RNAseq analysis of γδ T-APCs generated in the presence of IL-2, IL-15 or IL-21, aiming to define the molecular mechanism underlying selective induction of IL-10 in naïve and memory CD4+ T-cells
RNAseq analysis of γδ T-APCs generated in the presence of IL-2, IL-15 or IL-21, aiming to define the molecular mechanism underlying selective induction of IL-10 in naïve and memory CD4+ T-cells
After three days in culture, CD3+ Vδ2+ T-cells were enriched by cell sorting using a FACSAria II (BD Biosciences), to purities >99.8%. Monocytes were freshly isolated and irradiated at 50 Gy prior to co-culture with freshly isolated autologous at a monocyte to γδ T-cell ratio of 1:10.  Cultures were treated with 10 nM HMB-PP alone, or in combination with 100 U/ml IL-2, 20 ng/ml IL-15 or 20 ng/ml IL-21.  RNA was extracted from cell pellets using the RNeasy Micro Kit (Qiagen) and examined qualitatively and quantitatively using the TapeStation 2200 system (Agilent) and a Qubit fluorometer (Thermo Fisher Scientific).  Ribosomal RNA was depleted as part of the Illumina Stranded Total RNA Prep protocol using coated magnetic beads; the remaining RNA was fragmented and denatured, and first and second strand cDNA synthesis and anchor ligation was carried out according to the manufacturer's instructions.