Mossu, A. | bioRxiv Science

bioRxiv2025

Vitamin D differentially modulates effector and regulatory T-cell migration across the blood-brain barrier

Multiple sclerosis (MS) is an inflammatory disease of the CNS influenced by a combination of genetic predisposition and environmental factors. Vitamin D (VitD) deficiency is considered a major risk factor for MS. While VitD is associated with immunomodulatory roles, the exact mechanisms by which VitD protects from disease are still largely unknown. CD4 T cells play a key role in MS pathogenesis with autoimmune effector T cells (Teff) infiltrating the CNS across the blood-brain barrier (BBB) and regulatory T cells (Treg) displaying impaired functions. Here we show that treatment of human CD4 T cells with the active form of VitD (1,25-Dihydroxyvitamin D3; 1,25(OH)2D3) decreased cell-surface expression of 4{beta}1- and L{beta}2-integrins on Teff but not Treg and reduced Teff adhesion to their endothelial ligands VCAM-1 and ICAM-1. By employing live cell imaging, we observed that VitD treatment reduced arrest of Teff but not Treg to the BBB as well as ICAM-1 and VCAM-1 under physiological flow in vitro and differentially affected post-arrest behaviour of Teff versus Treg on the BBB under physiological flow. Furthermore, VitD treatment favoured the migration of Treg over Teff across the BBB under static and flow conditions in vitro. In vivo live cell imaging showed that VitD reduced T cell arrest on the inflamed BBB during autoimmune neuroinflammation. Finally, VitD also reduced expression of integrins mediating CNS homing on pathogenic CD4 T cells isolated from the CSF of persons with MS (PwMS). As VitD treatment did not alter barrier properties or adhesion molecule profile of our BBB model we propose a beneficial effect of VitD supplementation in PwMS by reducing CNS trafficking of pro-inflammatory T cells while leaving CNS entry of Treg unaffected.

immunology↗

bioRxiv2023

UFMTrack: Under-Flow Migration Tracker enabling analysis of the entire multi-step immune cell extravasation cascade across the blood-brain barrier in microfluidic devices

The endothelial blood-brain barrier (BBB) strictly controls immune cell trafficking into the central nervous system (CNS). In neuroinflammatory diseases such as multiple sclerosis, this tight control is, however, disturbed, leading to immune cell infiltration into the CNS. The development of in vitro models of the BBB combined with microfluidic devices has advanced our understanding of the cellular and molecular mechanisms mediating the multi-step T-cell extravasation across the BBB. A major bottleneck of these in vitro studies is the absence of a robust and automated pipeline suitable for analyzing and quantifying the sequential interaction steps of different immune cell subsets with the BBB under physiological flow in vitro. Here we present the Under-Flow Migration Tracker (UFMTrack) framework for studying immune cell interactions with endothelial monolayers under physiological flow. We then showcase a pipeline built based on it to study the entire multi-step extravasation cascade of immune cells across brain microvascular endothelial cells under physiological flow in vitro. UFMTrack achieves 90% track reconstruction efficiency and allows for scaling due to the reduction of the analysis cost and by eliminating experimenter bias. This allowed for an in-depth analysis of all behavioral regimes involved in the multi-step immune cell extravasation cascade. The study summarizes how UFMTrack can be employed to delineate the interactions of CD4+ and CD8+ T cells with the BBB under physiological flow. We also demonstrate its applicability to the other BBB models, showcasing broader applicability of the developed framework to a range of immune cell-endothelial monolayer interaction studies. The UFMTrack framework along with the generated datasets is publicly available in the corresponding repositories. Author summaryImmune cells continuously travel through our body to perform immune surveillance. They travel within blood vessels at a very high speed and slow down upon reaching their target organ by the sequential interaction with different adhesion and signaling molecules on the vascular endothelial cells. The study of molecular mechanisms mediating this multi-step extravasation of immune cells has been significantly advanced by in vitro cultures of microvascular endothelial cell monolayers. The dynamic interaction of the immune cells with endothelial monolayers can be imaged over time in vitro in microfluidic devices under physiological flow. The 2-dimensional structure of the endothelial monolayer allows for reliable visualization of the extravasation process required for the study of the molecular mechanisms involved. The manual analysis of the acquired imaging data is time- consuming and prone to experimenter error. Analysis automation is, however, hampered by the similar appearance of the unlabeled immune and endothelial cells and by the flow causing rapid immune cell displacement. Here we introduce UFMTrack, the under-flow migration tracker framework allowing for automated analysis of immune cell interactions with microvascular endothelial cells under flow in vitro. UFMTrack performs comparably to the manual analysis of an experienced researcher, eliminates experimenters bias, and improves the accuracy of the immune cell tracking. Taken together, UFMTrack sets the stage for scalability of in vitro live cell imaging studies of immune cell extravasation.

immunology↗