INTRODUCTION Introduction

Autoimmunity may be defined as a rupture of tolerance with a failure of the immune system to recognize as "self" its own cells and tissues. Consequently, immune cells react if those were foreign or invading bodies. Rose and Bona proposed three evidences at the origin of an autoimmune human disease (Rose and Bona, 1993). The first criteria for autoimmunity is a direct disease transfer from one human to another (or to animal) in which the immune system is competent. For instance, immune thrombocytopenic purpura (ITP) has been identified as an autoimmune disease since the serum transfer of patients with ITP into healthy subject induces the disease (Van Loghem Jj et al., 1959;van LOGHEM et al., 1962). Grave's disease, myasthenia gravis or microscopic polyangiitis (MPA) present with similar disease transfers from mother to child (Scott, 1976;Bansal and Tobin, 2004). Hashimoto's thyroidis or type 1 diabetes could also transfer during bone marrow allograft (Rossini et al., 1989;Karthaus et al., 1997). However, iwhen direct transfer is not identifying, the capacity to develop the disease in healthy animal upon transfer from a pathological generated model is also considered as autoimmune (Rose and Bona, 1993). For example, the collagen-induced arthritis mouse model of rheumatoid arthritis is a robust autoimmune disease easily transferable to healthy animals (Stuart and Dixon, 1983). Finally, evidences for the production of specific autoantibodies and activation of oligoclonal T cells against a defined autoantigen could also allowed definition an autoimmune disease. This is the case for granulomatosis with polyangiitis (GPA) in which we could detect anti-neutrophil cytoplasm antibodies (ANCA) and activation of T cells against membrane bound PR3 (Millet et al., 2015).

Autoimmune vascular diseases A. General introduction

Clinicians and scientists have deciphered a number of mechanisms that pont out the importance of latent and persistent inflammation of vessels in disease progression. While a global classification of vascular diseases is not easy to set up, several specificities are used in this characterization. For instance, atherosclerosis, which corresponds to deposits of cholesterol in the lumen of aorta and other large arterial vessels, presents similarities with vasculitis, which is characterized by an inflammation of the walls of arteries and/or veins and finally, or other vasculopathies associated with vascular remodelling (Figure 1). The causes and consequences of the three types of inflammated diseases are multiple but are frequently associated with a dysregulation of the immune system.

Atherosclerosis

Atherosclerosis is characterized by an inflammation within the arterial wall leading to severe vascular events, including coronary artery disease, myocardial infraction, stroke and peripheral artery diseases (Insull, 2009;Taleb, 2016). The iniatial event is due to lipid retention, oxidation, and alteration, which provoke chronic inflammation, ultimately causing thrombosis or stenosis.

Atherosclerosis develops progressively through the continuous evolution of arterial wall lesions caused by the accumulation of the cholesterol-rich lipids and the subsequent inflammatory response [START_REF] Woolf | The origins of atherosclerosis[END_REF].

Both innate and adaptive immunities play important roles in the development of atherosclerosis; notably implicating monocytes/macrophages, which are the surrogate cells of innate immunity. Also, on the other hand, T-helper cell type 1 (Th1) response has a potent proatherogenic effect, the pathogenic roles of other T cell subsets, such as the Th2 and Th17 dependant mechanisms, remain controversial. However, the anti-atherosclerotic protective roles of regulatory T cells and several Th2-related cytokines, such as interleukin-5, have been clearly established.

In addition, since a number of reports based on animal models argue for the important role of inflammatory cells in atherosclerosis and its complications, treatment of cardiovascular diseases with anti-inflammatory drugs is an attractive option to investigate.

However, atherosclerosis has mostly been described as resulting from the development and evolution of low density lipoprotein (LDL) particles deposits. These particules are enzymatic modified and are oxidized into proinflammatory particles, which trigger innate immune system reaction within the intima [START_REF] Mayadas | Mechanisms of immune complexmediated neutrophil recruitment and tissue injury[END_REF]. Fat droplets may accumulate in the cytoplasm of vascular smooth muscle cells (VSMC), leading to mechanic and inflammatory stress responses into the cells [START_REF] Schwartz | Replication of smooth muscle cells in vascular disease[END_REF].

Inflammation begins when endothelial cells become activated and express adhesion molecules, while smooth muscle cells secrete chemokines and chemoattractants, which induce the migration of neutrophils, monocytes and lymphocytes into the arterial wall. Intimal smooth muscle cells also secrete, within the extracellular matrix, proteoglycans, collagen, and elastic fibers triggering a complex remodelling of the whole vessel [START_REF] Hansson | Immune mechanisms in atherosclerosis[END_REF].

As the disease develops with lipids accumulation and inflammation, the processes and histologic changes become increasingly complex and can vary considerably both between individuals and for any given individual [START_REF] Ross | Atherosclerosis -An Inflammatory Disease[END_REF]. All of these changes may be significantly influenced by risk factors, notably stresses of local hemodynamics and blood flow patterns, hypertension, smoking, and diabetes, as well as genetically determined arterial susceptibility or resistance to atherosclerosis [START_REF] Smithies | Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension[END_REF]. The mechanisms involved for influencing atherosclerosis by these risk factors are the subject of an intensive investigation by molecular pathologists, along with proteomics and genomics, conducted to determine the exact molecular biological processes involved in their development.

In this work, we will not focus on atherosclerosis since this pathology is mainly influenced by hemodynamic and endothelium resistance [START_REF] Borissoff | The Hemostatic System as a Modulator of Atherosclerosis[END_REF].

Vasculitis:

Vasculitis are a group of rare diseases characterized by inflammation and infiltration of immune cells within vessel walls. If inflammation is not always observed during the course of vasculitis, it is still a common defining feature of all categories of vasculitis. Some categories of vasculitis also have a characteristic tissue injury unrelated to vasculitis. Features that vary among different forms of vasculitis and can be used for categorization include etiology, pathogenesis, the type of vessel affected, the inflammation process, the main organ affected, clinical manifestations, genetic predispositions, and distinctive demographic characteristics (e.g., in regards to age, sex, ethnicity, and geographic distribution) [START_REF] Jennette | Small-Vessel Vasculitis[END_REF][START_REF] Salvarani | Polymyalgia Rheumatica and Giant-Cell Arteritis[END_REF]. Disease categorization based on etiology is often a preferred approach; however, this remain undoable for most vasculitis for which the etiology is unknown.

Jennette and colleagues (Jennette et al., 2013) have proposed to discriminate vasculitis between:

-infectious vasculitis, caused by direct invasion and proliferation of pathogens within the vessel wall with a resulting inflammation but also several vasculitis induced indirectly by the presence of a pathogen (ex: cryoglobulinemic vasculitis due to an autoimmune response initiated by a hepatitis C virus infection [START_REF] Vassilopoulos | Hepatitis C virus infection and vasculitis: implications of antiviral and immunosuppressive therapies[END_REF] or HIV associated vasculitis). These vasculitis are commonly associated with the presence of circuliating immune complex. (Lidar et al., 2009) -non-infectious vasculitis, not known to be caused by direct vessel wall invasion by pathogens Many studies have shown the implication of both innate (monocytes in giant cell arteritis, neutrophils in granulomatosis with polyangiitis…) and adaptive immune cells (presence of auto-antibodies, T cell imbalance…) at the onset of the disease and along the entire course of vasculits [START_REF] Savill | The fate of the neutrophil in vasculitis[END_REF][START_REF] Barratt | Treatment of IgA nephropathy[END_REF][START_REF] Ly | Pathogenesis of giant cell arteritis: More than just an inflammatory condition?[END_REF]. However, as observed for atherosclerosis, inflammation subsequent to vasculitis also induces important vascular damage and stress and could, upon long time, enhance vessel remodelling and atheroma rupture.

Diseases associated with vascular remodelling

Vascular dysfunction due to vascular remodelling, such as microvascular complications in both insulin-independent and insulin-dependent diabetes, are major contributors to morbidity, mortality and therapeutic costs [START_REF] Emery | Vascular remodelling in the lung[END_REF][START_REF] Spinetti | Diabetes and vessel wall remodelling: from mechanistic insights to regenerative therapies[END_REF]. As previously mentioned, patients with diabetes have high risk to develop atherosclerotic disease (Ladeia et al., 2014).

Endothelial dysfunction has been identified as an early marker of vascular disease in type 1 diabetes. Alterations of the microcirculation involve small arteries, arterioles, capillaries and post capillary venules. Mechanisms involved in vascular alterations in diabetes are unclear and could be linked to hyper-glycosylation of both circulating proteins and collagen fibrins, metabolic disorders which induce endothelial cells activation (notably protein kinase C pathway) or to growth factors and probably insulin itself [START_REF] Schena | Pathogenetic mechanisms of diabetic nephropathy[END_REF][START_REF] Porter | The vascular smooth muscle cell: a therapeutic target in Type 2 diabetes?[END_REF]. In these mechanisms of vascular remodelling, inflammation is pointed out in both type 1 and type 2 diabetes (Arroyo and Iruela-Arispe, 2010).

Pulmonary hypertension (PH) is a cardio-pulmonary disorder that may involve multiple clinical conditions and can upgrade the majority of cardiovascular and respiratory diseases [START_REF] Ryan | The WHO classification of pulmonary hypertension: A case-based imaging compendium[END_REF]. The classification of PH is complex and discriminates groups of patients which share similar pathophysiological and hemodynamic characteristics upon treatments [START_REF] Huertas | Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: a complex interplay[END_REF]. Pulmonary arterial hypertension (PAH) corresponds to the first group of PH and is characterized by an increase in mean pulmonary artery pressure over 25 mmHg at rest, leading to right heart failure and ultimately death while low capillary pressure is lower than 15mmHg.

PAH may be associated with connective tissue diseases, notably systemic sclerosis (SSc), systemic lupus erythematosus (SLE), mixed connective tissue diseases (MCTD) (Sharp syndrome) and Sjögren's syndromes [START_REF] Bussone | IgG from patients with pulmonary arterial hypertension and/or systemic sclerosis binds to vascular smooth muscle cells and induces cell contraction[END_REF][START_REF] Kherbeck | The role of inflammation and autoimmunity in the pathophysiology of pulmonary arterial hypertension[END_REF]. In these different pathologies, PAH present one of the most severe complication showing the importance to study these patients subsets (Legendre and Mouthon, 2014). Clinical evolution of these patients is particularly constant; all patients present at early time a connective tissue disease preceeding PAH. These observations suggest that PAH could be associated with autoimmune and immunological events [START_REF] Rabinovitch | Inflammation and immunity in the pathogenesis of pulmonary arterial hypertension[END_REF]. (Smith et al., 2012) SSc is a rare connective tissue disease characterized by vascular involvement responsible for vascular hyperactivity and remodelling, together with fibroblasts activation and extra-cellular matrix synthesis (Dumoitier et al., 2014). SSc is often responsible for internal organ involvement as a consequence of remodelling of arterioles. In the lung, SSc is associated with the development of PAH and/or pulmonary fibrosis (Figure 2) [START_REF] O'callaghan | Pulmonary veno-occlusive disease: the bête noire of pulmonary hypertension in connective tissue diseases[END_REF][START_REF] Wells | Interstitial lung disease in connective tissue disease-mechanisms and management[END_REF]. In recent years, significant progress has been made in the understanding of mechanisms contributing to the occurrence of vasculopathy and fibrosis during the course of SSc, both in human and animal models (Denton, 2015). However, despite these scientific achievements, no universal treatment has been found yet and the elucidating of the complex immune response involved in this disease could be a real breakthrough in the future.

Both SSc, PAH and PAH associated SSc have a poor prognosis and an interesting overlap of their respective immune context.

B. Systemic vasculitis Classification

A first classification of systemic vascultis has been established by the American Society of Rheumatology in 1990 [START_REF] Hunder | The American College of Rheumatology 1990 criteria for the classification of vasculitis: Introduction[END_REF]. This important study has proposed to study 127 clinical manifestations associated to one or more of the 7 vasculitis: polyarteritis nodosa, Churg-Strauss syndrome, Wegener's granulomatosis, hypertensitivity vasculitis, Henoch-Schönlein purpura, giant cell arteritis and Takayasu arteritis. However, if this first classification has led to important improvements in the diagnosis of patients with vascultis, the characterization of microscopic polyangiitis (which was commonly and wrongly diagnosed as polyarteritis nodosa) and the development of etiology studies have allowed the scientific community to further increase the list to 10 vasculitis under the Chapel Hill Conference Consensus, first in 1994 and more recently in 2012, 11 vasculitis have been overviewed [START_REF] Zashin | Microscopic polyarteritis: a forgotten aetiology of haemoptysis and rapidly progressive glomerulonephritis[END_REF][START_REF] Jennette | Nomenclature of systemic vascultis. Proposal of an international consensus conference[END_REF]Jennette et al., , 2013a)). Chapel Hill Conference Consensus categorized non-infectious vasculitis by integrating knowledge about etiology, pathogenesis, pathology, demographics, and clinical manifestations. The first categorization level is based on the predominant type of vessels involved: large, medium and small-sized vessel vasculitis (Figure 3).

These terms refer to vessels that differ not only in size, but also in structural and functional attributes. There are further distinctions within each vessel type, for example, capillaries in different organs and different segments of the aorta have different biochemical and functional properties that make them susceptible to different pathogenic mechanisms (Jennette et al., 2013). On the other hand, large vessel vasculitis affects large arteries more often than medium vessels, medium vessel vasculitis affects predominantly medium arteries, and small vessel vasculitis affects predominantly small arteries, vessels and capillaries. and immune complex vasculitis (Panel D) - (Jennette et al., 2013) In this classification, a subgroup of vascultis has emerged among others: ANCA-associated vascultis (AAV) which are the granulomatosis with poliangiitis (GPA), the microscopic polyangiitis (MPA) and the eosinophilic granulomatosis with polyangiitis (EGPA). These three vascultis are pauci-immune necrotizing vascultis which present with a wide range of clinical manifestations from granuloma formation to renal damage or asthma. The detection of ANCA could be helpful for the diagnosis of GPA and microscopic polyangiitis MPA. The latter have been recently described as pathogenic, conversely to other vasculitis which present a wide broad of auto-antibodies or a more specific clinical presentation (Jennette et al., 2013b).

ANCA associated Vascultis

AAV are a heterogeneous group of diseases corresponding to necrotizing inflammation of small vessels with a wide range of clinical presentations (Millet et al., 2013). AAV are pauci-immune necrotizing vasculitis involving small-sized vessels which include GPA (formerly named Wegener's granulomatosis), EGPA (formerly named Churg-Strauss syndrome), and MPA.

While granuloma presence seems to discriminate between GPA plus EGPA and MPA, only GPA and MPA seem to have a clear lymphocyte dysregulation as these two pathologies have been linked to the presence of anti-proteinase 3 (PR3) and anti-myeloperoxydase (MPO) respectively. The ANCA signature in EGPA is quite debated in the field and has lead the scientific community to separate EGPA from the GPA/MPA subgroup of vascultis which are mostly treated with the same immunosuppressive strategy (Chaigne et al., 2016a). However, multiple other aspects of vascultis allow us to differentiate GPA and MPA patients and to highlight the matter of a different immunological disorder in these patients.

i) Epidemiology

Epidemiological data have challenged the notion that GPA and MPA could be grouped under the so-called AAV entity (Millet et al., 2013) (Figure 6). Notably, marked geographical disparities have been found. For instance, GPA is infrequent in the Japanese population and rarely occurs in African Americans. This is not the case for MPA. In Europe, GPA is more frequent in the north countries, and a reverse tendency has been found for MPA. Also, a periodical (7.5 years) non-random pattern of GPA occurrence has been recently described, but, it was not observed for MPA. Further implication of differential factors has been documented with fro instance the occurrence of anti-MPO-associated vasculitis upon propylthiouracil or hydralazine treatment while the treatment does not led to anti-PR3-associated vasculitis. In GPA, a link between infection and autoimmunity has been suggested since Staphylococcus aureus chronic carriage was found in patients exhibiting a higher relapse rate. Moreover, genome wide associated studies (GWAS) have shown a differential pattern between GPA and MPA. GPA is associated with several single nucleotide polymorphisms in PR3 (PRTN3), SERPINA1 and HLA-DP genes. AAT: α1-antitrypsin; AAV: ANCA-associated vasculitis; ENT: ear nose throat; HLA: human leukocyte antigen; MPO: myeloperoxidase; PR3: proteinase 3; PRTN3: gene coding for PR3; PTU: propylthiouraciladapted from (Millet et al., Ann Rheum Dis 2013) Finally, the third AAV, EGPA, has a prevalence of 11 cases per million of inhabitants which is lower than GPA (50 cases per million of inhabitants) or MPA (25 cases per million of inhabitants) (Chaigne et al., 2016b). Genetic susceptibility/predisposition has been suggested for EGPA with associations between EGPA and HLA. An association was found for HLA DRB1*04 and HLA-DRB1*07 twice, while a protective effect was reported with HLA-DRB3 and HLA-DRB1*13 (Vaglio et al., Secondary and Primary Vasculitis Study Group, 2007;Wieczorek et al., 2008a). Of note, a study on HLA-DRB1 locus in 403 patients with GPA and 103 with EGPA linked the extended interleukin 10 (IL-10)-3575/-1082/-592 TAC haplotypes with ANCA-negative EGPA (Wieczorek et al., 2008b). Even though IL-10 level was reported to be elevated in EGPA, we lack a post-transcription analysis of the haplotypes .Genes related to the costimulatory molecule CD226 (DNAX accessory molecule 1), the intracellular signaling-involved molecule PTPN22 and eotaxin-3 single nucleotide polymorphisms were investigated but were not associated with EGPA [START_REF] Wieczorek | Novel association of the CD226 (DNAM-1) Gly307Ser polymorphism in Wegener's granulomatosis and confirmation for multiple sclerosis in German patients[END_REF].

While EGPA is commonly associated with asthma, no clear link was found between the frequency of asthma nor the seasonal cycle and the prevalence of EGPA [START_REF] Bottero | The common allergens in the Churg-Strauss syndrome[END_REF].

Moreover, only several case reports and a limited number of studies have linked EGPA with infectious triggering, antigen inhalation or uses of drugs [START_REF] Guillevin | Churg-Strauss syndrome. Clinical study and long-term follow-up of 96 patients[END_REF][START_REF] Mouthon | Systemic small sized vessel vasculitis after massive antigen inhalation[END_REF].

ii) Clinical and immunological presentation Inflammation of small-sized vessels in AAV is responsible for organ damage with granulomatous involvement of upper and lower respiratory tract in GPA and EGPA, and glomerulonephritis, intra-alveolar hemorrhage, and/or peripheral neuropathy in both GPA, MPA, and EGPA (Figure 7) (Millet et al., 2013;Soussan et al., 2014;Chaigne et al., 2016b). Detection and characterization of ANCA strongly support the implication of B-cells in the pathophysiology of AAV (Figure 8). The prevalence of ANCA among patients with AAV differs according to the type of vasculitis [START_REF] Wiik | Autoantibodies in vasculitis[END_REF]Millet et al., 2013). In GPA, PR3-ANCA are detected in 85% of patients while MPO-ANCA are identified in less than 10% of the cases [START_REF] Van Der Woude | ANTICYTOPLASMIC ANTIBODIES IN WEGENER'S GRANULOMATOSIS[END_REF][START_REF] Hagen | Antineutrophil cytoplasmic autoantibodies: a review of the antigens involved, the assays, and the clinical and possible pathogenetic consequences[END_REF]. MPO-ANCA are detected in 60% of the patients with MPA and in 31-38% of patients with EGPA [START_REF] Wiik | Autoantibodies in vasculitis[END_REF]. Until recently, the pathogenesis of AAV was reported as involving predominantly neutrophils, eosinophils, T cells, and ANCA as antibodies. Importantly, data supporting B-cell implication in this setting was recently shown. One of the first reports on the implication of B cells in AAV focused on the analysis of B-cell infiltrations proximal to PR3-positive cells and on plasma cells within endonasal inflammatory lesions of patients with GPA (Voswinkel et al., 2006). Within these lesions, Voswinkel et al. reported on the activation and maturation of aggregated B-cells, arguing for a key role of B lymphocytes in the pathogenesis of AAV (Figure 9). This concept was reinforced by the results of two prospective randomized trials demonstrating the efficacy of rituximab, an anti-CD20 monoclonal antibody, in the induction of remission in patients with GPA or MPA, and more recently for the maintenance of remission in the same diseases, with the superiority of rituximab compared to azathioprine (Stone et al., RAVE-ITN Research Group, 2010;Specks et al., RAVE-ITN Research Group, 2013;Guillevin et al., 2014a;Dumoitier et al., 2015). (Voswinkel et al., 2006) Following these new insights due to clinical trials on rituximab in AAV, several studies on B lymphocytes subpopulations in patients with AAV have been reported. A correlation was found between the importance of B-cell infiltration within granulomatous lesions in GPA and the occurrence of relapses, despite peripheral B-cell depletion in few patients treated with rituximab (Ferraro et al., 2008).

iii) Pathophysiology

A lot of actors implicated in the pathophysiology of AAV have already been identified. The detection of ANCA underlined the importance of the neutrophils in AAV as well as the potential involvement of these antigens. However, the expression of both PR3 and MPO, as well as the modulation of the inflammatory process by their respective ANCA is still unclear (Figure 10). MPO and PR3 are two granule-stored microbicidal proteins expressed by neutrophils and monocytes and possess immunomodulatory properties [START_REF] Witko-Sarsat | Neutrophils: Molecules, Functions and Pathophysiological Aspects[END_REF]. Recent studies have demonstrated the importance of neutrophils and monocytes through the expression of PR3 and MPO in both human and animal models [START_REF] Roth | Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis[END_REF]Millet et al., 2015).

Besides the pathogenic role of ANCA, previous studies have investigated the role of genetic predisposition [START_REF] Alberici | Genetics of ANCA-associated vascultis: HLA and beyond[END_REF], silica exposure [START_REF] Gómez-Puerta | The association between silica exposure and development of ANCA-associated vasculitis: systematic review and metaanalysis[END_REF] or autoimmune overlaps (Guellec et al., CRI (Club Rhumatismes et Inflammation) and the French Vasculitis Study Group, 2015) and have demonstrated the implication of T cells in AAV, showing a predominant Th1 polarization in the granuloma form of GPA contrasting with a predominant Th2 polarization in the vasculitis form of EGPA (Abdulahad et al., 2011;Chaigne et al., 2016a).

Interestingly, in a cohort of patients with AAV, including GPA and MPA patients, tertiary lymphoid structures (TLS) have been identified in the tissues (Steinmetz et al., 2008). TLS were identified in glomerulonephritis lesions, composed of a germinal center containing follicular dendritic cells and memory as well aw non-memory B cells at the periphery of the germinal center, in contact with the T cells. Interestingly, highly proliferative B-cells have been found in the center of TLS, providing an important source of antigen-presenting cells enhancing T cells maturation at the periphery. However, there is no evidence has been obtained for antibody secretion or maturation of antibody-producing cells in renal lesions of patients with AAV.

These results suggest an antibody-independent role of the B-cells in the pathological site and suggest a complex interplay between adaptive and innate immune cells in GPA and MPA (Figure 9). Several B cell abnormalities have been found in patients with GPA and have been recently reviewed in our study (Dumoitier et al., 2015, ANNEXE 2). However, hardly any rich documentation has been published regarding the implication of B lymphocytes in MPA or EGPA while anti-MPO have been found clearly pathogenic in vivo [START_REF] Tsurikisawa | Decreases in the numbers of peripheral blood regulatory T cells, and increases in the levels of memory and activated B cells, in patients with active eosinophilic granulomatosis and polyangiitis[END_REF]Xiao et al., 2002).

In EGPA, the main immune dysfunction is linked to the presence of eosinophils as these cells present a dramatically increased proliferation, decreased apoptosis and increased tissue toxicity (Figure 11) (Chaigne et al., 2016b). Increased expression of activation markers have been described in both neutrophil in GPA and eosinophil in EGPA [START_REF] Klion | Eosinophil-Associated Disorders, An Issue of Immunology and Allergy Clinics of North America[END_REF]Millet et al., 2015). Moreover, specific cytokine cascades have been found in the three AAV [START_REF] Guilpain | Pathogenesis of primary systemic vascultis (I): ANCA-positive vascultis[END_REF]. Polymorphonuclear cells; ROS: Reactive oxygen species; TARC: thymus and activation regulated chemokine; Th: T helper cells -adapted from (Chaigne et al., 2016b) iv) Prognosis AAV causes considerable morbidity and mortality, with end-stage renal failure developing in over 20% of the patients at five years [START_REF] Booth | Outcome of ANCAassociated renal vasculitis: a 5-year retrospective study[END_REF]. Prior to the introduction of steroids and cyclophosphamides, in the early 1970s, survival was uniformly dismal, with a median survival of five months for patients with GPA [START_REF] Walton | Giant-cell Granuloma of the Respiratory Tract (Wegener's Granulomatosis)[END_REF]. Nowadays, use of immunosuppressive treatments leads to an induction of remission in 90% of the patients by six months [START_REF] Jayne | A randomized trial of maintenance therapy for vasculitis associated with antineutrophil cytoplasmic autoantibodies[END_REF] and a five-year survival rate around 75% [START_REF] Matteson | Long-term survival of patients with Wegener's granulomatosis from the American College of Rheumatology Wegener's Granulomatosis Classification Criteria Cohort[END_REF]. However, although progress has been made in the management of patients with AAV, there are still refractory and frequently relapsing cases after treatment (Specks et al., RAVE-ITN Research Group, 2013).

