Abstract
There is increasing interest in how immune cells in the meninges—the membranes that surround the brain and spinal cord—contribute to homeostasis and disease in the central nervous system1,2. The outer layer of the meninges, the dura mater, has recently been described to contain both innate and adaptive immune cells, and functions as a site for B cell development3,4,5,6. Here we identify organized lymphoid structures that protect fenestrated vasculature in the dura mater. The most elaborate of these dural-associated lymphoid tissues (DALT) surrounded the rostral-rhinal confluence of the sinuses and included lymphatic vessels. We termed this structure, which interfaces with the skull bone marrow and a comparable venous plexus at the skull base, the rostral-rhinal venolymphatic hub. Immune aggregates were present in DALT during homeostasis and expanded with age or after challenge with systemic or nasal antigens. DALT contain germinal centre B cells and support the generation of somatically mutated, antibody-producing cells in response to a nasal pathogen challenge. Inhibition of lymphocyte entry into the rostral-rhinal hub at the time of nasal viral challenge abrogated the generation of germinal centre B cells and class-switched plasma cells, as did perturbation of B–T cell interactions. These data demonstrate a lymphoid structure around vasculature in the dura mater that can sample antigens and rapidly support humoral immune responses after local pathogen challenge.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data supporting the findings of this study are available from the corresponding authors on request. There are no restrictions on data availability. scRNA-seq data are available in the NCBI Gene Expression Omnibus (GEO) under accession code GSE254265. The BCR-seq data are available under GEO accession code GSE253135. Source data are provided with this paper.
References
Kipnis, J. Multifaceted interactions between adaptive immunity and the central nervous system. Science 353, 766–771 (2016).
Rua, R. & McGavern, D. B. Advances in meningeal immunity. Trends Mol. Med. 24, 542–559 (2018).
Korin, B. et al. High-dimensional, single-cell characterization of the brain’s immune compartment. Nat. Neurosci. 20, 1300–1309 (2017).
Brioschi, S. et al. Heterogeneity of meningeal B cells reveals a lymphopoietic niche at the CNS borders. Science 373, eabf9277 (2021).
Schafflick, D. 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).
Wang, Y. et al. Early developing B cells undergo negative selection by central nervous system-specific antigens in the meninges. Immunity 54, 2784–2794 (2021).
Liu, Y. J., Zhang, J., Lane, P. J., Chan, E. Y. & MacLennan, I. C. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur. J. Immunol. 21, 2951–2962 (1991).
Fang, Y., Xu, C., Fu, Y. X., Holers, V. M. & Molina, H. Expression of complement receptors 1 and 2 on follicular dendritic cells is necessary for the generation of a strong antigen-specific IgG response. J. Immunol. 160, 5273–5279 (1998).
Hase, H. et al. BAFF/BLyS can potentiate B-cell selection with the B-cell coreceptor complex. Blood 103, 2257–2265 (2004).
Cyster, J. G. et al. Follicular stromal cells and lymphocyte homing to follicles. Immunol. Rev. 176, 181–193 (2000).
Renshaw, B. R. et al. Humoral immune responses in CD40 ligand-deficient mice. J. Exp. Med. 180, 1889–1900 (1994).
Han, S. et al. Cellular interaction in germinal centers. Roles of CD40 ligand and B7-2 in established germinal centers. J. Immunol. 155, 556–567 (1995).
Zotos, D. et al. IL-21 regulates germinal center B cell differentiation and proliferation through a B cell-intrinsic mechanism. J. Exp. Med. 207, 365–378 (2010).
Linterman, M. A. et al. IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses. J. Exp. Med. 207, 353–363 (2010).
Shulman, Z. et al. Dynamic signaling by T follicular helper cells during germinal center B cell selection. Science 345, 1058–1062 (2014).
Victora, G. D. et al. Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 143, 592–605 (2010).
Gitlin, A. D., Shulman, Z. & Nussenzweig, M. C. Clonal selection in the germinal centre by regulated proliferation and hypermutation. Nature 509, 637–640 (2014).
