The choroid plexus (ChP) is a highly vascularized tissue in each central nervous system (CNS) ventricle. It comprises a monolayer of cuboidal epithelial cells resting on a basal lamina, forming a blood-cerebrospinal fluid barrier (BCSFB). The ChP was proposed as an alternative CNS entry site specifically for CCR6+ Th17 cells in experimental autoimmune encephalomyelitis (EAE). We speculated that these cells accumulate in the ChP stroma and may follow different cues to reach the CNS. We aim to define the precise anatomical entry routes of encephalitogenic Th17 cells from the ChP stroma into the CNS.

Starting to explore the potential connections of the subarachnoid space (SAS) and the ChP, immunostainings for E-cadherin, a marker for arachnoid barrier cells, were performed on brain sections of VE-cadherin-GFP mice, allowing us to identify vascular, pial, and arachnoid adherens junctions. To study anatomical routes of Th17 cells entry from the ChP stroma into the CNS, we developed a fluorescent brain barrier reporter (BBR) mouse where the blood-brain barrier and BCSFB of the ChP are visible based on claudin-5 and FoxJ1-driven fluorescent reporter molecules. Using confocal and light sheet microscopy, we compared the distribution of infiltrating Th17 cells during EAE induced by adoptive transfer of in vitro polarized encephalitogenic Th17 cells into syngeneic BBR mice. Additionally, we employed two-photon intravital microscopy (2P-IVM) to investigate whether Th17 cells can reach the ventricular space from the ChP stroma through the BCSFB in vivo.

Immunostainings showed E-cadherin and VE-cadherin signals folding in from the brain's surface into the brain, as an extension of the leptomeninges between the cerebral cortex and midbrain, lining the third ventricle, suggesting a close spatial relation to the SAS and ChP stroma. Preliminary observations indicate that at the peak of EAE, Th17 cells accumulate along CSF-filled subarachnoid cisterns of the forebrain and midbrain. 2P-IVM, achieved by cannula implantation above the lateral ventricle in BBR mice, has not yet identified fluorescently labeled Th17 cells crossing the BCSFB during EAE in vivo.

Our data suggest that Th17 cells may use an undiscovered "secret" route to reach the SAS from the ChP stroma in neuroinflammation. This insight could improve understanding of how T cells invade the CNS in neuroinflammation.