Fig. 8

Exosomal circCCAR1 protects HCC cells from CD8⁺ T cells by stabilizing PD-1. A Representative images of the internalization of PKH67-labeled HCCLM3 exosomes (red) by CD8 + T cells. B CD8 + T-cell-pretreated exosomes were cocultured with HCC cells, and then CD8 + T-cell-mediated elimination of HCC cells was determined by FACS analysis. ***p < 0.001 vs. vector-exo; ###p < 0.001 vs. sh-NC-exo. C Perforin and granzyme-B levels in CD8 + T cells. D-E Secreted IFN-γ and TNF-α by CD8 + T cells were measured by ELISA. ***p < 0.001 vs. vector-exo; ###p < 0.001 vs. sh-NC-exo. F circCCAR1 and PD1 mRNA expression in CD8 + T cells. ***p < 0.001 vs. vector-exo; ##p < 0.01 vs. sh-NC-exo. G PD1 protein expression in CD8 + T cells. H The interaction strength between circCCAR1 and PD1 was determined using the RPISeq program. I The binding of circCCAR1 and PD1 was confirmed by RIP assay. ***p < 0.001. J PD1 levels in pulldown assays using a biotinylated antisense oligomer targeting the junction of circCCAR1 in CD8 + T cells. K PD1 expression in CD8 + T cells after circCCAR1 depletion or overexpression. L PD1 protein levels in CD8 + T cells treated with MG132 after circCCAR1 depletion or overexpression. M Stability analysis of PD1 protein in CD8 + T cells treated with CHX after circCCAR1 depletion or overexpression. N Ubiquitination assay of PD1 in CD8 + T cells