The CNS is tightly regulated to maintain immune surveillance and efficiently respond to injury and infections. The current appreciation that specialized “brain-adjacent” regions in the CNS are in fact not immune privileged during the steady state, and that immune cells can take up residence in more immune-privileged areas of the CNS during inflammation with consequences on the adjacent brain parenchyma, beg the question of what cell types support CNS immunity. As they do in secondary lymphoid organs, we provide evidence in this review that stromal cells also underpin brain-resident immune cells. We review the organization and function of stromal cells in different anatomical compartments of the CNS and discuss their capacity to rapidly establish and elaborate an immune-competent niche that further sustains immune cells entering the CNS from the periphery. In summary, we argue that stromal cells are key cellular agents that support CNS-compartmentalized immunity.

The CNS, which is composed of the brain and spinal cord, has been traditionally viewed as being composed of neurons and glial cells (microglia, astrocytes, oligodendrocytes). We seldom think of immune cells as being part of the brain except during severe inflammation caused by infection or chronic disease such as multiple sclerosis (MS). As such, the brain has long been considered as “immunoprivileged.” However, we now know that there are compartments of the brain, so-called “brain-adjacent regions,” that contain dense networks of immune cells (14). From an immunosurveillance perspective, this makes a great deal of sense—having an army of immune cells close by and ready to quickly protect the most vital organ of the body is a wise evolutionary move. However, we also know that immune cells do not exist in a vacuum but rather are supported by scaffold cells, which can be collectively referred to as “stroma.” In secondary lymphoid organs, fibroblastic reticular cells (FRC) are a major subset of stromal cells. FRCs in the lymph node (LN) express podoplanin (PDPN/gp38) and platelet-derived growth factor receptor-α (PDGFRα) (5). PDPN+ PDGFRα+ stromal cells have also been observed in nonlymphoid tissues, including the inflamed meninges of the brain (6, 7). In this review, we call these “FRC-like” cells and acknowledge that, like in the LN where there are at least five subsets of FRC (8), the same may be true in the brain. For the purposes of this review, when we refer to CNS “stroma,” this will encompass FRC-like fibroblasts, lymphatic endothelial cells (LEC), epithelial cells of the choroid plexus, and last, blood endothelial cells and pericytes, which line brain barriers.

We know that the function and phenotype of stromal cells in nonlymphoid tissues differ considerably depending on the tissue of origin. As will be detailed below, CNS stromal cells play critical roles in maintaining a healthy homeostatic state (resting stroma) but are also quickly and dramatically altered during inflammation (active stroma), including expression of cytokines, chemokines, and the production of extracellular network elements (9). Stromal cell niches in the inflamed brain were reviewed in The Journal of Immunology in 2017 (4). We update the findings presented in the 2017 review, with particular focus on brain-adjacent regions where stromal cells likely play a key role in influencing immune cell behavior during homeostasis (the leptomeninges, the choroid plexus, and the Virchow-Robin perivascular spaces of the brain parenchyma) as well as review what we know about lymphatic flow out of the CNS. We will also discuss how inflammation impacts stromal cell niches during infection, injury, and inflammation and the consequences for the underlying brain parenchyma. Last, we speculate on what we can learn from new techniques that have the power to dissect stromal cell phenotypes and functions in the healthy and diseased brain.

It is well known that stromal cells are vital to the anatomical and metabolic support of the CNS. Stromal cells have emerged as key players in the regulation of immune responses within the CNS by 1) maintaining the integrity of the barriers between the brain-penetrating blood vessels and the brain parenchyma at the blood–brain barrier (BBB) and between the peripheral circulation and the meninges at the blood meningeal barrier, 2) supporting homeostatic immune surveillance of the CNS, and 3) supporting lymphatic drainage of macromolecules and cells away from the healthy CNS. The anatomy and immunology of the brain parenchyma and the role of stromal cells in supporting the BBB have been well described in other reviews (14). In this review, we will focus on the stromal cells of the meninges and their role in the healthy and inflamed CNS.

Stromal cells in the highly specialized meninges: The dura.

Once considered an inert structure, the meninges lining the brain are now recognized as key players of immune responses that can affect the brain parenchyma (3). The meninges consist of three layers: the dura mater, the arachnoid mater, and the pia mater (Fig. 1). The arachnoid and pia together are termed the “leptomeninges.” The dura has received recent attention because of the fact that it is an incredibly rich source of immune cells in a compartment that is intimately close to the brain itself; thus, we take this opportunity to update recent findings on this interesting brain-adjacent region.

FIGURE 1.

Meningeal stromal cell remodeling during acute and chronic neuroinflammatory processes. The meninges covering the CNS parenchyma consist of the dura mater adjacent to the skull, the arachnoid mater, and the pia mater. The dura mater, made up of dural fibroblasts, contains lymphatic vessels and fenestrated vasculature, and contains a large repertoire of immune cells, including DCs (data not shown), mast cells (data not shown), innate lymphoid cells (data not shown), MMs, T cells, and B cells. The arachnoid mater represents the first impermeable barrier to the CNS parenchyma, with arachnoid fibroblasts joined by tight junctions. The SAS, consisting of arachnoid trabeculae made up of fibroblast-like cells, is filled with cerebrospinal fluid and traversed by nonfenestrated veins and arteries. The layer of pial fibroblasts, composing the pia mater, and the underlying glia limitans (formed by astrocytic endfeet) cover the surface of the brain parenchyma. During acute infection with a pathogen, meningeal and submeningeal fibroblasts elaborate a reticular cell network and upregulate expression of CCL19 and CCL21 (CCL19+/CCL21+ stromal cells in green) to recruit and position T cells (in orange) at the site of infection. Prolonged stromal cell activation under chronic inflammatory conditions triggers further remodeling and immune-stimulating fibroblast maturation that upregulate expression of CXCL13 (CXCL13+ stromal cells in red) to recruit and position B cells (in blue) at the site of infection, creating tertiary lymphoid structures with organized microenvironmental niches for T and B cell activation.

