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, Liaoning Province, China

https://doi.org/10.7554/eLife.96868.1

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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 and stimulated with anti-CD3 (2 µg/mL, plate coated) and anti-CD28 (1 µg/mL) for 12 hours. Quantitative PCR analysis was employed to determine the mRNA levels of cytokines, including Ifng, Il4, and Il2. The experiments were conducted four times with duplicates for each sample, and the data are presented as mean±SD.
(B) Sorted CD4SP thymocytes, CD4⁺ RTEs and CD4⁺ naive T cells from Cd11c- p28^f/fmice and WT littermates 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 were showed on the left. The statistical data collected from three pairs of mice were shown on the right as mean±SD.
(C) Cells sorted as described in (B) were cultured under Th0 conditions for 3 days, and the concentrations of IFN-γ and IL-4 in the supernatants of CD4SP thymocytes, RTEs, and naive T cells were measured by ELISA. The statistical data collected from three pairs of mice were shown as mean±SD.
(D) Sorted cells were treated as described in (A). The mRNA levels of transcription factors Tbx21 and Gata3 were determined by quantitative PCR. The experiments were repeated 4 times with duplicates for each sample and the data are presented as mean±SD.
(E) Western blotting was performed after a 3-day culture under Th0 conditions. The experiments were repeated three times with consistent results. The statistical data showed the relative protein expression levels of T-bet, quantified by densitometry and normalized to β-actin levels (mean±SD).
(F) Freshly sorted cells were dissolved in Trizol. The mRNA levels of Ifng and Tbx21 were determined by quantitative PCR. The experiments were repeated four times with triplicates for each sample and the data are presented as mean±SEM. Statistical differences between groups were determined by the Student’s t-test. * P < 0.05, ** P< 0.01, *** P<0.001.

Enhanced IFN-γ production and T-bet expression in Il27ra^-/- mice initiated from CD4SP thymocytes stage.
(A) CD4SP thymocytes and naive CD4⁺ T cells were isolated from Il27ra^-/- mice and WT littermates and stimulated with anti-CD3 (2 µg/mL, plate coated) and anti-CD28 (1 µg/mL) for 12 hours. The mRNA levels of cytokines including Ifng, Il4 and Il2, and transcription factors Tbx21 and Gata3 were determined by quantitative PCR. The experiments were conducted three times with duplicates for each sample and the data are presented as mean±SEM.
(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 determined by intracellular staining. Representative dot plots were showed on the left. The statistical data collected from three pairs of mice were shown on the right as mean±SD. Statistical differences between groups were determined by the Student’s t-test. * P< 0.05, ** P< 0.01.

Distinct methylation patterns of DNA and H3K4 at Ifng and Tbx21 promoter regions in CD4SP thymocytes from IL27p28-deficient mice.
(A) DNA methylation analysis of nine sites in the Ifng promoter with sodium bisulfite- treated genomic DNA from GFP⁺CD4⁺CD8^-CD44^lo CD4SP thymocytes. Each row represents a cloned and sequenced allele. Analyses included 10 clones from one of the three independent experiments. Filled circles (●) represent methylated cytosine, and open circles (○) the unmethylated residue.
(B) The left panel graphs show the percent of methylation at each individual site from one representative experiment, while the right panel graphs show the average percentage of methylation of three adjacent sites (group1: −205, −190, −170; group2: − 53, −45, −34; group3: +16, +96, +120) and all CpG sites from the three independent experiments. The statistical data were shown as mean±SEM.
(C) DNA methylation analysis for five CpG site upstream of the transcription start site of Il4 with sodium bisulfite-treated genomic DNA from GFP⁺CD4⁺CD8^-CD44^lo CD4SP thymocytes. Each row represents a cloned and sequenced allele. Analyses included 10 clones from the two independent experiments. Filled circles (●) represent methylated cytosine, and open circles (○) the unmethylated residue.
(D) Graphs show the percentage of methylation at each individual site (left panel) or all CpG sites (right panel).
(E-G) Histone trimethylation analysis on freshly isolated CD4SP thymocytes from IL27p28-deficient mice and littermates. ChIP-qPCR was performed using H3K4me3 (E), H3K27me3 (F) and H3K9me3 (G) specific antibodies. Quantitative PCR was conducted using primers specific for the promoters and trans-regulatory regions of Ifng, Tbx21, Il4, and Gata3. The experiments were repeated three times with duplicates for each sample and the data are presented as mean±SEM. * P<0.05; ** P<0.01 determined by Student’s t-test. Abbreviation: pro., promoter.