Cyclophosphamide, an alkylating agent, is one of the major anti-cancer treatments [START_REF] Cramblett | Experiences with cyclophosphamide in treatment of childhood tumors[END_REF]. This drug is also associated with major immunosuppressive effects and has been reported to improve the outcome of patients with systemic autoimmune diseases [START_REF] Taylor | IDIOPATHIC AUTOIMMUNE HEMOLYTIC ANEMIA. RESPONSE OF A PATIENT TO REPEATED COURSES OF ALKYLATING AGENTS[END_REF]Silva et al., 2011;Monach et al., 2013). Following these observations, cyclophosphamide was shown to improve the outcome of patients with GPA [START_REF] Novack | Cyclophosphamide therapy in Wegener's granulomatosis[END_REF], and cyclophosphamide has become the standard of care of AAV patients with severe disease.

Although cyclophosphamide is known to deplete both B cells and T cells in peripheral blood, depletion is more pronounced in B cells than in T cells [START_REF] Ziff | B-and T-lymphocytes in rheumatoid synovitis and the effect of cyclophosphamide[END_REF]. In vitro and in vivo experiments document the effect of cyclophosphamide on antibody production and secretion, in AAV and SLE, suggesting a possible impact on autoreactive B cells and autoantibody secretion [START_REF] Casey | Immunosuppression by cyclophosphamide in NZB X NZW mice with lupus nephritis[END_REF][START_REF] Buckley | Cyclophosphamide therapy of Behcet's disease[END_REF].

Anti-CD20 antibodies, and particularly rituximab (Rituxan® or Mabthera®), were designed to target B cells and treat B cell lymphoproliferative disorders, particularly B cell lymphomas and obtained an FDA agreement in 1997an FDA agreement in in this indication (1994)). Then, rituximab was tested in a number of autoimmune conditions such as cold agglutin disease [START_REF] Lee | Rituxan in the treatment of cold agglutinin disease[END_REF], immune thrombocytopenia [START_REF] Shvidel | Successful treatment with Rituximab for relapsing immune thrombocytopenic purpura (ITP) associated with low-grade non-Hodgkin's lymphoma[END_REF] or thrombotic thrombocytopenic purpura [START_REF] Faurschou | Sustained remission of platelet counts following monoclonal anti-CD20 antibody therapy in two cases of idiopathic autoimmune thrombocytopenia and neutropenia[END_REF]. Specks et al., reported in 2001 the first patient with GPA treated with rituximab in whom complete remission was documented at one year [START_REF] Specks | Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy[END_REF].

Limited numbers of patients with refractory MPA and EGPA were also successfully treated with rituximab in the middle of the 2000's decade [START_REF] Langford | New insights into the immunopathogenesis and treatment of small vessel vasculitis of the kidney[END_REF][START_REF] Eriksson | Nine patients with anti-neutrophil cytoplasmic antibody-positive vasculitis successfully treated with rituximab[END_REF].

The similar efficacy of rituximab and cyclophosphamide was documented in two prospective randomized trials, in the induction of remission for the treatment of GPA and MPA (Jones et al., 2010;Stone et al., 2010;Specks et al., RAVE-ITN Research Group, 2013). More recently, the superiority of rituximab compared to azathioprine was demonstrated in the maintenance of remission in GPA and MPA patients (Guillevin et al., 2014b).

v) Non classical treatments targeting B cells

In autoimmune diseases, rituximab treatment leads to rapid and complete depletion of naïve B cells and nearly all peripheral B cell subsets [START_REF] Szodoray | Apoptotic Effect of Rituximab on Peripheral Blood B Cells in Rheumatoid Arthritis[END_REF]. Interestingly, an in vitro nondepleting study also demonstrated a better efficacy of rituximab on CD27 -naïve B cells compared to CD27 + memory B regarding B cell activation, proliferation and viability [START_REF] Kamburova | In vitro effects of rituximab on the proliferation, activation and differentiation of human B cells[END_REF]. Few months after the first injection of rituximab, transitional B cells repopulation is observed and plasma cells recirculate in the peripheral blood of treated patients [START_REF] Leandro | Reconstitution of peripheral blood B cells after depletion with rituximab in patients with rheumatoid arthritis[END_REF][START_REF] Teng | Residual inflammation after rituximab treatment is associated with sustained synovial plasma cell infiltration and enhanced B cell repopulation[END_REF]. Moreover, notably in rheumatoid arthritis patients, a high proportion of circulating memory B cells is associated with increased risk of relapse in patients treated with rituximab [START_REF] Leandro | Reconstitution of peripheral blood B cells after depletion with rituximab in patients with rheumatoid arthritis[END_REF][START_REF] Kamburova | In vitro effects of rituximab on the proliferation, activation and differentiation of human B cells[END_REF]. In AAV, Bunch et al. suggested that after treatment by rituximab, the number of CD5 + B lymphocytes, a B cell subset that seems to be poorly affected by anti-CD20 monoclonal antibody, could correlate with the risk of relapse and/or disease severity [START_REF] Bunch | Decreased CD5 + B cells in active ANCA vasculitis and relapse after rituximab[END_REF].

Ten years ago, infliximab, a humanized anti-TNF-α monoclonal antibody was proposed for reduced the persistent inflammation and endothelial activation in AAV (Booth et al., 2004a;[START_REF] Wilkinson | Infliximab as rescue therapy in three cases of paediatric Wegener's granulomatosis[END_REF]. Several studies have suggested interesting results for refractory patients [START_REF] Josselin | Infliximab efficacy and safety against refractory systemic necrotising vascultis: long-term follow-up of 15 patients[END_REF][START_REF] Wilkinson | Infliximab as rescue therapy in three cases of paediatric Wegener's granulomatosis[END_REF]. Recently, infliximab was evaluated in addition to classical treatment with cyclophosphamide for GPA patients with similar results when compared to cyclophosphamide treated patients in the first 12 months [START_REF] Morgan | Addition of infliximab to standard therapy for ANCA-associated vasculitis[END_REF].

Targeting the bioactive form of TNF-α, secreted mostly by activated monocytes but also by activated B lymphocytes, is well known and benefits are described in other autoimmune pathologies and especially in RA [START_REF] Atzeni | Immunogenicity and autoimmunity during anti-TNF therapy[END_REF]. However, treatments with TNF-a blockers seem to be associated with increased risk of solid malignancy in patients with AAV (Silva et al., 2011).

Increased levels of BAFF, TACI and IL-21, three major cytokines which participate to B lymphocytes activation, have been found in the serum of patient with active GPA and MPA (Krumbholz et al., 2005). As previously indicated, BAFF seems to increase autoreactive B cells selection and promote the activation and survival of pathogenic B cells [START_REF] Mackay | B-cell stage and context-dependent requirements for survival signals from BAFF and the B-cell receptor[END_REF]Liu and Davidson, 2011). This pathogenic role of BAFF in AAV has been also suggested because of an increase secretion of BAFF by in vitro ANCA-stimulated neutrophils (Holden et al., 2011). Evidences of plasma cell selection by TACI have also been published in rheumatic diseases [START_REF] Faurschou | Anti-B cell antibody therapies for inflammatory rheumatic diseases[END_REF]. Belimumab, a humanized monoclonal antibody targeting BAFF, have been developed several years ago. BAFF-targeting therapies have been successfully tested in patients with SLE [START_REF] Manzi | Effects of belimumab, a B lymphocyte stimulator-specific inhibitor, on disease activity across multiple organ domains in patients with systemic lupus erythematosus: combined results from two phase III trials[END_REF][START_REF] Strand | Improvements in health-related quality of life with belimumab, a B-lymphocyte stimulator-specific inhibitor, in patients with autoantibody-positive systemic lupus erythematosus from the randomised controlled BLISS trials[END_REF] and patients with rheumatoid arthritis [START_REF] Stohl | Efficacy and safety of belimumab in patients with rheumatoid arthritis: a phase II, randomized, double-blind, placebo-controlled, dose-ranging Study[END_REF], with good tolerance profile in both cases. FDA approved belimumab in treatment of active form of SLE in 2011. The BREVAS study was started in 2012 to evaluate belimumab for the maintenance of remission in patients with AAV, but the study will ne be completed because of an inappropriate design.

Based on IL-6 overexpression in patients with AAV (Catanoso et al., 2011), the role of this cytokine was also suggested in AAV pathophysiology. Stimulation of monocytes by anti-PR3 leads to increase secretion of IL-6 and other pro-inflammatory cytokines (Hattar et al., 2002).

Similar results were obtained with endothelial cells stimulated with antibodies from patients with GPA and MPA, leading to an increased secretion of IL-6 and E-selectin (Muller Kobold et al., 1999). IL-6 is also involved in enhancing the maturation of B cells (Kishimoto, 2005a), particularly, in the induction of antibody secretion by B cells [START_REF] Hirano | Purification to homogeneity and characterization of human B-cell differentiation factor (BCDF or BSFp-2)[END_REF][START_REF] Suematsu | IgG1 plasmacytosis in interleukin 6 transgenic mice[END_REF]. Tocilizumab, a humanized monoclonal antibody targeting the IL-6 receptor, have been recently developed and tested effectively in rheumatoid arthritis [START_REF] Maini | Double-blind randomized controlled clinical trial of the interleukin-6 receptor antagonist, tocilizumab, in European patients with rheumatoid arthritis who had an incomplete response to methotrexate[END_REF]. However, tocilizumab is also evaluated in other inflammatory conditions, such as giant cell arteritis, Behçet disease or Takayasu arteritis. Tocilizumab could also be an interesting alternative therapy in AAV.

Bortezomib, a proteasome inhibitor, have demonstrated promising results compared to cyclophosphamide for the treatment of glomerulonephritis in the anti-MPO mice model [START_REF] Bontscho | Myeloperoxidase-specific plasma cell depletion by bortezomib protects from anti-neutrophil cytoplasmic autoantibodies-induced glomerulonephritis[END_REF]. Bortezomib significantly reduced plasma cells in bone marrow of treated mice, notably anti-MPO specific plasma cells, finding that was not observed in cyclophosphamide-treated group. Thus, bortezomib induced normalization of urinal abnormalities, crescent glomerulonephritis and finally neutrophils and macrophages [START_REF] Bontscho | Myeloperoxidase-specific plasma cell depletion by bortezomib protects from anti-neutrophil cytoplasmic autoantibodies-induced glomerulonephritis[END_REF].

C. Systemic sclerosis i) Epidemiology

SSc mostly occurs in females (3 to 8 females for 1 male). Its prevalence varies between 30 and 240 per million inhabitants, being higher in North America and Australia than in Japan (Ranque and Mouthon, 2010) (ANNEXE 1). In Europe, the prevalence varies from 50 to more than 150, with 158 per million in France (Le Guern et al., 2004). Reports of sporadic clusters of higher prevalence suggest the existence of environmental risk factors.

A number of those environmental factors that may contribute to the occurrence of SSc has been investigated. Various activities, including manufacturing and rural activities have been associated with the occurrence of SSc, notably the exposure to silica, dust and/or hydrocarbons (Ranque and [START_REF] Ly | Pathogenesis of giant cell arteritis: More than just an inflammatory condition?[END_REF]. The first reports of the association between silica and SSc were made of limited numbers of patients followed by retrospective comparative studies which confirmed that exposure to silica conferred a high risk to develop SSc (Nietert et al., 1998).

Odds Ratio of 3.93 (1.84-8.54) and 5.57 (1.69-18.37) were calculated in studies conducted in Australia and France, respectively, leading a number of countries including France, Germany, Canada and South Africa to consider SSc as an occupational disease (Englert et al., 2000;Diot et al., 2002). In addition to silica, case control studies pointed out that past exposure to solvents was associated with SSc (Nietert et al., 1998), with discrepancies among studies regarding the types of solvents involved and/or patient gender. Thus, paint thinner or removers, mineral spirits, trichloroethylene, trichloroethane, perchloroethane, gasoline, aliphatic hydrocarbons, halogenated hydrocarbons, benzene, toluene or xylene-solvent shave been proposed as the most at risk solvents, although discrepancies were identified among studies. Nietert et al. found that SSc males were more frequently exposed to organic solvents (in particular trichloroethylene) than controls (OR 2.9 [1.1-7.6]), which was not the case for females (Nietert and Silver, 2000).

In a recent meta-analysis of the literature, the occurrence of SSc was associated with increased ORs for silica, chlorinated solvents, trichloroethylene and welding fumes for male patients, aromatic solvents and ketones for female patients and white spirit for both (Marie et al., 2014).

Several others toxic products including epoxy resins and pesticides, as well as paints, adhesive, dyeing hair, contact lenses and fabric dyes have been investigated in SSc case controls studies but were never found significantly at risk for SSc. Finally, although silicone breast implants were suspected as potentially responsible for SSc induction (Varga et al., 1989), two metaanalysis (Janowsky et al., 2000;Marie et al., 2014) concluded to the absence of association between SSc and breast implants.

In addition, outbreaks of SSc-like disorders have clearly been linked to chemical exposure, such as polyvinyl chloride intoxication (Garabrant et al., 2003) or toxic oil syndrome (Nietert and Silver, 2000).

Several viruses or certain chemicals, such as solvents, may also play a triggering role in the initiation and maintenance of the disease ( SSc. Interestingly, this peptide shows an important homology with UL94, a cytomegalovirus (CMV) late protein that can be recognized by purified IgG from patients (Lunardi et al., 2000).

In vitro, patient antibodies targeting UL94 also seem to induce apoptosis of EC similarly to SSc specific antibodies suggesting a possible role of CMV in the triggering and the maintenance of SSc. Thus, it turns out that a cascade of extrinsic and intrinsic events seems necessary for the induction of the disease (Halenius and Hengel, 2014).

Multiple case series

Many studies have been conducted in recent years in an attempt to identify genetic risk factors in SSc. From three U.S. cohorts, involving 703 families, including 11 multiplex SSc, first degree relatives were identified for an increased susceptibility to develop SSc, with a relative risk close to 13 (10-16 across cohorts), with an recurrence rate of 1.6% versus 0.026 % in the general population (Arnett et al., 2010a). For siblings, the relative risk in siblings (λs) is increased to 15 (10-27 across cohorts). For comparison the risk (λs) varies between 3 and 15 in rheumatoid arthritis (RA) and between 20 and 29 in systemic lupus erythematosus (SLE). Only one study has been performed in twins. Based on the analysis of 42 twin pairs (24 monozygotic) this study demonstrates a poor agreement with the clinical expression of the disease (4.7%) (Feghali-Bostwick et al., 2003). However, there is a higher concordance for the presence of anti-nuclear antibodies (ANA): 40% for dizygotic and 90% for monozygotic. These results suggest that genetic predisposition alone is not sufficient to develop SSc but might influence the autoantibody profile production.

Following the development of molecular biology, many data have been published on association studies. Unfortunately, a number of them were not repeated in large cohorts and/or could not be independently replicated by another group, which is essential for the validation of a genetic association signal. This was particularly the case for genes suspected to be involved in the regulation of fibrosis such as SPARC (encoding the secreted protein acid rich in cysteine), CTGF (encoding connective tissue growth factor), TGFB (encoding TGF-β) or FBN1

(encoding fibrillin 1) (Allanore et al., 2007) or vascular factors including KCNA5 (encoding the potassium voltage-gated channel 5) or uPAR (encoding the urokinase-type plasmogen activator receptor) (Wipff et al., 2008;Manetti et al., 2011). Interestingly, uPAR extinction in a mouse model led to dermal and pulmonary fibrosis together with microvasculopathy (Manetti et al., 2013). In parallel, a decrease of full-length uPAR protein expression in dermal biopsies of patients with SSc as compared to normal skin has been reported, suggesting a protective role of this protein in SSc. In addition, the potential influence of KCNA5 SNP on SSc prevalence remains controversial (Bossini-Castillo et al., 2012).

The tumor necrosis factor ligand superfamily 4 gene (TNFSF4) encodes OX40L (CD252), the ligand of OX40 (CD134). OX40L, involved in antigen presentation in addition of T and B lymphocyte activation, is known as a susceptibility factor for SLE (Manku et al., 2013).

Interestingly, a study performed in 1,059 patients with SSc and 698 controls showed an association between OX40L polymorphism and SSc and the major disease subgroups (Gourh et al., 2010). This result has been confirmed in replication and meta-analysis studies (Bossini-Castillo et al., 2011;Coustet et al., 2012).

Type I interferons (IFN) are key mediators of innate immunity and antimicrobial defense. IRF5

(interferon-regulatory factor 5 encoded by IRF5) is a transcription factor involved in the signaling of toll-like receptors (TLRs) and activation of target genes of interferon. Candidate genes studies have shown its association with the disease and its contribution to pulmonary fibrosis associated with SSc, independently of other risk factors identified (Dieudé et al., 2009a;Ito et al., 2009). These studies permanently identified IRF5 as a susceptibility factor to SSc.

Moreover, recent updates highlighted the major role of IRF5 in the progression and in the occurrence of interstitial lung disease (ILD) in SSc (Sharif et al., 2012;Lindahl et al., 2013).

Toward documented role of IRF5 in SSc patients, recent update have identified in IRF4 gene a common susceptibility locus for SSc and rheumatoid arthritis, reinforcing the interest in IFN regulator factor genes in SSc (López-Isac et al., 2016a).

Signal transducer and activator of transcription 4 (STAT4) is a transcription factor of the STAT family that induces the expression of type I IFN, interleukin (IL)-12 and IL-23, and stimulates the synthesis of IFN-γ and IL-17. Thus, a variant of the gene located in the third intron (rs7574865), without specific biological function, has independently been found to be associated with several autoimmune diseases in two European, one U.S. and a Japanese cohorts (Watford et al., 2004). However, STAT4 is probably not directly responsible for the autoimmune state, since the combination of IRF5 and STAT4 (Dieudé et al., 2009a) variants was associated with an increased risk of developing pulmonary fibrosis in patients with SSc (OR 2.72 [1.86-3.99], p=3.07×10-7 in presence of 3 or 4 at-risk alleles). In that study, STAT4 was found to be the second outside association major histocompatibility complex (MHC) signal (rs3821236 p=3.37×10-9, OR 1.30 [1.18-1.44]). Interestingly, Stat4 -/-mice exposed to bleomycin developed reduced dermal fibrosis as compared to controls, as assessed by a 65 % decrease of dermis thickness, 68±5% decrease of hydroxyproline content and 71±6% decrease of myofibroblast numbers (Avouac et al., 2011). In addition, leukocyte infiltration was also reduced (63±5%) in skin lesions, as well as the pro-inflammatory cytokines production including TNF-α, IL-2 and IL-6. Thus, STAT4, which plays a major role in an inflammatory model of fibrosis, might represent a potential therapeutic target in SSc.

Two genes, PTPN22, encoding the protein tyrosine phosphatase called Lyp, and CSK encoding the c-src tyrosine kinase also called CSK, which modulates signaling through the T cell receptor (TCR), have been found to increase susceptibility to SSc. PTPN22 was one of the first susceptibility genes associated with several autoimmune diseases. Its main variant (rs2476601, 1858C>T, R620W) has been reported to be modestly associated with SSc (Gourh et al., 2006;Diaz-Gallo et al., 2011), particularly in patients with anti-centromere autoantibodies; however this association is attenuated by the fact that a significant proportion of patients with SSc also develop autoimmune thyroiditis, which is associated with polymorphisms of PTPN22.

Furthermore, the genome wide association studies (GWAS) identified an association of a CSK variant (rs1378942) with SSc frequency which was unrelated to auto-antibody specificities (p=5.04 x 10-12; OR=1.20) (Martin et al., 2012). Recent study has also identified the TYK2 gene as a potential susceptibility gene in SSc through IL-12 pathway (López-Isac et al., 2016b) Interestingly, in the 2 GWAS performed, CD247 (rs2056626; OR 0.86 [0.81 to 0.90] p=2.09×10-7), which encodes a subunit of the TCRζ (component of TCR/CD3 complex), was significantly associated with a risk of developing SSc (Radstake et al., 2010;Dieudé et al., 2011).

Several studies confirmed the major role of B cells in SSc. Thus, two variants (rs10516487 and rs3733197) of B cell-specific scaffold protein with ankyrin repeats (BANK1) gene, a promoter of the tyrosine kinase LYN phosphorylation, were found to be associated with dSSc (Dieudé et al., 2009b;Rueda et al., 2010), whereas another B lymphocytes specific gene, BLK (B lymphocyte kinase), which transduces the signal downstream of the BCR and is also involved in SLE (C8orf13-BLK region, rs13277113 and rs2736340) was associated with subgroups of lSSc and anti-centromere positive patients (Ito et al., 2010;Coustet et al., 2011).

The variant of TNFAIP3 (6q23) was also identified through the GWAS as a susceptibility factor for autoimmune diseases (Dieudé et al., 2010). TNFAIP3 is an intracellular protein that regulates negatively the NF-κB signaling pathway (by deubiquitination) downstream to the (Gilchrist et al., 2001).

Genetics might help to identify new biomarkers that could allow better stratification of patients at risk of developing visceral manifestations and offering them targeted therapies before the occurrence of severe organ involvement. However, identified OR are often weak even if they combine several gene polymorphisms. Thus, new genetic approaches might probably be helpful in the near future to go further in this direction.

ii) Pathophysiology

Collagen accumulation in the dermis is one of the hallmarks of SSc. Extra-cellular matrix (ECM) accumulation is mostly the consequence of fibroblasts activation. In vitro, fibroblasts isolated from the involved skin of patient with SSc produce more collagen IV, proteoglycan and fibronectin than those obtained from the skin of healthy controls. Phenotypical changes of these fibroblasts have also been described, with the expression of alpha-smooth muscle actin (α-SMA), a marker of differentiation toward myofibroblast (Krieg et al., 2007). These myofibroblasts are known to secrete more collagen and display vascular smooth muscle cell (SMVC) properties including migration phenotype. Distinctly from EC, which experience increased apoptosis, fibroblasts isolated from SSc patients have a defect in Fas/FasL induced apoptosis pathway which probably participates to the maintenance of the disease process (Santiago et al., 2001). Interestingly, a defect in metalloproteinase synthesis, which regulates formation of ECM, has also been reported (van der Slot et al., 2003).

In addition to intrinsic phenotype modifications of fibroblasts, extrinsic factors also contribute to explain excessive fibroblast activation in patients with SSc (Figure 10).

Transforming growth factor β (TGF-β) has been proposed as an utmost important factor in fibroblast activation (Denton and Abraham, 2001). Thus, fibroblasts phenotype modification in patients with SSc could be easily reproduced with incubation of normal fibroblasts with TGFβ (Smith and LeRoy, 1990). As in TGF-β-dependent experimental models, in patients with SSc, the expression of TGF-β receptors at the fibroblast surface has been found increased as compared to healthy control fibroblasts (Kawakami et al., 1998). This result, together with the observation of increased levels of endogenous TGF-β in lung and dermal SSc lesions suggests an over stimulation of this pathway, leading to fibroblast phenotypical modifications including over proliferation, ECM production and differentiation into myofibroblasts. In addition, the expression of endogenous inhibitors of TGF-β pathway, including Fli-1 and SMAD7, is also defective in SSc patient's fibroblasts (Chen et al., 1999;Bhattacharyya et al., 2005). However, the persistence of phenotypical modifications of fibroblasts from patients with SSc despite multiple passages, in vitro, and recent updates on epigenetic and DNA methylation mechanisms, suggest the existence of other mechanisms independent of TGF-β stimulation (Varga and Abraham, 2007).

In addition to TGF-β, IL-4, a cytokine secreted by T helper 2 (Th2) cells has been identified as a key cytokine in SSc. IL-4 express pro-fibrotic properties on fibroblasts and increased IL-4 production has been documented in the spleen of ROS-induced SSc mouse model as well as in the serum of patients with SSc (Postlethwaite et al., 1992) T lymphocytes that present an activated phenotype and expression of mRNA encoding IL-4

and latent TGF-β mRNA (Luzina et al., 2003). Another important local source of IL-4 could also be a subset of double positive CD4/CD8 T lymphocytes, present in dermal lesions of patients with SSc (Parel et al., 2007). In addition to IL-4, other growth factors have been identified in patients with SSc, including connective tissue growth factor (CTGF) or PDGF (Leask and Abraham, 2006).

Alternatively to these growth factors, autoantibodies may activate fibroblasts. Anti-fibroblast antibodies (AFA) are not specific to patients with SSc and could lead to fibroblasts activation by direct targeting surface adhesion molecules. Two studies notably reported that exposure of normal fibroblasts to AFA leads to the internalization of the antibody that could interact with caveolin pathway and induce the secretion of pro-fibrotic chemokines including CCL2, CXCL1, CXCL8, CKLF and ECGF1 (Chizzolini et al., 2002;Fineschi et al., 2008).

AFA directed at the PDGF receptor have been detected in patients with SSc. These autoantibodies have been reported to enhance in vitro the activation and pro-fibrotic phenotype of isolated fibroblast (Chizzolini et al., 2002;Svegliati et al., 2005). Unfortunately, these results remain controversial (Classen et al., 2009).

In patients with SSc, arterial and arteriolar hyperreactivity and myointimal proliferation can lead to vascular occlusion occurring in digital arteries, as well as in heart, lung and kidney arteries. The equilibrium of vascular endothelium in physiological conditions results from the balance between vasodilator and vasoconstrictor molecules, as well as the expression of adhesion molecules at the surface of EC. In patients with SSc, the vascular tone is dysregulated, leading to perturbed interactions between EC, vascular smooth muscle cells (VSMC) and ECM components participating in vascular remodeling and occlusion (Bhattacharyya et al., 2012). In patients with SSc, EC lesions are associated with increased plasma levels of factor VIII, decreased angiotensin converting enzyme production, increased activity of von Willebrand factor (vWf) and detection of circulating platelet aggregates (Kahaleh et al., 1981). At an early stage of the disease, EC involvement in SSc is characterized by increased apoptosis, loss of physiological barrier with permeabilisation of blood vessels and abnormal vascular tone regulation.