FIGURE 1.

Meningeal stromal cell remodeling during acute and chronic neuroinflammatory processes. The meninges covering the CNS parenchyma consist of the dura mater adjacent to the skull, the arachnoid mater, and the pia mater. The dura mater, made up of dural fibroblasts, contains lymphatic vessels and fenestrated vasculature, and contains a large repertoire of immune cells, including DCs (data not shown), mast cells (data not shown), innate lymphoid cells (data not shown), MMs, T cells, and B cells. The arachnoid mater represents the first impermeable barrier to the CNS parenchyma, with arachnoid fibroblasts joined by tight junctions. The SAS, consisting of arachnoid trabeculae made up of fibroblast-like cells, is filled with cerebrospinal fluid and traversed by nonfenestrated veins and arteries. The layer of pial fibroblasts, composing the pia mater, and the underlying glia limitans (formed by astrocytic endfeet) cover the surface of the brain parenchyma. During acute infection with a pathogen, meningeal and submeningeal fibroblasts elaborate a reticular cell network and upregulate expression of CCL19 and CCL21 (CCL19+/CCL21+ stromal cells in green) to recruit and position T cells (in orange) at the site of infection. Prolonged stromal cell activation under chronic inflammatory conditions triggers further remodeling and immune-stimulating fibroblast maturation that upregulate expression of CXCL13 (CXCL13+ stromal cells in red) to recruit and position B cells (in blue) at the site of infection, creating tertiary lymphoid structures with organized microenvironmental niches for T and B cell activation.

Close modal

The anatomy and immunology of the dura mater differ greatly from that of the leptomeninges and parenchyma. The dura mater is attached to the bony skull and houses dural sinuses, large dilations of the dura allowing for venous blood drainage (2). The dura mater contains arteries arising from the carotid artery and veins that drain into the dural sinuses (2). Dura mater blood vessels are fenestrated and lack tight junctions, allowing for extravasation of large molecules such as HRP (∼43 kDa) (10). In addition, dural blood vessels are innervated and responsive to signaling from peripheral nerves. For example, plasma extravasation from rat dural, but not parenchymal blood vessels, was shown to increase following electrical stimulation of the trigeminal ganglion or through i.v. injection of capsaicin, substance-P, neurokinin A, serotonin, and bradykinin (11). These data show that neural innervation of dural blood vessels has a direct impact on vascular permeability in this layer of the meninges and thereby potentially also immune cell extravasation into the dura. Moreover, Alves de Lima et al. (12) have proposed that calcitonin gene-related peptide–positive neurons in the meninges may modulate immune cell function in a similar manner as in peripheral organs; for example, neural release of calcitonin gene-related peptide in the skin induces a Th17 response. Overall, the role of innervation in the dura is not well understood.

The increased permeability of dural blood vessels compared with brain parenchymal blood vessels allows for homeostatic accumulation of immune cells within the dura, including macrophages, neutrophils, dendritic cells (DCs; including both plasmacytoid and conventional subsets), NK cells, T cells, B cells, monocytes (both classical and nonclassical subsets), and mast cells (1315) (Fig. 1). DCs, which are potent APCs, are more prevalent in the dura mater as compared with the leptomeninges and the perivascular spaces of the brain (15). Some dural DCs express genes associated with migration to LN (Ccr7 and Nudt17), suggesting that these cells may be migratory and can drain into LN (15). Recently, dural tissue-resident meningeal macrophages (MM) have been noted to be localized adjacent to blood vessels and along dural sutures (16). Dural macrophages express CCR2 and have been shown to be replaced by bone marrow–derived cells, likely CCR2+ monocytes (14, 15). In the noninflamed CNS, MMs are Lyve-1+ and express Ag-presenting machinery (MHC class I and MHC class II), adhesion molecules (ICAM-1), scavenging molecules (CD64), costimulatory molecules (CD80), receptors for engulfment and apoptotic cell clearance (MerTK), and high levels of a marker of alternatively activated macrophages (CD206) (see Table I) (16). MMs have active processes, suggesting that they continually monitor the dura akin to microglia and tissue-resident macrophages within the brain parenchyma (16). MHC class IIhi macrophages decrease but do not disappear in the dura of germ-free mice compared with conventionally housed or specific-pathogen-free mice, suggesting a role for the microbiome in shaping dural macrophage development, recruitment, or residence (15). Collectively, these studies shed light on the variety of immune cells residing in the dura at steady state. How these immune cells interact with stromal cells in the dura remains completely unexplored. Stromal cells within the dura may be an important source of secreted factors involved in immunity. Interestingly, Szerlip et al. (17) studied the impact of byproducts produced by dural fibroblasts. They found that dural fibroblasts express Tgfb, Cxcl12, and other secreted factors, and that these factors influenced the phenotype of at least myeloid cells and potentially other immune cells within the bone marrow overlying the spinal cord and brain (17). Therefore it is possible that dural fibroblasts may likewise influence dura-resident immune cells, although this remains to be studied.

