CAV (Table

Table 2 )

 2 . A majority of patients who developed CMV infection also developed CAV. Indeed 14 out of the 18 patients who developed CMV infection after heart transplant had a diagnosis of CAV (3 grade 3, 1 grade 2 and 10 grade 1). It is of note that 50% of these patients who developed CMV and CAV were FCGR3A-VV, while none of the patients that developed

	4.92 CI: 1.6-15.1), whereas the FCGR3A-FF genotype

Table 2

 2

), independent of other CAV risk factors such as time since transplantation, donor age, recipient age and BMI, occurrence of post-transplant viral and bacterial infections, CKD and immunosuppressive regimen. We further characterized the risk associated with the FCRGR3A genotype at the time of transplant by constructing a predicting multivariate model that restricts the adjusting variables to pretransplant features The FCGR3A-VV genotype remained a strong predictor of CAV development independent of other risk factors such as donor age, hypertension, and male

Table 1 Patient characteristics

 1

	Patients Without CAV, n=51	Patients With CAV, n=52	p Value

CKDEPI, ml/min/1.73m 2 ) 63.9 ± 3.6 51.8 ± 3.1 0,021 CKD ( CKDEPI<60) 47.1%

  Data are presented as the median (25-75 percentiles) or mean ± SD or values n (% within group).

			66.7%	0,046
	history of Acute cellular rejection	53%	52%	0.918
	Therapies
	Induction therapy (% Basiluximab vs ATG )	51%	25%	0.007
	Group A: Tritherapy MMF, steroids, CNI	76.5%	55.8%	0.027
	Group B: Everolimus , steroids ,CNI	11.8%	17.3%	0.425
	Group C : Bitherapy/monotherapy	11.8%	26.9%	0.052
	Immunisation status
	DSA positive (n)	9 (18%)	14 (28%)	0.235
	DSA MFI >10000	4 (8%)	7 (14%)	0.303
	DSA C1q binding (n)	5 (10%)	9 (18%)	0.249

Table 2 Multivariate predictors of cardiac allograft vasculopathy Risk factor for CAV

 2

	Odds
	Ratio	p	95% CI

Table 3 : Multivariate analysis for the prediction of CAV risk at time of transplant

 3

	Risk factor for CAV at
	transplant	Odds Ratio	p	95% CI
	CD16VV	4.7	0.023	1.244 -17.778
	Recipient EBV+ at Transplant	0.24	0.059	0.055 -	1.057
	Donor Age at Transplant	5.93	0.006	1.677 -20.995
	Donor Stroke	1.06	0.012	1.013 -	1.110
	Sex Mismatch MaleD/FemaleR	3.9	0.049	1.005 -15.116
	Recipient Hypertension	1.56	0.476	0.457 -	5.358

cell target Immune surveillance Recipient FCRG3A genotype Antibody Target cells Potential impact for HTR

	NK-cell antibody and CD16 mediated	CAV:VV>VF/FF Enhanced levels of NK cell CD16	Rituximab	Autologous CD20+ B-cells	High Rituximab responsiveness enhanced efficacy of AMR therapy
	Inflammation Cytotoxicity (ADCC)	expression associate to CAV	DSA Anti Endothelial Antibodies	Heart allograft endothelial cells	Enhanced alloimmune response and CAV Risk in FCGR3A -VV
	Viral Infection	VV>VF/FF	Enhanced Humoral Response	Infected Targets cells	Enhanced viral Inflammation in FCGR3A -VV HTRs
	Tumors	Skin cancer FF>VV/VF	-	Tumor cells	Lowered Tumor Immunosurveillance in FCGR3A -FF HTRs

Time at First CAV diagnostic (time post transplant, yrs)

	A				CAV-	>15000	B	>15000	Class I and II	>15000	>15000	FF
	99	2	Class I and II	5	CAV-	12000		>15000	Class I and II	7000	>15000	FF
	122	3	Class I and II	14	CAV-	7000		>15000	Class II	0	>15000	FF
	17	4	Class I and II	1	CAV grade 1	4000		2500	negative	0	0	FF
	24 32 41 92 10 78 60	5 6 7 8 9 10 11	Class I and II Class I Class I Class I Class I Class I Class II	1 14 1 3 4 3 7	CAV grade 3 CAV-CAV-CAV grade 1 CAV grade 2 CAV grade 2 CAV-	3000 survival CAV 2000 3000 free 3000 1400 1000 Percent 0		8000 0 0 0 0 0 >15000	negative negative negative Class I Class I negative Class II	0 0 0 600 7000 0 0	0 0 0 0 0 0 >15000	VV FF VF VV FF FF FF
	35	12	Class II	9	CAV-	0		10000	Class II	0	5000	VF
	15	13	Class II	1	CAV-	0		10000	negative	0	0	VF
	113	14	Class II	5	CAV-	0		2000	negative	0	0	VF
	108	15	Class II	8	CAV grade 1	0		>15000	Class II	0	>15000	FF
	77	16	Class II	3	CAV grade 1	0		>15000	Class II	0	>15000	FF
	21	17	Class II	1	CAV grade 1	0		>15000	Class II	0	>15000	FF
	5	18	Class II	1	CAV grade 1	0		>15000	Class II	0	>15000	VF
	98	19	Class II	3	CAV grade 1	0		12000	Class II	0	8000	VV
	20	20	Class II	3	CAV grade 1	0		14000	Class II	0	3000	VF
	9	21	Class II	1	CAV grade 3	0		5000	Class II	0	2000	VF
	26	22	Class II	8	CAV grade 1	0		1200	negative	0	0	VF
	25	23	Class II

