Fetal naive T cells are primed for preferential regulatory T cell differentiation through increased chromatin accessibility and expression at the Helios locus.

Abstract Human fetal CD4+ naïve T cells are primed to differentiate into CD25hiFOXP3hi regulatory T (Treg) cells upon T cell receptor (TCR) stimulation without TGFβ supplementation, thus contributing to the generation of immunotolerance in the developing human fetus. In addition to expression of the lineage-determining factor FOXP3, commitment to the Treg cell fate is preceded by the acquisition of permissive epigenetic modifications at Treg-specific enhancers associated with the transcriptional control of Treg signature genes. Through ATACseq (Assay for Transposase-Accessible Chromatin Sequencing) and H3K27ac ChIPseq (Chromatin Immunoprecipitation Sequencing), we reveal that this intrinsic predisposition for Treg differentiation in fetal naïve T cells is correlated with increased chromatin accessibility and H3K27ac enrichment at 906 and 109 Treg-specific enhancers respectively. We show by RNA sequencing that fetal naïve T cells subsequently have increased transcription of the underlying Treg-associated genes such as Helios (IKZF2). Using flow cytometry, we confirmed that Helios expression is higher in fetal naïve T cells relative to adult naïve T cells. CRISPR (clustered regular interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) mediated knockdown of Helios expression in primary human fetal naïve T cells reduced induction of CD25hiFOXP3hi Treg cells in response to TCR stimulation alone, indicating that high baseline Helios expression contributes to the priming of Treg differentiation in fetal naïve T cells. Overall, a novel Treg-associated epigenome and transcriptome within fetal naïve T cells establishes preferential Treg differentiation as a mechanism to maintain immunotolerance in utero.