It has been proposed that EC activation and proliferation might occur as a consequence of the binding of anti-endothelial cell antibodies (AECA). Thus, in patients with SSc, AECA have been reported to induce EC apoptosis through antibody dependent cell cytotoxicity, involving NK cells and Fas/FasL pathway. However, although of interest, these in vitro data do not preclude the pathogenic role of these antibodies in vivo. In addition, AECA are not specific for SSc and can be found in various auto-immune diseases, binding to a relatively large number of EC antigens (Bordron et al., 1998). Finally, AECA failed to be identified in a number of experimental models of SSc including the Tight skin (Tsk)-1 mouse model, a genetic model of SSc. In the same line, the prevalence of antiphospholipid syndrome is very low in SSc, although anti-cardiolopin antibodies and increased vWf activity have been associated with the detection of PAH, suggesting that anti-cardiolipin antibodies could represent a marker of EC damage in PAH associated with SSc (Liberati et al., 2010).

Other mechanisms are involved in the perturbation of the vascular tone in patients with SSc.

Thus, increased endothelin-1 (ET-1) levels, which are known to enhance vasoconstriction, inflammation, and mediate fibrosis and vascular remodeling, have been detected in the serum of SSc patients (Kahaleh, 1991). ET-1 is known to also activate fibroblasts and induce in vitro ECM production (Marsault et al., 1992). In patients with SSc, ET-1 levels have been shown to correlate with the severity of PAH. Moreover, ET-1 has been found to be increased in the lung at the mRNA and protein levels in patients with idiopathic PAH as compared to healthy controls. In kidney biopsies from SSc patients who experienced scleroderma renal crisis, immunohistochemistry staining demonstrated the presence of ET-1 in glomeruli, arterioles and arcuate arteries, which was not the case in other vascular diseases involving the kidney [START_REF] O'callaghan | Pulmonary veno-occlusive disease: the bête noire of pulmonary hypertension in connective tissue diseases[END_REF]. Interestingly, ET-1 receptors (ETR) inhibitors, including bosentan, ambrisentan and more recently macitentan, have demonstrated efficacy in the treatment of idiopathic PAH and PAH associated with SSc (Kovacs et al., 2012). In addition, defective prostacyclin synthesis and perturbed NO synthesis have also been reported to a possible key elements to explain EC dysfunction in patients with SSc (Schachna and Wigley, 2002).

Finally, vascular epidermal growth factor (VEGF) levels have been reported to be decreased in patients with SSc as compared to healthy control (Distler et al., 2004). Increased synthesis of MCP-1 and VCAM-1 by EC could also increase the recruitment of lymphocytes and promote the local and systemic inflammation in patients with SSc (Denton et al., 1998). Moreover, common classification allow clinicians to discriminate between: limited cutaneous SSc (lcSSc) defined by skin thickening in areas solely below to the elbows and knees, with or without facial involvement and diffuse SSc (dSSc) defined by the presence of skin thickening proximal, as well as distal, to the elbows and knees, with or without facial or truncal involvement (LeRoy et al., 1988).

Both subtypes of SSc evolved differentially as patients with dSSc present rapid and dramatic succession of severe symptoms (pulmonary fibrosis, renal involvement) and increased mRSS at the early stage of the disease with a decrease of mRSS and improvement of patients after this early stage. On the other hand, lcSSc patients present a really progressive disease without a "peak" of disease activity. However, patients with lcSSc also showed severe presentations of the disease usually a decade after SSc diagnosis. These autoantibodies argue for a pathogenic role of B cells (Kraaij and van Laar, 2008). Thus, circulating B cells from SSc patients differ in their phenotype as compared to healthy controls, with increased proportions of naïve B cells and decreased numbers of memory B cells and plasma cells (Sato et al., 2001). CD19 and CD21, two activation co-receptors of the B-cell receptor (BCR), are overexpressed in these naïve and memory B cells. The activation receptors CD80, CD86 and CD95 are upregulated on memory B cells, suggesting their participation to the pathogenetic process.

High levels of B cell activating factor (BAFF) have been measured in the serum of patients with SSc, together with an overexpression of BAFF-R at the surface of peripheral B cells from SSc patients (Hasegawa et al., 2006). BAFF activates the NF-κB pathway, promotes B cells survival and participates to the differentiation of autoreactive B cells. In addition, it has been documented that B cells infiltrate the dermis of SSc patients (Sunderkötter and Riemekasten, 2006;Chizzolini, 2008), that circulating levels of IL-6 are increased in SSc patients as compared to healthy controls and correlated with the extent of skin fibrosis. In Tsk-1 mouse, the depletion of B cells leads to a decrease of total IL-6 mRNA and improvement of fibrotic lesions (Sato et al., 2001). Finally, IL-6 is known to stimulate collagen secretion by fibroblasts and represent a potential "B cell link" to fibroblast activation.

T cells are also involved in the pathogenesis of SSc. Thus, a Th2 bias has been documented in SSc patients (Chizzolini, 2008). Increased levels of IL-4 and IL-13 have been measured in the serum of SSc patient. These two cytokines have been classically described as synergic Th2 polarization cytokines via STAT-6. Although the trigger contributing to the initiation of secretion of these cytokines is not identified, it has been documented that mature and activated Th2 secrete IL-4, IL-6 and IL-13, enhancing a positive loop of Th2 polarization and B cells stimulation. Interestingly enough, the treatment of Tsk-1 mice with an anti-IL-4 monoclonal antibody prevented the induction of skin lesions (Ong et al., 1998), underlining the major role this cytokine in SSc physiopathology.

In addition to B and T lymphocytes, dendritic cells appear of utmost importance in SSc. Thus, a recent proteome-wide analysis of the culture supernatant of plasmacytoid dendritic cells (pDC) isolated from patients with SSc allowed identification of increased levels of CXCL4, with higher levels in diffuse than in limited forms, and increased levels in early diffuse vs late diffuse SSc patients (van Bon et al., 2014). Interestingly, CXCL4 was also detected in the skin of SSc patients and not in healthy control skin. Serum levels of CXCL4 correlated with clinical status of SSc patients. Notably, in patients with lung fibrosis and/or PAH, high levels of CXCL4 correlated with a poor prognosis. Finally, delivery of CXCL4 leads to increased leukocytes infiltration, skin thickening and CCL2 mRNA expression in the bleomycin-induced model of SSc. Taken together, these observations argue for a possible role of CXCL4 in the pathogenesis of SSc. Further studies will aim at confirming the interest of targeting CXCL4 in SSc patients.

iv) Prognosis

More than half patients with systemic sclerosis finally die from the disease (Denton, 2015).

However, systemic sclerosis is one of the most heterogeneous disease with internal organ manifestations occurring in both dSSc and lcSSc while the kinetic of the two subforms is quite different. Thus, dSSc patients present a dramatic decrease of overall survival at the early stage of the disease with increased occurrence of interstitial lung diseases and renal crisis compared to patients with lcSSc. In contrast, patients with lcSSc will finally show pulmonary arterial hypertension and renal complications. Thus, prognosis of patients with SSc is highly correlated to clinical outcomes. For instance, patients developing renal crisis (5 to 10% of all) have a mortality of 30 to 40% while patients presenting associated PAH have a median survival inferior to 2 years (Denton et al., 2009;Le Pavec et al., 2010).

v) Treatment

Several approaches targeting respectively fibroblast activation pathway, endothelial cell activation and immune system have been tested in patients with SSc.

PDGF can be targeted by an inhibitor of the PDGF receptor, imatinib, a serine-proteine kinase receptor inhibitor, that has been developed in the treatment of chronic myeloid leukemia (Barst, 2005;Ghofrani et al., 2005). Imatinib binds to c-Abl, a signaling protein downstream of the TGF-β receptor, and blocks both TGF-β and PDGF receptor. Because of promising results in the bleomycin induced model of SSc (Aono et al., 2005), clinical trials investigated the efficacy of imatinib in patient with SSc (Sabnani et al., 2009;Spiera et al., 2011). However, the results

were disappointing in terms of tolerance, with a large proportion of dropouts and poor efficacy (Mouthon, 2012).

Immune suppression using cyclophosphamide is also classically used in patients with active systemic sclerosis. However, novel therapeutic strategies have been proposed in experimental models of SSc and fibroblasts from patients with SSc in culture including inhibitors of Src kinase family, Rho associated kinases, Fos related antigen-2 (Fra2) or histone acetylases and DNA methyl transferases (Bagnato et al., 2013;Maurer et al., 2013). Several of these strategies will be investigated in the treatment of patients with SSc in the near future.

Direct targeting of TGF-β in SSC, using a monoclonal antibody anti-TGF-β (Frexolimumab) has been recently evaluated in a limited number of patients (Rice et al., 2015). Improvement of skin fibrosis, decreased myofibroblast infiltration together with decreased SERPINE1 and CTGF in the skin of treated patients suggested promising results of this therapeutic. However, the importance of the TGF-β in both immune and metabolic systems do not allow a larger clinical trial.

Taken together, targeting B cells (anti-CD20), IL-6, IL-4 and also T cells and mature B lymphocytes (anti-CD52) may leads to substantial clinical improvement in mouse models and in open series of patient with SSc (Isaacs et al., 1996;Ong et al., 1998;Stratton et al., 2001;Khanna et al., 2016). These findings argue for the important role of B and T cells in the pathogenesis of SSc.

Toward targeted therapies, autologous hematopoietic stem cell transplantation have been recently evaluated in patients with diffuse systemic sclerosis (Laar et al., 2014). Promising results have been published in a limited number of cases with important benefits for patients compared to cyclophosphamides treatment. However, the important issue of bone marrow transplantation impact significantly the development of this treatment in SSc.

D. Pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) constitutes a heterogeneous group of clinical entities sharing similar pathologies that have been subcategorized as idiopathic pulmonary arterial hypertension (IPAH), familial PAH, pulmonary hypertension (PH) associated with other diseases such as connective tissue diseases, (for instance systemic sclerosis), portopulmonary hypertension, and PH related to human immunodeficiency virus (HIV) infection, drugs, and toxins (Simonneau et al., 2004).

All subsets of patients received specific treatments regarding the origin of the disease. For instance, blood vessels dilatators, endothelin receptor antagonists and high dose calcium blockers are particularly used. However, if medical treatment is not efficient, an open-heart surgery might be an option for an atrial septostomy or, in several cases, a lung or heart-lung transplantation can be used.

Regarding inflammation processes, patients with PAH also present increased activation of both vascular endothelial cells and fibroblasts (Tuder et al., 1994). Similar secretion of collagen as observed in SSc was described in patients with connective tissue disease associated with PAH (Dorfmüller et al., 2007). Furthermore, increased releases of PDGF and EGF were also detected which participate to disease progression and severity (Humbert et al., 1998;Schermuly et al., 2005). Finally, endothelin pathway is critical in patients with PAH as it works as a powerful vasoconstrictor and mitogen for smooth muscle cells which mediates the vasoconstriction of pulmonary artery (Ehrenreich et al., 1990;Galié et al., 2004).

In a subset of patients, particularly those with IPAH, PAH associated with connective tissue diseases or HIV infection, inflammation plays a pivotal role in the development and maintenance of the vascular remodelling. Thus, several autoantibodies, together with increased levels of pro-inflammatory cytokines have been detected in serum from patients with IPAH and connective tissue-PAH (Isern et al., 1992;Humbert et al., 1995;Le Pavec et al., 2010).

Moreover, innate immune cells also secrete higher levels of chemokines such as CX3CL1 and RANTES which could participate in the recruitment of activated B and T cells (Balabanian et al., 2002;Dorfmüller et al., 2002). However, only 5% of the population presents with an autoimmune pathology, which demonstrate the existence of regulatory mechanisms to eliminate or control autoreactive B cells production. Thus, one of the previously described checkpoints, has been bypassed inducing a rupture of tolerance.

In the bone marrow, mice deficient for the Bcl-2 interacting mediator of cell death (BIM)

produce spontaneously increased levels of anti-dsDNA in a SLE-like model (Enders et al., 2003). Indeed, BIM is engaged during the V(D)J recombination process and allows the elimination of cells with higher affinity for auto-antigens present at the central system.

In the germinal center, an absence of Fas/FasL signalling is associated with increased autoimmune symptoms in both human and mouse suggesting that this pathway is highly implicated in the elimination of autoreactive clones upon somatic hypermutation process (Wang et al., 1996;Strasser et al., 2009). Moreover, dysregulation of Tfh cells might also be associated with loss of tolerance and increased activation of Tfh has been observed in patients with SLE (Craft, 2012) (Sawaf et al., 2016). Thus, the Roquin san/san mouse model has shown that excess of activated Tfh induces the formation of spontaneous germinal center and SLE-like syndrom with production of anti-DNA antibodies together with a glomerulonephritis and immune complex deposits (Linterman et al., 2009). Also, at the germinal center level, aberrant activation of AID has been found in patients with autoantibodies suggesting an important role of this enzyme in the control of normal germinal center response and humoral immunity (Zaheen and Martin, 2011).

The B-cell activation factor (BAFF) and A proliferation-inducing ligand (APRIL) are two pro-B cells cytokines that play critical role in several models of SLE and notably the NZB/NZW F1 and the MRL/lpr mice models. This specific point will be discussed bellow. If the presence of auto-reactive B cells is a key event in the manifestation of autoimmune diseases, it is not the exclusive event. Indeed, a major part of the auto-antibodies is not pathogenic such as the anti-SSA in the Sjogren syndrom. However, several antibodies, like anti-platelets in immune thrombocytopenic purpura or anti-dsDNA in SLE or the ANCA in ANCA associated vasculitis, are clearly linked to disease pathogenesis and progression. These differences suggest a differential interplay between B-cells and their environment regarding the specificity of the autoantigens and the immunological context. This could be explained by both in situ differentiation and by the antigens specificity.

Tertiary lymphoid structure

Autoreactive plasmocytes producing autoantibodies are localized in both bone marrow or in secondary lymphoid organs like the spleen but also in tertiary lymphoid structures (TLS).

In systemic autoimmune diseases, such as rheumatoid arthritis and Sjögren's syndrome, target tissue infiltration by B cells correlates with disease activity and progression (Guellec et al., 2015;Mo et al., 2011). In GPA patients, B cells infiltrating granulomatous lesions might be activated. Voswinkel and colleagues performed the first BCR sequencing for biopsies of endonasal lesions of active GPA patients (Voswinkel et al., 2008). These authors and others suggested an in situ activation and selection of B cells in contact with PR3 at the surface of neutrophils. In another study, a correlation was found between the importance of B cell infiltrates within granulomatous lesions in GPA and the occurrence of relapses, in spite of the peripheral B cell depletion in some patients treated with rituximab (Ferraro et al., 2008).

Interestingly, in a cohort of patients with AAV, including GPA and MPA patients, TLS have been identified in the tissues involved (Steinmetz et al., 2008); notably, in glomerulonephritis lesions. They were composed of a germinal center containing follicular dendritic cells and memory and non-memory B cells at the periphery of the germinal center, in contact with T cells.

CXCL13, a B cell specific chemokine, was detected in TLS and CXCR5, the CXCL13 receptor, was expressed by infiltrated B lymphocytes (Mueller et al., 2014). Similar results were found regarding CCL21, expressed by perivascular stromal cells, and CCR7, expressed by B and T lymphocytes infiltrating ectopic lymphoid structure (Mueller et al., 2014). .Zhao et al. also investigated the expression of CCR10 at the surface of circulating and tissue infiltrating B cells.

However, they found no difference of expression, suggesting a chemokine specific mechanism for the migration of B cells within nasal granulomatous lesions in GPA.

Autoantigens

In SLE, autoreactive plasmocytes have been observed in bone marrow, spleen and kidney (Hiepe and Radbruch, 2016). Moreover, specific anti-dsDNA plasmocytes have been found

preferentially in the kidney of NZB/W mice model of SLE (Hoyer et al., 2004;Hutloff et al., 2004). Furthermore, the level of IgG anti-dsDNA is positively correlated with the amount of plasmocytes in kidney of these mice and disease severity suggesting a critical role for these plasmocytes in situ. These results also suggest a possible differentiation directly in the inflamed tissue under propitious microenvironment.

These data are supported by the previous detection of increased levels of BAFF in the serum of active GPA patients as compared to healthy donors (Krumbholz et al., 2005). Detection of Bcell Activating Factor of the Tumor Necrosis Factor (TNF) Family (BAFF) has also been reported in endonasal lesion of GPA patients. These results support the potential activation of B cells both in peripheral blood and tissue lesions, possibly through BAFF-BAFF-R interaction resulting in increased survival and activation of B cells and emergence of autoreactive B-cells.

Morevover, ANCA activated neutrophils secrete increased amounts of BAFF in vitro, suggesting a potential crosstalk between neutrophils and B lymphocytes in patients (Holden et al., 2011a).

Soluble factors associated with B cell activation i) Multiple roles of Interleukin-6 in inflammation

Interleukin (IL)-6, one of the first cytokines identified, has recently been recognized as a potential actor in inflammatory diseases [START_REF] Rincon | Interleukin-6: from an inflammatory marker to a target for inflammatory diseases[END_REF][START_REF] Tanaka | Targeting Interleukin-6: All the Way to Treat Autoimmune and Inflammatory Diseases[END_REF]. This cytokine originally described as a marker of inflammation is now considered as an essential agent controlling inflammation.

IL-6, initially designated as a B cell differentiation factor, is representative of the redundancy and pleiotropic activities of cytokines, particularly in inflammation [START_REF] Hirano | Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin[END_REF]. First reports have shown that IL-6 is produced by monocytes and macrophages immediately after stimulation of Toll-like receptors (TLRs) with distinct pathogen-associated molecular patterns (PAMPs), in the early phase of infection-linked inflammation [START_REF] Kishimoto | Interleukin-6: from basic science to medicine--40 years in immunology[END_REF]. Then, IL-6 was found to be produced by a number of immune and non-immune effector cells, including T and B cells, fibroblasts, keratinocytes, mesangial, glial, endothelial cells as well as many tumour cells (Figure 17). Moreover, IL-6 can induce strong release of a broad spectrum of acuteinflammation proteins, such as C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen or hepcidin [START_REF] Heinrich | Interleukin-6 and the acute phase response[END_REF]. In addition to this stress and the transient acute response to infection or aggression of the host, IL-6 plays a pivotal and deleterious role in the development of chronic inflammation diseases [START_REF] Akira | Interleukin-6 in biology and medicine[END_REF][START_REF] Kishimoto | Interleukin-6: from basic science to medicine--40 years in immunology[END_REF]. Excessive or persistent production of IL-6 is clearly associated with the development of arthritis, Castleman disease or hyper γ-globulinemia [START_REF] Yoshizaki | Pathogenic significance of interleukin-6 (IL-6/BSF-2) in Castleman's disease[END_REF][START_REF] Hirano | Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin[END_REF][START_REF] Ohshima | Interleukin 6 plays a key role in the development of antigen-induced arthritis[END_REF]. Nowadays, numerous IL-6 impacts on immune cells have been reported demonstrating the broad spectrum of modifications that could be induced by IL-6 dysregulation (Table 2) A major role of IL-6 was demonstrated in the T cell polarization toward Th9 or Th17 depending on the cytokines present in the microenvironment (Omenetti and Pizarro, 2015;Schütze et al., 2016). Furthermore, IL-6 inhibited the polarization of T cells into Treg, bypassing the regulation by TGF-β and resulting in a loss of immunoregulation. IL-6 production has also been associated to the microenvironment remodelling with an increase of matrix production by fibroblast together with an increase proliferation and migration, particularly during chronic inflammation (Figure 18). Endothelial cells also can be stimulated by IL-6secretion resulting in an increased VEGF release which induces hyper-vascular activation in situ. The B-cell activation factor (BAFF) and A proliferation-inducing ligand (APRIL), are two member of the TNF cytokine family. These cytokines are commonly secreted by neutrophil, dendritic cells and other innate immune cells (Scapini et al., 2008). The first function of BAFF and APRIL is to induce the survival of B lymphocytes during the maturation process of B cells through BAFF-R and TACI, respectively (Schneider, 2005). Moreover, several studies have demonstrated the role of BAFF and APRIL in the induction of antibody production at the late stage of differentiation by B lymphocytes (Matsushita and Sato, 2005;Bosello et al., 2007). Also, stimulation by these two cytokines triggers increased secretion of pro-inflammatory cytokines by B cells together with decreased apoptosis level (Mackay and Schneider, 2009).

Since a number of autoimmune diseases presents with a wide range of auto-antibodies and dysregulation of B lymphocytes, many studies have addressed the role of BAFF and APRIL in autoimmunity.

In SLE, many studies have evaluated BAFF and APRIL roles in the stimulation of B cells and the induction of autoantibody secretion (Groom et al., 2007;Treml et al., 2007). High serum levels of BAFF preferentially support the survival of B cells and induce class switching of autoreactive cells that recognize nucleic acids (Stohl et al., 2003;Groom et al., 2007).

Moreover, basophils, cells with an emergent role in SLE, have been described as a potential important booster of lupus through the secretion of IL-4 and BAFF (Davidson and Diamond, 2010). Thus, two phase III clinical trials have been conducted in 2010-2011 and showed that addition of anti-BAFF monoclonal antibody (belimumab), results in better outcome for patients with moderately active SLE (Merrill et al., 2010;Navarra et al., 2011). These results led to the US Food and Drug Administration (FDA) approval for belimumab in SLE.

In AAV, increased production of BAFF has been reported in neutrophils from patients with GPA, which could contribute to the maturation of B cells particularly in situ where, in close contact with neutrophils, B cells could be educated to link the PR3 (Scapini et al., 2008).

B cell signalling abnormalities in autoimmunity

Following BCR stimulation, SRC family kinases are activated, leading to the phosphorylation of immune receptor tyrosine activation motifs (ITAMs) of the CD79ab (Ig-α, Ig-β) complex (Vargas et al., 2013). Similarly, PI3K is activated to catalyse the conversion of membraneassociated PIP2 to PIP3 leading to BTK recruitment to the plasma membrane through the interaction of its PH domain with PIP3 (Tomlinson et al., 2004). Concomitantly, the phosphorylation of Ig-α and Ig-β ITAMs leads to the recruitment of SYK kinase via SYK SH2 domains. Following activation, BTK initiates multiple downstream pathways generating pleiotropic effects: PLCγ activation, generation of second messengers, such as inositoltriphosphate (IP3), diacylglycerol (DAG) and calcium flux, cell proliferation, differentiation, apoptosis or survival (Reth and Brummer, 2004).

To date, almost only B cell-derived tumour cells have been treated with the newly developed inhibitor of BTK, ibrutinib. The rationale for this, is that lymphoma cells, similarly to nontransformed B cells, may be dependent on BCR signalling for their survival. Although animal models of autoimmunity indicate very promising outcomes after ibrutinib treatment, the drug

has not yet been used in important clinical trials in human (Chang et al., 2011). However, one study has shown promising results for ibrutinib in Waldenström's macroglobulinemia (Treon et al., 2015).

Several other studies have described signalling abnormalities of various kinases such as increased activation of SYK in SLE, rheumatoid arthritis or immune thrombocytopenic purpura (Iwata et al., 2015a(Iwata et al., , 2015b)). Interestingly, fostamatinib, an inhibitor of SYK has also been effective in a phase III clinical trial with active rheumatoid arthritis patients showing an improvement of ACR20 response over placebo (Weinblatt et al., 2014).

C. B lymphocytes: pro-inflammatory cells? Pro-inflammatory secretions of B lymphocytes

In autoimmune conditions, B cells can produce IL-6, IL-21 and TNF-α, notably under the influence of type I interferon (IFN-α and IFN-) or TLR ligands (Rieckmann et al., 1997;Yehudai et al., 2012). These cytokines exert deleterious effects within inflammatory lesions, especially in rheumatoid arthritis for which patients have large improvement upon anti-TNF-α blocking agents. Interestingly, IL-6 and TNF-α have been reported as increased in the serum of patients with GPA and MPA (Muller Kobold et al., 1999;Jónasdóttir et al., 2001).

In AAV, TNF-α is known to increase the expression of PR3 at the surface of neutrophils (Csernok et al., 1994). Thus, Reumaux et al. reported the increased binding of ANCA to PR3 expressed on neutrophil surface after priming with TNF-α (Reumaux et al., 2006). Interestingly, triggering by TNF-α also activates NADPH oxydase in neutrophils, suggesting a possible amplification of the immune response against neutrophil and maintenance of inflammation in AAV patients (Kettritz et al., 2002). Two open studies suggested a beneficial effect of infliximab, a chimeric monoclonal anti-TNF-α antibody in GPA patients refractory to cyclophosphamide (Bartolucci et al., 2002;Booth et al., 2004). However, this finding was not confirmed in the maintenance of remission in a prospective randomized study using etanercept, a soluble dimerized TNF-α receptor (Silva et al., 2011).

B-cell depletion therapy using rituximab demonstrated decreased IL-6 and TNF-α serum levels in rituximab-treated patients compared to glucocorticoid-treated patients in AAV (Monach et al., 2013), suggesting that B cells could be an important source of pro-inflammatory cytokines in AAV. However, evidences are still lacking whether these inflammatory cytokines are directly produced by B cells.

Regulatory B cells

Following the peak of inflammation, the hallmark of an effective immune response is the recruitment or the expansion of immune cells with immunosuppressive functions that leads to a correct resolution of inflammation. If unresolved, this inflammatory response causes injury to the host tissues leading to the development of autoimmune pathologies (Medzhitov, 2008).

In order to keep a healthy balance, the immune system needs to control the activity of these numerous components in a very precise way. A first report on impairment of T cell response by regulatory B cells (Bregs) was described in 1974 where a delayed hypersensitivity response was observed (Katz et al., 1974). However, only the study on absence of B cells in experimental autoimmune encephalomyelitis (EAE) mouse model in 1996 has proved the necessity of B cells to negatively regulate hyperactivation of the immune system, responsible for chronic diseases (Wolf et al., 1996). Following this report, various teams have identified what is now characterized as Bregs, a heterogeneous population of B lymphocytes that suppress effector T cell responses (Rosser and Mauri, 2015).

