Transmembrane TNF-TNFR2 axis promotes generation of human T cell precursors

The goal of this study is to understand how low level of IRF8 expression promotes the differentiation of human hematopoietic stem and progenitor cells (HSPCs) into T cell precursors. To achieve this, we utilized a construct that encodes IRF8-ERT2 fusion protein where IRF8 activity is tamoxifen-dependent. We extracted total RNA from transduced HSPCs that had been co-cultured with OP9-DLL4 stromal cells and treated with or without 4-hydroxytamoxifen (4-OHT) for sequencing. This allowed us to determine the impact of low level of IRF8 activity on the transcriptome profile of differentiating HSPCs. Our analysis found significant increase of TNF expression in the presence of low level of IRF8 activity. HSPCs from 3 donors were transduced with empty vector control or IRF8-ERT2. No treatment was performend on the control cells whereas IRF8-ERT2-transduced cells were treated with 4-hydroxytamoxifen (4-OHT) or compound solvent. For all conditions, transduced HSPCs were sorted at day 4 post co-culture to obtain CD45+GFP+CD7+ cells for RNA extraction and sequencing.