Blimp1 Prevents Methylation of Foxp3 and Loss of Regulatory T Cell Identity at Sites of Inflammation

Abstract

Foxp3⁺ regulatory T (Treg) cells restrict immune pathology in inflamed tissues; however, an inflammatory environment presents a threat to Treg cell identity and function. Here, we establish a transcriptional signature of central nervous system (CNS) Treg cells that accumulate during experimental autoimmune encephalitis (EAE) and identify a pathway that maintains Treg cell function and identity during severe inflammation. This pathway is dependent on the transcriptional regulator Blimp1, which prevents downregulation of Foxp3 expression and "toxic" gain-of-function of Treg cells in the inflamed CNS. Blimp1 negatively regulates IL-6- and STAT3-dependent Dnmt3a expression and function restraining methylation of Treg cell-specific conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Consequently, CNS2 is heavily methylated when Blimp1 is ablated, leading to a loss of Foxp3 expression and severe disease. These findings identify a Blimp1-dependent pathway that preserves Treg cell stability in inflamed non-lymphoid tissues.

Keywords: Blimp1; CNS; CNS2; DNA methyltransferases; Foxp3; Interleukin-6; epigenetic regulation; inflammation; regulatory T cells.

Graphical abstract

Figure 1

CNS Treg Cells are Stable and Express Blimp1 in Response to Proinflammatory Cytokines (A) Mononuclear cells were isolated from the CNS of EAE mice at the peak of disease and were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin to analyze the expression of IL-10, IL-17, and IFN-γ in Treg cells by flow cytometry. Mean of eight biological replicates ± SD, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). (B) CD4⁺Foxp3⁺ Treg cells were sorted from the CNS and spleen (SPL) of Foxp3 (GFP) reporter mice at the peak of EAE and subjected to RNA-seq. Principal-component analysis. (C) Foxp3 expression by intracellular staining of splenic Treg cells and CNS Treg cells at the peak of EAE. Mean of six biological replicates ± SD, derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD), t test, p < 0.00005. (D and E) Enrichment of CNS Treg “signature” genes in gene sets upregulated in T cells in response to IL-12 (Agarwal et al., 2009) or IL-27 or IFN-γ (Hall et al., 2012). Overlap of core enriched genes of the IL-12, IL-27, and IFN-γ signatures in CNS Treg cells as venn diagram (D) and volcano plot (E). (F) Enrichment of Blimp1 signature genes (Cretney et al., 2011) within CNS Treg cells and spleen Treg cells. (G) Blimp1 expression was analyzed within Foxp3⁺ cells in naive spleen and in indicated compartments at the peak of EAE in Blimp1 (YFP) reporter mice. Fraction of Blimp1 (YFP)⁺ cells in Treg cells, mean of nine biological replicates ± SD, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). Significance was calculated using ANOVA plus Tukey’s post test, p < 0.05. (H) Cytokines inducing Blimp1 in Treg cells in vitro. CD4⁺CD25⁺GITR⁺Blimp1 (YFP)⁻ Treg cells were sorted from the spleen and lymph nodes (LNs) of naive Blimp1 (YFP) reporter mice and cultured with anti-CD3 and anti-CD28 dynabeads in the presence of the indicated cytokines. On day 4, Blimp1 (YFP) expression was analyzed by flow cytometry. Data are summarized from two independent experiments (three technical replicates per experiment) (mean ± SD). (I and J) Blimp1 is induced in a STAT1-dependent manner in vivo. Tconv and Treg cells were fluorescence-activated cell sorting (FACS) sorted from the spleen and CNS of immunized mixed bone marrow chimeras generated with wild-type plus Stat1^−/− bone marrow. The expressions of Prdm1 (encoding Blimp1) (I) and Il10 (J) in Tconv and Treg cells were analyzed by qPCR of re-sorted congenically marked control and knockout cells. Data were summarized from two independent biological replicates. Symbols depict individual biological replicates (bars, mean ± SD). See also Figures S1 and S2 and Table S1.