The absence of a common phenotype for Bregs led to multiple debates in the scientific community with a lack of reproducible data in all inflammatory models or diseases. This lack of constant phenotype focused new researches on the immunosuppressive mechanisms of these cells. Table 3.

Unfortunately, phenotypes of mouse and human Bregs are still not fully defined and very heterogeneous regarding the pathological background or the tissue. Blair et al. reported the identification of CD24 hi CD38 hi B cells with regulatory functions and IL-10 production in SLE patients (Blair et al., 2010), this phenotype was also found in lymphoma patients and in artherosclerosis (Ait-Oufella et al., 2010). Furthermore, in mice, other markers have also been associated with Bregs phenotype, such as upregulation of CD1d, CD5 or CD22 (Yanaba et al., 2008).

Multiple studies have demonstrated the ability of Bregs to produce and secrete IL-10 that leads to suppress inflammatory responses. In systemic sclerosis (SSc) patients, recent studies have also highlighted the potential pathogenic role of B lymphocytes that can activate fibroblasts by increasing collagen production (François et al., 2013). Up to now, the deleterious effect of B lymphocytes from patients with SSc has not been has not been. Moreover, one could hypothesize that B lymphocytes might be differentially activated depending on the heterogeneity of the disease and the severity of clinical manifestation as pulmonary arterial hypertension (PAH) or interstitial lung disease.

Therefore, the aim of this study was to provide comprehensive and comparative analysis of both phenotype and function of features B lymphocytes in three diseases characterized by an important inflammation and vascular remodelling: ANCA associated vasculitis, systemic sclerosis and pulmonary arterial hypertension associated with systemic sclerosis. 2,3,4,5, *, Jonathan London 1,2,3,6, *, Sébastien Lofek 1,2,3 , Jérémie Dion 1,2,3,6 , Benjamin Chaigne 1,2,3,6 , Julie Mocek 1,2,3 , Véronique Witko-Sarsat 1,2,3,4 , Pascal Cohen 6 , Claire Le Jeunne 6 , Nadine-Varin-Blank 4,7,8 , Loïc Guillevin 6 , Benjamin Terrier

RESULTS

Results

Conclusion:

Skewed T-cell polarization toward Th2, Th9 and Th17 responses characterizes GPA, whereas B cell populations are dysregulated in both GPA and MPA with an activated phenotype and decreased BAFF-R expression. Finally, inflammatory B cells producing IL-6 are dramatically increased in GPA providing an additional mechanism by which rituximab could be effective.

Introduction

Primary systemic vasculitis constitute a heterogeneous group of rare diseases characterized by inflammation of blood vessels and classified according to the size of involved vessels [START_REF] Jennette | revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides[END_REF][START_REF] Millet | Antineutrophil cytoplasmic antibodyassociated vasculitides: is it time to split up the group?[END_REF].

Anti-neutrophil cytoplasm antibodies (ANCA)-associated vasculitis (AAV) are pauciimmune necrotizing vasculitis involving small-sized vessels which include granulomatosis with polyangiitis (GPA), eosinophilic granulomatosis with polyangiitis (EGPA) and microscopic polyangiitis (MPA) [3]. The immunofluorescence pattern of ANCA distinguishes between cytoplasmic ANCA (c-ANCA), targeting proteinase 3 (PR3) in most of the cases, and perinuclear ANCA (p-ANCA), usually targeting myeloperoxidase (MPO).

Major advances have been done during the last fifteen years in the understanding of the pathophysiology of the disease. In vitro studies have demonstrated induction of respiratory burst in TNF-α primed neutrophils and monocytes after incubation with either PR3or MPO-ANCA [START_REF] Brooks | IL-1 beta production by human polymorphonuclear leucocytes stimulated by anti-neutrophil cytoplasmic autoantibodies: relevance to systemic vasculitis[END_REF][START_REF] Hattar | Wegener's granulomatosis: antiproteinase 3 antibodies induce monocyte cytokine and prostanoid release-role of autocrine cell activation[END_REF]. Xiao and colleagues published in 2002 an experimental model of MPO-ANCAassociated vasculitis, demonstrating the pathogenic role of MPO-ANCA in vivo [START_REF] Xiao | Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice[END_REF]. For PR3-ANCA, although a recent model of humanized severe combined immunodeficiency (SCID) mice suggested that transfer of human PR3-ANCA could induce vasculitis, the direct pathogenic effect of these antibodies has not been clearly established in vivo [START_REF] Surmiak | Expression profile of proinflammatory genes in neutrophil-enriched granulocytes stimulated with native anti-PR3 autoantibodies[END_REF]. In contrast, other components of the immune system could be involved, in particular neutrophils expressing increased amounts of PR3 at their membrane that could play an important role in the pathogenesis of GPA [START_REF] Millet | Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis[END_REF] Although GPA and MPA share a number of common clinical features, the clinical presentation varies, with prominent granulomatous involvement of upper and lower respiratory tracts in GPA [START_REF] Soussan | FDG-PET/CT in patients with ANCA-associated vasculitis: case-series and literature review[END_REF], and glomerulonephritis, intra-alveolar haemorrhage and/or peripheral neuropathy in MPA [START_REF] Millet | Antineutrophil cytoplasmic antibodyassociated vasculitides: is it time to split up the group?[END_REF]. Both conditions are associated with relapses, although they are more frequent in patients with PR3-ANCA than in those with MPO-ANCA [START_REF] Specks | Efficacy of remission-induction regimens for ANCA-associated vasculitis[END_REF].

The treatment of systemic vasculitis relies on glucocorticoids and cyclophosphamide in severe forms of MPA and in systemic GPA. Interestingly, rituximab, a monoclonal anti-CD20 antibody has been found to be effective in both conditions in the induction of remission and [10:11:12] more recently in its maintenance [START_REF] Guillevin | Rituximab versus azathioprine for maintenance in ANCA-associated vasculitis[END_REF].

Several studies reported on the role of T cells or B cells in the pathogenesis of AAV [14,[START_REF] Wilde | T cells in ANCA-associated vasculitis: what can we learn from lesional versus circulating T cells?[END_REF],

whereas only few studies analyzed the potential role of both B and T lymphocytes subpopulations in active GPA and MPA patients [START_REF] Dumoitier | Implication of B lymphocytes in the pathogenesis of ANCA-associated vasculitides[END_REF]. Herein, we report disturbed B-and Tcell phenotypes in untreated active GPA or MPA patients with dramatic increase of Th2, Th9 and Th17 cells in GPA patients, perturbed B cell populations in both GPA and MPA and dramatic increase of IL-6 producing B cells in GPA.

Patients and Methods

Patients

Peripheral blood was obtained from patients with active GPA and MPA fulfilling the 2012

Revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides criteria for the diagnosis of GPA and MPA [START_REF] Jennette | revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides[END_REF], included in the multicenter and prospective Neutrovasc study (AOM 10055 -NI 10017). Active patients were defined as having either GPA or MPA and a Birmingham vasculitis activity score (BVAS) above 3. None of them received glucocorticoids and/or immunosuppressants at the time of blood sampling. In parallel, peripheral blood was obtained from 22 healthy controls (blood donors) (HC) who were recruited among blood donors at the "Etablissement Français du Sang", Saint Antoine Hospital, Paris, France.

All patients and HC gave written informed consent. Sera and peripheral blood mononuclear cells (PBMC) were collected with approval of the ethics committee of the "Groupe hospitalier Pitié-Salpêtrière" and the study conforms to the principles outlined in the Declaration of Helsinki.

Lymphocyte culture

PBMC were isolated from peripheral blood on Ethylenediamine tetraacetic acid (EDTA)anticoagulated tubes using a Ficoll-Hypaque density gradient centrifugation (Eurobio, Courtaboeuf, France). PBMC were cultured in Roswell Park Memorial Institute medium 1640 (RPMI) obtained from Gibco (Life Technologies, Saint-Aubin, France), with 10% heatinactivated fetal calf serum (FCS) (Eurobio) at 37°C in 5% CO2 atmosphere.

Quantification of cytokine expression and production

Expression of interferon-gamma (IFN-γ), interleukin (IL)-4, IL-9, IL-17 and IL-21 from T cells and IL-6 and Tumor necrosis factor alpha (TNF-α) from B cells were assessed after PBMC stimulation for 4 hours with 50 ng/mM of PMA and 1 µg/ml of ionomycin, with 1 µl/million of cells of GolgiStop solution. Cells were then washed and stained.

Flow cytometry analysis

We performed flow cytometry analysis on PBMC after staining with: anti-CD19-Brillant Violet 605 (BV605), anti-CD27-AlexaFluor700 (BD Biosciences), anti-CD21-Peridin-chlorophyll protein-Cyanin5.5 (PerCP-Cy5.5) (Biolegend, London, UK). For B lymphocyte subpopulations, we further investigated membrane expressions of markers within CD19 + B cells as previously described [START_REF] Kaminski | Advances in human B cell phenotypic profiling[END_REF].

Anti-CD3-AlexaFluor 700, anti-CD4-BV605 from BD Biosciences and anti-CD8-PERCP-Cy5.5 from Biolegend were used to define T lymphocyte subpopulations. In combination with these markers, various antibodies were used to stain extracellular or intracellular proteins of B and T lymphocytes and are listed in Supplemental Table 1.

In longitudinal experiments, cytometer settings were standardized using BD Cytometer setup, SPHERO Rainbow Calibration Particles (BD) and BD Combeads. Flow cytometry acquisition was assessed using a BD LSR Fortessa at the cytometry plateform of Cochin Institute (Cybio) and data were analyzed using FlowJo analysis software (Treestar, Ashland, OR, USA).

Statistical analysis

All analysis were carried out using Prism 6 (GraphPad, La Jolla, CA, USA) with statistical significance defined as: * p-value < 0.05; ** p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001. Non parametric tests including Mann-Whitney U-test were used to compare median values. Correlations were performed using Spearman test.

Results

Differential activation of T cells in patients with AAV

Clinical and immunological characteristics of patients with active GPA and MPA are depicted in Supplemental table 2. Number of total lymphocytes and of B-and T-cells were similar between GPA and MPA patients and HC (data not shown). Expression of IFN-γ from T helper (Th)1, IL-4 from Th2, IL-9 from Th9 and IL-17A from Th17 cells are presented in Figure 1 (Panel A). Increased proportions of Th2 and Th9 cells were observed in AAV patients compared to HC (1.8 vs.1% and 1.1 vs. 0.2%; p=0.02 and p<0.001 respectively) (Figure 1, Panel B), whereas a decreased proportion of Th1 cells was observed in AAV patients (8.5 vs.

13.2% in HC; p=0.03) and Th17 cells were comparable between groups. Consequently, Th2/Th1 (0.16 vs. 0.07; p=0.006) and Th17/Th1 (0.14 vs. 0.06; p<0.001) ratios were increased in patients with AAV as compared to HC (Figure 1, Panel C). Patients with active GPA showed increased percentage of peripheral Th9 (1.1 vs. 0.2; p<0.001), but both GPA and MPA expressed an increased Th9/Th1 ratio compared to HC (0.12 and 0.13 vs. 0.01; p<0.0001 and p=0.05 respectively).

A remarkable heterogeneity was observed regarding the proportion of CD25 hi Foxp3 + regulatory T cells (Treg) in AAV patients, particularly in those with active MPA (Figure 2, Panels A and B). However, increased percentages of activated T cells (Tact) (Foxp3 low CD25 + )

were found in both GPA and MPA (5.8 and 17.9 vs. 4%; p=0.08 and p=0.03, respectively). As a consequence, Treg/Tact ratio was decreased in both AAV subtypes (0.12 vs. 0.21; p=0.03), although it failed to reach significance in the case of MPA patients (Figure 2, Panel B).

A decreased proportion of CXCR5 + circulating follicular T helper (Tfh) cells was found in patients with AAV vs. HC (10.3% vs. 15%; p=0.006). Interestingly, only patients with GPA showed a decreased proportion of PD-1 + CXCR5 + competent Tfh cells (3.1% vs. 4.5; p=0.03).

However, we observed a decreased proportion of PD-1 + cells within CXCR5 + T cells in patients with MPA compared to HC (50.7% vs. 66.7%; p=0.03), suggesting Tfh dysregulation in both MPA and GPA patients.

We further discriminated patients according to ANCA specificity or clinical manifestations, especially for the presence of granuloma in GPA, and failed to identify differences that had not been previously underlined using GPA/MPA comparison (Supplemental figure 1 and2).

Perturbation B cell maturation in patients with active MPA

We further analyzed B cell maturation using the expression of CD21, CD27, CD38 and IgD (Figure 3 and Supplemental figure 3). Patients with active MPA showed increased proportions of naïve B lymphocytes (CD19 + IgD + CD27 -) (59.8% vs. 50.7%; p=0.01) and decreased proportions of memory B cells (CD19 + IgD -CD27 + ) vs. HC (9.3% vs. 31.1%; p=0.003) (Figure 3, Panel C). Similar results were obtained when we analyzed the mature B cell classification (Bm) as we found increased proportions of Bm2 (CD19 + IgD + CD38 + ) (60.8 vs. 42.5%; p=0.02) and decreased early and late Bm5 (CD19 + IgD -CD38 + and CD19 + IgD -CD38 -respectively) (11.3 vs. 24 and 12.1 vs. 4.2; p=0.01 and p=0.02 respectively) in patients with active MPA compared to HC (Supplemental figure 3). Notably, a slight decrease in percentage of early Bm5 was also observed in patients with active GPA (13.6% vs. 24%; p=0.01). More interestingly, according to ANCA specificity, patients with MPO-ANCA showed increased CD21 -B lymphocytes (24.1% vs. 13.4%; p=0.01) and decreased peripheral memory B cells (14.5% vs.

31.1%; p=0.005) which was not the case for patients with PR3-ANCA.

Peripheral B cell activation in patients with active AAV

We further phenotyped B cells from patients with AAV and found increased expression of CD40 in patients with active GPA compared to HC using MFI ratio analysis (46.2 vs. 20.6; p=0.05) which was not the case for patients with active MPA (Figure 4, Panel A). Expression of CD69 on B cells was increased in patients with both GPA and MPA vs. HC (3 and 5.9 vs.

1.5%; p=0.03 and p=0.03 respectively), but no significant change of expression of other activation markers (CD20, CD86, CD95) and inhibitory receptors (CD22, CD32, CD72) was noted in AAV patients as compared to HC (Figure 4 and Supplemental figure 5), as well as for the expression of IL-6R or IL-21R. In contrast, a dramatic decrease of B-cell activating factor (BAFF) receptor expression was observed in both GPA and MPA patients compared to HC (MFI ratio 11.8 and 13.7 vs. 45.2; p<0.0001 and p=0.003 respectively) (Figure 4, Panel B).

Similar results were obtained when AAV patients were compared according to MPO or PR3 specificity (Supplemental figure 6).

B cells from patients with active GPA produce increased amounts of IL-6

We finally analyzed the expression of two proinflammatory cytokines, i.e. IL-6 and TNF-α, by B cells (Figure 5, Panel A). B cells from GPA patients showed a strikingly increased expression of IL-6 compared to HC (25.8 vs. 14.9%; p<0.0001), whereas B cells from MPA patients had dramatically decreased expression of IL-6 (4.6 vs. 14.9%; p=0.005) (Figure 5, Panel A). In contrast, similar decrease in TNF-α expression by B cells was noted in both AAV compared to HC (14.1% vs. 30%; p=0.001). Similar results were obtained when AAV patients were compared according to anti-MPO or anti-PR3 ANCA specificity (Supplemental Figure 7

Discussion

In the present work, we provide evidence for a dysregulation of lymphocytes compartments in patients with active untreated AAV. We observed skewed T-cell polarization toward Th2, Th9 and Th17 responses in GPA, whereas B cell populations were dysregulated in both GPA and MPA with an activated phenotype and decreased BAFF-R expression. We also established that IL-6-producing B cells were dramatically increased in GPA patients whereas TNF-α-producing B cells were decreased in both GPA and MPA patients.

First, we observed specific T-cell polarization toward Th2, Th9 and Th17 responses in GPA patients which is in agreement with previous studies [START_REF] Abdulahad | Persistent expansion of CD4+ effector memory T cells in Wegener's granulomatosis[END_REF][START_REF] Abdulahad | T-helper cells as new players in ANCA-associated vasculitides[END_REF][START_REF] Wilde | Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity, immune regulation and therapy[END_REF], as well as an increased percentage of peripheral activated T cells (Tact) (CD25 + Foxp3 low ) conversely to previous report [START_REF] Ikeda | Reduced CD4-CD8 T cell ratios in patients with Wegener's granulomatosis[END_REF]. These findings in human are similar to what was observed when naive mouse T cells were co-cultured with apoptotic neutrophils expressing PR3 [START_REF] Millet | Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis[END_REF]. In addition, in the same mouse model, PR3-ANCA induced an increased expansion of Th17 cell in agreement with what we observed in GPA patients. Moreover, we confirmed our previous report showing that patients with active GPA displayed Th9 and Th17 expansions whereas no change was observed for Th1 lymphocytes [START_REF] Millet | Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis[END_REF]. Since IL-17 is commonly detected in vasculitis lesions in the kidney, these findings suggest the potential interest of targeting Th17 and IL-17A in GPA patients [START_REF] Wilde | Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity, immune regulation and therapy[END_REF].

Unlike what has been published in the literature [START_REF] Müller | Localized Wegener's granulomatosis: predominance of CD26 and IFN-gamma expression[END_REF], we failed to identify any significant difference according to clinical manifestations, particularly for the presence of granuloma in GPA patients.

In parallel, we observed a decreased proportion of Th1 cells in patients with MPA or MPO-ANCA resulting in similar Th2/Th1, Th9/Th1 and Th17/Th1 ratios to what was observed in GPA patients, whereas no expansion of Th2, Th9 or Th17 cells was observed in MPA. These data are important since very few studies were dedicated to the analysis of T cells in patients with MPA, even if these data should be interpreted with caution because of the small number of MPA patients. Overall, our data suggests that T helper cells are particularly disrupted in GPA patients and not in the limited number of MPA patients, supporting the existence of differences in the activation of T helper cells between GPA and MPA patients as previously suggested [23].

We did not identify any diminution of Tregs in our cohort of AAV patients, although other groups did, suggesting it could be used as a possible disease activity marker [START_REF] Morgan | Patients with Wegener's granulomatosis demonstrate a relative deficiency and functional impairment of T-regulatory cells[END_REF]. However, we found a significantly decreased Treg/Tact ratio in patients with AAV which could contribute to explain the functional limitation of Treg previously observed in AAV patients [START_REF] Morgan | Patients with Wegener's granulomatosis demonstrate a relative deficiency and functional impairment of T-regulatory cells[END_REF]. In addition,

Tregs from GPA patients may convert into IL-17A-secreting cells as it was observed in other systemic autoimmune diseases and suggested in a recent review [28,29]. Finally, Zhao and colleagues recently reported an increased proportion of Tfh in patients with either active or inactive GPA under cyclophosphamide treatment [START_REF] Zhao | Circulating T follicular helper cell and regulatory T cell frequencies are influenced by B cell depletion in patients with granulomatosis with polyangiitis[END_REF]. In our study, we found a decreased percentage of Tfh in AAV patients, particularly in patients with MPA or patients with anti-MPO ANCA who also presented decreased PD-1 expression which could participate in the B-T cell cooperation and the expansion of autoreactive B cells [START_REF] Sage | PD-1 controls Lymph Node and Blood T Follicular Regulatory Cells[END_REF][START_REF] Yao | PD-1 as an Immune Modulatory Receptor[END_REF][START_REF] Mesquita | Follicular helper T cell in immunity and autoimmunity[END_REF].

Second, we observed that B-cell compartment was dysregulated in MPA with increased proportion of naïve B cells and decreased circulating memory B cells, whereas no significant difference was found in GPA patients. This result is in agreement with previous studies [16] [34]. Besides B-cell maturation perturbation, we identified for the first time that MPA patients had an increased proportion of CD21 -/lo B lymphocytes, which have previously been shown to be enriched in autoreactive B cells in Sjögren's syndrome, hepatitis C virus-related cryoglobulinemia vasculitis (mettre ma ref SVP), systemic lupus erythematosus and rheumatoid arthritis [START_REF] Isnardi | Complement receptor 2/CD21-human naive B cells contain mostly autoreactive unresponsive clones[END_REF][START_REF] Saadoun | Expansion of autoreactive unresponsive CD21-/low B cells in Sjögren's syndrome associated lymphoproliferation[END_REF]. This difference between GPA and MPA patient reinforced the paradigm of GPA and MPA toward autoimmunity. B cells from MPA patients are thus potentially enriched in autoreactive B cells while B cells from GPA patients displayed classical maturation subpopulations contrasting with markedly deregulated neutrophils and innate immunity [START_REF] Millet | Antineutrophil cytoplasmic antibodyassociated vasculitides: is it time to split up the group?[END_REF].

In addition, we observed in both GPA and MPA a B-cell activated phenotype with increased expression of CD69 and CD40. Both of these activation markers are known to play a role in Band T-cell cooperation and were found to be increased in B cells from patients with SLE or rheumatoid arthritis [START_REF] Zhang | Aberrant CD40-induced NF-κB activation in human lupus B lymphocytes[END_REF][START_REF] Medgyesi | The protein tyrosine phosphatase PTP1B is a negative regulator of CD40 and BAFF-R signaling and controls B cell autoimmunity[END_REF]. These receptors also influence the development of autoreactive B cells in several experimental models [START_REF] Bonomo | Pathogenesis of post-thymectomy autoimmunity. Role of syngeneic MLR-reactive T cells[END_REF][41][42]. We also found that BAFF receptor expression on B cells was dramatically decreased in both GPA and MPA which could be related to the increased activation of the BAFF pathway in AAV [43]. This finding also revealed a potential interplay between neutrophils and B cells, as neutrophils highly express BAFF in patients with GPA, which could participate in B cell activation [START_REF] Scapini | Regulation of B-cell-activating factor (BAFF)/B lymphocyte stimulator (BLyS) expression in human neutrophils[END_REF][START_REF] Holden | ANCA-stimulated neutrophils release BLyS and promote B cell survival: a clinically relevant cellular process[END_REF].

Then, we analysed the ability of B cells to produce proinflammatory cytokines, in particular IL-6 and TNF-α. Similarly to previous data in monocytes, B cells from active GPA patients produced less TNF-α than HC [START_REF] Park | Decreased tumour necrosis factor-α production by monocytes of granulomatosis with polyangiitis[END_REF]. In the present work, we also provide similar results in patients with MPA. In contrast, B cells producing IL-6 were strikingly increased in GPA, whereas the opposite was found in MPA patients. Recently, Wright and colleagues have shown that in vivo and in vitro stimulations of neutrophils by IL-6 enhanced neutrophil migration through IL-8, one of the well described neutrophil activation pathway in GPA [START_REF] Richter | Neutrophil chemotaxis in granulomatosis with polyangiitis (Wegener's) and idiopathic pulmonary fibrosis[END_REF][START_REF] Wright | Effects of IL-6 and IL-6 blockade on neutrophil function in vitro and in vivo[END_REF].

Furthermore, increased IL-6 production by B cells could induce Th2, Th9 and Th17, which is consistent with our findings in patients with GPA [START_REF] Diehl | The two faces of IL-6 on Th1/Th2 differentiation[END_REF][START_REF] Omenetti | The Treg/Th17 Axis: A Dynamic Balance Regulated by the Gut Microbiome[END_REF][55]. Thus, we found a positive correlation between the percentage of IL-6 producing B cells and the proportions of Th2 and Th9 cells in patients with GPA which was not the case for Th1, Th17 or Treg. These data highlight the interest of using anti-IL-6R monoclonal antibody in patients with AAV [START_REF] Catanoso | New indications for biological therapies[END_REF][START_REF] Sumida | Complete remission of myeloperoxidase-antineutrophil cytoplasmic antibody-associated crescentic glomerulonephritis complicated with rheumatoid arthritis using a humanized anti-interleukin 6 receptor antibody[END_REF][START_REF] Berti | Interleukin-6 in ANCA-associated vasculitis: Rationale for successful treatment with tocilizumab[END_REF]. Moreover, in the RAVE study, patients with PR3-ANCA (mostly GPA) responded very well to rituximab, which could suggest that the depletion of IL-6 producing B-cells could participate in the induction of remission [START_REF] Specks | Efficacy of remission-induction regimens for ANCA-associated vasculitis[END_REF].

Finally, our study has some limitations. The number of patients with MPA was low, therefore findings in this group should be interpreted with caution. The NeutroVasc study was a prospective study in which consecutive patients with active AAV who did not receive glucocorticoid or immunosuppressive treatments were included. These very restrictive inclusion criteria explain the low number of MPA patients. However, because of these restrictive inclusion criteria, the data from our analysis provide new insights in B and T cells dysregulations in untreated and active AAV.

