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Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease

Abstract

Regulatory T (Treg) cells suppress the development of inflammatory disease, but our knowledge of transcriptional regulators that control this function remains incomplete. Here we show that expression of Id2 and Id3 in Treg cells was required to suppress development of fatal inflammatory disease. We found that T cell antigen receptor (TCR)-driven signaling initially decreased the abundance of Id3, which led to the activation of a follicular regulatory T (TFR) cell–specific transcription signature. However, sustained lower abundance of Id2 and Id3 interfered with proper development of TFR cells. Depletion of Id2 and Id3 expression in Treg cells resulted in compromised maintenance and localization of the Treg cell population. Thus, Id2 and Id3 enforce TFR cell checkpoints and control the maintenance and homing of Treg cells.

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Figure 1: Ablation of Id2 and Id3 expression in Treg cells leads to the early onset of fatal inflammatory disease.
Figure 2: Spontaneous inflammation in the lung, eyelid, skin and esophagus in mice depleted for the expression of Id2 and Id3 in Treg cells.
Figure 3: TH2 cell–mediated inflammation in the lungs of mice depleted for Id2 and Id3 expression in Treg cells.
Figure 4: Spontaneous germinal center formation and TH2 cell–mediated inflammation in mice depleted for the expression of Id2 and Id3 in Treg cells.
Figure 5: Depletion of Id2 and Id3 expression in Treg cells modulates the expression of CXCR5, Foxp3 and Nrp1.
Figure 6: Impaired homeostasis of Treg cells deleted for Id2 and Id3 expression after transfer into Rag1−/− mice.
Figure 7: Id2 and Id3 modulate the homeostasis and survival of Treg cells.
Figure 8: Unique patterns of gene expression in Treg cells depleted for Id2 and Id3.

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Acknowledgements

We thank A. Bortnick for critical reading of the manuscript, S. Kuan, B. Lin and Z. Ye for help with Illumina DNA sequencing, B. Ren for access to the Illumina Hi-Seq instrumentation, A. Goldrath (University of California San Diego) for Id2-YFP mice, Y. Zhuang for Id3fl/fl mice (Duke University), A. Lasorella (Columbia University) for Id2fl/fl mice, Y. Zheng (Salk Institute) for Foxp3-deficient organs, C. Katayama, M. Suzukawa, L. Deng, P. Rosenthal, T. Katakai, A. Beppu and A. Coddington for technical advice, J. Lee (Mayo Clinic) for providing the MBP antibodies, and members of the University of California, San Diego Histology Core for performing histology. This work was supported by US National Institutes of Health (AI 38425, AI 70535 and AI 7211 to D.H.B. and CA054198, CA78384 and 1P01AI102853 to C.M.).

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Authors

Contributions

M.Miy., K.M. and S.C. performed the majority of experiments. K.M. performed RNA-seq analysis. M.I. performed immunostaining of spleen and lymph nodes. N.V. contributed to the analysis of pathology. M.Mil. contributed to the analysis of lung tissue. L.-F.L. suggested key experiments and edited the manuscript. A.N.C. analyzed RNA-seq data. M.Miy. and C.M. wrote the manuscript. D.H.B. and C.M. supervised the study.

Corresponding author

Correspondence to Cornelis Murre.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Normal peripheral CD4 T cell homeostasis in mice depleted for the expression of either Id2 or Id3 in Treg cells.

(a) Representative flow cytometric analysis of CD44 and CD62L expression gated on CD4+TCRβ+YFPCD25 T cells derived from Id2+/+, Id2fl/fl, Id2fl/flFoxp3Cre mice. (b) Representative flow cytometric analysis of CD44 and CD62L expression gated on CD4+TCRβ+YFPCD25 T cells derived from Id3fl/–, and Id3fl/–Foxp3Cre mice. Graphs show the proportions of the compartments. Data represent the mean ± SD. (n=3-4).

Supplementary Figure 2 Heterozygous female Id2fl/flId3fl/flFoxp3Cre/+ mice do not develop inflammatory disease.

(a) Development of inflammatory disease in 8-week-old Id2fl/flId3fl/flFoxp3Cre and littermate control mice. Blue arrows indicate swelling of eyelids. Representative images of spleen, subcutaneous and mesenteric lymph nodes derived from 8-week-old littermate control (Id2fl/flId3fl/+Foxp3Cre/+) and Id2fl/flId3fl/flFoxp3Cre/Cre mice are shown. (b) Cell numbers of spleen, subcutaneous lymph nodes (sLN), and mesentric lymph nodes (mLN) derived from control or Id2fl/flId3fl/flFoxp3cre mice are indicated.Data represent the mean ± SD (n=5-6). *, P <0.05 **, P <0.01 (Student’s t test). (c) Histology of eyelid (H&E) and lung (PAS) derived from Id2fl/flId3fl/flFoxp3Cre/Cre and littermate heterozygous female Id2fl/flId3fl/flFoxp3Cre/+ mice. (d) Flow cytometric analysis of CD44 versus CD62L (upper) and CXCR5 versus PD1 expression gated on CD4+TCRβ+CD25YFP nonTreg cells and CD8+TCRβ+ cells.