Table I.
Stromal cell subsets of the CNS at steady state
Stromal Cell SubtypePhenotypeFunction and CharacteristicsReferences
Dura    
 Dural fibroblasts Vimentinhigh Secrete cytokines capable of modulating the immune microenvironment (17
 Endothelial cells of dural blood vessels CD31+ Fenestrated and lack tight junctions (3, 9, 10, 20
Neural innervation of vascular permeability 
 Smooth muscle cells and pericytes of dural blood vessels SMA+ Neural innervation of vascular permeability (88
 Diploic vein endothelial cells CD31+ Enriched in laminin Conduit for immune cells from the skull bone marrow to the dura during pathology (18, 19
 Dural lymphatic vessel endothelium PDPN+ CD31 Run along transverse and superior sagittal venous sinuses as well as nerve roots within spinal cord meninges (20, 88
Lyve-1+ Support immune cell drainage into CLN 
Prox1+ At least a proportion contain tight junctions 
CCL21+ Distinct gene signature compared with peripheral lymphatics 
VE-cadherin+ and claudin-5+  
Leptomeninges    
 Arachnoid mater epithelial cells E-cadherin+, plectin+, laminin α5+ Tight junctions create a barrier between the dura and SAS (2, 22, 24, 89, 90
Occludin+ Efflux drug transporter and cytochrome p450 enzyme expression facilitates drug removal from cerebrospinal fluid (P-gp+ and BCRP+
AKAP12+ Coexpression of cytokeratin, desmoplakin, and vimentin; PG D2 synthase+ 
 Pia mater epithelial cells E-cadherin, plectin+, laminin α1+ Lack tight junctions (22, 23, 90
Covers the brain parenchyma and is continuous with penetrating blood vessels 
 Endothelial cells of leptomeningeal blood vessels P-selectin+, E-selectin+, and ICAM-1+ Nonfenestrated and contain tight junctions (6, 24, 25, 3739
VCAM-1 Facilitate neutrophil influx to the leptomeninges following stroke 
CD31+  
 Fibroblastic reticular-like cells At least a proportion are CD31 and PDPN+ Supports immune cell infiltration during viral infection, sometimes with the formation of a fibroblastic reticular network (6, 44, 45, 50, 51, 90
ERTR7+ Provide a niche for brain-resident memory immune cells 
May express collagen type I and III  
 Smooth muscle cells and pericytes of leptomeningeal blood vessels SMA+ laminin α4+ and α5+ BM  (90
Stromal Cell SubtypePhenotypeFunction and CharacteristicsReferences
Dura    
 Dural fibroblasts Vimentinhigh Secrete cytokines capable of modulating the immune microenvironment (17
 Endothelial cells of dural blood vessels CD31+ Fenestrated and lack tight junctions (3, 9, 10, 20
Neural innervation of vascular permeability 
 Smooth muscle cells and pericytes of dural blood vessels SMA+ Neural innervation of vascular permeability (88
 Diploic vein endothelial cells CD31+ Enriched in laminin Conduit for immune cells from the skull bone marrow to the dura during pathology (18, 19
 Dural lymphatic vessel endothelium PDPN+ CD31 Run along transverse and superior sagittal venous sinuses as well as nerve roots within spinal cord meninges (20, 88
Lyve-1+ Support immune cell drainage into CLN 
Prox1+ At least a proportion contain tight junctions 
CCL21+ Distinct gene signature compared with peripheral lymphatics 
VE-cadherin+ and claudin-5+  
Leptomeninges    
 Arachnoid mater epithelial cells E-cadherin+, plectin+, laminin α5+ Tight junctions create a barrier between the dura and SAS (2, 22, 24, 89, 90
Occludin+ Efflux drug transporter and cytochrome p450 enzyme expression facilitates drug removal from cerebrospinal fluid (P-gp+ and BCRP+
AKAP12+ Coexpression of cytokeratin, desmoplakin, and vimentin; PG D2 synthase+ 
 Pia mater epithelial cells E-cadherin, plectin+, laminin α1+ Lack tight junctions (22, 23, 90
Covers the brain parenchyma and is continuous with penetrating blood vessels 
 Endothelial cells of leptomeningeal blood vessels P-selectin+, E-selectin+, and ICAM-1+ Nonfenestrated and contain tight junctions (6, 24, 25, 3739
VCAM-1 Facilitate neutrophil influx to the leptomeninges following stroke 
CD31+  
 Fibroblastic reticular-like cells At least a proportion are CD31 and PDPN+ Supports immune cell infiltration during viral infection, sometimes with the formation of a fibroblastic reticular network (6, 44, 45, 50, 51, 90
ERTR7+ Provide a niche for brain-resident memory immune cells 
May express collagen type I and III  
 Smooth muscle cells and pericytes of leptomeningeal blood vessels SMA+ laminin α4+ and α5+ BM  (90

BCRP, breast cancer resistance protein; P-gp, P-glycoprotein; SMA, smooth muscle actin.

Another portal of entry of immune cells into the meninges is through diploic veins, which travel directly from the skull bone marrow to drain into meningeal veins. In a mouse model of stroke, neutrophils were shown to migrate into the dura through diploic veins (18). These dural veins are enriched in laminin in mice, a property that is exploited by α6-integrin+ acute lymphoblastic leukemia cells as a means of entering the meninges (19). However, the homeostatic relevance of immune cell movement from the skull bone marrow into the meninges via diploic veins and the source of laminin in dural vein structures have yet to be elucidated.

