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Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function

Nature Immunology volume 16, pages 517–524 (2015)Cite this article

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Abstract

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.

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Figure 1: Expression of let-7 miRNAs in wild-type and LIN28-transgenic thymocytes.
Figure 2: Analysis of thymocytes from LIN28-transgenic mice.
Figure 3: let-7 miRNAs target Zbtb16 mRNA.
Figure 4: Effect of let-7 and PLZF on NKT thymocytes.
Figure 5: let-7 deficiency prevents the generation of IFN-γ-producing NKT cells.
Figure 6: Effect of let-7 on NKT effector cell lineages.
Figure 7: Signals that upregulate let-7 expression in developing NKT thymocytes.

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Acknowledgements

We thank N. Taylor and J.-H. Park for critical reading of the manuscript; G.Q. Daley (Harvard Medical School) for iLet7ΔLIN28 and M2rtTA double-transgenic mice; A. Bendelac (University of Chicago) for Zbtb16+/LU mice; the Tetramer Core Facility of the US National Institutes of Health for tetramer reagents; A. Adams and L. Granger for flow cytometry; and J.A. Williams (National Cancer Institute) for cDNA reagents. Supported by the Intramural Research Program of the US National Institutes of Health, the National Cancer Institute, Center for Cancer Research.

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  1. Leonid A Pobezinsky

    Present address: Present address: Veterinary and Animal Sciences Department, University of Massachusetts, Amherst, Massachusetts, USA.,

Authors and Affiliations

  1. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Leonid A Pobezinsky, Ruth Etzensperger, Susanna Jeurling, Amala Alag, Tejas Kadakia, Tom M McCaughtry, Motoko Y Kimura, Susan O Sharrow, Terry I Guinter & Alfred Singer

  2. SAIC-Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland, USA

    Lionel Feigenbaum

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Contributions

L.A.P. designed the study, performed experiments, analyzed data and contributed to the writing of the manuscript; R.E., S.J., T.K., T.M.M., M.Y.K., S.O.S. and T.I.G. performed experiments and analyzed data; A.A. and L.F. generated experimental mice; and A.S. designed the study, analyzed data and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Schema for construction of mixed–donor bone marrow chimeras.

Mixed donor bone marrow chimeras were constructed by injecting equal mixtures of wild-type and iLet7ΔLIN28 Tg bone marrow cells into lethally irradiated CD45.1 B6 host mice. Beginning on day 2 after chimera construction and continuing for 8 weeks when the mice were analyzed, mice were given either plain or doxycycline-supplemented drinking water.

Supplementary Figure 2 Effect of let-7 on NKT effector cell lineages in the periphery.

NKT cells from lymph nodes, spleen and liver were assessed for transcription factor expression by intracellular staining for PLZF vs RORγt and PLZF vs T-bet. Data represent a single experiment out of 3 independent experiments.

Supplementary Figure 3 Schematic model of NKT cell development in the thymus.

Upon positive selection of NKT cell precursors, PLZF expression is up-regulated to high levels and NKT differentiation proceeds. While CD44hiNK1.1-PLZFhi thymocytes are defined to be stage 2 thymocytes, our study suggests that stage 2 thymocytes actually consist of two different subsets at distinct stages of differentiation which we separate into immature intermediate cells and mature effector cells. Stage 2 intermediate cells terminally differentiate into IL-4-producing NKT2 effector cells and IL-17-producing NKT17 effector cells, or, alternatively, differentiate into IFN-γ-producing NKT1 effector cells. Differentiation of bi-potential stage 2 intermediate cells into NKT2, NKT17, or NKT1 cells depends on whether or not PLZF expression is down-regulated by let-7 miRNAs. If PLZF expression is not down-regulated, stage 2 intermediate cells terminally differentiate into NKT2 and NKT17 cells. If PLZF expression is down-regulated by let-7 miRNAs, stage 2 intermediate cells terminally differentiate into NKT1 cells. let-7 miRNAs can be up-regulated in the thymic medulla by IL-15, Vitamin D, and Retinoic Acid.

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Pobezinsky, L., Etzensperger, R., Jeurling, S. et al. Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function. Nat Immunol 16, 517–524 (2015). https://doi.org/10.1038/ni.3146

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