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HEB Is Required for the Specification of Fetal IL-17-Producing Γδ T Cells

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HEB Is Required for the Specification of Fetal IL-17-Producing Γδ T Cells ARTICLE DOI: 10.1038/s41467-017-02225-5 OPEN HEB is required for the specification of fetal IL-17- producing γδ T cells Tracy S.H. In1,2, Ashton Trotman-Grant1,2, Shawn Fahl3, Edward L.Y. Chen1,2, Payam Zarin1,2, Amanda J. Moore1,2, David L. Wiest3, Juan Carlos Zúñiga-Pflücker1,2 & Michele K. Anderson 1,2 IL-17-producing γδ T(γδT17) cells are critical components of the innate immune system. However, the gene networks that control their development are unclear. Here we show that 1234567890 HEB (HeLa E-box binding protein, encoded by Tcf12) is required for the generation of a newly defined subset of fetal-derived CD73− γδT17 cells. HEB is required in immature CD24+CD73− γδ T cells for the expression of Sox4, Sox13, and Rorc, and these genes are repressed by acute expression of the HEB antagonist Id3. HEB-deficiency also affects mature CD73+ γδ T cells, which are defective in RORγt expression and IL-17 production. Additionally, the fetal TCRγ chain repertoire is altered, and peripheral Vγ4 γδ T cells are mostly restricted to the IFNγ- producing phenotype in HEB-deficient mice. Therefore, our work identifies HEB-dependent pathways for the development of CD73+ and CD73− γδT17 cells, and provides mechanistic evidence for control of the γδT17 gene network by HEB. 1 Sunnybrook Research Institute, 2075 Bayview Avenue, Toronto, ON M4N 3M5, Canada. 2 Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada. 3 Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. Correspondence and requests for materials should be addressed to M.K.A. (email: [email protected]) NATURE COMMUNICATIONS | 8: 2004 | DOI: 10.1038/s41467-017-02225-5 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02225-5 – nterleukin-17 (IL-17)-producing γδ T(γδT17) cells are the and psoriasis7 9. Furthermore, γδT17 cells can promote tumor – major source of early IL-17 production in mucosal tissues, such progression10 12, whereas interferon-γ (IFNγ)-producing γδ T I 1–4 γδ 13,14 γδ as the lung, gut, and reproductive tract . Although T17 cells (γδT1) cells have potent anti-tumor activity . T17 cell pro- are critical for clearing many bacterial and fungal infections2,5,6, gramming involves high-mobility group (HMG) box family aberrant regulation of these cells influences the courses of many members, such as Sox13 and Sox4, and the transcription factor autoimmune diseases, including multiple sclerosis, type 1 diabetes, RORγt15. Furthermore, T-cell receptor (TCR) signal strength can a FTOC Day 1 FTOC Day 2 FTOC Day 4 FTOC Day 7 FTOC Day 10 Immature Immature 58.6 41.4 45.0 35.4 20.1 7.66 4.30 4.51 3.58 0.89 uncommitted committed Mature committed CD24 0 0 2.72 16.9 7.46 64.8 17.0 74.2 33.7 61.9 CD73 b E17.5 Day 1 Day 7 Week 4 Week 7 Week 12 55.3 23.1 50.5 7.69 35.2 13.1 80.1 10.2 79.8 10.9 39.3 41.9 CD24 11.3 10.3 23.5 18.3 12.7 39.0 3.13 6.60 2.76 6.51 3.82 15.0 CD73 c E17.5 Day 1 Day 7 FTOC Day 7 FTOC Day 10 All 30.1 39.4 34.2 29.7 25.1 γδ T-cells t γ 62.0 58.1 49.5 61.3 63.2 ROR TCRγδ d 63.1 3.77 26.2 2.07 31.1 14.5 9.09 4.01 7.90 3.01 RORγt+ γδ T-cells CD24 30.0 3.14 66.2 5.52 18.7 35.7 70.6 16.3 65.1 24.0 CD73 e 47.9 34.4 59.1 8.39 35.5 11.6 9.17 13.2 4.04 5.05 RORγt– γδ T-cells CD24 2.31 15.5 5.03 27.5 7.66 45.2 4.17 73.5 6.06 84.9 CD73 FTOC Day 10 f E17.5 Day 1 60 Day 7 FTOC Day 7 80 p < 0.05 80 NS 100 100 RORγt+ p = 0.003 NS p < 0.001 p < 0.001 NS γδ T cells 80 p < 0.001 80 60 60 γ – 40 p < 0.001 ROR t p <0.05 γδ T cells NS 60 60 40 p < 0.05 p < 0.01 40 NS NS 40 40 20 NS 20 20 p < 0.001 % cells/subset 20 NS 20 NS p < 0.001 0 0 0 0 0 24+ 24+ 24– 24– 24+ 24+ 24– 24– 24+ 24+ 24– 24– 24+ 24+ 24– 24– 24+ 24+ 24– 24– 73– 73+ 73+ 73– 73– 73+ 73+ 73– 73– 73+ 73+ 73– 73– 73+ 73+ 73– 73– 73+ 73+ 73– g Spleen Lungs Gut 3.04 30.3 2.93 41.1 0.32 26.1 Week 12 CD24 10.4 56.3 12.4 43.5 0.24 73.3 CD73 2 NATURE COMMUNICATIONS | 8: 2004 | DOI: 10.1038/s41467-017-02225-5 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02225-5 ARTICLE influence the γδT17 cell fate16,17, and the E protein antagonist Id3 essential for the generation of Th17 cells, are dispensable for – is upregulated in response to TCR signalling18,19, suggesting a