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The current study is the first, to our knowledge, to demonstrate a correlation between the presence of meningeal TLOs, and CXCL13 meningeal gene expression and concentration in post-mortem CSF, and confirms speculation of a link by previous studies (Krumbholz et al., 2006). Our finding that CXCL13 gene expression was substantially upregulated in F+SPMS compared to F-SPMS cases and controls, and CSF concentration was increased in F+SPMS compared to F-SPMS, suggests that CXCL13 may play a significant role in meningeal TLO formation.

Although few studies have investigated CXCL13 concentration in SPMS CSF and none have investigated post-mortem CSF, our results support the many reports of increased CXCL13 in RRMS CSF and the correlation of CXCL13 concentration with markers of intrathecal B cell maturation and autoantibody production, which are suggested to occur in TLOs (Krumbholz et al., 2006; Kuenz et al., 2008; Sellebjerg et al., 2009; Villar et al., 2010). CXCL13 in RRMS CSF correlates with intrathecal Ig-production, and the presence of plasmablasts and B and T cells (Krumbholz et al., 2006) and with CSF leukocyte count in progressive MS (Sellebjerg et al., 2009). CXCL13 is known to play a key role in the organisation and recruitment of B cells to SLOs, and is thought to play a similar role in TLOs (Aloisi and Pujol-Borrell, 2006; Drayton et al., 2006), so the correlation found in the current study between the presence of meningeal TLOs and increased CXCL13 in the CSF agrees with previous data. The difference in structure and relative lack of organisation observed in TLOs compared to SLOs is suggested to contribute to autoimmunity by promoting the mishandling of autoantigens, resulting in the breaking of tolerance (Weyand et al., 2001). TLOs arise in areas close to autoantigen production, and are suggested to optimise autoimmune responses by production of increasingly specific autoantibodies through affinity maturation. In addition, TLOs are postulated to contribute to B cell (Pollinger et al., 2009; Cornaby et al., 2015) and T cell (Kuerten et al., 2012) epitope spreading, which is suggested to occur in MS, resulting in a range of intrathecal autoantibody specificities per patient (Quintana et al., 2012), and correlates with the development and progression of EAE (Yu et al., 1996; Pollinger et al., 2009). We suggest that in F+SPMS cases the autoimmune response is optimised by the formation of TLOs, which may be initiated and maintained by LTα and CXCL13 among other cytokines and chemokines, resulting in increased pathology compared to F-SPMS cases. The significant increase in CXCL13 CSF concentration in females observed in the current study, when the outlier case was excluded from analysis, has not been previously reported. However, as with other autoimmune diseases (Selmi et al., 2012), MS affects approximately twice as many women as men, a fact that was reflected by our selection of cases for CSF samples in the current study, (males n=14, females n=24), suggesting that the correlation between gender and CXCL13 concentration may be influenced by the inherent sex bias in studying MS cases.

Our findings of a correlation between CXCL13 and F+SPMS, as well as increased CXCL13 correlating with a younger age at first wheelchair use, (and a younger age at progression when the outlier was removed) support the suggested role of CXCL13 in the exacerbation of Chapter 3 – Expression of lymphoid cytokines and chemokines in SPMS 107

disease, since F+SPMS is associated with a more severe disease course compared to F- SPMS (Magliozzi et al., 2007; Howell et al., 2011). The involvement of CXCL13 in relapses and intrathecal Ig production was suggested following observation of a significant increase in CXCL13 CSF concentration during relapse in patients with oligoclonal bands against myelin antigens in the CSF, compared to those without (Villar et al., 2010). Increased CXCL13 CSF concentration also correlates with measures of disease activity, including an increased number of gadolinium-enhancing MRI brain lesions in RRMS and CIS, increased relapse rate and EDSS score (Sellebjerg et al., 2009; Khademi et al., 2011). In addition, elevated CXCL13 concentrations were associated with increased rate and speed of conversion from CIS to MS, leading the authors to suggest that CXCL13 could be used as a prognostic marker in the early stages of disease (Khademi et al., 2011). The findings of the current study also support reports of wide ranging CSF concentrations between SPMS cases. The authors of a previous study which quantified CXCL13 CSF concentration noted that SPMS cases could be roughly divided into two subsets, one with low or undetectable CXCL13 CSF concentrations and the other with high concentrations, which they speculated might be correlated with the presence of meningeal TLOs (Krumbholz et al., 2006). Our study confirms this speculation.