Fitzpatrick, Z. et al. Gut-educated IgA plasma cells defend the meningeal venous sinuses. Nature 587, 472–476 (2020).
Serafini, B., Rosicarelli, B., Magliozzi, R., Stigliano, E. & Aloisi, F. Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis. Brain Pathol. 14, 164–174 (2004).
Magliozzi, R. et al. Meningeal B-cell follicles in secondary progressive multiple sclerosis associate with early onset of disease and severe cortical pathology. Brain 130, 1089–1104 (2007).
Peters, A. et al. Th17 cells induce ectopic lymphoid follicles in central nervous system tissue inflammation. Immunity 35, 986–996 (2011).
Kuerten, S. et al. Tertiary lymphoid organ development coincides with determinant spreading of the myelin-specific T cell response. Acta Neuropathol. 124, 861–873 (2012).
Rustenhoven, J. et al. Functional characterization of the dural sinuses as a neuroimmune interface. Cell 184, 1000–1016 (2021).
Rosenblum, J. S. et al. Non-invasive in situ visualization of the murine cranial vasculature. Cell Rep. Methods 2, 100151 (2022).
Eberl, G. et al. An essential function for the nuclear receptor RORγt in the generation of fetal lymphoid tissue inducer cells. Nat. Immunol. 5, 64–73 (2004).
Bird, D. J. et al. Olfaction written in bone: cribriform plate size parallels olfactory receptor gene repertoires in Mammalia. Proc. Biol. Sci. 285, 20180100 (2018).
Hangartner, L. et al. Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies. Proc. Natl Acad. Sci. USA 100, 12883–12888 (2003).
Zhang, X. et al. BAFF supports human B cell differentiation in the lymphoid follicles through distinct receptors. Int. Immunol. 17, 779–788 (2005).
Allen, C. D. et al. Germinal center dark and light zone organization is mediated by CXCR4 and CXCR5. Nat. Immunol. 5, 943–952 (2004).
Igarashi, K., Ochiai, K., Itoh-Nakadai, A. & Muto, A. Orchestration of plasma cell differentiation by Bach2 and its gene regulatory network. Immunol. Rev. 261, 116–125 (2014).
Han, S., Zheng, B., Schatz, D. G., Spanopoulou, E. & Kelsoe, G. Neoteny in lymphocytes: Rag1 and Rag2 expression in germinal center B cells. Science 274, 2094–2097 (1996).
Hikida, M. et al. Reexpression of RAG-1 and RAG-2 genes in activated mature mouse B cells. Science 274, 2092–2094 (1996).
Wang, Q. et al. The Allen Mouse Brain Common Coordinate Framework: a 3D reference atlas. Cell 181, 936–953 (2020).
Gossa, S., Nayak, D., Zinselmeyer, B. H. & McGavern, D. B. Development of an immunologically tolerated combination of fluorescent proteins for in vivo two-photon imaging. Sci. Rep. 4, 6664 (2014).
Moseman, E. A., Blanchard, A. C., Nayak, D. & McGavern, D. B. T cell engagement of cross-presenting microglia protects the brain from a nasal virus infection. Sci. Immunol. 5, eabb1817 (2020).
Rosenblum, J. S. et al. Developmental vascular malformations in EPAS1 gain-of-function syndrome. JCI Insight 6, e144368 (2021).
Lesciotto, K. M. et al. Phosphotungstic acid-enhanced microCT: optimized protocols for embryonic and early postnatal mice. Dev. Dyn. 249, 573–585 (2020).
Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).
Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902 (2019).
Wolock, S. L., Lopez, R. & Klein, A. M. Scrublet: computational identification of cell doublets in single-cell transcriptomic data. Cell Syst. 8, 281–291 (2019).
Popescu, D. M. et al. Decoding human fetal liver haematopoiesis. Nature 574, 365–371 (2019).
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate—a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289–300 (1995).
Traag, V. A., Waltman, L. & van Eck, N. J. From Louvain to Leiden: guaranteeing well-connected communities. Sci. Rep. 9, 5233 (2019).
