Immunology and Inflammation

Thymic dendritic cell-derived IL-27p28 promotes the establishment of functional bias against IFN-γ production in newly generated CD4⁺ T cells through STAT1-related epigenetic mechanisms

Jie Zhang
Hui Tang
Haoming Wu
Xuewen Pang
Rong Jin author has email address
Yu Zhang author has email address

Department of Immunology, School of Basic Medical Sciences, NHC Key Laboratory of Medical Immunology, Medicine Innovation Center for Fundamental Research on Major Immunology-related Diseases, Peking University, Beijing, China
Institute of Life Sciences, Jinzhou Medical University, Jinzhou, China

https://doi.org/10.7554/eLife.96868.2

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Figures and data

Elevated IFN-γ production and T-bet expression in Cd11c-p28^f/f mice initiated from CD4SP thymocytes stage.
(A) CD4SP (GFP⁺CD4⁺CD8^-CD44^lo) thymocytes, CD4⁺ RTEs (GFP⁺CD4⁺CD8^-CD25^-CD44^lo) and CD4⁺ naive (GFP^-CD4⁺CD8^-CD25^-CD44^lo) T cells were sorted from 6-8-week-old Cd11c-p28^f/f mice and WT littermates, stimulated with plate coated anti-CD3 (2 µg/mL) and soluble anti-CD28 (1 µg/mL) for 12 hours. mRNA levels of Ifng, Il4, and Il2 were determined by qPCR. Data: mean ±SD (n=4, duplicates).
(B) Sorted cells were cultured under Th0 conditions for 3 days. The frequency of IFN-γ-producing CD4⁺ T cells were measured by intracellular staining. Representative dot plots (left) and statistical data (right, mean ±SD, n=3).
(C) Supernatants from 3-day cultures were analyzed for IFN-γ and IL-4 by ELISA. Data: mean ±SD (n=3).
(D) mRNA levels of Tbx21 and Gata3 in sorted cells were determined by qPCR. Data: mean ±SD (n=4, duplicates).
(E-F) T-bet protein levels were assessed by Western blot (E) and flow cytometry (F) after 3-day culture. Data: mean ±SD (n=3).
(G) Freshly sorted cells were lysed in Trizol, and Ifng and Tbx21 mRNA levels were determined by qPCR. Data: mean ±SD (n=4, duplicates). Statistical differences: * P < 0.05, ** P< 0.01, *** P<0.001 (Student’s t-test).

Enhanced IFN-γ production and T-bet expression in Il27ra^-/- mice initiated at the CD4SP thymocytes stage.
(A) CD4SP thymocytes and naive CD4⁺ T cells were isolated from Il27ra^-/- and WT mice, stimulated with plate-coated anti-CD3 (2 µg/mL) and soluble anti-CD28 (1 µg/mL) for 12 hours. mRNA levels of Ifng, Il4, Il2, Tbx21, and Gata3 were determined by qPCR. Data: mean ±SD (n=3, duplicates).
(B) CD4SP thymocytes and CD4⁺ naive T cells were cultured under Th0 conditions for 3 days. The frequency of IFN-γ-producing CD4⁺ T cells were analyzed by intracellular staining. Representative dot plots (left) and quantification (right, mean ± SD, n=3). Significance: * P< 0.05, ** P< 0.01 (Student’s t-test).

Distinct DNA and H3K4 methylation patterns at Ifng and Tbx21 promoter regions in CD4SP thymocytes from IL27p28-deficient mice.
(A) DNA methylation analysis of nine CpG sites in the Ifng promoter using sodium bisulfite-treated genomic DNA from GFP⁺CD4⁺CD8^-CD44^lo CD4SP thymocytes. Each row represents a sequenced allele (n=10 clones from one of the three independent experiments). Filled (●) and open (○) circles denote methylated and unmethylated cytosine, respectively.
(B) Left: Percent methylation at individual CpG sites from one representative experiment. Right: Average methylation of three adjacent site groups (group1: −205, − 190, −170; group2: −53, −45, −34; group3: +16, +96, +120) and all CpG sites (mean ± SD, n=3).
(C) DNA methylation analysis of five CpG sites upstream of the Il4 transcription start site using sodium bisulfite-treated genomic DNA from GFP⁺CD4⁺CD8^-CD44^lo CD4SP thymocytes. Each row represents a sequenced allele (n=10 clones from the two independent experiments). Filled (●) and open (○) circles denote methylated and unmethylated cytosine, respectively.
(D) Left: Graphs show the percentage of methylation at each individual site (left panel) or all CpG sites (right panel).
(E-G) Histone trimethylation analysis in freshly isolated CD4SP thymocytes from IL27p28-deficient and WT mice. ChIP-qPCR was performed using antibodies against H3K4me3 (E), H3K27me3 (F), and H3K9me3 (G). qPCR primers targeted promoter and trans-regulatory regions of Ifng, Tbx21, Il4, and Gata3. Data: mean ±SEM (n=3, duplicates). Significance: * P<0.05; ** P<0.01 (Student’s t-test). Abbreviation: pro., promoter.

