Genome-wide methylation analysis of murine CD4+ Th1, Th2, and Th17 cells

T helper cells play an essential role in supporting immune reactions as a part of the adaptive immune system. During activation, naive CD4+ T cells can differentiate into various types of effector T cells, each defined by the expression of lineage-specific transcription factors and secretion of specific cytokines. The three main effector/memory cells were classified as T helper 1 (Th1), 2 (Th2) or 17 (Th17) cells, expressing T-bet, Gata3 or Ror gamma t, respectively. Differentiation into a specific T helper lineage is accompanied with durable transcriptional changes partially regulated by epigenetic mechanisms like changes in DNA methylation. Comparing the methylomes of CD4+ memory T cells opens up the chance to identify accessible, functionally relevant gene loci that would have been overlooked in an RNA-seq approach because their transcription depends on inflammatory stimuli. Because studies have shown that in vitro induced T helper cells do not completely match in vivo generated cells, we sorted ex vivo Th1, Th2, and Th17 cells from spleen and lymph nodes of healthy mice, and performed a bisulfite sequencing (Bi-Seq). The pairwise comparison of the methylomes identified differential methylated regions (DMRs) in known epigenetically regulated genes like Tbx21, Ifng, Gata3, Il4, and Ccr6, as well as in loci not known to be epigenetically regulated like Ccr8, Lck or NfkB1. Overall design: Spleens and lymph nodes of numerous healthy mice were prepared and pooled. Then, Th1, Th2 and Th17 cells were sorted by flow cytometry and used for Bi-seq. Differentially methylated regions were identified by pairwise comparison of the samples with the software metilene.