a.iv. Immunopathophysiology of MS and EAE

It has not been demonstrated whether MS is initiated in the periphery or in the CNS, although the consensus among scientists classifying MS as an autoimmune disease assumes that MS is initiated in the periphery (Dendrou et al., 2015). MS is considered as a CD4⁺ T cell-mediated autoimmune disease, based on the fact that certain MHC-II alleles, such as HLA-DRB1*1501, are major risk alleles for MS and also since EAE can be induced in mice by injecting MHC-II-restricted myelin antigens (Dendrou et al., 2015; Dendrou et al., 2018; International Multiple

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Sclerosis Genetics et al., 2007; International Multiple Sclerosis Genetics et al., 2011; Rangachari and Kuchroo, 2013).

In the EAE model, autoreactive T cells are activated by APCs in the periphery, in SLOs (Becher and Greter, 2012; Dendrou et al., 2015). The idea that autoreactive T cells are primed in the LNs is supported by the efficacy of Fingolimod, used to treat MS, which sequesters T cells in the LNs by blocking their egress (Chun and Hartung, 2010). Encephalitogenic T cells migrate to the CNS and enter the subarachnoid space, which contains CSF, after having crossed the BBB at the choroid plexus (Becher and Greter, 2012; Dendrou et al., 2015). Autoreactive T cells are subsequently re-activated by APCs in the CNS and produce pro-inflammatory cytokines, leading to immune cell recruitment in the parenchyma, such as monocytes, macrophages, neutrophils and other T cells, which leads to the destruction of the myelin sheath (Becher and Greter, 2012;

Dendrou et al., 2015). These processes are detailed in the following paragraphs.

Priming of cells in the periphery Role of DCs and other APCs

The old dogma stipulates that, after subcutaneous immunization with CFA and myelin antigen for EAE induction, DCs residing at the injection site capture the antigen and migrate to the dLNs, where they prime naïve autoreactive T cells that have escaped central tolerance (Becher and Greter, 2012; Dendrou et al., 2015; Huang et al., 2012).

Regarding the role of cDCs, this dogma has been challenged by studies using transgenic mice in which DCs are depleted, such as the CD11c-DTR (diphtheria toxin receptor) mouse model (Becher and Greter, 2012; Birnberg et al., 2008; Isaksson et al., 2012; Ohnmacht et al., 2009;

Yogev et al., 2012). Some studies concluded that cDCs were not required for T cell priming or that they played a tolerogenic role in EAE, raising the question whether other APCs primed encephalitogenic T cells in the SLOs (Becher and Greter, 2012; Ohnmacht et al., 2009; Yogev et al., 2012). However, these conflicting results might be partly explained by depletion protocols that were not fully optimized, by distinctive roles of cDCs in SLOs in the periphery and in the CNS, and finally, by a failure to distinguish the functions of different cDC subsets (Becher and Greter, 2012). Indeed, as described in the general introduction, there are several subsets of cDCs, which have distinct roles (Anderson et al., 2018; Dalod et al., 2014; Guilliams et al., 2014; Merad et al., 2013). For example, monocytes could be recruited to the site of immunization and differentiate into inflammatory monocyte-derived (mo)DCs that would subsequently prime encephalitogenic T cells (Becher and Greter, 2012). Indeed, human inflammatory DCs, which share characteristics with in vitro-differentiated moDCs were demonstrated to induce the

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differentiation of Th17 cells, a T cell subset known to play a pathogenic role in MS (Segura et al., 2013b). Dermal CD103⁺ DCs or macrophages could also prime encephalitogenic T cells (Becher and Greter, 2012). Macrophages might also be involved in the priming, while other cDC subsets play an anti-inflammatory role (Becher and Greter, 2012; Marta et al., 2008; Ohnmacht et al., 2009; Yogev et al., 2012).