Finally, dural lymphatic vessels run along the transverse and superior sagittal venous sinuses of the dura. Recently, dural lymphatic vasculature has also been observed to extend into the subarachnoid space (SAS)—solutes and cells injected into the cerebrospinal fluid of the SAS were found to drain into dural lymphatic vessels and then into superficial and deep cervical LNs (CLNs). Moreover, some meningeal LECs were found to produce CCL21 and interact with CCR7+ T cells injected into the cerebrospinal fluid at steady state, thereby potentially guiding T cell egress into the CLN. Endothelial cells of these dural lymphatics were found to be Lyve-1+ and Prox1+, and at least a proportion contain VE-cadherin and claudin-5 tight junctions. Interestingly, endothelial cells of the dural lymphatics exhibited a distinct transcriptomic signature compared with peripheral lymphatics of the diaphragm or ear skin, showing a gene signature consistent with decreased inflammation-induced lymphanogenesis (20). An alternative hypothesis for lymphatic drainage of the cerebrospinal fluid to the CLNs has been proposed to be through perineural routes alongside cranial nerves or through nasal lymphatics (1, 2, 21). Differences in the speed, volume, and location of injection of tracers into the cerebrospinal fluid of animal models have been criticized as artificially causing drainage over the arachnoid mater into dural lymphatics (3). However the study by Louveau et al. (20) showed that ablation of dural lymphatics leads to a lack of macromolecule and cell drainage from the cerebrospinal fluid into superficial CLNs and deep CLNs, supporting meningeal lymphatics as a potential route for immune cell and macromolecule drainage to CLN from the cerebrospinal fluid during steady state. Ultimately, more research is required to understand the relative contributions of dural lymphatics and perineural drainage in cerebrospinal fluid clearance. Regardless of the drainage patterns of CNS lymphatics, the impact of inflammation on stromal LECs themselves and the role of LEC in influencing brain-adjacent regions are relatively unknown.

Stromal cells in the highly specialized meninges: The SAS.

Inferior to the dura are the leptomeninges. The arachnoid mater is composed of squamous epithelial cells connected by tight junctions and contains collagenous trabecular projections that form the SAS, through which cerebrospinal fluid flows (22). The tight junctions of the arachnoid mater epithelium create a physiologic barrier between the dura and the SAS (22) (Fig. 1). Moreover, various efflux drug transporters and cytochrome p450 metabolizing enzymes have been identified in arachnoid epithelial cells and are thought to contribute to increased macromolecule removal from the cerebrospinal fluid (22). Below the arachnoid mater is the pia mater, which lies immediately above the brain parenchyma and is continuous along the blood vessels that penetrate the brain (22, 23).

In contrast to the dura, blood vessels within the leptomeninges are nonfenestrated and have tight junctions, limiting extravasation of immune cells and macromolecules from the blood into the cerebrospinal fluid (24) (Fig. 1). However, P-selectin, E-selectin, and ICAM-1 expression on endothelial cells of leptomeningeal blood vessels in the noninflamed brain is thought to facilitate some leptomeningeal steady-state immune cell traffic (6, 25), and in human cerebrospinal fluid, CCL19 protein, which has the capacity to attract CCR7-expressing leukocytes, is constitutively present even without inflammation (26). In contrast, parenchymal blood vessels lack P-selectin, VCAM-1 (which is also absent on leptomeningeal blood vessels), and E-selectin but sometimes express ICAM-1 under steady state (25).

There is conflicting literature on whether vessels within the SAS and cerebral cortex have perivascular spaces and whether these spaces are continuous with the Virchow-Robin spaces that surround blood vessels within the white matter (1, 27, 28). Given that Virchow-Robin spaces are populated by immune cells involved in immunological surveillance, the presence of perivascular spaces around arachnoid vessels would provide a potential space for immune cell traffic and residence, even if temporary, within the SAS (1, 2, 27, 29).

Unique to the leptomeninges is a network of FRC-like cells that can potentially attract, retain, and modulate peripheral immune cells. Interestingly, at least a proportion of meningeal FRC-like cells are CD31 and PDPN+ in the noninflamed brain (see Table I) (6). This network of resting stroma may allow for the recruitment and support of immune cells within the SAS. As evidence that this is occurring, flow cytometric analysis identified steady-state T cells (largely memory CD4+ T cells) and a few B cells in the cerebrospinal fluid of humans, whereas histological studies revealed the presence of macrophages, DCs, neutrophils, and mast cells in the meninges of the steady-state mouse CNS (1315, 25). NK cells, T cells, and B cells—but not neutrophils—were also identified in the leptomeninges of mice via RNA sequencing (15). These data suggest that, like the dura, the SAS may be populated by immune cells; however, nothing is known about their length of residence in this environment, and the density of immune cells in the SAS is likely far lower than that of the dura. A better understanding of the gene expression profile of subarachnoid FRC-like cells will likely provide clues as to if and how immune cells transit through the SAS.

