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

Abstract

Getting around the blood–brain barrier The meninges comprise three membranes that surround and protect the central nervous system (CNS). Recent studies have noted the existence of myeloid cells resident there, but little is known about their ontogeny and function, and whether other meningeal immune cell populations have important roles remains unclear (see the Perspective by Nguyen and Kubes). Cugurra et al. found in mice that a large proportion of continuously replenished myeloid cells in the dura mater are not blood derived, but rather transit from cranial bone marrow through specialized channels. In models of CNS injury and neuroinflammation, the authors demonstrated that these myeloid cells have an immunoregulatory phenotype compared with their more inflammatory blood-derived counterparts. Similarly, Brioschi et al. show that the meninges host B cells that are also derived from skull bone marrow, mature locally, and likely acquire a tolerogenic phenotype. They further found that the brains of aging mice are infiltrated by a second population of age-associated B cells, which come from the periphery and may differentiate into autoantibody-secreting plasma cells after encountering CNS antigens. Together, these two studies may inform future treatment of neurological diseases. Science, abf7844, abf9277, this issue p. eabf7844, p. eabf9277; see also abj8183, p. 396 Bone marrow niches adjacent to the brain and spinal cord supply B cells and myeloid cells to the meninges and central nervous system. INTRODUCTION The meninges are a three-layer membrane that covers the central nervous system (CNS): The outermost layer, the dura mater, is attached onto the skull periosteum; the two inner layers, the arachnoid and the pia mater, cover the brain cortex. The meningeal compartment hosts both innate and adaptive immune cells, providing constant immunosurveillance of the CNS border regions. Given its strategic location, meningeal immunity is a key component of neuroimmune disorders. Recent studies have investigated the origin and dynamics of meningeal myeloid cells. However, little is known about meningeal B cells. RATIONALE To investigate the phenotype of meningeal B cells, we used different high-throughput techniques, such as single-cell RNA sequencing (scRNA-seq), cytometry by time of flight (CyTOF), and single-cell B cell receptor sequencing (scBCR-seq). We further corroborated our finding by flow cytometry and confocal imaging. We determined the origin of meningeal B cells by bone marrow transplantation (BMT) and parabiosis experiments. Lastly, we investigated how meningeal B cells change during aging. RESULTS We characterized the mouse meninges using scRNA-seq, which revealed that meningeal B cells encompass multiple stages of their development, spanning pro-B to mature B cells. Identical subsets were found in the bone marrow (BM), but not in the blood. CyTOF and flow cytometry further confirmed this result, demonstrating that early B cell subsets that are normally present in the BM are also found in the meninges under homeostasis. BMT with selective reconstitution of the skull BM showed that meningeal B cells are derived from the calvaria; namely, the hematopoietic region located within the cranial flat bones. We then performed parabiosis between wild-type and CD19-Tomato mice, which express the tdTomato fluorescent protein specifically in B cells. This experiment demonstrated that circulating B cells minimally infiltrated the mouse meninges under homeostasis. Using confocal imaging, we showed that B cells migrate from the calvaria to the meninges through specialized vascular channels traversing the inner skull bone. Interactome analysis of our scRNA-seq data highlighted a network of molecular communications between meningeal B cells and dura fibroblast-like cells (FLCs). Notably, we showed that FLCs express high levels of Cxcl12, whereas dura early B cells express its receptor Cxcr4. Expression of these molecules in the respective populations was validated by imaging and flow cytometry. The CXCL12–CXCR4 axis is required for the survival and differentiation of early B cells in the BM, and the same mechanism may be present in the dura. We also showed that aged mice (~2 years of age) accumulate age-associated B cells (ABCs) and plasma cells in the dura. Compared to naïve B cells, ABCs featured important transcriptional changes, as well as reduced diversity of the V-region repertoire and accumulation of somatic mutations, thus indicating antigen experience. Analysis of B cells clones by scBCR-seq showed that dura ABCs infiltrated from the periphery. On the basis of BCR clonality, we also suggest that dura ABCs may locally undergo terminal differentiation into immunoglobulin (Ig)–secreting plasma cells. CONCLUSION This study reveals that mouse meninges harbor a lymphopoietic niche specific for the CNS borders. B cell development in the meninges may induce immune tolerance against CNS antigens, thus preserving immune privilege within the CNS. However, blood-derived ABCs accumulate in the meninges over time. Peripheral ABCs are not educated by the CNS antigens and may locally differentiate into CNS-reactive plasma cells. This condition may endanger the immune-privileged CNS environment during aging. Composition of meningeal B cells in young and aged mice. Meningeal B cells originate from the calvaria and migrate to the meninges through skull vascular channels. These B cells complete their development locally, where fibroblast-like cells (FLCs) provide critical factors for survival and differentiation of early B cells (i.e., CXCL12). At this stage, CNS-antigen experience induces immune tolerance. In aged mice, age-associated B cells (ABCs) infiltrate the meninges from the periphery and differentiate into Ig-secreting plasma cells upon encounter with CNS antigens. 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.