Transcriptional control of invariant NKT cell development

Abstract

Invariant natural killer T (iNKT) cells comprise a rare lymphocyte sublineage with phenotypic and functional properties similar to T and NK cells. Akin to conventional αβ T cells, their development occurs primarily in the thymus, where they originate from CD4(+) CD8(+) double positive (DP) progenitors. However, the selection of iNKT cells is unique in that it is mediated by homotypic interactions of DP cells and recognition of glycolipid antigen-CD1d complexes. Additionally, iNKT cells acquire an activated innate-like phenotype during development that allows them to release cytokines rapidly following antigen exposure. Given their hybrid features, it is not surprising that the developmental program of iNKT cells partially overlaps with that of T and NK cells. Several recent reports have provided new and exciting insights into the developmental mechanisms that direct natural killer T (NKT) cell lineage commitment and maturation. In this review, we provide a discussion of the NKT cell developmental program with an emphasis on the signaling mechanisms and transcription factors that influence the ontogeny of this lineage. Continued investigations into the complex interplay of these transcription factors and their relationship with other extracellular and intracellular signaling molecules will undoubtedly provide important clues into the biology of this unusual T-cell lineage.

Fig.1. Schematic view of iNKT cell versus conventional αβ T-cell development

NKT cell precursors diverge from conventional αβ T cells at the CD4⁺CD8⁺ double positive (DP) stage. The invariant TCR α chain (iTCR) on iNKT cell precursors recognizes glycolipid antigens presented by the CD1d molecule expressed at the surface of ‘selecting’ DP thymocytes. In contrast, CD4⁺ and CD8⁺ T cells express diverse TCRs that recognize peptide antigens presented by MHC class II or class I molecules, respectively, expressed by thymic cortical epithelial cells (TEC). Mature thymic emigrants of the conventional αβ T cell lineage have a naive phenotype, as opposed to iNKT cells, which acquire a memory/effector phenotype during thymic development. Immature NKT cells can acquire the NK1.1 marker after emigration to peripheral tissues or within the thymus. While a subset of NK1.1⁺ iNKT cells remain in the thymus as ‘long term residents’, some also migrate to the periphery.

Fig. 2. The SLAM-SAP-Fyn signaling axis during iNKT cell development

‘Selecting’ DP thymocytes and iNKT ‘precursor’ cells communicate via invariant TCR-CD1d and SLAM family (SLAM, Ly108) interactions. Engagement of the SLAM family receptors at the cell surface leads to recruitment of the adaptor molecule SAP and the Src family tyrosine kinase Fyn. The SLAMf receptor-SAP-Fyn signaling complex triggers activation of NFκB via protein kinase θ (PKCθ) and Bcl10. Additionally, SLAMf receptor-SAP-Fyn signaling also phosphorylates and recruits the SH2 domain–containing inositol phosphatase (SHIP), Dok1/2 adaptor proteins, and the Ras GTPase-activating protein (RasGAP). By binding to RasGAP, Dok1/2 inhibits Ras-MAPK activation. It is possible that SLAMf receptor-induced signals cooperate with those induced by the invariant TCR to modulate NFκB-Ras pathway activity in a manner required for iNKT cell development.

Fig. 3. Stages of NKT cell development

NKT cell development occurs in the thymus from a common precursor pool of CD4⁺CD8⁺ DP thymocytes. Thymocytes that express the canonical invariant TCR are selected following interaction with CD1d⁺ SLAMf receptor-expressing DP thymocytes and commit to the NKT cell lineage. After positive selection, immature iNKT cells undergo lineage expansion and acquire a memory phenotype and markers of the NK cell lineage. The intermediate stages of the differentiation are characterized by differences in expression of CD24, CD44 and NK1.1. Although most immature NKT cells emigrate from the thymus at Stage 2 and acquire NK1.1 expression in the periphery, a subset of Stage 2 cells acquire this marker in the thymus and remains as long-term residents.

Fig. 4. Transcriptional regulation of different stages of iNKT cell development

The developmental program of iNKT cells is controlled by the activity of several transcriptional factors, some of which regulate multiple steps of this process. The definitive stages at which some of these factors are expressed during iNKT cell development remains unclear. Based on recent data, we have placed specific factors along the iNKT cell developmental program at the earliest stage where the activity of a given transcription factor appears critical for iNKT cell development. It is likely that these factors could also be regulating the later stages of ontogeny or differentiation. As individual members of the NFκB family of transcription factors impact different stages, they are placed at multiple points in the developmental pathway.

Fig. 5. Relationship between the SLAMf receptor-SAP-Fyn signaling axis and the transcriptional activity of c-Myb and HEB

c-Myb and HEB regulate the earliest steps in NKT cell development, just prior to and perhaps following the point of positive selection. c-Myb and HEB both facilitate the prolonged survival of DP thymocytes to allow for distal Va14-Ja18 TCR rearrangements. RORγt is a direct downstream target of HEB, but not of c-Myb, and CD1d appears to be a target of c-Myb, but not HEB. c-Myb and HEB function at a similar point in iNKT cell ontogeny as the SLAMf receptor-SAP-Fyn signaling complex. Remarkably, c-Myb regulates the expression of SLAM, Ly108 and SAP while HEB might modulate the expression of Fyn and SLAM. Thus, these transcription factors likely promote iNKT cell development by inducing the expression of proteins involved in the SLAMf receptor-SAP-Fyn signaling complex. It is unknown if c-Myb and HEB are expressed simultaneously or if they regulate one another’s activity at this point in iNKT cell development.