Figure 2

Ablation of Blimp1 in Treg Cells Results in Failure to Control CNS Inflammation Blimp1^flox/flox (Blimp1^WT) or Foxp3 Cre mice and Blimp1^flox/flox × Foxp3 Cre (Blimp1^ΔFoxp3) were immunized with MOG(35-55) in CFA to induce EAE. Mononuclear cells were isolated at the peak of EAE. (A and B) EAE scores of Blimp1^ΔFoxp3 mice in comparison to Blimp1^WT controls (A) and Blimp1^ΔFoxp3 mice in comparison to Cre control mice (Blimp1^wt/wt x Foxp3 Cre) (B). Data were summarized from three independent experiments. (Blimp1^WT, n = 20; Foxp3 Cre, n = 4; Blimp1^ΔFoxp3, n = 31) (mean ± SEM). (two-way ANOVA, Sidak post test, p < 0.05). (C) Survival curve. Data were summarized from three independent experiments (Blimp1^WT, n = 8; Blimp1^ΔFoxp3, n = 10). Log-rank test (Mantel-Cox) (^∗∗p < 0.01). (D) Frequencies and absolute cell numbers of live CD4⁺ T cells and CD4⁺Foxp3⁺ Treg cells isolated from the CNS at the peak of EAE. Data were summarized from three independent experiments (mean ± SD). (E) Mononuclear cells were isolated from the draining lymph node (dLN), spleen, and CNS and stimulated with PMA and ionomycin to analyze IL-10, IL-17, and IFN-γ in Treg cells by flow cytometry. Data are summarized from twelve biological replicates, derived from three independent experiments. Symbols depict individual mice (bars, mean ± SD). Mann-Whitney test (^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001). (F–H) Wild-type and Il10^flox/flox × Foxp3 Cre (Il10^ΔFoxp3) mice were immunized with MOG(35-55) in CFA to induce EAE. Wild-type, n = 6; Il10^ΔFoxp3, n = 6 (mean ± SD) (F). (G) Mononuclear cells were isolated from the CNS and analyzed for Foxp3 expression by flow cytometry. The graph represents the frequency and absolute cell numbers of Treg cells. Symbols depict individual mice (bars, mean ± SD). (H) The isolated mononuclear cells from the CNS were stimulated with PMA and ionomycin to analyze IL-17 expression in Foxp3⁺ Treg cells by flow cytometry. Data are summarized from six biological replicates. Symbols depict individual mice (bars, mean ± SD).

Figure 3

Blimp1 Governs Treg Cell Identity in CNS Treg Cells Mixed bone marrow chimeras (MBMCs) were generated by reconstituting Rag1^−/− hosts with (CD45.1, wild-type) and Blimp1^ΔFoxp3 (CD45.2) bone marrow at a ratio of 1:1. The mixed bone marrow chimeras were immunized with MOG(35-55) in CFA to induce EAE. The mice were analyzed at the peak of EAE. (A) Chimerism between wild-type and Blimp1^ΔFoxp3 cells in the live CD4⁺ gate of spleen and CNS mononuclear cells and analysis of Foxp3 expression by flow cytometry in control (wild-type) Treg cells and Blimp1^ΔFoxp3 Treg cells isolated from the mixed bone marrow chimeras at the peak of EAE. Data are representative and summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test (^∗p < 0.05). (B) Expression of IL-10 and IL-17 in Foxp3⁺ Treg cells after ex vivo stimulation with PMA and ionomycin. (C) Data were summarized from three biological replicates. Symbols depict individual mice (bars, mean ± SD). Student’s t test (^∗p < 0.05). (D–F) Treg cells and conventional CD4⁺ T cells (Tconv) were sorted at the peak of EAE from the spleen and CNS of immunized mixed bone marrow chimeras (CD45.1 × Foxp3^GFP, wild-type; CD45.2, Blimp1^ΔFoxp3 × Foxp3^GFP) and subjected to RNA-seq. (D) Principal-component analysis of control Treg cells (wild-type), Blimp1^ΔFoxp3 Treg cells, and Tconv cells isolated from mixed bone marrow chimeras, calculated for all genes with greatest difference according to gene expression, with log₂ transformed row and column standardized data. (E) Genes upregulated (top) or downregulated (bottom) in CNS Treg cells as compared to spleen Treg cells (see Figure 1) were assessed as to their expression in Blimp1^ΔFoxp3 versus wild-type Treg cells and displayed in volcano plots. (F) Enrichment of genes that are normally suppressed by Foxp3 (Williams and Rudensky, 2007) in Blimp1^ΔFoxp3 CNS Treg cells as compared to wild-type Treg cells. See also Figure S3 and Table S2.

