Fig. 8: Internalisation of E-cadherin^hi CD8⁺ T cells into BEC is more frequent than E-cadherin^lo CD8⁺ T cells.

Peripheral blood CD8⁺ T cells derived from healthy volunteers were activated by α-CD3/CD28 stimulation and cultured for 48 h. E-cadherin^hi CD8⁺ T cells and E-cadherin^lo CD8⁺ T cells were then isolated using fluorescence activated cell sorting (FACS). T cells were then rested for 24 h, labelled with CellTracker™ Red, and then co-cultured with CellTracker™ Green-labelled biliary epithelial cells (BEC) for 4 h. a FACS-associated scatter plot showing the sorting strategy for activated CD8⁺ T cells into E-cadherin^lo (orange) or E-cadherin^hi (purple) populations. Full gating strategy shown in Supplementary Fig. 10B. b Representative images of BEC (CellTracker™ Green; yellow) co-cultured with either E-cadherin^lo or E-cadherin^hi CD8⁺ T cells (CellTracker™ red, cyan). c–e Quantification of CD8⁺ T cells attached to BEC (c), as well as the size (d) and number per 100 BEC (e) of internalised CD8⁺ T cells for E-cadherin^lo and E-cadherin^hi sorted cells. Mean values/technical repeat are plotted. Statistics were derived from unpaired two-tailed Student’s t-tests. n = 2 biologically independent patient samples. df=10. c, d Error bars represent median and interquartile range. c t = 2.785. d t = 2.406. e Error bars represent standard error of the mean (SEM). t = 3.224. f Quantification of internalised E-cadherin^hi CD8⁺ T cells per 100 BEC in which T cells were treated with 1 μM wortmannin or 5 nM H-1152 (ROCK1 inhibitor). T cells were pretreated with inhibitors for 30 min and then co-cultured for 4 h, whilst maintaining inhibitor concentrations. Nine fields of view were analysed from triplicate wells. Mean values of number of internalised CD8⁺ T cells/technical repeat are plotted. Values are normalised and displayed as a fold change from DMSO (vehicle) treated cells. n = 1 biologically independent experiment. Error bars represent SEM. p-values are displayed in the figure for each statistical comparison made.