Regarding pDCs in EAE, the role of IFN-I production is controversial, as this cytokine can have anti- or pro-inflammatory effects (Guery and Hugues, 2013; Isaksson et al., 2009; Kasper and Reder, 2014; Loschko et al., 2011a). Inded, EAE severity is increased in IFNAR^-/- mice, and IFN-β, the first-line treatment of MS, delays relapses and dampens the severity of the disease in MS patients, while pDC depletion and IFN-I neutralization have been shown to ameliorate early phase of EAE (Guery and Hugues, 2013; Isaksson et al., 2009; Kasper and Reder, 2014).

Regarding APC functions of pDCs, we have demonstrated that pDCs, as MHC-II-restricted APCs, have a tolerogenic role in EAE and promote Treg generation (Duraes et al., 2016; Guery and Hugues, 2013; Irla et al., 2010; Lippens et al., 2016). Furthermore, antigen-targeting to pDCs via Siglec-H dampens EAE severity (Loschko et al., 2011a).

Other APCs, beside monocytes, macrophages and DCs, could be involved in the priming of encephalitogenic T cells, such as B cells (Li et al., 2018). B cell depletion ameliorates EAE independently of antibody production, suggesting that their APC functions may play a pathogenic role (Fillatreau and Anderton, 2007). For instance, it was recently demonstrated that memory B cells from the at-risk allele HLA-DR15 MS patients present antigens to autoreactive Th1 cells, inducing their proliferation (Jelcic et al., 2018). Moreover, it was recently shown that B cells were the major APCs implicated in naïve CD4⁺ T cell activation after immunization with a monoparticle antigen derived from a virus, showing that DCs are not necessarily the primary initiator of CD4⁺ T cell responses (Hong et al., 2018).

Th1/Th17

Th1 and Th17 cells are strongly implicated in the immunopathophysiology of EAE. The frequencies of myelin-antigen specific CD4⁺ T cells are similar in MS patients and healthy donors, however, they are functionally different, with increased production of IFN-γ, IL-17 and GM-CSF in MS patients (Cao et al., 2015).

However, EAE severity in IFN-γ^-/- and IL-17^-/- mice was increased or similar, compared with control mice, respectively, showing that IFN-γ and IL-17 are not crucial cytokines for EAE induction (Ferber et al., 1996; Haak et al., 2009). On the contrary, IL-1β and IL-23, which drive

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Th17 differentiation, are essential cytokines, without which mice are resistant to EAE (Cua et al., 2003; El-Behi et al., 2011; Langrish et al., 2005). Using GM-CSF^-/- mice, it has been demonstrated that this cytokine is essential to induce EAE (McQualter et al., 2001). In addition, Th17 cells expansion and GM-CSF expression by T cells are increased in MS patients, while these phenomena are reduced by IFN-β therapy (Durelli et al., 2009; Rasouli et al., 2015). In EAE, encephalitiogenic or non-encephalitogenic Th17 can be distinguished based on their production of GM-CSF (Codarri et al., 2011; El-Behi et al., 2011). Moreover, the production of GM-CSF by IL-17^-/- IFN-γ^-/- CD4⁺ T cells is sufficient to induce EAE (Codarri et al., 2011). Although Th17 cells play an important role, it does not exclude a role for Th1 cells, which had long been thought to be the main player in EAE, before the discovery of Th17 cells (Bettelli et al., 2004; Lovett-Racke et al., 2004). Indeed, similarly to Th17 cells, the adoptive transfer of Th1 cells can induce EAE (Jager et al., 2009). Nonetheless, Th1 and Th17 do not use the same mechanisms to infiltrate the CNS (Stromnes et al., 2008). Th1 cells, via the production of TNF-α and IFN-γ, induce the expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1), facilitating the transmigration of these cells into the CNS, preferentially the spinal cord (Stromnes et al., 2008). Th17 cells, by their production of IL-17, enhance the permeabilization of the BBB and preferentially infiltrate the brain (Huppert et al., 2010; Stromnes et al., 2008).

