Effect of BCL11B knockdown on transcriptome of human T-cell precursors

To investigate the role of BCL11B in the initial stages of human thymopoiesis, we performed loss of function (knockdown) studies in an in vitro human thymopoiesis model (cord blood CD34+ cells co-cultured on OP9DLL1 stromal cell line). Gene expression profiling by RNA-Seq demonstrated that BCL11B knockdown resulted in downregulation of T-lineage genes and upregulation of stem cell, myeloid and NK genes, indicating BCL11B is required for the establishment of a T-lineage commitment transcriptional program. Cord blood CD34+ cells were transduced with a BCL11B knockdown or scrambled control shRNA lentivral vector containing a GFP reporter. GFP+ cells were isolated by fluorescence activation cell sorting (FACS) at 48 hours post transduction and cultured on OP9DLL1 stroma in the presence of FLT3 ligand (5 ng/ ml) and IL-7 (5 ng/ ml). On day 14 of co-culture, CD45+GFP+CD7+CD1a- and CD45+GFP+CD7+CD1a+ T-cell precursors were isolated from control and knockdown co-cultures by FACS and analyzed by RNA-Seq to determine the effect of BCL11B knockdown on the transcriptome of T-cell precursors. N=3 experiments, each done with a different pool of cord blood donors.