Fig. 5. CRISPR/Cas9-RNP KO reveals that BATF regulates human activated T_regs by multiple signaling pathways.

(A and B) Human primary T_regs isolated from cord blood were CRISPR-edited and then cultured with TCR stimulation for 3 days. (A) Representative flow staining of T_regs showing BATF protein level in nontargeting scrambled control and unperturbed control T_regs. (B) The BATF expression by median fluorescence intensity (MFI) in T_regs (n = 8) is shown. Each dot indicates an individual replicate. Bars indicate the median of expression, and error bars represent 1 SD. (C to G) RNA-seq was conducted on BATF KO activated T_regs (n = 6) and scrambled control from the same donor. (C) PCA of the transcriptome of the scrambled control and BATF KO activated T_regs. Human activated T_regs were stratified by PC1. (D) Weightings of the genes that were the strongest drivers of PC1 were visualized on a bar plot. (E) Selected gene sets that were enriched in human BATF KO activated T_regs were visualized on a dot plot. Genes are differentially expressed in both TNFR⁺ T_regs from scRNA-seq and BATF KO activated T_regs in bulk RNA-seq selected as input to the gene set enrichment analysis. Dots are colored by the false discovery rate (FDR)–adjusted P value, and dot sizes are scaled by the number of significantly up-regulated genes within each gene set (<0.1% FDR). (F) The relative expression of the top 20 up/down-regulated genes differentially expressed in both TNFR⁺ T_regs and BATF KO activated T_regs were visualized on a heatmap. The gene expression is scaled by row. (G) Dot plots showing the expression levels of BCL2 and BID in BATF KO activated T_regs and scrambled controls from eight individual replicates. Data in (B) were analyzed by a ratio paired t test, and the FDR P values in (G) were calculated by Wald test. P values: **P < 0.01; ***P < 0.001, ****P < 0.0001. ns, not significant.