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Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells - PubMed

️Tue Jan 01 2019

Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells

Susanne Herppich et al. Front Immunol. 2019.

Abstract

Regulatory T (Treg) cells mainly develop within the thymus and arise from CD25⁺Foxp3^- (CD25⁺ TregP) or CD25^-Foxp3⁺ (Foxp3⁺ TregP) Treg cell precursors resulting in Treg cells harboring distinct transcriptomic profiles and complementary T cell receptor repertoires. The stable and long-term expression of Foxp3 in Treg cells and their stable suppressive phenotype are controlled by the demethylation of Treg cell-specific epigenetic signature genes including an evolutionarily conserved CpG-rich element within the Foxp3 locus, the Treg-specific demethylated region (TSDR). Here we analyzed the dynamics of the imprinting of the Treg cell-specific epigenetic signature genes in thymic Treg cells. We could demonstrate that CD25⁺Foxp3⁺ Treg cells show a progressive demethylation of most signature genes during maturation within the thymus. Interestingly, a partial demethylation of several Treg cell-specific epigenetic signature genes was already observed in Foxp3⁺ TregP but not in CD25⁺ TregP. Furthermore, Foxp3⁺ TregP were very transient in nature and arose at a more mature developmental stage when compared to CD25⁺ TregP. When the two Treg cell precursors were cultured in presence of IL-2, a factor known to be critical for thymic Treg cell development, we observed a major impact of IL-2 on the demethylation of the TSDR with a more pronounced effect on Foxp3⁺ TregP. Together, these results suggest that the establishment of the Treg cell-specific hypomethylation pattern is a continuous process throughout thymic Treg cell development and that the two known Treg cell precursors display distinct dynamics for the imprinting of the Treg cell-specific epigenetic signature genes.

Keywords: Foxp3; IL-2; TSDR; Treg cell; Treg cell precursors; demethylation; epigenetic signature; thymus.

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Figures

**Figure 1**
Newly generated CD25⁺Foxp3^hCD2+ Treg cells progressively mature within the thymus. Thymocytes were isolated from Foxp3^hCD2xRag1^GFP reporter mice and analyzed by flow cytometry or prepared for subsequent methylation analysis. **(A)** Representative dot plots show the gating of CD4SP thymocytes among living cells (LD⁻, LIVE/DEAD⁻), newly generated Rag^GFP+ cells among CD4SP thymocytes, CD25⁺Foxp3^hCD2+ Treg cells among Rag^GFP+CD4SP thymocytes, and CD24^hi/int/low cells among CD25⁺Foxp3^hCD2+Rag^GFP+CD4SP thymocytes. Numbers specify frequencies of cells in indicated gates. **(B,C)** (Left) Representative histograms depict the expression of CD25 **(B)** and Foxp3^hCD2 **(C)** among CD24^hi/int/low subsets of CD25⁺Foxp3^hCD2+Rag^GFP+CD4SP thymocytes. (Right) Scatter plots summarize the data from three independent experiments and bars indicate mean ± SD. Each symbol represents an individual mouse. The significance was calculated using Kruskal-Wallis with Dunn's test (^**p < 0.01). Data are given relative to the maximal span (x_max-x_min) per experiment. **(D,E)** Genomic DNA was isolated from sorted CD24^hi/int/low subsets of CD25⁺Foxp3^hCD2+Rag^GFP+CD4SP thymocytes for analysis of the methylation status of TSDR, *Helios, Eos, Ctla-4*, and *Gitr*. Cells were pooled from four independent sorts. **(D)** Each bar represents an individual CpG motif. Percentage of methylation is color-coded according to the scale. **(E)** Bar graph summarizes changes in the CpG methylation ratio of the Treg cell-specific epigenetic signature genes in CD24^hi/int/low subsets of CD25⁺Foxp3^hCD2+Rag^GFP+CD4SP thymocytes (bars indicate mean ± SD of all CpG motifs). The significance was calculated using Kruskal-Wallis with Dunn's test (^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

