Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders

Abstract

The meninges contain adaptive immune cells that provide immunosurveillance of the central nervous system (CNS). These cells are thought to derive from the systemic circulation. Through single-cell analyses, confocal imaging, bone marrow chimeras, and parabiosis experiments, we show that meningeal B cells derive locally from the calvaria, which harbors a bone marrow niche for hematopoiesis. B cells reach the meninges from the calvaria through specialized vascular connections. This calvarial-meningeal path of B cell development may provide the CNS with a constant supply of B cells educated by CNS antigens. Conversely, we show that a subset of antigen-experienced B cells that populate the meninges in aging mice are blood-borne. These results identify a private source for meningeal B cells, which may help maintain immune privilege within the CNS.

Conflict of interest statement

Competing interests: All authors declare no conflict of interest.

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1.. B cells represent a main immune cell type in mouse meninges and are capable of trafficking through meningeal lymphatics.

(A) Cartoon representing the structural organization of the meninges. (B) Representative flow cytometry plot showing the proportion of B cells within the overall CD45+ population in mouse dura (average of n=4 mice, data generated from a single experiment). (C) Schematic depiction of the experimental approach to perform in vivo two-photon imaging in the subdural space of CD19-Tomato mice. (D) Representative two-photon image of extravascular B cells in CD19-Tomato mouse meninges (scale bar=50μm). (E) Two-photon time-lapse imaging in the meninges of a CD19-Tomato mouse. Intravascular B cell: yellow arrowhead; extravascular B cells: white arrowhead (scale bar=20μm). (F) Schematic depiction of the experimental approach (ICM: Intra-Cisterna Magna). (G) Flow cytometry analysis of donor (CD19-Tomato) derived B cells in inguinal lymph nodes (iLN) and cervical lymph nodes (cLN) 24h post-injection. (H) Frequency and absolute number of donor-derived B cells in iLN and cLN (mean ± SEM; n=4 mice; Mann Whitney U test *P<0.05; data generated from a single experiment). (I) Representative confocal image of donor (CD19-Tomato) B cells trafficking through the dura lymphatics 24h post-injection (low magnification image scale bar=50μm; high magnification image scale bar=20μm). (J) Representative confocal image of donor (CD19-Tomato) derived B cells in cLN 24h post-injection (low magnification image scale bar=100μm; high magnification image scale bar=50μm).

Fig. 2.. Mouse meninges harbor a heterogeneous B cell population that encompass multiple developmental stages.

(A) Uniform manifold approximation and projection (UMAP) of CD45+ brain cells from a deposited scRNA-seq dataset (GSE98969). Normalized expression for Ighm, Igkc and Iglc2 was color-coded for transcript counts. The cell cluster highly enriched for the gene set was inferred as B cells. (B) Enrichment for transcripts linked to different B cell maturation stages. Pan-B cell: Cd79b and Cd19; mature B cell: H2-Aa and Ms4a1; immature B cell: Rag1 and Cd93. (C) Flow cytometry analysis of bone marrow (BM), brain, dura, blood and spleen B cell in C57BL/6 mice. B cells were gated as CD45+CD19+ and lineage (CD3, CD11b, F4/80 and Gr-1) negative (left column) and were further divided into early (B220loCD43hi), late (B220loCD43lo), and mature (B220hiCD43−) subset (middle column). CD93 and IgM staining is shown for the three subsets (right column). (D) Flow cytometry analysis of Rag1−/− mice. B cells derived from BM, brain, and dura were gated as CD19+ and lineage (CD3, CD11b, F4/80 and Gr-1) negative (left column) and were further divided into early (B220loCD43hi), late (B220loCD43lo), and mature (B220hiCD43−) subsets (right column). (E) Quantification of B cell subsets in bone marrow, brain and dura of C57BL/6 and Rag1−/− mice (n=8 and 7 mice respectively; data generated from two independent experiments). (F) Representative confocal images of the dura mater from Rag1-GFP mice (scale bar=100μm). (G) Representative confocal image of IgM+ (yellow arrowhead) and IgM− B cells (red arrowhead) in CD19-Tomato mice (low magnification image scale bar=1mm; high magnification image scale bar=50μm).

Fig. 3.. scRNA-seq analysis reveals a similar transcriptomic pattern in dura and BM B cells.

(A) Schematic depiction of the experimental design related to scRNAseq. (B) Expression of featured genes denoting mature and developing B cells in blood, BM and dura. (C) UMAP of 13,281 B cells aggregated from blood, BM, and dura collected from three C57BL6 mice and colored by cluster (left) or tissue origin (middle) (data generated from a single experiment). (D) UMAP plots split by tissue showing the distribution of blood, BM, and dura B cell. E) Proportional contribution of the three tissues (blood, BM, and dura) to the ten B cell clusters. (F) Gene expression heatmap of the top 10 signature genes per cluster. (G) Frequency of C-region usage per tissue determined by scBCRseq. (H) Developmental trajectory displayed on PCA map and colored by slingshot pseudotime. (I) Enrichment of key transcripts differentially expressed throughout B cell maturation.

