scRNA-seq of LP CD4 ⁺ T cells in Rag1^w/w (W/W) and wild-type (+/+) mice. Total CD4⁺ T cells were sorted from the colonic LP of littermate +/+ or W/W at 3 and 12 wk of age and analyzed by scRNA-seq. Single-cell samples were obtained from four to five pooled mice/group. (A) The Uniform Manifold Approximation and Projection (UMAP) plot shows the expression profile of CD4⁺ T cells. Colors represent cells clustered together on the basis of similarity of global gene expression. (B) Dot plot shows percent and average expression of selected genes in each cluster. (C) Frequency and absolute count of CD4⁺FoxP3⁺ cells in the LP of +/+ or W/W at 12 wk of age (n = 6/group). Statistical analysis was performed using a Mann–Whitney test. (D and E) Selected gene ontology (GO) biological process terms enriched among gene sets differentially regulated (with >50% fold change and false discovery rate [FDR] = 0) in a cell type–specific manner between W/W and +/+ mice at 12 wk of age (D) and between W/W mice at 12 and 3 wk of age (E). The color of each circle represents the FDR value of the enriched GO term, whereas the size of each circle is proportional to the enrichment ratio, which is the number of differentially expressed genes associated with the GO term normalized by the number of such genes that one can encounter by random chance given the size of the total gene set in the GO database. Data are from at least two independent experiments. **P < 0.01.