Extended Data Figure 2. Acquisition of cytotoxic properties by intraepithelial lymphocytes requires IL-15 expression in both the lamina propria and epithelium.

(A-J) DQ8-D^d-IL-15tg, DQ8-villin-IL-15tg, and DQ8-D^d-villin-IL-15tg mice were maintained on a GFD (sham) or fed with gluten for 30 days (gluten).

(A-I) The intestinal epithelium was isolated and analyzed by flow cytometry. A subset of IELs was identified as TCRβ⁺ CD4⁻ CD8αβ⁺ cells. In parallel, total IELs were identified as TCRβ⁺ CD4⁻ CD8⁺ cells by flow cytometry and quantified among IECs on H&E stained ileum sections.

(A) Percentage of NKG2D⁺ NKG2⁻ CD8αβ⁺ IELs are indicated. Six independent experiments (DQ8-D^d-IL-15tg sham, n=11, gluten n=14; DQ8-villin-IL-15tg sham, n=20, gluten n=20; DQ8-D^d-villin-IL-15tg sham, n=17, gluten n=22); mean; ANOVA/ Tukey’s multiple comparison.

(B) Numbers of NKG2D⁺ NKG2⁻ CD8⁺ IELs / 100 IECs. Six independent experiments (DQ8-D^d-IL-15tg sham, n=11, gluten n=13; DQ8-villin-IL-15tg sham, n=20, gluten n=20; DQ8-D^d-villin-IL-15tg sham, n=16, gluten n=19); mean; ANOVA / Tukey’s multiple comparison.

(C) Percentage of CD94⁺ NKG2A⁻ CD8αβ⁺ IELs are indicated. Three independent experiments (DQ8-D^d-IL-15tg sham, n=10, gluten n=10; DQ8-villin-IL-15tg sham, n=11, gluten n=11; DQ8-D^d-villin-IL-15tg sham, n=9, gluten n=10); mean; ANOVA / Tukey’s multiple comparison.

(D) Numbers of CD94⁺NKG2A⁻ CD8⁺ IELs / 100 IECs. Three independent experiments (DQ8-D^d-IL-15tg sham, n=10, gluten n=10; DQ8-villin-IL-15tg sham, n=11, gluten n=11; DQ8-D^d-villin-IL-15tg sham, n=9, gluten n=10); mean; ANOVA / Tukey’s multiple comparison.

(E) Intracellular granzyme B⁺ IELs are indicated by percentage. Data are representative of five independent experiments shown as mean (DQ8-D^d-IL-15tg sham, n=9, gluten n=10; DQ8-villin-IL-15tg sham, n=14, gluten n=13; DQ8-D^d-villin-IL-15tg sham, n=12, gluten n=18). ANOVA / Tukey’s multiple comparison.

(F) Numbers of granzyme B⁺ IELs / 100 IECs. Five independent experiments (DQ8-D^d-IL-15tg sham, n=11, gluten n=13; DQ8-villin-IL-15tg sham, n=20, gluten n=20; DQ8-D^d-villin-IL-15tg sham, n=16, gluten n=19); mean; ANOVA / Tukey’s multiple comparison.

(G) Intracellular granzyme B mean fluorescence intensity (MFI) was measured. Four independent experiments (DQ8-D^d-IL-15tg sham, n=9, gluten n=9; DQ8-villin-IL-15tg sham, n=14, gluten n=13; DQ8-D^d-villin-IL-15tg sham, n=10, gluten n=15); mean; ANOVA / Tukey’s multiple comparison.

(H) The percentages of CD8αβ⁺ CD107a⁺ IELs are indicated.

(I) Numbers of CD8⁺ CD107a⁺ IELs / 100 IECs are shown. Two independent experiments (DQ8-D^d-IL-15tg sham, n=7, gluten n=6; DQ8-D^d-villin-IL-15tg sham, n=8, gluten n=5; DQ8-D^d-villin-IL-15tg sham, n=5, gluten n=4). ANOVA / Tukey’s multiple comparison.

(J) Expression of Prf1 in the intestinal epithelium was measured by qPCR. Relative expression levels in sham and gluten-fed mice for each strain are shown (DQ8-D^d-IL-15tg sham, n=16, gluten n=16; DQ8-villin-IL-15tg sham, n=15, gluten n=15; DQ8-D^d-villin-IL-15tg sham, n=20, gluten n=26). Unpaired, two-tailed, t-test.