Background and Objectives Invariant Natural Killer T (iNKT) cells are able to produce large amounts of cytokines within hours of activation. Disease outcome is known to be influenced depending on the type of cytokines produced by iNKT cells. Recently, three functionally and transcriptionally defined subsets of iNKT cells have been described; TBET + NKT1 cells produce IFNg and IL-4, while PLZF + NKT2 cells produce IL-4 and IL-13, and NKT17 cells are positive for RORγt and produce IL-17. However, the factors governing the development of these unique iNKT sublineages during thymic development are unknown. Here we explored the mechanism by which E protein transcription factors and their negative regulators, the Id proteins control the development of iNKT sublineages after positive selection.
Materials and Methods We generated Vα14-Jα18 transgenic (Vα14tg) mice conditionally deficient for the E proteins Tcf3 (E2A) and Tcf12 (HEB). Id2-YFP and Id3-GFP reporter mice were used to analyse expression of Id proteins. Functional relevance of these findings was confirmed in Id2 and Id3 knock-out mice. iNKT cells were analysed using flow cytometry, real-time PCR and chromatin immunoprecipitation.
Results We found that E proteins directly bound the PLZF promoter and were required for expression of this lineage-defining transcription factor and for the maturation and expansion of thymic iNKT cells. Moreover, expression of the negative regulators of E proteins, Id2 and Id3, defined distinct iNKT cell sublineages. Id3 was expressed in PLZFhigh NKT2 cells and loss of Id3 allowed for increased thymic iNKT cell expansion and abundance of the PLZF+ NKT2 sublineage. Id2 was expressed in TBET+ NKT1 cells and both Id proteins were required for the formation of this sublineage.
Conclusions We provide insight into E and Id protein regulation of iNKT cell proliferation and differentiation to specific sublineages during development in the thymus.