Acknowledgements

We thank Dr. E. Jouve and L. Boyer in reviewing the statistical methods of the manuscript; Sophie Morange and Alain Vazy from the CIC, EFS technicians and Aurélie Larosa and Lauriane Leong-Feng for their assistance in the collection of the data.

Sources of Funding: The work was supported by an INSERM-DGOS-AP-HM translational research grant under the ref 2010-A01145-34/ NCT01569334

Disclosures: The authors declare that they had no financial relationships with any organizations that might have an interest in the submitted work.

Supplementary data


Pete Sinclair
email: peter.sinclair@ecole-art-aix.fr

Roland Cahen
email: roland.cahen@ensci.com

Jonathan Tanant
email: jonathan@free.fr

Peter Gena
email: pgena@artic.edu

New Atlantis: Audio Experimentation in A shared Online World

Keywords: audiographic creation, audio for virtual environments, sound spatialisation, networked music

Computer games and virtual worlds are "traditionally" visually orientated, and their audio dimension often secondary. In this paper we will describe New Atlantis a virtual world that aims to put sound first. We will describe the motivation, the history and the development of this Franco-American project and the serendipitous use made of the distance between partner structures. We explain the overall architecture of the world and discuss the reasons for certain key structural choices. New Atlantis' first aim is to provide a platform for audio-graphic design and practice, for students as well as artists and researchers, engaged in higher education art or media curricula. We describe the integration of student's productions through workshops and exchanges and discuss and the first public presentations of NA that took place from January 2016. Finally we will unfold perspectives for future research and the further uses of New Atlantis.

Introduction

New Atlantis is a shared (multi-user) online virtual world dedicated to audio experimentation and practice. Unlike most online worlds where image is the primary concern, in NA sound comes first. NA provides a context for new-media students to showcase research projects that explore the relationship between sound, virtual 3D image and interactivity. It offers a pedagogical platform for audiographic animation, real-time sound synthesis, object sonification and acoustic simulation. It is a place to organize virtual sound installations, online concerts, Soundwalks and other audiovisual art experiences.

The name New Atlantis comes from the title of an unfinished 1628 utopian novel by philosopher Francis Bacon [START_REF] Bacon | The New Atlantis[END_REF], which describes a legendary island somewhere in the ocean, doted with extraordinary audio phenomena that might be considered as premonitory of today's electronic and digital audio techniques. We have adopted some of Bacon's ideas and nomenclature to create classes for the virtual world such as "Sound Houses", "Sound Pipes", "Trunks" and "Helps". In NA all elements are intended to have audio qualities: spaces resonate, surfaces reflect and collisions activate the multiple sounds of the objects involved. A collection of purpose built scripts implement low level sound synthesis and multiple parameter interactivity, enabling the creation of complex sound sources and environments linked to animation or navigation in the visual scene.

NA can be accessed via a web viewer or as a standalone application. It is organized as "spaces" that can be accessed independently but that share the same basic principles of navigation and specific scripts. Multi-user, it can be shared by several players at the same time making it suitable for group playing in both the gaming and the musical sense of the word. Every registered user can create and host individual or shared spaces which he or she can decide to make persistent or not. At the time of writing, we are working on a limited number of public "spaces" that contain multiple "Sound Houses" (architectural elements with specific acoustics) and other sound objects. These can be visited by navigating through the scene or created within the scene. The audio "mix" of these different sources varies with distance, so placing and navigating between sound objects can become a musical experience. In public spaces, players can interact with one another and with shared objects potentially playing together.

New Atlantis project is not only about creating a multi user virtual universe, but also about making it together while learning. It is experimental, creative and educational. New opportunities for further development in NA are created through the organization of workshops, courses or events that group art students in different locations, working together at a distance. It is a way to encourage ubiquitous working groups of students to share immaterial and non-local (international) art projects. Most sound and music education schemes tend to be oriented towards established disciplines. NA on the other hand encourages experimental design projects and the exploration of new or emerging creative fields. Francis Bacon's New Atlantis proposed a model for the role of science and art in society that placed education at the heart of culture. Our project emphasizes discovery, cultural exchange, experimentation, learning and furthering of knowledge in an educational and creative environment.