If we assume that there are immune cells that even transiently populate the SAS, one question is their function in this compartment. One could be immunosurveillance, although immune cells in the dura presumably can serve this function. Radjavi et al. (30) showed that brain myelin oligodendrocyte glycoprotein (MOG) reactive T cells localized to the meninges and improved learning behavior in T cell–deficient mice. In addition, through their production of IL-17, γδ+ T cells in the meninges have been shown to promote cognition and short-term memory (31). Furthermore, Baruch et al. (32) found that the choroid plexus of naive mice was populated by CD4+ memory T cells with TCR sequences resembling those of T cells within the spleens of mice immunized with CNS Ags. These findings imply a homeostatic role for T cells within the SAS and the choroid plexus.

Changes to stromal cells in the CNS under circumstances of acute inflammation are best exemplified in the context of acute injuries, viral infection, parasitic infection, and autoimmunity. In peripheral LNs, both endothelial and FRC-like cells can be induced to express CCR7 ligands through LTβR ligation (33, 34). This type of feed-forward loop may also take place in the meninges, where cytokine expression may similarly induce meningeal endothelial cells and FRC-like cells to express proinflammatory proteins (7). In addition, cultures of unfractionated rat meninges with gp120, an HIV Ag, have been shown to secrete inflammatory molecules such as TNF, IL-1β, and IL-6, although it is unknown which cells (immune or stromal) are involved (35). In this article, we review evidence for the role of stromal cells during stroke, infection, and experimental autoimmune encephalomyelitis (EAE). These models help clarify the role of stromal cells in mounting and maintaining acute immune responses in human diseases.

Stroke.

Ischemic brain damage from stroke triggers a rapid inflammatory response consisting of macrophage, lymphocyte, and DC infiltration of the infarcted area followed by a large influx of neutrophils (36). In this article, we briefly review the role that meningeal stromal cells play in the recruitment of peripheral immune cells following stroke as well as the contribution of recruited meningeal immune cells into the infarcted parenchyma.

Most of the literature has looked at the source and role of neutrophil recruitment to the infarcted parenchyma. Given the role of P-selectin in the recruitment of neutrophils, it has been suggested that meningeal endothelial cells, which constitutively express P-selectin, facilitate a strong neutrophil influx first to the leptomeninges rather than the parenchyma, which lacks homeostatic endothelial expression of P-selectin (2, 25, 3739). γδ+ T cells are recruited from the small intestine to the meninges, and mast cells resident within the meninges are activated following ischemic brain injury; both promote neutrophil recruitment to the CNS in an IL-17– and IL-6–facilitated manner, respectively (13, 40, 41). Neutrophils exit the leptomeninges and Virchow-Robin space to enter the brain parenchyma in the presence of matrix metalloproteinases, likely secreted by microglia, astrocytes, and stromal cells, such as blood endothelial cells and pericytes (2, 42). Further research is required to elucidate the beneficial versus deleterious effects of inflammation following stroke; however, there is a large body of evidence for inflammation as a driver of secondary brain damage. For example, meningeal mast cells, IL-17+ γδ+ T cells, and parenchymal infiltration of neutrophils have all been implicated in worsening stroke pathology (2, 13, 41, 43). These data shed light on the importance of the meninges as an important reservoir of immune cells and conduit between the peripheral immune system and the CNS parenchyma during stroke. Ultimately, the role of meningeal stromal and resident immune cells during stroke remains understudied.

Pathogen infection.

Stromal cells in the leptomeninges rapidly respond to viral infection. Infection with neurotropic mouse hepatitis virus induces ICAM-1, VCAM-1, and MHC class I upregulation and CCR7 ligand (CCL21 and CCL19) expression by endothelial cells of meningeal blood vessels and meningeal PDPN+ER-TR7+ FRC-like cells (Fig. 1). Migration of CCR7+ T cells supported by these changes in endothelial cells and FRC-like cells was shown to be necessary for effective viral clearance (6). Others have reported the formation of an FRC-like network producing laminin in the meninges and in the Virchow-Robin space of parenchymal vessels following mouse hepatitis virus infection (6, 44). Toxoplasma gondii infection of the CNS similarly induces the formation of a reticular network within the cortex of the brain (see Table I) that supports CD8+ T cell migration along the CCL21-coated reticular fibers (45).

In addition to playing a role in the acute response to CNS infection, the meninges also provide a niche for brain-resident memory immune cells to persist. Tissue-resident memory T (TRM) cells are noncirculating cells that persist in tissues previously infected by pathogens and are sentinels of long-term immune protection (46). The presence of these cells has been observed in many peripheral tissues, such as skin and mucosal tissues, as well as the CNS in the meninges, choroid plexus, periventricular areas, and the parenchyma (4749). These cells show a transcriptional signature distinct from circulating T memory populations and have been observed to outnumber effector memory cells (46, 47, 50).

