Extended Data Fig. 4: A GFD decreases the anti-gluten antibody response and the number of cytotoxic IELs.

a–h, k, l, DQ8-D^d-villin-IL-15tg mice raised on a GFD were maintained on a GFD, fed gluten for 30 days, or fed gluten for 30 days and then reverted to a GFD for 30 days. a, Left, CD3ε⁺ T cells (red) and CD138⁺ plasma cells (green) were distinguished by immunohistochemistry staining of frozen ileum sections. Scale bars, 50 μm. Right, the number of CD138⁺ cells per section, normalized to lamina propria area (sham, n = 8 mice; gluten, n = 8 mice; gluten→GFD, n = 9 mice). b, c, Serum levels of anti-gliadin IgG (b) and anti-gliadin IgA (c) were measured by ELISA. Serum was collected sequentially in the same mice before gluten feeding (untreated), 30 days after gluten feeding (gluten d30), and 30 days after reversion to a GFD (GFD d60) (n = 12 or 13 mice per group for anti-gliadin IgG or IgA, respectively). d, Mucosal IgA deposits (red) and TG2 (green) were identified by immunohistochemistry staining of frozen ileum sections. Scale bars, 100 μm. e, Serum levels of anti-TG2 IgG antibody measured by ELISA 30 days after gluten feeding (n = 13 mice per group). f, Quantification of IELs among IECs was performed on H&E-stained ileum sections (sham, n = 8 mice; gluten, n = 12 mice; gluten→GFD, n = 9 mice). g, Left, granzyme B staining by immunohistochemistry on paraffin-embedded ileum sections. Scale bars, 20 μm. Right, number of granzyme B⁺ IELs per 100 IECs per mouse (sham, n = 6 mice; gluten, n = 10 mice; gluten→GFD, n = 9 mice). h, Expression of Gzmb in the intestinal epithelium was measured by qPCR. Relative expression levels in gluten and gluten→GFD groups were normalized against the expression levels observed in sham-fed DQ8-D^d-villin-IL-15tg mice (n = 6 mice per group). i, j, DQ8-D^d-villin-IL-15tg mice raised on a GFD were maintained on a GFD, fed gluten for 30 days, or fed gluten for 30 days and then reverted to a GFD for 60 or 90 days. The intestinal epithelium was isolated and analysed by flow cytometry, and IELs were identified as TCRβ⁺CD4⁻CD8αβ⁺ cells. Granzyme B⁺ IELs are indicated by percentage (i) and MFI (j) (sham, n = 6 mice; gluten, n = 8 mice; gluten→GFD, n = 8 mice). k, Expression of Prf1 in the intestinal epithelium was measured by qPCR. Analysis was performed as in h (gluten, n = 6 mice; gluten→GFD, n = 7 mice). l, The intestinal epithelium was isolated and analysed by flow cytometry. IELs were identified as TCRβ⁺CD4⁻CD8⁺ cells. In parallel, IELs were quantified among IECs on H&E-stained ileum sections. NKG2D⁺NKG2⁻ IELs are indicated by absolute number per 100 IECs (sham, n = 8 mice; gluten, n = 11 mice; gluten→GFD, n = 9 mice). Data are mean ± s.e.m. (b, c, g–l) or mean values (a, e, f) from three (a, d, g), four (b, c, e, f) or two (h–l) independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test (a–c, f, g, i, j, l) or unpaired, two-tailed, t-test (e, h, k).

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