Tregs

The Treg compartment has been shown to be altered in MS patients; tTregs isolated from MS patient peripheral blood exhibit impaired suppressive abilities (Viglietta et al., 2004). It has been suggested that MHC-II risk alleles, such as HLA-DR15, might be implicated in dysfunctional negative selection in the thymus (Dendrou et al., 2018). It has been recently shown, in patients suffering from Goodpasture disease, a CD4⁺ T cell-mediated autoimmune disease in which HLA-DR15 is also a risk allele, that HLA polymorphism shapes the abundance or lack of self-specific Tregs, leading to protection or susceptibility to the disease (Ooi et al., 2017). The same conclusions were drawn using HLA-DR15 transgenic mice in a model of Goodpasture disease (Ooi et al., 2017). It is possible that this discovery also applies for MS. Other phenomenon such as Treg instability and Treg plasticity could be implicated in MS pathogenesis (Dominguez-Villar and Hafler, 2018).

It has been demonstrated, in mice, that the adoptive transfer or depletion of Tregs before EAE induction decreases or exacerbates EAE severity, respectively (Irla et al., 2010; Lippens et al., 2016; McGeachy et al., 2005).

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Immune cell migration into the CNS

The entry of T cells in the CNS is believed to occur in two phases (Engelhardt and Ransohoff, 2012). A first wave of Th17 cells entry, for which the expression of CCR6 is important to reach the subarachnoid space (Reboldi et al., 2009). CCR6 ligand, CCL20, is expressed by choroid plexus epithelial cells. This is followed by a second wave of immune cell infiltration, including Th1 cells, which enter the CNS parenchyma, independently of CCR6. The infiltration of Th1 rather depends on the expression of CXC-chemokine receptor (CXCR)3 (Lalor and Segal, 2013).

The entry of Th1 and Th17 cells in the spinal cord, as well as the entry of Th1 in the brain, is dependent on the interaction of the integrin very late antigen 4 (VLA-4) with VCAM-1, expressed by BBB endothelial cells (Baron et al., 1993; Glatigny et al., 2011). Of note, Natalizumab is a monoclonal antibody used in the treatment of MS that blocks the VLA-4/VCAM-1 interaction (Polman et al., 2006; Schwab et al., 2015; Shirani and Stuve, 2017). On the contrary, the entry of Th17 cells in the brain does not depend on VLA-4 but on the expression of lymphocyte function-associated antigen 1 (LFA-1) (Glatigny et al., 2011;

Rothhammer et al., 2011).

Moreover, it has recently been demonstrated, using a lewis rat EAE model, that the leptomeninges act as a checkpoint from where activated myelin-specific T cells can enter the CNS parenchyma, while non-activated T cells are released in the CSF (Bartholomaus et al., 2009;

Schlager et al., 2016). The attachment of activated T cells to the leptomeninges is due to the interaction of VLA-4 and LFA-1 with their ligands expressed by resident macrophages, CXCR5/CXCR3 signaling and antigen presentation by macrophages, while non-activated T cells are flushed by the CSF (Schlager et al., 2016).

In addition to these well-established routes for T cell entry into the CNS, a putative role of meningeal LVs could also be implicated. As mentioned previously, the recent (re)-discovery of LVs in rodent CNS, also visualized by MRI in human and non-human primate, has induced a reassessment of the immune privilege dogma, in which the BBB and blood-leptomeningeal barrier (BLMB), at the surface between the brain and spinal cord, separating the blood and the CSF, were thought to prevent the entry of immune cells into the CNS (Absinta et al., 2017;

Aspelund et al., 2015; Engelhardt et al., 2017; Louveau et al., 2015a; Louveau et al., 2017;

Louveau et al., 2015b; Mezey and Palkovits, 2015). These finding are of major importance for MS research. Indeed, meningeal LVs, which link the CNS and deep cervical LNs, might be involved in CNS inflammatory processes that take place in MS (Meyer et al., 2017).

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Furthermore, endothelial cells of the BBB are strategically positioned and are implicated in the control of adaptive immune responses in the CNS, especially for the entry of T cells in the CNS (Galea et al., 2007; Meyer et al., 2017).

