---
### **The Glymphatic System Mediates Targeted Immune Surveillance of the Central Nervous
System via a Novel Meningeal Lymphatic-CNS Antigen Portal**
**Running Title:** CNS-Immune Connectivity via Glymphatics
**Authors:** Elena A. Vasquez¹†, Benjamin R. Croft²†, Akiko Tanaka³, Samuel L. O’Hara², Isabelle
Moreau⁴, Kenji Sato³, Maria A. Popović¹, David Chen²*
**Affiliations:**
¹ Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA
² Department of Immunology, Yale School of Medicine, New Haven, CT 06510, USA
³ RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan
⁴ Brain and Mind Institute, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, 1015,
Switzerland
†These authors contributed equally.
*Lead Contact: david.chen@yale.edu
---
### **SUMMARY**
The central nervous system (CNS) has historically been considered an immunologically privileged
site, with limited interaction with the peripheral immune system. While the discovery of meningeal
lymphatic vessels challenged this dogma, the mechanisms by which parenchymal CNS antigens are
sampled and presented to the adaptive immune system remain enigmatic. Here, we report that the
glymphatic system, a brain-wide perivascular network responsible for waste clearance, functions as
a critical conduit for the selective transport of CNS-derived antigens to meningeal lymphatic vessels.
We identify a population of dural-resident, CD11c⁺ antigen-presenting cells that actively sample
glymphatic efflux at specific "antigen portals" at the confluence of paravenous spaces and meningeal
lymphatics. Using in vivo two-photon microscopy and a novel transgenic mouse model for cellspecific antigen tracing, we demonstrate that this pathway is essential for the generation of antigenspecific CD4⁺ and CD8⁺ T-cell responses against intracerebrally introduced model antigens and latent
viral infections. Disruption of glymphatic flow, either genetically or via sleep deprivation, ablates
subsequent adaptive immune activation. This glymphatic-immune axis represents a fundamental
mechanism of CNS immune surveillance, with profound implications for understanding
neuroinflammation, brain tumor immunotherapy, and the pathogenesis of autoimmune diseases
like multiple sclerosis.
---
### **INTRODUCTION**
The concept of the CNS as an "immune-privileged" site, largely sequestered from peripheral immune
surveillance, was established decades ago (Medawar, 1948). This privilege was attributed to the
blood-brain barrier (BBB), the apparent absence of conventional lymphatic drainage, and an
immunosuppressive microenvironment. However, this view has been progressively refined. The
presence of immune cells in the meninges and choroid plexus, and the capacity for activated T-cells
to cross the BBB, indicated a more nuanced relationship (Ransohoff & Engelhardt, 2012). A paradigm
shift occurred with the (re)discovery of functional lymphatic vessels lining the dural sinuses,
providing a direct anatomical connection for the drainage of cerebrospinal fluid (CSF) and immune
cells to the deep cervical lymph nodes (dcLNs) (Aspelund et al., 2015; Louveau et al., 2015).
Despite this breakthrough, a critical question remained: how are antigens derived from the deep
brain parenchyma, far removed from the CSF-filled subarachnoid space, delivered to these
meningeal lymphatics? The recently characterized glymphatic system offers a potential solution (Iliff
et al., 2012). This macroscopic waste clearance system facilitates the convective influx of CSF along
para-arterial channels, its mixing with interstitial fluid (ISF), and the efflux of solutes along
paravenous spaces. It is primarily active during sleep and is dependent on astrocytic aquaporin-4
(AQP4) water channels (Xie et al., 2013).
We hypothesized that the glymphatic system is not merely a passive waste-clearance pathway but
an active immunological conduit. We proposed that CNS-derived proteins, including potential neoantigens from tumors or pathogen-associated antigens, are carried within the glymphatic flow to
specific exit points at the brain's surface, where they are intercepted by the peripheral immune
system.
In this study, we provide definitive evidence for this hypothesis. We identify a direct anatomical and
functional link between the paravenous glymphatic efflux and the meningeal lymphatic network. We
characterize a novel population of dural antigen-presenting cells (APCs) that patrol these efflux sites
and demonstrate their necessity for initiating adaptive immune responses. Our findings establish a
new framework for understanding CNS immune communication, where the glymphatic system
serves as the "afferent arm" of neuro-immune surveillance, fundamentally reshaping our view of
brain-immune interactions in health and disease.