Increased expression of STAT1-activated genes in CD4SP thymocytes from Cd11c-p28^f/f mice.
RNA-seq was performed to analyze the transcriptome of CD4SP thymocytes from Cd11c-p28^f/fand WT mice.
(A) Top 10 enriched Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for up-regulated differentially expressed genes (DEGs) in Cd11c-p28^f/fmice.
(B) Overlap between DEGs in Cd11c-p28^f/f mice and STAT1-activated and suppressed genes. Numbers indicate overlapping genes in each category.
(C) Validation of RNA-Seq results by qPCR for representative up-regulated genes. Data: fold change in Cd11c-p28^f/fversus WT mice (mean ± SEM, n=3, duplicates).
(D) Gene Set Enrichment Analysis (GSEA) showing coordinated upregulation of STAT1-activated genes in Cd11c-p28^f/fCD4SP thymocytes.
(E) Protein-protein interaction network of DEGs. Nodes represent proteins; edges indicate interactions. Larger nodes denote higher interaction degrees. Red: up-regulated; green: down-regulated; gray: non-DEGs connected to the network (added by Network Analyst).

Enhanced STAT1 activation in CD4SP thymocytes from Cd11c-p28^f/f mice.
(A) Intracellular staining of freshly isolated thymocytes from Cd11c-p28^f/f and WT mice using antibodies against phosphorylated STAT1 (Y701), STAT3 (Y705), and STAT4 (Y693). Representative histograms for CD4SP thymocytes (left) and mean fluorescence intensity (MFI) from three independent experiments (right, mean ±SD).
(B) Western blot analysis of total and phosphorylated STAT1 (Y701), STAT3 (Y705), and STAT4 (Y693) in purified CD4SP thymocytes, CD4⁺ RTEs, and naive CD4⁺ T cells from Cd11c-p28^f/f and WT mice. Representative blots (left) and relative protein levels quantified by densitometry and normalization to β-actin (right, mean ±SD, n=3).
(C) Increased STAT1 binding to promoter and regulatory regions of Tbx21 and Ifng loci in Cd11c-p28^f/f mice.
(D) Correlation between STAT1 binding and H3K4me3 levels at Tbx21 and Ifng loci. Significance: * P<0.05; ** P< 0.01.

Exacerbated autoimmune responses in Aire^-/- mice in the absence of IL27p28.
(A) CD44 and CD62L expression in CD4⁺ T cells from splenocytes of 6-8 week-old WT (n=6) and Cd11c-p28^f/f (n=6) mice. Representative dot plots (left) and percentages of CD44^loCD62L⁺ naive and CD44^hiCD62L^- activated T cells are shown (right, mean ±SD).
(B-D) WT, Cd11c-p28^f/f, Aire^-/-, and Cd11c-p28^f/fAire^-/- mice (24-30 weeks old) were analyzed. Each symbol represents one mouse (mean ±SD).
(B) Serum anti-dsDNA antibody levels (ELISA).
(C) H&E staining (left) and histological scores (right) of the lung and stomach. Arrows mark lymphocytic infiltrates. Scale bar = 100 μm.
(D) Percentages of IFN-γ⁺, IL-4⁺, and IL-17A⁺ CD4⁺ T cells in lung tissue after PMA/ionomycin stimulation.
(E) Splenocytes from 12-week-old mice were stained for CD44, Foxp3, CD73, and FR4. Percentages of anergic (CD4⁺Foxp3⁻CD44^hiCD73^hiFR4^hi), effector/memory (CD4⁺Foxp3⁻CD44^hiCD73^loFR4^lo), and regulatory (CD4⁺Foxp3⁺) T cells are shown. * P<0.05; ** P<0.01; ns, not significant.

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