Helios enhances the preferential differentiation of human fetal CD4+ naïve T cells into regulatory T cells [RNA-Seq - induced Treg cells].

RNA sequencing was used to compare the transcriptome of in vitro differentiated adult and fetal induced Treg (iTreg) cells that were culture with IL-2 alone or supplemented with additional TGFb. Additionally, fetal iTreg cells also had Helios knocked out by CRISPR-Cas9 editing, with paired samples treated with the Helios guide RNA1 (HeliosKO) or with the non-targeting control (HeliosWT). Differential gene expression was assessed using generalised linear model designs. This revealed that fetal iTreg cells retain a expression of Treg-specific signature previously identified to be unregulated in primary ex vivo fetal naive T cells, and this signature was not expressed by adult iTreg cells. Helios expression then regulates the Treg-specific transcriptome within fetal iTreg cells by enhancing upregulation of genes associated with Treg differentiation and function, and repressing genes associated with effector specification and pro-inflammatory function. RNA sequencing for 4 biological replicates of ex vivo sorted CD4+CD25-CD45RA+CD27+ adult peripheral blood and fetal splenic naïve T cells differentiated in vitro for 6 days with IL-2 alone or in the presence of added TGFb. 6 biological replicates across two sequencing runs (3 biological replicates each) of ex vivo sorted CD4+CD25-CD45RA+CD27+ fetal naive T cells subjected to CRISPR-Cas9 mediated editing with the Helios gRNA1 (HeliosKO) or non-targeting control (HeliosWT). Cells were differentiated after resting for 6 days in the presence of IL-2 alone or with added TGFb.