Figure 5.

The supernatant from CD8αβ⁺ IELs can inhibit the formation of bacterial colonies directly. (A,B) CD8αβ⁺ IELs and CD8αβ⁺ SPLs were sorted and cultured for 24 h, with activation by PMA and ionomycin during the final 6 h. After 24 h, the supernatants were collected for an antimicrobial-activity assay. Iscove’s modified Dulbecco’s medium (IMDM) with streptomycin (0.1 mg/mL) was added to the middle Oxford cup as a positive control (labeled as “Str,” middle). Supernatants from CD8αβ⁺ SPLs (labeled as “SPLs,” left) and CD8αβ⁺ IELs (labeled as “IELs,” right) were added into Oxford cups, respectively, on the left and right side of the agar plate (A). Inhibition rings were observed after 18 h. The diameter of the inhibition rings was measured and is shown in (B). One-way ANOVA followed by Dunnett’s test. Each cell sample was sorted from 5 wild-type mice. Data are representative of three independent experiments; error bars represent the mean ± SEM. ****P < 0.0001. (C) IELs (1 × 10⁶ cells) and increasing concentrations of Escherichia coli (CFU/mL), indicated in the Figure, were co-cultured with or without IL-15. The color of the medium indicated the acidity produced by bacterial proliferation. Data are representative of three independent experiments. (D,E) CD8αβ⁺ IELs were sorted and cultured with or without the IL-15/IL-15R complex for 24 h, after which the supernatants were collected for an antimicrobial-activity assay. IMDM with streptomycin (0.1 mg/mL) was added to the middle Oxford cup as a positive control (labeled as “Str,” middle). Oxford cups containing supernatants from CD8αβ⁺ IELs activated without (labeled as “Ctrl,” left) or with (labeled as “IL-15,” right) the IL-15/IL-15R complex were located, respectively, on the left and right side of the agar plate (D). Inhibition rings were observed after 18 h. The diameter of the inhibition rings was measured and is shown in (E). Unpaired t-test. Each cell sample was sorted from 5 wild-type mice. Data are representative of two independent experiments. Error bars represent the mean ± SEM. *P < 0.05. Abbreviations: ns, not significantly different; IEL, intraepithelial lymphocyte; SPL, splenocyte; Str, streptomycin.