Figure 4

Blimp1 Regulates Genes in CNS Treg Cells in a Direct and Indirect Manner (A) ATAC peaks in splenic wild-type and Blimp1^ΔFoxp3 Tconv and Treg cells were quantified. Very few peaks were differentially enriched in Tconv cells (left), whereas substantial dysregulation could be identified in Treg cells (right). (B) Transcription factor motif prediction in differential ATAC peaks of splenic wild-type versus Blimp1^ΔFoxp3 Treg cells was performed with Homer by using known transcription factor motifs. Heatmap displays the log of the transcription factor (TF) prediction p value (left) and the expression of the respective transcription factors (right). For some transcription factors, multiple motifs were tested (denoted by numbers after the transcription factor name). (C) Homer was used for de novo motif prediction in differential ATAC peaks of splenic wild-type versus Blimp1^ΔFoxp3 Treg cells. The top 3 motifs of this analysis are shown. (D) Blimp1 peaks and differential ATAC peaks between wild-type and Blimp1^ΔFoxp3 Treg cells in CNS were identified within 50 kb of the transcriptional start sites of differentially expressed genes. Heatmap displays Blimp1 binding and ATAC up- or downregulated peaks in the top 75 differentially expressed genes between wild-type and Blimp1^ΔFoxp3 Treg cells in CNS (red bars). (E) Intergrative genomics viewer (IGV) screenshots of Blimp1 ChIP-seq (Mackay et al., 2016) and ATAC-seq tracks for CNS wild-type and Blimp1^ΔFoxp3 Treg cells. The scale of the Blimp1^ΔFoxp3 Treg ATAC-seq track was adjusted to display comparable intensities for non-changed ATAC peaks due to the overall reduced signal intensity in this dataset.

Figure 5

Blimp1 in Treg Cells Is Required to Maintain the Demethylated State of CNS2 Treg cells and Tconv cells from both the wild-type and Blimp1^ΔFoxp3 compartments of mixed bone marrow chimeras were sorted from the spleen and the CNS according to experimental set up of Figure 3, and their genomic DNA was analyzed for the methylation status of CNS2 (TSDR). The data are representative of three independent biological replicates. Each line represents one CpG motif (left). The degree of methylation at each CpG motif is represented according to the color code. Cumulative quantification of the methylation of all CpG islands in CNS2 (TSDR) in Tconv and Treg cells as indicated (right). One-way ANOVA with Holm-Sidak’s post-test (^∗p < 0.0001).