---
### **RESULTS**
#### **A Direct Anatomical Connection Exists Between Paravenous Glymphatic Spaces and
Meningeal Lymphatics**
To investigate the interface between glymphatic efflux and the immune system, we first sought to
define the anatomical relationship between paravenous pathways and dural lymphatic vessels. We
performed high-resolution ex vivo confocal microscopy on whole-mount meningeal preparations
from Prox1-GFP mice (which label lymphatic endothelial cells) following intracisternal infusion of a
red-fluorescent tracer (Texas Red-dextran, 3kDa).
**Figure 1 (Page 1 of Supplementary Data):**
**(A-C)** 3D reconstruction of dural lymphatics (Prox1-GFP, green) at the transverse sinus shows
tracer (red) within a Prox1⁺ lymphatic vessel (arrow) after intracisternal infusion. Scale bar, 50µm.
**(D-F)** Correlative light and electron microscopy (CLEM) of a paravenous space (PVS) at the
rostral rhinal vein. A tracer-filled PVS (red, asterisk) is shown in direct apposition to a Prox1⁺
lymphatic capillary (green). The boxed region in (E) is shown at the EM level in (F), demonstrating a
discontinuous basement membrane (arrowheads) between the PVS and the lymphatic lumen (Ly).
Scale bars, 10µm (E), 1µm (F).
We identified specific, reproducible "hotspots" where tracer-laden paravenous spaces converged
with and emptied directly into the initial lymphatic capillaries of the meningeal network (Figures 1A1F). These junctions, which we term "antigen portals," were characterized by a thin, often
fenestrated basement membrane, facilitating the exchange of solutes. Tracer was consistently
observed within the lumen of Prox1⁺ lymphatic vessels at these portals, confirming direct efflux from
the CNS parenchyma into the lymphatic system.
#### **A Novel Population of CD11c⁺ MHC-II⁺ APCs Resides at Antigen Portals and Captures CNSDerived Antigens**
We next asked which immune cells interact with CNS-derived antigens at these portals.
Immunostaining of dural whole mounts from Cx3cr1[GFP/+] mice (labeling myeloid cells) revealed a
dense network of CD11c⁺ MHC-II⁺ cells with a dendritic morphology specifically enriched at the
antigen portals (Figure 2A-2C). Flow cytometric analysis of enzymatically digested dura mater
confirmed a distinct population of CD45⁺ CD11b⁺ CD11c⁺ MHC-II⁺ F4/80[int] cells, which we termed
"Portal-associated Dendritic Cells" (PADCs) (Figure 2D).
**Figure 2 (Page 2 of Supplementary Data):**
**(A-C)** Dural whole mount from a Cx3cr1[GFP/+] mouse (green) immunostained for CD11c
(magenta) and LYVE-1 (lymphatics, cyan). PADCs (arrow) are closely associated with a LYVE-1⁺ vessel
at a portal. Scale bar, 20µm.
**(D)** Flow cytometry gating strategy for PADCs from dura mater
(Live/CD45⁺/CD11b⁺/CD11c⁺/MHC-II⁺).
**(E)** In vivo two-photon image of a PADC (YFP, yellow) extending a dendrite (arrow) into a lumen
of a meningeal lymphatic vessel (LYVE-1, blue) containing a CSF-derived tracer (red). Scale bar,
10µm.
To visualize antigen capture in real-time, we performed in vivo two-photon microscopy through a
chronic cranial window over the transverse sinus in CD11c-EYFP mice. Following intracisternal
injection of Qdot655-labeled ovalbumin (OVA), we observed PADCs actively extending dendrites into
the lumen of the lymphatic capillaries, directly sampling the flowing antigen (Figure 2E,
Supplementary Video 1). This "trans-lymphatic dendritosis" represents a previously unknown
mechanism of antigen surveillance.
#### **Development of a Transgenic Model to Trace CNS Antigen Fate: The CNS-TRAP Mouse**
To definitively trace the fate of a defined CNS antigen, we generated a novel transgenic mouse
model, which we call CNS-TRAP (CNS-Targeted Recombinant Antigen Presentation). This system uses
a Cre-inducible, membrane-tethered form of OVA (LSL-mTOmato-OVA) crossed with a GFAP-Cre
driver, resulting in the specific and permanent expression of the model antigen OVA in astrocytes
and their released exosomes (Figure 3A).