Supplementary Figure 3 Spontaneous inflammatory disease in the eyelids and lung in mice depleted for Id2 and Id3 expression in Treg cells.

(a) Representative images of Haematoxylin and eosin (H&E) staining of eyelid, lachrymal gland, and lung from littermate control and Id2fl/flId3fl/flFoxp3Cre/Cre mice are shown. (b) B220 and CD3 immunostaining of lung tissue derived from littermate control and Id2fl/flId3fl/flFoxp3Cre/Cre mice. (c) MBP immunostaining of hilar LN derived from littermate control and Id2fl/flId3fl/flFoxp3Cre/Cre mice are shown. (d) Representative of H&E staining of pancreas from Id2fl/flId3fl/flFoxp3Cre/Cre mice. Blue arrows indicate eosinophils. (e) Representative images of Haematoxylin and eosin (H&E) staining of the esophagus isolated from control, Foxp3 or Id2fl/flId3fl/flFoxp3Cre/Cre mice are shown. Three independent mice showed similar results.

Supplementary Figure 4 Chronic inflammatory disease in aged mice depleted for Id2 and Id3 expression in Treg cells.

(a) Representative images of Haematoxylin and eosin (H&E) staining of colon, liver, stomach, and lung derived from 16-week-old Id2fl/flId3fl/flFoxp3Cre mouse. Blue arrows indicate eosinophilic foamy macrophages. (b) Trichrome staining of lung tissue.Increased collagen surrounds the bronchioles. Blue arrows indicate eosinophilic crystals. (c) Representative of MBP staining of colon.Three Id2fl/flId3fl/flFoxp3Cre mice that survived beyond 12-weeks showed similar results.

Supplementary Figure 5 Elevated levels of chemokine receptors in peripheral T cells isolated from female Id2fl/flId3fl/flFoxp3Cre/+ and Id2fl/flId3fl/flFoxp3Cre/Cre mice.

(a) The expression of CXCR4, CXCR5, and CXCR3 in YFP+ Treg, CD25+ Treg cells derived from female Id2fl/flId3fl/flFoxp3Cre/+ and IId2fl/flId3fl/flFoxp3Cre/Cre mice is shown. (b) CD62L expression in CD4+Foxp3-YFP+ Treg cells from control Foxp3Cre, Id2fl/flId3fl/flFoxp3Cre, heterozygous control Foxp3Cre/+, or Id2fl/flId3fl/flFoxp3Cre/+ mice are indicated. (c) ICOS and CTLA4 expression in Foxp3+ Treg cells. (d) Down-regulation of Foxp3 protein in Id2- and Id3-deficient Treg cells under non-inflammatory conditions. Foxp3 expression in YFP+Foxp3+ (blue) and YFPFoxp3+ (red) cells in the thymus, lymph nodes and spleen derived from heterozygous female Id2fl/flId3fl/flFoxp3Cre/+ mice (n=3). (e) Foxp3 expression in YFP+Foxp3+ cells derived from heterozygous female Id2+/+Id3+/+Foxp3Cre/+ (red) or Id2fl/flId3fl/flFoxp3Cre/+ (blue) mice. RNA was isolated from YFP+ or CD25+ Treg cells in LN derived from heterozygous female Id2+/+Id3+/+Foxp3Cre/+ (red) or Id2fl/flId3fl/flFoxp3Cre/+ (blue) mice. The expression of Foxp3 was analyzed by real-time PCR and is presented relative to Hprt1 abundance. (f) Nrp1 expression gated on thymic Foxp3+Helios+YFP+ or YFP Treg cells (left) or Foxp3+ YFP+ or YFP Treg cells in lymph nodes derived from heterozygous female Foxp3Cre/+ or Id2fl/flId3fl/flFoxp3Cre/+ mice.

Supplementary Figure 6 Depletion of Id2 and Id3 expression in Treg cells leads to aberrant patterns of localization.