**Figure 2**
Foxp3⁺ TregP already display a partially demethylated Treg cell-specific epigenetic signature. **(A,B)** Thymocytes were isolated from Foxp3^hCD2xRag1^GFP reporter mice and analyzed by flow cytometry. **(A)** Representative dot plot shows the gating of CD25⁺Foxp3^hCD2− (CD25⁺ TregP), CD25⁻Foxp3^hCD2+ (Foxp3⁺ TregP), and CD25⁺Foxp3^hCD2+ (Treg cells) cells among Rag^GFP+CD4SP thymocytes. Numbers specify frequencies of cells in indicated gates. **(B)** (Left) Representative histograms depict CD24 expression among CD25⁺ TregP, Foxp3⁺ TregP and Treg cells. (Right) Scatter plot summarizes the data from three independent experiments and bars indicate mean ± SD. Each symbol represents an individual mouse. The significance was calculated using Kruskal-Wallis with Dunn's test (^*p < 0.05; ^****p < 0.0001). Data are given relative to the maximal span (x_max-x_min) per experiment. **(C)** Thymocytes were isolated from Foxp3^GFPCrexROSA26^RFP fate-mapping mice and analyzed by flow cytometry. (Left) Representative histograms depict RFP expression among CD25⁺ TregP, Foxp3⁺ TregP and Treg cells. (Right) Scatter plot summarizes the data from three independent experiments and bars indicate mean ± SD. Each symbol represents an individual mouse. The significance was calculated using Kruskal-Wallis with Dunn's test (^***p < 0.001). **(D)** Thymocytes were isolated from Foxp3^hCD2 reporter mice, and indicated populations pre-gated on newly generated CD73^lowCD4SP thymocytes were sorted. Genomic DNA was isolated from sorted cells for analysis of the methylation status of TSDR, *Helios, Eos, Ctla-4*, and *Gitr*. Cells were pooled from two independent sorts. Each bar represents an individual CpG motif. Percentage of methylation is color-coded according to the scale.

**Figure 3**
CD25⁺ TregP and Foxp3⁺ TregP show distinct responsiveness toward IL-2. Thymocytes were isolated from Foxp3^hCD2 reporter mice, CD25⁺ TregP and Foxp3⁺ TregP pre-gated on newly generated CD73^lowCD4SP thymocytes were sorted, and sorted Treg cell precursors were cultured in the presence of rmIL-2. At day 3 or 5 of the culture, CD25⁺Foxp3^hCD2+ Treg cells were FACS-sorted and analyzed by flow cytometry or prepared for subsequent methylation analysis. **(A)** (Left) Representative dot plots show the gating of CD25⁺Foxp3^hCD2+ Treg cells from the indicated cultures. Numbers specify frequencies of cells in indicated gates. (Right) Scatter plot summarizes the data from six independent experiments and bars indicate mean ± SD. Each symbol represents an individual experiment. The significance was calculated using Mann-Whitney test (^**p < 0.01). **(B)** (Left) Representative histograms depict Foxp3^hCD2 expression among Treg cells sorted from indicated cultures. (Right) Scatter plots summarize the data from six independent experiments and bars indicate mean ± SD. Each symbol represents an individual experiment. The significance was calculated using Mann-Whitney test (ns = not significant; ^**p < 0.01). **(C,D)** Genomic DNA was isolated from sorted CD25⁺Foxp3^hCD2+ Treg cells of the indicated cultures for analysis of the methylation status of TSDR, *Helios, Eos, Ctla-4*, and *Gitr*. Cells were pooled from six independent experiments. **(C)** Each bar represents an individual CpG motif. Percentage of methylation is color-coded according to the scale. **(D)** Bar graph summarizes changes in the CpG methylation ratio of the Treg cell-specific epigenetic signature genes in sorted Treg cell precursors (input) and sorted CD25⁺Foxp3^hCD2+ Treg cells from the indicated cultures (bars indicate mean ± SD of all CpG motifs). The significance was calculated using Kruskal-Wallis with Dunn's test (^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

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References

1. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. (2003) 4:330–6. 10.1038/ni904 - DOI - PubMed
1. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. (2003) 299:1057–61. 10.1126/science.1079490 - DOI - PubMed
1. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al. . The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet. (2001) 27:20–1. 10.1038/83713 - DOI - PubMed
1. Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, et al. . Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet. (2001) 27:68–73. 10.1038/83784 - DOI - PubMed
1. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol. (2003) 4:337–42. 10.1038/ni909 - DOI - PubMed

Dynamic Imprinting of the Treg Cell-Specific Epigenetic Signature in Developing Thymic Regulatory T Cells - PubMed