A comparison of the transcriptional profiles of DP CD24highCD44lowNK1.1-CD1d-tet+TCRa+ cells (i.e. very immature NKT cells) with those of more mature NKT cell subsets

Type 1 NKT cells play a critical role in controlling the strength and character of adaptive and innate immune responses. Their functional characteristics are distinct from conventional T cells, and are induced by a transcriptional program initiated by positive selection on CD4+CD8+ (double positive, DP) thymocytes. Here we examined transcriptional events in four immature thymic NKT cell subsets in a novel Vα14 TCR transgenic strain bearing greatly expanded numbers of CD24+CD44- NKT cells. We used a transcriptional regulatory network approach to map TCR validation to the transition from DP T to DP NKT cells, and positive selection and lineage commitment to the transition from DP NKT to CD4 NKT Thymocytes from NOD.Va14tg mice, a strain with greatly increased numbers of NKT cells with the characteristics of pre-selection and Stage 0 NKT cells, were subjected to FACS sorting to isolate four subsets: DPhighCD24highNK1.1-CD1d-tet-TCRa+ cells (immature DP conventional T cells), DP CD24highCD44lowNK1.1-CD1d-tet+TCRa+ cells (DP NKT cells), CD4+CD8-CD24high NK1.1-CD1d-tet+TCRa+ cells (CD4 NKT cells) and CD4-CD8-CD24 highNK1.1-CD1d-tet+TCRa+ cells (DN NKT cells). RNA was isolated and Affymetrix Mouse Gene_1.0ST arrays utilised to compare gene expression levels of the sorted T and NKT cell subsets (7 samples/group) in order to allow us to dissect the transcriptional programs involved in NKT cell positive selection and lineage commitment.