Context and History

"We have also sound-houses, where we practise and demonstrate all sounds and their generation. We have harmony which you have not, of quartersounds and lesser slides of sounds. Divers instruments of music likewise to you unknown, some sweeter than any you have; with bells and rings that are dainty and sweet. We represent small sounds as great and deep, likewise great sounds extenuate and sharp; we make divers tremblings and warblings of sounds, which in their original are entire. We represent and imitate all articulate sounds and letters, and the voices and notes of beasts and birds. We have certain helps which, set to the ear, do further the hearing greatly; we have also divers strange and artificial echoes, reflecting the voice many times, and, as it were, tossing it; and some that give back the voice louder than it came, some shriller and some deeper; yea, some rendering the voice, differing in the letters or articulate sound from that they receive. We have all means to convey sounds in trunks and pipes, in strange lines and distances." [START_REF] Bacon | The New Atlantis[END_REF] The origins of the NA project go back to 2005 when the Locus Sonus, ESA-Aix (Ecole Superieur d'Art d'Aix-En-Provence) and SAIC (School of the Art Institute of Chicago) were awarded FACE [START_REF]French-Americain Cultural Exchange[END_REF] funding for an academic and research exchange program. The original impulse occurred through collaboration between teams of the 3d and sound departments of the partner establishments, which led to the observation that there was scope for research into the area of audio in games and other virtual environments.

That NA refers to a Utopian model can be interpreted in several different ways. Firstly as the above citation demonstrates, the original text by Francis Bacon describes an island territory that was home to numerous extraordinary audio phenomena. Beyond this the novel predicts the principles of the contemporary research university as seen in the description of "Salomon's House" which promotes international exchange: "For the several employments and offices of our fellows, we have twelve that sail into foreign countries ... who bring us the books and abstracts, and patterns of experiments of all other parts" [START_REF] Bacon | The New Atlantis[END_REF] and trans-disciplinary research: "We have three that collect the experiments of all mechanical arts, and also of liberal sciences, and also of practices which are not brought into arts" [START_REF] Bacon | The New Atlantis[END_REF]. These ideas, combined with the fact that we are indeed engaged in creating a utopian world that might be considered as situated (albeit in our imagination) somewhere in the oceanbetween Europe and America -combine to make NA a suitable reference.

Since the ESA-Aix/SAIC partnership was separated by the Atlantic Ocean, we rapidly adopted networked solutions for our collaborations: video conferencing and remote desktop were used to exchange lessons and conferences and an interconnected 3d cave were set up with an interface in both Aix and Chicago. The first experiments in 3d audio-graphy took place in Second Life [START_REF]Second Life official site[END_REF], using Pure Data [START_REF]Pure Data -Pd community site[END_REF] as an audio engine.

The process involved sending html commands from second life to an external server that did the audio synthesis and streamed the result back to second life; the project was presented in 2009 at the "Second Nature" festival in Aix-En-Provence [START_REF] Nature | [END_REF]. This system worked well enough to convince us that it was worthwhile pursuing the development of sophisticated audio features for virtual environments, however, it was difficult to implement and the delay due to streaming was problematic.

The decision was made to build our own multi user world using Panda 3d [START_REF]Panda3D -Free 3D game Engine[END_REF] with Pure Data [START_REF]Pure Data -Pd community site[END_REF] bundled as an audio engine. Ecole Nationale Superieure de Creation Industrielle (ENSCI, les ateliers) became associated with the project at this point. This first version of NA was developed during multiple workshops that took place in Chicago and Aix en Provence between 2007 and 2011. The project involved a relatively complex path finding system used to calculate acoustics and custom-built client server software [7]. Although a working version was tested successfully during a workshop at ENSAB in 2011 [START_REF] Laforet | New Atlantis, un monde virtuel sonore[END_REF], it was decided to abandon the development based on Panda/Pd in favor of Unity3d[9], a more recent and efficient platform offering greater scope for audio programming and possessing built in networking capabilities.

New Atlantis Project Aims

There are multiple aims associated with the NA project: as mentioned above the project emerges from an international exchange program and one of its first ambitions is to provide a platform for international academic, artistic, cultural and scientific exchange. The more specific aim is to further research into audio for virtual environments with the precise goals of providing an educational tool for students and a synchronized platform for remote music and sound art practices.

3.1

Research Into Audio for Virtual and networked Environments, background.

