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Thymic iNKT cell differentiation at single-cell resolution

Cellular & Molecular Immunology volume 18, pages 2065–2066 (2021)Cite this article

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Recently, Baranek et al. performed single-cell RNA sequencing (scRNA-seq) analysis and confirmed some of the transcriptomic results by in vitro cell culture with IL-7/IL-15 and flow cytometric analysis.2 The authors defined several intermediate stages, including iNKT2a (CD24−NK1.1−CD138−FR4−), iNKT2b (CD24−NK1.1−CD138−FR4+), and iNKT1a (CD24−Sca-1hiCD138-NK1.1dim), with various degrees of potential to differentiate into iNKT1, iNKT2, and iNKT17 subsets (Fig. 1). The transcription cofactor FHL2 was further found to be required for iNKT1 lineage commitment, and mice lacking Fhl2 showed total loss of the iNKT1a subpopulation and a slight increase in the iNKT2b subpopulation.2 In addition, iNKT2a, iNKT17 (CD24−Sca-1hiCD138+), and iNKT1c (CD24−Sca-1−CD138−NK1.1hi) subpopulations were shown to express high levels of thymic egress-related transcripts, including Klf2 and its regulated genes, and low levels of retention-related transcripts, including Cd69 and Cxcr3. These subsets were specifically enriched when thymic egress was blocked with FTY720. Interestingly, the iNKT1c subpopulation with egress potential expressed high levels of NK1.1 and cytotoxicity-related genes, indicating that previously defined stage 3 (CD44hiNK1.1hi) thymus-resident iNKT cells are heterogeneous, with a subpopulation capable of leaving the thymus. While it remains to be further clarified whether these cells are generated de novo or iNKT cells returning to the thymus from the circulation, in-depth analysis of thymic iNKT cells at the single-cell level2,3,4 opens the door to research on the spatiotemporal regulation of iNKT differentiation with more accurately defined precursor stages.

As recent progress in iNKT development is still largely based on the more simplified four-stage model defined by CD24, CD44, and NK1.1 (stage 0, 1, 2, and 3, with the phenotypes CD24+CD44lo, CD24−CD44lo, CD44hiNK1.1−, and CD44hiNK1.1+, respectively), integration of these results with scRNA-seq data and reexamination of some of the regulators at newly defined precursor stages are clearly warranted. For instance, a detailed study of Foxn1CreLTβRfl/fl mice showed that thymic epithelial cells in the medulla, including CCL21+CD104+mTEClow cells with their transpresentation of IL-15 (for iNKT1 and iNKT17 cells) and tuft cells with their secretion of IL-25 (for iNKT2 cells), provide important support for the survival/proliferation, master transcription factor expression, and functional maturation of iNKT subsets at stages 2 and 3.5 Mice with T cell-specific deficiency of PDCD5 showed reduced expression of T-bet and selective abolition of iNKT1 differentiation at stage 1,6 while T cell-specific deletion of the protein arginine methyltransferase PRMT5 revealed decreased levels of common γc/JAK3 and blockade of the differentiation of all iNKT subsets at stage 1.7 YY1 interacts with the master regulator PLZF to drive functional maturation processes, such as IL-4 and IFN-γ expression, in iNKT cells. The few remaining iNKT cells in PLZFCreYY1fl/fl mice showed failure of NK1.1 expression upregulation and cytokine production.8

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Fig. 1

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Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2017YFA0104500), the National Natural Science Foundation of China (31671244, 31872734, Q.G.), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81621001), the Beijing Natural Science Foundation (7202079), and the Non-Profit Central Research Institute Fund of the Chinese Academy of Medical Sciences (2018PT31039).

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  1. Department of Immunology, School of Basic Medical Sciences, Peking University, NHC Key Laboratory of Medical Immunology (Peking University), Beijing, China

    Ke Wang, Weijia Zhao, Rong Jin & Qing Ge

  2. Department of Integration of Chinese and Western Medicine, School of Basic Medical Sciences, Peking University, Beijing, China

    Qing Ge

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Correspondence to Qing Ge.

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Wang, K., Zhao, W., Jin, R. et al. Thymic iNKT cell differentiation at single-cell resolution. Cell Mol Immunol 18, 2065–2066 (2021). https://doi.org/10.1038/s41423-021-00697-6

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