Or PBMC. For co-culture 16105 CFSE-labelled donor PBMC were co-cultured or not

Or PBMC. For co-culture 16105 CFSE-labelled donor PBMC were co-cultured or not with a confluent monolayer of either resting or 10 ng/ml TNF+50 ng/ml IFNc pre-stimulated HBEC cells. PBMC were either subjected to resting conditions or stimulation with aCD3 or aCD3/CD28 mAbs. Vitamin D2 web Following 6 days of culture, cells were harvested and stained with CD4 and CD8 mAbs to Title Loaded From File identify proliferating cell populations. CFSE histograms depict the number of events (y-axis) and the fluorescence intensity (x-axis) with proliferating cells displaying a progressive 2-fold loss in fluorescence intensity following cell division, indicative of proliferating cells. Histograms are representative of four independent experiments with the same donor. Graphical representation of the percentage of CD4+ (B) and CD8+ (C) PBMC proliferating following 6 days of culture either alone (white bars) or in the presence of resting (grey bars) or cytokine stimulated (black bars) HBEC as outlined above. Data is pooled from four independent experiments with the same donor. * indicates statistically significant differences between control PBMC and respective co-culture conditions using a non-parametric Mann-Whitney test (p,0.05). doi:10.1371/journal.pone.0052586.gbetween HBEC and T cells is required for HBEC-mediated support 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 1527786 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 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 c.Or PBMC. For co-culture 16105 CFSE-labelled donor PBMC were co-cultured or not with a confluent monolayer of either resting or 10 ng/ml TNF+50 ng/ml IFNc pre-stimulated HBEC cells. PBMC were either subjected to resting conditions or stimulation with aCD3 or aCD3/CD28 mAbs. Following 6 days of culture, cells were harvested and stained with CD4 and CD8 mAbs to identify proliferating cell populations. CFSE histograms depict the number of events (y-axis) and the fluorescence intensity (x-axis) with proliferating cells displaying a progressive 2-fold loss in fluorescence intensity following cell division, indicative of proliferating cells. Histograms are representative of four independent experiments with the same donor. Graphical representation of the percentage of CD4+ (B) and CD8+ (C) PBMC proliferating following 6 days of culture either alone (white bars) or in the presence of resting (grey bars) or cytokine stimulated (black bars) HBEC as outlined above. Data is pooled from four independent experiments with the same donor. * indicates statistically significant differences between control PBMC and respective co-culture conditions using a non-parametric Mann-Whitney test (p,0.05). doi:10.1371/journal.pone.0052586.gbetween HBEC and T cells is required for HBEC-mediated support 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 1527786 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 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 c.