Suppression of ILC2 differentiation from committed T cell precursors by E protein transcription factors

Current models propose that group 2 innate lymphoid cells are generated in the bone marrow. Here we demonstrate that subsets of these cells can differentiate from multipotent progenitors and committed T cell precursors in the thymus, both in vivo and in vitro. These thymic ILC2s can exit the thymus, circulate in the blood and home to peripheral tissues. Ablation of E protein transcription factors greatly promotes the innate lymphoid cell fate at the expense of B and T cell development. Consistently, a transcriptional network centered on the ZBTB16 transcription factor and IL-4 signaling pathway is highly up-regulated due to E protein deficiency. Our results show that ILC2 can still be generated from what are normally considered to be committed T cell precursors, and that this alternative cell fate is restrained by high levels of E protein activity in these cells. Overall design: RNA-seq was performed on freshly isolated ILC2 cells from the thymus, mesenteric lymph nodes and lung of different strains of mice. Cells cultured from DN1 or DN3 thymocytes on OP9-DL1 stromal cells in the presence IL-2, IL-7 and Stem cell factor were also analyzed. The genotypes of the mice where DN1 and DN3 cells were obtained are ROSA26-CreERT2;ROSA26-stop-tdTomato and ROSA26-CreERT2;ROSA26-stop-tdTomato;E2Af/f/HEBf/f. Addition of tamoxifen on Day 4 to the cultures induces deletion of the HEB and E2A gene as well as the expression of tdTomato. On Day 5 and Day7 of the culture, tdTomato positive cells were sorted and used for RNA isolation.