Abstract
The CNS is ensheathed by the meninges and cerebrospinal fluid, and recent findings suggest that these CNS-associated border tissues have complex immunological functions. Unlike myeloid lineage cells, lymphocytes in border compartments have yet to be thoroughly characterized. Based on single-cell transcriptomics, we here identified a highly location-specific composition and expression profile of tissue-resident leukocytes in CNS parenchyma, pia-enriched subdural meninges, dura mater, choroid plexus and cerebrospinal fluid. The dura layer of the meninges contained a large population of B cells under homeostatic conditions in mice and rats. Murine dura B cells exhibited slow turnover and long-term tissue residency, and they matured in experimental neuroinflammation. The dura also contained B lineage progenitors at the pro-B cell stage typically not found outside of bone marrow, without direct influx from the periphery or the skull bone marrow. This identified the dura as an unexpected site of B cell residence and potentially of development in both homeostasis and neuroinflammation.
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Data availability
All raw single-cell sequencing data are available in the Gene Expression Omnibus (GEO) repository: GSE165153. All processed, unmodified scRNA-seq data (differential expression data, cell abundances) and technical scRNA-seq information are included as supplementary tables. An interactive version of the single-cell sequencing data created with cerebroApp51 is available at https://osmzhlab.uni-muenster.de/shiny/cerebro_meninges_mouse/ and https://osmzhlab.uni-muenster.de/shiny/cerebro_meninges_rat/. Reference datasets are publicly available: Tabula Muris bone marrow scRNA-seq data (GSM2967055); cell migration Gene Ontology term (GO:0016477); murine dura scRNA-seq data from Van Hove et al. (GSE128855). Source data are provided with this paper.
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Acknowledgements
A.H. was funded by grant no. RTI2018-095497-B-I00 from Ministerio de Ciencia e Innovacion (MICINN). The CNIC is supported by MICINN and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MICINN award no. SEV-2015-0505). G.M.z.H. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG; grant nos. ME4050/4-1 and ME4050/12-1) and DFG grant no. ME4050/8-1, under the frame of E-Rare-3, the ERA-Net for Research on Rare Diseases G.M.z.H. was also supported by the Heisenberg program of the DFG (grant no. ME4050/13-1), by the Grant for Multiple Sclerosis Innovation (Merck) and by a grant from the Ministerium für Innovation, Wissenschaft und Forschung (MIWF) des Landes Nordrhein-Westfalen. M. Heming and G.M.z.H. were supported by the Interdisciplinary Center for Clinical Research (IZKF) of the Medical Faculty of Münster (grant no. MzH3/020/20 to G.M.z.H. and grant no. SEED/016/21 to M. Heming). A.L. received funding from the European Research Council (ERC-StG grant no. 802305) and the DFG under Germany’s Excellence Strategy (EXC 2145 SyNergy—grant ID 390857198) and grant no. LI-2534/5-1. Servier Medical Art, licensed under a Creative Common Attribution 3.0 Generic License (https://smart.servier.com/), was used for Figs. 1a and 4a,b, Extended Data Fig. 5b and Supplementary Figs. 14 and 15.
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D.S., J.W., M. Heming, C.T., M. Hartlehnert, X.L., S.M.-S., A.R., A.-L.B., J.-K.S., J.M. and T.S. performed experiments. M. Heming and I.-N.L. performed computational analyses. D.S. and J.W. performed data analysis and created/edited figures. A.H., A.L., S.G.M. and H.W. cosupervised the study. M.P. revised the manuscript. G.M.z.H. conceived and supervised the study, and G.M.z.H. and D.S. wrote the manuscript. All authors critically revised the manuscript and agree with its contents.
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Extended data
Extended Data Fig. 1 Perfusion is not sufficient to deplete intra-vascular leukocytes in the meninges.
a, Graph depicting the percentage of remaining peripheral blood leukocytes of all leukocytes isolated from CNS-associated border tissues (CNS, SDM, Dura, choroid plexus (CP)) after intracardial perfusion as shown in Fig. 1b. Four healthy C57BL/6 mice were intravenously injected with a fluorescent coupled antibody against CD45 (CD45iv). 10 minutes later, blood was taken, mice were intracardially perfused and leukocytes were isolated from the CNS-associated border tissues and blood. Leukocytes were then stained with a second CD45 antibody and were measured on a flow cytometer. Data represented as mean ± SD, n = 4 (b) Dotplot of marker genes defining the different tissue-resident leukocyte (TRL) cell cluster in the merged dataset from Fig. 1c. Dot size encodes percentage of cells expressing the gene, color encodes the average per cell gene expression level. c, Feature plots of selected marker genes used to identify cluster identities in the merged dataset from Fig. 1c. d, Uniform Manifold Approximation and Projection (UMAP) plots of the merged dataset from Fig. 1c split by tissue of origin. Corresponding cluster frequencies are shown in Fig. 1d.
