Global epigenetic stability of liver-resident invariant natural killer T cells in response to cues capable of inducing transcriptional re-programming

Unlike conventional CD4+ T cells, which are phenotypically and functionally plastic, invariant NKT (iNKT) cells generally exist in a terminally differentiated state. Although peripheral CD4+ T cells can acquire alternative epigenetic states in response to different cues, it remains unclear whether peripheral iNKT cells are epigenetically stable or maleable. Repetitive encounters of liver-resident iNKT cells (LiNKTs) with galactosylceramide (aGalCer)/CD1d-coated nanoparticles (NPs) can trigger their differentiation into a LiNKT cell subset (LiNKTR1) expressing a T regulatory type 1 (TR1)-like transcriptional signature. Here we use a broad range of omics tools to dissect the epigenetic underpinnings of the LiNKT-LiNKTR1 transdifferentiation process as compared to those underlying the T-conv-to-T-follicular helper (TFH) and TFH-to-TR1 differentiation pathways. We show that LiNKTR1 cell generation is partially associated with treatment induced hypo-methylation of gene bodies and regulatory elements of upregulated genes, in the absence of significant changes in chromatin exposure, deposition of expression-promoting histone marks, or removal of their repressive counterparts from bivalent regulatory elements. In contrast, the Tconv-TFH differentiation process involves extensive remodeling of the chromatin and the acquisition of a broad repertoire of epigenetic modifications that are then inherited by TFH cell-derived TR1 cell progeny. These results suggest that peripheral tissue-resident iNKT cells, unlike conventional T cells but like TFH cells, are epigenetically stable and that such stability contributes to their phenotypic and functional stability. Overall design: Liver-resident aGalCer/CD1d tetramer+ cells isolated from both aGalCer/CD1d-NP-treated and untreated NOD mice were processed for bulk ATAC-seq, ChIP-seq for H3K27Ac, H3K4me3, and H327me3, whole genome methylome, as well as single-cell scRNA-seq + scATAC-seq multiome, in order to underpin the epigenetic changes during their transdifferenation into LiNKTR1 induced by aGalcer/CD1d-NP .