In conclusion, skewed T-cell polarization toward Th2, Th9 and Th17 responses characterizes GPA but not MPA, whereas B cell populations are dysregulated in both GPA and MPA with an activated phenotype and decreased BAFF-R expression. B cells cytokine production disturbances were identified with a dramatic increase of IL-6-producing B cells in GPA and decreased TNF-α producing B cells in both GPA and MPA, providing an additional mechanism by which rituximab could be effective in GPA and raising the question of the potential interest of anti-IL-6 in GPA. Interestingly, circulating B lymphocytes from SSc patients differ from those of HC, with increased proportions of naïve B cells and decreased numbers of memory B cells and plasma cells (Sato et al., 2004). In addition, signaling alterations, including overexpression of both CD19 and CD21, two activating co-receptors of the B-cell antigen receptor (BCR), have been detected both in the Tsk-1 mouse model of SSc and in naive and memory B lymphocytes from patients with SSc. Moreover, the activation receptors CD80, CD86 and CD95 are upregulated in memory B cells from SSc patients, suggesting their participation to the pathogenic process (Sato et al., 2004). High levels of B cell activating factor (BAFF) have been measured in the serum of SSc patients, together with an overexpression of BAFF-R at the surface of peripheral B cells from those patients (Matsushita and Sato, 2005). BAFF through the NF-κB pathway, promotes B cells survival and participates to autoreactive B cells differentiation (Liu and Davidson, 2011). In addition, B cells infiltrates have been detected in the dermis of SSc patients [7; 8] and serum levels of IL-6 are increased and correlate with the extent of skin fibrosis both for SSc patients and Tsk-1 mouse model [9; 10]. IL-6 stimulates collagen secretion by fibroblasts and therefore might be an intermediate for fibroblast activation (Chizzolini, 2008).

Legend to Figures

Supplemental

More recently, depletion of B cells using a chimeric anti-CD20 monoclonal antibody allowed improving skin fibrosis and prevented loss of pulmonary function in patients with SSc (Bosello et al., 2015). Moreover, targeting IL-6 receptor (IL-6R) with tocilizumab showed promising effect on skin involvement in a prospective randomized trial [13;14].

Herein, we describe abnormal phenotypic patterns of peripheral B lymphocytes in patients with SSc, notably between lcSSc and dSSc, together with increased cytokine production leading to fibroblast activation.

Patients and Methods

Patients

Peripheral blood was obtained from 90 patients with SSc fulfilling the American College of Rheumatism (ACR) / European League Against Rheumatism (EULAR) criteria for the diagnosis of SSc (van den Hoogen et al., 2013d), followed at Cochin Hospital in the department of Internal Medicine. Patient's clinical characteristics are summarized in Supplemental Table 1. LcSSc was defined by skin thickening in areas solely distal to the elbows and knees, with or without facial involvement. Diffuse SSc was defined by the presence of skin thickening proximal, as well as distal, to the elbows and knees, with or without facial or truncal involvement (LeRoy 1988). Early systemic sclerosis was defined as the first twelve months after the first non-Raynaud's symptom of systemic sclerosis. Peripheral blood was obtained from 30 healthy controls (blood donors) (HC) who were included at the "Etablissement Français du Sang", Saint Antoine Hospital, Paris, France.

All patients and HC gave a written informed consent. Sera and peripheral blood mononuclear cells (PBMC) were collected with approval of the ethics committee of the "Groupe hospitalier Pitié-Salpêtrière" and the study conforms to the principles outlined in the Declaration of Helsinki.

Fibroblast culture and functional studies

Skin biopsies were performed in involved skin from 4 patients with SSc. Normal human dermal fibroblasts were cultured from skin biopsies obtained from 4 HC. Biopsy specimen were cut into small pieces and seeded into Petri dishes and then into 175cm Promokine (Promocell GmBH, Heidelberg, Germany), proliferation was quantified using the Cell proliferation ELISA BrdU from Roche (Boulogne-Billancourt, France) and indirect immunofluorescence of Ki67 evaluated using an anti-Ki67 (Abcam, Cambridge, UK) and an anti-rabbit fluorescein isothiocyanate-coupled secondary antibody (Jackson Immunoresearch).

Collagen quantification was assessed after five days of culture using Sircol assay kit from Biocolor (Carrickfergus, UK) according to manufacturer protocol. Real Time-PCR (RT-PCR)

were assessed using 2.5 10 4 fibroblasts cultured in a 24 well plate at the same concentration as described above. RNA extracted using RNeasy mini kit from Qiagen (Hilden, Germany)

according to the manufacturer protocol, were quantified using NanoVue (GE Healthcare Life Science, Little Chalfont, UK). Four hundred nanograms of total RNA was retrotranscripted in cDNA using iScriptTMcDNA Synthesis kit from Biorad (Marnes-la-Coquette, France)

following the manufacturer protocol. Alpha-Smooth muscle actin (α-SMA), Collagen type I, Snail and Vimentin mRNA expressions were quantified compared to β2-microglobulin as housekeeping gene. Real-time PCR was performed using Power SYBR®Greenq-PCR kit on a

StepOnePlus Real Time PCR System TM (Applied Biosystem, Life Technology, Saint-Aubin, France). Primers obtained from Eurogentec (Liège, Belgium) were referenced in Supplemental Table 3.

Immunohistochemical study

Immunohistochemistry was performed on paraffin embedded lung samples: 4 μm sections were dewaxed and rehydrated progressively. EDTA buffer (pH 9.0) or Citrate buffer (pH 6.0) were used for antigen retrieval and endogenous peroxidase was quenched with hydrogen peroxide.

Sections were blocked with 5% bovine serum albumin and incubated for 32 minutes with polyclonal antibody against CD20 (reference BSB5195, as recommended by the manufacturer;

Bio SB, Santa Barbara, USA) or CD69 (reference ab202909, as recommended by the manufacturer; Abcam, Cambridge, USA). After incubation with a biotinylated secondary antibody, its detection was performed with HRP-streptavidin and DAB-solution.

Statistical analysis

All analysis were carried out using Prism 6 (GraphPad, La Jolla, CA, USA) with statistical significance defined as:* p-value < 0.05; ** p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001. Non parametric tests including Mann-Whitney U-test were used for two group independent comparisons. Correlations of protein expressions were performed using Spearman test. Comparisons between fibroblast responses to B lymphocyte stimulation were analyzed using paired t-tests.

Results

Differential peripheral B lymphocytes maturation in patients with SSc

The overall number of B and T cells was similar between HC and SSc patients, despite a larger dispersal for the later, mainly observed in patients with lcSSc (Supplemental figure 2).

Similarly, B lymphocyte subpopulations counts including naïve, effector memory, Breg cells and plasmablasts were equivalent among the cohort between HC, total SSc, dSSc and lcSSc patients (Figure 1). In contrast, the proportion of IgD -CD27 + memory B lymphocytes was significantly decreased in patients with SSc as compared to HC (p=0.04) (Figure 1), with similar distribution between dSSc and lcSSc. An additional differential proportion of marginal zone B cells was found between patients with dSSc and lcSSc that was compensated when global SSc was compared to HC. Specific analysis of mature B cells (Bm) classification identified the CD38 + IgD -early Bm5 subpopulation as the predominantly decreased memory B cell subset, irrespective to lcSSc or dSSc disease form (p=0.001) (Supplemental figure 3).

Peripheral B lymphocytes from patients with SSc are activated

Patients with SSc exhibited increased proportions of B lymphocytes expressing activation markers CD95 (p=0.0004), CD69 (p=0.002) and CD86 (p=0.001) as compared to HC (Figure 2A). Similar results were obtained using mean fluorescence intensity (MFI) quantification (Supplemental Figure 4A). We also identified a B cell subpopulation expressing both CD69 and CD95 in SSc patients that was absent in HC (p=0.0002) (Figure 2B). Interestingly, subpopulations expressing CD69 were correlated to those expressing CD95 in SSc patients, both in dSSc and lcSSc forms (r=0.76; p<0.0001) (Figure 2C). Interestingly, percentages of CD95 + B cells were higher in patients with early lcSSc compared to patients with non-early lcSSc (p=0.0027) (Figure 2D). In contrast, the proportion of peripheral CD69 + B lymphocytes was higher in patients with early dSSc as compared to those with non-early dSSc (p=0.048).

Furthermore, when we combine these two activation markers, the percentage of CD69 + CD95 + B cells was higher in early SSc patients than in non-early SSc patients (p<0.0001). Association between other activation markers were not found for both SSc patients and HC.

Additionally, the proportions of cells expressing CD86 differed significantly between dSSc and lcSSc (p=0.0007). Similar difference was also observed for CD5 (p=0.03) (Figure 2A), whereas CD86 and CD5 B cell subsets were not associated with autoantibody status (data not shown).

Since these two receptors play a critical role within B/T lymphocyte synapse, we wanted to investigate the potential correlations between the proportion of CD5 + and CD86 + B lymphocytes with the proportions of T helper (Th)1 (CD3 + CD4 + IFN-γ + ), Th2, (CD3 + CD4 + IL-4 + ), Th9 (CD3 + CD4 + IL-9 + ) and Th17 (CD3 + CD4 + IL-17A + ) (Supplemental Figure 5). No correlation of the percentage of CD5 or CD86 with these T cell subsets was found except a correlation between CD86 and the percentage of Th2. Similar results were obtained when we analyzed separately patients with dSSc and lcSSc.

In contrast, the proportion of cells expressing the inhibitory receptors CD22, CD32 and CD72

did not differ significantly between HC and SSc patients, except for CD32 between dSSc and lcSSc (p=0.02) (Figure 3A). Regarding membrane expression, B lymphocytes from SSc patients, showed increased levels of the constitutively expressed activating co-receptor CD20 as compared to HC cells (p=0.001), whereas no significant variation was found for the expression of the inhibitory receptors CD22, CD32, CD72 or of HLA-DR (Supplemental figures 4B and 6A). Finally, concerning the crosstalk of B cells with the microenvironment, a slight decrease of BAFF-receptor (BR3) expressing cells was detected for SSc patients compared to HC (p=0.04); however, this difference was not significant at the cellular level (Supplemental figure 6B). Noteworthy, a remarkable increase of the populations expressing either IL-6R (p=0.01) or IL-21R (p=0.02) was observed in SSc patients with major differences for IL-21R expression between dSSc and lcSSc (p<0.0001) regarding both subpopulation percentages and MFI analysis (Figure 3B and Supplemental figure 6C).

In order to identify clinical manifestations of SSc that could be associated with B cell subsets, expression of activation markers and receptors were evaluated in the presence or not of pulmonary arterial hypertension (PAH), interstitial lung disease (ILD), arthralgia (joint) and digital ulcers (DU) (Supplemental Table 2). An increased proportion of B cells expressing CD86 was slightly associated with PAH and digital ulcers in patients with SSc while increased subpopulation of CD95 + B cells was associated with arthralgias. More interestingly, patients with either arthralgias, PAH and/or ILD had increased proportions of B cells expressing the IL-6R. For these two late manifestations, similar proportions of B lymphocytes expressing IL-6R

were observed which allowed discriminating between patients with PAH and/or ILD, and those without documented pulmonary involvement with lower proportion of peripheral IL-6R + B cells (Supplemental figure 7).

Overall, no significant difference was found in B cell phenotypes on the basis of autoantibody specificity (data not shown).

B lymphocytes are detected in the lungs from patients with SSc-associated PAH

We performed immunostaining in lung samples from three patients with SSc-associated PAH and three controls (normal lung tissue from patients with non-small cell lung carcinoma). In SSc-patients we observed a number of lymphoid follicles, mostly in the vicinity of bronchioles/small airways, whereas only scattered B-lymphocytes are perceived in the lung parenchyma of the control patient, as assessed by anti-CD20 immunostaining. In lymphocyterich areas, scattered CD69 + cells can be observed, whereas only faint staining of sparse cells is observed in control patients (Supplemental figure 8).

Phenotypical characteristics of peripheral B cells are consistent with higher production of IL-6 and TGF-β in SSc patients

A major increase in IL-6 producing cells was evidenced for SSc patients as compared to HC (p<0.0001) (Figure 4A). This difference was also found for both IL-6 + CD27 -IgD + naïve B cells (ratio 1:2 p<0.0001) and IL-6 + CD27 + IgD -memory B cells (ratio 1:1.8 p=0.0003) but not for marginal zone or double negative effector B cells (data not shown). We further analyzed the intracellular staining of IL-6 in both T lymphocytes (defined as CD27 + CD19 -cells) and monocytes (using FCS/SSC parameters in CD19 -CD27 -cells) as shown in Supplemental figure 9 (Panel A). No difference was observed for percentages of IL-6 + T cells between patients with SSc and HC (Supplemental figure 9 Panel B), whereas a decreased proportion of monocytes expressed IL-6 in SSc patients as compared to HC (Panel C). Thus, in patients with SSc, we identified the B cells as the main source of IL-6 compared to T lymphocytes and monocytes, which was not the case in HC (Supplemental figure 9 Panel D). We also investigated IL-10 and TGF-β (Figure 4B and 4C) production by purified B lymphocytes after three days of culture upon stimulation or not with CpG. Number of IL-10 producing B cells did not differ significantly between our various samples in such conditions and their phenotype corresponded to CD19 + CD24 hi CD27 +/-CD38 hi predominantly CD5 -(Supplemental figure 10A). More interestingly, peripheral B cells isolated from patients with SSc expressed increased proportion of TGF-β + CD19 + B lymphocytes as compared to HC, even in the absence of further stimulation of TLR9 through CpG incubation (p=0.0002) (Figure 4C). TGF-β expressing B lymphocytes were CD19 + CD5 -CD24 low CD27 + CD38 hi both in HC and patients with SSc and did not overlap with IL-10 + B lymphocytes (Supplemental figure 10B).

B cells from SSc patients secrete higher amounts of IL-6 and TGF-β and lower amounts of IL-10 upon stimulation

We detected higher levels of IL-6 in the serum of SSc patients as compared to HC (p=0.01) (Figure 5A, left panel). Conversely, no significant difference was found for IL-10 or TGF-β (Figure 5A, middle and right panel). Interestingly, purified B cells from SSc patients, incubated3 days with CpG or for 4 hours with a mix of PMA/ionomycin secreted increased amounts of IL-6 whereas those from HC remained low (p=0.0007) (Figure 5B).We also detected slightly increased IL-10 concentration in B cell supernatant from SSc patients as compared to HC after stimulation with PMA/ionomycin (p=0.01) (Figure 5C). Unexpectedly, while CpG stimulation promoted a work increase of IL-10 secretion for SSc patients a strong increase was observed for HC in such condition. According to the increased proportion of TGFβ producing cells, we evidenced increased amount of TGF-β in the supernatant of CpG stimulated SSc B cells as compared to HC (p=0.001) while PMA/ionomycin treatment did not influence significantly the release as compared to unstimulated cells (Figure 5D and Supplemental figure 11).

A correlation was found between modified Rodnan Skin Score (mRSS) and the concentration of TGF-β in culture supernatant from B cell stimulated with CpG (r=0.60; p=0.04) (Figure 5E).

Similar correlation was between mRSS and the concentration of IL-6 within supernatants of B cells stimulated with PMA/ionomycin cocktail for 4 hours (r=0.71; p=0.01), suggesting a potential pro-fibrotic role of B lymphocytes in SSc patients.

In vitro stimulation of HC and SSc dermal fibroblasts by B lymphocyte supernatant increase proliferative and fibrotic phenotype

Culture of fibroblasts from HC or fibroblasts isolated from involved skin of SSc patients for 3 days in the presence of supernatants of B cells from HC did not influence significantly their metabolic activity (Figure 6A) and their proliferation rate as assessed by immunofluorescence using Ki67 labeling (Supplemental figure 12). A randomly increased viability was observed for all fibroblasts when cultured in the presence of supernatant from isolated B cell from SSc patients from without reaching significance (Figure 6A). Previous stimulation of HC or SSc B cells did not provide additional effect on viability. However, supernatant from CpG or PMA stimulated SSc B cells significantly enhanced to variable extents the proliferation of both HC and SSc fibroblasts (Figure 6B). In agreement with their increased proliferation rate, both HC and SSc fibroblasts showed poor production of collagen in the presence of PMA stimulated B cell supernatant from SSc patients compared to HC (Figure 6C). When supernatants of CpG stimulated purified B cells from HC and SSc patients were used to stimulate fibroblasts in culture, collagen production was increased differentially in both HC and SSc fibroblasts (Figure 6C).

Furthermore, we analyzed by q-PCR, expression levels of α-Smooth muscle actin (SMA), Snail and vimentin mRNA within HC and SSc stimulated fibroblasts (Supplemental figure 13).

Unfortunately, no consistent detection of α-SMA was obtained with either HC or SSc fibroblasts using both q-PCR and immunofluorescence (data not shown). However, when both HC and SSc fibroblasts were stimulated with either CpG stimulated or PMA/ionomycin stimulated purified B cell supernatant from SSc patients, an increased expression of vimentin was observed. Increased expression of the transcription factor Snail and of collagen mRNA levels were also detected when fibroblasts from either HC or SSc patients were stimulated with CpG stimulated B cell supernatant from SSc patients. These increases were abrogated when supernatants were pre-incubated with anti-TGF-β antibody for CpG supernatant, or anti-IL-6R antibody for PMA supernatant. Furthermore, incubation with anti-TGF-β antibody or anti-IL-6R antibody induced a drastic decrease in fibroblast proliferation without any difference in viability assay, reinforcing the potential pathophysiological role of B lymphocytes in SSc (Supplemental figure 13).

Discussion

In the present work, we provide evidence for an increase in SSc patients as compared to HC of a peripheral B cell subpopulation that express both activation markers and cytokine receptors as well as high levels of intracellular IL-6 and TGF-β. We also establish that B lymphocytes supernatants from SSc patients activate at higher levels than those from HC both normal and scleroderma human fibroblasts.

In SSc patients, we observed an increased expression of CD86 and CD95 on B cells altogether with a lower proportion of peripheral memory B cells, in agreement with results obtained by Sato et al. (Sato et al., 2004). We further restricted this decrease to the early Bm5 memory peripheral B cell subpopulation as in peripheral blood of patients with Sjögren's syndrome [START_REF] Dumoitier | Implication of B lymphocytes in the pathogenesis of ANCA-associated vasculitides[END_REF].

Interestingly, we also found in patients with SSc, an increased expression of CD69, IL-6R and IL-21R on peripheral B lymphocytes, and further correlated the increased proportions of CD69 + cells within decreased CD95 + B lymphocytes. Furthermore, we showed a major differential proportion of CD5 + , CD86 + , IL-6R + or IL-21R + B cells between dSSc and lcSSc patients. Indeed patients with dSSc had higher proportion of these B cells compared to both lcSSc patients and HC. In contrast, both lcSSc and dSSc patients showed increased proportions of CD69 + and CD95 + B cells compared to HC. Moreover, we found several correlations between the activation of B lymphocytes and disease duration in SSc patients, with higher expression of CD95 within the subgroup of early lcSSc patients, increased percentage of CD69 + for patients with early dSSc. In addition, we detected the double positive CD69 + CD95 + peripheral B cells subpopulation in patients with early SSc, suggesting a possible link between B cells activation at an early stage of the disease and disease severity. Of note, IL-6R expression is strongly associated with arthralgia, PAH and/or ILD. Similar proportions of B lymphocytes expressing IL-6R were observed for PAH and ILD. This finding allowed discriminating patients with or without lung involvement, the later exhibiting lower proportions of peripheral IL-6R + B cells. In lung tissue from patients with SSc-PAH, we observed important numbers of B cells, with a number of lymphoid follicles, mostly in the vicinity of bronchioles/small airways, whereas only scattered B-lymphocytes were perceived in the lung parenchyma of control patients, providing further evidence in the implication of B cells in lung involvement in patients with SSc. Altogether, our data argue for an activation profile of B lymphocytes in SSc patients, probably due to BCR engagement via autoantigens for both lSSc and dSSc [17; 18]. But, it also suggests a differential activation between dSSc and lSSc B lymphocytes as for patients with PAH and/or ILD vs absence of lung involvement; this differential activation could potentially occur through cytokine modulation as suggested in a recent clinical study [START_REF] Abdulahad | T-helper cells as new players in ANCA-associated vasculitides[END_REF]. No correlation was found between B lymphocytes activation and autoantibody status, probably arguing that they represent additional markers of disease activity/severity. Since B cells from patients with dSSc, with PAH and/or ILD are activated, these cells could be proposed as biomarkers.

We also provide novel evidences regarding the production and secretion of cytokines. We identified both naïve and memory B lymphocytes derived from SSc patients as major IL-6 secreting cells. However, a direct correlation between IL-6 levels in the supernatant and IL-6R expression at the membrane of B lymphocytes was not observed. This result does not argue for an autocrine activating loop in SSc B lymphocytes, similar to the autostimulatory IL-6-driven B cell differentiation in patients with systemic lupus erythematosus [START_REF] Wilde | Th17 expansion in granulomatosis with polyangiitis (Wegener's): the role of disease activity, immune regulation and therapy[END_REF]. The mechanisms leading to B lymphocytes overactivation might rather be due to the increased concentration of serum IL-6 in the serum of SSc patients vs HC as previously reported by Needleman et al. [START_REF] Millet | Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis[END_REF].

Moreover, we shown that IL-6 levels in the supernatant of purified B cells isolated from SSc patients also correlate with mRSS, suggesting a critical role of B lymphocyte in the induction of fibrosis. Monocytes have been proposed as the unique immune cells producing IL-6 in patients with SSc [22;23]. Herein, we provide new evidences for the characterization of IL-6 producing B lymphocytes in patients with SSc. These B lymphocytes might represent interesting targets for SSc treatment since the elevated levels of IL-6 in patients sera correlate with disease severity (Sato et al., 2001), Additionally, in contrast with a previous study, we report elevated TGF- production and release by peripheral B lymphocytes from SSc patients as compared to HC both at steady state and upon specific TLR stimulation [START_REF] Ikeda | Reduced CD4-CD8 T cell ratios in patients with Wegener's granulomatosis[END_REF]. These high levels of TGF-β in supernatants of sorted B cells also correlate with mRSS, suggesting a role of B cells in fibrosis development. Indeed

Hasegawa et al. reported a lack of substantial increase in the release of TGF- by regulatory B cells, as well as activated T cells [START_REF] Bordin | Idiopathic CD4+ lymphocytopenia and systemic vasculitis[END_REF] or monocytes [START_REF] Villa-Forte | Substitution of methotrexate for cyclophosphamide in Wegener granulomatosis: a 12-year single-practice experience[END_REF] in SSc patients, in line with a nonimmune production of TGF-β in those patients. Herein, we identified a specific B lymphocyte subpopulation that secretes high level of TGF-β and exhibits a phenotype similar to those recently described for GVHD [START_REF] Morgan | Patients with Wegener's granulomatosis demonstrate a relative deficiency and functional impairment of T-regulatory cells[END_REF]. The absence of IL-10 secretion by SSc B lymphocytes might also contribute to the loss of immunoregulation observed in SSc [28; 29]. Unfortunately, we

were not able to perform co-culture experiments using B and T cells in order to evaluate these immunomodulatory roles or potential interplay with T cells due to the limitation in the amount of biological samples available.

Pathogenic effect of the B lymphocytes in SSc remains poorly understood (Arnett, 2006).Recent reports have addressed interesting therapeutic targeting both in mouse models and in open studies on limited populations of patients with SSc [30; 31; 32; 33]. Notably,

François and colleagues reported a direct implication of normal B cells in increased proliferation and extracellular matrix production by HC and SSc fibroblasts [START_REF] Todd | Regulatory B cells are numerically but not functionally deficient in anti-neutrophil cytoplasm antibody-associated vasculitis[END_REF]. In the present study, we demonstrate that supernatants of B lymphocytes purified from SSc patients induce fibroblast activation even in absence of direct cell-to-cell interaction or autoantibodies secretion, but more as a consequence of an increased secretion of IL-6 and TGF-β. We also found that supernatants of purified B lymphocyte with the higher level of TGF-β (CpG supernatant) induce an increased expression of vimentin and the transcription factor Snail, two proteins that have been associated with the differentiation of fibroblasts into myofibroblasts [35; 36; 37]. Moreover, incubation with either anti-TGF-β or anti-IL-6R antibodies induce a dramatic decrease in fibroblast proliferation without any difference in viability assay, suggesting an implication of these pathways in B cell/fibroblast crosstalk in SSc. These data further underline a possible interplay between B lymphocytes and fibroblasts through new secretory pathways that need to be further investigated in SSc pathogenesis. Furthermore, the recent results obtained with IL-6 targeted therapy in the bleomycin induced-scleroderma mouse model and in patients with SSc highlight the prevalent role of this cytokine in SSc pathophysiology and reinforce the interest for evaluating IL-6 secretion capacity of peripheral B cells [21; 33;38; 39]. Finally, these data also argue for the interest of investigating the effects of IL-6 targeted therapy in patient with SSc as the faSScinate study has shown interesting results using a monoclonal anti-IL-6R antibody (tocilizumab) (Khanna et al., 2016). Our study, based on a consistent number of phenotypically characterized SSc patients who did not receive glucocorticoid or immunosuppressive treatments, provide new insights in the functional implication of B lymphocytes in SSc.

Conclusion

Clinical studies have shown promising results for anti-CD20 and anti-IL-6R based therapies in patients with SSc [21; 39; 40]. We observed an increased activation of peripheral B cells in patients with dSSc as compared to lcSSc patients. In vitro, B lymphocytes from SSc patients secrete high amounts of IL-6 and TGF-β which induce fibroblast proliferation, collagen secretion and differentiation into myofibroblasts in both healthy individuals and SSc fibroblasts. This report highlights for the first time differential B lymphocyte subpopulations between the two different SSc subtypes. Viability of fibroblasts was quantified using XTT reduction assay kit and results were expressed as percentage of XTT reduction in stimulated vs unstimulated fibroblasts (Panel A).

Proliferation of fibroblasts was quantified using BrdU incorporation dosage (Panel B). Results

were expressed as percentage of BrdU incorporation by stimulated vs unstimulated fibroblast.

Collagen production by fibroblasts was quantified using Sircol Biocolor assay and expressed as percentage of collagen production of stimulated vs unstimulated fibroblasts (Panel C). All experiments were performed twice with similar results. Comparisons between groups were performed using paired t-test and represented in histogram (mean ± standard deviation (SD).

Statistical significance was defined as:* p-value < 0.05; ***p-value < 0.001.

