scRNAseq defines 2 transcriptionally discrete CD4⁺ GM-CSF⁺ T-cell populations in the colon. Lethally irradiated Balb/c mice were transplanted with B6 bone marrow (5 × 10⁶) plus spleen cells (adjusted to yield an αβ T-cell dose of 8 × 10⁵). (A) Uniform manifold approximation and projection (UMAP) dimensional reduction of scRNAseq data of flow-sorted, donor-derived T cells from the colons of transplant recipients 21 days posttransplantation. Unsupervised clustering using Seurat revealed 7 transcriptionally distinct clusters using a resolution of 0.4. (B) Heat map depicting z-scored expression of the top 10 overexpressed genes in CD4⁺ T-cell clusters (clusters 2-5) relative to all other cells. (C-D) Violin plots showing log-normalized expression of the indicated proinflammatory cytokines (C) and CD4⁺ T-cell lineage-defining transcription factors (D). (E-F) Representative flow cytometry dot plots and summary data depicting T-bet (E) and Gata3 (F) expression on donor CD4⁺ GM-CSF⁺ IFN-γ⁺ or CD4⁺ GM-CSF⁺ IFN-γ^– T cells in the colon. (G) Violin plots showing log-normalized expression of the indicated Th2-associated genes in CD4⁺ T-cell clusters. Data are from 2 to 3 experiments. In UMAP and violin plots, each dot represents a single cell. ****P < .0001. Log. Norm. Expr., log-normalized expression, SSC-A, side scatter area.