To systematically investigate the earliest phases of iNKT cell development and subsequent subset differentiation, we generated a genetic system to induce a timed wave of iNKT cell generation upon 4-hydroxytamoxifen (4-OHT) administration in mice bearing CD4-CreERt2 (Sledzinska et al., 2013) and Va14iStopF (Vahl et al., 2013) knock-in transgenes. We globally investigated the transcriptional changes that guide early iNKT cell development by bulk 3'-sequencing of poly(A)-RNA of induced developing iNKT cells between 12h and 5d after induction in comparison to DP thymocytes, stage 0 CD24+ iNKT cells as well as NKT1, NKT2 and NKT17 mature subsets. Briefly, CD4-CreERt2 Va14iStopF (± Traj18KO) mice were administered with 4-OHT and sacrificed at different timepoints after administration (between 12h and 120h). 1000 cells of the following samples were FACS purified for RNA-seq analyses: 9 timepoints of induced iNKT cells (from 12h to 120h) (gated as mCD1d-PBS57- Tetramer+ CD44low NK1.1-); DP

Innate-like T cell populations expressing conserved TCRs play critical roles in immunity through diverse effector functions acquired as intrinsic part of their development. However, many aspects of this process remain unclear and controversial. Here, we present a differentiation roadmap obtained by the temporal analysis of a genetically induced developmental wave of NKT cells, a prototypical innate-like T lineage. We define the precise timing of positive selection, lineage commitment, acquisition of cytokine secretion potential, proliferation and thymic egress. We find that a short period of homogenous TCR signaling triggers highly synchronous and uniform NKT cell development, strongly arguing against widely favored models of TCR-instructed effector subset diversification. These effector subsets emerge simultaneously from highly proliferating progenitors but follow dramatically different fates. Our results indicate that differences in NKT cell generation rates can influence steady state effector subset composition, offering an alternative interpretation of experimental results involving genetically altered NKT cell differentiation.