Figure 6

Lack of Blimp1 in Treg Cells Results in Instability of Treg Cells through Epigenetic Changes (A) Schematic scheme of experimental set up. (B and C) Total Treg cells, namely, both control Foxp3 Treg cells (Foxp3 Cre) (B) or wild-type (C) and Blimp1^ΔFoxp3 Tre cells together, were sorted from spleen and draining lymph node of immunized mixed bone marrow chimeras on day 8 post-immunization and were transferred into Rag1^−/− recipient mice along with congenically marked CD25^-CD44⁻CD90.1⁺ naive conventional CD4⁺ T cells (Thy 1.1 Tconv). On day 1 post-transfer, the recipient mice were immunized with MOG(35-55) and CFA. Mononuclear cells were isolated from the spleen and draining lymph node for analysis 8 days post-immunization. (C) Ki67 staining in wild-type and Blimp1^ΔFoxp3 Treg cells re-isolated from the spleen of the secondary hosts. Representative of three independent biological replicates. (D) Foxp3 expression was analyzed by intracellular staining. Frequency and geomean of Foxp3⁺ cells within the transferred Foxp3 Cre control Treg cells and Blimp1^ΔFoxp3 Treg cells isolated from the spleen of secondary hosts. Cumulative data of five biological replicates derived from two independent experiments. Symbols depict individual mice (bars, mean ± SD). Student’s t test (^∗p < 0.05). (E) Wild-type and Blimp1^ΔFoxp3 Treg cells were sorted from the secondary hosts, and genomic DNA was analyzed for methylation status of CNS2 (TSDR), Foxp3 promoter, and Ctla4. Representative of three independent biological replicates. Each line represents one CpG motif. The degree of methylation at each CpG motif is depicted according to the color code. (F) Gene expression of Dnmt1 and Dnmt3a by qPCR in wild-type and Blimp1^ΔFoxp3 Treg cells sorted from the spleen of secondary host mice. The expression of each gene was normalized to the control gene Actb and was plotted relative to the mean expression value of replicates for each gene. Cumulative data from five biological replicates, derived from two independent experiments. Symbols depict individual biological replicates (bars, mean ± SD). Student’s t test (^∗p < 0.05). (G) Methylation status of known Dnmt3a target genes in control Treg cells (Foxp3 Cre × Blimp1^wt/wt) and Blimp1^ΔFoxp3 Treg cells (Foxp3 Cre × Blimp1^flox/flox) re-isolated from secondary hosts. Representative of three independent biological replicates. The degree of methylation at each CpG motif is depicted according to the color code. See also Figure S4.

Figure 7

IL-6 Promotes the Loss of Foxp3 in Treg Cells in the Absence of Blimp1 by Inducing DNA Methylation (A) Treg cells sense IL-6 in the inflamed CNS. The CNS Treg cell transcriptome (see Figure 1) is enriched for IL-6 signature genes as assessed by gene set enrichment analysis (GSEA). (B) KLRG1⁻Foxp3 (GFP)⁺ and KLRG1⁺Foxp3 (GFP)⁺ Treg cells were sorted from spleen by flow cytometry of wild-type mice and tested for Prdm1 (encoding Blimp1) expression by qPCR. Data are summarized from two biological replicates. KLRG1⁻Blimp1⁻Foxp3 (GFP)⁺ Treg cells were sorted from spleen and lymph nodes of wild-type and Il6^−/− mice and tested for Dnmt3a expression by qPCR. Data are summarized from three biological replicates. Symbols depict individual biological replicates (bars, mean ± SD). Student’s t test (^∗p < 0.05). (C) Blimp1 (YFP) ⁻ and Blimp1 (YFP)⁺ Treg cells were sorted from the spleen and LNs of unmanipulated Blimp1 (YFP) reporter mice and stimulated with anti-CD3 and anti-CD28 dynabeads in the presence of either IL-2 alone or IL-2 and IL-6. On day 4, cultured Treg cells were analyzed for Foxp3 expression by intracellular staining. Cumulative data of three independent experiments, (mean ± SD). Two-way ANOVA (Sidak’s multiple comparison test), p < 0.05. (D) Lack of Dnmt3a reduces loss of Foxp3 in Treg cells in response to IL-6. KLRG1⁻ (Blimp1⁻) Foxp3⁺ Treg cells were purified from the spleen and LNs of control mice or inducible Dnmt3a-deficient animals (Dnmt3a^ΔMx1), cultured in the absence or presence of IL-6, and assessed for Foxp3 expression by flow cytometry after 4 days. Representative histograms of Foxp3 expression in IL-2 alone or IL-2 and IL-6 cultured control or Dnmt3a^ΔMx1 Treg cells. ΔMFI (mean fluorescence intensity; Foxp3) in individual mice (bars, mean ± SD). Student’s t test (^∗p < 0.05). (E) In some cultures, the Blimp1 (YFP)⁻ Treg cells were additionally treated with SGI-1027 at indicated concentrations to block Dnmt3a activity and analyzed for Foxp3 expression level on day 5 of culture. Cumulative data from two independent experiments. See also Figure S5.