Studies by Steinbach et al. (50) and Wakim et al. (48) have shown that antiviral cytotoxic T cells infiltrate the CNS parenchyma at sites of infection and form persisting clusters of TRM cells within the meninges and brain parenchyma, acting as an independent network of CNS defense. In mice infected intracranially with an attenuated strain of lymphocytic choriomeningitis virus (LCMV), whereas the total number of T cells contracted following viral clearance, TRM populations persisted in stable numbers and frequency from 6 wk onwards. Moreover, LCMV-induced TRM preferentially associated with FRC-like cells in the meninges and choroid plexus, suggesting the importance of stromal-derived factors in maintenance of this population (50). As homeostatic proliferation of memory T cells is known to rely heavily on STAT5 signaling via IL-7 and IL-15 and FRC in secondary lymphoid organs are major producers of these cytokines, it is possible that FRC-like cells in the brain may act similarly, providing niches crucial for memory cell persistence. The authors further found that if mice were infected with LCMV at 1 wk of age (but not 3–4 wk of age), TRM cells cluster into microenvironments within the periventricular areas of the brain and the choroid plexus at sites of previous infection and remain in the brain following viral clearance. These microenvironments express CCL5 and recruit CCR5+ self-reactive cells during EAE, ultimately leading to worsened clinical disease (51). Although this study did not probe the role of stromal cells in the generation of this proinflammatory CNS microenvironment, meningeal FRC-like cells may also be implicated in CNS autoimmune pathology by creating a niche for immune cell survival and activation. Moreover, Wakim et al. (48) found that when they intranasally infected mice with vesicular stomatitis virus, clusters of virus particles were found in the cortex, ventral striatum, caudale putamen, thalamus, hypothalamus, and midbrain in association with Ag-specific CD8+ T cells that persisted for 120 d postinfection. These CD8+ T cells were found to die quickly after dissociation from their tissue environment, suggesting that the microenvironments in which they reside are essential for persistence (48, 50).

In contrast to the leptomeninges, the role of stromal cells within the dura during infection has not yet been delineated, but changes in this environment following infection may hint at a role for stromal cell imprinting of dural-resident immune cells. For example, during LCMV infection, MMs become virally infected and are killed, likely by viral-specific CD8+ T cells. MMs killed after LCMV infection are replaced by peripherally derived monocytes that then differentiate into MHC class II+ macrophages in response to INF-γ and engraft for at least 30 d following infection. The newly engrafted MMs exhibit altered immune responses, such as a decreased ability to recruit neutrophils following LPS challenge and a deficiency in mounting an immunoregulatory response to acetylcholine (16). It is possible that stable alterations of the dural cellular environment (including stroma) follow viral infection with implications for future immune responses. More research is required to further understand what changes occur in dural stromal cells following viral infection.

Experimental autoimmune encephalomyelitis.

EAE is a model that reproduces key aspects of MS pathology. Studies by Kuchroo and colleagues (52) have shown that adoptive transfer of Th1 and Th17 cells expressing a MOG-specific transgenic TCR into wild-type C57BL/6 recipient mice was sufficient to induce EAE. However, further analysis revealed that disease induced by each T cell subset differed in terms of pathological and histological phenotype. Mice receiving Th17 cells developed more-severe EAE, accompanied by more inflammation, increased lesion burden, and induction of tertiary lymphoid tissue (TLT) formation in the leptomeninges (52). Subsequent immunohistochemical analyses of these structures have revealed the presence of collagen fibers encapsulating B cell clusters surrounded by T cells. Flow cytometry further revealed the expression of the GL7 glycoprotein on CNS-derived B cells, indicating these B cells may be participating in a germinal center reaction. CXCR5, ICOS, and Bcl6 were also expressed by CCR6-positive Th17 cells, indicating that these may have acquired attributes of T follicular helper cells in the CNS that can provide help to B cells. Last, follicular DCs (FDCs), a type of stromal mesenchymal cell that is found in B cell follicles and germinal centers, were detected in the CNS of Th17 recipients, further indicating the presence of an ectopic lymphoid structure capable of supporting an immune response (53). Thus, in this model it appears that stromal cell remodeling in the meninges promotes a niche that is underpinned by FDC-like stroma.

Interestingly, Th17 cells infiltrating the meninges during the C57BL/6 EAE model were found to have the highest expression of the surface molecular PDPN, and PDPN immunoreactivity was detected in and around the TLTs and on T cells, macrophages, and collagen fibers (54). This observation, combined with its constitutive expression in the meninges, highlights its potential role in anchoring essential components of the lymphoid structure (54). mAb-mediated blockade of PDPN in mice receiving Th17 cells resulted in significantly reduced numbers of TLTs compared with mice receiving the isotype control (54). Together, these data suggest a unique role for Th17 cells in mediating the formation of these lymphoid structures in a manner partially dependent on stromal anchoring via PDPN.

One variant of EAE involves immunization of donor SJL/J mice against a myelin Ag (in this case, proteolipid protein [PLP]), followed by adoptive transfer of PLP-primed T cells from the donor mouse into naive SJL/J recipient mice (7). The recipient SJL/J mice typically develop clinical disability 8–9 d following adoptive transfer. Unlike C57BL/6 models of EAE, the SJL/J model exhibits brain meningeal inflammation and subpial cortical pathology. Histopathology studies of brains from mice sacrificed at the acute phase showed infiltration of CD4+ T cells, B220+ B cells, and CD11c+ myeloid cells in the meninges, with lymphocytes occasionally organized in distinct zones (7). These brains were also profiled for demyelination by histology, and regions of myelin loss were found near meningeal inflammation (7). Additionally, immunohistochemistry staining for astrocytes using glial fibrillary acidic protein (GFAP) also identified regions of astrogliosis in the cortex adjacent to areas of leptomeningeal infiltrates (7). However, in addition to the astrogliosis, meningeal infiltrates were also associated with a disrupted glial limitans, microgliosis, and oxidative injury in neurons (55) (Fig. 1). Further examination of these meningeal TLT revealed an expansion of CD45GP38+CD31PDGFRα+PDGFRβ+ FRC-like cells associated with an ERTR7+Fibronectin+ network that expressed mRNA for chemokines such as Cxcl13, Ccl19, and Ccl21 and cytokines including Il23. The impact of inhibiting the lymphotoxin pathway, a key TNF superfamily member that plays an important role in the development of LNs, was tested in this model. Although inhibition of this pathway had no impact on the establishment of meningeal TLTs during EAE, it did blunt the expression of Cxcl13 and Il23 by meningeal FRC-like cells concomitant with a reduction in Th17 cell accumulation in the meninges. In contrast, Th17 cell–derived cytokines IL-17 and IL-22 promoted the initiation of FRC-associated extracellular matrix deposition within the meninges. Thus, although the LT pathway is not required for the initiation of meningeal TLTs in this model, it is important for the elaboration of an immunocompetent stromal cell niche (7).

