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Additional file 7: Table S1. Antibody list for fluorescence-activated cell sorting of different CD4+ Th subsets. Table S2. Antibody list for flowcytometry analysis of adhesion molecules surface expression of BLEC, HBMEC and EECM-BMEC-like cells. Table S3. Antibody list for flowcytometry analysis of integrins surface expression of different CD4+ Th subsets. Table S4. Antibody list for immunofluorescence staining of adhesion molecules surface expression of BLEC, HBMEC and EECM-BMEC-like cells.

Additional file 1: Figure S1. Flow cytometry analysis of Th1* cell-bound NTZ, bNTZ, mNTZ, and anti-β2-antibody at different concentrations: 50 μg/mL, 10 μg/mL, 1 μg/mL, 0.1 μg/mL, 0.01 μg/mL, and 0.001 μg/mL. Isotype controlis shown in grey. ΔMFI is indicated next to each peak of detection. Percentage of human CD4+ Th1 adhesion inhibited by titrated NTZ , bNTZ and mNTZ to immobilized recombinant VCAM-1 under static conditions. Red dotted line shows 95% limit of T cell adhesion inhibition. Number of adhered Th1/Th1*/Th2/Th17 cells on immobilized recombinant VCAM-1 under static conditions grouped by treatment condition. Th1/Th1*/Th2/Th17 cells were treated with the minimal inhibitory concentration shown in Fig. 2 –C for the respective natalizumab constructsprior to the experiment. An isotype control antibody was used as internal control. Each figure shows the mean ± SEM of 3 experiments. Gating strategy for the multicolor flow cytometry analysis of α4-, β1- and β7-integrin cell-surface expression on Th1, Th1*, Th2and Th17 cells. Isotype control condition is shown in grey. Percentage and ΔMFI of α4-, β1- and β7-integrins high and low expressing Th1, Th1*, Th2and Th17 cellsis shown. Figure S2. Number of adhered human effector/memory CD4+ Th1* cells on immobilized recombinant JAM-B and fibronectin under static conditions . Th1* cells were incubated with 1 μg/mL natalizumabor with 1 μg/mL of an isotype control antibodyprior experiment. Each figure shows the mean ± SD of 2 independent experiments done in triplicates. Statistical analysis: unpaired T-test. Figure S3. Number of adhered human CD4+ Th1* cells on titrated concentrations of immobilized recombinant VCAM-1 and ICAM-1 under flow conditions. Each figure shows the mean ± SEM of 3 independent experiments. Figure S4. Mean crawling speed , distance and Euclidian distance of human CD4+ Th1* cells on immobilized recombinant VCAM-1, 10-times higher molecular concentrations of ICAM-1and combined VCAM-1/ ICAM-1under physiological flow condition. Th1* cells were treated with 1 μg/mL natalizumab and/or with 1 μg/mL of functional anti-β2-integrins blocking antibody prior experiment. An isotype control antibody was used as internal control. Each figure shows the mean ± SEM of one representative experiment per group. Statistical analysis: one-way ANOVA followed by Dunn’s multiple-comparison test. x/y diagrams of Th1*-cell crawling tracks on immobilized recombinant VCAM-1, ICAM-1, and combined VCAM-1/ ICAM-1 under physiological flow conditions are depicted for one representative experiment per group. For each track, the site of arrest was set to the center point of the respective diagram. End points of tracks are indicated by dots. Flow direction is illustrated by an arrow. Figure S5. Percentage of human CD4+ Th1* adhesion inhibited by titrated bNTZ and mNTZ to immobilized recombinant VCAM-1, and combined VCAM-1/ ICAM-1when β2-integrins are blocked under flow condition. Red dotted line shows 95% limit of T cell adhesion inhibition. Each figure shows the mean ± SEM of 3 experiments.

International audience; Vitamin D deficiency has been associated with the risk of multiple sclerosis, disease activity and progression. Results from in vitro experiments, animal models and analysis of human samples from randomized controlled trials provide comprehensive data illustrating the pleiotropic actions of Vitamin D on the immune system. They globally result in immunomodulation by decreasing differentiation of effector T and B cells while promoting regulatory subsets. Vitamin D also modulates innate immune cells such as macrophages, monocytes and dendritic cells, and acts at the level of the blood-brain barrier reducing immune cell trafficking. Vitamin D exerts additional activity within the central nervous system reducing microglial and astrocytic activation. The immunomodulatory role of Vitamin D detected in animal models of multiple sclerosis has suggested its potential therapeutic use for treating multiple sclerosis. In this review, we focus on recent published data describing the biological effects of Vitamin D in animal models of multiple sclerosis on immune cells, blood-brain barrier function, activation of glial cells and its potential neuroprotective effects. Based on the current knowledge, we also discuss optimization of therapeutic interventions with Vitamin D in patients with multiple sclerosis, as well as new technologies allowing in-depth analysis of immune cell regulations by vitamin D.

Background The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood–brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). Objective Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. Methods Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. Results While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. Conclusions Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB., Fluids and Barriers of the CNS, 17 (1), ISSN:2045-8118

BACKGROUND The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). OBJECTIVE Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. METHODS Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. RESULTS While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. CONCLUSIONS Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.