Transcriptional profiling of Th1, Th2, Th9, and Th17 T cell clones

We set out to explore whether "Th9" cells represent a distinct T-helper (Th) cell subset in humans by comprehensively characterizing the lineage-defining properties of human IL-9-producing Th cells. In particular, to investigate the transcription factor expression in Th9 clones as compared to Th1, Th17, and Th2 clones, we selected three to five representative T cell clones of each subset and performed gene expression analysis by using RNAseq . Peripheral Blood Mononuclear Cells (PBMC) were isolated by Ficoll-Plaque Plus (GE Healthcare, UK) density gradient centrifugation. Human CD4+ T cells were isolated from PBMC by EasySep positive selection kit (Stemcell Technologies) according to manufacturer’s instruction. Positively selected CD4+ T cells were washed with PBS and stained for subsequent Th cell subset sorting. Memory Th cell subsets were sorted to over 90% purity according to their expression of chemokine receptors from CD45RA-CD25-CD8-CD3+ cells: Th1(CXCR3+CCR8-CCR6-CCR4-), Th2 (CXCR3-CCR8-CCR6-CCR4+), Th17 (CXCR3-CCR8-CCR6+CCR4+), Th9 (CXCR3-CCR8+CCR6-CCR4+). Single cell Th subset clones were directly sorted into 96well plate according to their expression of chemokine receptors (see above). Single cell clones were expanded and maintained by periodic restimulation with PHA (phytohaemaglutinine, 1 µg/ml, Sigma-Chemicals) and irradiated allogenic feeder cells (5x104/well) in culture medium.