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. 2020 Jul;30(4):779-793.
doi: 10.1111/bpa.12841. Epub 2020 Apr 26.

B cell rich meningeal inflammation associates with increased spinal cord pathology in multiple sclerosis

Camilla Reali  1   2 , Roberta Magliozzi  1   3 , Federico Roncaroli  1   4   5 , Richard Nicholas  1 , Owain W Howell  1   6 , Richard Reynolds  1
Affiliations

B cell rich meningeal inflammation associates with increased spinal cord pathology in multiple sclerosis

Camilla Reali et al. Brain Pathol. 2020 Jul.

Abstract

Increased inflammation in the cerebral meninges is associated with extensive subpial cortical grey matter pathology in the forebrain and a more severe disease course in a substantial proportion of secondary progressive multiple sclerosis (SPMS) cases. It is not known whether this relationship extends to spinal cord pathology. We assessed the contribution of meningeal and parenchymal immune infiltrates to spinal cord pathology in SPMS cases characterized in the presence (F+) or absence (F-) of lymphoid-like structures in the forebrain meninges. Transverse cryosections of cervical, thoracic and lumbar cord of 22 SPMS and five control cases were analyzed for CD20+ B cells, CD4+ and CD8+ T cells, microglia/macrophages (IBA-1+), demyelination (myelin oligodendrocyte glycoprotein+) and axon density (neurofilament-H+). Lymphoid-like structures containing follicular dendritic cell networks and dividing B cells were seen in the spinal meninges of 3 out of 11 F+ SPMS cases. CD4+ and CD20+ cell counts were increased in F+ SPMS compared to F- SPMS and controls, whilst axon loss was greatest in motor and sensory tracts of the F+ SPMS cases (P < 0.01). The density of CD20+ B cells of the spinal leptomeninges correlated with CD4+ T cells and total B and T cells of the meninges; with the density of white matter perivascular CD20+ and CD4+ lymphocytes (P < 0.05); with white matter lesion area (P < 0.05); and the extent of axon loss (P < 0.05) in F+ SPMS cases only. We show that the presence of lymphoid-like structures in the forebrain is associated with a profound spinal cord pathology and local B cell rich meningeal inflammation associates with the extent of cord pathology. Our work supports a principal role for B cells in sustaining inflammation and tissue injury throughout the CNS in the progressive disease stage.

Keywords: B-cell follicle; axon loss; demyelination; lymphoid-like structures.

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Conflict of interest statement

RR has received speaking honoraria from Roche, Novartis and ECTRIMS and grant funding from MedImmune plc. OWH has received travel reimbursement or speaking honoria (paid to Swansea University) from Roche, the Neurology Academy and ECTRIMS. All other authors have no relevant disclosures.

