This is a prospective non interventional study including patients with Relapsing-Remitting Multiple Sclerosis (RRMS) or with Neuromyelitis Optica Spectrum Disorders (NMOSD) and healthy subjects, who are enrolled within the routinely programmed clinical examinations at the IRCCS Neuromed (Pozzilli, Italy), IRCCS Polyclinic Hospital San Martino (Genoa, Italy) and Sant'Andrea Hospital - University of Rome La Sapienza (Rome, Italy).

Specifically, the study investigates how ozanimod may contrast neurodegenerative mechanisms triggered by both arms of the adaptive immune response (T and B cells) and by their suboptimal regulation in MS. Overall, the project aims at assessing by in vitro experiments (there will be no patients on treatment with ozanimod and the drug will be only used in vitro):

AIM1: ozanimod ability to modulate the synaptotoxic effect of T-cells derived from patients with MS relapse in a MS-chimeric ex-vivo model and to identify possible mediators (IRCCS Neuromed-Pozzilli, in collaboration with Synaptic Immunopathology Laboratory Dep. Systems Medicine, Tor Vergata University of Rome); AIM2: ozanimod ability to reduce the cytokine-mediated breakdown of the BBB and the migration of the here studied immune cells through ex vivo models of BBB (IRCCS Polyclinic Hospital San Martino); AIM3: ozanimod ability to affect the migration properties of Epstein Barr virus (EBV) infected B cells in MS (Sant'Andrea Hospital); AIM4: ozanimod ability to modulate the number and/or function of regulatory T cells (Treg), a lymphocyte population playing a key role in the control of pathogenic adaptive immune responses (Treg Cell Laboratory, Università degli Studi di Napoli "Federico II", Naples, Italy, receiving blood samples from Neuromed Hospital and Sant'Andrea Hospital; not recruiting unit).

The work of the four labs is conceptually and operationally integrated: the labs at IRCCS Neuromed-Pozzilli/Tor Vergata University (Aim1) and at Polyclinic Hospital San Martino (Aim2) will investigate the effects of ozanimod on well-known mechanisms of damage in MS, inflammatory synaptopathy and BBB damage and immune cell migration. The lab at Sant'Andrea Hospital (Aim3), will verify whether B cells infected by different EBV genotypes are involved in BBB migration, and how ozanimod may interfere with this mechanism. The Treg Cell Laboratory (Aim4) will investigate whether ozanimod can also act "upstream" of these mechanisms by regulating the adaptive immune response.

Multiple sclerosis (MS) is a multifactorial and complex disease with several and tangled pathophysiological mechanisms, responsible for the inflammatory and neurodegenerative damage. Therapies targeting inflammatory pathways have brought substantial progress in the treatment of relapsing-remitting MS. Disappointingly, the impact of these treatments on neurodegeneration is limited. To tackle the complexity of the problem, combination therapies may be necessary. However, safety, tolerability and cost issues have so far limited this option. Single therapies that target at once inflammation and neurodegeneration are an appealing alternative. However, a comprehensive understanding of their effects is essential to maximize their impact.

Ozanimod is a molecule that targets simultaneously inflammation and neurodegeneration. Its impact on the latter is less well understood. The drug produces its effects by acting on two (S1P1 and S1P5) of the five sphingosine-1-phosphate G protein-coupled receptor subtypes. While the molecular targets of the drug are precisely defined, much remains to be learned about the many biological effects that may follow receptors' modulation.

A collaborative and integrated approach is proposed to deepen our understanding of ozanimod's effects on the complex pathophysiology of MS.

The research will focus on crucial aspects for MS biology. Specifically, it will be investigated how ozanimod may contrast neurodegenerative mechanisms triggered by both arms of the adaptive immune response (T and B cells) and by their suboptimal regulation in MS. Overall, the project aims at investigating:

AIM1: ozanimod ability to modulate the synaptotoxic effect of T-cells derived from active MS patients in a MS-chimeric ex-vivo model and to identify possible mediators (IRCCS Neuromed-Pozzilli, in collaboration with Synaptic Immunopathology Laboratory Dep. Systems Medicine, Tor Vergata University of Rome). Inflammatory synaptopathy, namely synapse loss and synaptic dysfunction, is emerging as an important hallmark of grey matter pathology in MS and its mouse model experimental autoimmune encephalomyelitis (EAE). In the past years, evidence has been provided of a direct role of T cell, derived from MS patients or EAE mice, in inducing glutamate-mediated excitotoxicity, a prominent form of synaptopathy detected in the MS/EAE brain. Notably, several lines of evidence indicate that limiting glutamate-excitotoxicity is an attractive therapeutic strategy to treat MS.

