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Alternative pathway for the development of Vα14+ NKT cells directly from CD4–CD8– thymocytes that bypasses the CD4+CD8+ stage

Nature Immunology volume 18, pages 274–282 (2017)Cite this article

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Abstract

Although invariant Vα14+ natural killer T cells (NKT cells) are thought to be generated from CD4+CD8+ double-positive (DP) thymocytes, the developmental origin of CD4–CD8– double-negative (DN) NKT cells still remains unresolved. Here we provide definitive genetic evidence obtained, through studies of mice with DP-stage-specific ablation of expression of the gene encoding the recombinase component RAG-2 (Rag2) and by a fate-mapping approach, that supports the proposal of the existence of an alternative developmental pathway through which a fraction of DN NKT cells with strong T-helper-type-1 (TH1)-biased and cytotoxic characteristics develop from late DN-stage thymocytes, bypassing the DP stage. These findings provide new insight into understanding of the development of NKT cells and propose a role for timing of expression of the invariant T cell antigen receptor in determining the functional properties of NKT cells.

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Figure 1: Demonstration of the DN-stage-thymocyte origin of NKT cells by a novel fate-mapping approach using E8III-Cre+Rosa26-YFP mice, in which YFP expression is controlled by the DP-stage-specific expression of E8III-Cre.
Figure 2: DN-stage-restricted expression of Rag2 in E8III-Cre+Rag2fl/fl mice.
Figure 3: Detection of Trav11Traj18 transcripts and NKT cells of DN-stage origin in the thymus of E8III-Cre+Rag2fl/fl mice.
Figure 4: Cytokine-secretion patterns and gene-expression profiles of NKT cells of DN-stage origin from E8III-Cre+Rag2fl/fl mice.
Figure 5: Peripheral tissue distribution of NKT cells of DN-stage origin from E8III-Cre+Rag2fl/fl mice and NKT cells of DP-stage origin from E8III-Cre+Rosa26-YFP mice.
Figure 6: Cytotoxic-lineage characteristics of liver NKT cells of DN-stage origin.

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Acknowledgements

We thank A. Singer (US National Institutes of Health) for E8III-Cre mice; F. Constantini (Columbia University) for Rosa26-YFP mice; D. Littman (New York University) for Rorc–/– mice; P. Burrows for critical reading of this manuscript; T. Hasegawa for support with animal facility services; and N. Takeuchi for secretarial assistance. Supported by the Japan Society for the Promotion of Science (KAKENHI grants 24790490 to N.D. and 23229005 to M.T.).

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Authors and Affiliations

  1. Laboratory for Immune Regulation, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Nyambayar Dashtsoodol, Tomokuni Shigeura, Minako Aihara, Ritsuko Ozawa, Satoshi Kojo, Michishige Harada & Masaru Taniguchi

  2. Core Research Laboratory, Mongolian National University of Medical Sciences, Ulaanbaatar, Mongolia

    Nyambayar Dashtsoodol

  3. Laboratory for Integrative Genomics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan

    Takaho A Endo, Takashi Watanabe & Osamu Ohara

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  1. Nyambayar Dashtsoodol
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Contributions

N.D. conceived of the study, performed experiments, analyzed data and wrote the paper; T.S., M.A. and R.O. assisted with experiments; S.K. and M.H. discussed experiments; T.A.E. did bioinformatics analyses; T.W. and O.O. performed next-generation sequencing experiments; M.T. directed the study and wrote the paper.

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Integrated supplementary information

Supplementary Figure 1 Confirmation of the DN-stage origin of NKT cells with the Cd4-Cre+Rosa26-YFP mouse.

(a) YFP reporter expression within gated viable B220-TCRβ+ splenic CD4+, CD8+, and DN fractions obtained from Cd4-Cre+Rosa26-YFP mice. Numbers on flow cytometry plots show the frequency of cells within indicated gates. (b) Detection of CD1d dimer+ NKT cells within gated YFP-negative and YFP-positive TCRβ+ DN splenocytes from Cd4-Cre+Rosa26-YFP mice. Prior to the cell staining, DN splenocytes were purified by AutoMACS using anti-CD4 and anti-CD8 microbeads. Numbers on flow cytometry plots show the percentage and absolute number of CD1d dimer+ NKT cells within gated YFP-negative and YFP-positive DN TCRβ+ cells. Results shown are from two mice done in one experiment.

Supplementary Figure 2 TCR α-chain joining-region gene-segment usage among out-of-frame Trav11-containing sequences detected in DN thymocytes from E8III-Cre+Rag2fl/fl mice.

High-throughput sequencing of Trav11Trac transcripts was carried out using the MiSeq system, where cDNA was prepared from sorted CD4-CD8- DN thymocytes from E8III-Cre+Rag2fl/fl mice. Bars depict the frequency (mean ± SEM, n = 3 biological replicates) of Traj gene segment usage among 3,002 out-of-frame Trav11 (CDR3) sequences detected when a total of 55,056 Trav11+ sequences were analyzed as shown in Table 1. The data are from one sequencing experiment.

Supplementary Figure 3 The TCR Vβ repertoire of NKT cells of DN-stage-thymocyte origin.

