**9. Meningeal tertiary lymphoid organs may play a role in B‐cell traffic and maturation**

Meningeal TLOs are ectopic follicles with germinal centers aggregating a reticulum of CD35+ and CXCL13+ stromal/dendritic follicular cells, proliferating CD20+/Ki67+ B‐cells, Ig+ plasma/ plasmablast cells, and CD138+ plasma cells [168]. The mantle zone is often lacking, whereas CD4+ and CD8+ T‐cells infiltrate follicles [169]. CXCL13 immunoreactivity is confined to dendritiform cells inside intra‐meningeal B‐cell follicles [163]. The abundance of plasma cells is variable and proliferating cells in B‐cell follicles, mostly CD20+ cells, are observed at rates varying from 0 to 43% [163]. CCL21 and adhesion molecule peripheral node addressin (PNAd), which selectively binds to naïve T and B lymphocytes and allows their homing to secondary lymphoid organs, are absent [163], suggesting that the homing is dependent on various markers specific to brain tissues (see reference [170]). TLO demonstration is associated with higher CSF cytokines (TNFα and IFNγ) [171] but further studies are needed. The functions of TLO have been well described in various models of peripheral inflammation. TLOs are able to mount a T‐cell memory response in connection with the other lymphoid compartments [172]. In these models, the amount of Ig synthesis occurring in TLO is completely uncoupled from the blood response, meaning that local Ig‐secreting cells are highly specific, although a nonspecific response is associated [173, 174]. In fact, TLOs commonly occur during EAE; AID is locally expressed in association with SHM and B‐cell switching occurs [175–177]. The incidence of TLO in response to age insults is genetically determined in mice by two genes [178], but data are lacking in human and in EAE models.

in the CLN [149]. Cell lineage analysis demonstrated that most of the founder cells originated in the CLN [149]. A tentative interpretation is that antigen‐driven affinity maturation of B‐cells takes place in the CLN, which drains CNS antigens [164], then B‐cells migrate to colonies populating the CNS and continue to traffic between the CNS and the periphery, notwith‐ standing the possibility that a bidirectional traffic occurs in association with clonal expansion in both compartments (**Figure 4**) [149]. Clonal populations of CSF IgM‐ and IgG‐secreting B‐ cells do not overlap and seem to have matured independently from each other [158]. This has been interpreted as a failure of IgM switching to IgG despite aberrant AID activity. However, only a partial overlapping of IgG and IgM populations has been demonstrated in various

Naïve B‐cells are probably not randomly recruited in the inflammatory CNS since the VH family is biased [159]. Although the fate of naïve B‐cells emigrating to the CNS is still not clear, local maturation in the TLO structures and/or emigration to CLN are both probable (see below).

Most of the Ig peptides recovered from OCB by mass spectrometry match Ig‐secreting CSF B‐ cells [14]. Moreover, clusters of related B‐cells present in the CSF are also sometimes recovered from blood, the largest bi‐compartmental proportion being observed in association with a recent relapse [14]. This observation may explain the existence of OCB "mirror" patterns 3 and

Inflammatory lesions are observed in the eye in association with MS and perivenous lympho‐ cyte cuffings (i.e., periphlebitis) are commonly observed, reminiscent of lymphoid aggregates [165]. Therefore, besides the classical intrathecal Ig synthesis, local intraocular synthesis may also affect the eyes and glands. The MRZ reaction occurs with single specificity in up to 76% of cases and with ≥2 specificities in 82% [166, 167], but the ratio of *Fs* between Fuchs hetero‐ chromic uveitis syndrome (FHUS) and MS is about 40‐fold, which is in the same range of ITS ratios as observed between MS and viral encephalitis. OCBs are always found in the CSF and aqueous humor of MS patients. Importantly, both OCB and MRZ patterns mismatch in most

**9. Meningeal tertiary lymphoid organs may play a role in B‐cell traffic and**

Meningeal TLOs are ectopic follicles with germinal centers aggregating a reticulum of CD35+ and CXCL13+ stromal/dendritic follicular cells, proliferating CD20+/Ki67+ B‐cells, Ig+ plasma/ plasmablast cells, and CD138+ plasma cells [168]. The mantle zone is often lacking, whereas CD4+ and CD8+ T‐cells infiltrate follicles [169]. CXCL13 immunoreactivity is confined to dendritiform cells inside intra‐meningeal B‐cell follicles [163]. The abundance of plasma cells is variable and proliferating cells in B‐cell follicles, mostly CD20+ cells, are observed at rates varying from 0 to 43% [163]. CCL21 and adhesion molecule peripheral node addressin (PNAd),

studies but it now needs to be replicated.

