Microvessels of the blood-brain barrier (BBB) regulate transport into the brain. The highly specialized brain microvascular endothelial cells, a major component of the BBB, express tight junctions and efflux transporters which regulate paracellular and transcellular permeability. However, most existing models of BBB microvessels fail to exhibit physiological barrier function. Here, using (iPSC)-derived human brain microvascular endothelial cells (dhBMECs) within templated type I collagen channels we mimic the cylindrical geometry, cell-extracellular matrix interactions, and shear flow typical of human brain post-capillary venules. We characterize the structure and barrier function in comparison to non-brain-specific microvessels, and show that dhBMEC microvessels recapitulate physiologically low solute permeability and quiescent endothelial cell behavior. Transcellular permeability is increased two-fold using a clinically relevant dose of a p-glycoprotein inhibitor tariquidar, while paracellular permeability is increased using a bolus dose of hyperosmolar agent mannitol. Lastly, we show that our human BBB microvessels are responsive to inflammatory cytokines via upregulation of surface adhesion molecules and increased leukocyte adhesion, but no changes in permeability. Human iPSC-derived blood-brain barrier microvessels support quantitative analysis of barrier function and endothelial cell dynamics in quiescence and in response to biologically- and clinicallyrelevant perturbations.

IntroductionAmong the numerous molecules that regulate T-cell function, the inhibitory receptor PD-1 and its 2 B7 family ligands, B7-H1(PD-L1) and B7-DC (PD-L2), seem to play important roles in microbial immunity, tumor immunity, and autoimmunity. 1-6 PD-1 contains a classic immunoreceptor tyrosine-based inhibitory motif (ITIM) and binds the phosphatase SHP-2. 7 PD-1 engagement has been shown to induce T cell apoptosis and to inhibit proliferative responses and cytokine release in vitro in response to TCR engagement. 8,9 Definitive evidence that PD-1 represents an inhibitory receptor comes from analysis of PD-1 knockout mice, which develop strain-specific autoimmune syndromes later in life. 10,11 The more delayed and tissue-specific autoimmune phenotypes of PD-1 knockout mice contrast markedly with the multiorgan autoimmunity observed within the first few weeks of birth for CTLA-4 knockout mice. [12][13][14] These findings support the notion that PD-1 is part of a system that fine-tunes immune responses, in contrast to the "on-off switch" mediated by the B7-1/B7-2-CD28/CTLA-4 system. Most of the inhibitory roles of PD-1 on T-cell responses have been attributed to its interaction with B7-H1. In vivo, B7-H1:PD-1 interactions have been documented to inhibit T-cell-effector responses to both tumors as well as to normal peripheral tissues. It is highly likely that the up-regulation of B7-H1 by tumors that serves to protect them from immune attack is a reflection of the normal expression of B7-H1 within peripheral tissues that represents a natural mechanism for regulating tissue injury by effector T cells. B7-H1 is expressed constitutively on subsets of macrophages, B cells, and thymocytes as well as certain nonhematopoietic cells within many organs. 1,15 This PD1 ligand is inducible on dendritic cells and all lymphocytes, as well as endothelial cells, epithelial cells, myocytes, and mesenchymal cells. 1,15 B7-H1 plays a well-documented role in down-modulating T-cell responsesparticularly CD8 responses-within the liver, where it is expressed on cells lining the sinusoids. 16 B7-H1 knockout mice display decreased apoptosis of intrahepatic CD8 cells as well as enhanced CD8 responses within the liver after infections with microbes such as adenovirus and Listeria monocytogenes. 16,17 The role of B7-H1: PD-1 interactions extends beyond the liver. For example, autoimmune diabetes is exacerbated in diabetes-prone mouse strains crossed onto a B7-H1-null background. 18 Further evidence for the role of the B7-H1:PD-1 interaction in effector T-cell responses has come from recent studies demonstrating that "exhausted" CD8 cells from mice chronically infected with the clone 13 strain of LCMV express high levels of PD-1. 6 This inactivated functional phenotype can be reversed in mice treated with blocking antibodies to B7-H1, emphasizing the important role of the B7-H1:PD-1 interaction in down-modulating effector immune responses. To date, however, the earliest point at which a role for B7-H1/PD-1 in the CD8 T cell response has b...

Feedback regulation of adaptive immunity is a fundamental mechanism for controlling the overall output of different signal transduction pathways, including that mediated by the T-cell antigen receptor (TCR). Calcineurin and Ras are known to have essential functions during T-cell activation. However, how the calcineurin signalling pathway is terminated in the process is still largely unknown. Although several endogenous inhibitors of calcineurin have been reported, none fulfils the criteria of a feedback inhibitor, as their expression is not responsive to TCR signalling. Here we identify an endogenous inhibitor of calcineurin, named Carabin, which also inhibits the Ras signalling pathway through its intrinsic Ras GTPase-activating protein (GAP) activity. Expression of Carabin is upregulated on TCR signalling in a manner that is sensitive to inhibitors of calcineurin, indicating that Carabin constitutes part of a negative regulatory loop for the intracellular TCR signalling pathway. Knockdown of Carabin by short interfering RNA led to a significant enhancement of interleukin-2 production by antigen-specific T cells in vitro and in vivo. Thus, Carabin is a negative feedback inhibitor of the calcineurin signalling pathway that also mediates crosstalk between calcineurin and Ras.