**Figure 3 (Page 3 of Supplementary Data):**
**(A)** Schematic of the CNS-TRAP system. GFAP-Cre drives excision of a Lox-Stop-Lox (LSL)
cassette, leading to astrocyte-specific expression of membrane-tethered OVA and TdTomato.
**(B)** Immunostaining of brain section from CNS-TRAP mouse shows TdTomato (red) co-localizing
with GFAP (green). Scale bar, 25µm.
**(C)** Flow cytometry plot of dcLNs from a wild-type mouse adoptively transferred with OVAspecific CD8⁺ T-cells (OT-I) and immunized with CNS-TRAP or control brain homogenate. Proliferation
(CFSE dilution) is only observed in the CNS-TRAP group.
In CNS-TRAP mice, OVA is a bona fide CNS-derived protein. When we isolated PADCs from these
mice and co-cultured them with OVA-specific CD8⁺ T-cells (OT-I), they potently stimulated T-cell
proliferation and IFN-γ production (Figure 3C). This demonstrates that PADCs can acquire and
present a naturally expressed, parenchymal CNS antigen.
#### **Glymphatic Transport is Necessary and Sufficient for CNS Antigen Presentation in the
dcLNs**
We next tested whether glymphatic flow was required for antigen presentation. We administered
Evans Blue-conjugated OVA (EB-OVA) intracortically to three groups of mice: (1) wild-type (WT)
controls, (2) Aqp4⁻/⁻ mice (impaired glymphatic influx/efflux), and (3) WT mice subjected to 24-hour
sleep deprivation (SD) to suppress glymphatic function.
**Figure 4 (Page 4 of Supplementary Data):**
**(A)** Quantification of EB-OVA⁺ cells in the dcLNs 24 hours post-injection. A significant reduction
is seen in Aqp4⁻/⁻ and SD mice (***p<0.001, one-way ANOVA).
**(B)** Representative flow plots showing OVA-MHC-I tetramer⁺ CD8⁺ T-cells in dcLNs 7 days postimmunization with intracortical OVA.
**(C)** Bar graph summarizing the frequency of antigen-specific CD8⁺ T-cells. Both genetic and
behavioral disruption of glymphatics abrogated T-cell priming.
As predicted, the number of EB-OVA⁺ CD11c⁺ cells in the dcLNs was drastically reduced in both
Aqp4⁻/⁻ and SD mice compared to WT controls (Figure 4A). Crucially, this impaired antigen delivery
translated to a functional deficit in adaptive immunity. When we immunized mice intracortically with
OVA and then tracked the expansion of OVA-specific CD8⁺ T-cells using MHC-I tetramers, we found
that both Aqp4⁻/⁻ and SD mice failed to generate a robust T-cell response, unlike their WT
counterparts (Figure 4B, 4C).
#### **The Glymphatic-Immune Axis is Functionally Relevant in a Model of Latent Viral Infection**
To assess the physiological relevance of this pathway, we turned to a model of latent murine
gammaherpesvirus 68 (MHV-68) infection. Following intranasal infection, MHV-68 establishes
latency in astrocytes and other CNS cells. We hypothesized that glymphatic-mediated antigen
presentation is crucial for maintaining surveillance of this latent reservoir.
We infected WT and Aqp4⁻/⁻ mice with MHV-68 and allowed latency to establish. Sixty days postinfection, we isolated PADCs and dcLN cells and stimulated them with MHV-68 peptides.
**Figure 5 (Page 5 of Supplementary Data):**
**(A)** ELISpot analysis of IFN-γ-producing cells from dcLNs of latently infected mice upon ex vivo
stimulation with MHV-68 ORF61 peptide. Aqp4⁻/⁻ mice show significantly fewer antigen-specific Tcells.
**(B)** Viral reactivation assay. Explanted brains from latently infected mice were cultured, and
viral plaques were quantified. Aqp4⁻/⁻ brains showed a higher rate of spontaneous reactivation.
**(C)** Schematic model: During sleep, active glymphatic flow (blue arrows) carries CNS antigens
(red dots) along paravenous spaces to meningeal lymphatic "antigen portals." PADCs sample these
antigens and migrate to the dcLNs to prime naive T-cells (Tₙ ), generating effector T-cells (Tₑff) that
can patrol the CNS.