The history of audio development in game environments is relatively short and, at least when this project was initiated, somewhat lacking in substance. Arguably, this might be put down to competition between audio and visual requirements in terms of processing power on personal computers or game boxes. If in recent years companies such as AudioGaming [START_REF] Audiogaming | [END_REF] that are specialized in audio for game environments have started to appear, for the essential they provide sound design services for the commercial game market, rather than considering the virtual world as a possible audio interface. Therefore the historical origins of this project might be considered from several other angles. One approach is that of visual interfaces for musical compositions such as UPIC [11] originally developed by Iannis Xenakis or navigable scores such as Fontana Mix 1958 by John Cage [START_REF] Cage | Fontana Mix[END_REF]. Distant listening, remote performance and other streamed and networked art forms are another thread that has been largely investigated by Locus Sonus [START_REF] Sinclair | Locus Sonus 10 ans d'expérimentation en art sonore[END_REF]. Experiments with remote musical presence started as early as 1992 when during an event organized by Michel Redolfi; Jean Claude Risset and Terry Riley in Nice played with David Rosenboom and Morton Subotnick in Los Angeles using Disklaviers and a satellite connection [START_REF] Polymeneas-Liontiris | The Disklavier in Networked Music Performances In[END_REF]. In 1967, the late Maryanne Amacher conceived of and produced City Links, in Buffalo, NY. The 28-hour performance took live microphone feeds from five different locations in the city. Victor Grauer and Max Neuhaus were involved in the Buffalo production and Neuhaus subsequently did similar work that came to be known as "telematic performance". The rapidly developing discipline of sonification [START_REF] Sinclair | Sonification -What Where How Why[END_REF] is equally useful when reflecting on the sound of virtual objects. In effect the different members of the research team have pursued these different lines of investigation over the past decades.

We should not however ignore early forays into audio-graphic creation an example being Ivan Chabanaud1 and co-author Roland Cahen's Icarus [START_REF]Installation Icare de Ivan Chabanaud[END_REF]. When this virtual reality project was started in 1995, sound synchronization techniques where cumbersome: a Silicon graphics machine rendering visual objects was connected to Opcode Max 3 (MIDI only) using an external MIDI expander and the IRCAM's spatialisation system. More recent audio-graphic projects that R. Cahen has been involved in include BANDONEON [START_REF] Bandoneon | [END_REF], PHASE [START_REF] Cahen | Sound Navigation in the PHASE installation: producing music as performing a game using haptic feedback[END_REF], ENIGMES [START_REF] Enigmes | [END_REF], and TOPOPHONIE [START_REF] Topophonie | [END_REF].

Other recent research initiatives focusing on shared virtual environments such as UDKOSC [START_REF] Udkosc | [END_REF] use the OSC protocol to associate external audio engines such as Pd or Supercollider with a virtual environment. Although the original version of NA followed this line of investigation we have switched to the using the audio engine incorporated in Unity for the sake of simplicity and versatility on the user side (see section 6.). The Avatar Orchestra Metaverse, formed in 2007, is another Interesting experimental project led by American Sound Artist, Pauline Oliveros: "The Avatar Orchestra Metaverse is a global collaboration of composers, artists and musicians that approaches the virtual reality platform Second Life as an instrument itself" [START_REF] Kanaga | Proteus -A game by Twisted Trees[END_REF]. However this approach is very different to that adopted for New Atlantis in the sense that it is dependent on the resources of the existing virtual world Second Life. Proteus by Ed Key and David Kanaga [23] is a rare example of a virtual world where the combination of low-resolution graphics and generative composition create an everevolving landscape/soundscape, offering a delicate interaction between the audio and visual components.

Fields of Investigation

Interactive sound and music with virtual objects

Audiographic design is a multimodal approach that consists of coordinating graphical form and behavior with auditory events in the design of virtual objects. These objects, although they are visual images, can incorporate physical simulation so they can interact with us and between themselves. While artists and designers often have a limited culture of multimodality, creating audiographic objects or compositions requires interconnections between various domains of expertise such as sound design, graphic design, 3D animation, real time interaction and coding. New Atlantis has been conceived to provide an adequate platform for this type of training. Designing multimodal interactions and implementing them in a virtual world is more complex than creating simple visual representations or video. It obliges the author to resolve more design issues and to give objects deeper consistency, bringing them close to a physical reality, even if they remain immaterial. At a certain point, the simulated object becomes the object in its own right and the notion of "virtual" is modified. Arguably, virtual objects are somehow non-material objects that incarnate their own reality. This has long been the case in music as well as with other abstract artistic activities, when an initial manipulation of representation ceases to be the finality.

Since the audiographic relationship is constructed, it can also be fictitious. An object's sound can be modified to become quite different from the "real life" original, while paradoxically appearing very real or possibly more than real (hyperreal). An early example of this phenomenon can be found in Jacques Tati's film Mon Oncle where "retouched" audio recordings focus attention unnaturally on specific banal visual objects [24]. We consider that this bending, schematizing or detournement of the relation between the visual object and its associated sound is a fruitful terrain for investigation. Virtual composition also allows us to interact with sound objects using a variety of expressive devices such as physical modeling, avatar form and behavior, fictional scenarios and gameplay, opening a multitude of new forms of narration.

We are currently developing new tools for New Atlantis in collaboration with FRE PRISM that will incorporate LMA's intuitive synthesizer control system. The system is based on a principal of control by metaphorical description of material qualities and physical actions. It is thus highly suitable for the creation of virtual objects in particular since the simulated object does not necessarily require a real-world model.