Recently, the modulatory effects of ozanimod on inflammatory glutamate-mediated excitotoxicity was investigated and the different involvement of sphingosine receptor subtypes (S1P1 and S1P5) were evaluated in the EAE model. Two main results emerged: first, the S1P1/S1P5 modulator ozanimod has central neuroprotective effects likely mediated by an action on microglia cells and infiltrating lymphocytes, resulting in a reduced release of proinflammatory cytokines, the main players of inflammatory synaptopathy. Second, the central delivery of a selective S1P1 modulator showed neuroprotective effects, in terms of both EAE clinical score and inflammatory synaptopathy, suggesting a primary involvement of this receptor subtype in ozanimod- induced neuroprotection also in MS.

In the present project, the MS chimeric ex-vivo model recently developed in Tor Vergata University' lab, will be used. T cells isolated from active relapsing remitting MS patients but not from non-active (naMS) and healthy subject (HS), induced an increase of the spontaneous glutamatergic currents, reminiscent of the alterations induced by EAE T cells.

In this specific aim of the project, the following aspect will be assessed: i) to investigate if ozanimod is able to modulate the synaptotoxic effect of T dell derived from active MS patients in the MS-chimeric ex-vivo model and ii) to identify sphingosine receptor subtypes and iii) soluble molecules potentially involved.

A role for ozanimod in dampening excitotoxic damage caused by synaptotoxic T-cell circulating in the MS brain, independently of a prevention of T cell trafficking, is expected to be revealed.

AIM2: ozanimod ability to reduce the cytokine-mediated breakdown of the BBB and the migration of the here studied immune cells through ex vivo models of BBB (IRCCS Polyclinic Hospital San Martino). B and T lymphocytes, and NK cells are involved in MS pathogenesis (Lassmann, 2019). They differentially use S1P1 and S1P5 receptors to regulate their traffic across secondary lymphoid tissues, and to enter the CNS through the BBB (Sallusto et al., 2012). In turn, these receptors are important in maintaining the integrity and in restoring the function of BBB (Chun 2021).

Modulators of S1P1 and S1P5 receptors are widely used to treat MS, but their mode of action is only partially known due to the complex downstream effects of the combined interactions among receptors localized at the membrane surfaces of immune cells and at the endothelial cells and podocytes of the BBB. These modulating features have been mainly studied for fingolimod, the first drug of this class used in MS, whereas ozanimod's mechanisms of action need in-depth investigations. Indeed, this drug has chemical structure different from that of fingolimod, a binding restricted to S1P1 and S1P5 receptors vs S1P1, S1P3, S1P4 and S1P5 of fingolimod, different pharmacokinetics, and shorter time of elimination half-life (Chun et al., 2021).

The purpose of this specific aim is to evaluate, in ex vivo models of the human BBB exploited through transwell-based systems and organoids, the effects of ozanimod on: i) maintaining of BBB integrity in a context of cytokine-mediated barrier breakdown; ii) impairing the migration of T cells and NK cells across BBB.

The ex-vivo exposure of peripheral blood mononuclear cells (PBMCs) from MS patients to ozanimod is expected to reduce the damage of the BBB mediated by pro-inflammatory cytokines, as well as to mitigate ability of these immune cells across the BBB. PBMCs of patients with NMOSDs will be also used as inflammatory controls, since in these disorders Th17 cells have a major pathogenetic role, together with the BBB-targeting AQP-4 antibodies.

AIM3: ozanimod ability to affect the migration properties of Epstein-Barr virus (EBV) infected B cells in MS (Sant'Andrea Hospital).

In the general population, the memory subset of B lymphocytes, proved to be decreased during therapy with S1P receptors modulators, is the one that selectively harbors EBV as a latently infecting agent. Several studies on EBV genotypes demonstrated an association of specific Epstein- Barr nuclear antigen 2 (EBNA2) gene variants with MS. EBNA2 acts as a transactivator on both viral and cellular gene promoters/enhancers by obligatory interactions with host cell transcription factors. Taking into consideration the complex interplay between EBNA2 and cellular components, it is likely that EBNA2 variants may affect the processes of early infection and participate in the dysregulated virus-host interactions in MS. Also, MS-associated EBNA2 variants seem to interfere with B cell migration and maturation within germinal centers. Despite the presence of EBV infection in the MS brain is controversial, some observations support evidence of an intrathecal reactivation and virus-driven immunopathogenic response in MS. The hypothesis of this specific aim is that ozanimod can have immunomodulatory effects on B cells and EBV infected cells in a MS context. As already mentioned, ozanimod is a potent S1P1R modulator, that inhibits the egress of lymphocytes from lymphoid tissues. B cells and lymphoblastoid cell lines [spontaneously outgrowing (spLCLs) or in vitro infected with the EBV laboratory strain B95.8 (95.8LCLs)], will be used to study how EBV infection affects migration capacity and how this capacity is affected by ozanimod.

