Fig. 3. Nod2 negatively regulates hyper-pathogenic Th17 cells.

Splenocytes from immunized WT and Nod2^−/− mice were stimulated in vitro with IRBP_1–20 (a–e). a [³H] thymidine incorporation after 72 h. Data are combined from 3 independent experiments, n = 12 mice/group; ***p < 0.0001. b Gene expression of CD4⁺ T cells 18 h was assessed by qPCR. Heatmap indicates expression relative to IRBP_1–20-stimulated CD4⁺ T cells from adjuvant-control mice. c Cytokine production after 48 h was assessed by ELISA. Data are combined from 4 independent experiments, n = 16 mice/group. d Frequencies of IL-17⁺ or IFNγ⁺ of CD4⁺Thy1.2⁺ T cells was determined by flow cytometry. Data are combined from 2 independent experiments, n = 5 (unstimulated) or 6 (IRPB-stimulated) mice/group. IL-17: **p = 0.0043 (WT unstimulated vs. WT peptide, Nod2^−/− unstimulated vs. Nod2^−/− peptide^), **p = 0.0022 (WT peptide vs. Nod2^−/− peptide^). IFNγ: **p = 0.0043 (WT unstimulated vs. WT peptide), **p = 0.0025 (Nod2^−/− unstimulated vs. Nod2^−/− peptide⁾. e Proportion of Th17 cells (IL-17⁺CD4⁺Thy1.2⁺) co-producing cytokines. Data are combined from 2 independent experiments, n = 6 mice/group; **p = 0.0022. f–h Ocular cells from immunized mice were stimulated in vitro with IRBP_1–20 for 18 h. Data are combined from 3 independent experiments, n = 6 mice pooled/genotype. f Representative contour plot showing IL-17⁺CD4⁺ T cells. g IL-17⁺ cells from gated live CD4⁺Thy1.2⁺ T cells. ^**p = 0.0013 (%), ***p = 0.0001 (#). h CD4⁺Thy1.2⁺ T cells co^-expressing TNFα, IFNγ, or Foxp3. i–k The effect of cytokine neutralization on uveitis was evaluated clinically and histopathologically 21 days post-immunization. Data are combined from 3 independent experiments, n = 18 (WT IC, WT αIL-17), 19 (WT αIFNγ, Nod2^−/− IC), 17 (Nod2^−/− αIL-1⁷, or 23 (Nod2^−/− αIFNγ) mice. i Representative images of fundi and H&E-stained retinal sections 21 days post-immunization. j Uveitis scores from histopathology. *p = 0.0095 (WT IC vs. WT IL-17 Ab), ***p < 0.0001 (Nod2^−/− IC vs. Nod2^−/− IL-17 Ab, WT IC vs. Nod2^−/− IC). k Uveitis scores (from j) were normalized to the IC Ab group within each genotype (baseline = 0); **p = 0.0035, ***p < 0.0001. Data are box-whisker plots showing median with 25th–75th percentile and min–max range (a, d–e, j, k), floating bars showing median with min–max range (g, h), or mean ± SEM (dots represent individual values) (c). Data were analyzed by two-tailed Mann–Whitney U test (a, d–e, h, j) or unpaired, two-tailed Student’s t-test (c, g, k). Source data are provided as a Source Data file.