Figure 7. Ablation of Helios in fetal iT_reg results in downregulation of T_reg-specific genes, and the concurrent upregulation of pro-inflammatory genes.

CRISPR-Cas9 editing was carried out in fetal naïve T cells (n=6) with Helios gRNA1 (Helios^KO) or the non-targeting control guide (Helios^WT). Edited cells stimulated with αCD3/αCD28/αCD2 tetramers in the absence or presence of TGF-β for 6 days, after which changes in their overall transcriptome was assessed by RNAseq.

(A) Schematic showing hypothesized expression levels (dotted lines) of a T_reg-specific gene that is upregulated with IL-2 in the absence or presence of TGF-β signaling, with proposed changes in transcription level given one of the three proposed scenarios of transcriptional control by Helios and TGF-β. Gene expression that is driven by Helios is shown, with the arrows denoting the corresponding decrease in gene expression occurring with Helios knockout.

(B, C) Scatterplots show log 2 fold change (log₂FC) values comparing the absence or presence of exogenous TGFβ during the differentiation process (y-axis) and Helios^KO against Helios^WT iTreg (x-axis) that underwent differentiation in IL-2 alone (B) or with exogenous TGF-β added (C). Dotted lines in grey denote log₂FC cut-offs. Only genes upregulated (dark purple) or downregulated (dark orange) in Helios^KO relative to Helios^WT iT_reg cells are colored and shown (fold change, FC>1.1, false discovery rate, FDR<0.05). Genes associated with T_reg or pro-inflammatory immune functions are outlined and labeled.

(D, E) Same scatterplots as in (B,C) for IL-2 only iT_reg cells (D) and IL-2 + TGF-β-iT_reg (E) now colored to only show shared genes meeting the same cutoffs that were upregulated (light purple) or downregulated (light orange) in Helios^KO relative to Helios^WT iT_reg cells. Genes associated with T_reg or pro-inflammatory immune functions within the shared Helios-regulated transcriptome are outlined and labeled.