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T cell development requires constraint of the myeloid regulator C/EBP-α by the Notch target and transcriptional repressor Hes1

Nature Immunology volume 14, pages 1277–1284 (2013)Cite this article

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Abstract

Notch signaling induces gene expression of the T cell lineage and discourages alternative fate outcomes. Hematopoietic deficiency in the Notch target Hes1 results in severe T cell lineage defects; however, the underlying mechanism is unknown. We found here that Hes1 constrained myeloid gene-expression programs in T cell progenitor cells, as deletion of the myeloid regulator C/EBP-α restored the development of T cells from Hes1-deficient progenitor cells. Repression of Cebpa by Hes1 required its DNA-binding and Groucho-recruitment domains. Hes1-deficient multipotent progenitor cells showed a developmental bias toward myeloid cells and dendritic cells after Notch signaling, whereas Hes1-deficient lymphoid progenitor cells required additional cytokine signaling for diversion into the myeloid lineage. Our findings establish the importance of constraining developmental programs of the myeloid lineage early in T cell development.

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Figure 1: Hes1 expression is upregulated in the thymus and is reciprocal to Cebpa expression.
Figure 2: Hes1-deficient MPPs, but not lymphoid progenitor cells, divert to myeloid fates in T cell–inductive conditions.
Figure 3: Hes1-deficient lymphoid progenitor cells undergo apoptosis in T cell–inductive conditions lacking myeloid cytokines.
Figure 4: Hes1 inhibits myeloid development and Cebpa expression.
Figure 5: Deletion of C/EBP-α restores the development of T cells from Hes1-deficient progenitor cells in vitro.
Figure 6: Deletion of C/EBP-α restores the development of T cells from Hes1-deficient progenitor cells in vivo.

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Acknowledgements

We thank L. Raetzman (University of Illinois at Urbana-Champaign) for Hes1−/− mice (used with permission from R. Kageyama (Kyoto University Institute for Virus Research)); P. Zweidler-McKay (University of Texas MD Anderson Cancer Center) for mutant Hes1 constructs; A. Chi for advice on the design of luciferase reporter constructs; and N. Speck, D. Allman, S. Reiner, Q. Yang, S. Zhang, W. Bailis and D. Northrup for critical comments on the manuscript. Supported by the US National Institutes of Health (AI059621 and AI098428 to A.B., AI047833 to W.S.P., T32 GM-007229 and T32 CA-009140 to M.E.D., 1-F32-AI-080091-01A1 to J.J.B. and F30-HL-099271 to D.A.Z.).

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  1. Department of Pathology and Laboratory Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Maria Elena De Obaldia, J Jeremiah Bell, Xinxin Wang, Christelle Harly, Yumi Yashiro-Ohtani, Jonathan H DeLong, Daniel A Zlotoff, Dil Afroz Sultana, Warren S Pear & Avinash Bhandoola

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  1. Maria Elena De Obaldia
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Contributions

M.E.D., J.J.B. and A.B. designed the study and did the experiments; X.W., C.H., Y.Y.-O., D.A.Z., D.A.S. and J.H.D. did experiments; W.S.P. contributed conceptual expertise and provided input into the design of the study; and M.E.D. and A.B. wrote the paper.

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Correspondence to Avinash Bhandoola.

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Integrated supplementary information

Supplementary Figure 1 Hes1-deficient progenitors show defective T cell development after intravenous injection and intrathymic injection.

(a) Photographs of Hes1−/− or Hes1+/+ embryos and flow cytometry analysis of fetal thymi (E15.5) (b) Flow cytometry of mixed irradiation chimeras made by intravenously injecting a mixture of either CD45.2+ Hes1−/− or Hes1+/+ littermate control FL (E15.5) and competitor CD45.1+ BM. Recipient mice were examined 8-10 weeks post reconstitution. (c) Flow cytometry of thymus from CD45.1+ recipient mice injected intrathymically with a mixture of CD45.2+ FL progenitors (Lin−Kit+Flt3+) sorted from Hes1−/− or Hes1+/+ littermate control embryos (15.5 dpc) and sorted CD45.1+ BM progenitors (Lin−Sca1+Kit+Flt3+) and intrathymically injected into sublethally irradiated CD45.1+ recipient mice. Recipient mice were examined 17 days post-injection.

