Fig. 4: Activated CD8+ T cells undergo morphological changes and actin cytoskeleton rearrangements to form discrete junctions with, and consequently invade, BEC.
From: Expression of E-cadherin by CD8+ T cells promotes their invasion into biliary epithelial cells
a Transmission election microscopy (TEM) micrograph showing a CD8+ T cell contacting the surface of a BEC. Right panel shows magnified image corresponding to the inset (blue box) showing pseudopodia-like elongated contacts made between the CD8+ T cell surface and BEC membrane (blue arrows). b Airyscan confocal image of phalloidin-labelled (grey) co-cultured cells showing CD8+ T cell (CellTracker™ Red; magenta) polarising towards a BEC (CellTracker™ Green; yellow) and forming actin-rich filopodia at the BEC surface. c Scanning electron microscopy (SEM) image showing CD8+ T cells on the BEC surface. Insets demonstrate T cell polarisation (i) and initial pseudopodia-like contacts (ii) between the cells. d SEM image showing CD8+ T cells beginning to flatten out whilst formed multiple discrete contacts with BEC. e SEM image showing two CD8+ T cells; one internalised into the BEC (white arrow) and the second one in contact with the surface. f, g Effect of small molecular inhibitor treatment of CD8+ T cells on their internalisation into BEC. f Representative images of co-cultured BEC (CellTracker Green™; yellow) and 48 h activated CD8+ T cells (CellTracker Red™; cyan), all labelled with Alexa Fluor 680-conjugated wheat germ agglutinin (WGA680) comparing the frequency of internalisation of T cells into BEC following treatment with 5 µM wortmannin. g Quantification of internalised 48 h activated CD8+ T cells per 100 BEC in which T cells were treated with 1 μM wortmannin, 5 nM H-1152, or 5 μM cytochalasin D prior to co-culture. Nine fields of view were analysed from triplicate wells. Mean numbers of internalised T cells/100 BEC/technical repeat are plotted. Values are normalised and displayed as a fold change from vehicle-treated cells. n = 5 biologically independent experiments. Statistics were derived from unpaired two-tailed Student’s t-tests. Vehicle control=0.001% DMSO. Error bars represent standard error of the mean (SEM). df = 22. Wortamannin t = 3.411, H-1152 t = 0.4783, cytochalasin D t = 2.321. p-values are displayed in the figure for each statistical comparison made.
