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TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells

Nature Immunology volume 18, pages 45–53 (2017)Cite this article

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Abstract

TET proteins oxidize 5-methylcytosine in DNA to 5-hydroxymethylcytosine and other oxidation products. We found that simultaneous deletion of Tet2 and Tet3 in mouse CD4+CD8+ double-positive thymocytes resulted in dysregulated development and proliferation of invariant natural killer T cells (iNKT cells). Tet2-Tet3 double-knockout (DKO) iNKT cells displayed pronounced skewing toward the NKT17 lineage, with increased DNA methylation and impaired expression of genes encoding the key lineage-specifying factors T-bet and ThPOK. Transfer of purified Tet2-Tet3 DKO iNKT cells into immunocompetent recipient mice resulted in an uncontrolled expansion that was dependent on the nonclassical major histocompatibility complex (MHC) protein CD1d, which presents lipid antigens to iNKT cells. Our data indicate that TET proteins regulate iNKT cell fate by ensuring their proper development and maturation and by suppressing aberrant proliferation mediated by the T cell antigen receptor (TCR).

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Figure 1: Simultaneous deletion of Tet2 and Tet3 severely compromises T cell homeostasis, leading to iNKT cell expansion and disease.
Figure 2: Tet2-Tet3 DKO iNKT cells in the thymus of young (3- to 4-week-old) mice are skewed toward the NKT17 cell lineage.
Figure 3: Gene-expression profiles of thymic wild-type and Tet2-Tet3 DKO iNKT cells.
Figure 4: Antigen stimulation promotes the expansion of Tet2-Tet3 DKO iNKT cells and disease development in fully immunocompetent recipients.
Figure 5: 5hmC shows enrichment in the bodies of genes with high expression in iNKT cells and those expressing key iNKT-cell-lineage-specifying factors.
Figure 6: TET proteins regulate DNA modification in iNKT cells.
Figure 7: Characterization of chromatin regions with differential accessibility in wild-type iNKT cells versus Tet2-Tet3 DKO iNKT cells.
Figure 8: Ectopic expression of ThPOK or T-bet in Tet2-Tet3 DKO iNKT cells can suppress the aberrant increase in RORγt expression.

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Acknowledgements

We thank C. Kim, K. van Gunst, L. Nosworthy, D. Hinz and R. Simmons at the LJI Flow Cytometry Core for help with fluorescence-activated cell sorting; G. Seumois and J. Day at the LJI Functional Genomics Center for assistance with next-generation sequencing (Illumina HiSeq 2500); M. Kronenberg, I. Engel and C.-W. Lio (LJI) for discussions, the LJI Bioinformatics Core for routine analysis; Z. Mikulski and B. Kiosses at the LJI microscopy core, M. Chadwell at the LJI Histology core, and the Histology Core at the University of California at San Diego Moores Cancer Center; and R. Bosselut (National Cancer Institute) for pMX-ThPOK-IRES-GFP. Supported by US National Institutes of Health (R01 AI44432, CA151535 and R35CA210043), the Leukemia and Lymphoma Society (Translation Research Project grant 6187-12 to A.R.), the Academy of Finland Centre of Excellence in Molecular Systems Immunology and Physiology Research (H.L. and. S.R.), an Albert Billings Ruddock Professorship at Caltech (E.V.R.), the Cancer Research Institute (Irvington Institute postdoctoral fellowship to A.T.), the Fraternal Order of Eagles Fellow of the Damon Runyon Cancer Research Foundation (DRG-2069-11 to J.P.S.-B.) and the National Science Foundation (W.A.P.).

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Author notes

  1. William A Pastor

    Present address: Present address: Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, California, USA.,

  2. Edahí González-Avalos and Sini Rautio: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Signaling and Gene Expression, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Ageliki Tsagaratou, Edahí González-Avalos, James P Scott-Browne, Susan Togher, William A Pastor & Anjana Rao

  2. Department of Computer Science, Aalto University School of Science, Aalto, Finland

    Sini Rautio & Harri Lähdesmäki

  3. Sanford Consortium for Regenerative Medicine, La Jolla, California, USA

    James P Scott-Browne & Anjana Rao

  4. Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA

    Ellen V Rothenberg

  5. Division of Pediatric Neurooncology, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Lukas Chavez

  6. Department of Pharmacology and Moores Cancer Center, University of California at San Diego, La Jolla, California, USA

    Anjana Rao

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Contributions

A.R. and A.T. designed the study; A.T. performed all of the experiments, and A.R. and A.T. wrote the manuscript. E.G.-A. analyzed WGBS and ATAC-seq data sets under the supervision of L.C. and J.P.S.-B., respectively. S.R. analyzed RNA-seq and CMS-IP data sets under the supervision of H.L. S.T. helped with in vivo adoptive transfer experiments. W.A.P. generated the Tet3fl/fl mice. E.V.R. provided critical input and suggestions during the course of this study and helped write the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Characterization of Tet2-3 DKO mice

(a) Tet1, Tet2 and Tet3 mRNA expression assessed by quantitative RT-PCR. PolyA+ RNA was isolated from DP, CD4 SP and CD8 SP T cells from wild type (WT) and Tet2-/-Tet3fl/fl CD4cre (Tet2-3 DKO) mice. Relative mRNA expression levels were normalized to Gapdh.