Fig. 4.. Mass cytometry confirms developmental heterogeneity of dura B cells.

(A) Schematic depiction of the experimental design related to CyTOF analysis. (B) Unsupervised clustering by means of t-distributed stochastic neighbor embedding (t-SNE) of 6,000 live singlet CD45+CD19+ B cells obtained from concatenation of three tissues (blood, BM, and dura) collected from three C57BL6 mice (data generated from a single experiment). Cells are displayed by pseudocolor (left) and contour plots (right). (C) Heatmap showing the surface protein expression of selected B cell markers among the six B cell clusters (cluster 7 was arbitrarily excluded due to low cell number). (D) Staining enrichment of representative surface markers for mature and developing B cells. (E) Merged tSNE plot colored by cluster (left) and tSNE plot split by tissue (middle). Frequency of each cluster in blood, BM, and dura (right).

Fig. 5.. Skull BM chimeras demonstrate that dura B cells originate from the calvaria.

(A) Schematic depiction of the experimental design of calvaria BM transplantation. (B) Representative flow cytometry plots of B cells (gated on CD19+CD11b− cells) and CD4 T cells (gated on CD19−CD11b−Thy.1+CD4+ cells) from multiple compartments in recipient mice (left). Percentage of donor-derived (CD45.1+) and host-derived (CD45.2+) B and CD4 T cells are shown (right). (C) Frequency of donor-derived B cells and CD4 T cells per compartment (mean ± SEM; n=9 mice; Mann Whitney U test ***P<0.001; data generated from two independent experiments). (D) Representative confocal image of an IgM− B cell trafficking from the calvarial BM towards meninges through a skull vascular channel.

Fig. 6.. Ligand-receptor interactions between CXCL12+ fibroblasts and CXCR4+ early B cells in the dura.

(A) Dot plot presenting the expression level of featured genes in fibroblasts and B cells at different maturation stages. (B) NicheNet analysis displaying the putative interaction score between fibroblasts-derived ligands and B cell receptors. (C) Representative confocal images of freshly prepared dura explant from a Cxcl12-DsRed reporter mouse (Tomato-lectin-488 injected iv. before perfusion). (D) FACS analysis of dura fibroblasts from Cxcl12-DsRed reporter mice (representative of three mice; data generated from a single experiment). (E) FACS analysis of CXCR4 expression in early, late, and mature B cells from dura and BM. (F) frequency of CXCR4+ B cells in the three B cells subsets from dura and BM (mean ± SEM; n=4 mice; data generated from a single experiment).

Fig. 7.. Age-associated B cells disseminate throughout the dura of aged mice.

(A) Schematic depiction of the experimental design of scRNAseq comparing young and aged mice. UMAP of 9,352 B cells aggregated from seven 12-week-old and seven 25-month-old C57BL6 female mice (data generated from two independent experiments). (B) Distribution of B cells from young and aged mice. (C) Contribution of young versus aged mice to each cluster. (D) Differential gene expression analysis of ABCs versus mature B cells. (E) Violin plots showing top up-regulated genes in ABCs compared to mature B cells. (F) Dura ABCs gated as the B220hiCD23−CD2+Sca1+ cells. (G) ABC population is significantly increased in the dura of aged mice compared to young mice (mean ± SEM; n=5 mice; unpaired Student’s t test **P<0.01; data generated from two independent experiments). (H) Flow cytometry histogram showing increased levels of Syk protein in dura ABCs (representative of three 18-month-old female mice; data generated from a single experiment). (I) Ig heavy chains transcript counts per cell in ABCs and mature B cells. (J) Frequency of heavy chains usage determined by BCRseq in ABCs and mature B cells (violin plot; n=11-14 mice per group; two-ways ANOVA and Bonferroni post-hoc test ***P<0.001).

Fig. 8.. Age-associated B cells in the aged dura originate from the periphery.

(A) Venn diagrams representing the proportion of detected B cell clones shared between dura mature B cells and blood in young and aged mice, or between dura ABCs and blood. The connected pie chart represents the percentage of dura B cells (mature or ABCs) belonging to shared clonotypes. (B) Venn diagrams representing the proportion of detected plasma cell (PC) clones shared between dura and blood in young and aged mice, or between dura plasma cells and dura ABCs. The connected pie chart represents the percentage of dura plasma cells belonging to shared clonotypes. (C) UMAP showing the distribution of the ten most frequent clones in dura.