Compositional and transcriptional changes in NK cells of Aire-deficient rats. (A) A graph of miloR NK cell neighborhoods superimposed on the UMAP embedding of the data. Color represents log fold change (LFC) estimated with differential neighborhood abundance testing (FDR < 0.05), node size represents the size of the neighborhood, and edge width represents the amount of overlap between neighboring neighborhoods. Empty circles in white represent neighborhoods with statistical significance above 0.05. (B) UMAP embedding of NK cells showing a subpopulation of activated NK cells in orange. (C) Dot plot showing the expression of activated NK cell marker genes. (D) Heatmap showing expression of top 30 marker genes for the two NK subpopulations. Left annotation shows diminished numbers of activated NK cells in Aire-deficient rats. (E) Representative flow cytometry plots of splenic NK cells (above) and activated splenic NK cells from >7-mo-old Aire-deficient (KO) and control (HE) rats. (F) Mean values of splenic NK cells (above) and activated splenic NK cells (below) from 1–2-mo-old (young) and >7-mo-old (old) Aire-deficient (KO) and control (HE) rats. Data in A–D are obtained from a single experiment with three Aire-deficient (KO) and three control (HE) rats per group. In F symbols indicate individual animals and horizontal lines with whiskers indicate mean value with SEM (n = 5–8, combined from two independent experiments). P values are based on t tests. P values are adjusted for multiple comparisons using the Holm–Bonferroni method.