Thymic Treg Cell Development Research Articles

NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells.

The transcription factors of the nuclear factor of activated T cell (NFAT) family play a crucial role in multiple aspects of T cell function. It has recently been reported that NFATs play an important role in the suppressive function of CD4+CD25+Foxp3+ regulatory T (Treg) cells. In this study, we have investigated the role of NFATs in the thymic development of Treg cells in mice. We show that NFAT factors are dispensable for the development of Foxp3+ Treg cells in the thymus but are critical for the maintenance of both the phenotype and survival of Treg cells in the thymus as well as in peripheral lymphoid organs. Specifically, the homeostasis of CD4+CD25+Foxp3+ but not the CD4+CD25−Foxp3+ fraction is severely perturbed when NFAT signaling is blocked, leading to a strongly reduced Treg population. We underscored this intriguing effect of NFAT on CD4+CD25+Foxp3+ Treg cells to the disruption of survival signals provided by interleukin 2 (IL-2). Accordingly, blocking Treg cell death by abolishing the activity of pro-apoptotic Bcl-2 family member Bim, compensated for the survival defects induced due to a lack of NFAT-IL-2-IL-2R signaling. Inhibition of NFAT activity led to a strong reduction in the number of Foxp3+ Treg cells; however, it did not influence the level of Foxp3 expression on an individual cell basis. In addition, we show a differential effect of IL-2 and IL-7 signaling on Foxp3+ Treg versus CD4+CD25− T cell development, again underlining the dispensability of NFAT signaling in the development, but not in the maintenance of Foxp3+ Treg cells.

RANK links thymic regulatory T cells to fetal loss and gestational diabetes in pregnancy.

Successful pregnancies rely on adaptations within the mother1, including marked changes within the immune system2. It has long been known that the thymus, the central lymphoid organ, changes markedly during pregnancy3. However, the molecular basis and importance of this process remain largely obscure. Here we show that the osteoclast differentiation receptor RANK4,5 couples female sex hormones to the rewiring of the thymus during pregnancy. Genetic deletion of Rank (also known as Tnfrsf11a) in thymic epithelial cells results in impaired thymic involution and blunted expansion of natural regulatory T (Treg) cells in pregnant female mice. Sex hormones, in particular progesterone, drive the development of thymic Treg cells through RANK in a manner that depends on AIRE+ medullary thymic epithelial cells. The depletion of Rank in the mouse thymic epithelium results in reduced accumulation of natural Treg cells in the placenta, and an increase in the number of miscarriages. Thymic deletion of Rank also results in impaired accumulation of Treg cells in visceral adipose tissue, and is associated with enlarged adipocyte size, tissue inflammation, enhanced maternal glucose intolerance, fetal macrosomia, and a long-lasting transgenerational alteration in glucose homeostasis, which are all key hallmarks of gestational diabetes. Transplantation of Treg cells rescued fetal loss, maternal glucose intolerance and fetal macrosomia. In human pregnancies, we found that gestational diabetes also correlates with a reduced number of Treg cells in the placenta. Our findings show that RANK promotes the hormone-mediated development of thymic Treg cells during pregnancy, and expand the functional role of maternal Treg cells to the development of gestational diabetes and the transgenerational metabolic rewiring of glucose homeostasis.

Autocrine TGF-β1 Maintains the Stability of Foxp3+ Regulatory T Cells via IL-12Rβ2 Downregulation.

Transforming growth factor beta 1 (TGF-β1) is an immunosuppresive cytokine that plays an essential role in immune homeostasis. It is well known that regulatory T (Treg) cells express TGF-β1; however, the role of autocrine TGF-β1 in the development, function, and stability of Treg cells remains poorly understood. We found that Treg cell-derived TGF-β1 was not required for the development of thymic Treg cells in mice, but played a role in the expression of latency-associated peptide and optimal suppression of naïve T cell proliferation in vitro. Moreover, the frequency of Treg cells was significantly reduced in the mesenteric lymph nodes of the Treg cell-specific TGF-β1-deficient mice, which was associated with increased frequency of IFN-γ-producers among Treg cells. TGF-β1-deficient Treg cells were more prone to express IFN-γ than TGF-β1-sufficient Treg cells in a dendritic cell-mediated stimulation in vitro as well as in an adoptive transfer study in vivo. Mechanistically, TGF-β1-deficient Treg cells expressed higher levels of Il12rb2 and were more sensitive to IL-12-induced conversion into IFN-γ-producing Treg cells or IFN-γ-producing exTreg cells than TGF-β1-sufficient Treg cells. Our findings demonstrate that autocrine TGF-β1 plays a critical role in the optimal suppressive activity and stability of Treg cells by downregulating IL-12R on Treg cells.

