JunB regulates homeostasis and suppressive functions of effector regulatory T cells

Foxp3-expressing CD4+ regulatory T (Treg) cells need to differentiate into effector Treg (eTreg) cells to maintain immune homeostasis. T-cell receptor (TCR)-dependent induction of the transcription factor IRF4 is essential for eTreg differentiation, but how IRF4 activity is regulated in Treg cells is still unclear. Here we show that the AP-1 transcription factor, JunB, is expressed in eTreg cells and promotes an IRF4-dependent transcription program. Mice lacking JunB in Treg cells develop multi-organ autoimmunity, concomitant with aberrant activation of T helper cells. JunB promotes expression of Treg effector molecules, such as ICOS and CTLA4, in BATF-dependent and BATF-independent manners, and is also required for homeostasis and suppressive functions of eTreg. Mechanistically, JunB facilitates the accumulation of IRF4 at a subset of IRF4 target sites, including those located near Icos and Ctla4. Thus, JunB is a critical regulator of IRF4-dependent Treg effector programs, highlighting important functions for AP-1 in Treg-mediated immune homeostasis.

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Software and code
Policy information about availability of computer code Data collection N/A Data analysis Flow Jo_V10 was used for analysis of flow cytometric data. Excel 2016 and GraphPad Prism 7.01 was used for data analysis. Bowtie2 was used for ChIP-seq and RNA-seq analysis. Homer 4.10 was used for ChIP-seq analysis. Tophat2 .2.1 and cuflinks 2.2.1. were used for RNAseq analysis.
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Data
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Life sciences study design
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Sample size
Sample sizes were determined according to the basis of previous publications in the immunology field and our previous experience. In most of the experiments, 3 -8 samples per condition was considered as enough to identify the deferences. Exact sample sizes were indicated in the figure legend.
Data exclusions No exclusion criteria was used.

Replication
Data are representative of two or more independent experiments with similar results (statistical significance).
Randomization No randomization method was used.

Blinding
Histology scoring and weight measurement were performed in blind conditions.
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Laboratory animals
Junbfl/fl mice were described previously42. Foxp3Cre (Foxp3YFP-Cre; stock# 016959), Cd4Cre (stock# 017336), Rag1-/-(stock# 002216), and B6SJL (stock# 002014) mice were obtained from the Jackson Laboratory. All mice were maintained on a C57BL/6 background under specific pathogen-free conditions. Sex-matched, 6-12-week-old mice were used for experiments. All animal experimental protocols were approved by the Animal Care and Use Committee at Okinawa Institute of Science and Technology Graduate University.

Wild animals
The study did not involve wild animals.

Field-collected samples
The study did not involve samples collected from the field.

ChIP-seq Data deposition
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Data quality
We repeatedly performed ChIP-PCR to comfirm ChIP-seq data quality.

Software
Bowtie2, Homer Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Methodology
Sample preparation Cells were isolated from spleens, lymph nodes, and thymuses by mashing them through cell strainers (BD; 352340). Cells were Instrument BD LSRFortessa, BD FACSAria II, BD FACSAriaIII Software BD FACS software was used for collection and FlowJo v10 was used for analysis.

Gating strategy
For the all data analysis, FSC-A/SSC-A gating was performed for cell size and characteristics determination and exclusion of debris. In addition, FSC-H/FSC-W gating and SSC-H/SSC-W gating were performed for the exclusion of cell doublets. Then dead cells were excluded by Zombie-NIR staining. Positive gate and negative gate were determined by using isotype control or knockout control. Gating strategies are provided in Supplementary Figure 10.
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