Fig. 3: CD8⁺ T cells internalised within BEC are larger and more irregularly shaped than those situated around the bile ducts. α-CD3/CD28 activation induces similar morphological changes in CD8⁺ T cells in vitro and increased their rate of internalisation into BEC.

a Immunohistochemistry staining of a liver tissue section from a patient with primary biliary cholangitis (PBC), showing larger, irregularly shaped CD8⁺ T cells (yellow) invading the biliary epithelial cells (BEC). Right panels show 3D-reconstructed versions (top) and 3D-volume renders (bottom) of Z-stack images acquired for inset in left panel (red box) showing eccentric CD8⁺ T cells surrounded by cytokeratin-19 (CK19; magenta). Further magnified images of CD8⁺ T cells are also shown (far right; yellow boxes). b Representative flow cytometry gating strategy for phenotyping peripheral blood-derived live, CD3⁺ CD4⁺ and CD8⁺ T cells. c Representative flow cytometry contour plots of side and forward scatter areas (SSC-A/FSC-A), showing the difference in granularity and size, respectively, of CD8⁺ T cells. Cells were phenotyped in the absence of activation stimuli (black) or following 24 h (red) or 48 h (cyan) after α-CD3/CD28-mediated activation. d Representative images of BEC (CellTracker™ Green; yellow) co-cultured with 24 h or 48 h-activated CD8⁺ T cells (CellTracker™ Red; cyan). Cells were labelled with wheat germ agglutinin conjugated to Alexa Fluor 680 (WGA680, grey) after co-culture. e–g Quantification of number per 100 BEC (e), size (f), and eccentricity (g) of internalised CD8⁺ T cells in either non-activated, 24 h activated, or 48 h activated prior to co-culture. Nine fields of view were analysed from triplicate wells. Mean values/technical repeat are plotted. n = 5 biologically independent experiments. Statistics for all graphs are derived from unpaired two-tailed Student’s t-tests. p-values are displayed for each statistical comparison made. e Values are normalised and displayed as a fold change from non-activated cells. Non-activated v 24 h activated t = 2.068, df = 28; Non-activated v 48 h activated t = 3.137, df = 28; 24 h activated v 48 h activated t = 2.294, df = 28. Error bars represent standard error of the mean (SEM). f t = 0.1335, df=26. Error bars represent median and interquartile range. g t = 2.068, df = 28. Error bars represent median and interquartile range.