(b) 5hmC levels in genomic DNA of thymocyte subsets from WT (n=2) and Tet2-3 DKO (n=2) mice, estimated by anti-CMS dot blot after bisulfite treatment of genomic DNA to convert 5hmC to CMS. One representative experiment out of 2 is shown.

(c) Haematoxylin and eosin staining of spleen, lung and liver sections from 6 week-old control (upper panel) and Tet2-3 DKO (lower panel) mice.

(d) iNKT cells in lymph nodes (LN) of WT versus Tet2-/-, Tet3 KO and Tet2-3 DKO iNKT cells as defined by GalCer-CD1d and TCRβ staining.

(e) Percentage (left) and number (right) of iNKT cells in lymph nodes. n=6 (WT), n=3 (Tet2 KO), n=at least 2 (Tet3 KO), n=6 (Tet2/3 DKO).

Data are mean ± SEM. *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired t test)

Supplementary Figure 2 Analysis of CD4 and CD8 T cell development in wild type and Tet2-3 DKO mice.

(a) Thymic cellularity in wild type (n=9) and Tet2-3 DKO (n=10) mice at 3-4 weeks of age.

(b) Top, Analysis of DN, DP, CD4 and CD8 SP thymic subsets defined by surface staining for CD4 and CD8 markers. Bottom, Representation of these subsets after gating out iNKT cells as defined by GalCer-CD1d and TCRβ staining.

(c) Percentage and (d), Number of DP, CD4 SP and CD8 SP cells in the thymus of WT (n=6) and Tet2-3 DKO (n=5) mice at 3-4 weeks.

(e) Representation of CD4 and CD8 T cells in lymph nodes (LN, top) and spleen (bottom) of WT and Tet2-3 DKO mice at 3-4 weeks.

(f) Total number of cells in lymph nodes (LN) of 3-4 week old WT (n=3) and Tet2-3 DKO (n=3) mice.

(g) Percentage and number of CD4 and CD8 cells in the LN of WT (n=6) and Tet2-3 DKO (n=3) mice analyzed at 3-4 weeks old.

(h) Percentage and number of CD4 and CD8 cells in the spleen of WT (n=6) and Tet2-3 DKO (n=3) mice analyzed at 3-4 weeks old.

Data are mean ± SEM. *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired t test).

At least 3 independent experiments were performed.

Supplementary Figure 3 Profound loss of TET proteins is required for dysregulated expansion and function of iNKT cells.

(a) iNKT cells in the thymus of representative 20 day-old WT versus Tet2-/-, Tet3 KO and Tet2-3 DKO mice, defined by staining with GalCer-CD1d tetramer and anti-TCRβ.

(b) Increased percentages (left) and numbers (right) of iNKT cells in spleens isolated from 4 week-old WT (n=6), T2-/- (T2 KO, n=3), T3 KO (n=3) and Tet2-3 DKO (n=10) mice.

(c) Histogram evaluating the expression of RORγt (left); PLZF (center); and CD4 (right) in WT, single Tet2-/- (Tet2 KO), Tet3 KO, Tet2-3 DKO thymic iNKT cells.

(d, e) Histogram evaluating the expression of RORγt in the spleen (d) and lymph nodes (e) of WT, single Tet2-/-, Tet3 KO, Tet2-3 DKO iNKT cells.

Data are mean ± SEM. *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired t test)

Supplementary Figure 4 Cytokine production in Tet2-3 DKO iNKT cells.

(a) Representative flow cytometric analysis of IL17 secretion by GalCer-CD1d tetramer+ TCRβ+ cells.

(b) Percentage of IL17-secreting iNKT cells among total iNKT cells.

(c) Percentage of IL17-secreting iNKT cells among total thymocytes.

(d) Representative flow cytometric analysis of IFNγ secretion by GalCer-CD1d tetramer+ TCRβ+ TCR cells.

(e) Percentage of IFNγ-secreting iNKT cells among total iNKT cells.

(f) Percentage of IFNγ-secreting iNKT cells among total thymocytes.

(g) Representative flow cytometric analysis of IL4 secretion by GalCer-CD1d tetramer+ TCRβ+ cells.

(h) Percentage of IL4-secreting iNKT cells among iNKT cells.

(i) Percentage of IL4-secreting iNKT cells among total thymocytes.

In all cases (a-i) 3 mice per genotype were evaluated in 2 independent experiments.

(j) CD8 SP thymocytes were assessed by flow cytometry for the expression of markers that characterize memory-like CD8 cells: surface markers CD122, CD44, CXCR3 and the transcription factor Eomes.