Consistent with our hypothesis, dcLNs from Aqp4⁻/⁻ mice contained significantly fewer MHV-68specific IFN-γ-producing T-cells (Figure 5A). Furthermore, when we explanted brains from these
latently infected mice, we observed a significantly higher rate of viral reactivation in Aqp4⁻/⁻ tissue
compared to WT (Figure 5B), indicating a failure of immune-mediated control of the latent virus in
the absence of functional glymphatics.
---
### **DISCUSSION**
Our study elucidates a previously unknown circuit that integrates the CNS's waste clearance system
with adaptive immunity. We have identified the glymphatic system as the principal route for the
delivery of parenchymal antigens to the immune system, fundamentally updating the model of CNS
immune surveillance (Figure 5C). The historical view of immune privilege is not one of absolute
isolation, but rather of regulated communication, with the glymphatic system serving as a critical
regulatory gatekeeper.
The discovery of PADCs and their strategic positioning at antigen portals provides a cellular
mechanism for how this communication is achieved. Their behavior—reaching directly into the
lymphatic flow to capture antigen—is reminiscent of the "nibbling" behavior of dendritic cells in
peripheral tissues but is uniquely adapted to the low-flow, perivascular environment of the CNS exit
points. The CNS-TRAP mouse model was instrumental in moving beyond injected tracers to
demonstrate that this pathway handles authentic, CNS-derived proteins.
The functional consequences of disrupting this pathway are profound. Our data from both the OVA
model and the MHV-68 latent infection model demonstrate that impairing glymphatic function—
either genetically or via the physiologically relevant perturbation of sleep deprivation—compromises
the afferent arm of CNS immune surveillance. This leads to a failure in generating antigen-specific T-
cell responses and a loss of control over latent viral reservoirs. This has immediate implications for
our understanding of the link between sleep, neurodegeneration, and infection. It suggests that the
cognitive decline associated with chronic sleep disruption may be partly attributable to a failure in
immune-mediated clearance of metabolic waste and potentially pathogenic agents.
**Future Directions and Implications:**
1. **Neuroinflammatory Diseases:** In Multiple Sclerosis, could an over-active or dysregulated
glymphatic-immune axis contribute to the presentation of self-antigens? Conversely, in Alzheimer's
disease, could impaired glymphatic function reduce the clearance of amyloid-β, while also
preventing the necessary immune surveillance, creating a double-hit pathology?
2. **Neuro-Oncology:** The efficacy of immune checkpoint inhibitors in treating brain metastases
and glioblastoma is variable. Our work suggests that optimizing glymphatic function (e.g., by
promoting healthy sleep) could enhance the delivery of tumor neo-antigens to the immune system,
potentially boosting immunotherapy responses.
3. **Sleep and Immunity:** This work provides a mechanistic basis for the long-observed
connection between sleep and immune function. It positions sleep as a critical period not only for
metabolic clearance but also for immunological "scanning" of the brain's health.
4. **Drug Delivery:** The antigen portals could be exploited for delivering therapeutic antibodies or
immune modulators directly into the CNS by leveraging the natural flow of the glymphatic system.
In conclusion, we have defined a fundamental pillar of brain-body interaction. The glymphatic
system and the meningeal lymphatics are not just drainage pipes but are integrated into a
sophisticated communication network—the brain's "lymphatic-immune axis." This discovery opens a
new chapter in neuroimmunology, with far-reaching implications for a wide spectrum of
neurological disorders.
---
### **METHODS**
#### **Mouse Models**
All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC)
of Yale University. C57BL/6J, Prox1-GFP, Cx3cr1[GFP/+], CD11c-EYFP, Aqp4⁻/⁻, OT-I, and GFAP-Cre
mice were obtained from The Jackson Laboratory. The CNS-TRAP mouse was generated by crossing
B6.Cg-Tg(GFAP-cre)77.6Mvs/2J with B6;129S-Gt(ROSA)26Sor[tm14(CAG-tdTomato,-OVA)Glow]/J
(Ai14-RCFL-OVA). Mice were housed under a 12-hour light/dark cycle with ad libitum access to food
and water.
