Fig. 3. Epithelial HVEM affects CD8αα⁺ IET survival.

(A) Frequencies of EdU ⁺ cells from proximal SI IET of Hvem^fl/fl (n=4) and Hvem^ΔIEC (n=4) mice. Mice were administered EdU once per day for 6 days. Cells were isolated from proximal SI and EdU⁺ cells were measured at either day 6 or day 20. (B) Representative flow cytometry images detecting apoptotic cells in TCRαβ⁺CD8αα⁺ IET from proximal SI in Hvem^fl/fl and Hvem^ΔIEC mice.

(C, D) Representative flow cytometry showing expression of pro-apoptotic Bcl family proteins (Bax and Bim) on TCRαβ⁺CD8αα⁺ IET from proximal SI of Hvem^fl/fl (n=4) and Hvem^ΔIEC (n=4) mice (C) and compiled MFI from multiple individual mice (D). (E) Absolute cell number of TCRαβ⁺CD8αα⁺, TCRαβ⁺CD8αβ⁺, and TCRγδ⁺CD8αα⁺ IET subsets from Hvem^fl/fl (n=18), Hvem^ΔIEC (n=14), Lck^pr-Bcl-xL^TgHvem^fl/fl (n=5) and Lck^pr-Bcl-xL^TgHvem^ΔIEC (n=5) mice. Statistical analysis was performed using an unpaired t-test (A, D) or 2 way ANOVA with Bonferroni’s multiple comparison test (E). Statistical significance is indicated by *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data in A and C show means ± SEM. In A, D and E, each symbol represents a measurement from a single mouse. Data are representative results of one of at least two independent experiments with at least four mice in each experimental group. (F) Sorted TCRαβ⁺CD8αα⁺ IET from SI of Hvem^fl/fl and Hvem^ΔIEC mice were analyzed by RNA-seq. The top 50 most differentially expressed genes with respect to P-value. (G) Volcano plots showing mean log₂-transformed fold change (x axis) and significance (−log₁₀ (adjusted P value)) of differentially expressed genes between the TCRαβ⁺CD8αα⁺ IET from the SI of Hvem^fl/fl and Hvem^ΔIEC mice. In F and G, genes in blue font or blue symbol are associated with cell survival or proliferation and those designated with green are associated with cell migration. Groups of co-housed littermates (A-D) or cohoused mice (E) were analyzed.