Fig. 1: An epigenome-defined LT stem-like preinfusion CAR T cell subset shows BACH2 activation.
From: BACH2 dosage establishes the hierarchy of stemness and fine-tunes antitumor immunity in CAR T cells
a–i, In vitro-expanded anti-mouse CD19 CAR T cells derived from C57BL/6 mice were analyzed with scATAC-seq + scRNA-seq. a, CD8+ CAR T cells displayed in a transcriptome-based UMAP plot (RNA UMAP). Effector-like (RNA 0 Teff-like), stem-like (RNA 1 Tstem-like) and proliferating (RNA 2 and RNA 3) clusters are defined by single-cell transcriptomes and color coded accordingly. b, Heat map of the top marker genes in each transcriptome-defined cluster. Columns represent individual cells. Rows represent genes. c, Single-cell expression of Bach2, Sell, Id2 and Mki67 shown in a UMAP plot. Color reflects the mRNA level of each gene. d, CD8+ CAR T cells projected on UMAP plots based on scATAC-seq (left, ATAC UMAP) and scRNA-seq profiles (right, RNA UMAP). Cells are color coded based on cluster IDs defined by the scATAC-seq profile, including transitory stem-like (ATAC 0 Ttrans), effector-like (ATAC 1 Teff-like), LT stem-like (ATAC 2 LT-Tstem-like) and proliferating (ATAC 3) clusters. e, Heat map of epigenetically activated genes in each scATAC-seq-defined cluster. The color code is defined by scATAC-seq reads in the promoter and gene body. Clusters are represented by columns and organized based on hierarchical clustering. f, Volcano plot showing genes with differential chromatin accessibility between transitory stem-like and LT stem-like subsets. Adjusted P values (Padj) were calculated using a two-sided Wilcoxon rank-sum test with Bonferroni correction to account for multiple comparisons. g, GSEA showing increased chromatin accessibility at published LT stem-like signature genes (GSE199839) in cluster ATAC 2 compared to cluster ATAC 0; NES, normalized enrichment score. The adjusted P value was calculated using the Benjamini–Hochberg procedure to correct for multiple testing. The red dashed line in the GSEA plot indicates the location of the maximum running enrichment score (ES). h, Heat map highlighting the differentially enriched transcription factor motifs in open chromatin regions across the clusters defined by the scATAC-seq profile shown in d. Color represents the degree of motif enrichment. i, Feature plots depicting the single-cell enrichment of BACH2, LEF1, TCF1 and TBX21 (T-bet) binding motifs in the open chromatin regions of CD8+ CAR T cells. Color intensity indicates the degree of motif enrichment.
