Fig. 2: The follicular to GC-like developmental transition in Tfr cells is sensitive to costimulatory blockade.

a Diagram of single-cell RNA sequencing experiment. Balb/c kidneys were transplanted into Foxp3^IRES-GFP mice with or without CTLA4Ig treatment. Tfr (CD4⁺CXCR5⁺FoxP3⁺) and Treg (CD4⁺CXCR5^-FoxP3⁺) cells from dLN were sorted and scRNAseq performed. b UMAP plot showing unsupervised clustering of all post-filter cells. The total number of cells analyzed is shown. c Cells in UMAP plot marked by cell type. d Feature plots showing the expression levels of indicated genes of interest across clusters. e Clusters annotated based on gene expression states, including Naïve-like, Follicular-like, and GC-like. f Monocle pseudotime analysis utilizing cluster 0 as the starting node and indicating developmental trajectories by lines and overall pseudotime by color, plotted within UMAP space. g. UMAP plot showing clustering (top) and cell type (bottom) for cells in no treatment or CTLA4Ig treated mice. Number of cells per group is indicated. h Cluster distribution changes analyzed with miloR package. Red dots indicate a reduced cell population while blue dots indicate an increased cell population with the CTLA4Ig treatment. i Heatmap showing top marker genes for each cluster compared to all clusters. j Volcano plot showing differentially expressed genes (DEGs) between clusters 3 and 4. DEGs were identified using a two-sided Wilcoxon rank-sum test, as implemented by the Seurat FindMarkers function (logFC threshold=0.1, min.pct=0.1). p-values were adjusted for multiple comparisons using the Bonferroni method. k Venn diagram showing differentially expressed genes between Tfr and Treg cells in control or CTLA4Ig treated group. Portions created in BioRender. Zhang, H. (2025) https://BioRender.com/b29q775.