Figure 3. RORγt⁺ IL-17A⁺ T cells in thyroid immune infiltrates of ICI-treated mice.

a, Comparison of intrathyroidal gene expression in isotype vs. Dual ICI-treated mice for Type 3 immunity (RORγt-pathway) associated genes Rorc, Il23r, Il17a, and cytokines associated with IL-17A pathway differentiation (IL6, Tgfb1, Tnf, Il1b) measured by qRT-PCR. n=12 animals/group; thyroid tissue pooled and run in 3 experiments, in triplicate. b, Comparison of thyroid-infiltrating RORγt⁺ CD3⁺ (αβ and γδ) T cells by flow cytometry in isotype (n=6) vs. Dual ICI-treated (n=8) mice. c, Representative flow cytometry plots and gating strategy and quantification of intrathyroidal IL-17A⁺ CD3⁺ T cells (d) and IL-17A⁺ T cell subsets (e) from ICI-treated mice at 4 weeks; isotype (n=12), anti-PD-1 (n=8), anti-CTLA-4 (n=8), and Dual ICI (n=7). Data are mean ±SEM shown. f, Schematic of thyroid tissue processing for scRNAseq from Dual ICI or isotype-treated mice. g, UMAP plot of thyroid-infiltrating CD45⁺ immune cells from Dual ICI-treated (n=16) or isotype-treated (n=10) mice. Cluster analysis yields 18 distinct clusters comprising CD4, CD8, and γδ T cells, natural killer (NK) cells, innate lymphoid cells type 2 (ILC2), B cells, macrophages, monocytes, dendritic cells (DC), as well as clusters with mixed cell populations clustered by markers of cell proliferation (prolif). T effector memory, TEM. T central memory, TCM. T regulatory cells, Treg. h, Split plot showing distribution of cells by condition. i, Feature plots showing Rorc⁺ and Il23r⁺ cells associated primarily with γδ T cell clusters in intrathyroidal immune cells from isotype or ICI-treated NOD mice by scRNAseq. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. two-tailed, unpaired t test with Welch correction, assuming unequal s.d. (a, b) and Holm-Sidak method correction for multiple comparisons (a); Brown-Forsythe ANOVA, assuming unequal s.d., followed by Dunnett’s multiple comparisons test (d-e).