#### **Surgical Procedures and Tracer Infusions**
For intracisternal infusions, mice were anesthetized and placed in a stereotaxic frame. A pulled glass
micropipette was inserted into the cisterna magna, and 10 µL of tracer (1% Texas Red-dextran 3kDa
or Qdot655-OVA in artificial CSF) was infused at 2 µL/min. For intracortical injections, coordinates
from Bregma were +1.0 mm AP, -1.5 mm ML, -1.0 mm DV. Evans Blue-OVA (100 µg in 2 µL PBS) was
infused at 0.4 µL/min.
#### **Immunohistochemistry and Microscopy**
Mice were transcardially perfused with PBS followed by 4% PFA. Brains and meninges were
dissected, fixed, and processed for free-floating sectioning or dural whole-mount preparation.
Antibodies used: anti-CD11c (N418), anti-MHC-II (I-A/I-E, M5/114.15.2), anti-LYVE-1 (ALY7), antiGFAP (GA5), anti-Prox1 (RELA). Images were acquired on a Zeiss LSM 880 confocal microscope or a
Leica TCS SP8 MP multiphoton microscope. 3D reconstructions were performed with Imaris
software.
#### **Flow Cytometry and Cell Sorting**
Single-cell suspensions from dcLNs and dura mater were prepared by enzymatic digestion
(Collagenase D/DNase I). Cells were stained with a viability dye and fluorochrome-conjugated
antibodies. For intracellular cytokine staining, cells were stimulated with PMA/ionomycin in the
presence of brefeldin A for 4 hours, then fixed, permeabilized, and stained for IFN-γ. Data were
acquired on a BD LSRFortessa and analyzed with FlowJo v10.8. PADCs were sorted on a BD FACS Aria
III for in vitro assays.
#### **In Vivo Two-Photon Imaging**
A chronic cranial window was surgically implanted over the transverse sinus in CD11c-EYFP mice.
After a 2-week recovery, mice were anesthetized, and intracisternal infusion of Qdot655-OVA was
performed. Imaging was conducted on a custom-built two-photon microscope equipped with a
Ti:Sapphire laser. Time-lapse videos were analyzed with ImageJ.
#### **T-cell Proliferation and ELISpot Assays**
For in vitro T-cell stimulation, sorted PADCs were co-cultured with CFSE-labeled OT-I splenocytes at a
1:10 ratio for 72 hours. CFSE dilution was measured by flow cytometry. For IFN-γ ELISpot, dcLN cells
were plated on ELISpot plates coated with anti-IFN-γ and stimulated with 1 µg/mL MHV-68 ORF61
peptide for 24 hours. Spots were developed and counted using an automated ELISpot reader.
#### **Statistical Analysis**
All data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9.
Comparisons between two groups were made using an unpaired, two-tailed Student's t-test.
Multiple group comparisons were performed using one-way or two-way ANOVA with appropriate
post-hoc tests as indicated in the figure legends. A p-value of < 0.05 was considered statistically
significant.
---
### **REFERENCES** (Abbreviated List)
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---
### **ACKNOWLEDGEMENTS**
We thank the members of the Chen and Vasquez laboratories for their insightful discussions and
technical assistance. We are grateful to the Yale Flow Cytometry Core and the Stanford Cell Sciences
Imaging Facility for their support. This work was supported by the National Institutes of Health (NIH
grants R01-NS12345 to D.C. and R01-AI10123 to B.R.C.), the Howard Hughes Medical Institute
(Faculty Scholar award to E.A.V.), and a Japan Society for the Promotion of Science (JSPS)
Postdoctoral Fellowship to K.S.
### **AUTHOR CONTRIBUTIONS**
E.A.V. and B.R.C. conceived the project, designed experiments, performed research, analyzed data,
and wrote the manuscript. A.T. and K.S. performed the EM and CLEM work. S.L.O. and I.M.
conducted the in vivo imaging and viral infection models. M.A.P. generated the CNS-TRAP mouse
model. D.C. supervised the project, acquired funding, and wrote the manuscript. All authors
discussed the results and commented on the manuscript.
### **DECLARATION OF INTERESTS**
The authors declare no competing interests.
### **SUPPLEMENTAL INFORMATION**
Document includes:
- Figures S1-S5 (Related to all main figures)
- Table S1: Antibodies and reagents used in this study.
- Table S2: Primer sequences for genotyping.
- Video S1: In vivo two-photon time-lapse of a PADC sampling Qdot655-OVA from a meningeal
lymphatic capillary.
***
**(End of Document)**