γδΤ17 development50 52. Furthermore, Sox13 does not operate in function for Id and E proteins in the γδT17/γδT1 cell fates. Here αβ Th17 cells15,53. Given these two models, a critical question is we investigate the role of the E protein HEB (HeLa E-box binding whether E protein activity positively affects the Sox-RORγt net- protein, encoded by Tcf12)inγδT17 cell development. work, within the context of developing γδ T cells, and whether γδ T cells are functionally programmed in the thymus, unlike Id3 activity can inhibit it. conventional αβ T cells that acquire effector function in the Here we study mice with targeted deletions in the Tcf12 locus to – periphery15,20 23.SpecificTCRγ chains are linked to distinct effector investigate a possible function for HEB factors in γδT17 develop- − programs, but the nature of these links is not clear23,24. γδT1 cells ment. We identify a new type of CD73 HEB-dependent γδT17 − are primarily CD27+CD44 cells21, and typically express Vγ5, Vγ1, cell subset that arises early in the fetal thymus, prior to the − or Vγ4 (nomenclature of Tonegawa and colleagues25,26). γδT17 cells appearance of CD73+ γδT17 cells. Whereas CD73 γδT17 cells are − are mostly CD27 CD44+ cells, and express either Vγ6orVγ427. absent in the fetal thymus of HEB-deficient mice, CD73+ Vγ6+ Additionally, some Vγ1+ cells are capable of producing IL-4 and cells are present. However, they are compromised in RORγt IFNγ 28.Thegenerationofdifferent types of γδ T cells occurs in expression, and in their ability to make IL-17. We also show that successive waves during fetal thymic development. Vγ5+ γδ T cells Vγ4+ γδT17 cells, but not Vγ4+ γδT1 cells, are dependent on HEB. appear first in the fetal thymus and migrate to the skin29.Vγ6+ γδ HEB can directly regulate Sox4 and Sox13, and Id3 can inhibit T cells arise next, and home to mucosal tissues and secondary expression of these transcription factors in γδ T cell precursors. lymphoid organs4.Thesetwoγδ T cell subsets are produced only in These results place HEB at the top of the known gene network that the fetal thymus, followed by lifelong residence in their respective regulates γδT17 cell programming, and provide a new link between tissues. Vγ4+ and Vγ1+ cells, which first appear in late fetal and TCR signalling and γδT17 cell fate determination. neonatal life, continue to be generated throughout adult life30. Functional programming of γδ T cells occurs after surface 31 Results expression of the γδ TCR, but prior to exit from the thymus .One − − fi γδ Identification of CD24 CD73 γδT17 cells in the fetal thymus. model posits that TCR signal strength instructs speci cation of T γδ 22 cell effector fate choice, with a stronger signal inducing the γδT1 Most T17 cells are generated in the fetal thymus . We there- cell fate20,32,33. TCR signalling involves activation of the PKCθ fore evaluated fetal thymic organ cultures (FTOCs) as a model κ system for studying γδ T-cell development. This approach pathway, leading to activation of NF B; the PI3K pathway, leading fi to activation of NFAT; and the MAPK signalling pathway (in allowed us to analyze precursors from germline HEB-de cient (HEBko) mice, which undergo embryonic lethality46, and to particular, ERK), which is upstream of many inducible transcrip- γδ tion factors, including Egr334. MAPK signalling and Egr3 activity capture successive waves of developing T cells without the αβ γδ 19 γδ γδ complications of migration. Development of γδ T cells in the have been linked to the / T cell and T17/ T1 cell lineage 32 choices17,35,36, in agreement with the signal strength model. adult thymus can be tracked using CD24 and CD73 . CD73 is upregulated in response to TCR signalling and marks commit- However, other experiments have indicated that strong TCR signals γδ are needed for the development of at least some γδT17 cells16. ment to the T cell lineage, whereas CD24 downregulation indicates maturity (Fig. 1a)32. We performed a time course for Thus, controversies remain regarding the function of TCR signal- γδ + ling in γδT17/γδT1 specification. CD24 and CD73 expression on all TCR cells from FTOCs to Id3 is one of the genes that is upregulated downstream of TCR- assess the CD24/CD73 subset distribution in a fetal context mediated ERK activity in developing thymocytes18,19,37,38.Id3acts (Fig.
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