Fig. 1: Live activated CD8+ T cells are internalised into BEC in human livers.

a Immunohistochemistry (IHC) staining of liver tissue from a primary biliary cholangitis (PBC) patient. Red arrows indicate CD3⁺ (yellow) CD8⁺ (magenta) T cells invading cytokeratin-19⁺ (CK19; grey) biliary epithelial cells (BEC). Right panel shows magnified view of area outlined by yellow box in left panel and further magnified image of CD8⁺ T cell (blue arrow) surrounded by CK19 staining (magenta box). b Orthographical confocal micrograph showing peripheral blood-derived CD8⁺ T cells (CellTracker™ Red; CTR, cyan) co-cultured with BEC (CellTracker™ Green; CTG, yellow) stained for CK19 (magenta) using immunocytochemistry (ICC). Blue arrows show CD8⁺ T cells attached to the surface of the BEC. White arrows show flatter internalised CD8⁺ T cells. c Percentage of lysosome-associated CD8⁺ T cells after 4 or 24 h co-culture with BEC. Mean values of the number of Lysotracker™-associated T cells/technical repeats are plotted. n = 2 biologically independent experiments. Error bars represent standard error of the mean (SEM). d Representative orthographical micrograph showing BEC labelled with CellTracker™ Green (yellow) and Lysotracker™ Blue (LTB; cyan) following 24 h co-culture with CD8⁺ T cells (CellTracker™ Red, magenta). White arrows show internalised T cells lacking lysosome association. Blue arrow shows T cell associating with lysosomes. e IHC staining of PBC liver showing CD4⁺ T cells (yellow; yellow arrow), and a CD8⁺ T cell (magenta, white arrow) associated with the CK19⁺ BEC (grey). Right panel shows magnified view of area outlined by orange box in left panel. f 3D-volume rendered version of Z-stack acquired from same region as (e). White arrow identifies internalised CD8⁺ T cell. Yellow arrows indicate adhered and infiltrating CD4⁺ T cells. g Representative images of BEC (yellow) co-cultured with α-CD3/CD28-activated CD4⁺ or CD8⁺ T cells (cyan). h Quantification of internalised non-activated (black bars) or activated (blue bars) T cells per 100 BEC. Mean values from triplicate technical repeats are plotted. n = 3 biologically independent experiments. Statistics were derived from paired two-tailed Student’s t-tests. Error bars represent standard error of the mean (SEM). Left: t = 4.098e-007, df = 16. Right: t = 3.015. df = 8. p-values are displayed for each statistical comparison made.