Extended Data Fig. 2 Transcriptome analysis of dura-resident leukocytes during neuroinflammation in mice.
a, Merged Uniform Manifold Approximation and Projection (UMAP) plot of tissue-resident leukocytes (TRL) isolated from the dura merged of naive and EAE mice corresponding to the split UMAP plot depicted in Fig. 1h. b, Dotplot of marker genes defining the different cell clusters identified in the dura of naive and EAE mice as shown in A and in Fig. 1h. Dot size encodes percentage of cells expressing the gene, color encodes the average per cell gene expression level. c, Gene score feature plots depicting the expression of gene sets identified in ILC subtypes previously (Vivier et al. 2016; Robinette et al. 2015). d, Dot plot depicting the expression of plasma cell related genes in the Bc, cswBc, and BcProg cluster in healthy and EAE mice merged. Dot size encodes percentage of cells expressing the gene, color encodes the average per cell gene expression level. e, Stacked bar plots depicting cell cluster proportions in the scRNA-Seq dataset from naive vs. EAE mice as shown in Fig. 1h. f, Plots corresponding to Fig. 1i quantifying the change of cluster proportions in naive (left) vs. EAE (right) depicted on a linear scale to facilitate the log to linear transformation. g, Dot plot depicting the expression of plasma cell related genes in the Bc and cswBc cluster in naive vs. EAE mice. Dot size encodes percentage of cells expressing the gene, color encodes the average per cell gene expression level.
Extended Data Fig. 3 Neuroinflammation specifically alters T helper cell subsets in the dura.
a, Dot plot of selected marker genes to identify different T helper cell subsets in the Dura of naive vs. EAE mice, corresponding to Fig. 1h. Dot size encodes percentage of cells expressing the gene, color encodes the average per cell gene expression level. b, Representative flow cytometry gating strategy to identify Th1, Th17 and Treg cells isolated from Dura of naive (top row) and EAE mice at peak of disease (bottom row). c, Quantification of the percentage of cells in the respective gates as shown in B. Data represented as mean ± SD, n = 5, two-sided Mann-Whitney U test was used to calculate statistical significance, *p < 0.05 **p < 0.01 ***p < 0.001 (d) Representative flow cytometry gating strategy showing the Ki67 staining in CD19+B220+ Bc in Dura of naive (top row) vs. EAE mice at peak of disease (bottom disease). e, Quantification of the percentage of Ki67+ cells in the representative gates as shown D. Data represented as mean ± SD, n = 5 (c), n = 4 e, two-sided Mann-Whitney U test was used to calculate statistical significance, *p < 0.05 **p < 0.01 ***p < 0.001. f, Heatmap depicting the expression of gene signature (GSE28237) from follicular vs. early germinal center (GC) vs. late GC Bc in the Bc and cswBc cluster. Higher values mean higher overlap of the respective gene signature in cells located in the individual cluster.
Extended Data Fig. 4 Characterization of CSF and IgA+ B cells from naive vs. EAE mice.
a, Proportion (%) of Bc clones expressing any Ighg (for example Ighg2b, Ighg2c, Ighg3) gene and Igha, Ighd, Ighm genes as identified by BCR reconstruction from 3′ single cell RNA-sequencing libraries from naive vs. EAE mice (Methods). Bc clones were classified based on the number of cells expressing identical clonotype sequences into categories Single (0–1 cells), Small (2–5 cells), Medium (6–10 cells), Large (11–25 cells). Related to Supplementary Table 12. b, Total number of IgA+CD19+ Bc and percentage of IgA+CD19+ Bc of tissue-resident leukocytes identified by flow cytometry in the dura from naive vs. EAE mice. Data represented as mean ± SD, n = 6, two-sided Mann-Whitney U test was used to calculate statistical significance, *p < 0.05 **p < 0.01. c, Representative flow cytometry gating strategy to identify IgA expressing Bc in the dura of naive mice (top row) and in the dura of EAE mice (bottom row). d, Representative flow cytometry gating strategy to identify lymphocytes in the CSF of naive mice (top row) and EAE mice (bottom row). e, Quantification of Bc and Tc proportions in the CSF of naive mice corresponding to D. mean ± SD, n = 5, two-sided Mann-Whitney U test was used to calculate statistical significance (f) Quantification of Bc, Tc and CD4+ Tc in CSF of naive and EAE mice corresponding to D. mean ± SD, n = 5 naive, n = 6 EAE, two-sided Mann-Whitney U test was used to calculate statistical significance, P-value not depicted = not significant.
Extended Data Fig. 5 B cell progenitors identified in Dura.
a, Additional zoomed feature plots of key B cell (Bc) progenitor markers in the dura of naive (WT) Lewis rats as shown in Fig. 3c. b, Schematic representation of marker gene expression during B cell maturation stages. Figure created using Servier Medical Art templates. c, Heatmap of cells from the ‘pre Bc’ and the ‘late pro Bc’ clusters shown in Fig. 3a submitted to the mouse cell atlas (MCA). All cell types from the MCA reference dataset that did not contain ‘B cell’ in their cell type annotation were excluded. Each column represents one cell barcode, each row one MCA reference dataset. Colors indicate Pearson correlation coefficient between the filtered MCA reference dataset and the submitted cells.