More recently, the mechanism by which Th17 cells infiltrate the CNS and induce meningeal inflammation and stromal cell remodeling was explored. Lymphocytes are known to rely on the sphingosine-1-phosphate receptor (S1PR) to egress out of lymphoid organs, and agonists of the S1PR, such as fingolimod, serve to desensitize lymphocytes to the effects of S1P, resulting in sequestration of lymphocytes within LNs (56, 57). For this reason, S1PR modulators such as fingolimod and siponimod are used as MS therapies (56). Siponimod was found to attenuate the clinical signs of EAE in the same model that was used to study brain meningeal inflammation and cortical injury. Following siponimod therapy, meningeal stromal cells were less able to form fibronectin networks, and immune cell recruitment to the meninges was blunted. EAE mice receiving siponimod also exhibited less cortical demyelination, reduced microglia proliferation, and an intact glial limitans compared with mice receiving a control treatment. Interestingly, the ability of the adoptively transferred Th17 cells to make IL-17 and IL-22 in the brain was reduced with treatment, thus correlating Th17 cells with meningeal inflammation and cortical pathology (55). These data highlight the potential role of S1P in lymphocyte trafficking, IL-17 and IL-22 production, and creation of an immune-competent niche within the CNS stromal cell compartment.

In MS, autoreactive lymphocytes are primed against a CNS-reactive Ag in the periphery, enter the CNS via the vasculature, and are reactivated in the perivascular spaces of postcapillary venules and meningeal vessels (58). At these sites of preferred lymphocyte accumulation, changes to the endothelium (59), upregulation of chemokines (60), and the elaboration of a fibroblastic reticular network (61) have been described. Importantly, these changes in the perivascular Virchow-Robin space are associated with demyelinating lesions in the parenchymal white matter (62). Moreover, meningeal aggregates of leukocytes overlay cortical gray matter lesions, which display a gradient of demyelination, myeloid cell activation, and neuronal loss extending inwards from the pia mater into the cortex (63, 64). In this section, we will review the evidence that supports the concept that an immunocompetent stromal cell niche has the capacity to harbor immune cells and express mediators that attract and retain leukocytes, supporting their proliferation and production of pro- and anti-inflammatory molecules that can shape pathological processes in the brain during MS.

The first demonstration of a lymphoid tissue–like microenvironment in the CNS of MS patients came from Prineas in 1979 (65). He found thin-walled, lymphatic capillary-like channels and plasma cells associated with fibroblastic cells in the Virchow-Robin space adjacent to demyelinated MS lesions. These clusters of immune cells displayed an organization reminiscent of the LN medulla. Further studies also reported a meshwork of extracellular matrix closely interacting with infiltrating lymphocytes at the core of perivascular white matter lesions in MS (61). Although TLTs have not been observed in perivascular white matter lesions, their presence has been extensively demonstrated in the brain meninges of patients with early MS (66, 67) and progressive MS (63, 64, 6872). Imaging studies using postcontrast fluid-attenuated inversion recovery with 3 Tesla magnetic resonance imaging found focal leptomeningeal enhancement in 25% of 299 MS patients analyzed (73), and histological studies on autopsy CNS tissue from progressive MS cases showed evidence of TLT in the leptomeninges lining the brain (63, 64, 68, 69, 71, 72) and the spinal cord (70). Although variable in their size and organization (63, 64, 6872, 74), meningeal TLT comprise B cells, T cells, DCs, macrophages, monocytes, plasma cells, and stromal cells that resemble FDCs normally found in B cell follicles (63, 67, 6971, 75). The additional evidence that within TLT, B cells proliferate and FDC-like stromal cells produce the chemokine CXCL13 suggests that an immunocompetent stromal cell niche is formed and maintained by attracting and retaining leukocytes, supporting their proliferation at these sites (71) (Fig. 1C). In terms of T cells, RORγt+ lymphocytes with variable coexpression of IL-17 were preferentially found at sites of meningeal TLT, suggesting that Th17 cells may contribute to formation of these niches (76).

Because cells that reside within the meninges likely do not penetrate the glial limitans, the soluble byproducts secreted by these cells have been posited as being potentially inflammatory, cytotoxic, and myelinotoxic mediators that, by circulating within the cerebrospinal fluid, may diffuse freely throughout the SAS, cross the pial membrane, enter the adjacent gray matter, and mediate subpial gray matter injury in the MS brain (77, 78). In support of this concept, significant associations have been found between high cortical lesion load and proinflammatory cytokines (IFN-γ, TNF, IL-2, and IL-22) (79, 80), molecules related to sustained B cell activity and lymphoid neogenesis (CXCL13, CXCL10, LTα, IL-6, and IL-10) (79), B cell survival factors (BAFF) (81), factors indicative of BBB leakage (fibrin, complement, and coagulation factors) (81), and iron-related proteins (free hemoglobin and haptoglobin) (81) in the cerebrospinal fluid.

