Research Achievements Research Achievements Multistep T cell extravasation across the BBB Making use of primary mouse brain microvascular endothelial cells (pMBMECs) we have shown in collaboration with Ruth Lyck that CD4 T cell arrest under physiological flow is mediated by endothelial VCAM-1 and ICAM-1, while endothelial ICAM-1 and ICAM-2 are essential for subsequent T-cell polarization and extended crawling against the direction of flow to sites permissive for diapedesis (Abadier et al., EJI, 2015). We identified BBB tricellular junctions as novel sites for T-cell diapedesis across the BBB (Casto Dias et al., J Cell Sci, 2021). In further studies we showed that high levels of endothelial ICAM-1 combined with de novo expression of the atypical chemokine receptor 1 (ACKR1) shuttling chemokines from the abluminal to the luminal side of the inflamed BBB, and/or impaired BBB junctional integrity lead to increased transcellular T-cell diapedesis across the inflamed BBB (Minten et al., Brain 2014; Wimmer et al., Front Immunol, 2019; Marchetti et al., EJI, 2021). Our data thus underscore that BBB junctional integrity is regulated independently of the cellular pathway of T-cell diapedesis across the BBB. This is supported by our observation that inducible endothelial expression of the tight junction sealing claudin-1 reduces BBB leakiness and EAE severity while leaving inflammatory cell recruitment into the CNS undisturbed (Pfeiffer et al., Acta Neuropathol, 2011). Molecular composition of BBB tight junctions Analyzing the molecular makeup of BBB tight junctions we clarified that claudin-3 is not expressed in brain endothelial cells (Castro Dias et al., Sci Reports, 2019), and that claudin-12 is not required for BBB integrity (Castro Dias et al., FBCNS, 2019). The choroid plexus as immune cell entry site into the CNS Since our original discovery that during experimental autoimmune encephalomyelitis (EAE) the adhesion molecules ICAM-1 and VCAM-1 are upregulated at the BCSFB of the choroid plexus in parallel to their upregulation at the BBB (Steffen et al., Am J Pathol, 1996), we have speculated on a function of the choroid plexus in CNS immunity. In collaboration with Federica Sallusto (Bellinzona/Zürich) we demonstrated that Th17 cells may enter the CNS in a CCR6/CCL20 dependent manner via the ChP to initiate EAE (Reboldi et al., Nat Immunol, 2009). We have succeeded to establish a valid in vitro model of the mouse BCSFB allowing for in depth modeling of the immune function of the BCSFB (Lazarevic et al., FBCNS, 2016). In addition, by using human in vitro BBB and BCSFB models we have shown that human T-cell subsets differ in their ability to cross the BBB versus the BCSFB in vitro (Nishihara et al, FBCNS, 2020). Drug delivery across the BBB Making use of nanoparticle chemistry in collaboration with Harm-Anton Klok (EPFL, Lausanne) we identified T cells as novel cellular targets to be explored for cell mediated drug delivery into the CNS (Ayer et al., Bioconjug Chem, 2021; Thomsen et al., Biomacromolecules, 2021; Ayer et al., Adv Healthc Mater, 2021) Human models of the BCSFB and the BBB We have established human models for the BCSFB and stem-cell derived in vitro models of the human BBB allowing to study the migration of human immune cells across these barriers (Nishihara et al., FBCNS, 2020). In collaboration with James Mc Grath (Rochester University, NY, USA) we have developed a breakthrough microfluidic device (mSIM-CVB) to investigate the migration of rare patient derived immune cell subsets across the BBB under physiological flow by live cell imaging (Mossu et al, JCBFM, 2019). In collaboration with Renaud Du Pasquier (Lausanne, CH) and Eric Shusta (Madison-Wisconsin, USA) we developed the extended endothelial-cell culture method (EECM) by differentiating human induced pluripotent stem cells (hiPSC) to brain microvascular endothelial (BMEC)-like cells. These BMEC-like cells display an inherent endothelial adhesion molecule phenotype making them the first uniquely suitable model to study human immune cell interactions with the BBB (Nishihara et al., FASEB J, 2020; Nishihara et al., STAR Prot, 2021). Establishing EECM-BMEC-like cells from hiPSC derived from MS patients and healthy controls (HC), we found that MS-derived BMEC-like cells display impaired barrier properties and an inflammatory phenotype with increased immune cell interaction compared to their HC counterparts. Thus hiPSC-derived EECM-BMEC-like cells are suitable to explore the molecular underpinnings of BBB dysfunction in neurological disorders and are thus eligible to study drug delivery across the BBB in neurological diseases.