Spatialization, Sound Navigation and Virtual Acoustics

Sound navigation consists of browsing through different sound sources placed in a spatialized sound scene, thereby composing a musical form from the mix produced by movements relative to these sources. It is an artistic transposition of our sound experience in the physical world [25]. In virtual worlds, sounds are mainly spatialized through orientation and distance. The simple act of navigation allows us to create spatial mixes or canons as well as subject and object motion sequences. Spatial acoustics are another important part of our audio perception and indeed they participate in our natural audio interactions as we activate reverberant spaces through our own actions such as footsteps or vocalizations [START_REF] Sinclair | Inside Zeno's Arrow: Mobile Captation and Sonification Audio Mobility[END_REF]. This praxeology [START_REF] Thibaud | Towards a praxiology of sound environment[END_REF] of sound space can also be developed in virtual environments and the juxtaposition of different acoustic responses as we navigate while generating sounds or listening to sounds generated by other players, provides an original approach to real-time audio signal processing. Short simple sound sources such as clicks or collision sounds can be used to activate and compare virtual acoustics and continuous sound sources can be multiplied to create complex harmonics. Such indeterminate forms of navigation, inspired by SoundWalking or other Soundscape related activities (see The Tuning of the World by R.Murray Schafer [START_REF] Schafer | The Tuning Of The World[END_REF]), are alternatives too more permanently authored audiographic forms.

A Synchronized Platform for Remote Music and Sound Art Practices

Networked Playing and Performance.

In the case of NA "playing" may mean "gameplay" i.e. in general video games parlance "the specific way in which players interact with a game" [START_REF] Key | Proteus -A game by Twisted Trees[END_REF], or it can equally be used to designate musical playing. In the preceding section, we approached NA as a compositional tool but it can also be considered as an audio "sandpit", as a shared instrument, or as a stage for public performances. The fact that these activities are synchronized online means that NA also opens a line of investigation into remotely shared musical and sound art practices. This in turn invites speculation as to the possibility of a creating a new paradigm in musical practice and distribution, a question that we wish to investigate in future research.

Networked music performances have existed since the beginning of the Internet and even before through the use of telephone and radio (see examples in section 3.1.). Until now however, such activities have, for the most part, focused on mixing networked (streamed, midi or OSC controlled sounds) with locally sounding instruments an example is Jean Claude Risset's use of the Disc Clavier, another is

French musician Eric M's practice of using Locus Sonus' "open microphone" live audio streams [30] to introduce indeterminate sounds into his DJ sets. With NA however, the virtual sound space is distributed, and co-users share events, functions, triggers, controllers and audio recordings (made on the fly) through a dedicated server. The fact that all connected users share the "current state" of a space (see technical explications in section 3) means that as long as an instance of a space is active, they experience the same evolving situation from wherever they are (within the limits of the network's capabilities). There is no distinction per se between musician and audience, this allows for inventive approaches to shared performance (for example, if one player introduces a sound source object or makes a recording, other players can hear it, move it or modify its parameters).

An Educational Tool

Locus Sonus [START_REF]Locus Sonus[END_REF] is a research unit based at ESA -Aix and funded by the French ministry for culture. Its main concern is practice-based research into sound art. Locus Sonus' research investigates virtual and acoustic sound space in a permanent exploration of "new auditoriums" [START_REF] Sinclair | Locus Sonus 10 ans d'expérimentation en art sonore[END_REF]. ENSCI les Ateliers, have worked on numerous sound design projects including sounding objects, industrial sound design, audiography, sound spatialization, auditory interfaces but also sound art, electronic music, radio broadcasts, soundtracks for films, and various research projects [START_REF] Cahen | Teaching Sound--Design @ENSCI les Ateliers, Sound-Design and innovation: a virtuous circle[END_REF]. The School of the Art Institute of Chicago's Art and Technology department teach virtual and augmented reality, gaming, 3d modeling, computer imaging, live performance audio and immersive audio [33]. Between them, these three structures provide the multidisciplinary expertise, creative and educational context that the NA project is founded on. The recent addition of the FRE PRISM as a new partner adds elements of fundamental research in the domain of digital audio to this rich consortium.

Teaching Sound in Art and Design Schools

Over the last 20 years sound education in France has progressively shifted from the conservatoires (music academies) to schools of art and design. The main reason for this is that, apart some rare exceptions, conservatoires were designed to teach solely classical music theory and instrumental practice and have therefore experienced difficulty in adapting to the challenges of XXth and XXIst century music and sound art production. As a consequence sound arts, sound design, some performing arts and sound for multimedia are now taught in many art and design schools including ENSCI and ESA-Aix. Most of the students concerned are consequently visual artists and rarely trained musicians. On the one hand this is a drawback, because they have a limited sonic culture, and on the other it is an advantage because they don't have preconceived models or cultural bias. Similarly, unlike traditional composers who arguably consider music as a result of relationships between pitch and rhythm, artists tend to use sound as material. Since their culture is primarily visual, these students have a tendency to create sounds in relation to images and to be concerned with multimedia and interaction.

