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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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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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.
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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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DOI: https://doi.org/10.1038/nature11581
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