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Novel Foxo1-dependent transcriptional programs control Treg cell function

Nature volume 491, pages 554–559 (2012)Cite this article

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Abstract

Regulatory T (Treg) cells, characterized by expression of the transcription factor forkhead box P3 (Foxp3), maintain immune homeostasis by suppressing self-destructive immune responses1,2,3,4. Foxp3 operates as a late-acting differentiation factor controlling Treg cell homeostasis and function5, whereas the early Treg-cell-lineage commitment is regulated by the Akt kinase and the forkhead box O (Foxo) family of transcription factors6,7,8,9,10. However, whether Foxo proteins act beyond the Treg-cell-commitment stage to control Treg cell homeostasis and function remains largely unexplored. Here we show that Foxo1 is a pivotal regulator of Tregcell function. Treg cells express high amounts of Foxo1 and display reduced T-cell-receptor-induced Akt activation, Foxo1 phosphorylation and Foxo1 nuclear exclusion. Mice with Treg-cell-specific deletion of Foxo1 develop a fatal inflammatory disorder similar in severity to that seen in Foxp3-deficient mice, but without the loss of Treg cells. Genome-wide analysis of Foxo1 binding sites reveals ∼300 Foxo1-bound target genes, including the pro-inflammatory cytokine Ifng, that do not seem to be directly regulated by Foxp3. These findings show that the evolutionarily ancient Akt–Foxo1 signalling module controls a novel genetic program indispensable for Treg cell function.

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Figure 1: Foxo1 expression and TCR-induced Foxo1 nuclear exclusion in T reg cells.
Figure 2: An essential role for Foxo1 in the control of T reg cell function.
Figure 3: Foxo1-dependent transcriptional programs in T reg cells.
Figure 4: Restoration of Foxo1-deficient T reg cell function in the absence of IFN-γ.

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Accession codes

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Gene Expression Omnibus

Data deposits

The expression and ChIP-seq data have been deposited in the NCBI GEO database under accession number GSE40657.

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Acknowledgements

We thank R. Flavell for the Foxp3-IRES-RFP mouse strain, K. Rajewsky and C. Xiao for the Rosa26 targeting vector and T. Unterman for the HA-hFoxo1(AAA) construct. This work was supported by the Starr Cancer Consortium (13-A123 to M.O.L., M.Q.Z. and G.A.), the Rita Allen Foundation (M.O.L.), National Bio Resource Project (NBRPC) (2012CB316503 to M.Q.Z.) and National Institutes of Health (HG001696 to M.Q.Z.).

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Authors and Affiliations

  1. Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA

    Weiming Ouyang, Chong T. Luo, Na Yin, Morgan Huse, Myoungjoo V. Kim, Min Peng, Pamela Chan, Qian Ma, Alexander Y. Rudensky & Ming O. Li

  2. Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, New York, USA

    Will Liao, Yifan Mo & Gurinder Atwal

  3. Department of Applied Mathematics & Statistics, Stony Brook University, Stony Brook, 11794, New York, USA

    Will Liao & Yifan Mo

  4. Louis V. Gerstner, Jr Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA

    Chong T. Luo

  5. Department of Cell Biology and Genetics, Erasmus University Medical Center, 3000 DR Rotterdam, The Netherlands,

    Dies Meijer

  6. Systems Biology Center, NHLBI, National Institute of Health, Bethesda, 20892, Maryland, USA

    Keji Zhao

  7. Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, 10065, New York, USA

    Alexander Y. Rudensky

  8. Department of Molecular and Cell Biology, Center for Systems Biology, The University of Texas at Dallas, Richardson, 75080, Texas, USA

    Michael Q. Zhang

  9. Bioinformatics Division, Center for Synthetic and Systems Biology, TNLIST, Tsinghua University, Beijing 100084, China,

    Michael Q. Zhang

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  1. Weiming Ouyang
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Contributions

W.O., W.L., C.T.L., N.Y., M.H., G.A., M.Q.Z. and M.O.L. designed the research and analysed the data; W.O., W.L., C.T.L., N.Y., M.H., M.V.K., M.P., P.C., Q.M. and Y.M. did the experiments; D.M. and A.Y.R. provided birA and Foxp3cre mouse strains; K.Z. supervised the ChIP-seq experiment; and W.O., W.L. and M.O.L. wrote the manuscript.

Corresponding authors

Correspondence to Michael Q. Zhang or Ming O. Li.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-14. (PDF 5377 kb)

Supplementary Table 1

This file contains a complete list of candidate binding sites detected in antibody and biotin Foxo1 ChIP-Seq (overlapping peaks have been merged). (XLSX 253 kb)

Supplementary Table 2

This file shows differentially expressed genes in Foxo1-deficient Tregs (1.5X or greater). (XLSX 232 kb)

Supplementary Table 3

This file contains a complete list of candidate binding sites detected in antibody and biotin Foxo1 ChIP-Seq (selecting for peaks nearby genes that are differentially expressed in Foxo1 KO and rescued by Foxo1AAA). (XLSX 91 kb)

Supplementary Table 4

This file shows gene ontology analysis of Foxo1 direct target genes. (XLSX 47 kb)

Foxo1 nuclear exclusion in response to high-dose CD3 antibody

CD4+ cells were isolated from Foxo1tag/tag Foxp3-IRES-RFP mice, and stimulated with plate-bound anti-CD3 (0.1 μg/ml) and anti-CD28 (1.0 μg/ml). GFP, RFP and bright-field images were acquired every 2 min after addition of cells using a fluorescence videomicroscope. (MOV 68 kb)

Foxo1 nuclear exclusion in response to low-dose CD3 antibody

CD4+ cells were isolated from Foxo1tag/tag Foxp3-IRES-RFP mice, and stimulated with plate-bound anti-CD3 (0.01 μg/ml) and anti-CD28 (1.0 μg/ml). GFP, RFP and bright-field images were acquired every 2 min after addition of cells using a fluorescence videomicroscope (MOV 110 kb)

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Ouyang, W., Liao, W., Luo, C. et al. Novel Foxo1-dependent transcriptional programs control Treg cell function. Nature 491, 554–559 (2012). https://doi.org/10.1038/nature11581

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