Working and Creating Online in Virtual Workshops, Complementary Skills & Delocalized Working Groups

Creating and playing music together and by extension audiographic composition and performance are a great way to link people; this holds for artists, designers and students as well as the game audience. NA "bridges people with and through sound" since it is a group project within which participants with different skills and different degrees of expertise can come together and cooperate. Thus although the permanent team of NA is constituted of experienced developers, researchers, artists and teachers, this team has been completed from the outset of the project by successive generations of post graduate students. The architecture of NA has been designed in such a way as to encourage a variety of artistic approaches and methodologies, a creative participant can easily build a new and unique space (or level) however different propositions can also be easily combined into a single space. This method of creating content is gradually enlarging and enriching the scope of the project through a process of experimentation, feedback and exchange. On another level, as described above, creating content for the world is also be a challenging pedagogical exercise. Students are encouraged to work in teams pooling skill sets and sharing experience. The NA team is spread over several geographic locations, rather than this becoming a handicap it has turned into a fundamental characteristic of the project. NA is progressively becoming the place where we meet to work and remote collaboration is in the process of becoming second nature.

Architecture and Development

The particular aims of New Atlantis, described above, imply that certain decisions have been made regarding the architecture and other parameters of the world. To give some examples: the world needs to be shared online, it should be easy to use by a wide range of players (including those creating content) and audio performanceincluding synchronization between players -is paramount.

System Architecture

New Atlantis consists of three main software components:

The first is a MySQL database / PHP backend. The second is a standalone app (Mac OS X/Windows), named the Viewer, which allows the player to navigate the world, host multiplayer sessions, manage his account (spaces, assets…) and upload content. The third (optional) component is the SDK, which allows the advanced participant to build a viewer and to create components and contents using Unity3D authoring software.

Entities and Related Concepts

A Space is an independent New Atlantis world. It is not necessarily related to the Bacon Sound Houses concept: several Sound Houses could exist in one Space, or one Sound House could be split in several Spaces. The absolute rule is that one Space is totally independent: nothing communicates with, exits to or enters from the outside.

An Object is a composite-audio-graphic-3d-interactive object created by a participant (designer/artist/developer) in Unity and uploaded to a Space in the form of an "Asset Bundle". Objects have qualities (in particular audio capabilities) and consist of a data packages with associated states. These include the updating position, orientation, and other related parameters in the currently activated space. When not in a space, an object is referred to as an asset.

A player (user or visitor) is a human user of the New Atlantis platform. The system holds parameters related to the players, such as email, login, password and a list of assets.

The viewer is the multi-purpose application that is the main tool a player has to use to visit and navigate a space.

Synchronization and Data Persistence

As a shared audio world, New Atlantis has stringent needs when it comes to synchronization since, as described above, we wish players to be able to play together musically. This requires that what each player hears on different connected computers is as close as is possible to the same thing. A MySQL database / PHP backend holds the main state of objects and a web service allow the access to this database, including creation, modification and deleting of objects. Any player is able to host a session on his/her personal computer and accept other players running the same viewer application, from virtually anywhere in the world (within the limits of firewall and network structures). This is implemented using Unity's built-in advanced network features (NAT punchthrough, Master server, Facilitator server) -so instead of running a dedicated server, we decided to take this simpler and more flexible approach where any user is able to host a New Atlantis session with a chosen Space (this method is used by most multiplayer video games).

We have had to define and implement the way Unity's network system manages synchronization, when several players are connected together in a session. We decided to use an authoritative server scheme, meaning that the server runs the entire simulation and sends objects states to the clients that are running the scene without the logic (solely the rendering). The only exception to this is the avatar position, which is not sent by the server but generated by each client to guarantee good local fluidity (to avoid lag in the avatar's own position). The number of objects that need to be synchronized has a direct effect on performance and we tried several variations on "depths" of synchronization, from every single object to only a few main ones. There is inevitably a compromise that has to be made between performance and accuracy in simultaneity between clients (largely dependent on bandwidth). We should insist here on the special needs of working with sound: principally good temporal synchronization. To sum up, in the viewer application, two types of synchronization occur:

1. A synchronization of the objects contained in a given space: the viewer app connects to the web service and downloads an XML manifest (list) of all objects in the space with their initial state (position, orientation, name). Each object is an Asset Bundle that is then downloaded by the viewer and held in cache for future retrieval.

2. A client/server synchronization with Unity Network engine: synchronization of avatars positions, created objects, audio recorded during playing... The Unity Network engine implementation works with TCP/IP and UDP/IP messages sending with a proprietary format including synchronized parameters serialization. We have had to implement RPCs (Remote Procedure Calls) functions to handle special cases and audio-graphic synchronization.

Content Creation: Tradeoff Between Simplicity and Features

As described in section 2, NA has several levels of usage. Technically, the system needs to be used by a wide range of players with skills that range between basic gaming and professional design and programming. Advanced players, such as students in workshops, can use the NA SDK package in Unity, while a player without such skills is still be able to run the viewer app and access the spaces without doing any programming. It should be mentioned that some of the students during the workshops and throughout the duration of project development have even programmed substantial improvements and add-ons to the platform.

