Fig. 6: Functional assays for exhausted CD8⁺ T cells.

a Detection of PD1⁺CD8⁺ T cells and XBP1⁺CD8⁺ T cells in BTC tissue by multiplex immunohistochemistry staining. Representative data from eighteen patients were shown. b Quantitation of XPB1⁺CD8⁺ T cells and PD1⁺CD8⁺ T cells as a percentage of total exhausted CD8⁺ T cells in two different response groups. c–f CD8⁺ T cells were stimulated with anti-CD3 and anti-CD28 for 96 h and cultured with the supernatant from HIBEpiC, HCCC-9810 and GBC-SD for 48 h. c the protein level of both IRE1α and XBP1in cocultured CD8⁺ T cells tested by western blotting. β-actin were used as loading control. (representative plots on left; quantitation on right). d Representative FASC plots gated on XBP1⁺ cells, with positive cells (XBP1⁺CD8⁺) boxed (left). Shown is the quantitation of positive cells as a percentage of total CD8⁺ T cells (right). e, f. 10 µM 4μ8C was added to the above medium for 24 h before harvesting. Quantitation of FACS plots of positive TIGIT⁺ T cells (e) and PD1⁺ T cells (f) as a percentage of total CD8⁺ T cells. g CD8⁺ T cells were stained with ER tracker (red) and nuclei were stained using DAPI (blue). Morphologies of endoplasmic reticulum were analyzed by laser scanning confocal microscopy from control, GBS-SD, and HCCC-9810 cell lines. Error bars indicate mean ± SEM. Two-tail paired t tests, ****p < 0.0001, ***p < 0.001, **p < 0.01, *p ≤ 0.05, no significance.