Supplementary Figure 2 Developmental potential of Hes1-deficient progenitors in OP9 and OP9-DL4 cultures.

(a) Flow cytometry of OP9 cultures using FL Lin-Kit+Flt3+IL-7Rα- multipotent progenitors or Lin-Kit+Flt3+IL-7Rα+ lymphoid progenitors sorted from Hes1-/- or Hes1+/+ littermate control embryos (E12.5-13.5). Cultures were harvested after 6 days to assay for the presence of myeloid cells and B cells (b-c) Flow cytometry of OP9 -DL4 cultures using FL Lin-Kit+Flt3+IL-7Rα- multipotent progenitors or Lin-Kit+Flt3+IL-7Rα+ lymphoid progenitors sorted from Hes1-/- or Hes1+/+ littermate control embryos (E12.5-13.5). Cultures were harvested 7 days after plating to assay for the presence of (b) T cells, and (c) myeloid cells. Results are representative of three independent experiments.

Supplementary Figure 3 Hes1-deficient prethymic T cell progenitors show defective T cell development in OP9-DL4 cultures.

(a) Flow cytometry of fetal liver from Hes1-/- or Hes1+/+ littermate control embryos (E12.5-13.5). (b) Flow cytometry of OP9-DL4 cultures using prethymic T cell progenitors (Lin-Kit+Flt3+IL-7Rα+PIR+ or PIR-) sorted from Hes1-/- or Hes1+/+ littermate control fetal liver (E12.5-13.5) (200 cells/well). Cultures were analyzed by flow cytometry after 6 days. Triplicate wells were set up for each experimental group. Bar graph represents mean cell number per well. Error bars represent SEM. Results are representative of two experiments.

Supplementary Figure 4 Hes1-deficient multipotent fetal liver progenitors cultured with Notch ligands have higher expression of Cebpa mRNA.

Flow cytometry and qPCR of OP9-DL4 cultures using FL progenitors (Lin-Kit+Flt3+) sorted from Hes1-/- or Hes1+/+ littermate control embryos (E15.5). After 6 days, Mac1-CD25- cells were sorted for qPCR analysis. Shown are mean mRNA expression levels normalized based on GAPDH levels.

Supplementary Figure 5 C/EBP-α deletion restores early and late stages of T cell development in vivo after intravenous injection.

Flow cytometry of hematopoietic lineages in mixed FL chimeras made by intravenously injecting a mixture of CD45.2+Hes1-/- Cebpafl/flVav1Cre, Hes1-/-Cebpafl/+Vav1Cre, or Hes1+/-Cebpafl/flVav1Cre FL and CD45.1+ competitor BM cells into CD45.1+ lethally irradiated recipient mice. After 8-10 weeks, the CD45.2+ donor contribution to each population was assessed.

Supplementary Figure 6 Hes1-mediated constraint of myeloid gene expression programs is essential for T cell development; the thymus is settled by a myelo-lymphoid progenitor from the bone marrow.

Early thymic progenitors (ETP) develop under the influence of Notch signaling in the thymus. Shortly after thymic entry, Notch induces the expression of the transcriptional repressor Hes1, which directly inhibits C/EBP-α, a critical myeloid regulator and perhaps other myeloid lineage genes. Hes1 is expressed in CD4/CD8 double-negative (DN) thymocytes, including ETP, DN2, and DN3 progenitors, but is no longer expressed at the CD4/CD8 double-positive (DP) stage. DN3 and DP thymocytes are committed to the T-cell lineage.

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De Obaldia, M., Bell, J., Wang, X. et al. T cell development requires constraint of the myeloid regulator C/EBP-α by the Notch target and transcriptional repressor Hes1. Nat Immunol 14, 1277–1284 (2013). https://doi.org/10.1038/ni.2760

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