We have identified the following users:

1. Simple player/visitor -visits and navigates a space. 2. Advanced player/visitor -creates spaces with existing assets.

3. Builder -builds new assets to be used and shared. 4. Core developer -creates new standard NA components.

Unity is used both as a content creation platform and as the game engine. This means that while it is tempting to allow the builder to use Unity's authoring tool with as few restrictions as possible, it is necessary to forbid practices that could lead to issues, such as bad synchronization or malfunctioning components.

The need to standardize accepted scripts

For security and design reasons, builder created scripts cannot be included in Asset Bundles. User-created Objects can reference existing scripts, but a new script (at the time of writing), needs to be included in the viewer to be available in the system. A workaround would be to build a .NET assembly using Mono Develop or Microsoft Visual Studio and use .NET reflection to make it available at runtime. However this is still in development and is not a perfect solution since it would also enable potentially unlimited access to scripting with the implied security problems.

Performance considerations

Resources are a major concern when it comes to user-generated content: a player could potentially build a highly complex object that is too "greedy" with the resources of visiting computers (including video rendering performance, download time, CPU processing and memory). We have decided to manage this in two ways. Firstly, by defining "good practice" guidelines, these include: limiting the number of simultaneously playing sound sources; reducing the number of physical collisions and rigid bodies; reducing the size of audio clips; avoiding triggering too many audio sources too frequently; being careful with the use of custom DSP processing. In addition to these guidelines, we have limited the Asset Bundle upload size to a maximum of 7MB. The second approach is via the implementation of active optimization schemes, such as dynamic audio sources management as well as dynamic components (distant audio sources and objects in general are deactivated).

The Viewer App and the Standard Tools and Navigation

As we aim for high coherency between spaces and the player's experience, we decided to introduce some standard concepts.

1. Navigation: after a few early discussions and tests concerning a userdefinable avatar and navigation system, we finally decided to provide a standard navigation system, that includes a few cameras and standard controls (with keyboard and mouse or with a dedicated gamepad). 2. Interactions and tools: we designed all interactions as "tools", that are selectable in the viewer by the player. A large number of these tools have been suggested many of which concern variations on modes of audio interaction (rubbing or dragging as well as simply colliding). At the time of writing roughly half of these ideas are implemented including sound-playing tools, an object thrower, physical interactions, flashlight, sunlight, trunk creation (audio recorder)...

Audio Components

We have built a library of audio components to be used on audiographic objects in New Atlantis, these include: 1. Audio synthesis: noise generation, oscillators, FM synthesis, loopers, wave terrain synthesis... 2. Audio triggering: play/stop an audio source based on specific events, such as a collision, a volume intersection (named a trigger), a threshold distance with the listener… 3. Audio parameter modulation: pitch, volume, panning... 4. Experimental audio filtering: implementation of Bacon's audio helps. 5. Audio recording: with content synchronization over the network (the "trunk").

This list of audio components continues to augment with the ultimate intention of providing a comprehensive toolbox for DSP interaction and sound generation.

Other standard components

Because in New Atlantis designers can only use built-in scripts, it has also been necessary to provide a wide range of other standard components, including animations, visuals, move and rotate, teleport, GUI, particles triggering, physics…

Audio Spatialisation and Virtual Acoustics

A standard approach to audio spatialisation divides perception into three classes: the direct path, the early reflections and the late reverberation. As most 3D game engines today, Unity has a simple 3D audio sources management, which we used to spatialize an audio source's direct path. Unity also has a concept of Reverb zones with dedicated parameters (such as presets, level, reverb time....), but the Reverb effect is applied to the final mix and is triggered by the listener position -this means that by default, it is not possible to have sources in different spaces and have each source /space resonate independently since all sources are reverberated simultaneously. To compensate for this we have introduced the possibility for an Audio source to make its immediately surrounding space resonate (NAReverbEffector / Resonator) by using volume triggering, this allows us to have more complex shapes than standard Unity reverb zones (simple spherical volumes). This is a workaround since instead of the listener it is the audio source that is reverberated (we found this to be a better compromise).

First Reflections

We have conducted some experiments concerning first reflections, that consisted of sending rays in all directions around the audio source, determining which rays hit a colliders (corresponding to an audio reflecting surface in our approximation), and then calculating the distance source-to-collider and collider-to-listener to define the audio delay for this path. The audio delay for each ray is then introduced in a multitap delay System (a FIR (Finite Impulse Response) filter) and applied to the audio source (using C# OnAudioFilterRead() custom DSP code). Although still an approximation, this gave interesting results -a sense of scale, small spaces (with a short slap back echo) and big spaces (cliffs, canyons etc.). However at the time of writing we have not yet included this as a standard feature in NA.