In summary, many studies have reported meningeal TLT and soluble byproducts that have the potential to shape pathological processes in the underlying cortex as suggested by their close association with sites of subpial demyelination, microglia/macrophage activation, and neurodegeneration (64, 6772). Although we have begun to uncover the nature of the soluble molecules associated with the chronically inflamed CNS microenvironment, we still know very little about which cells, including stromal cells, actually produce these byproducts. In situ characterization of immune cells and stromal cells phenotypes as well as an assessment of the soluble factors they produce will be critical in understanding their role in chronic immune-mediated diseases such as MS.

Moving forward, to properly characterize the function of immune cells and how their behavior changes during autoimmune disease, the development of a comprehensive, multiparameter method that has the potential to be translated across archived tissue from well-curated cohorts is necessary (82). Imaging mass cytometry (IMC) combines principles of mass cytometry and immunofluorescence. In IMC, the tissue of interest is fixed on a slide and stained with a mixture of metal-conjugated Abs in a process similar to an immunofluorescence staining protocol; however, in this case, >40 analytes can be measured at a time (83). A region of interest on the slide is then passed through an ablation chamber, where a laser vaporizes the tissue 1 μm2 at a time. The vaporized tissue is carried by an inert stream of gas through a detection portal, where the mass of ions detected is measured and mapped retroactively back onto the tissue, creating pseudo colored images (83). The laser ablation technology provides the multiparameter benefits of mass cytometry without compromising the structural information from the tissue. IMC is especially useful for studying “precious” human tissues, which are often quite small, have a complex structure, and can be difficult to procure (83). We have recently applied IMC for a multidimensional look at MS lesions. We were able to not only identify different lesion types but also characterize and quantify the immune cells present in the tissue (84). Because our panel allowed for many markers, we were able to use thresholds of positivity/negativity for each marker to determine cell phenotypes, analyzing the distribution of immune cells in the MS brain based on anatomical landmarks, such as blood vessels. For example, we were able to show that CD8+ T cells were on average farther away from blood vessels compared with CD4+ T cells in the active core of a so-called “smoldering” MS lesion (also called “active-inactive demyelinating” lesion). However, CD4+ and CD8+ T cells showed equal distance from blood vessels in an active lesion (84). This type of phenotypic and positional information will be well-suited to understanding what types of stromal cells occupy brain-adjacent regions, such as the perivascular Virchow Robbins space, the SAS, the dura, and the choroid plexus. When complemented by positional transcriptomics, much will be learned about brain-resident stroma, in particular whether the diversity of FRC-like cells matches that observed for FRC in LN.

Moreover, an understanding of what supports TRM cells is critical, as their appearance deeper in the brain parenchyma poses many unanswered questions (85, 86). What supports their continued presence in a tissue that is thought to only contain glial cells and neurons? Could there be extracellular matrix tendrils that extend beyond the Virchow Robbins space into the parenchyma to create microniches for such cells? Clearly, we are only at the beginning of understanding such niches in the brain. Single-cell RNA sequencing will provide some insights. However, it is imperative that this be performed on relatively fresh material. The ability to do this on donors who are undergoing planned euthanasia has begun in countries such as Canada (84, 87). We await what these studies will tell us about brain-associated stromal cells.

Last, we typically assume that stromal cell remodeling is promoting an inflammatory process as the brain prepares to deal with a pathogen invasion, and such a process is maladaptive during chronic inflammatory diseases such as MS. However, we do not know enough about the changes in brain-resident stromal cells to assume that these are always generating a proinflammatory niche. Indeed, significant data not only support a role for stromal cells in setting up and supporting immune-competent niches but also that stromal cells suppress immune responses, as they have been found to do in tumor microenvironments (9). Indeed, Magliozzi et al. (81) failed to identify a singular signature in cerebrospinal fluid–associated cytokines from MS patients with meningeal TLT. Because this study was by necessity cross-sectional, this suggests that there may be various types or stages of TLT that are shaped by the phase of one’s MS disease sequelae (81). If we can learn more about the types of molecules produced by stromal cells at different stages of infection or disease, we may have a better idea on what their true function is in influencing local immune cells. Indeed, in our view, stromal cells are key cellular players in the brain-compartmentalized immune system, and we anxiously await the elucidation of their functional attributes in this new age of multiparameter analysis.

This work was supported by the Multiple Sclerosis Society of Canada (Grant 3194) and the Canadian Institutes of Health Research (Grant 15992).

Abbreviations used in this article:

BBB

blood–brain barrier

CLN

cervical LN

DC

dendritic cell

EAE

experimental autoimmune encephalomyelitis

FDC

follicular DC

FRC

fibroblastic reticular cell

IMC

imaging mass cytometry

LCMV

lymphocytic choriomeningitis virus

LEC

lymphatic endothelial cell

LN

lymph node

MM

meningeal macrophage

MS

multiple sclerosis

PDGFRα

platelet-derived growth factor receptor-α

PDPN

podoplanin

SAS

subarachnoid space

S1PR

sphingosine-1-phosphate receptor

TLT

tertiary lymphoid tissue

TRM

tissue-resident memory T.

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The authors have no financial conflicts of interest.