Chronic CNS inflammation and damages Reactivation of T cells in the CNS

Autoreactive T cells that infiltrate the CNS are re-activated by APCs, the nature of which is widely debated (Dendrou et al., 2015). In the steady-state, several types of myeloid cells co-exist in the CNS, including microglia and different subsets of macrophages and DCs (Mrdjen et al., 2018). T cell reactivation might occur in the cervical LNs, in the CNS parenchyma by microglia or astrocytes, or in the perivascular and meningeal spaces by macrophages or DCs (Dendrou et al., 2015). The role of antigen presentation by astrocytes and microglia in MS and EAE is a matter of debate and will not be developed here (Colombo and Farina, 2016; Goldmann and Prinz, 2013; Ponath et al., 2018). Conventional DCs have been recently demonstrated to be of crucial importance for the reactivation of self-reactive T cells in the CNS (Giles et al., 2018).

Chronic CNS inflammation

In MS, pro-inflammatory cytokines are secreted by leukocytes that have infiltrated the CNS (Becher et al., 2017). As mentioned earlier, GM-CSF produced by re-activated T cells play a detrimental role in MS and EAE (Croxford et al., 2015b). It has been shown to be implicated in the recruitment of myeloid cells in the CNS (Spath et al., 2017). Among these cells, monocytes and CD103⁺ DCs are recruited (Croxford et al., 2015a; King et al., 2009; King et al., 2010).

IL-17 has been found in CNS lesions and in the CSF of MS patients (Matusevicius et al., 1999).

IL-17 is involved in the induction of cytokine and chemokine secretion, as well as the production of reactive oxygen species (ROS) due to microglial activation, which leads to demyelination and axonal loss (Gilgun-Sherki et al., 2004; Kawanokuchi et al., 2008). IL-17 was also implicated in the formation of tertiary lymphoid structures (TLS) (Becher, 2015; Pikor et al., 2015).

Myeloid cells recruited to the CNS produce IL-1β, which is critical for EAE induction via the differentiation of Th17 cells (Levesque et al., 2016). This cytokine can be produced by Th17 themselves, inducing an autocrine loop of Th17 cell maintenance (El-Behi et al., 2011; Martin et al., 2016).

Altogether, these inflammatory processes result in white and grey matter damages (Dendrou et al., 2015).

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Tregs in the CNS

During EAE, Tregs infiltrate the CNS and dampen the inflammation (Kohm et al., 2002). In addition, in EAE mice, remitting phases are associated with an accumulation of IL-10-producing Tregs within the CNS, although Tregs in the inflamed CNS environment were shown to have impaired suppressive capacity (Korn et al., 2007; Zhang et al., 2004). Finally, neurons (MHC-II-negative cells) were demonstrated to induce the conversion of encephalitigenic CD4⁺ T cells into Tregs via the production of TGF-β (Liu et al., 2006).

Beyond CD4⁺ T cells

Although MS is considered as a CD4⁺ T cell-mediated disease, CD8⁺ T cells and B cells also have been shown to be implicated in the immunopathophysiology of MS and EAE (Li et al., 2018;

Rangachari et al., 2017). However, their role will not be developed here. Briefly, B cells are involved in the production of auto-antibodies that target the myelin sheath, and are APCs, with paradoxical effects, as the outcome can be either encephalitogenic T cell priming/re-activation or Treg generation (Fillatreau and Anderton, 2007; Jelcic et al., 2018; Li et al., 2018). Nonetheless, a rationale for the pathogenic role of B cells in MS is that the use of monoclonal antibodies depleting B cells induces a delay in relapses in RRMS patients (Gasperini et al., 2013; Hauser et al., 2008; Sorensen and Blinkenberg, 2016).

Although CD8⁺ T cells have a higher frequency than CD4⁺ T cells in MS lesions, their role has been underestimated and is still debated, with studies concluding for a pathogenic role while others for a regulatory role in MS (Salou et al., 2015; Sinha et al., 2015)