Fig. 10: Internalisation of PBC-derived CD8⁺ T cells into BEC is driven by E-cadherin expression by CD8⁺ T cells.

a Multichannel image of immunohistochemistry (IHC) staining of PBC biopsy for CD8 (yellow), E-cadherin (white) and EpCAM (magenta), showing CD8⁺ T cells (white arrows) within the biliary epithelial cells (BEC) of a ruptured bile duct. b 3D-reconstructed Z-stack of internalised CD8⁺ T cells highlighted in (a) (green box) which also expressed E-cadherin (colocalised signal in grey; right panel). c Airyscan super-resolution orthographical confocal micrographs of PBC patient blood-derived CD8⁺ T cells and BEC, following 4 h co-culture, stained intracellularly for cytokeratin-19 (CK19; grey) and α-tubulin (yellow). Image shows both attached and internalised T cells, including one in mitosis. d Comparison between haemochromatosis (HFE; control) and PBC patients for frequency of E-cadherin expression amongst blood-derived CD8⁺ cells 48 h post-activation, determined by flow cytometry. p-value was generated using a two-tailed Mann-Whitney test. Error bars represent median and interquartile range. n = 7 biologically independent patient samples per condition. e Median fluorescence intensity (MFI) values, determined by flow cytometry, of E-cadherin staining performed for PBC patient-derived CD8⁺ T cells treated with different CDH1-targeting siRNAs (siRNA1-4). Values are expressed as fold-changes from matched control (Silencer Select negative control 1; SS1). Statistics were derived from paired two-tailed Student’s t-tests. n = 4 biologically independent experiments. t = 10.66, df = 3. f Representative images of BEC (CellTracker™ Green; yellow) co-cultured with siRNA- treated CD8⁺ T cells (CellTracker™ red; cyan). Red arrows show internalised CD8⁺ T cells. g Quantification of internalised CD8⁺ T cells per 100 BEC for siRNA-treated CD8⁺ T cells. Mean values/technical repeat are plotted, expressed as fold-changes from matched control values (SS1). Statistics were derived from unpaired two-tailed Student’s t-tests. n = 3 biologically independent experiments. t = 4.184, df = 10. h Linear regression analysis comparing matched E-cadherin MFI determined by flow cytometry (x) with mean frequency of CD8⁺ T cell internalisation (y). Plot point colour corresponds to mean values of the same conditions in (g). e, g Error bars represent standard error of the mean (SEM). p values are displayed in the figure for each statistical comparison made.