Imulation [23]. The expression of this co-stimulatory molecule on brain EC provides

Imulation [23]. The expression of this co-stimulatory molecule on brain EC provides key evidence for their potential role as APC as the binding of CD40L on helper T cells to CD40 activates `APCs’ to upregulate the expression of more co-stimulatory molecules, increase cytokine expression and promote T cell differentiation [24]. Finally, inducible co-stimulator ligand (ICOSL) expression was detectedon HBEC following TNF stimulation (Fig. 1). ICOS and its ligand, ICOSL are members of the CD28 family of co-stimulators mediating effector T cell differentiation [25]. Previously, ICOSL has been detected not only basally on human umbilical vein ECs but also upregulated by cytokine stimulation [25,26].HBEC take up antigens using macropinocytosis and clathrin-coated pitsA recent study from our 3PO web laboratory demonstrated that during malaria, the transfer of parasite antigens to ECs can take place [3], however, the precise mechanisms behind this remain unclear. The ability of our HBEC to take up soluble antigens was assessed in vitro using fluorescently labeled antigens in a classic antigen uptake experiment. The ability of HBEC to take up antigen via macropinocytosis and clathrin-coated pits was assessed using Lucifer yellow and FITC-OVA respectively. The amount of fluorescence incorporated into the cells at 37uC was measured by flow cytometry with nonspecific surface binding subtracted following incubation on ice. Interestingly, HBEC were able to take up FITC-OVA via clathrin-coated pits and macropinocytose Lucifer yellow (Fig. 2A, C respectively). To further prove that the uptake of antigen by HBEC was not an experimental artifact, a specific inhibitor of macropinocytosis and other actin-dependent mechanisms, cytochalasin D (CCD; 10 mM) was employed [27]. Indeed, following pre-incubation with CCD, both the uptake of FITC-OVA and Lucifer yellow was significantly inhibited (Fig. 2 B, D) indicating that HBEC have the capacity to take up soluble antigen in a similar manner as professional APC.HBEC support the proliferation of activated T cellsAs optimal T-cell activation and differentiation in vivo requires long-lasting T PC interaction, a classical in vitro conjugate forming assay was adapted to assess the ability of HBEC to form conjugates with T cells [28]. Red fluorescently labeled (PKH26) CD4+ or CD8+ T cells were incubated in suspension with green fluorescently labeled (PKH67) HBECs with the adherence between HBEC and T cells examined using flow cytometry.Figure 1. Expression of markers relevant to antigen presentation and T cell activation on HBEC. Histograms represent flow cytometry results from unstimulated and cytokine stimulated HBEC cells 18 h following stimulation. HBEC were stimulated with either 10 ng/ml TNF (blue line), 50 ng/ml IFNg (green line), or 10 ng/ml TNF+50 ng/ml IFNg (orange line) and compared to unstimulated cells (red line). Cells were stained with mAbs against CD54 (ICAM-1), Endoglin (CD105), MHC II (HLA-DR), ICOSL (CD275), CD40, CD80 and CD86 as per manufacturers instructions. Data are representative of four INCB039110 biological activity independent experiments. doi:10.1371/journal.pone.0052586.gBrain Endothelium and T Cell ProliferationFigure 2. HBEC take up fluorescently labelled antigen via actin-dependent mechanisms and form conjugates with T cells. Flow cytometry histograms depicting level of uptake of FITC-OVA (A) and Lucifer yellow (C) by HBEC at 37uC (blue line) vs background uptake at 4uC (red line). Data are representative of three independen.Imulation [23]. The expression of this co-stimulatory molecule on brain EC provides key evidence for their potential role as APC as the binding of CD40L on helper T cells to CD40 activates `APCs’ to upregulate the expression of more co-stimulatory molecules, increase cytokine expression and promote T cell differentiation [24]. Finally, inducible co-stimulator ligand (ICOSL) expression was detectedon HBEC following TNF stimulation (Fig. 1). ICOS and its ligand, ICOSL are members of the CD28 family of co-stimulators mediating effector T cell differentiation [25]. Previously, ICOSL has been detected not only basally on human umbilical vein ECs but also upregulated by cytokine stimulation [25,26].HBEC take up antigens using macropinocytosis and clathrin-coated pitsA recent study from our laboratory demonstrated that during malaria, the transfer of parasite antigens to ECs can take place [3], however, the precise mechanisms behind this remain unclear. The ability of our HBEC to take up soluble antigens was assessed in vitro using fluorescently labeled antigens in a classic antigen uptake experiment. The ability of HBEC to take up antigen via macropinocytosis and clathrin-coated pits was assessed using Lucifer yellow and FITC-OVA respectively. The amount of fluorescence incorporated into the cells at 37uC was measured by flow cytometry with nonspecific surface binding subtracted following incubation on ice. Interestingly, HBEC were able to take up FITC-OVA via clathrin-coated pits and macropinocytose Lucifer yellow (Fig. 2A, C respectively). To further prove that the uptake of antigen by HBEC was not an experimental artifact, a specific inhibitor of macropinocytosis and other actin-dependent mechanisms, cytochalasin D (CCD; 10 mM) was employed [27]. Indeed, following pre-incubation with CCD, both the uptake of FITC-OVA and Lucifer yellow was significantly inhibited (Fig. 2 B, D) indicating that HBEC have the capacity to take up soluble antigen in a similar manner as professional APC.HBEC support the proliferation of activated T cellsAs optimal T-cell activation and differentiation in vivo requires long-lasting T PC interaction, a classical in vitro conjugate forming assay was adapted to assess the ability of HBEC to form conjugates with T cells [28]. Red fluorescently labeled (PKH26) CD4+ or CD8+ T cells were incubated in suspension with green fluorescently labeled (PKH67) HBECs with the adherence between HBEC and T cells examined using flow cytometry.Figure 1. Expression of markers relevant to antigen presentation and T cell activation on HBEC. Histograms represent flow cytometry results from unstimulated and cytokine stimulated HBEC cells 18 h following stimulation. HBEC were stimulated with either 10 ng/ml TNF (blue line), 50 ng/ml IFNg (green line), or 10 ng/ml TNF+50 ng/ml IFNg (orange line) and compared to unstimulated cells (red line). Cells were stained with mAbs against CD54 (ICAM-1), Endoglin (CD105), MHC II (HLA-DR), ICOSL (CD275), CD40, CD80 and CD86 as per manufacturers instructions. Data are representative of four independent experiments. doi:10.1371/journal.pone.0052586.gBrain Endothelium and T Cell ProliferationFigure 2. HBEC take up fluorescently labelled antigen via actin-dependent mechanisms and form conjugates with T cells. Flow cytometry histograms depicting level of uptake of FITC-OVA (A) and Lucifer yellow (C) by HBEC at 37uC (blue line) vs background uptake at 4uC (red line). Data are representative of three independen.