Density of 107 cells/ml) were incubated for 10 min at 37uC in

Density of 107 cells/ml) were Title Loaded From File incubated for 10 min at 37uC in 5 mM CFSE in serum free RPMI. The labelling reaction was stopped by the addition of serum. Cells were then washed 3 times prior to use. For the quantification of cell proliferation, cells were analysed by flow cytometry with 1379592 a reduction in CFSE MFI indicative of cell division.MHC II expression on HBEC following co-cultureTo assess the expression of MHC II on HBEC following coculture with PBMC, HBEC were removed by trypsinisation following 6 d of co-culture. HBEC were then stained with antihuman MHC II (HLA-DR; eBioscience). For flow cytometric analysis CFSE positive cells (PBMC) were excluded by gating to ensure MHC II analysis was conducted on HBEC only.Flow cytometryFor multicolor flow cytometric analysis, HBEC were incubated in the presence of fluorochrome-conjugated mAbs against CD105 (SN6), CD106 (STA), CD80 (2D10.4), CD86 (IT2.2), CD40 (5C3), HLA-DR/MHC II (LN3) and CD275 (MIH12) (all from eBioscience), CD54 (5.6E; Beckman Coulter) and b2-microglobu?lin/MHC I (TU99; BD Biosciences) as per manufacturer’s instructions.Results HBEC express key molecules for antigen presentation and T cell activationFor this study we employed a particular line of immortalized human microvascular EC (HBEC; hCMEC/D3) that recapitulates many of the key characteristics of primary brain EC and thus hasAntigen uptake analysisThe ability of HBEC to take up fluorescently labeled protein was assessed using flow cytometry after the cells were Ollection (group II) (Fig 8). RT-PCR was performed using total RNA extracted incubatedBrain Endothelium and T Cell Proliferationbeen validated as an excellent model of the BBB for in vitro studies [18?0]. A number of adhesion molecules known to be expressed by brain endothelium are involved, under inflammatory conditions, in the migration of activated leukocytes across the BBB. Flow cytometric analysis of HBEC cells not only confirmed the strong basal expression of ICAM-1, but also demonstrated a marked upregulation following stimulation with TNF and/or IFNc (Fig. 1). Endoglin (CD105), an EC marker predominantly expressed by proliferating EC was expressed at high levels basally, with no regulation in expression seen following pro-inflammatory cytokine stimulation (Fig. 1). Similarly, MHC I (b2-microglobulin) was expressed at high levels basally on HBEC with no increase observed following cytokine stimulation (Fig. 1). This is in contrast to previous results whereby MHC I expression has been shown to be upregulated by stimulation with IFNa, -b or [21]. Despite this, our results provide evidence that HBEC, like most cell types, possess the minimal requirement for antigen presentation to CD8+ T cells. In contrast to MHC I, despite the low basal expression of MHC II on HBEC cells, its expression was 15826876 greatly increased upon the addition of IFNc or TNF+IFNc (Fig. 1), highlighting a potential role for these cells in antigen presentation to CD4+ T cells. Previous analysis of MHC II on EC has proved difficult in vivo, with constitutive expression only detected in post-capillary venules [22]. Whilst the expression of the co-stimulatory molecules CD80/CD86 (B7-1/B7-2) was not detected on resting or cytokinestimulated HBEC cells, the co-stimulatory molecule, CD40 was detected following stimulation with IFNc or TNF+IFNc (Fig. 1), indicating that like MHC II the expression is regulated by IFNc. Previously, CD40 has been demonstrated to be constitutively expressed on primary human brain ECs, with this expression being upregulated following cytokine st.Density of 107 cells/ml) were incubated for 10 min at 37uC in 5 mM CFSE in serum free RPMI. The labelling reaction was stopped by the addition of serum. Cells were then washed 3 times prior to use. For the quantification of cell proliferation, cells were analysed by flow cytometry with 1379592 a reduction in CFSE MFI indicative of cell division.MHC II expression on HBEC following co-cultureTo assess the expression of MHC II on HBEC following coculture with PBMC, HBEC were removed by trypsinisation following 6 d of co-culture. HBEC were then stained with antihuman MHC II (HLA-DR; eBioscience). For flow cytometric analysis CFSE positive cells (PBMC) were excluded by gating to ensure MHC II analysis was conducted on HBEC only.Flow cytometryFor multicolor flow cytometric analysis, HBEC were incubated in the presence of fluorochrome-conjugated mAbs against CD105 (SN6), CD106 (STA), CD80 (2D10.4), CD86 (IT2.2), CD40 (5C3), HLA-DR/MHC II (LN3) and CD275 (MIH12) (all from eBioscience), CD54 (5.6E; Beckman Coulter) and b2-microglobu?lin/MHC I (TU99; BD Biosciences) as per manufacturer’s instructions.Results HBEC express key molecules for antigen presentation and T cell activationFor this study we employed a particular line of immortalized human microvascular EC (HBEC; hCMEC/D3) that recapitulates many of the key characteristics of primary brain EC and thus hasAntigen uptake analysisThe ability of HBEC to take up fluorescently labeled protein was assessed using flow cytometry after the cells were incubatedBrain Endothelium and T Cell Proliferationbeen validated as an excellent model of the BBB for in vitro studies [18?0]. A number of adhesion molecules known to be expressed by brain endothelium are involved, under inflammatory conditions, in the migration of activated leukocytes across the BBB. Flow cytometric analysis of HBEC cells not only confirmed the strong basal expression of ICAM-1, but also demonstrated a marked upregulation following stimulation with TNF and/or IFNc (Fig. 1). Endoglin (CD105), an EC marker predominantly expressed by proliferating EC was expressed at high levels basally, with no regulation in expression seen following pro-inflammatory cytokine stimulation (Fig. 1). Similarly, MHC I (b2-microglobulin) was expressed at high levels basally on HBEC with no increase observed following cytokine stimulation (Fig. 1). This is in contrast to previous results whereby MHC I expression has been shown to be upregulated by stimulation with IFNa, -b or [21]. Despite this, our results provide evidence that HBEC, like most cell types, possess the minimal requirement for antigen presentation to CD8+ T cells. In contrast to MHC I, despite the low basal expression of MHC II on HBEC cells, its expression was 15826876 greatly increased upon the addition of IFNc or TNF+IFNc (Fig. 1), highlighting a potential role for these cells in antigen presentation to CD4+ T cells. Previous analysis of MHC II on EC has proved difficult in vivo, with constitutive expression only detected in post-capillary venules [22]. Whilst the expression of the co-stimulatory molecules CD80/CD86 (B7-1/B7-2) was not detected on resting or cytokinestimulated HBEC cells, the co-stimulatory molecule, CD40 was detected following stimulation with IFNc or TNF+IFNc (Fig. 1), indicating that like MHC II the expression is regulated by IFNc. Previously, CD40 has been demonstrated to be constitutively expressed on primary human brain ECs, with this expression being upregulated following cytokine st.