By marking specific cell types, DNA methylation does not only identify stable cell phenotypes, but may also help in discovering and characterizing intermediate cell transitions, some of which may represent a pathological response. For example, plastic T cells exhibiting an intermittent phenotype between Th1/Th17 cells have been identified in chronic inflammatory conditions. However, a method that reliably and readily profiles DNA base modifications in immune cells is still not available. Simultaneous detection of cytosine modifications (including 5mC and 5hmC) has been made recently available by the next generation of nanopore long-read sequencers, that are able to provide such information in the native DNA context. Here, we present a new method to profile immune cell identity using targeted nanopore sequencing, and provide clear evidence for the use of 5mC and 5hmC as markers for cellular identity.
Using this novel approach, we identify important genes for understanding immune cell identity of CD4+T cell subsets. By profiling 5mC and 5hmC at single molecule resolution, we show that specific epigenetic footprints are able to distinguish some of the major groups of in vitro polarized T cells (Th0, Th1, Th2, Tregs, Th17s). Moreover, the intermediate Th1/Th17 phenotype was also identified, suggesting that this method may have clinical applicability. Mapping epigenetic landscapes is important to improve our understanding of T cell plasticity, an inherent characteristic of T cell responsiveness that provides advantages to the host for protective immunity and immune control. Moreover, clear maps of DNA methylation in T cell subsets is imperative to aid in the development of novel biomarkers based on these highly plastic cells.