Supplemental methods

Lymphocyte culture

PBMC were isolated from peripheral blood on Ethylenediaminetetraacetic acid (EDTA)anticoagulated tubes from HC or patients with SSc using a Ficoll-Hypaque density gradient

Lymphocyte stimulation

Purified B lymphocytes (5.10 5 ; purity>95%) were stimulated in a 48 well plate at a concentration of 1.10 6 cells/ml using 50 ng/ml of phorbolmyristate acetate (PMA), 1 µg/ml of ionomycin and, for generation of regulatory B cells, additional 10 µg/ml of CpG Oligodeoxynucleotide (ODN) 2006 (Invivogen, Toulouse, France). After three days of culture, supernatants were collected and 5.10 5 B lymphocytes were stimulated with PMA/ionomycine with 1 µl/million of cells of GolgiStop solution (BD Biosciences, Le Pont de Claix, France) for four hours and then stained for flow cytometry analysis. Cells were then washed, stained and then fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences). Finally, cells were stained with intracellular antibodies.

Interferon-gamma (IFN-γ), IL-4, IL-6, IL-9, IL-17, IL-21 and Tumor necrosis factor alpha (TNF-α) production by B or T lymphocytes were assessed after PBMC stimulation for 4 hours with 50 ng/ml of PMA, 1 µg/ml of ionomycin and 1 µl/million of cells of GolgiStop solution.

Cells were then washed and stained.

Quantification of cytokine production in cell culture supernatants, HC or SSc serum was assessed using a multiplex assay from MesoScaleDiscovery (MSD, Rockville, MD, USA).

Flow cytometry analysis

We performed flow cytometry analysis with peripheral blood of SSc patients and healthy controls after total PBMC staining with an anti-CD19-Brillant Violet 605 (BV605), an anti-CD27-AlexaFluor700 obtained from BD Biosciences and an anti-CD21-Peridin-chlorophyll protein-Cyanin5.5 (PERCP-Cy5.5) obtained from Biolegend (London, UK). For B lymphocyte subpopulations, we further investigated membrane IgD, CD21, CD27 and CD38 expressions within CD19 + B cells (Supplemental Figure 2) as previously described [41].

Anti-CD3-AlexaFluor 700, anti-CD4-BV 421 from BD Biosciences and anti-CD8-PERCP-Cy5.5 from Biolegend were used to define T lymphocyte subpopulations. In combination with these markers, various antibodies were used to stain extracellular or intracellular proteins of B and T lymphocytes and are listed in Supplemental Introduction:

In addition to our study on patients with systemic sclerosis (SSc) which develop pulmonary arterial hypertension (PAH), we intended to evaluate the impact of PAH on B cell phenotype independently of SSc. Thus, we selected only patients with IPAH only which present similar inflammatory phenotype to what those observed in patients with PAH-SSc.

Patients and methods

Patients

In collaboration with the team of the Pr. Marc Humbert at Kremlin Bicètre Hospital, we performed phenotypic analysis of B lymphocytes in patients with idiopathic pulmonary arterial hypertension (IPAH) (n=8) which did not receive any immunosuppressant. PAH was defined as an increase in the mean pulmonary artery pressure superior to 25 mmHg at rest evaluated at Kremlin Bicètre Hospital. All patients gave a written consent. We particularly focused on phenotypic dysregulations that have been underlined in patients with PAH associated with SSc (Paper 1).

Lymphocytes culture

PBMC were isolated from peripheral blood on Ethylenediamine tetraacetic acid (EDTA)anticoagulated tubes from HC or patients with IPAH using a Ficoll-Hypaque density gradient centrifugation (Eurobio, Courtaboeuf, France).

Interferon-gamma (IFN-γ), IL-4, IL-6, IL-9, IL-17, IL-21 and Tumor necrosis factor alpha (TNF-α) production by B or T lymphocytes was assessed after PBMC stimulation for 4 hours with 50 ng/ml of PMA, 1 µg/ml of ionomycin and 1 µl/million of cells of GolgiStop solution.

Cells were then washed and stained as described (Jung et al., 1993).

Flow cytometry analysis

We performed flow cytometry analysis with peripheral blood of SSc patients and healthy controls after total PBMC staining with an anti-CD19-Brillant Violet 605 (BV605), an anti-CD27-AlexaFluor700 obtained from BD Biosciences and an anti-CD21-Peridin-chlorophyll protein-Cyanin5.5 (PERCP-Cy5.5) obtained from Biolegend (London, UK). For B lymphocyte subpopulations, were analyzed for the expression of membrane IgD, CD21, CD27 and CD38 expressions within CD19 + B cells (Figure 1) as previously described (Kaminski et al., 2012).

Anti-CD3-AlexaFluor 700, anti-CD4-BV 421 from BD Biosciences and anti-CD8-PERCP-Cy5.5 from Biolegend were used to define T lymphocyte subpopulations. In combination with these markers, other antibodies were used to stain extracellular or intracellular proteins of B and T lymphocytes.

In longitudinal experiments, cytometer settings were standardized using BD Cytometer setup, SPHERO Rainbow Calibration Particles (BD) and BD Combeads. Flow cytometry acquisition was assessed using a BD LSR Fortessa at the cytometry plateform of Cochin Institute (Cybio) and data were analysed using FlowJo analysis software (Treestar, Ashland, OR, USA).

Statistical analysis

All analysis were carried out using Prism 6 (GraphPad, La Jolla, CA, USA) with statistical significance defined as:* p-value < 0.05; ** p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001. Non parametric tests including Mann-Whitney U-test were used for two group independent comparisons between these two groups.

Results

Peripheral B lymphocytes from patients with IPAH are moderately activated

The proportion and amounts of B or T lymphocyte weren't significantly modified in patients with IPAH compared to patients with PAH-SSc, SSc only or healthy controls (HC) (Figure 1).

Increased proportions of B lymphocytes expressing CD95 were observed in patients with IPAH compared to HC (43.8% vs. 24.5%; p=0.007) which is similar to what was observed in patients with either SSc or PAH-SSc (Figure 2). CD5, CD69 or CD86 percentages were not significantly different in patients with IPAH which is consistent to what was observed for patients with PAH-SSc for CD69 and CD86 while the proportion of CD5 was slightly increased in PAH-SSc patients without reaching significance in this small cohort. with PAH-SSc or SSc only (Figure 3). We did not evidence differences for expression of IL-21R or CD72 within the various groups of patients and HC. from HC. This resul is very different from patients with either SSc or PAH-SSc (Figure 3).

However, B lymphocytes from patients with IPAH produced higher levels of IL-6 than HC, PAH-SSc and SSc patients (Figure 4). (Denton, 2015;Dumoitier et al., 2014b). Patients' prognosis remains poor, mostly depending on lung involvement caused by both fibrotic mechanisms leading to interstitial lung disease (ILD) and/or vascular abnormalities leading to pulmonary arterial hypertension (PAH) (Launay et al., 2013;Steen and Medsger, 2007). Pathophysiological mechanisms need to be unraveled in order to identify biomarkers that could help both to detect and treat these complications at an early stage.

Significant progress has been made in understanding this condition's pathophysiology. The "ménage à trois" between immune cells (mainly Th2 cells and B cells), endothelial cells and fibroblasts is currently the main hypothesis leading to the disease's onset, which also requires exposure to environmental factors and genetic predisposition (Denton, 2015;Dumoitier et al., 2014b). Th2 cells are increased in the blood of patients with SSc and overrepresented in SSc tissues (Scaletti et al., 2002;Truchetet et al., 2011). Th2-polarized T cells release profibrotic mediators such as interleukine-4 (IL-4), IL-5, IL-6, IL-10, IL-13 which lead to fibrosis (O'Reilly et al., 2012). High levels of IL-4 and IL-13 were detected in serum and tissues from SSc patients (Hasegawa et al., 1997;Vettori et al., 2014). In SSc, B cells are characterized by polyclonal B cell activation and production of both specific autoantibodies and profibrotic cytokines such as IL-6 (Dumoitier et al., 2014b). Autoantibodies targeting endothelial cells and fibroblasts were also suggested to play a key role in the onset of vascular and fibrotic damages during SSc (Dib et al., 2012;Terrier et al., 2010). One of the key players involved in the genesis of SSc's fibrotic process is transforming growth factor-β (TGF-β), i.e. a potent growth factor which also promotes cell migration and differentiation of fibroblasts into myofibroblasts.

Targeting TGF-β seems to be a promising treatment option (Lafyatis, 2014;Rice et al., 2015).

Basophils are rare granulocytes that link innate and adaptive immune responses. They have long been associated with parasitic infections and allergy, and a strong level of evidence has been accumulated to support their ability to influence a Th2 oriented immune response (Karasuyama and Yamanishi, 2014). During the past decade, substantial efforts have been made

to better understand their role in the pathophysiology of various immunological disorders.

Noteworthy, they play an important role in innate immune responses by producing key mediators (e.g. histamine and IL-4) which can promote alternatively activated macrophages (Egawa et al., 2013) and stimulate B cells. Thus, they contribute to enhance antibody production in physiological conditions (Chen et al., 2009;Denzel et al., 2008) and autoimmune diseases (Charles et al., 2010). Recently, murine studies of basophils in graft-versus-host (GvH) disease have also emphasized basophils' possible profibrotic functions. In a cardiac GvH model, Schiechl G et al. reported that basophils trigger the development of fibrosis (Schiechl et al., 2016). Interestingly, it has also been shown that TGF-ß promote basophils' differentiation (Sillaber et al., 1992). Thus, we anticipated that basophils could be involved in SSc's pathophysiology.

Methods

Patients

Peripheral blood was obtained from 65 untreated patients with SSc fulfilling the American et al., 1988).

Peripheral blood was obtained from 38 healthy controls (HC) who were included at the "Etablissement Français du Sang", Saint Antoine Hospital, Paris, France.

All patients and HC gave a written informed consent. Blood was collected with approval of the ethics committee of the "Groupe hospitalier Pitié-Salpêtrière" and the study conforms to the principles outlined in the Declaration of Helsinki.

Basophil analysis and basophil activation test

For basophil baseline analysis and basophil activation test, 1 mL of whole blood was harvested in ethylenediamine tetraacetic acid (EDTA)-anticoagulated tubes from HC or patients with SSc.

Five hundred microliters of whole blood was used for baseline basophil analysis and 500 µL of whole blood was used for basophil activation test. Basophil activation test was performed using 50 ng/ml of phorbol myristate acetate (PMA), 1 µg/ml of ionomycin, 1 µg/ml of IL-3 (Sigma-Aldrich, Saint-Quentin Fallavier, France) and 1 µg/ml of immunoglobulin E (IgE) (Abcam, Paris, France) for 2 hours before staining for flow cytometry analysis.

Human basophilic KU812F cells

The basophilic cell line KU812F (Kishi, 1985;Okamura et al., 1989)

B lymphocyte functional study

B lymphocytes were sorted from peripheral blood using a Ficoll-Hypaque density gradient centrifugation (Eurobio) and a magnetic negative isolation with a pan B-cell isolation kit II from Milteny Biotec ® (Ref 130-101-638, Paris, France). We cultured 5.10 5 B lymphocytes from either HC or SSc patients at the concentration of 10 6 cells/mL in a 24-well plate for three days in presence or absence of KU812F cells at a concentration of 10 4 cells/ml. After three days, we performed flow cytometry analysis.

Fibroblasts functional study

Skin biopsies were performed in involved skin from patients with SSc. Normal human dermal fibroblasts were cultured from skin biopsies obtained from HC. Biopsy specimen were cut into small pieces and seeded into Petri dishes and then into 175 cm 2 plastic flasks. Patients and HC fibroblasts were grown in Dulbecco's modified Eagle's medium (DMEM) obtained from Gibco, with 10% heat-inactivated FCS at 37°C in 5% CO2.

At the third passage, fibroblasts from HC or SSc patients were plated in a 96-well plate at a concentration of 2500 cells/well in the presence or absence of human basophilic KU812F cells, with or without anti-TGF-ß monoclonal antibody (mAb) (Sigma-Aldrich). Triplicate wells were used for each concentration of KU812F including 625 cells/well 1250 cells/well, 2500cells/well and 5000 cells/well. Proliferation and viability of fibroblasts were measured using xCelligence real-time cell analyzer (ACEA Biosciences Inc., San Diego, CA, USA).

Flow cytometry analysis

We performed flow cytometry analysis with peripheral blood of SSc patients and healthy controls. We stained basophils with an anti-CD45-AlexaFluor700 (AF700) antibody ( Ab ). B lymphocytes were identified as CD19 + cells. To perform intracellular staining cells were washed, stained, then fixed and permeabilized using Cytofix/Cytoperm kit (BD Biosciences). In combination with these markers, various Abs were used to stain extracellular or intracellular proteins of basophils and B lymphocytes, as listed in Table E1.

In longitudinal experiments, cytometer settings were standardized using BD Cytometer setup, SPHERO Rainbow Calibration Particles (BD) and BD Combeads. Flow cytometry acquisition was assessed using a BD LSR Fortessa at the cytometry plateform of Cochin Institute (Cybio) and data were analyzed using FlowJo analysis software vX.0.7 (Treestar, Ashland, OR, USA).

Statistical analysis

Non-parametric tests including Mann-Whitney U-test were used for independent two group comparisons. Comparisons between fibroblast and B-lymphocyte responses to basophils were analyzed using paired t-tests. Correlations of protein expressions were performed using Spearman's rank correlation coefficient. All analyses were carried out using Prism 6

(GraphPad, La Jolla, CA, USA) with statistical significance defined as: * P < 0.05; ** P < 0.01; ***P < 0.001; ****P < 0.0001.

Results

Increased proportion of activated peripheral basophils in patients with systemic sclerosis

Sixty-five consecutive untreated patients with SSc, 53 lcSSc and 12 dSSc, were included in this study (Table E2). The proportion of peripheral basophils was higher in patients with SSc than HC as evaluated by the percentage of basophils among CD45 + Lin -CD11c -PMBCs (median

Basophils' functional markers in patients with systemic sclerosis

In order to analyze the potential role of basophils in patients with SSc, we checked for functional markers. We did not see any difference in the proportions of cells expressing CD178, CD44, CD62L, CD178, CD196, or HLA-DR. However, we found that the MFI quantification of CD154 (CD40 ligand) within basophils was significantly higher in patients with SSc than in HC (854 [541 -1334]

Basophils stimulate B cells of patients with systemic sclerosis

We further checked for cytokine production by basophils in patients with SSc. We found that basophils produced B-cell activating factor (BAFF) ( producing cells (21.2% [16.5 -29.9] vs 14.9 [12.3-16.7]; P < 0.05) (Figure 5E) and TGF-β We wondered whether basophils from SSc patients produce TGF-ß. Strikingly, we found that the proportion of TGF-ß-secreting basophils was higher in SSc patients as compared to HC We found that the proliferation of fibroblasts from SSc patients was higher when cultured with activated 1250 KU812F basophils than with unstimulated KU812F basophils (Fig 6 ,B).

Moreover, this proliferation was higher than that of fibroblasts from HC also cultured with 1250 KU812F activated basophils (Fig 6 ,A). Conversely, compared to their proliferation when cultured with unstimulated KU812F basophils, the proliferation of HC's fibroblasts was not increased when these were cultured with activated KU812F basophils (Fig E5 ,B). No difference between HC and SSc patients' fibroblast proliferation was found when KU812F basophils were not activated (Fig E5 ,A).

To confirm that this difference of proliferation was TGF-ß-mediated, we first confirmed that KU812F basophils were able to secrete TGF-ß (Fig 6 ,C). We then tested TGF-ß's inhibition on the proliferation of patients' dermal fibroblasts cultured with activated KU812F basophils and observed that the inhibition of TGF-ß decreased the proliferation rate of patients' fibroblasts to baseline values (Fig 6,D). Altogether these results demonstrate that activated basophils enhance the proliferation of fibroblasts of patients with SSc in a TGF-ß-dependent manner.

Discussion

In this study, we have identified in patients with SSc that peripheral basophils are activated and promote fibrosis through a TGF-ß-dependent manner compelling evidence for a key role of these cells in the pathophysiology of the disease.

To date, to our knowledge a very limited number of studies focusing on the role of basophils in SSc have been published. Previous works highlighted this cell's ability to release histamine which is increased in patients with SSc (de Paulis et al., 1991). An impairment of the inhibitory effect of sodium on basophil-related histamine release (Tedeschi et al., 1996) and a reduced priming effect of IL-3 on IgE-mediated histamine release (Tedeschi et al., 1998) have also been reported. These results suggest that the histamine release of PBMC's from SSc patients is altered. However, this is to our knowledge the first study to decipher basophils' activation status and functions in patients with SSc. Indeed, we herein provide a large description of basophils' biological functions in SSc patients and investigated basophils-lymphocytes' and basophilsfibroblasts interactions. Overall, our study demonstrates that basophils are activated in SSc patients, as characterized phenotypically by an increased expression of CD203c and, a downregulation of CCR3, and functionally by an increased production of both BAFF and TGFß.

Next, this is also the first report showing that there is a correlation between basophils' characteristics and SSc patients' clinical manifestations. Interestingly, we found that the prostaglandin D2 receptor CRTH2 was associated with disease features. CRTH2 is an identification marker of both basophils (Eberlein et al., 2015) and type 2 innate lymphoid cells (ILC2s) (Wohlfahrt et al., 2016), which have already been reported to be associated with disease fibrosis. Wohlfahrt et al. recently reported that ILC2 counts correlate with the extent of fibrosis as assessed by the mRSS (Wohlfahrt et al., 2016). Moreover, Boin et al reported that SSc patients with ILD had lower CCR5/CRTH2 T cell ratios than SSc patients without ILD (Boin et al., 2008). Our results may seem to be in contradiction with these studies. However, in our study, we found a correlation between disease manifestations and MFI, whereas disease manifestations correlated with CRTH2 + cell counts in the two previous studies. Altogether these two studies advocate for a Th2-cytokine environment favored by CRTH2 on T cells and ILC2s, which enhances fibrotic manifestations. Additionally, we showed that the expression of CRTH2 at the surface of basophils negatively correlates with vascular involvement of the disease, notably PAH, which is of interest as stable synthetic analogues of prostaglandin are used for the treatment of SSc associated-PAH (Badesch et al., 2000). Thus, the expression of CRTH2 at the surface of basophil appears to be a potential marker of disease severity.

Another original finding of our work is the evidence for a participation of basophils in the fibrotic mechanisms of SSc. Firstly, we showed that basophils stimulate B cells from SSc patients to induce IL-6 and TGF-β secretion. This result is in agreement with a recent report demonstrating that B cells secrete both IL-6 and TGF-β in SSc patients resulting in activation of normal and SSc fibroblasts (Dumoitier et al. unpublished data). We suggest that basophils interact with B cells through higher BAFF secretion, leading to increased B cell's survival and IL-6 and TGF-ß secretion. Basophils are already known to stimulate B cells in autoimmune and inflammatory diseases such as systemic lupus erythematosus (Meyer-Bahlburg and Dijkstra, 2016) or IgG4-related disease (Watanabe et al., 2013). Basophils' ability to stimulate B cells relies on the secretion of BAFF and IL-4 (Merluzzi et al., 2015). Although we failed to detect an increased secretion of IL-4, we provided evidence for an overexpression of CD154 that could increase B-cell activation in patients with SSc. Interestingly, an increased expression of CD154 at the surface of basophils has already been reported in asthma (Devouassoux, 2000) and allergy (Siegmund et al., 2000). We observed that B lymphocytes do not overexpress CD40 at their surface when cultured with KU812F basophils, which express CD154 (Hirano et al., 2006).

Still, the engagement of CD40 at the surface of B cells by CD154 is required for an optimal Bcell response (Lee et al., 2003) and auto-antibody production (Xu et al., 2015).

Here, we also demonstrate that basophils closely interact with fibroblasts. Interaction between fibroblasts and basophils has been previously discussed in a mouse model of atopic dermatitis (Jiao et al., 2015) and it has been shown that basophils express epidermal growth factor, which can induce proliferation and differentiation of fibroblasts (Qi et al., 2010;Shoyab et al., 1989).

The secretion of IL-4 by basophils, a well-known fibrogenic cytokine, also enhances this interaction. In response to this cytokine, human fibroblasts are able to synthesize elevated levels of extracellular matrix proteins which concur to fibrosis (Postlethwaite et al., 1992). In line with these findings, Schiechl et al. have recently reported that both basophils and IL-4 play crucial roles for the activation of fibroblasts and the development of organ fibrosis in a mouse model of cardiac GvH disease (Schiechl et al., 2016). Interestingly, we bring evidence that basophils' TGF-ß secretion is involved in organ fibrogenesis in patients with SSc (Fig 6). It is well established that TGF-ß is one of the key players in SSc pathophysiology and targeting TGF-ß is considered as a promising strategy (Rice et al., 2015). The ability of basophils to produce this cytokine has not been previously reported. The TGF-ß gene being expressed by the KU812F basophil cell line (Okamura et al., 1989), we believe that the model used in the present study is robust and able to explore the functionality of basophil activation and TGF-ß secretion on fibroblasts from patients with SSc. Previous studies have suggested that monocytes, macrophages, mast cells and fibroblasts produce TGF-ß in SSc (Hasegawa et al., 2004b;Stevenson et al., 2008;Hügle et al., 2011Hügle et al., , 2012)). Identifying additional cells that produce an excess of TGF-ß is therefore of major relevance in the context of this connective tissue disease.

In conclusion, this study provides evidence that basophils, through interactions with both B cells and fibroblasts exert a pro-fibrotic role in SSc's pathogenesis. Further studies are warranted in order to assess whether targeting these cells could be of interest in daily practice. particularly those with detectable autoantibodies in which B lymphocyte terminal differentiation is commonly altered. However, autoimmunity may also be defined as a rupture of tolerance independently of antibodies targeting the "self" or autoreactive plasmocytes expansion.

In AAV, we observed that B-cell compartment was unbalanced, notably in MPA patients, with increased proportion of naïve B cells and decreased circulating memory B cells, whereas no significant difference was found in GPA patients. This result is in agreement with previous studies that we recently reviewed (Dumoitier et al., 2015). Besides alteration of the B-cell maturation, we identified for the first time that MPA patients had an increased proportion of CD21 -/lo B lymphocytes, which were previously found enriched in autoreactive B cells in Sjögren's syndrome, hepatitis C virus-related cryoglobulinemia vasculitis, systemic lupus erythematosus and rheumatoid arthritis (Isnardi et al., 2010;Saadoun et al., 2013). Moreover, this subset of autoreactive B cells was more frequent in MPO-positive vasculitis in spite of a differentiation between GPA and MPA. This result argues for a very specific deregulation of B cells associated with MPO antigens while not in PR3 positive AAV. Furthermore, if ANCA pathogenicity suggests various roles for B lymphocytes in patients with AAV, this observation also reinforces the interest of screening antigen specificity which patently influences B lymphocyte response in AAV.

In our study, we did not observe important bias in the maturation status of B lymphocytes in patients with SSc conversely to what was found in MPA patients. However, we identified the CD69 + CD95 + subpopulation as the prevalent activated subset in patients with early SSc. This report is, to our knowledge, the first identifying an immune adaptive response correlated with disease progression in systemic sclerosis. We further identified increased expression of nearly all the activation markers that we tested, showing that B cells are particularly activated in SSc patients. Moreover, in SSc patients, expression of CD69, CD95 and IL-6-R allowed discrimination between the various forms of the disease, with particular emphasis in the most severe, diffuse forms, and in the associated PAH. These data constitute the first report suggesting a different activation of B lymphocytes regarding the form and the severity of the disease. It would be of high interest to evaluate the potential deleterious roles of these B cell subpopulations in subsequent studies.

In addition, we observed in both GPA and MPA a B-cell activated phenotype with increased expression of CD69 and CD40. Both of these activation markers are known to play a role in Band T-cell cooperation and were found increased in B cells from patients with SLE or rheumatoid arthritis (Zhang et al., 2012;Medgyesi et al., 2014). These receptors also influence the development of autoreactive B cells in several experimental models like EAE and rheumatoid arthritis (Bonomo et al., 1995;Sancho et al., 2003;Monneaux et al., 2000).

Differences between GPA and MPA patients reinforced the paradigm of GPA and MPA toward autoimmunity. B cells from MPA patients were thus potentially enriched in autoreactive B cells while B cells from GPA patients displayed classical maturation subpopulations contrasting with markedly deregulated neutrophils and innate immunity (Millet et al., 2013). In the present work, we provide new evidences for a deregulation of lymphocyte subpopulations that presents similarity to what was observed in innate immune cells compartment. For instance, in GPA, we can hypothesize that neutrophil and innate immunity are the first deregulated cells whereas both T and B lymphocytes were activated through the presentation of PR3 in patients' granuloma as previously reported by Voswinkel and colleagues (Voswinkel et al., 2006). Indeed, this was not the case for MPA patients. We also found that BAFF receptor expression on B cells was dramatically decreased in both GPA and MPA which could be related to the increased activation of the BAFF pathway in AAV (Vincent et al., 2013). This finding also revealed a potential interplay between neutrophils and B cells, as neutrophils highly express BAFF in patients with GPA, which could participate in B cell activation (Holden et al., 2011b;Scapini et al., 2008).

In SSc, presence of activated basophils also participates in the activation of B cells and fibroblasts through the secretion of IL-4, TGF-β1 and BAFF, together with high expression of CD40L. Moreover, the secretion of TGF-β1 by basophils could be an interesting new field of investigation in fibrotic diseases such as SSc. Altogether these data highlight the potential interplay between innate immune cells and B lymphocytes.

In line with these observations, patients affected by GPA presented with important activation of innate immune system altogether with an increased production of IL-6 by B lymphocytes correlated with T lymphocytes activation, and particularly for Th2 and Th9 subsets. These observations suggest that B and T cells deregulations could act as counterparts in patients with GPA which could enhance and maintain the inflamed context.

