Pport of T cell proliferation.and VCAM-1/VLA-4 on EC/T cells respectively in addition to interactions required for antigen presentation.MHC expression on HBEC is upregulated SC1 following 
coculture with allogeneic PBMCTo determine whether the interaction between T cells and HBEC occurs in a two-way fashion, the expression of MHC II on the HBEC monolayer was determined following 6 days of coculture with PBMCs. A significant increase in MHC II-positive cells was observed when HBEC were co-cultured with aCD3 oraCD3/aCD28 stimulated PBMCs when compared to HBEC cells alone (Fig. 4A, B) indicating that the donor PBMC were able to modulate the MHC II expression on the HBEC themselves. These conjugates likely involve interactions of ICAM-1/LFA-DiscussionIn this study, we provide for the evidence that microvascular brain EC are able to act as APCs. Our analysis of MHC and costimulatory molecule expression on HBEC show for the first time that brain EC are endowed a “professional” costimulatory ligand of the B7 family, ICOSL. This in conjugation with the expression of MHC II and CD40 following IFNc stimulation supports the notion of the brain endothelium being able to present antigens to and co-stimulate T cells promoting effector CD4+ T cell responses. Additionally, with constitutively high expression of MHC I,Brain Endothelium and T Cell ProliferationFigure 4. PBMC modulate MHC II expression on HBEC following co-culture. A, Histogram plots of HBEC depicting expression of MHC II (HLA-DR) 6 days following the start of the co-culture with donor PBMC. 16105 CFSE-labelled donor PBMC were co-cultured with a confluent monolayer of either resting (left panels) or 10 ng/ml TNF+50 ng/ml IFNc pre-stimulated (right panels) HBEC cells. PBMC were either subjected to resting conditions or stimulation with aCD3 or aCD3/CD28 23977191 mAbs (top, middle lower panels respectively). Histograms are representative of four Anlotinib biological activity independent experiments with the same donor. B, Percentage of MHC II+ HBEC in resting (white bars) vs TNF/IFNc stimulated (black bars) HBEC. Data is pooled from four independent experiments with the same donor. * indicates statistically significant differences between control HBEC and respective co-culture conditions using a non-parametric Mann-Whitney test (p,0.05). doi:10.1371/journal.pone.0052586.gHBEC, like most cell types, possess the minimal requirement for antigen presentation to CD8+ T cells. Antigen uptake is the first step in antigen-presenting pathways, and pinocytosis is the major means by which cells sample soluble protein antigen. Here we show that HBEC are able to take up soluble antigen using both macropinocytosis and clathrin-coated pits as pathways for antigen uptake. Whilst liver sinusoidal EC have been demonstrated to be fully efficient APC in that they express co-stimulatory molecules [30], take up antigen via the mannose receptor [31] and are able to cross present exogenous antigen [32], no previous studies have been conducted on the ability of HBEC to take up and process antigens. The data presented here shows for the first time that HBEC are able to take up soluble antigen using actin-dependent mechanisms, in a manner similar to `professional’ APCs. In the co-culture assays presented here, HBEC were able to support and promote the proliferation of TCR-stimulated CD4+ and CD8+ T cells. In these assays, an MLR occurs and the T cells proliferate due to an MHC mismatch [33]. The demonstration of antigen-specific activation of human T cells b.Pport of T cell proliferation.and VCAM-1/VLA-4 on EC/T cells respectively in addition to interactions required for antigen presentation.MHC expression on HBEC is upregulated following coculture with allogeneic PBMCTo determine whether the interaction between T cells and HBEC occurs in a two-way fashion, the expression of MHC II on the HBEC monolayer was determined following 6 days of coculture with PBMCs. A significant increase in MHC II-positive cells was observed when HBEC were co-cultured with aCD3 oraCD3/aCD28 stimulated PBMCs when compared to HBEC cells alone (Fig. 4A, B) indicating that the donor PBMC were able to modulate the MHC II expression on the HBEC themselves. These conjugates likely involve interactions of ICAM-1/LFA-DiscussionIn this study, we provide for the evidence that microvascular brain EC are able to act as APCs. Our analysis of MHC and costimulatory molecule expression on HBEC show for the first time that brain EC are endowed a “professional” costimulatory ligand of the B7 family, ICOSL. This in conjugation with the expression of MHC II and CD40 following IFNc stimulation supports the notion of the brain endothelium being able to present antigens to and co-stimulate T cells promoting effector CD4+ T cell responses. Additionally, with constitutively high expression of MHC I,Brain Endothelium and T Cell ProliferationFigure 4. PBMC modulate MHC II expression on HBEC following co-culture. A, Histogram plots of HBEC depicting expression of MHC II (HLA-DR) 6 days following the start of the co-culture with donor PBMC. 16105 CFSE-labelled donor PBMC were co-cultured with a confluent monolayer of either resting (left panels) or 10 ng/ml TNF+50 ng/ml IFNc pre-stimulated (right panels) HBEC cells. PBMC were either subjected to resting conditions or stimulation with aCD3 or aCD3/CD28 23977191 mAbs (top, middle lower panels respectively). Histograms are representative of four independent experiments with the same donor. B, Percentage of MHC II+ HBEC in resting (white 
bars) vs TNF/IFNc stimulated (black bars) HBEC. Data is pooled from four independent experiments with the same donor. * indicates statistically significant differences between control HBEC and respective co-culture conditions using a non-parametric Mann-Whitney test (p,0.05). doi:10.1371/journal.pone.0052586.gHBEC, like most cell types, possess the minimal requirement for antigen presentation to CD8+ T cells. Antigen uptake is the first step in antigen-presenting pathways, and pinocytosis is the major means by which cells sample soluble protein antigen. Here we show that HBEC are able to take up soluble antigen using both macropinocytosis and clathrin-coated pits as pathways for antigen uptake. Whilst liver sinusoidal EC have been demonstrated to be fully efficient APC in that they express co-stimulatory molecules [30], take up antigen via the mannose receptor [31] and are able to cross present exogenous antigen [32], no previous studies have been conducted on the ability of HBEC to take up and process antigens. The data presented here shows for the first time that HBEC are able to take up soluble antigen using actin-dependent mechanisms, in a manner similar to `professional’ APCs. In the co-culture assays presented here, HBEC were able to support and promote the proliferation of TCR-stimulated CD4+ and CD8+ T cells. In these assays, an MLR occurs and the T cells proliferate due to an MHC mismatch [33]. The demonstration of antigen-specific activation of human T cells b.
