Meningeal ectopic lymphoid tissues (mELTs): Composition, organization and expression revealed by imaging and in situ transcriptomics

ABSTRACT While relapsing-remitting multiple sclerosis (RRMS) can effectively be managed by a pantheon of disease modifying drugs with anti-inflammatory activities, the transition to secondary progressive MS (SPMS) is met with refractoriness to therapeutics and a downward spiral of neurodegeneration. This dire scenario has called into question the role of inflammation in SPMS, and cast SPMS treatment as a pressing unmet clinical need! However, meningeal ectopic lymphoid tissues (mELTs), which appear during SPMS in pathognomonic fashion, could drive atypical inflammatory activity and provide novel targets for therapeutic intervention. These indeterminate structures are B cell-rich aggregates that accumulate in the subarachnoid space of the meninges, and resemble tertiary lymphoid organs (TLOs) observed elsewhere in the body during various autoimmune and infectious conditions. In both MS patients and animal models, mELTs are conspicuously located over activated sub-pial microglia in areas of extensive cortical demyelination and neuronal loss, raising speculation they rain down as yet unknown incendiary factors that spark “compartmentalized inflammation” and neurodegeneration. Their role in SPMS etiopathogenesis nevertheless remains ambiguous, as the composition, compartmentalization, and gene expression of the varied cell types comprising mELTs have not been evaluated in relation to disease status and locale along the CNS axis – factors that can significantly impact the meninges and, thus, potentially mELTs. Technical hurdles had precluded examining these issues, but methodological advances by this group now allow for interrogation of mELTs – histologically and transcriptionally – within their native meningeal habitat. Two broad Aims are thus proposed to fill these major gaps, and test the hypothesis The cellular composition, organization and gene expression of meningeal Ectopic Lymphoid Tissues (mELTs) depend on CNS locale and disease status. Aim 1 will analyze animals at early and late stages of secondary progressive disease, and characterize the cell composition of mELTs in brain and spinal cord. Emphasis will be placed on identifying immune, stromal cell and vascular cell types found in known TLOs, and detailing their 3D organization with respect to each other and meningeal elements. Attention to meninges will be critical, as meningeal trabeculae might nucleate mELT assembly and/or segregate mELT cells into functional domains. High-resolution, 3D fluorescence microscopy and immuno-scanning electron microscopy will be used to view mELT cell organization at the light microscopic and ultrastructural levels, respectively, and imaging mass cytometry to reveal the full repertoire of cells in context with meningeal structures. Aim 2 will dissect the respective transcriptomes of the different mELT cell populations in brain and spinal cord in situ during early and late disease, using laser capture microdissection coupled to RNA-seq to preserve context-dependent gene expression patterns. This deliberative and methodical spatiotemporal analysis will yield foundational information necessary for resolving the contribution of mELTs to SPMS and highlighting new therapeutic prospects.