McInnes, L., Healy, J. & Melville, J. UMAP: uniform manifold approximation and projection for dimension reduction. Preprint at arxiv.org/abs/1802.03426 (2018).
Watson, S. J. et al. Viral population analysis and minority-variant detection using short read next-generation sequencing. Philos. Trans. R. Soc. Lond. B 368, 20120205 (2013).
Lefranc, M. P. IMGT, the international ImMunoGeneTics information system. Novartis Found. Symp. 254, 126–136 (2003).
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Brochet, X., Lefranc, M. P. & Giudicelli, V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 36, W503–W508 (2008).
Gupta, N. T. et al. Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data. Bioinformatics 31, 3356–3358 (2015).
Bashford, G. R., Burnfield, J. M. & Perez, L. C. Physical activity discrimination improvement using accelerometers and wireless sensor network localization. Biomed. Sci. Instrum. 49, 243–250 (2013).
Acknowledgements
D.B.M., Z.F., N.G.Z., M.L.N.-D., P.M., P.C., D.M., M.B. and D.D. were supported by the intramural program of The National Institute of Neurological Disorders & Stroke, National Institutes of Health. Z.Z. was supported by the National Cancer Institute. Z.K.T. and M.R.C. were supported by a Medical Research Council Research Project Grant (MR/S035842/1) and M.R.C. by a Medical Research Council New Investigator Research Grant (MR/N024907/1). M.R.C. is supported a Wellcome Strategic Scientific award (WT211276/Z/18/Z) and a Wellcome Investigator Award (220268/Z/20/Z). J.R.F. and M.R.C. are supported by the National Institute of Health Research (NIHR) Cambridge Biomedical Research Centre and the NIHR Blood and Transplant Research Unit. The content of this Article does not necessarily reflect the views, policies or opinions of the US Department of Health and Human Services. The mention of commercial products, their source or their use in connection with material reported herein is not to be construed as an actual or implied endorsement of such products by the US government.
Author information
Authors and Affiliations
Contributions
D.B.M. and M.R.C. wrote the manuscript. D.B.M., M.R.C., Z.F. and N.G.Z. designed experiments and interpreted results with input from all of the co-authors. N.G.Z. and D.D. collected micro-CT data. J.S.R. and V.C. analysed the micro-CT data with support from D.B.M. and Z.Z.; J.R.F., A. Penalver and E.G. performed the BCR-seq. Z.F., M.L.N.-D., N.G.Z. and M.B. performed scRNA-seq experiments. Z.K.T. and C.Y.C.L. analysed all sequencing data. Z.F., N.G.Z., M.L.N.-D., M.B., A.P.S., D.A.P. and A. Portet performed mouse experiments and collected data. K.S.J.A., P.M., P.C., D.M., A.H. and T.T. participated in the collection and/or staining of human meningeal tissue.
Corresponding authors
Ethics declarations
Competing interests
J.S.R. and V.C. are affiliated with NeuroSimplicity, which is a medical device and technology company focused on medical image processing.
Peer review
Peer review information
Nature thanks the anonymous reviewers for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 DALT stromal cell populations, immune aggregate expansion with aging and absence in RORγt-/- mice.
