Regulatory T cells protect the neonatal liver and secure the hepatic circadian rhythm

Regulatory T cells (Treg cells) play a crucial role in the immune system by controlling the establishment and maintenance of immune tolerance and homeostasis. Treg cells have been described to perform additional non-immunological functions beyond their classical functions in peripheral tissues. In this context, best examples are the specialized Treg cell population in visceral adipose tissue (VAT). The VAT-resident Treg cells gain the ability to control metabolic parameters. To further extent the concept of tissue-resident Treg cells, we aimed to analyze the phenotype and function of Treg cells in the liver tissue. We observed a significant accumulation of hepatic Treg cells in neonatal mice at around day 10 after birth. With progressive maturation of the mice the Treg frequency normalized. The neonatal hepatic Treg cells were highly proliferative as demonstrated by cell cycle measurements. Furthermore, the cells showed the characteristic Treg cell methylation pattern of the Foxp3 locus. The Treg cell accumulation in the neonatal liver occurred in an immature liver with strongly proliferating hepatocytes, a low-grad inflammatory signature and changes in key gene expression. Depletion of Treg cells with the associated loss of Treg-mediated immune control induced major changes in the liver, including increased expression of immune-related genes and genes regulating the circadian clock such as Rev-Erb-a or Per1. The circadian expression of approximately 400 genes in the liver was affected by the ablation of Treg cells. Our results indicate that Treg cells are important to secure the circadian rhythm of genes regulating the hepatic clock as well as clock-controlled genes. Furthermore, the presence of Treg cells is required for a normal expression of genes involved in liver metabolism, especially in the neonatal phase of the mice. Therefore, we propose that Treg cells do not only control the inflammatory state of the liver, but are also critical for the establishment and maintenance of liver homeostasis

Premature Expression of Foxp3 in Double-Negative Thymocytes

<div><p>Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tT_reg) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the T_reg lineage defining transcription factor Foxp3 in developing tT_reg cells depends on TCR signaling during the thymic selection process of these T cells. In this study, we surprisingly identify Foxp3⁺ immature thymocytes at the double-negative (DN) stage in transcription factor 7 (Tcf7)-deficient mice. These Foxp3⁺ cells did not express a TCR (β or γδ chains), CD3 or CD5 and therefore these cells were true DN cells. Further investigation of this phenomenon in a transgenic TCR model showed that Foxp3-expressing DN cells could not respond to TCR stimulation <i>in vivo</i>. These data suggest that Foxp3 expression in these DN cells occurred independently of TCR signaling. Interestingly, these Foxp3⁺ DN cells were located in a transition state between DN1 and DN2 (CD4^-CD8^-CD3^-TCR^-CD44^highCD25^low). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.</p></div

Analysis of Foxp3⁺ DN cells in TEa-Tcf7-deficient mice.

<p>(A) Representative plots showing TCRVβ6 and TCRVα2 expression on CD4SP thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the presence or absence of cognate antigen (Ag). The Tg TCR population is divided into TCR^high and TCR^low populations. (B-C) Quantification of the percentage of total (B) or TCR^high (C) TCRVβ6⁺TCRVα2⁺ cells among CD4SP thymocytes (n = 8). (D) Representative plots showing Foxp3 expression in DN TCRVβ6⁺TCRVα2⁺ thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the absence of Ag. (E) Quantification of Foxp3⁺ DN TCRVβ6⁺TCRVα2⁺ thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the presence or absence of Ag (n = 8). (F-G) Representative plots showing TCRVβ6 and TCRVα2 expression on DN Foxp3⁺ (F) or CD4SP Foxp3⁺ (G) thymocytes from TEa-Tcf7^+/+ and TEa-Tcf7^-/- mice in the presence or absence of Ag. Cells are pre-gated on TCRVβ6⁺TCRVα2⁺. Each dot represents one individual animal and mean is shown for all quantified data. Numbers show percentages of cells within the indicated box. NS, not significant, *** P < 0.001, **** P < 0.0001 (unpaired t-test).</p

Foxp3 expression at the DN cell stage in Tcf7-deficient mice.

<p>(A) Representative plots and quantification of Foxp3 staining in CD4^-CD8^- (DN) thymocytes from Tcf7^+/+ and Tcf7^-/- mice (n = 8). (B) Left panels: Representative plots showing Foxp3 and intracellular (IC) TCRβ staining in DN thymocytes from Tcf7^+/+ and Tcf7^-/- mice. Middle panels: TCRγδ and CD3 staining on DN Foxp3⁺TCRβ^- cells (gate R1). Right panel: Quantification of DN Foxp3⁺TCRβ^-TCRγδ^-CD3^- cells (gate R2) depicted as the percentage of total DN cells (n = 6). (C) Left panel: Representative histograms showing CD5 staining on Foxp3⁺ DN, Foxp3⁺ DP, and Foxp3⁺ CD4SP cells from Tcf7^-/- mice. Right panel: Quantification of CD5 geometric mean from DN, DP, and CD4SP Foxp3⁺ or Foxp3^- populations (n = 3). Mean + SD are shown for all quantified data. Numbers show percentages of cells within the indicated box. Each symbol represents an individual animal. ** P < 0.01 (unpaired t-test).</p

Genome-wide DNA-methylation landscape defines specialization of regulatory T cells in tissues

Regulatory T cells (T-reg cells) perform two distinct functions: they maintain self-tolerance, and they support organ homeostasis by differentiating into specialized tissue T-reg cells. We found that epigenetic modifications defined the molecular characteristics of tissue T-reg cells. Tagmentation-based whole-genome bisulfite sequencing revealed more than 11,000 regions that were methylated differentially in pairwise comparisons of tissue T-reg cell populations and lymphoid T cells. Similarities in the epigenetic landscape led to the identification of a common tissue T-reg cell population that was present in many organs and was characterized by gain and loss of DNA methylation that included many gene sites associated with the T(H)2 subset of helper T cells, such as the gene encoding cytokine IL-33 receptor ST2, as well as the production of tissue-regenerative factors. Furthermore, the ST2-expressing population was dependent on the transcriptional regulator BATF and could be expanded by IL-33. Thus, tissue T-reg cells integrate multiple waves of epigenetic reprogramming that define their tissue-restricted specialization

Quantitative Proteomics Identifies TCF1 as a Negative Regulator of Foxp3 Expression in Conventional T Cells

Regulatory T cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. Here, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Using viral over-expression, we identified TCF1 as a repressor of Foxp3 expression. In TCF1-deficient animals, increased levels of Foxp3(intermediate)CD25(negative) T cells were identified. CRISPR-Cas9 knockout studies in primary human and mouse conventional CD4 T (T-conv) cells revealed that TCF1 protects T-conv cells from inadvertent Foxp3 expression. Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells

Rbpj expression in regulatory T cells is critical for restraining TH2 responses

Transcriptional regulator Rbpj is involved in T-helper subset differentiation. Here the authors show that expression of Rbpj in regulatory T cells is required to both regulate TH2 responses and regulate Treg TH2 differentiation potential