Extended Data Fig. 6 B progenitor cells populate the dura of naive mice.
a-e, 4% PFA fixed, whole mount dura of naive C57BL/6 mice stained for Cd19 (a) or Cd79a b, for Cd19 and Dntt c, for Cd19, Dntt and Igll1 d, and for Cd19, Dntt and Ezh2 e, by RNA ISH as described in the methods. Nuclei were stained with DAPI. Please note that each dot represents a single RNA molecule. An indication of where pictures were taken is depicted in each individual picture. Scale bars 20 μm (left) and 10 μm (insets). One representative staining of n = 2 mice a,b, and n = 3 mice c-e, is shown.
Supplementary information
Supplementary Information
Supplementary Figs. 1–16 and Supplementary Table 14.
Supplementary Table 1
Technical information on scRNA-seq samples and sequencing. STDEV, standard deviation; SEM, standard error of the mean.
Supplementary Table 2
Marker genes of merged six tissue rat dataset (corresponding to Fig. 1c,d). Marker genes were calculated in a one versus all comparison. Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. Only genes with a positive average log FC are shown. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction). Cluster names correspond to Fig. 1.
Supplementary Table 3
Cell cluster proportions of the merged six tissue rat dataset (corresponding to Fig. 1c,d). Relative cell cluster proportions are given in percentages.
Supplementary Table 4
Marker genes of individual six tissue rat dataset (corresponding to Supplementary Figs. 1a–f and 2a–f). Marker genes were calculated in a one versus all comparison. Average log FC threshold was set to 0.25 for all tissues except for ‘cns’ (average log FC set to 0.1 here due to lower differential gene expression). Genes with an adjusted P value above 0.05 were removed. Only genes with a positive average log FC are shown. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Supplementary Table 5
Cell cluster proportions of individual six tissue rat dataset (corresponding to Supplementary Figs. 1a–f and 2a–f). Relative cell cluster proportions are given in percentages. Each excel sheet shows cell cluster proportions of one tissue.
Supplementary Table 6
DE genes of merged six tissue rat dataset one versus all tissue. Differentially expressed genes of all cells in one tissue versus all cells of all remaining tissues are shown. Each sheet shows the comparison of one tissue versus all remaining tissues. A positive average log FC value demonstrates a higher expression in the respective tissue compared with the remaining tissues. Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Supplementary Table 7
DE genes of merged six tissue rat dataset one versus one tissue. Differentially expressed genes of all cells in one tissue (CNS, dura or SDM) versus all cells of another tissue (SDM, blood or CP) are shown. Each sheet shows the comparison of one tissue versus another tissue. A positive average log FC value demonstrates a higher expression in the first tissue compared with the second tissue. Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Supplementary Table 8
DE genes of merged six tissue rat dataset one versus one tissue in cluster Bc 1. Differentially expressed genes of cluster Bc 1 in CNS-associated border compartments versus blood or dura versus all remaining clusters are shown. A positive average log FC value demonstrates a higher expression in the respective first group (border compartments or dura) compared with the second group (blood or all remaining tissues). Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Supplementary Table 9
Marker genes of EAE dataset (corresponding to Fig. 1h and Extended Data Fig. 2e). Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. Only genes with a positive average log FC are shown. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Supplementary Table 10
Absolute and relative cell cluster proportions of the EAE dataset (corresponding to Fig. 1h and Extended Data Fig. 9e). Relative cell cluster proportions are given in percentages.
Supplementary Table 11
DE genes of EAE dataset EAE versus naive. Differentially expressed genes of EAE versus naive. Clusters with less than ten cells were removed. Each sheet shows the DE genes of one cluster. A positive average log FC value demonstrates a higher expression in EAE compared with control. Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Supplementary Table 12
Abundance of BCR clonotypes in naive versus EAE (corresponding to Extended Data Fig. 4a). Number of the individual BCR clonotypes identified by BCR sequencing in the naive versus EAE dataset. CTgene, BCR gene sequence; Abundance, number of B cells with the respective CTgene expressed; Condition, condition of mouse (naive versus EAE) the respective clone was found in.
Supplementary Table 13
Marker genes of a published dura dataset (corresponding to Supplementary Fig. 12a). Published dura-derived single-cell transcriptomes8 were downloaded and clustered and marker genes were calculated in one versus all comparisons. Average log FC threshold was set to 0.25; genes with an adjusted P value above 0.05 were removed. Only genes with a positive average log FC are shown. avg_log_FC, log fold change of the average expression between the cluster versus all remaining clusters; pct.1, percentage of cells with the gene detected in the cluster; pct.2, percentage of cells with the gene detected in all remaining clusters; p_val_adj, adjusted P values (based on Bonferroni correction).
Source data
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Unprocessed IHC images.
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Unprocessed IHC images.
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Source Data Extended Data Fig. 3
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Statistical source data.
Source Data Extended Data Fig. 6
Unprocessed IHC images.
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Schafflick, D., Wolbert, J., Heming, M. et al. Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges. Nat Neurosci 24, 1225–1234 (2021). https://doi.org/10.1038/s41593-021-00880-y
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DOI: https://doi.org/10.1038/s41593-021-00880-y
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