a, Confocal image of the rostral-rhinal hub from a representative naïve mouse shows staining of podoplanin (yellow) and CD45 (red) (scale bar, 50 µm) (n = 3). b, Representative flow cytometric plot of stromal cell populations in a representative rostral-rhinal hub sample. c, Quantification of fibroblastic reticular, blood endothelial and lymphatic endothelial cells in steady-state rostral-rhinal hub samples (n = 5 mice; mean ± SD). d, Representative flow cytometric plots show the percentage of CD45+ cells isolated from the rostral-rhinal hub versus the meningeal lobes of naïve B6 mice at 13 weeks of age. Bars depict the frequency and absolute number of CD45+ cells from each location. UMAPs show the immune subsets found in the rostral-rhinal hub and lobes combined (centre) versus each individual location (far right). B cells (TCRβ-B220+CD19+), CD4+ T cells (TCRβ+CD3+CD8-CD4+), CD8+ T cells (TCRβ+CD3+CD8+CD4-), type 2 innate lymphoid cells (ILC2s) (TCRβ-CD3-CD127+ST2+), plasmacytoid dendritic cells (pDCs) (CD11c+Ly6C+B220+), monocyte-derived DCs (moDCs) (CD11b+F4/80loCD11c+Ly6Clo), inflammatory monocytes (CD11+CX3CR1loLy6Chi), patrolling monocytes (CD11+CX3CR1hiLy6Clo), and neutrophils (CD11b+Ly6G+) are labelled (n = 5 mice per group; **p = 0.0018, unpaired two-tailed Student’s t-test; one of two independent experiments; mean ± SEM). e, Representative confocal images of rostral-rhinal hubs (scale bar, 70 µm) and SSS (scale bar, 50 µm) immunolabeled for CD4 (green), CD8 (white), B220 (red) and CD45 (blue) from 8-, 26-, and 60-week-old naïve mice (yellow arrows denote immune clusters f, Quantification of CD4+, CD8+ and B220+ cells in meningeal whole-mount tissue from indicated age groups (n = 4-5 mice per group; 8-week-old vs. 60-week-old mice, CD4: **p = 0.0047; CD8: ***p = 0.0004; B220: ****p = 0.00002, unpaired two-tailed Student’s t-test; mean ± SD). g, Quantification of total lymphoid cluster number (n = 4-5 mice per group; 8-week-old vs. 60-week-old mice, ***p = 0.0006, unpaired two-tailed Student’s t-test) and average area (n = 4-5 mice per group; 8-week-old vs. 60-week-old mice, **p = 0.0014, unpaired two-tailed Student’s t-test; mean ± SD) in indicated age groups (one of two replicated experiments). h, Confocal images of rostral-rhinal hubs from wild-type (WT) and RORγt-/- mice (scale bar, 50 µm) (n = 4 WT and n = 3 RORγt-/-).
Extended Data Fig. 2 Development of the rostral-rhinal venolymphatic hub.
Representative images are shown. Gross evaluation of the mouse head at post-natal day 9 (P9 mouse, gross) under 2X magnification reveals that the superior sagittal sinus has not yet developed. Rather, the rostral rhinal confluence (red box) is the main venous drainage of the head at this stage, connecting to the olfactory sinus, rostral rhinal veins, developing diploic veins, and developing superior sagittal sinus. Micro-computed tomography (Micro-CT) of the intact mouse head at post-natal day 8 (P8) following terminal vascular casting with Microfil® polymer, fixation, decalcification, and 3-day immersion in phosphotungstic acid (PTA), which binds protein, shows the rostral rhinal venous hub (red box) (n = 3 mice per group). The developing superior sagittal sinus and olfactory sinus are connected to this hub. The developing caudal confluence of sinuses, median vein, and tentorial sinuses, all providing venous drainage to the posterior aspect of the head are appreciated. In this Micro-CT of the P8 mouse head, the polymer is hyper-dense, as is the bone, which has bound PTA. Volumetric reconstruction and three-dimensional render of the Micro-CT of the P8 mouse head (3D visualization of Micro-CT of polymer-casted P8 mouse) shows the relationships of this anatomy. Gross evaluation of the mouse head at post-natal day 12 (P12) (n = 3 mice per group), shows that the rostral rhinal hub (red box) is the main site of continued post-natal initial development and organization of veins bridging from the intracranial space, e.g., dural sinuses, to the diploic veins entering the calvarial bone marrow, which can be seen throughout the anterior aspect