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Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling

Nature Immunology volume 17, pages 565–573 (2016)Cite this article

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Abstract

Positive selection occurs in the thymic cortex, but critical maturation events occur later in the medulla. Here we defined the precise stage at which T cells acquired competence to proliferate and emigrate. Transcriptome analysis of late gene changes suggested roles for the transcription factor NF-κB and interferon signaling. Mice lacking the inhibitor of NF-κB (IκB) kinase (IKK) kinase TAK1 underwent normal positive selection but exhibited a specific block in functional maturation. NF-κB signaling provided protection from death mediated by the cytokine TNF and was required for proliferation and emigration. The interferon signature was independent of NF-κB; however, thymocytes deficient in the interferon-α (IFN-α) receptor IFN-αR showed reduced expression of the transcription factor STAT1 and phenotypic abnormality but were able to proliferate. Thus, both NF-κB and tonic interferon signals are involved in the final maturation of thymocytes into naive T cells.

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Figure 1: Three major SP stages defined by function.
Figure 2: Transcriptome analysis of late stages of thymocytes.
Figure 3: TAK1 is required for the SM-to-M1 transition.
Figure 4: Blockade of TNF activity restores maturation but not proliferation.
Figure 5: IKK activity restores proliferation and maturation but not licensing or survival.
Figure 6: Interferon-regulated genes and STAT1 levels are not 'rescued' by IKKCA, and the interferon receptor IFN-αR is required for Qa2 expression but not for maturation.

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Acknowledgements

We thank H. Chi (St. Jude Children's Hospital) for Rag2GFP mice; M. Mescher (University of Minnesota) for Ifnar1−/− mice; A. Strasser (Walter and Eliza Hall Institute) for Bcl2l11−/− mice; K. Hayakawa (Fox Chase Cancer Center) for monoclonal antibody SM6C10; and M.A. Farrar for comments and review of the manuscript. Supported by the NIH (R01 AI088209 and P01 AI35296 to K.A.H.).

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  1. Department of Laboratory Medicine & Pathology, The Center for Immunology, University of Minnesota, Minneapolis, Minnesota, USA

    Yan Xing, Xiaodan Wang, Stephen C Jameson & Kristin A Hogquist

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  1. Yan Xing
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Contributions

Y.X. designed and performed experiments, analyzed data, and wrote the manuscript; X.W. performed experiments and analyzed data; S.C.J. provided reagents, animals and input for the preparation of the manuscript; and K.A.H. directed the research, analyzed data and wrote the manuscript.

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Correspondence to Kristin A Hogquist.

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Integrated supplementary information

Supplementary Figure 1 Comparison of staining methods for SP thymocyte subsets.

(a) Flow cytometric analysis of thymocytes from Rag2GFP mice (n=4). Gating strategy for exclusion of re-circulating mature T cells (GFP−), γδ T cells (GL3+), iNKT (CD1dTet+) and Treg (CD25+) cells. (b) Flow cytometric analysis of thymocytes from H2-Ab1−/− mice (n=3). (c) Flow cytometric analysis of CCR7+TCRβ+ thymocytes from Klf2GFP mice (n=3). (d) Comparison of staining methods for CD4SP and CD8SP thymocyte subsets from Rag2GFP mice (n=3).

Supplementary Figure 2 Gene-expression changes during thymocyte maturation.

(a) Dump gates for cell sorting using Rag2GFP mice (n=3). Red numbers indicate frequency of cells in adjacent gates among total CD4SP thymocytes. (b) The numbers of genes whose expression changes in MHC class II restricted T cells during positive selection and maturation. Gene expression changes ≥ 2 fold, with P ≥ 0.05 (unpaired, t-test, n=3)

Supplementary Figure 3 A TAK1 signaling model, and ‘agonist-selected’ populations strongly affected by TAK1 deficiency.

(a) A schematic model showing TAK1 signaling transduction pathway components that potentially regulate T cell functional maturation. (b) Flow cytometric analysis and cell numbers of iNKT cells, Treg cells, IEL precursor (IELp) thymocytes and CD8aa gut intraepithelial lymphocytes (IEL) in Tak1fl/fl (n=5) and Tak1fl/flCd4Cre mice (n=5). Error bars indicate the mean ± s.d.; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, two-tailed unpaired Student’s t-test.

Supplementary Figure 4 Ingenuity pathway analysis of thymocytes.

Ingenuity pathway analysis defined upstream regulators of genes expressed differentially in “Early”, “Late” changed genes, and TAK1 dependent genes. Upstream regulators with p-values < 1.0E-08 are shown. Activation z-scores > 2.0 or < -2.0 are considered evidence of “Activated” or “Inhibited” pathways. Yellow highlighting indicated upstream regulators activated late in maturation that are lost with TAK1 deficiency. Likewise green highlighting indicates regulators that are inhibited late in maturation, but gained with TAK1 deficiency.

Supplementary Figure 5 Expression of genes encoding members of the TNFRSF superfamily during thymocyte maturation.

(a) Graphs show microarray gene expression signal values for all TNF receptor super family members (Tnfrsf) in each of the four populations indicated. (b) Protein expression of TNF receptor super family members was assessed by flow cytometry. Representative data from two independent experiments are shown. (c) Flow cytometric analysis of thymocytes from Tak1fl/fl, Tak1fl/flCd4Cre and Tak1fl/flCd4Cre Bcl2l11−/− mice. (d) Cell numbers of total thymocytes or indicated populations from Tak1fl/fl (n=10), Tak1fl/flCd4Cre (n=10) or Tak1fl/flCd4CreBcl2l11−/− mice (n=3). Error bars indicate the mean values ± s.d. in d. NS, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (unpaired t-test).

Supplementary Figure 6 IFN-αR is dispensable for TNF licensing and the proliferation competency of SP thymocytes.

(a) Flow cytometric analysis of TNF production of M2 CD4SP cells from Ifnar1−/− (n=3) and control mice (n=3). The cells were stimulated with plate-coated anti-CD3 plus anti-CD28 for 4 hours. Numbers indicate frequency of cells in adjacent gates and all plots have the same axis scales. (b) CellTrace Violet cell proliferation analysis of SM, M1 and M2 CD4SP cells from the indicated mice. Those cells were labeled with CellTrace Violet and then stimulated via CD3 and CD28 for 3 days. Representative data from three independent experiments are shown.

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Xing, Y., Wang, X., Jameson, S. et al. Late stages of T cell maturation in the thymus involve NF-κB and tonic type I interferon signaling. Nat Immunol 17, 565–573 (2016). https://doi.org/10.1038/ni.3419

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