Lipid antigen stimulation of iNKT cells triggers PC degranulation in an IFN-γ–dependent manner. (A) Experimental strategy. Cell culture SNs were collected from activated iNKT cells and transferred to the culture medium of small intestinal organoids. iNKT activation in the presence of the lipid antigen α-GC and HeLa-CD1D antigen presentation. SNs were preincubated with neutralizing anti–IFN-γ antibody or control IgG. (B) ELISA quantification of IFN-γ levels in SNs of control iNKT cells and after α-GC stimulation (both in presence of HeLa-CD1D cells). Means of quadruplicate measurement is shown (±SD; ***, P < 10−4; Student’s t test). (C) Small intestinal organoids were cultured for 2 d in presence of iNKT SNs, followed by UEA-1 lectin staining; blue arrowheads label unaffected PCs and blue arrows show discharged PCs. Bars, 100 µm. (D) Western blot analysis of epithelial lysozyme content; conditions as in C. Organoid lysates from three parallel cultures; α-tubulin was probed for normalization. (E and F) Quantification of lysozyme expression from Western blot data shown in D by densitometric analysis. Mean normalized signals ± SD are shown; *, P < 0.05; ***, P < 0.001; Student’s t test. Data (B–F) are from one representative experiment of two independent experiments.