In the present work, we observed skewed T-cell polarization toward Th2, Th9 and Th17 responses in GPA, whereas patients with MPA presented with decreased percentages of Th1 within T cells. Moreover, in both AAV, we reported deregulation of Tfh compartment, with significant differences between MPA and GPA patients, which could explain both different B lymphocyte response and maturation status in AAV.

Patients with systemic sclerosis showed several antibodies targeting mostly fibroblasts and endothelial cells. However, B lymphocytes implication in SSc is poorly documented while evidences for a deleterious role of B cells on fibroblasts have been recently reported (François et al., 2013). We reported for the first time, increased production of IL-6 and TGF-β1 in patients with SSc. Moreover, we highlighted the potential role of the IL-6/IL-6R pathway in the SSc context, as IL-6R seems particularly expressed at the membrane of B cells from patients with PAH associated with SSc. This observation is of peculiar interest since IL-6 is strongly implicated in endothelial cells remodeling.

Unexpectedly, B cells from patients with iPAH did not present similar phenotype as patients with PAH associated with SSc. However, deregulation of the IL-6/IL-6R pathway was observed in our small cohort of patients. These data highlight the critical role of IL-6 in both iPAH and SSc-PAH (Ricard et al., 2014).

In SSc, a basal activation of the B cells was observed, allowing an important secretion of TGF-ß1. The latter contributed to the proliferation of fibroblasts and to the secretion of collagen, responsible for the induction of fibrosis as observed in the pathology. This report reinforces the interest of B cell/fibroblast interplay which was previously highlighted in rheumatoid arthritis (Störch et al., 2016). These data also suggest that drugs targeting this cytokine pathway could be an interesting opportunity in systemic sclerosis while B cells could be preferentially targeted in diffuse SSc.

Vascular autoimmune pathologies regroup a vast amount of diseases related or not to B lymphocytes deregulation. However, in most of the cases, targeting B lymphocytes using anti-CD20 monoclonal antibody or B cell physiology (BAFF, IL-6, IL-4…) through immunomodulation drugs, induce promising results in animal models and clinical studies. Our To conclude, in these settings, evaluation of anti-cytokine antibodies efficiency should be an additional therapeutic strategy in addition to the direct targeting using anti-B cell monoclonal antibodies. This is particularly the cases for severe manifestation of SSc, like SSc-PAH or early diffuse SSc, in which both B cells, IL-6 and TGF-β1, seem particularly implicated. These strategies could also interrupt potential interplays between innate immune cells, T cells and pathological B cells like in GPA. This could allow the management of the various roles of B lymphocytes and not only those of auto-antibody production.

Figure 1 .

 1 Figure 1. Histopathological pattern of various human vascular disorders: normal aorta (Panel A), atheromatous aorta (Panel B), temporal artery from giant cell arteritis (Panel C) and vascular remodelling of pulmonary arterioles (idiopathic pulmonary arterial hypertension) (Panel D).

Figure 2 .

 2 Figure 2. Nailfold capillaroscopy of a healthy individual (Panel A) and in a patient with systemic sclerosis with rarefaction of vascular loops and giant capillaries (Panel B) (Smith et

Figure 3 .

 3 Figure 3. Revision of the Chapel Hill nomenclature of primary systemic vasculitis in 2012: systemic vasculitis may affect large vessels (Panel A), medium vessels (Panel B) or small vessels (Panel C); the revised Chapel Hill nomenclature proposed to discriminate between pauci-immune vasculitis (Anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitis)

Figure 6 .

 6 Figure 6. Compared features of granulomatosis with polyangiitis (GPA), and microscopic polyangiitis (MPA). The comparison between GPA and MPA includes different levels from genetic to pathophysiological characteristics and clinical manifestations illustrate the major differences between these two conditions -ANCA: antineutrophil cytoplasmic antibodies;

Figure 7 .

 7 Figure 7. Clinical manifestations during Anti-neutrophil cytoplasm antibodies (ANCA)

Figure 8 .

 8 Figure 8. Immunofluorescence pattern of Anti-neutrophil cytoplasm antibodies (ANCA) against normal neutrophil. Panel A. Perinuclear ANCA pattern (p-ANCA); Panel B. Cytoplasmic ANCA pattern (c-ANCA)kindly provided by Pr. L. Mouthon

Figure 9 .

 9 Figure 9. Immunohistochemistry of endonasal granuloma in a case of granulomatosis with polyangiitis (GPA): CD20 + B cell (Panel A) infiltrate in close contact of neutrophils expressing

Figure 10 .

 10 Figure 10. Pathophysiology of granulomatosis with polyangiitis (GPA) -ANCA: Antineutrophil cytoplasm antibodies; BAFF: B-cell activating factor;; EC: Endothelial cells; IL: Interleukin; MHC: Major histocompatibility complex; MPO: Myeloperoxydase; PR3: Proteinase 3; ROS: Reactive oxygen species; Th: T helper cell; TNF-α: Tumor necrosis factoralpha; Treg: Regulatory T cells.

Figure 11 .

 11 Figure 11. Pathophysiology of eosinophilic granulomatosis with polyangiitis (EGPA) -ANCA: Anti-neutrophil cytoplasm antibodies; DC: Dentritic cells; ECP: Eosinophil cationic protein; EPO: Eosinophil peroxidase; IFN: Interferon; IL: Interleukin; MBP: Major basic protein; MHC: Major histocompatibility complex; MPO: Myeloperoxydase; PMN:

Figure 10 .

 10 Figure 10. Physiopathology of systemic sclerosis -AECA: Anti-endothelial cell antibodies; AFA: Anti-fibroblast antibodies; BAFF: B-cell activating factor; CD: Cluster of differentiation; CTGF: Connective tissue growth factor; CXCL: Chemokine C-X-C motif ligand; ECM: Extracellular matrix; ET: Endothelin; IL: Interleukin; IRF: Interferon responsible factor; MCP: Monocyte chemotactic protein; MHC: Major histocompatibility complex; pDc: Plasmacytoid dendritic cell; PDGF: Platelet derived growth factor; ROS: Reactive oxygen species; TGF: Transforming growth factor; Th: T helper cell; (Dumoitier et al., Presse Med 2014) ANNEXE 1

Figure 11 .

 11 Figure 11. Clinical manifestations during systemic sclerosis: Superinfected digital ulcer (Panel A), Raynaud's phenomenon (Panel B), limitation of mouth opening (Panel C), computed tomography scanner showing pulmonary fibrosis (Panel D), left hand Xray with calcinosis of the pulp of the third phalange of the third finger (Panel E)kindly provide by Pr. L. Mouthon

Figure 11 .

 11 Figure 11. Summary of pulmonary arterial hypertension (PAH) pathophysiology associated with connective tissue diseases -CCL: CC chemokine ligand; CD: Cluster of differentiation; CTGF: Connective tissue growth factor; EC: Endothelial cell; Fb: Fibroblast; Ig:

Figure 13 .

 13 Figure 13. General scheme of B lymphopoiesis. The main organs and maturation steps of B-

Figure 14 .

 14 Figure 14. Early development of human B lymphocytes in the bone marrow -CLP: common Lymphoid progenitor cell; CSH: Hematopoietic stem cell; EBF: Early B-cell factor; E2A: E2A immunoglobulin enhancer-binding factors E12/E47; Pax-5: Paired box protein-5adapted from (Busslingeret al.,2004.)

Figure 15 .

 15 Figure 15. V(D)J rearrangement of immunoglobulin genes; Ig: Immunoglobulin; mRNA:

Figure 16 .

 16 Figure 16. Structure of the B-cell receptor (BCR)-CD: Cluster of differentiation; CDR: Complementary determined region; CH: Constant heavy chain domain; CL: Constant light chain domain; Ig: Immunoglobulin; VH: Variable heavy chain domain; VL: Variable light chain domain -adapted from (Burger and Chiorazzi, 2013)

Figure 17 .

 17 Figure 17. Overview of Interleukin (IL)-6 impacts in the inflammation context -CRP: Creactive protein; M1: Macrophage 1; TGF-β: Transforming growth factor beta. Th: T helper cell; Treg: regulatory T cell; VEGF: Vascular endothelial growth factoradapted from[START_REF] Rincon | Interleukin-6: from an inflammatory marker to a target for inflammatory diseases[END_REF]

Figure 18 .

 18 Figure 18. IL-6 activation pathways in fibroblasts -JAK: Janus activation kinase; SHP: small

  For years, only T lymphocytes have been proposed to correctly suppress hyperactivation of the immune system notably in the context of infection, allergy or transplantation. Thus, one of the first immunosuppressive functions reported for T cells was the homeostatic control of antibody responses by B cells(Moore et al., 2001). Regulatory T cells (Treg) exert notably their suppressive effects by producing immunoregulatory cytokines such as transforming growth factor beta (TGF-β), Interleukin (IL)-10 or IL-35. These cytokines are able to increase T lymphocyte anergy, induce Treg expansion and on the other hand to decrease T helper (Th)1 and Th17 differentiation and downregulate the expression of costimulatory molecules including CD80, CD86 and CMH class II, which affect the ability of antigen presenting cells (APCs) to activate T cells(Moore et al., 2001).Secretion of these cytokines has been associated with the expression of the transcription factor Forkhead box P3 (Foxp3) in Treg together with higher expression of CD25(Ohkura et al., 2013). Recent reports highlighted the relationship between expression of Foxp3 and IL-10 signaling as FoxP3 acts as a cotranscription factor of STAT3 in association with epigenetic modifications of IL-10 promoter leading to a ''conditional'' gain of function ofFoxP3 (Hossain et al., 2013). Also, Foxp3+ and Foxp3-precursor cells might allow the development of peripheral IL-10-expressing Treg cells by a mechanism dependent on TGF-β but independent of IL-10 showing a significant contextual cross-talk betweenIL-10 and TGF-β (Maynard et al., 2007). These findings argue for a complex interplay between IL-10, TGF-β and Foxp3 in Tregs.Recent updates have highlighted the emergent role of B cells in the secretion of both IL-10 and TGF-β and thus in Treg expansion in several autoimmune models. Thus, due to our better understanding of the mechanisms of Treg emergence, maintenance and suppressive effects, the unilateral suppressive effect of T cells over B cells has been eluded and replaced by the notion of a complex modulation interplay(Rosser and Mauri, 2015).

  CD71 hi Van de Veen et al., 2013 B10 : IL-10 producing B cells ; Br1 : regulatory B cell 1 ; CD : Cluster of differentiation If IL-10 was the only commonly used marker of Bregs, the presence in normal amounts of these cells in most of autoimmune or inflammatory models suggest also IL-10 independent pathways for the regulation by Bregs that could be impaired in these contexts. Recent reports demonstrated novel immunoregulatory function for unclassical Bregs that secrete or express other immunosuppressive molecules.The TGF-β1 is another immunosuppressive cytokine, known to promote T cell survival by inhibiting activation-induced cell death and to block their proliferation by inhibiting IL-2 production via Smad3(Li et al., 2006). This factor also promotes Treg while inhibiting Th1 and Th2 development via the blockage of the transcription factors required for expression of IFN-γ (T-bet) and IL-4 (GATA-3)(Heath et al., 2000; Horwitz et al., 2003).Several studies have shown that IL-10 and TGF-β1 cooperate to downregulate immune responses especially within regulatory T cells(Levings et al., 2002). The production and action of these two cytokines are interrelated and involve a positive feedback loop in which each cytokine enhances the expression of the other. Indeed, lymphocytes from mice deficient for IL-10 produce less TGF-β1 and exogenous IL-10 was shown to stimulate TGF-β1 production in vitro(Seder et al., 1998; De Winter et al., 2002). IL-10 not only enhances the production of TGF-β1, but can also control the ability of the cells to respond to the cytokine(Cottrez and Groux, 2001). These findings therefore revealed an interrelation between TGF-β1 and IL-10 in immune tolerance.A subset of B-cells secreting TGF-β1 named Br3, has been described for the first time by Lotzet al. in 1994. TGF-β1 is produced by both CD5+ and CD5-B cells from autoimmune-prone NZB mice(Douglas et al., 1997) and is also expressed by normal human B cells. High levels of TGF-β1 secretion by ex vivo LPS stimulated splenocytes has been associated with their ability to down-regulate pathogenic responses in autoimmune diabetes through an induction of CD4+ apoptosis(Tian et al., 2001). Thus, this TGF-β1 production has been correlated with the Bregs suppressive function. Indeed, TGF-β1 is critical for the induction of Tregs(Chen et al., 2003).Singh et al. demonstrated in vitro that Bregs could induce Foxp3 expression by activated T cellsin a TGF-β1-dependent manner(Singh et al., 2008) .Taking together, TGF-β1 may have an important role in Bregs control of autoimmunity(Mauri and Ehrenstein, 2008).There are also descriptions of human IL-10 and TGF-β1 producing CD25 hi CD27 hi CD86 hi CD1d hi B lymphocytes able to suppress autologous T cell proliferation and induce the generation of FoxP3 + CTLA4 + Tregs in a cell-to-cell contact-mediated and TGF-β1 dependent mechanism(Kessel et al., 2012). In contrast to IL-10, TGF-β1 promotes apoptosis of CD5 + B cells(Vitale et al., 2010) and could act as a negative regulator of B10 cells.Interestingly, TGF-β1 is well known to transform naive T-cells into inducible Tregs(Chen et al., 2003; Marie et al., 2005) enhancing the regulatory environment, but, in combination with IL-6, TGF-β1 could skew naive T-cells toward IL-17-secreting cells and push the balance towards effector T-cells(San Segundo et al., 2013). These similarities between Treg and Bregs led the researchers to investigate other T cells cytokines with regulatory functions that could be secreted by B cells.Besides TGF-β1 and IL-10, which have been described for years, new regulatory cytokines have emerged in the B and T cells interface. Conversely to IL-12 and IL-23, which have been associated with chronic inflammatory diseases, IL-35, a member of the same cytokine family, was recently discovered and linked to autoimmune diseases(Vignali and Kuchroo, 2012).Similarly to other signals described in this review, IL-35 biological activity was first observed in Treg with decreased expression of pro-inflammatory cytokines by activated T cells in an infectious context(Collison et al., 2007) while, IL-35 producing B cells seem critical cells in autoimmune diseases(Shen et al., 2014). This specific subpopulation of Bregs represents an interesting overlapping population which secrete both IL-10, TGF-β1 but also Granzyme B (GrzB)(Shen et al., 2014; Wang et al., 2014). Morevover, this population presents with a plasmocyte-like phenotype like several other Bregs subpopulation already described(Rosser and Mauri, 2015).

Furthermore, secretion

  of IL-35 by Tregs and Bregs has been found highly correlated to EAE, multiple sclerosis, SLE and IBD activity and to the response to treatment(Cai et al., 2015; Fonseca-Camarillo et al., 2015). Interestingly, recent publications in animal models have shown that depletion of IL-35 does not result in loss of tolerance(Choi et al., 2015). These findings argue for a powerful balance in B cells between IL-6/TNF-α on one side and IL-10/IL-35 on the other, which could play a key role in both chronic inflammation and autoimmune diseases(Shen and Fillatreau, 2015).Toward cytokine secretions, regulatory functions of B cells have also been found associated with the expression of several membrane markers inducing cell-death like PD-L1, CTLA-4 or Fas-L, together with Granzyme B and TRAIL expression. These findings argue for multiple suppressor mechanisms notably in autoimmunity (Table4).AIM OF THE STUDYAim of the StudyAutoimmune vascular diseases are characterized by the production of autoantibodies by activated B cells. A number of studies has addressed the characterization of this specific production and therapies, targeting B cells, have demonstrated promising strategies in several diseases such as ANCA associated vasculitis. In contrast, several other pathologies indicate resistance to these treatments while the activation of B cells is demonstrated by its implication in vascular remodelling, inflammatory mechanisms and fibrosis related to the disease. In AAV patients, activation of innate immune cells is accompanied by important amounts of different B cells in granulomatous lesions. These B cells are activated and their BCR have been rearranged. Clinical trials have demonstrated that, in AAV patients, Rituximab (anti-CD20) is as efficient as cyclophosphamide in the primary attack treatment of AAV. Despite treatment efficacy, no consistent study has analysed the phenotype of these B lymphocytes and characterized their promising role in vascular and systemic inflammation.

Paper 1 : 3 :

 13 Phenotypical and functional analysis of peripheral B and T lymphocytes in granulomatosis with polyangiitis and microscopic polyangiitis Paper 2: Peripheral B lymphocytes secrete both interleukin 6 and transforming growth factorbeta and potentiate fibroblasts activation in systemic sclerosis Préliminary results: Analysis of B cell activation in idiopathic pulmonary arterial hypertension Paper Basophils are activated and promote fibrosis through TGF-ß secretion in systemic sclerosis Paper 1: Skewed peripheral B and T lymphocytes compartments in patients with ANCA associated vasculitis Will be submitted before thesis defense Skewed peripheral B-and T-cell compartments in patients with ANCA-associated vasculitis Nicolas Dumoitier 1,

  analyzed the percentage of IL-6 + B cells in parallel with Th/Treg subsets in patients with GPA. A positive correlation was found between IL-6 + B cells and Th2 (r=0.63; p=0.03) and Th9 (r=0.54; p=0.05) cells, but not with Th1, Th17 or Treg cells (Figure 5, Panel B and Supplemental figure 7, Panel B). Using similar analysis, we found only one negative correlation between the proportion of TNF-α + B cells and the proportion of Th1 within CD4 + T cells in patients with GPA (r=-0.68; p=0.01) (Figure 5, Panel C and Supplemental figure 7, Panel B).

Figure 1 .

 1 Figure 1. Analysis of T lymphocytes distribution in patients with active ANCA associated

Figure 2 .

 2 Figure 2. Analysis of regulatory T cell and follicular T helper cell distributions in patients

Figure 3 .

 3 Figure 3. Analysis of B lymphocytes distributions in patients with active ANCA associated

Figure 4 .

 4 Figure 4. Analysis of B lymphocytes activation in patients with active ANCA associated

Figure 5 .Supplemental Figure 7 .

 57 Figure 5. Analysis of cytokine productions by B lymphocytes in patients with active ANCA

Figure 1 .Figure 2 .

 12 Figure 1. Maturation status of B lymphocytes populations from healthy controls (HC) and

Figure 3 .

 3 Figure 3. Receptor expression at the membrane of peripheral CD19 + B lymphocytes from healthy controls (HC) and patients with systemic sclerosis (SSc). Inhibitory (CD22 + (left), CD32 + (middle) and CD72 + (right)) (Panel A), IL-6R + (lower left) and IL-21R + (lower right)) (Panel B) receptors was analyzed at the surface of CD19 + B lymphocytes from 20 HC (•) and

Figure 4 .

 4 Figure 4. Intracellular cytokine staining of peripheral B lymphocytes from healthy controls

Figure 5 .

 5 Figure 5. Cytokine concentrations in sera and supernatants of peripheral stimulated B cells

Figure 6 .

 6 Figure 6. Impact of purified B lymphocyte supernatants from healthy controls (HC) and

Figure 1 :

 1 Figure 1: Analysis of B and T lymphocytes amounts per milliliter of blood

Figure 2 :

 2 Figure 2: Analysis of activation marker expression within CD19 + B lymphocytes

Figure 3 :

 3 Figure 3: Analysis of cytokine receptor expressions within CD19 + B lymphocytes

Figure 4 :

 4 Figure 4: Analysis of IL-6 secretion within CD19 + B lymphocytes

  ), an anti-lineage (Lin) cocktail-Pacific Blue (PB) Ab, an anti-CD11c-PB Ab obtained from Biolegend (London, UK) an anti-FCƐRI-Fluorescein isothiocyanate (FITC) Ab obtained from eBioscience (Paris, France) and an anti-CD123-Phycoerythrin Cyanin 7 (PE-Cy7) Ab obtained from BD Biosciences (Le Pont-de-Claix, France). Basophils were identified in peripheral blood mononuclear cells (PBMC) as Lin -CD11c -CD45 + FCƐRI + CD123 + cells (Fig E1). We stained sorted B lymphocytes with an anti-CD19-Brillant Violet 605 (BV605) Ab and an anti-CD21-Peridin-chlorophyll protein-Cyanin5.5 (PERCP-Cy5.5) obtained from Biolegend (London, UK

[

  interquartile range] 3.5%[2.0 -6.4] versus (vs) 2.6%[1.2 -4.1]; P < 0.05) without any difference between patients with lcSSc or dSSc. There was no difference in the absolute count of basophils between patients and HC (FigE1). Interestingly, basophils of patients with SSc exhibited more activation markers than those of HC (Fig[START_REF] Jennette | revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides[END_REF]. Compared to HC, we found in patients with SSc a higher proportion of CD203c + cells among basophils(37.5 % [24.3 -59.0] vs 25.6%[13.6 -41.7]; P < 0.001) (Fig1, A), an increased quantification of this marker's mean fluorescence intensity (MFI) (624.5 [462.3 -897.3] vs 440 [324.3 -650]; P < 0.001) (Fig1, C) and a lower proportion of C-C chemokine receptor type 3 (CCR3) + cells (70.2%[35.3 -88.2] vs 92.8% [77.3 -97.5]; P < 0.001) (Fig 1, B). Expression of activation markers did not differ between patients with lcSSc or dSSc. Upon activation, the proportion of basophils between patients with SSc and HC was similar for both CD203c + cells and CCR3 + cells (FigE2). As for HC, other identification markers of basophils including CD22 and CD38 were also expressed by basophils of patients with SSc without any difference upon activation(Fig E3).

  Finally, MFI quantification of CRTH2 among basophils positively correlated with patients' diffusing lung capacity of carbon monoxide (r = 0.60, P < 0.05) (Fig 3, C) and negatively correlated with patients' modified Rodnan skin score (r = -0.77, P < 0.01) (Fig 3, F).

Fig 4 )

 4 , since the MFI quantification of BAFF within basophils was higher in SSc patients than inHC (1551HC ( [1001 -4373] -4373] vs 857 [722 -1498]; P < 0.05) (Fig 4, D).To further decipher the impact of basophils on B lymphocytes from SSc patients, we looked at the functional properties of B cells isolated from the peripheral blood of patients with SSc or HC when cultured with KU812F basophils(Fig 5). After three days of culture, we found that activated KU812F basophils were able to decrease the proportions of CD95 + cells(9.4% [5.4 - 14.1] vs 10.4%[6.0 -14.8]; P < 0.05) (Fig 5, D) and to increase the proportion of IL-6

( 5 .

 5 6% [4.7 -7.2] vs 4.1[2.6-5.4]; P < 0.05) (Fig 5, F) among B cells isolated from patients with SSc. Such differences were only seen when KU812F basophils were activated (Fig5).On the contrary, we failed to observe similar discrepancies when we compared the proportion of CD40 + (Fig5, A), CD86 + (Fig 5, C) or BAFF-receptor + (Fig 5, B) B cells of patients with SSc vs HC. Basophils enhance the proliferation of fibroblasts of patients with systemic sclerosis in a TGF-ß dependent manner.

( 21 .

 21 9% [15.1 -35.1] vs 9.7%[5.0 -17.5]; P < 0.05) (Fig4, C). To further analyze the role of this secretion, we next tested whether KU812F basophils were able to influence fibroblasts' proliferation(Fig 6).

Figure legends FIG 1 .FIG 2 .FIG 3 .

 legends123 Figure legends FIG 1. Expression of activation markers of peripheral basophils from patients with SSc

FIG 4 .FIG 5 .FIG 6 .

 456 FIG 4. Intracellular cytokine staining of peripheral basophils from patients with SSc.

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6

 123456 Figure 1

  results place B lymphocytes, besides their role in antibody production, as important pathophysiological players through the secretion of pro-inflammatory and pro-fibrotic cytokines such as IL-6 and TGF-ß1 which are both implicated in endothelial cells activation in autoimmune vascular diseases. Based on these findings, we might now propose a new classification of autoimmune vascular diseases: those with deregulation in B cells maturation and differentiation (MPA), those with strong pro-inflammatory response related to innate immune cells (SSc and GPA), those with important activation of IL-6 pathway and endothelial cell remodeling (SSc-PAH and iPAH) and finally those with important pro-fibrotic secretions (SSc).

Table

Table 1 .

 1 Systemic sclerosis (SSc) and SSc-like disorders following specific environmental exposuresadapted from(Varga et al., 2016)

	Exposure	Disease		Evidence
	Crystalline silica/silica dust	SSc		Meta-analysis
	Solvents	SSc		Meta-analysis
	Vinyl chloride monomer	Vinyl chloride disease	Investigation of outbreak
	Adulterated cooking oil	Toxic oil syndrome		Investigation of outbreak
	Tryptophan	Eosinophilic	myalgia	Investigation of outbreak
		syndrome
	Gadolinium	Nephrogenic	systemic	Multiple case series
		fibrosis
	Drugs
	Bleomycin	Pulmonary fibrosis		Multiple case series
	Pentazocine	Localized dermal fibrosis at
		injection site

Figure 11 and Table 2) (

  van den Hoogen et al., 2013).

	Thus, both fibroblasts and endothelial cells participate in the activation of innate and adaptive
	immune cells which could induce specific clinical and immunological presentation of SSc.
	iii) Clinical and immunological presentation
	First classification of SSc criteria in the 1980' were developed in patients with longstanding
	SSc (Masi and Subcommittee For Scleroderma Criteria of the American Rheumatism
	Association Diagnostic and Therapeutic Criteria Committee, 1980). However, according to the
	broader spectrum of SSc clinical manifestation, including skin thickening (measured using the
	modified Rodnan Skin Score (mRSS), puffy finger, digital ulcers, telangiectasia, abnormal
	naifold capillaroscopy, PAH, interstitial lung disease, Raynaud's phenomenon or auto-
	antibodies, a new table of criteria was published in 2013 that would enable identification of
	individuals with SSc (

Table 2 .