(a) Localization of Foxp3+ Treg cells in spleens and lymph node are shown. Frozen sections of spleen and lymph nodes derived from littermate control and Id2fl/flId3fl/flFoxp3Cremice were processed for immunostaining using antibodies directed against B220, CD4 and Foxp3. DAPI-marked nuclei are shown in blue. The lines indicate the border between B cell follicle and T cell area. Scale bar, 100mm. Graph shows quantification of Foxp3+ cells in B cell area. Six similarly sized white pulps were chosen. Foxp3+ cells in B cell area were calculated in more than three mice in each group. (b) Treg cells deleted for Id2 and Id3 expression failed to localize to GCs. Representative images of PNA and Foxp3+ cells in spleens derived from SRBC-immunized wild-type control mice and unimmunized Id2fl/flId3fl/flFoxp3Cre mice. The images are representative for the spleens derived from three different control and Id2fl/flId3fl/flFoxp3Cre mice. *, P <0.05 (Student’s t test).

Supplementary Figure 7 Impaired homeostasis and homing in lymphoid organs and lung tissues derived from mice depleted for the expression of Id2 and Id3 in Treg cells.

(a) Representative flow cytometric analysis of Foxp3 and CD25 expression, gated on CD4 T cells (CD4+CD8TCRβ+Lineage) in subcutaneous LNs and lung tissues derived from control, 4-week-old or 7-8-week-old Id2fl/flId3fl/flFoxp3Cre mice. Graph shows the frequency of Foxp3+ cells in CD4 T cell compartment. Data represent the mean ± SD. Results represent four independent experiments. **, P <0.01 (Student’s t test). (b) Moderately affected migration into the lung of Treg cells deleted for Id2 and Id3. Purified CD4+CD25+ Treg cells from heterozygous female Id2fl/flId3fl/flFoxp3Cre/+ mice (CD45.2) were injected into CD45.1 recipient mice. 20 h after transfer, mice were dissected and YFP expression in transferred Treg cells was analyzed in cells derived from LNs, spleen, and lung. Two independent experiments yielded similar results.

Supplementary Figure 8 Id2- and Id3-deficient Treg cells display normal in vitro immunosuppressive function.

CFSE-labeled, or unlabeled CD4+CD25 T cells (responder T cells; TR) were co-cultured with Treg cells at indicated ratios (Treg: TR) and stimulated with anti-CD3 antibody in the presence of T-depleted splenocytes treated with mitomycin C for 3 d. The proliferation of responder cells was assessed by CFSE dilution (a) or [3H]thymidine incorporation (b). Three independent experiments produced with similar results.

Supplementary Figure 9 Gene expression patterns for Treg cells depleted for Id2 and Id3 expression.

Differentially expressed genes (P-value <0.05, >2-fold change, >10 abundance values) in Treg cells depleted for the expression of Id2 and Id3. RNA-seq analysis was analyzed at a depth of 20-30 million reads per sample using an Illumina Hiseq2500 sequencer. A single-stranded, strand-specific protocol was used (50nt). Raw sequencing files were quality controlled using FastQC. Alignment and trimming of reads was performed using the OSA algorithm against the mm10 mouse genome reference in Arraystudio. RNA transcripts were quantified using RSEM methods as implemented in Arraystudio. Principal component analysis was then performed to check for possible batch effects. Abundance values (counts) were normalized and compared between control and Id2fl/flId3fl/flFoxp3Cre mice using DESeq. GO enrichment analysis was performed using HOMER. (a) Selected biological process GO terms and their associated P-values are shown. (b) Heatmaps are displayed for significantly differentially expressed genes in each category. Displayed data are derived from three replicas using sorted Treg cells isolated from 3-week-old Id2fl/flId3fl/flFoxp3Cre mice. Three different lanes represent three different samples that were derived from three distinct Id2- and Id3-deficient Treg cells.

Supplementary Figure 10 Target gene expression, E2A occupancy and deposition of H3K4me1 in B-lineage and T-lineage cells.

(a) Selected group of genes that showed elevated or decreased transcript abundance in Treg cells depleted for Id2 and Id3 expression versus control cells are depicted. Values indicated on the vertical axis show fold change (log2). (b) Depostion of E2A and H3K4me1 across a selected group of genes. E2A occupancy is indicated in blue, H3K4me1 is shown in red. Black arrow indicates transcriptional start site and direction of transcription. (c) Model depicting the roles of Id2 and Id3 in developing Treg cells

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Rapid development of dermatitis in mice depleted for the expression of Id2 and Id3 in Treg cells. (MOV 12098 kb)

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Miyazaki, M., Miyazaki, K., Chen, S. et al. Id2 and Id3 maintain the regulatory T cell pool to suppress inflammatory disease. Nat Immunol 15, 767–776 (2014). https://doi.org/10.1038/ni.2928

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