Increased expression of STAT1-activated genes in CD4SP thymcoytes from Cd11c-p28^f/f mice.
RNA-seq was performed to probe the transcriptome of CD4SP thymcoytes from Cd11c-p28^f/fand WT mice.
(A) The top 10 hits from Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses are shown for Differentially Expressed Genes (DEGs) up-regulated in Cd11c-p28^f/fmice.
(B) Comparison of genes up- or down-regulated in the absence of IL27p28 (Cd11c- p28^f/f) and those activated or suppressed by STAT1. The numbers of overlapping genes in different categories are indicated.
(C) Validation of the expression levels of representative genes up-regulated by RNA- Seq using quantitative PCR. The experiments were repeated three times with duplicates and data are presented as fold changes in cKO versus WT mice (mean±SEM).
(D) Coordinated upregulation of STAT1-activated genes in CD4SP thymcoytes from Cd11c-p28^f/f mice revealed by Gene Set Enrichment Analysis (GSEA).
(E) Interaction network of the proteins encoded by DEGs. Nodes represent individual proteins and edges between two nodes represent interactions between proteins. Larger nodes indicate higher degrees of interaction. Red and green nodes represent up- regulated and down-regulated genes, respectively. Gray nodes were not in our DEG datasets but were inserted by Network Analyst as they are connected to the network.

Heightened activation of STAT1 in CD4SP thymocytes from Cd11c-p28^f/f mice.
(A) Intracellular staining was performed with freshly isolated thymocytes from Cd11c- p28^f/f mice and WT littermates using antibodies against phosphorylated STAT1 (Y701), STAT3 (Y705) and STAT4 (Y693). Representative histograms are shown for CD4SP thymocytes (left). The mean fluorescence intensity (MFI) from three independent experiments are presented as mean±SD (right).
(B) Cell lysate was prepared from purified CD4SP thymocytes, CD4⁺ RTEs and naive CD4⁺ T cells from WT and Cd11c-p28^f/fmice. The levels of total and phosphorylated STAT1 (Y701), STAT3 (Y705) and STAT4 (Y693) were examined using Western blotting. The experiments were repeated three times with similar results. Representative blots are shown on the left, and the relative expression levels of phosphorylated and total STATs are presented as mean±SD following quantification by densitometry and normalization to β-actin.
(C) Increased binding of STAT1 onto the promoter and regulatory regions of Tbx21 and Ifng loci.
(D) Correlation of the levels of STAT1 binding and H3K4me3 at the Tbx21 and Ifng loci. * P<0.05; ** P< 0.01.

Exacerbated autoimmune responses in Aire^-/- mice in the absence of IL27p28.
(A) Splenocytes from 6-8 week-old WT (n=6) and Cd11c-p28^f/f (n=6) mice were analyzed for CD44 and CD62L expression after gating on CD4⁺ T cells. Representative dot plots are shown on the left. The percentage of CD44^loCD62L⁺ naive and CD44^hiCD62L^- activated T cells are presented as mean±SD on the right
(B-D) WT, Cd11c-p28^f/f, Aire^-/-, and Cd11c-p28^f/fand Aire^-/- double knockout mice were sacrificed at 24-30 weeks of age and subjected to various analyses. Each symbol represents an individual mouse. The horizontal bars denote the average and SD.
(B) The serum levels of anti-dsDNA antibodies as measured by ELISA.
(C) Representative images of H&E staining (left) and histological scores (right) of the lung and stomach. The arrows point to lymphocytic infiltrates. Scale bar equals 100 μm.
(D) The percentage of IFN-γ⁺, IL-4⁺, and IL-17A⁺ cells in CD4⁺ T cells isolated from the lung tissue as determined by intracellular staining following stimulation with PMA/ionomycin.
(E) Splenocytes from 12-week-old mice were stained for the expression of various cellular markers. CD44 and Foxp3 expression was analyzed by gating on CD4⁺ T cells, and Foxp3⁻CD44^hi population was further analyzed for CD73 and FR4 staining. The percentage of anergic (CD4⁺Foxp3⁻CD44^hiCD73^hiFR4^hi), effector/memory (CD4⁺Foxp3⁻CD44^hiCD73^loFR4^lo), and regulatory (CD4⁺Foxp3⁺) T cells are presented on the right. Each symbol represents an individual mouse. The horizontal bars denote the average and SD. * P<0.05; ** P<0.01; ns, not significant.

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