Transcriptional programs of in vitro-expanded HSPCs with acute disruption of Lmo2 before and after co-culture with OP9-DLL1

Though Lmo2 is a well-established member of the hematopoietic stem/progenitor program, its function in normal T cell development has remained unclear and difficult to study. Lmo2’s quick downregulation in thymic-seeding progenitor cells makes it particularly challenging to precisely perturb for mechanistic analyses; however, a recent in vitro HSPC expansion method provides a platform for probing a greater number of progenitor cells during an extended window of time through the T cell DN stages. Consequently, we performed loss-of-function experiments in expanded HSPCs and analyzed subsequent gene expression with bulk RNA-seq and single cell RNA-seq to reveal Lmo2’s previously unknown role in early T cell development. Remarkably, premature loss of Lmo2 in expanded HSPCs results in the specific upregulation of the T cell program. Bulk RNA-seq at day 5 of OP9-Dll1 co-culture reveals that the DN1 (CD44+CD25-) population derived from expanded HSPCs with the Lmo2 knockout (KO) have higher expression of T cell-related genes, such as the TCR signaling genes Lat, Itk, Lck, Zap70, Cd3e, and Cd3g, and higher transcriptional activation of the T cell receptor (TCR)-C beta locus when compared to DN1 controls. These findings are supported by single cell RNA-seq (scRNA-seq), where Lmo2-KO cells have higher expression of T cell program members like Tcf7 and higher transcriptional activation of the (TCR)-C gamma locus by the day 3 timepoint. Interestingly, before entering a Notch environment, there is minimal difference between Lmo2 KO and their control counterparts by scRNA-seq. Furthermore, the d3 and d5 datasets do not strongly indicate that Lmo2 KO causes a downregulation of the stem/progenitor program. The scRNA-seq dataset also introduces the impact of Flt3L pre-treatment for 4 days of expansion (Flt3L-priming) before seeding onto OP9-DLL1 co-cultures. Differences due to Flt3L-priming can only be seen in a subpopulation of samples before entering a Notch signaling environment; by the day 3 timepoint, cells are virtually indistinguishable by priming, suggesting that developmental acceleration caused by Flt3L-priming is distinct from Lmo2-related mechanisms. Taken together, these datasets provide gene expression profiles of control-derived and Lmo2 KO-derived DN populations to present Lmo2 as an important controller of early T cell developmental kinetics.