T experiments. buy SC1 Inhibition of FITC-OVA (B) and Lucifer yellow (D) uptake by HBEC cells pre-incubated with 10 mM Cytochalasin D (CCD). C, Flow cytometry histogram depicting level of uptake of Lucifer yellow by HBEC at 37uC (blue line) vs background uptake at 4uC (red line). Data are representative of three independent experiments Percentage increase in mean fluorescence intensity (MFI) is calculated as follows: (MFI following uptake at 37uC/MFI following uptake at 4uC)6100. Data are pooled from three independent experiments (n = 3 per experiment) and are expressed as mean +/2 SD. ** and *** indicates statistically significant differences between control and CCD treatment as assessed by Student t test (p, 0.001, p,0.001 respectively). Representative flow cytometry plots indicating the levels of conjugation between HBEC and CD4+ (E) and CD8+ (F) cells. HBEC were labeled with PKH67 and isolated T cells labeled with PKH26 and equal numbers of cells were co-cultured for 30 min prior to flow cytometric analysis. doi:10.1371/journal.pone.0052586.gConjugates were BIBS39 determined to be cells positive for both PKH26 and PKH67. Interestingly, both CD4+ and CD8+ T cells form conjugates, i.e. cell doublets in suspension, with control (data not shown) and cytokine activated HBECs, as shown by flow cytometry (Fig. 2E, F respectively). There were higher percentages of T cell/HBEC conjugates seen when the HBEC were cytokine activated (3.6 vs 1.4 for CD4+ and 6.3 vs 2.1 for CD8+). After determining that HBEC were capable of binding to both CD4+ and CD8+ T cells, the ability of HBEC to support T cell proliferation and present alloantigens was assessed by co-culturing CFSE-labelled donor PBMCs with a confluent monolayer of either resting or cytokine stimulated HBECs. In addition, the agonistic antibodies aCD3/aCD28 were also 18325633 added to the assay to mimic T cell receptor (TCR) stimulation and co-stimulation respectively [29]. Six days following co-culture the percentage of CD4+ and CD8+ T cells proliferating was determined by measuring the reduction in CFSE MFI (Fig. 3A). While the presence of soluble aCD3 and aCD28 resulted in a modest increase in proliferatingCD8+ cells, the only significant increase in proliferation was observed when the PBMC were co-cultured with TNF+IFNcactivated HBEC and aCD3/aCD28 (Fig. 3B), indicating that HBEC support the proliferation of CD8+ T cells, however, the CD8+ cells must also be activated via their TCR. Interestingly, CD4+ T cell proliferation was significantly increased in the presence of both resting and cytokine-stimulated HBEC (Fig. 3C), however, the CD4+ cells also must be stimulated via their TCR with aCD3 or aCD3/aCD28 to observe the HBEC-mediated support of proliferation. It is most likely that the modest increase in proliferation for both CD4+ and CD8+ 1527786 T cells following aCD3 stimulation is indicating that the cells were not stimulated using a solid phase activation, i.e. plate bound aCD3. Experiments using transwells have indicated that when the PBMC were physically separated from the HBEC monolayer during co-culture, the increase in proliferation over control samples were greatly reduced (Fig. S1). This was observed for both CD4+ and CD8+ T cells suggesting that direct interactionBrain Endothelium and T Cell ProliferationFigure 3. HBEC support the proliferation of CD4+ and CD8+ T cells. A, CFSE histogram plots of gated CD4+ (left panel) and CD8+ (right panel) 6 days following the start of the co-culture of HBEC and don.T experiments. Inhibition of FITC-OVA (B) and Lucifer yellow (D) uptake by HBEC cells pre-incubated with 10 mM Cytochalasin D (CCD). C, Flow cytometry histogram depicting level of uptake of Lucifer yellow by HBEC at 37uC (blue line) vs background uptake at 4uC (red line). Data are representative of three independent experiments Percentage increase in mean fluorescence intensity (MFI) is calculated as follows: (MFI following uptake at 37uC/MFI following uptake at 4uC)6100. Data are pooled from three independent experiments (n = 3 per experiment) and are expressed as mean +/2 SD. ** and *** indicates statistically significant differences between control and CCD treatment as assessed by Student t test (p, 0.001, p,0.001 respectively). Representative flow cytometry plots indicating the levels of conjugation between HBEC and CD4+ (E) and CD8+ (F) cells. HBEC were labeled with PKH67 and isolated T cells labeled with PKH26 and equal numbers of cells were co-cultured for 30 min prior to flow cytometric analysis. doi:10.1371/journal.pone.0052586.gConjugates were determined to be cells positive for both PKH26 and PKH67. Interestingly, both CD4+ and CD8+ T cells form conjugates, i.e. cell doublets in suspension, with control (data not shown) and cytokine activated HBECs, as shown by flow cytometry (Fig. 2E, F respectively). There were higher percentages of T cell/HBEC conjugates seen when the HBEC were cytokine activated (3.6 vs 1.4 for CD4+ and 6.3 vs 2.1 for CD8+). After determining that HBEC were capable of binding to both CD4+ and CD8+ T cells, the ability of HBEC to support T cell proliferation and present alloantigens was assessed by co-culturing CFSE-labelled donor PBMCs with a confluent monolayer of either resting or cytokine stimulated HBECs. In addition, the agonistic antibodies aCD3/aCD28 were also 18325633 added to the assay to mimic T cell receptor (TCR) stimulation and co-stimulation respectively [29]. Six days following co-culture the percentage of CD4+ and CD8+ T cells proliferating was determined by measuring the reduction in CFSE MFI (Fig. 3A). While the presence of soluble aCD3 and aCD28 resulted in a modest increase in proliferatingCD8+ cells, the only significant increase in proliferation was observed when the PBMC were co-cultured with TNF+IFNcactivated HBEC and aCD3/aCD28 (Fig. 3B), indicating that HBEC support the proliferation of CD8+ T cells, however, the CD8+ cells must also be activated via their TCR. Interestingly, CD4+ T cell proliferation was significantly increased in the presence of both resting and cytokine-stimulated HBEC (Fig. 3C), however, the CD4+ cells also must be stimulated via their TCR with aCD3 or aCD3/aCD28 to observe the HBEC-mediated support of proliferation. It is most likely that the modest increase in proliferation for both CD4+ and CD8+ 1527786 T cells following aCD3 stimulation is indicating that the cells were not stimulated using a solid phase activation, i.e. plate bound aCD3. Experiments using transwells have indicated that when the PBMC were physically separated from the HBEC monolayer during co-culture, the increase in proliferation over control samples were greatly reduced (Fig. S1). This was observed for both CD4+ and CD8+ T cells suggesting that direct interactionBrain Endothelium and T Cell ProliferationFigure 3. HBEC support the proliferation of CD4+ and CD8+ T cells. A, CFSE histogram plots of gated CD4+ (left panel) and CD8+ (right panel) 6 days following the start of the co-culture of HBEC and don.
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