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 Vα14iStopF (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 Vα14iStopF (± 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 = DP thymocytes (TCRβ− mCD1d-PBS57-Tetramer− CD4+ CD8+ CD69−); CD24+ = early wildtype iNKT cells (mCD1d-PBS57-Tetramer+ CD44− CD24+); NKT1 (mCD1d-PBS57-Tetramer+ CD44+ NK1.1+ CD27+ CD138−); NKT2 (mCD1d- PBS57-Tetramer+, NK1.1− PLZFeGFP+ ICOS+ IL17RB+ CD138−); NKT17 (mCD1d- PBS57-Tetramer+ CD19− ICOS+ CD138+). For each condition, four biological replicates were sequenced (except for 96h timepoint, where two samples of the same mouse where sequenced). For the NKT17 samples and two NKT2 samples, 2 mice were pooled. For CD24+ samples, 4-6 mice were pooled. Library preparation for bulk 3’-sequencing of poly(A)-RNA was done as described previously (Parekh et al., 2016). The library was sequenced on a NextSeq 500 (Illumina) with 16 cycles for the barcodes and UMIs and 50 cycles for the cDNA.
- Project data type: Other
- Scope:
- Monoisolate
- Klinikum rechts der Isar, II. Medizinische Klinik