Fig. 4

eCIRP-induced cytotoxic CD4CD8αα cells induce intestinal epithelial cell death. (A) A schematic of cytotoxicity assay using CD4CD8αα cells differentiated from splenic naïve CD4⁺ T cells and IECs derived from intestinal organoids. CD4CD8αα cells were pretreated with PBS or eCIRP for 4 h in the presence and absence of GrB (50 μM) and Prf (100 nM) inhibitors for the last 30 min. IECs and CD4CD8αα cells were then cocultured for 4 h, and cell death of IECs was evaluated by flow cytometry using a Zombie Aqua Fixable Viability Kit. (B) Representative flow cytometry plots and histogram showing CD45⁻EpCAM⁺ intestinal epithelial cells’ death rate. (C) Cytotoxicity of CD4CD8αα cells measured by the frequency of dead IECs. Experiments were performed 2 times, and all data were used for analysis. Data represent the mean ± SEM. The groups were compared by one-way ANOVA followed by a Tukey’s multiple comparisons test. *p < 0.05 vs. CD4CD8αα(-) eCIRP(-) inhibitors(-), ^#p < 0.05 vs. CD4CD8αα(+) eCIRP(-) inhibitors(-), ^†p < 0.05 vs. CD4CD8αα(+) eCIRP(+) inhibitors(-)