Flow cytometry analysis of TCR Vβ chain usage analysis of thymic CD1d dimer+CD3ɛ+ DN NKT cells from E8III-Cre+Rag2fl/fl (black bars) and littermate control mice (gray bars). Bars represent mean percentages ± SEM of three independent experiments with at least two mice per group.

Supplementary Figure 4 Detection of Trav11Traj18 transcripts and DN NKT cells in the thymus of Cd4-Cre+Rag2fl/fl mice.

(a) Viable 7-AAD-B220-CD1d dimer-TCRβ-/lo DP and DN thymocyte fractions that were electronically gated as CD5loCD69- cells were sorted from Cd4-Cre+Rag2fl/fl mice, and Trav11Traj18 mRNA expression was determined by qPCR analysis. Relative gene expression (mean ± SEM, n = 3) with Gapdh used as internal control. Results are representative of two experiments, (n.d.) not detected. (b) Detection of CD1d dimer+ DN NKT cells in the thymus, and (c) in the liver from Cd4-Cre+Rag2fl/fl mice, where littermate mice were used as controls. MACS depletion of CD4+, CD8+ and DP cells with magnetic microbeads was done prior to the cell staining. Numbers on flow cytometry plots show the percentage and the absolute number of CD1d dimer+TCRβ+ NKT cells among electronically gated DN cells. Results shown are from one experiment with two mice per genotype analyzed.

Supplementary Figure 5 Detection of Trav11Traj18 transcripts and mature DN NKT cells in the DN thymocyte fraction of Rorc–/– mice.

(a) Trav11Traj18 mRNA expression in sorted CD4+CD8+ DP and CD4-CD8- DN thymic fractions from Rorc-/- mice, as determined by qPCR. Bars show relative expression (mean ± SEM), where Gapdh was used as an internal control. Results are representative from three independent experiments with combined total of three to six mice per group, (n.d.) not detected. (b) Detection of CD1d dimer+ mature NKT cells within DN thymocytes of Rorc-/- mice. Prior to the cell staining, DN thymocytes were purified with AutoMACS by using anti-CD4 and anti-CD8 microbeads. Numbers on plots indicate the percentage and absolute number (mean ± SEM, n=6 mice) of CD1d dimer+ NKT cells within gated DN TCRβ+ cells. Unloaded CD1d staining of DN thymocytes from B6 mice was used as a staining control. Results shown are representative from three independent experiments with two mice per group.

Supplementary Figure 6 Lack of Trav11Traj 18 transcripts as well as mature DN NKT cells in the thymus of Ptcra–/– mice.

(a) The qPCR analysis of Trav11Traj18 mRNA expression in sorted thymic CD4+CD8+ DP, and CD44+CD25- DN1, CD44+CD25+ DN2, CD44-CD25+ DN3, CD44-CD25- DN4 fractions from Ptcra-/- mice. Bars show relative expression (mean ± SEM, n = 3 biological replicates), where Gapdh was used as an internal control. Results are representative from three independent experiments, (n.d.) not detected. (b) Detection of CD1d dimer+ NKT cells within DN thymocytes from B6 and Ptcra-/- mice. Numbers on flow cytometry plots indicate the percentage of CD1d dimer+TCRβ+ NKT cells within electronically gated DN cells. Prior to the cell staining, DN thymocytes were purified with AutoMACS by using anti-CD4 and anti-CD8 microbeads. Results shown are representative from three independent experiments with two mice per group.

Supplementary Figure 7 Surface phenotype of NKT cells of DN-stage-thymus origin is similar to that of B6 wild-type stage 3 NKT cells.

Viable 7-AAD-B220-CD1d dimer+TCRβ+ DN thymic NKT cells from B6 and E8III-Cre+Rag2fl/fl mice were analyzed for the expression of CD24, CD44, and NK1.1 surface antigens by flow cytometry. Numbers on the histograms show the percentage of CD24lo DN NKT cells, and numbers on flow cytometry plots represent the percentage of DN NKT cells within the indicated quadrant gates. Results shown are representative from three independent experiments, n = 3 mice per genotype.

Supplementary Figure 8 Cytotoxic killing assay with YFP– and YFP+ NKT cells from E8III-Cre+Rosa26-YFP mice.

FACS-sorted YFP-negative and YFP-positive thymic DN NKT cells from E8III-Cre+Rosa26-YFP mice were activated with anti-CD3 and anti-CD28 overnight, and then were co-cultured with CellTraceTM Violet labeled YAC-1 target cells at a E/T ratio of 5:1 for 4 hrs. Samples then were FACS analyzed using annexin V and 7-aminoactinomycin D. Bars depict the mean specific cell lysis percentage ± SEM of triplicate samples done in one experiment, and were calculated by the following formula: % cytotoxicity = (target-spontaneous/maximum-spontaneous) ×100.

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Dashtsoodol, N., Shigeura, T., Aihara, M. et al. Alternative pathway for the development of Vα14+ NKT cells directly from CD4–CD8– thymocytes that bypasses the CD4+CD8+ stage. Nat Immunol 18, 274–282 (2017). https://doi.org/10.1038/ni.3668

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