of the head here. The superior sagittal sinus at this stage is still developing, as is the caudal confluence of sinuses. The olfactory and rostral rhinal sinuses have also increased in this period from P9 to P12. Micro-CT of the adult mouse following vascular casting, decalcification, and immersion in PTA (2 weeks) allows visualization of bone, all soft tissues, and vessels. In the axial view, lymphatic vessels, which are hyper-dense due to the binding of PTA to protein within these vessels, within the wall of the superior sagittal and olfactory sinuses are seen coalescing at the dura lining the rostral rhinal venolymphatic hub (red box); this is the dural-associated lymphoid tissue (DALT). The sagittal view shows falcine lymphatic vessels (arrowhead) between the olfactory bulb and cortex connecting to the DALT (arrow) lining the rostral-rhinal venolymphatic hub (asterisk), which resides in the depression in the calvarium; this depression enters the diploic space between the inner and outer table of the bone. The coronal view also shows the falcine lymphatic vessels arising between the olfactory bulbs and travelling into the wall of the olfactory sinus; there are also falcine lymphatic vessels connecting to the dural lining the olfactory venous plexus inferiorly. Sagittal view of the three-dimensional visualization of vessels, brain, and bone from iterative micro-CT of the adult mouse head shows the rostral-rhinal venolymphatic hub (red box) within the depression in the calvarium (left) (n = 5 mice per group). The superior sagittal sinus has two components—one dural and one within the diploic space, which can be appreciated when the bone is removed (right). This visualization is obtained by registration of a bone mesh (blue) from micro-CT before processing, vessels (white) extracted from the micro-CT following decalcification, and brain extracted from micro-CT following immersion in PTA; the Allen Atlas CCF v.333 are registered to the brain and shown in multicolour.
Extended Data Fig. 3 Rostral-rhinal and basal olfactory venous hubs under homeostasis and bead acquisition by hub lymphatics.
a, Representative confocal image of a sagittal brain section from a VE-Cadherincre/+ Stopfl/fl TdTomato (red) mouse immunolabelled for Lyve1 (green), CD45 (white), and E-cadherin. Nuclei are blue. Bone marrow (BM), skull bone, bone recess, the rostral-rhinal hub (hub), arachnoid mater, and olfactory bulb are labelled in the merged image (scale bar, 100 µm) (n = 3 mice). b, Representative confocal images of the basal olfactory venous plexus immunolabeled for CD4 (blue), B220 (white), CD45 (red) in a naive CX3CR1gfp/+ (green) mouse (scale bar, 400 µm); right, high-magnification image of highlighted box (scale bar, 50 µm) (n = 3 mice). c, Confocal images of the rostral-rhinal hub of a mouse 6 and 16 hrs following intravenous injection of 0.2 µm beads (green). Hub is immunolabeled for CD45 (blue) and Lyve1 (red). White arrows denote beads inside Lyve1+ lymphatic vessels. (scale bar, 20 µm) (n = 3 mice per group; one of two independent experiments).
Extended Data Fig. 4 Identification of DALT in human dura samples.
a, Confocal images of human dural tissue immunolabelled for CD3 (magenta), CD20 (green), and CD31 (red), with nuclei shown in cyan (scale bar, 50 µm). b, Confocal images of human dural tissue immunolabelled for CD3 (magenta), and CD19 (green), with nuclei shown in blue (scale bar, 40 µm). c, Confocal image of human dural tissue immunolabelled for CD3 (red), CD20 (green), and Lyve1 (cyan), with nuclei shown in blue (scale bar, 25 µm). d, A representative section of human dura mater including the superior sagittal sinus (SSS) was immunolabeled with antibodies against IBA1 (myeloid cells), CD4, CD8, and CD20 (B cells). The fluorescent channel for each immunolabel is overlayed on a brightfield image of the tissue. The magnified images at the bottom are denoted with a yellow box in the lower power view above. (upper scale bar, 250 µm; lower scale bar, 31 µm). The images shown in panels a to d were captured from four different patient samples.