Specifically, the following aspect will be investigated: i) the migration properties of B cells, spLCLs and B95.8LCLs from MS patients and age- and sex-matched healthy donors (HD); ii) if ozanimod is able to modulate the migration properties of the above cells from patients and HD.

The knowledge of ozanimod influence on B cells and EBV-infected B cells migration through lymphoid organs and even in the brain, will be importantly improved by this research.

AIM4: ozanimod ability to modulate the number and/or function of regulatory T cells (Treg), a lymphocyte population playing a key role in the control of autoimmune responses (Treg Cell Laboratory, Università degli Studi di Napoli "Federico II", Naples, Italy, receiving blood samples from Neuromed Hospital and Sant'Andrea Hospital; not recruiting unit). Intracellular metabolic pathways are able to control the induction and function of different immune cellular subsets, indeed glycolysis is indispensable for the generation of human iTreg cells from Tconv cells. In addition, a glycolytic defect during the activation of Tconv cells in MS and T1D subjects, was associated with a reduced induction and suppressive function of iTreg cells in these patients suggesting a defective function of iTreg cells during autoimmunity. The effect of ozanimod on the cellular metabolic profile of T lymphocytes and of iTreg cells will be investigated. In particular, this specific aim will evaluate i) the capacity of ozanimod to affect T cell and iTreg cell function/activity (activation, proliferation, suppression, Foxp3 induction) and ii) the metabolic asset of circulating T cell populations (ie. measurement of glycolysis and oxidative phosphorylation) purified from healthy subjects and MS patients.

Ozanimod is expected to be able to control immunometabolism of Tconv and iTreg cells in RRMS patients. This aim will also assess whether ozanimod is able to modify the induction of iTreg cells in healthy controls and RRMS patients.

Inclusion Criteria:

Main inclusion criteria of patients with RRMS:

• Relapsing-remitting MS, as diagnosed by the revised 2010 McDonald Criteria
• EDSS score ≤ 5.5;
• Age between 18 and 55 years (exclusive);
• No disease modifying therapies for at least 3 months or treatment naïve;
• No corticosteroid administration in the previous month;
• Disease duration <10 years;
• Ability to provide written informed consent.

For the estimation of radiological variables, brain and spinal cord MRI will be performed according to clinical practice and lesions will be classified as symptomatic or asymptomatic if they were associated with clinical relapse or not.

Patient groups will be matched by gender, age, ethnicity and MS duration.

Main inclusion criteria of patients with NMOSD (Wingerchuk et al., 2015):

• Positive test for Aquaporin 4 IgG;
• Age between 18 and 55 years (exclusive);
• no immunosuppressive therapies for at least 3 months or treatment naïve
• no corticosteroid administration in the previous month
• disease duration <10 years
• Ability to provide written informed consent

Healthy subjects

• Age between 18 and 55 years (exclusive), matched by gender, age and ethnicity towards the MS groups.
• Ability to provide written informed consent

Exclusion Criteria:

Exclusion criteria patients with RRMS:

• Adverse effects to MRI imaging with i.v. gadolinium;
• Blood count basal alteration;
• Clinically significant medical condition other than MS, including latent infections (e.g. tuberculosis, viral hepatitis, HIV/AIDS) that might confound the results of the study.

This is a prospective non interventional study with MS patients enrolled within the routinely programmed clinical examinations at the IRCCS NeuromedPozzilli, IRCCS polyclinic hospital San Martino (Genoa, Italy), Sant'Andrea

Hospital - University of Rome La Sapienza (Rome, Italy). Study population:

Approximately 72 Relapsing-Remitting Multiple Sclerosis (RRMS - naïve or after 3 months wash out from DMT patients; 10 Neuromyelitis Optica Spectrum Disorders patients (NMOSD); 72 healthy subjects (HD) will be recruited from the hospitals involved in the study.