Figures

Figure 1
Figure 1
Characterization of ectopic lymphoid‐like structures in post‐mortem spinal cord tissue from SPMS cases previously characterized in the presence (F+) or absence (F−) of lymphoid‐like structures in the forebrain meninges (A). Immunostaining of serial spinal cord sections from an F+ SPMS case shows a meningeal lymphoid‐like structure comprising a dense aggregate of CD20+ B cells (B), a reticulum of CD35+ fibers consistent with the presence of follicular dendritic cells (C), CD3 and CD8+ T cells (D–F) and plasma cells labeled with an anti‐Ig‐G, ‐A, ‐M antibody (G). Proliferating B cells were identified by double immunofluorescence staining for CD20 (red) and Ki67 (green; H). CD20+ B cells were present in the meninges of F− SPMS cases but at lower densities and did not display any organised structure (I). Scale bar in B = 50µm applies to all images.
Figure 2
Figure 2
Quantifying meningeal, perivascular and parenchymal B cells in the spinal cord. Anti‐CD20 immunohistochemistry revealed moderate (A) to mild (B) infiltrates of B cells in the spinal meninges, alongside small numbers in the perivascular space and tissue parenchyma (C, D). The number of CD20+ B cells was greater in the F+ SPMS meninges compared to the F− SPMS and controls (E). The number of B cells was also increased in comparison to controls in perivascular and parenchymal tissues. Values represent total CD20+ cells per section, from three sections per case with median and interquartile range indicated. Kruskal–Wallis and Dunn's multiple comparison posttest. *P < 0.05, **P < 0.01. Scale bar: A, B = 40 µm; C, D = 30 µm. Ctrl, control; F+ SPMS, lymphoid‐like structure positive SPMS; F− SPMS, lymphoid‐like structure negative SPMS.
Figure 3
Figure 3
T cell infiltrates in the SPMS spinal cord. Immunohistochemical detection of CD4+ T cells in different compartments of the spinal cord of SPMS cases (A–C). The number of CD4+ T cells was greater in the meninges and in white and grey matter perivascular cuffs in F+ SPMS compared to controls (D). CD8+ T cells were occasionally seen as modest infiltrates in the meninges and at higher numbers than CD4+ T cells in perivascular spaces and parenchyma of the spinal cord white and grey matter (E–G). There was a large and variable extent of CD8+ infiltrates of the cord meninges, which differed between F+ SPMS and controls but not between F+ and F− SPMS (H). Scatter dot plot of total immunopositive cells per section, from three sections per case, with median and interquartile range indicated. Kruskal–Wallis and Dunn's multiple comparison posttest. *P < 0.05. Scale bar: 40 µm. Ctrl, control; F+ SPMS, lymphoid‐like structure positive SPMS; F− SPMS, lymphoid‐like structure negative SPMS.
Figure 4
Figure 4
Demyelination and axon loss in the SPMS cord. Anti‐MOG immunostaining revealed the variable extent of demyelination in the F+ and F− SPMS spinal cord. Multiple discrete white and grey matter lesions or extensive confluent demyelination was seen in F+ SPMS, whereas lesions tended to be smaller in the F− SPMS cord (A–C; arrows). MOG+ myelin in a control case for comparison (D). The percentage of total (Tot), white matter (WM) and grey matter (GM) demyelination showed a trend to being greater in the F+ SPMS cohort in comparison to the F− SPMS cases (E). Axon density was determined by quantifying the number of neurofilament H+ (NFil) structures in the anterior (a) and lateral (b) corticospinal tracts (CST) and the dorsal column (c; F, G). Axon density relative to control counts were compared for each tract across the three sampled levels (cervical, thoracic and lumber cord) and expressed as percent axon loss. Axon density (%) was reduced in comparison to control axon counts in F+ SPMS cases at all sampled levels of the cord. Total axon count correlated with the relative area of white matter demyelination in F+ SPMS (P < 0.002 for each comparison; H). Box‐and‐whiskers plot showing the mean (+), median (line), interquartile range (box) and 5‐95 percentiles (whiskers) for each group. Differences between groups were tested by (E) the non‐parametric Mann‐Whitney test or (F) Kruskal–Wallis and Dunn's multiple comparison posttest. *P < 0.05, **P < 0.01. Correlation analysis by Spearman comparison. Scale bars (A–D, G) = 1 mm, inset 40 µm. Ctrl, control; F+, lymphoid‐like structure positive SPMS; F−, lymphoid‐like structure negative SPMS.
Figure 5
Figure 5
Microglia/macrophage activation in the SPMS cord. Example of demyelination in the case of F+ SPMS (A). Large numbers of IBA‐1+ microglia/macrophages (green) were present in the white matter parenchyma (B) and, although lower in number, in the grey matter (C; IBA‐1+ cells and neurofilament‐H+ neurons/neurites). The relative area of anti‐IBA‐1 immunoreactivity was increased in F+ SPMS in comparison to controls but was not different between F+ and F− SPMS cases (D). Data plotted as the box‐and‐whiskers plot showing the mean (+), median (line), interquartile range (box) and 5‐95 percentiles (whiskers) and compared by Kruskal–Wallis and Dunn's multiple comparison posttest. *P < 0.01. Scale bars: A = 1mm B, C = 40µm. Ctrl, control; F+, lymphoid‐like structure positive SPMS; F−, lymphoid‐like structure negative SPMS.
Figure 6
Figure 6
The main findings of this study are highlighted. Meningeal inflammation and particularly B cell inflammation (red circles), is associated with greater CD4+ T cell (blue circles) and B cells in the connective tissue and cord parenchyma. CD20+ B cell density of the meninges also correlated with the extent of underlying white matter demyelination (blue sector) and axon loss (red sector). These correlations only existed in the F+ SPMS cohort characterized in the presence of lymphoid‐like structures in the forebrain meninges.
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