Epigenetic signature of human vitamin D3 and IL-10-conditioned regulatory DCs
To define the epigenetic signature of human DCregs generated in vitamin 14 D3 (vitD3) and IL-10 compared to immune stimulatory DCs (sDCs), we measured levels of DNA methylation by whole genome bisulfite sequencing (WGBS). Distinct DNA methylation patterns were acquired by DCregs compared to sDCs. These patterns were located mainly in transcriptional regulatory regions. Genes associated with these programs were enriched in STAT3-signaling and valine catabolism in DCregs; conversely, pro-inflammatory pathways e.g., pattern recognition receptor signaling, were enriched in sDCs. Further, DCreg differentially-methylated regions (DMRs) were enriched in binding motifs specific to the immunomodulatory transcription factor KLF11, while AP1 (Fos:Jun) transcription factor binding motifs were enriched in sDC DMRs. Using publicly-available data22 sets, we defined a common epigenetic signature shared between DCregs generated in vitD3 and IL-10, dexamethasone or vitD3 alone. These insights may help pave the way for design of epigenetic-based approaches to enhance production of DCregs as effective therapeutic agents. Overall design: Leukapheresis products were obtained under an IRB-approved protocol (Institute for Transfusion Medicine, Pittsburgh, PA). Monocyte-enriched, elutriated fractions recovered from 6 normal human donor leukapheresis products were used for generation of DCregs and monophosphoryl lipid A (MPLA; TLR4 ligand low toxicity derivative of bacterial lipopolysaccharide (LPS))-matured sDC. In instances where the elutriated monocyte fraction purity was <90%, CD14-specific immunobeads (Miltenyi, San Diego, CA) were used according to the manufacturer's protocol to increase monocyte purity to >90%. After 7 days culture, monocyte-derived DCreg and sDC were harvested. The viability of DCregs and sDCs, determined by trypan blue staining, was routinely > 95%. A portion of each DC group was snap-frozen in liquid nitrogen and stored at -80°C for Whole Genome Bisulfate Sequencing (WGBS) DNA Methylation analysis. Portions of the remaining DC were either antibody-stained for key surface molecules, HLA-DR, CD11c, T cell costimulatory and coinhibitory molecules and CD163 (clone RM3/1, Biolegend) and flow cytometry data analyzed using Flow Jo software. DNA was extracted from frozen cell pellets and underwent bisulfite treatment. The bisulfite modified DNA-sequencing library was generated using the Accel-NGS Methyl-Seq DNA library kit (Swift Biosciences) per the manufacturer's instructions. WGBS data were collected from 6 sDC libraries and 6 corresponding DCreg libraries from the same donors.