Decrease in butyrate-producing bacteria and alterations in metabolome correlate with skewed Tregs in partial Rag1 deficiency. (A) UMAP plot of subclustered Tregs from wild-type (+/+) and Rag1^w/w (W/W) mice at 12 wk of age. Colors represent cells clustered together on the basis of similarity of global gene expression. (B) Feature plots show single-cell gene expression levels of selected genes projected on the UMAP. (C) Treg UMAP split by genotype contribution. (D) Representative plots and quantification of RORγt⁺ Tregs in littermate +/+ (n = 5) and W/W mice (n = 7) at 12 wk of age. All flow plots were gated on live CD45⁺CD90.2⁺TCRβ⁺. (E) Scatter plot shows co-expression analysis of Rorc, Gata3, and Maf on single cells derived from the subclustered Tregs from +/+ and W/W mice at 12 wk of age. (F) TCR clonotype analysis projected on UMAP shows the distribution of different clonotype abundance ranges (single: 1 cell; small: 2–5 cells; medium: 6–20 cells; large: 21–100 cells; and hyperexpanded: 101–500 cells) of reclustered Tregs from the colonic LP of +/+ or W/W at 12 wk of age and analyzed by scRNA-seq. Single-cell samples were obtained from four to five pooled mice/group. Data are from at least two independent experiments. *P < 0.05.