Extended Data Fig. 5 Dextran uptake, DALT disorganization in LTα-deficient animals, IL21-expression and B cell distribution after VSV infection.
a, Super resolution confocal image of the vascular network (CD31, red) within the rostral-rhinal hub of a mouse following intravenous administration of 2,000 kDa dextran (green) (scale bar, 100 µm) (n = 6). b, Confocal image of the rostral-rhinal hub following 2,000 kDa dextran (green) administration (scale bar, 20 µm). Inset shows high power image of a macrophage (F4/80, red) with internalized dextran (scale bar, 4 µm) (n = 6). c, Representative confocal images of rostral-rhinal hubs immunostained for CD4 (red) and B220 (green) from IL21 reporter (white) mice 8 days post intranasal VSV infection (scale bar, 100 µm); bottom, higher magnification image of highlighted box (scale bar, 20 µm) (blue arrows indicate IL21-mCherry-expressing CD4+ cells within a B cell cluster) (n = 4 mice total from two independent experiments). d, Representative confocal images of rostral-rhinal hubs immunostained for CD4 (green) and B220 (red) from WT and LTα-/- animals 7 days after intranasal VSV inoculation (scale bar, 100 µm) (n = 4 mice per group; one of two independent experiments). e, Histo-cytometric dot plots of meningeal whole-mount tissue immunolabeled for IgA (blue) and IgG (red) from mice at the indicated timepoints post intranasal VSV inoculation. f, Quantification of IgA+ and IgG+ cells by meningeal whole-mount confocal imaging in rostral-rhinal hub and superior sagittal sinus at the indicated timepoints post-VSV infection (n = 4-5 mice per group; mean ± SD).
Extended Data Fig. 6 Analysis of gene expression in the rostral-rhinal hubs of naïve versus VSV-infected mice.
a, Mean expression dot plot of marker genes used to define cell type annotations in single cell RNAseq data from the rostral-rhinal hubs of naïve and d8 VSV-infected mice. See Fig. 4g and Extended Data Fig. 6c. Size of circles indicate proportion of cells expressing the gene, and increasing gradient from purple to blue to yellow corresponds to increasing mean expression (scaled from 0 to 1). b, Mean expression dot plot of B cell and Tfh related genes plotted for the denoted cell types. Size of circles indicate proportion of cells expressing the gene, and increasing gradient from white to blue corresponds to increasing mean expression (scaled from 0 to 1). c, Top: UMAP embedding of 10,839 cells from the rostral-rhinal hubs of naive and d8 VSV-infected mice coloured according to treatment group. Bottom: UMAP embedding of 812 developing B cells, plasmablast/plasma cell, GC B cells, Tfh cells, Tfr cells and B-T multiplets coloured according to experimental group. d, Mean expression dot plot of genes encoding surface receptors, BCR signalling molecules, Ig isotype, proliferation and transcriptions factors in B GC cells and B plasmablast/plasma cells from the rostral-rhinal hubs of naïve versus VSV-infected mice. A dot plot is also shown for Tfh associated genes in Tfh cells. Size of circles indicate proportion of cells expressing the gene and increasing gradient from white to blue corresponds to increasing mean expression (scaled from 0 to 1).
Extended Data Fig. 7 Analysis of BCR clones in the superior sagittal sinus and skull bone marrow.
a, Representative BCR clone network plots in sagittal sinus and skull bone marrow coloured by immunoglobulin isotype in naïve, week 1 and week 3 of VSV infection. Networks were down-sampled to the same sequencing depth (per tissue type; n = 2500 and n = 5000 UMI counts for sagittal sinus and skull bone marrow, respectively) before plotting. Each node represents a unique BCR, and each connected component represents a single clone. Edges between nodes are due to non-indel differences between pairs of BCRs. Sizes of nodes are scaled according to UMI count. b, Stacked bar charts of isotype usage amongst clones in the sagittal sinus and skull bone marrow in naïve, week 1 and week 3 of VSV infection. BCR networks were down-sampled to the same sequencing depth, and clone isotype usage was tabulated per sample and represented as a percentage out of 100%. (n = 4-5 mice per group).