(k) Percentage of CD8 SP thymocytes that express CD122, CD44, CXCR3 and Eomes. WT (n=3) and Tet2-3 DKO (n=3) mice. For (j) and (k) 3 independent experiments were performed.

Data are mean ± SEM. *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 (unpaired t test)

Supplementary Figure 5 Increase of NKT17 subset and reduction of NKT1 precursors in the NK1.1- Tet2-3 DKO iNKT cells.

(a) Representative flow cytometric analysis of transcription factors PLZF and RORγt in GalCer-CD1d tetramer+ CD24-TCRβ+ NK1.1- iNKT cells reveals dramatic increase of NKT17 subset.

(b) Percentage of iNKT cells expressing PLZF and Rorγt (NKT17 subset) among the NK1.1− iNKT cells. n=5 WT mice and 7 Tet2-3 DKO mice. Data are compiled from 4 independent experiments.

(c) Representative flow cytometric analysis of PLZF and T-bet expression in NK1.1− iNKT cells. The frequency of PLZF+Tbet+ NKT1 precursor cells is reduced. One representative experiment of 4 is shown.

(d) Percentage of iNKT cells expressing PLZF and T-bet (NKT1 precursor cells) among NK1.1− iNKT cells. p<0.0001. n=5 WT mice and 7 Tet2-3 DKO mice. Data are compiled from 4 independent experiments.

(e) Representative flow cytometric analysis of transcription factors PLZF and T-bet in α-GalCer-CD1d tetramer+ CD24- TCRβ+ NK1.1+ iNKT cells shows expression of RORγt and emergence of an aberrant PLZFhigh Tbet+ population.

(f) α-GalCer-CD1d tetramer+ TCRβ+ NK1.1+ WT and Tet2-3 DKO iNKT cells were stimulated ex vivo with PMA and ionomycin. Representative flow cytometric analysis of α-GalCer-CD1d tetramer+ cells that secrete IFNγ is shown.

(g) Percentage of WT and Tet2-3 DKO α-GalCer-CD1d tetramer+ CD24-TCRβ+NK1.1+ iNKT cells that secrete IFNγ. One of 2 independent experiments is shown. Cells isolated from 3 different mice/ genotype were evaluated.

Data are mean ± SEM. **P< 0.01, ****P< 0.0001 (unpaired t test)

Supplementary Figure 6 Correlation of DNA modifications (5mC and 5hmC) with gene expression

(a) Composite analysis of methylation (5mC+5hmC) in genes with decreased (left, downregulated) or increased (middle, upregulated) expression in young Tet2-3 DKO iNKT cells compared to WT. Right, Methylation in random genomic fragments.

(b) Methylation changes in the promoter (left) and the gene body (right) of individual differentially-expressed genes, plotted against the ratio of their expression in WT versus young Tet2-3 DKO iNKT cells. Each dot represents a gene. Up (red dots) 698 genes with higher expression in WT compared to Tet2-3 DKO iNKT cells; Down (blue dots), 649 genes with lower expression in WT compared to Tet2-3 DKO iNKT cells.

Supplementary Figure 7 Differentially modified regions (DMRs) and differentially accessible regions (DARs) in genes related to the iNKT cell specification program.

Genome browser views of DNA modification (5mC+5hmC) identified by WGBS and chromatin accessibility identified by ATAC-seq in (a) Tbx21; (b), Zbtb7b and (c) Rorc genes in common lymphoid progenitors (CLP, purple), WT iNKT cells (blue) and young Tet2/3 DKO iNKT cells (green). The arrow indicates the direction of transcription. Statistically significant DMRs (black) or DARs (purple for gain of accessibility in Tet2/3 DKO or black for more accessibility in WT iNKT cells) are indicated by horizontal bars. Selected regions in which modification or/and accessibility is altered from CLP to iNKT cells, and then progressively is affected in young iNKT cells are highlighted (salmon shading). For each gene, the gene expression level (RPKM) in each cell type is indicated.

Supplementary Figure 8 Portraits of DNA modification and gene expression of selected downregulated cytokines and transcription factors in Tet2-3 DKO iNKT cells.

(a)-(c), Genome browser views of DNA modification (5mC+5hmC, identified by WGBS) and chromatin accessibility (identified by ATAC-seq) in a, Il4; b, Ifng and c, Bcl11b genes in common lymphoid progenitors (CLP, purple), WT iNKT cells (blue) and young Tet2-3 DKO iNKT cells (green). The arrow indicates the direction of transcription. Statistically significant DMRs (black) or DARs (purple for gain of accessibility in DKO or black for more accessibility in WT) are indicated by horizontal bars. Selected regions in which DNA modification or/and accessibility is altered from CLP to iNKT cells, and then progressively affected in young iNKT cells, are highlighted (salmon shading). For each gene the expression (RPKM) in each cell type is indicated.

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Tsagaratou, A., González-Avalos, E., Rautio, S. et al. TET proteins regulate the lineage specification and TCR-mediated expansion of iNKT cells. Nat Immunol 18, 45–53 (2017). https://doi.org/10.1038/ni.3630

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