TNF-alpha regulates human plasmacytoid dendritic cells by suppressing IFN-alpha production and enhancing T cell activation

pDCs from healthy donors (n = 3) were purified by negative selection using the Diamond Plasmacytoid Dendritic Cell Isolation Kit II. pDCs were further sorted using an antibody to BDCA-4 . The pre-enriched pDCs (purity >85%) from each donor were divided into two aliquots before they were cultured in the presence or absence of human recombinant TNF-alpha (10 uL/mL) for 18 hours. After incubation, untreated and TNF-treated pre-enriched pDCs from all three donors (n = 6) were washed and sorted) based on CD304 (BDCA-4) expression to achieve purity > 99%. RNA libraries were made by using SMART-Seq V4 ultra low Input RNA Kit and sequenced on a HiSeq 3000 150 bp paired end lane.