Extended Data Fig. 8 Analysis of BCR clonal overlap in the rostral-rhinal hub, skull bone marrow, and superior sagittal sinus.
a, Unique rostral-rhinal hub BCRs in naïve, week 1 and week 3 of VSV infection (all isotypes considered) per sample (n = 4-5 mice per group; non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons correction; **p = 0.0327). Boxes capture the first to third quartiles, and whiskers span from minima to maxima on each side of the box, respectively. b, Pie charts of IgM, IgG, and IgA clone overlaps in rostral-rhinal hub, skull bone marrow and sagittal sinus in naïve, week 1 and week 3 of VSV infection. For each tissue type, the proportion in each pie chart is a proportion of clones within that tissue. The largest slice of each pie chart corresponds to the proportion of clones that are found unique to the main tissue type, and smaller slices represent the proportion of clones found overlapping between the main tissue type and the other respective tissues. A singular grey slice is used for clones that are found to overlap between more than 2 tissue types. c, Representative reconstructed BCR lineage trees for clones found to be shared between the rostral-rhinal hub and superior sagittal sinus. Each coloured node is a unique BCR sequence found in a clone, coloured according to tissue source. Black nodes are germline sequences and white nodes are inferred nodes. Numbers of edges indicate the distance (mutation count) away from the parent nodes.
Extended Data Fig. 9 Germinal centre B cell depletion following transcranial CD40L blockade.
a, Representative flow cytometric plots of GL7+Fas+ germinal centre B cells in rostral-rhinal hub samples from mice 10 days after intranasal VSV inoculation with a sub-scalp (transcranial) course of either isotype control or αCD40L neutralizing antibody. Graphs show quantification of percent GL7+Fas+ germinal centre B cells of total B cells in rostral-rhinal hubs following transcranial CD40L blockade versus control (n = 4-5 mice per group; **p = 0.0010, unpaired two-tailed Student’s t-test; mean ± SD) and total cell counts (n = 4-5 mice per group; *p = 0.0145, unpaired two-tailed Student’s t-test; mean ± SD) (one of two independent experiments). b, Quantification of percent GL7+Fas+ germinal centre B cells (n = 4-5 mice per group; ****p < 0.0001, unpaired two-tailed Student’s t-test) and total cell counts in spleens of mice treated transcranially with either isotype control or αCD40L (n = 4-5 mice per group; **p = 0.0081, unpaired two-tailed Student’s t-test; mean ± SD). c, Confocal images of rostral-rhinal hubs immunolabeled for IgA (blue), IgG (green) and B220 (red) from mice 8 days after intranasal challenge with VSV during transcranial CD40L blockade (scale bar, 100 µm). d, Quantification of IgA+ and IgG+ cells in rostral-rhinal hubs 8 days post intranasal challenge with VSV (n = 7 mice per group; IgA: **p = 0.0092, IgG: *p = 0.0150, unpaired two-tailed Student’s t-test; mean ± SD). e, Relative expression of VSV-specific mRNA in olfactory bulbs of animals treated transcranially with isotype control or αCD40L neutralizing antibody 8 days after intranasal VSV infection (n = 8–10 mice per group; ****p < 0.0001, two-tailed Mann-Whitney test; mean ± SD). f, Representative flow cytometric plots show the frequency of B220+TFP+ VSV-specific (VI10YEN) B cells in the rostral-rhinal hubs of uninfected (n = 6 mice per group) versus day 21 post-VSV infection mice. VSV infected mice received anti-LFA1/VLA4 (n = 4 mice per group) or isotype control (n = 5 mice per group) antibodies beginning at day 6 and VSV-specific B cells adoptively transferred at day 7. g, Bar graphs depict the absolute number of VI10YEN B cells (left; **p = 0.0024 and 0.0011, one-way ANOVA multiple comparisons) and of GL7+Fas+ GC VI10YEN B cells (right; **p = 0.0039 and 0.0015, one-way ANOVA multiple comparisons; mean ± SEM) (one of two independent experiments).
Extended Data Fig. 10 Gating strategy used to identify rostral-rhinal hub B cells and bead acquisition by hub immune cells.
a, Representative flow cytometry plots depict to the gating strategy used to identify and characterize B cells extracted from the rostral-rhinal hub. b, Representative flow cytometry plots show the gating strategy used to identify immune cells in the rostral-rhinal hub that acquired 0.2 µm beads injected intravenously. See Fig. 3g. c, Representative flow cytometry plots show the gating strategy used to identify GC B cells for the plots shown in Fig. 4k.