Audio Sources Directivity and Roll-off By default, the Unity audio engine does not provide management of audio source directivity. We have introduced the possibility to set a curve defining the source volume depending on the angle (similar to a polar diagram). Sound attenuation is implemented either by manual adjustment of roll off curves, where the builder defines how the sound attenuates over distance, or by using a physically accurate model for sound attenuation, (the well known inverse square distance law) where the builder simply defines the Sound Pressure Level at 1m in dB (dB SPL).

Presentations January 16 th 2016

For the performance on January 16th 2016 participants and audience were gathered in four different venues: Le Cube (Issy les Moulineaux, Paris France), the Vasarely Foundation (Aix en Provence), SAIC (Chicago) and Rensselaer Polytechnic Institute (Troy -New York state). The sound was played over 7.1 or 5.1 surround systems. As well as being connected via NewAtlantis the participants in different venues were connected via Skype during the performance and the audience was able listen to their commentaries and exchanges live. Five NA spaces were visited during the 80-minute performance that started at 9pm in Paris and 2pm in Chicago. The audience reaction was enthusiastic and there was a positive reaction towards the prospect of continuing development.

Conclusions and Further Research

One of the aims of NA is to provide a platform for international academic, artistic, cultural and scientific exchange. In this sense the project has indeed succeeded in creating a robust bridge between the American and French research teams and students. The remaining challenge is for New Atlantis to become a real-time tool or platform for international artistic and musical collaboration. Hopes are high however since music and sound are after all internationally comprehensible. Possibly the most important step, that of making the app publicly available has yet to be made.

Game architecture and Development

With the ultimate goal of making all things audio in NA (i.e. that all visual elements have an audio counterpart) the concept of acoustic materials, could allow us to define the way a 3D surface interact with incoming sound, with parameters such as absorption at several audio frequencies, audio diffusion and reflection. We intend to implement audio path-finding using Unity's ray casting capabilities -precisely calculating the contribution of each source to each space and applying a chain of audio effects based on the traversed space's characteristics. This was implemented in the previous Panda3D/Pd version of NA it now has to be ported to Unity. Other advanced topics will be addressed in the future, such as accurate sound occlusion (possibly using ray casting between the source and the listener), and more advanced audio spatialization algorithms with the Unity Spatialization SDK. It is our intention to incorporate audio streaming capabilities into NA permitting the real time inclusion "real world" captured sounds. This would enable a voice object for example whereby a visitor could detach his or her voice (from navigation) and use it to make a distant space resound.

Integration of and Intuitive synthesizer.

Locus Sonus has recently joined with the audio group of the CNRS Laboratory LMA, now FRE PRISM with a program (accord cadre) funded by MCC (Ministry for Culture and Communication) and CNRS (Centre Nationale pour la Recherche Scientifique). In this context it is planned to integrate the LMA's research into intuitive synthesizer interaction. In effect this synthesizer that is controlled by "semantic descriptions of sound events" [34][35], including non-existent ones, would appear to be an ideal solution for audio graphic experimentation in NA.

Interface and Graphic Design

Before releasing a public version of the New Atlantis app in is necessary to improve the ergonomics of the user interface. The design process is programmed and we hope to achieve this goal within the coming months.

Fig 1 .

 1 Fig 1. Students showcasing their projects in New Atlantis. ENSCI les ateliers, September 2015.

Fig 2 .

 2 Fig 2. System architecture overview.

Fig 3 .

 3 Fig 3. Synchronization and data persistence architecture overview.

Fig 4 .

 4 Fig 4. New Atlantis space "Ljubljana Island" showing reverb zones.

Fig 5 .

 5 Fig 5. New Atlantis performance synchronized between: Le Cube, Vasarely Foundation, SAIC and RPI. January 2015. Photo: Le Cube.

5 First Results -Student Workshops Public Presentations 5.1 Period September 2014 -January 2016 Description & Method New

  Atlantis design and development took place between September 2014 and September 2015, during workshops in Chicago and Aix-En-Provence and through regular online (skype) meetings. Subsequently a workshop was organized in ENSCI les Ateliers in Paris that included a group of 15 students and the whole team of researchers, artists from Aix, Chicago, Troy and Paris. Within a week, students who had never worked with either sound or 3D environments were able to create a set of audiographic objects. At this point we programmed the 2016 performance (see below). From October 2015 to January 2016 the team worked separately but connectedly to prepare this performance: in Paris, another group of 10 students created their own objects, helped to design graphic interface elements such as the avatar and prepared for the upcoming performance. At the same time a Masters student from ESA-Aix (Alex Amiel) was in residence in SAIC Chicago, helping to build the Chicago space, a group of Masters students were working in ESA-Aix to create the Aix space and Ben Chang was working in RPI-Troy to create his own space. From January 10 to 15 another workshop with a fresh group of students from design art and gaming schools took place at Le Cube. This time, the framework was more advanced and the group was able to work and produce results much faster.

Ivan Chabanaud, was a fascinating french digital artist who gave wings to his audience as they became icarus for the space of a visit. Our friend Ivan died the 4 th september

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New Atlantis Team 2016