 2 Summary of immunological and clinical impacts of Interleukin (IL)-6 in vivo -adapted

	from (Ho et al., 2015)
	Major targets	Main effects
	T-helper cells	Negatively regulates Th2 response
		Inhibition of TGF-β-induced Treg differentiation
		Together with TGF-β promotes differentiation of Th17 cells from naïve
		T cells
		In combination with IL-1β, TGF-β, and IL-23 allows polarization of
		naive Vγ9Vδ2 T cells to produce IL-17
	Hematopoietic	Controls proliferation and differentiation of the progenitor cells in many
	system	hematopoietic lineages
		Controls leukemic multipotent progenitor cell fate and regulates the
		development of chronic myelogenous leukemia
		Promotes or prevents lymphoma development by acting at distinct
		stages of hematopoietic development
		Induces activation of T and B cellsas well as hematopoiesis and lupus-
		like autoimmune manifestations in Lyn-deficient mice
	Nervous system Is required for myelin oligodendrocyte glycoprotein-induced
		experimental autoimmune encephalomyelitis
		B cell-specific IL-6 regulated Th17 responses in experimental
		autoimmune encephalomyelitis
		Maintains plasmablast-like B cells survival and increases autoantibody
		production in patients with neuromyelitis optica
		Induces CD4 + T follicular helper cell-differentiation program and
		exacerbates autoimmune reaction
	Cardiovascular	Is constitutively produced in cardiac myxomas
	system	Aggravates the severity of autoimmune myocarditis induced by cardiac
		α-myosin peptide immunization
		Increases the severity of angiotensin II-induced cardiac fibrosis
		Is protective in viral-induced autoimmune myocarditis
	Respiratory	Exacerbates pleural exudation and polymorphonuclear cell infiltration in
	system	immune-mediated lung damage
		Increases death rate in acute pancreatitis-associated acute lung injury in
		mice
		Induces Th17 cell differentiation within the lung and causes lung
		damage
	Liver	Induces production of a variety of acute phase proteins

Table 3 .

 3 Phenotypes of human Interleukin (IL)-10 producing regulatory B cells (Breg) subsets

	Subtype	Mechanism	Phenotype	References
		of suppression
	B10 cells	IL-10 production	CD24 hi CD27 +	Horikawa et al., 2013
				Yanaba et al., 2008
	Plasmablasts	IL-10 production	CD19 + CD24 hi CD27 int Matsumoto et al.,2014
	Immature cells	IL-10 production	CD19 + CD24 hi CD38 hi Blair et al.,2010
	Br1 cells	IL-10 production	CD19 + CD25 hi

Table 4 .

 4 Phenotypes of B cells associated with regulatory function

	Mechanism of regulation/suppression	Subtypes	Species Models		Phenotypes within CD19 +	References
		B10 cells	Human,	EAE,	RA,	CD1d hi CD5 +	Horikawa et al.,2013;
		Br1 cells	mouse	CHS,	islet	CD24 hi CD38 hi	Van de Veen et al.,2013
		Plasmablasts		allograft, solid	CD25 hi CD71 hi	Matsumoto et al.,2014
		Immature B cells		cancer, SLE,	CD24 hi CD27 int	Blair et al.,2010;
	IL-10 secretion	Fas-L + Breg		MS,	AAV,	CD24 hi CD38 hi PD-L1 +
		GrzB + Breg		HIV, healthy	CD5 + Fas-L +
				controls		CD5 + CD38 + CD1d + IgM + CD
		IL-35 + Breg				147 + CD86 +
						CD154 + CD25 + IDO + GrzB +
						B220 low CD5 + CD1d hi IL-35 +
		Br3	Human,	Islet transplant	TIM1 +	Lee K.M et al.,2014
			mouse	CLL		CCR6 + CXCR3 + IL10 +	Lotz M et al.,1994
	TGFβ1 secretion					IDO +	Lee JH et al., 2011
		iBreg	Human	Healthy
				controls		-
	IL-35 secretion	IL-35 + Breg	Mouse	EAE		B220 low CD5 + CD1d hi IL-10 +	Shen, Nature, 2014
							Cai, Lupus 2015
				SLE, MS, HIV,	CD5 + CD43 + CD86 + CD147 -
				solid cancer,	IL-10 -
	Granzyme B secretion	GrzB + Breg	Human	kidney transplantation	CD5 + CD27 + CD138 + CD38 + I gD -CD5 + CD38 + CD1d + IgM + CD	Blood 2006 JI 2009
						147 + CD86 +
						CD154 + CD25 + IDO + IL-10 +
			Human	DLBL, CLL,	CD5 + IL-10 +
				MM
	Fas-L expression	Fas-L + Breg	Mouse	SLE, diabetes,	RA,
				allergy,
				transplantation
	TRAIL expression	TRAIL + Breg	Human DLBL, CLL	Fas-L + ?	Sedger LM 2010 Blood
	PD-L1 expression	Immature B cells Human Kidney		CD24 hi CD38 hi IL-10 +	Khan 2015, Nature
				transplantation		Comm
	CTLA-4 expression	CTLA-4 + B cells Human AAV		-
	AAV:ANCA associated vasculitis; Breg: regulatory B cells; CD : Cluster of differntiation ; CHS: Contact hypersensitivity response;
	CLL: Chronic lymphocytic leukemia; CTLA-4: Cytotoxic T-lymphocyte-associated protein 4; DLBL: Diffuse large B cell
	lymphoma, EAE, Experimental autoimmune encephalomyelitis, GrzB: Granzyme B; HIV: Human immunodeficiency virus; IDO:
	Indoleamine-pyrrole 2,3-dioxygenase; Ig: Immunoglobulin; IL: Interleukin; MM: Multiple myeloma, MS, Multiple sclerosis, PD-
	L1: Programmed death ligand 1 ; RA: Rheumatoid arthritis; SLE: Systemic lupus erythematosus; TGF : Transforming growth factor ;
	TRAIL: TNF-related apoptosis-inducing ligand

IL-35 is able by its own to induce differentiation of both B and T cells in Bregs and Tregs respectively, through

Stat-1 and Stat-4 activation (Choi et al., 2015; Egwuagu et al., 2015)

.

Table 1 .

 1 Antibodies used for flow cytometry experiments.

	Target	Fluorochrome	Clone	Society
	BR3	Fluorescein isothiocyanate	11C1	BD biosciences
		(FITC)
	CCR6	Peridin-Chlorophyll protein-	G034E3	Biolegend
		Cyanin5.5 (PerCP-Cy5.5)
	CD3	AlexaFluor700	UCHT-1	BD biosciences
	CD4	Brillant Violet 605 (BV605) RPA-T4	BD biosciences
	CD5	BV421	UCHT2	BD biosciences

Table 2 .

 2 Clinical characteristics of ANCA-associated vasculitis (AAV) patientsIntroductionSystemic sclerosis (SSc) is a connective tissue disease characterized by fibroblasts activation, increased extra-cellular matrix synthesis and vascular remodeling(Dumoitier et al., 2014a).Activation of fibroblasts and endothelial cells, together with increased oxidative stress represent the hallmarks of SSc pathogenesis. Patients with SSc are classified depending on the extent of skin involvement: limited cutaneous SSc (lcSSc), with alterations essentially limited to the hands and face, and diffuse SSc (dSSc), with skin lesions proximal to the elbows and knees and frequent visceral involvement. Although autoantibodies have been detected in the serum of patients with SSc and are helpful for the diagnosis and evaluation of prognosis, B lymphocyte implication in the pathogenesis of SSc is still poorly defined[2; 3].

	at the time of blood sampling (n=37)
	Patient	Age	Disease	Previous	BVAS	Previous		CRP
						features		(mg/l)
	ID	(years)	duration	Treatment		during the	ANCA
			(days)			course of
						AAVs
	GPA 1	27	0	None	25	ENT, L,	PR3	66
						K
	GPA 2	24	2048	AZA, CYC	27	ENT, L,	PR3	72
						K, S
	GPA 3	48	0	None	17	ENT, L,	PR3	185
						K

AAV: ANCA-associated vasculitides, GPA: granulomatosis with polyangiitis, MPA: microscopic polyangiitis B: bowel; E: eye; ENT: ear, nose, throat; K: kidney; L: lung; PN: peripheral neuropathy; S: skin AZA: azathioprine; CYC: cyclophosphamide; MMF: mycofenolate mofetil; MTX: methotrexate; Ritux: rituximab ANCA: anti-neutrophil cytoplasmic antibody; Neg: ANCA negative; MPO: anti-myeloperoxydase; PR3: anti-proteinase 3

Table 4 .

 4

	Gender (female): n (%)

Table 2 .

 2 Association of B lymphocytes activation markers with clinical

	manifestations in systemic sclerosis patients †
	Marker/Clinical manifestation	PAH (n=7) vs no PAH	ILD (n=31) vs no ILD	PAH or ILD (n=38) vs no pulmonary involvement	Arthralgia (n=36) vs no arthralgia	Digital ulcers (n=32) vs no digital ulcers
	% CD5 + within CD19 +	14.12 vs 16.09 (p=0.82)	17.18 vs 13.50 (p=0.68)	16.21 vs 10.78 (p=0.35)	14.20 vs 14.80 (p=0.97)	14.21 vs 16.75 (p=0.64)
	% CD86 + within CD19 +	47.82 vs 19.26 (p=0.03)	34.20 vs 21.92 (p=0.49)	34.84 vs 19.42 (p=0.13)	29.75 vs 19.76 (p=0.36)	36.17 vs 18.72 (p=0.04)
	% CD95 + within CD19 +	49.78 vs 45.41 (p=0.62)	49.46 vs 45.31 (p=0.93)	50.95 vs 43.24 (p=0.47)	55.23 vs 38.16 (p=0.01)	44.79 vs 46.91 (p=0.70)
	% IL-6R + within CD19 +	46.92 vs 22.24 (p=0.01)	40.80 vs 20.99 (p=0.03)	42.12 vs 13.24 (p<0.0001)	32.47 vs 14.35 (p=0.007)	26.59 vs 24.35 (p=0.81)
	% IL-	15.77 vs	8.46 vs	11.90 vs	14.38 vs	15.77 vs
	21R + within	8.46	13.27	8.31	7.77	11.9
	CD19 +	(p=0.31)	(p=0.63)	(p=0.92)	(p=0.22)	(p=0.99)
	† % of B lymphocytes subpopulations in each clinical manifestation subgroups were
	compared using Mann-Whitney U-test.
	Significance was characterized by p-value<0.05 (light grey); p-value<0.01 (grey); p-
	value<0.001 (dark grey).
	ILD: interstitial lung disease; PAH: pulmonary arterial hypertension.

Table 3 .

 3 Primer sequences used for amplificationSupplemental

Table 4 .

 4 Antibodies used for flow cytometry experiments.

	Gene	Primer Forward			Primer Reverse
	Alpha-Smooth muscle actin	5'-CAATGGCTCTGGGCTCTGTAAG-3'	5'-TGTTCTATCGGGTACTTCAGGGTC-3'
	Collagene I	5'-CACACGTCTCGGTCATGGTA-3'	5'-CGGCTCCTGCTCCTCTTAG-3'
	Vimentin	5'-GGCTCGTCACCTTCGTGAAT-3'		5'-GCAGAGAAATCCTGCTCTCCT-3'
	Snail1	5'-CCAGTGCCTCGACCACTATG-3'	5'-CTGCTGGAAGGTAAACTCTGGA-3'
	Slug	5'-AAGGCGTTTTCCAGACCCTG-3'	5'-AAGAAAAAGGCTTCTCCCCCGT-3'
	β2microglobulin	5'-CTCCGTGGCCTTAGCTGTG-3'		5'-TTTGGAGTACGCTGGATAGCCT-3'
	Target	Fluorochrome	Clone	Society
	BR3	Fluorescein isothiocyanate	11C1		BD biosciences
		(FITC)
	CCR6	Peridin-Chlorophyll protein-	G034E3	Biolegend
		Cyanin5.5 (PerCP-Cy5.5)
	CD3	AlexaFluor700	UCHT-1	BD biosciences
	CD4	Brillant Violet 605 (BV605) RPA-T4	BD biosciences
	CD5	BV421	UCHT2	BD biosciences
	CD8	PerCP-Cy5.5	RPA-T8	BD biosciences
	CD19	BV605	SJ25C1	BD biosciences
	CD20	BV421	2H7		BD biosciences
	CD21	PerCP-Cy5.5	Bu32		Biolegend
	CD22	Phycoerythrin (PE)	HIB22	Biolegend
	CD24	PE	ML5		BD biosciences
	CD25	BV421	M-A251	BD biosciences

  College of Rheumatology (ACR) / European League Against Rheumatism (EULAR) criteria for the diagnosis of SSc (van den Hoogen et al., 2013c), followed at Cochin Hospital in the Internal Medicine department. Limited cutaneous SSc (LcSSc) was defined by skin thickening in distal areas (i.e. lower than elbows and knees), with or without facial involvement. Diffuse SSc (dSSc) was defined by the presence of proximal (i.e. upper than elbows and knees) and/or distal skin thickening, with or without facial or truncal involvement (LeRoy

  was cultured in Roswell Park Memorial Institute medium 1640 (RPMI) obtained from Gibco (Life Technologies, Saint-

	Aubin, France), with 10% heat-inactivated fetal calf serum (FCS) (Eurobio) at 37°C in 5% CO2
	atmosphere. Stimulation of 10 6 cells was performed in 1.5 ml micro tubes (Eppendorf,
	Montesson, France) with 50 ng/ml of PMA, 1 µg/ml of ionomycin, 1 µg/ml of IL-3 and 1 µg/ml
	of IgE for two hours.

  Upon stimulation, the proportion of CD54 + basophils was significantly lower in patients with SSc as compared to HC (68.3%[32.9 -77.1] vs 77.8% [71.1 -87.9]; P < 0.05) (FigE4).

	Interestingly, patients with SSc had a lower proportion of CRTH2 + basophils than HC (99.8%
	[98.9 -100] vs 100% [99.7-100]; P < 0.05) and this difference was maintained after basophil
	activation (99.8% [98.8 -100] vs 100 [100 -100]; P < 0.05) (Fig 3, A). We found that SSc
	patients with PAH had lower MFI quantification of CRTH2 among basophils (31476 [30213 -
	31633] vs 59576 [41312 -67788]; P < 0.001) (Fig 3, B). Similarly, SSc patient with gastro-
	intestinal tract involvement had a lower MFI quantification of CRTH2 among basophils (52046
	[36576 -62276] vs 69391 [57853 -73191]; P < 0.05) (Fig 3, E).

vs 485 [147 -871]; P < 0.05) (Fig 2 and Fig E4).

Table E1 .

 E1 Antibodies used for flow cytometry experiments.

	FCεRI	FITC	AER-37	eBioscience
	Target HLA-DR	Fluorochrome PerCP-Cy5.5	Clone Tu36	Society Biolegend
	BAFF IL-4	PE APC	1D6 8D4-8	ebioscience eBioscience
	BAFF-R	FITC	11C1	BD
	(BR3) IL-6	PE	MQ2-6A3	BD
	CD11c Lineage	PB PB	3.9 -	Biolegend Biolegend
	CD22 Livedead	PE BV421	HIB22 -	Biolegend LifeTechnologies
	CD38 TGF-β	PerCP-Cy5.5 APC	HIT2 TW7-16B4	BD Biolegend
	CD40	APC-Cy 7	5C3	BD
	CD44	BV 605	G44-26	BD
	CD45	AF 700	30-F11	Biolegend
	CD54	PE-Cy5	HA58	BD
	CD62L	PE-Cy5	DREG-56	BD
	CD86	BV 421	2331 (PUN-1)	BD
	CD95	PE	DX2	BD
	CD123	PE-Cy7	7G3	BD
	CD154	PE	TRAP1	BD
	CD178	APC	NOK-1	BD
	CD193	AF 647	5E8	BD
	CD196	PerCP-Cy5.5	G034E3	Biolegend
	CD203c	BV 605	NP4D6	Biolegend
	CD294
		PE	BM16	Biolegend
	(CRTH2)

Abbreviations: AF: alexa fluor; APC: allophycocyanin; APC-Cy 7: allophycocyanin-cyanin 7; BAFF: B-cell activating factor; BD: BD biosciences; BV: brilliant violet; CD: cluster of differentiation; FCεRI: receptor for the Fc region of immunoglobulin E; FITC: fluorescein isothiocyanate; HLA: human leucocyte antigen; IL: interleukine; PB: pacific blue; PE: phycoerythrin; PE-Cy5: PE-Cyanin 5; PE-Cy7: PEcyanin 7; PerCP-Cy5.5: peridin-chlorophyll protein-cyanin5.5; TGF-β: transforming growth factor-ß.

Table E2 .

 E2 Clinical characteristics of SSc patients at the time of blood sampling

	Characteristics	SSc (n=65)
	Clinical Features
	Gender (female),	(83)
	Age, median [IQR] years	58 [49-67]
	Disease duration, median [IQR] years	8 [4-13]
	Limited cutaneous SSc:	(82)
	Diffuse SSc:	(18)
	Skin sclerosis:
	-Modified Rodnan Skin Score: median [IQR]	2 [0-5]
	-Mouth opening (mm): median [IQR]	40 [33-47]
	Digital ulcers:	(31)
	Calcinosis:	(22)
	Raynaud's phenomenon:	(91)
	Telangiectasia:	(52)
	Sclerodactyly:	(38)
	Interstitial lung disease:	(45)
	DLCO, %: median [IQR]	66 [51-74]
	Pulmonary arterial hypertension:	7 (11)
	Renal involvement*:	1 (2)
	Gastro-intestinal involvement:	(55)
	Arthralgia:	(29)
	Muscle involvement:	(15)
	Anti-nuclear antibodies	(92)
	-Anti-centromere antibodies	(42)
	-Anti-topoisomerase 1 antibodies	(29)
	-Anti-RNP antibodies	4 (6)
	-Anti-PM/Scl antibodies	6 (9)
	-Anti-RNA Pol III	2 (3)
	Data are represented as n (%) unless indicated differently.
	* Renal involvement was defined as elevated serum creatinin level
	(>150μmoL/L) or renal crisis

SSc, systemic sclerosis; IQR, interquartile range, DLCO, diffusing capacity of the lung for carbon monoxide; RNP: ribonucleoprotein; PM/Scl: polymyositis/scleroderma.

Sancho D, Gómez M, Viedma F, et al. CD69 downregulates autoimmune reactivity through active transforming growth factor-beta production in collagen-induced arthritis. J ClinInvest 2003;112:872-82. doi:10.1172/JCI19112

Acknowledgments

Je tiens à remercier le Pr. Colette Kanellopoulos-Langevin d'avoir acceptée de présider mon jury de thèse et d'apporter, je suis sûr, un regard intéressant sur ce travail. Je tiens à remercier le Dr. Christophe Guignabert d'avoir accepté d'etre rapporteur de cette thèse malgré les nombreux problèmes de dates et administratifs. Votre grande expérience sur l'IL-6, les cellules endotheliales et l'hypertension artérielle pulmonaires seront un vrai plus pour ce travail et, je l'espère, permettront de mettre en place une collaboration avec notre équipe et celle du Pr.Marc Humbert. Un grand merci au Pr.Bernard Bonnotte d'avoir accepté d'être rapporteur de ce travail malgré son emploi du temps serré. Je suis ravi de pouvoir accueuillir à Paris une référence de la médecine interne et des vascularites en France et à l'étranger. I grattefully thanks the Dr. Martina Siefert for the reviewing of this thesis. Her great experience in the field of B lymphocyte dysregulation would be a great opportunity for our team. I'm honored to see you in Paris. AAV: ANCA-associated vasculitis ANCA: anti-neutrophil cytoplasm antibodies BAFF: B-cell activating factor BAFF-R: BAFF receptor BCR: B-cell receptor c-ANCA: cytoplasmic ANCA EGPA: eosinophilic granulomatosis with polyangiitis ENT: ear nose and throat GPA: granulomatosis with polyangiitis HC: healthy control IL: interleukin IFN: interferon MFI: median fluorescence intensity MPA: microscopic polyangiitis MPO: myeloperoxidase p-ANCA: perinuclear ANCA PR3: proteinase 3 SLE: systemic lupus erythematosus Th: T helper TNF-α: tumor necrosis factor-alpha Treg: regulatory T cells Acknowledgements: The authors are grateful to the Cochin Cytometry and Immunobiology Facility. The authors are grateful to the Unité de Recherche Clinique (URC) of Cochin hospital and particularly, Severine Ait el Ghaz-Poignant. ND received financial support from the Centre of Excellence, LabEx Inflamex, Université Paris-Diderot, Paris, France (ANR-11-IDEX-0005-02). JL received financial support from the Société Nationale Française de Médecine Interne (SNFMI) and from the Fondation pour la Recherche Médicale (FRM). This work was performed within the Département Hospitalo-Universitaire (DHU) AutHors (Autoimmune and Hormonal Diseases).

Acknowledgements:

The authors are grateful to the Cochin Cytometry and Immunobiology Facility. The authors thank Lionel Guitat for the primer designing. ND received financial support from the Centre of Excellence, LabExInflamex, Université Paris-Diderot, Paris, France (ANR-11-IDEX-0005-02). This work was performed within the Département Hospitalo-Universitaire (DHU) AutHors (Autoimmune and Hormonal Diseases).

Acknowledgements:

The authors sincerely thank the patients with systemic sclerosis and healthy volunteers for taking part in this study. We are also grateful to the Cochin Cytometry and Immunobiology Facility (Cybio).

Funding : BC received financial support from the INSERM (Poste d'accueil), from the Département Hospitalo-Universitaire AutHors (Autoimmune and Hormonal Diseases), and from the Société Nationale Française de Médecine Interne. ND received financial support from the Centre of Excellence, LabEx Inflamex, Université Paris-Diderot, Paris, France (ANR-11-IDEX-0005-02).

Abbreviations

ANA: anti-nuclear antibodies

Abbreviations

Legend to supplemental Figures Supplemental Figure 1. Analysis of T lymphocytes distribution in patients with active anti-proteinase 3 (PR3) and anti-myeloperoxydase (MPO) ANCA associated vasculitis (AAV) and healthy controls (HC). Percentages of Th1 (IFN-γ + ), Th2 (IL-4 + ), Th9 (IL-9 + ) and Th17 (IL-17A + ) were measured in total CD3 + CD4 + lived cells in 25 patients with PR3-ANCA (⎔), 5 patients with MPO-ANCA (Δ) and 17 HC (•) (Panel A). Th2/Th1, Th9/Th1 and Th17/Th1 T cells ratios were also calculated (Panel B). Comparisons between HC and either PR3-ANCA, MPO-ANCA or total AAV patients (■) were assessed using non-parametric Mann-Whitney test. Statistical significance was defined as: * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, **** p-value < 0.0001.

Supplemental Figure 2. Analysis of regulatory T cell and follicular T helper cell distributions in patients with active anti-proteinase 3 (PR3) and anti-myeloperoxydase (MPO) ANCA associated vasculitis (AAV) and healthy controls (HC). Percentages of

Foxp3 + , Treg (Foxp3 + CD25 hi ) and activated T cells (Foxp3 low CD25 + ) were quantified in 25 patients with PR3-ANCA (⎔), 5 patients with MPO-ANCA (Δ) and 17 HC (•) (Panel A).

Treg/activated T cells ratio within CD3 + CD4 + T cells was also calculated (Panel A). Follicular T helper cells (CXCR5 + ) and competent Tfh (PD-1 + CXCR5 + ) were assessed as depicted in gating strategy and represented in percentages of CD3 + CD4 + T cells and total Tfh cells (Panel B). Comparisons between HC and either PR3-ANCA, MPO-ANCA or total AAV patients (■)

were assessed using non-parametric Mann-Whitney test. Statistical significance was defined as: * p-value < 0.05, ** p-value < 0.01. 1,2,3,4,5 , Benjamin Chaigne* 1,2,3,6 , Alexis Régent* 1,2,3,6 ,Sébastien Lofek 1,2,3 ,Maïssa Mhibik 7,8 ,Peter Dorfmüller 9 ,Benjamin Terrier 1,2,3,6 ,Jonathan London 3,6 ,Alice Bérezné 6 ,Nicolas Tamas 1,2,3 , Nadine Varin-Blank 4,7,8 andLuc Mouthon 1,2,3,4,6 1 INSERM U1016, Institut Cochin, Paris, France ; In longitudinal experiments, cytometer settings were standardized using BD Cytometer setup, SPHERO Rainbow Calibration Particles (BD) and BD Combeads. Flow cytometry acquisition was assessed using a BD LSR Fortessa at the cytometry plateform of Cochin Institute (Cybio) and data were analyzed using FlowJo analysis software (Treestar, Ashland, OR, USA).

Supplemental

Legend to supplemental Figures

Supplemental Figure 1. Flow cytometry gating strategy of peripheral B and T lymphocytes.

Lymphocytes were identified using the side scatter (SSC) and forward scatter (FSC) parameters. B lymphocytes were identified as CD19 + CD21 +/-lymphocytes. Dot plots representative of the various markers used to characterize B lymphocytes are shown. T lymphocytes were defined as CD3 + cells expressing either CD4 + or CD8 + (panel B). T lymphocyte subpopulations were identified as follows: i) T helper (Th) 1 as viable CD3 + CD4 + IFN-γ + cells, ii) Th2 as viable CD3 + CD4 + IL-4 + cells, iii) Th9 as viable cells CD3 + CD4 + IL-9 + , iv) Th17 as viable CD3 + CD4 + IL-17 + cells and v) T CD8 effector cells as viable CD3 + CD8 + IFN-γ + cells.

Supplemental