Accumulation of meningeal lymphocytes correlates with white matter lesion activity in progressive multiple sclerosis

Subpial cortical demyelination is an important component of multiple sclerosis (MS) pathology contributing to disease progression, yet mechanism(s) underlying its development remain unclear. Compartmentalized inflammation involving the meninges may drive this type of injury. Given recent findings identifying substantial white matter (WM) lesion activity in patients with progressive MS, elucidating whether and how WM lesional activity relates to meningeal inflammation and subpial cortical injury is of interest. Using postmortem FFPE tissue blocks (range, 5–72 blocks; median, 30 blocks) for each of 27 patients with progressive MS, we assessed the relationship between meningeal inflammation, the extent of subpial cortical demyelination, and the state of subcortical WM lesional activity. Meningeal accumulations of T cells and B cells, but not myeloid cells, were spatially adjacent to subpial cortical lesions, and greater immune cell accumulation was associated with larger subpial lesion areas. Patients with a higher extent of meningeal inflammation harbored a greater proportion of active and mixed active/inactive WM lesions and an overall lower proportion of inactive and remyelinated WM lesions. Our findings support the involvement of meningeal lymphocytes in subpial cortical injury and point to a potential link between inflammatory subpial cortical demyelination and pathological mechanisms occurring in the subcortical WM.

and CD20 (c and d, arrows) in meninges adjacent to a subpial grey matter lesion (GML) or adjacent to normal appearing gray matter (NAGM) in MS donors with low CD3 + or CD20 + meningeal cell count. e., f. Representative immunostaining for CD3 and CD20 in Lymph Nodes used as technical positive controls. In a-d, scare bars represent 100μm. In e, f, scare bars represent 200μm.

Supplementary figure 4. Enrichment of meningeal T cells and B cells is not linked to disease
duration. Disease duration in MS donors with high vs low (a) CD3 + or (b) CD20 + meningeal cell count. Data shown as mean±SD. Statistically significant differences were tested by the nonparametric Mann Whitney test (p<0.05).

Supplementary figure 5. Enrichment of meningeal T cells and B cells is not linked to the proportion of cortical
subpial demyelinated grey matter lesions. Quantification of (a, b) number of cortical grey matter lesions (GMLs), (c, d) proportion of leukocortical (type I) GMLs, (e, f) proportion of intracortical (type II) GMLs and (g, h) proportion of subpial (type III) GMLs in MS donors with high vs low meningeal CD3 + T cells (a, c, e, g) or CD20 + B cell (b, d, f, h) count. Each data point represents the proportion of GMLs in all tissue blocks analyzed per case. Data shown as mean±SD. Statistically significant differences were determined by the non-parametric Mann Whitney test.

Supplementary figure 6. Enrichment of meningeal T cells and B cells is not linked to the total number of subcortical white matter lesions. Total number of subcortical white matter lesions in
MS donors with high vs low (a) CD3 + or (b) CD20 + meningeal cell count. Data shown as mean±SD. Statistically significant differences were tested by the non-parametric Mann Whitney test (p<0.05).

Supplementary figure 7. Characterization of mixed active-inactive white matter lesions (WLM) in progressive MS cases.
A mixed active-inactive WML in progressive MS case, showing loss of luxol fast blue (LFB) staining (a), loss of immunoreactivity for the proteolipid protein (PLP, in b) and loss of immunoreactivity for the myelin oligodendrocyte glycoprotein (MOG, in c) within the lesions, with intracellular inclusions of MOG+ myelin products inside ionized calcium-binding adapter molecule-1 (IBA1)+ macrophages at the lesion edge (d), indicative of demyelinating activity. Immunoreactivity for the CD68 lysosomal marker is abundant throughout the lesion and accumulates at the lesion edge (e) in a staining pattern consistent with enlarged lysosomes within phagocytic cells (f). Human leukocyte antigen (HLA-DR)+ myeloid cells accumulate at the lesion edge (g) and show enlarged morphology (h), consistent with the active phenotype. CD3+ T cells are found in the parenchyma (i) and as perivascular "cuffs", expanding within the Virchow-Robbin space of capillaries (j), located within the lesion or at the peri-lesional areas. CD20+ B cells are occasionally found in the parenchyma (k) and more often observed as perivascular "cuffs" (l), located within the lesion or at the peri-lesional areas.

Supplementary figure 8. Characterization of inactive WML in progressive MS cases.
Inactive WML in a progressive MS case, showing loss of luxol fast blue (LFB) staining (a), loss of immunoreactivity for the proteolipid protein (PLP, in b) and loss of immunoreactivity for the myelin oligodendrocyte glycoprotein (MOG, in c) with no evidence of intracellular inclusions of MOG+ myelin products inside ionized calcium-binding adapter molecule-1 (IBA1)+ macrophages (d), indicative of no demyelinating activity. Immunoreactivity for the CD68 lysosomal marker is scarce within the lesion and more obvious at the lesion edge (e) in a staining pattern consistent with inactive, gliotic cells (f). A paucity of human leukocyte antigen (HLA-DR)+ microglia can be found at the lesion edge (g) and show small ramified morphology (h), consistent with the inactive, resting phenotype. (i-l) CD3+ T cells and CD20+ B cells are occasionally found in the parenchyma (i, k) and perivascular space (j, l), located within the lesion or at the perilesional area. In a-d, scare bars represent 100μm.