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.
Figure 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 37°C (blue line) vs background uptake at 4°C (red line). Data are representative of three independent 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 37°C (blue line) vs background uptake at 4°C (red line). Data are representative of three independent experiments Percentage increase in mean fluorescence intensity (MFI) is calculated as follows: (MFI following uptake at 37°C/MFI following uptake at 4°C)×100. Data are pooled from three independent experiments (n = 3 per experiment) and are expressed as mean +/− 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.
Figure 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 donor PBMC. For co-culture 1×105 CFSE-labelled donor PBMC were co-cultured or not with a confluent monolayer of either resting or 10 ng/ml TNF+50 ng/ml IFNγ 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).
Figure 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. 1×105 CFSE-labelled donor PBMC were co-cultured with a confluent monolayer of either resting (left panels) or 10 ng/ml TNF+50 ng/ml IFNγ 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/IFNγ 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).