66 Trending Topics in Multiple Sclerosis

**8.1. Local synthesis also occurs in the eye**

cases.

**maturation**

4 with overlapping B‐cell lineage on both sides of the BBB.

**Figure 5. Schematic drawing of tertiary lymphoid organs (TLOs)**. A few TLOs are disseminated on the leptomening‐ es. At pathologic level, leptomeningeal TLOs are surrounded by subpial lesions (cell and myelin loss). The toxic mech‐ anism is less certain and might involve TNFα secretion. At the immunological level, TLOs recruit naïve T‐ and B‐cells that undergo antigen presentation, AID expression, affinity maturation, and proliferation of B‐cells. Local Ig synthesis depends on plasmablasts/plasma cells and remains the only easily accessible bedside parameter indirectly informing about the presence of TLO (adapted from reference [179]).

In MS, the presence of TLO is associated with a more intense subpial demyelination (cortical lesions type III) and cell loss [169]. A strong topographical relationship is observed between TLO and subpial inflammation and an incidental observation showed that acute subpial lesions are associated with active local neurodegeneration (**Figure 5**) [180]. Owing to their small size and low number (about six follicles in positive blocks [168]), TLOs are probably largely underestimated by the sampling process of pathological examinations. Nevertheless, it was recently demonstrated that late post‐contrast FLAIR sequences on 3T‐MRI sequences might sample few leptomeningeal lesions (one lesion in 65% of patients, ≤6 in all of them) in a third of progressive patients [181], and this prevalence could be higher with 7T‐MRI. Pathological examination of one of them confirmed the congruence of the MRI lesion with TLO. Given the massive underestimation of TLO lesions, the practical consequences of this technique are currently being examined for their predictive clinical value. The longitudinal evolution of these structures and the effect of immunosuppressive therapies on them are unknown. Up to now, TLO could only be examined in the removed CNS structures including brain and leptomeninges but excluding skull and dura mater. Therefore, the recently discov‐ ered dura mater lymphatic network, which drains CSF to CLN, has never been examined in the context of MS. Since lymphatics are closely associated with TLO in all other tissues, confirmation of these structures might open up unexpected avenues in studies of inflammatory cell studies trafficking and maturation [182].

#### **10. Epitope spreading**

Epitope spreading in EAE and MS is mainly documented for T‐cells where it occurs in association with TLO formation [176]. The contribution of B‐cells to epitope spreading, especially for their intrathecal counterpart, is less well known. The intimate mechanisms driving epitope spreading are speculative and it remains unclear to what extent SHM con‐ tributes to this process.

It was observed some years ago that the ITS of IgG becomes enriched during the early phase after MS onset [27, 42, 68–70, 74]. The frequency of the MRZ pattern increases, OCB‐negative patients become positive, and more OCBs occur in the few patients initially displaying a low number of OCB. Assessment of IgG synthesis with multiplex antigen arrays now allows epitope spreading against CNS antigens to be monitored over time. Serum studies have shown that both intramolecular and intermolecular spreading occurs early after the very first demyelinating index event in children but only in patients demonstrating a further clinical MS conversion [42]. By contrast, pediatric patients remaining monophasic fail to diversify their antigenic response. A failure to regulate the aberrant autoimmune response is tentatively thought to explain this observation. Studies on early harvested CSF would give rewarding clues about CNS B‐cell epitope spreading. A longitudinal description of the network of interactions in B‐ and T‐cell epitopes (functional immunomics) at both serum and CSF level, and their interaction with HLA polymorphisms, might shed light on the pathophysiology of MS [183].
