Meningeal lymphoid structures are activated under acute and chronic spinal cord pathologies

(A) Representative scanning electron microscopy (SEM) images showing separation of the three spinal cord meningeal layers. (B) Representative immunofluorescent whole-mount staining of the spinal cord pia and dura meningeal layers, for T cells and B cells under uninjured conditions. Scale bar: 20 μm. (C, D) Representative immunofluorescent whole-mount staining of the pia (C), and dura (D) meningeal B- and T-cell clusters at d 14 after SCI. Scale bar; 50 μm. (E) Whole-mount quantification of the number of B cells (black) or T cells (white) in each cluster present at the pia and dura layers of uninjured mice, and along different time points following SCI. (E) Results are pooled from three animals at each time point, and normalized to the indicated area.

(A) Gene expression evaluation of lymphogenesis factors in spinal cord meninges adjacent to the injury site, at different time points after the insult, and in uninjured mice, n = 2–4; each sample represents a pool of three animals. ANOVA_Ltα; F = 1.1, P = 0.43. ANOVA_Ltβ; F = 12.9, P = 0.01. ANOVA_Ccl21; F = 2.3, P = 0.18. ANOVA_Cxcl13; F = 0.7, P = 0.54. ANOVA_Bcl6; F = 0.11, P = 0.89. ANOVA_Ccr7; F = 10.3, P = 0.01. ANOVA_Ltβr; F = 14.5, P = 0.005. (B) Whole-mount staining of CD31 and CCL21 in brain meninges, at days 3 and 7 after SCI, and in uninjured mice. Uninjured and day 3 sections: scale bar; 50 μm; day 7 section: scale bar; 100 μm. (C) Immunofluorescent whole-mount staining of spinal cord meninges 14 d after SCI for reticular fibroblasts (ERTR7) and the T-cell chemoattractant molecule, CCL21. Scale bar: 50 μm. (D, E) Immunofluorescent whole-mount staining of spinal cord meninges 14 d after SCI for lymphatic vessels (LYVE-1) and endothelium (CD31, CD34) in the pia (D), and the dura (E). Scale bar; 50 μm. (F, G) Immunofluorescent whole-mount staining of dura mater at d 7 after SCI for B220⁺ B cells, and for the B cell-associated chemokine, CXCL13, and (G) quantification of the number of co-localized B cell clusters with CXCL13 expression, under uninjured conditions, and at acute (day 7 after SCI) and chronic (days 14 and 21 after SCI) phases of the response to the injury. Results are pooled from three animals at each time point, and normalized to the tissue area. Scale bar; 50 μm. Asterisk in (A) indicates statistically significant differences after ANOVA post-hoc analysis using Tukey’s honestly significant difference (*, relative to uninjured; ** in LTβr, relative to d 14 after SCI) *P < 0.05. **P < 0.01. Data are presented as mean ± SEM.

(A) Gene expression analysis of isolated B220⁺ B and CD4⁺ T cells, from the spinal cord meninges and cervical lymph node 14 d after SCI, for evaluation of cytokines and transcription factors associated with inflammation and lymphocyte activation. Tissues from 20 animals were pooled; data shown are representative of two independent experiments; red and blue indicate high and low relative expression, respectively. (B) Quantification of B220^low and B220^high cells at the meninges in uninjured animals (black) and at day 14 after SCI (white); n = 2–4; each sample represents a pool of four animals. Data shown are representative of four independent experiments. (C, D) Immunofluorescent whole-mount staining of spinal cord meninges for IgD⁺ (C), and IgM⁺ (D) B cells at d 14 after SCI. Scale bar; 50 μm. (E) Flow cytometry plots indicating composition of IgM⁺ and IgD⁺ cells out of B220^low and B220^high B-cell subsets at the spinal cord meninges, n = 2 samples; each sample represents a pool of two animals. Data shown are representative of two independent experiments. (F) Density flow cytometry plot demonstrating the presence of PNA⁺GL7⁺ GC B cells at the meninges at d 14 after SCI. Meningeal tissues were pooled from four animals. (G) Representative immunofluorescent whole-mount staining at d 14 after SCI, of dura mater samples for proliferating Ki67⁺ cells and B cells. Asterisks indicate non-proliferating B cells. Scale bar; 50 μm. (H) Representative immunofluorescent whole-mount staining of meninges, demonstrating the proliferating BrdU⁺CD3⁺ T cells. Asterisks indicate non-proliferating T cells. Scale bar; 50 μm. (I) Representative immunofluorescent whole-mount staining of meningeal CD11c⁺ DC in close proximity to CD3⁺ T cells at d 14 after SCI. Scale bar; 50 μm. (J) Flow cytometry density plots of CD4⁺ T cells isolated from cLN and meninges adjacent to the lesion site 14 d after SCI, comparing the composition of CD44^high memory T cells, and the subpopulations of central-like and effector-like memory T cells. Results are representative of two experiments, n = 2; each sample represents a pool of four animals. **P < 0.01; Data are presented as mean ± SEM.

(A) A scheme depicting the experimental design of Myelin Oligodendrocyte Glycoprotein (MOG) immunization after SCI. Naïve WT mice (EAE), or mice 14 d after SCI (SCI+EAE) were immunized with the encephalitogenic MOG peptide. Animals subjected to SCI-only served as controls (SCI); cells were sorted by fluorescence activated cell sorting 14 d after MOG immunization, or after SCI (controls). (B) Flow cytometry plots showing TCRβ⁺ T cells and B220⁺ B cells isolated from spinal cord meninges under the different conditions detailed in (A). (C, D) qRT-PCR results for selected genes expressed by meningeal CD4⁺ T cells (C), and B220⁺ B cells (D) derived from spinal cord meninges under the different conditions detailed in (A). Each condition represents a pool of 10 mice.