Supplementary information
Supplementary Video 1
Super-resolution confocal imaging of fenestrated vasculature in the rostral-rhinal hub. A mouse dural whole mount was imaged at super resolution using dense z stacks. Overview images of the rostral-rhinal hub (CD31, red) show extensive fenestrations (PV1, green) and diffuse immune clusters (CD45, cyan). z–y orthogonal projections are shown for the entire hub visualizing immune clusters in three dimensions. x–y positions are seen on the overview image with the white bar. A lymphoid cluster at the edge of the hub is displayed projecting through z stacks. Finally, z–x orthogonal projections of this region are shown with x–y position seen on the overview image with the white bar.
Supplementary Video 2
Rostral-rhinal hub and dural associated lymphoid tissue. A fly-through of a representative volume rendering of brain regions, bone and vessels extracted from micro-CT images of a C57BL/6 mouse head after sample iterative processing is shown (n = 7 mice per group). Bone (blue) is segmented from the micro-CT image of the sample acquired before sample processing. Vessels (white) are segmented from the micro-CT image of the same sample acquired after decalcification. Brain region parcellations from the Allen Atlas Common Coordinate Frame v3 (multicolour) are registered to the brain visualized by micro-CT after immersion in PTA. The axial fly-through (0:08–0:17) shows the rostral rhinal confluence of sinuses (red box at 0:14), which connects to the rostral rhinal sinuses, olfactory sinus, superior sagittal sinus and diploic veins. This rostral-rhinal hub is shown in an expanded inset panel (0:18–0:21). The coronal view shows cortical and falcine veins connecting to this hub (0:22–0:30); the sagittal view shows the falcine veins connecting to the olfactory venous plexus at the skull base (0:31–0:40). In the same sagittal view, the hub is seen connecting to the super sagittal sinus, which splits around the inner table of the skull into dural and diploic components. In the axial micro-CT images acquired after vascular polymer casting, decalcification and immersion in PTA, which is radio-dense and binds protein, linear hyperdensities consistent with lymphatic vessels are seen lining the walls of the superior sagittal sinus and olfactory sinus (0:41–0:56). These vessels coalesce within a thickened region of the dura of the rostral-rhinal venous hub, the dural associated lymphoid tissue (DALT). In the coronal view, lymphatic vessels are seen within the falx connecting to the DALT of the rostral-rhinal venous hub (0:57–1:06).
Supplementary Video 3
The anatomy of the rostral-rhinal venolymphatic hub. A representative fly-through of the anatomy of the rostral-rhinal venolymphatic hub after iterative sample imaging and processing is shown (n = 7). Bone (blue) is segmented from the micro-CT image of the sample acquired before sample processing. Vessels (white) are segmented from the micro-CT image of the same sample acquired after decalcification. Brain region parcellations from the Allen Atlas Common Coordinate Frame v3 (multicolour) are registered to the brain visualized by micro-CT after immersion in PTA. The coronal fly-through (0:05–0:20) shows the rostral rhinal confluence of sinuses (red box at 0:05), which connects to the rostral rhinal sinuses, olfactory sinus, superior sagittal sinus, falcine veins, cortical veins and diploic veins. The oblique and sagittal fly-through shows the connections to these structures as well as the olfactory venous plexus (0:20–0:38). In the sagittal view, the hub is seen connecting to the super sagittal sinus, which splits around the inner table of the skull into dural and diploic components. The vascular anatomy of the hub and its relationship to the brain is shown in the fly-through without bone in view (0:39–0:49).
Rights and permissions
About this article
Cite this article
Fitzpatrick, Z., Ghabdan Zanluqui, N., Rosenblum, J.S. et al. Venous-plexus-associated lymphoid hubs support meningeal humoral immunity. Nature 628, 612–619 (2024). https://doi.org/10.1038/s41586-024-07202-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-024-07202-9
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