The cellular source of CXCL13 was not identified in the current study. Large, dense meningeal aggregates were rarely found as mentioned previously, due to the relatively small study size and limited sampling. Little TLO tissue was available for CXCL13 IHC and optimisation of antibodies for use on snap frozen tissue proved extremely time-consuming. However, several other studies have been successful in demonstrating CXCL13 expression in cells thought to be FDCs within meningeal TLOs (Serafini et al., 2004; Magliozzi et al., 2007). DCs and macrophages are also known to express CXCL13 under inflammatory conditions (Carlsen et al., 2004; Perrier et al., 2004), and CXCL13+ cells have been described in the parenchyma and perivascular cuffs of active lesions (Krumbholz et al., 2006). Infiltrating macrophages have been suggested to be the source of CXCL13 in active lesions in acute inflammation, but meningeal TLOs may be the major source of CXCL13 in SPMS in chronic disease (Krumbholz et al., 2006). As discussed earlier, meningeal fibroblasts may be driven to differentiate into CXCL13-expressing FDCs by the high local concentrations of pro-inflammatory cytokines, including LTα and/or TNF, within sulci in F+SPMS. No data is available on whether meningeal cells themselves are capable of secreting CXCL13 without differentiation of fibroblasts into FDCs, although rat meningeal cultures have been observed to express high levels of CXCL13 in vitro (unpublished observations from this laboratory). In addition, CXCL13 appears to be Chapter 3 – Expression of lymphoid cytokines and chemokines in SPMS 108

expressed by meningeal cells adjacent to aggregates of immune cells and suspected TLOs in F+SPMS, associated with the concomitant expression of CXCR5 by astrocytes in the underlying GM (unpublished observations from this laboratory). This raises the intriguing possibility that the meninges themselves may be activated by infiltrating inflammatory cells and become a source of cytokines and chemokines, including TNF, which has been observed following inflammatory stimulation of fibroblasts in vitro (Wells et al., 2001; Barone et al., 2012; Fan et al., 2012), and would support observations from the current study that CXCL13 was highest in the F+SPMS cases associated with particularly prominent meningeal inflammation. Data from EAE studies found CXCL13 expression to be upregulated only in mice developing relapsing-remitting and chronic relapsing EAE, and not in mice with non-relapsing chronic EAE (Columba-Cabezas et al., 2004) also suggesting a role for CXCL13 in the exacerbation of CNS inflammation during relapse, as suggested by human studies. Depletion of CXCL13 in CXCL13-/- mice did not impact B cell accumulation in the CNS in EAE however, suggesting that CXCL13 plays a more important role in facilitating lymphocyte organisation and interaction within the CNS than recruiting lymphocytes from the periphery in this model (Bagaeva et al., 2006; Rainey-Barger et al., 2011). Ectopic expression of CXCL13 induces formation of TLO- like structures, but these lack germinal centres (Luther et al., 2000a), in contrast to ectopic expression of LTα (Kratz et al., 1996), suggesting that LTα may be more important for initiating fully functional TLO formation while CXCL13 may have other roles in the organisation of inflammation. Indeed, LTα has also been shown to induce ectopic expression of CCL21 and CXCL13 in vivo suggesting that it may be upstream of CXCL13 in the signaling cascade during TLO neogenesis (Hjelmstrom et al., 2000).

Studies of other inflammatory diseases suggest that CCL21 expression is also important for TLO formation. CCL21 and CXCL13 expression correlates with autoantibody titres and the presence of TLOs in thyroid AI (Armengol et al., 2003), and mice deficient in the receptors of these chemokines (CXCR5 and CCR7 respectively) show severely impaired formation of TLOs and reduced cellular and humoral responses in an in vivo model of RA (Wengner et al., 2007). Ectopic expression of CCL21 results in the formation of lymphocyte aggregates with FDCs, but since these structures lack functional GCs and often HEVs, they may not represent fully functional TLOs as demonstrated by ectopic LTα expression (Fan et al., 2000; Chen et al., 2002; Martin et al., 2004).

Our finding that CCL21 meningeal gene expression was increased in F+SPMS compared to F- SPMS supports a role for CCL21 in TLO formation in MS. However the increase in CCL21 expression was not statistically significant compared to control cases, and further study of a larger cohort is needed to confirm the reliability of these results. Although reports of CCL21 expression in the meninges are lacking, CCL21 CSF concentration is elevated in MS (Pashenkov et al., 2003) and it is postulated to play a role in recruitment of T cells to the CNS during MS, since nearly all T cells in the CSF were shown to express the CCL21 receptor, CCR7 (Kivisakk et al., 2004). In addition, CCL21 gene expression is upregulated during chronic EAE and IHC showed that expression was confined to cerebral blood vessels (Columba-Cabezas et al., 2003), supporting a role in immune cell recruitment to the CNS. These findings contrast with those of a relatively small study in which CCL21 was undetectable by IHC in meningeal tissue from MS cases (Kivisakk et al., 2004; Serafini et al., 2004). Since CCL21 is primarily expressed by HEVs and stromal cells in the T cell zones of SLOs, the lack of CCL21 expression in meningeal TLOs was suggested to be due to the apparent absence of HEV formation (Cyster, 1999; Luther et al., 2000b). Our results suggest that either HEVs are formed in F+SPMS and express CCL21, or support the findings of CCL21 expression by endothelial cells in intrameningeal vessels during chronic EAE (Columba- Cabezas et al., 2003). However, as CCL21 was not confirmed by the current study to be increased in SPMS compared to control cases, it was not investigated further by CSF or IHC analysis.