The Transcription Factor MAZR/PATZ1 Regulates the Development of FOXP3+ Regulatory T Cells

Forkhead box protein P3+ (FOXP3+) regulatory Tcells (Treg cells) play a key role in maintaining tolerance and immune homeostasis. Here, we report that a T cell-specific deletion of the transcription factor MAZR (also known as PATZ1) leads to an increased frequency of Treg cells, while enforced MAZR expression impairs Treg cell differentiation. Further, MAZR expression levels are progressively downregulated during thymic Treg cell development and during in-vitro-induced human Treg cell differentiation, suggesting that MAZR protein levels are critical for controlling Treg cell development. However, MAZR-deficient Treg cells show only minor transcriptional changes exvivo, indicating that MAZR is not essential for establishing the transcriptional program of peripheral Treg cells. Finally, the loss of MAZR reduces the clinical score in dextran-sodium sulfate (DSS)-induced colitis, suggesting that MAZR activity in Tcells controls the extent of intestinal inflammation. Together, these data indicate that MAZR is part of a Treg cell-intrinsic transcriptional network that modulates Treg cell development.

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

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.

Generation of Foxp3+CD25- Regulatory T-Cell Precursors Requires c-Rel and IκBNS.

Next to the classical developmental route, in which first CD25 and subsequently Foxp3 are induced to generate thymic regulatory T (Treg) cells, an alternative route has been described. This alternative route is characterized by reciprocal induction of Foxp3 and CD25, with CD25 induction being required to rescue developing Treg cells from Foxp3-induced apoptosis. NF-κB has been demonstrated to be crucial for the development of thymic Treg cells via the classical route. However, its impact on the alternative route is poorly characterized. Using single and double deficient mice for key regulators of the classical route, c-Rel and IκBNS, we here demonstrate that NF-κB is essential for the generation of alternative CD25−Foxp3+ precursors, as well. Thus, c-Rel and IκBNS govern both routes of thymic Treg cell development.

Tissue resident and follicular Treg cell differentiation is regulated by CRAC channels

T regulatory (Treg) cells maintain immunological tolerance and organ homeostasis. Activated Treg cells differentiate into effector Treg subsets that acquire tissue-specific functions. Ca2+ influx via Ca2+ release-activated Ca2+ (CRAC) channels formed by STIM and ORAI proteins is required for the thymic development of Treg cells, but its function in mature Treg cells remains unclear. Here we show that deletion of Stim1 and Stim2 genes in mature Treg cells abolishes Ca2+ signaling and prevents their differentiation into follicular Treg and tissue-resident Treg cells. Transcriptional profiling of STIM1/STIM2-deficient Treg cells reveals that Ca2+ signaling regulates transcription factors and signaling pathways that control the identity and effector differentiation of Treg cells. In the absence of STIM1/STIM2 in Treg cells, mice develop a broad spectrum of autoantibodies and fatal multiorgan inflammation. Our findings establish a critical role of CRAC channels in controlling lineage identity and effector functions of Treg cells.

In vitro Differentiation of Thymic Treg Cell Progenitors to Mature Thymic Treg Cells

Thymic Treg cell differentiation occurs via a two-step process. Step one generates Treg cell progenitors (TregP) and is driven by strong TCR interactions with antigens presented in the thymus. Step two is initiated by activation of STAT5 via IL-2, or IL-15, leading to mature Treg cells capable of emigrating from the thymus and mediating immune tolerance and homeostasis in peripheral tissues. Herein we describe an in vitro TregP cell differentiation assay that models the second, cytokine dependent, step of thymic Treg cell development. It can be utilized with relative ease to determine if a population of thymocytes represents a potential progenitor population for Treg cells as well as test how different cytokines or chemical inhibitors modulate the differentiation of known TregP cell populations into mature Treg cells.

A20 Restrains Thymic Regulatory T Cell Development.

Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell–specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3− thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell–mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.

Epigenetic orchestration of thymic Treg cell development.

Regulatory T cells develop in the thymus as a distinct lineage of T cells instructed by the lineage-specifying transcription factor Foxp3. Epigenetic imprinting by the genome organizer Satb1 precedes this cell-fate 'decision' during thymocyte development.

Thymus-Derived Regulatory T Cells Are Positively Selected on Natural Self-Antigen through Cognate Interactions of High Functional Avidity

Regulatory T (Treg) cells expressing Foxp3 transcripton factor are essential for immune homeostasis. They arise in the thymus as a separate lineage from conventional CD4(+)Foxp3(-) T (Tconv) cells. Here, we show that the thymic development of Treg cells depends on the expression of their endogenous cognate self-antigen. The formation of these cells was impaired in mice lacking this self-antigen, while Tconv cell development was not negatively affected. Thymus-derived Treg cells were selected by self-antigens in a specific manner, while autoreactive Tconv cells were produced through degenerate recognition of distinct antigens. These distinct modes of development were associated with the expression of Tcell receptor of higher functional avidity for self-antigen by Treg cells than Tconv cells, a difference subsequently essential for the control of autoimmunity. Our study documents how self-antigens define the repertoire of thymus-derived Treg cells to subsequently endow this cell type with the capacity to undermine autoimmune attack.

Excessive miR-27 expression impairs regulatory T cell-mediated immunological tolerance

Abstract MicroRNAs (miRNAs) are tightly regulated in the immune system as aberrant expression of miRNAs often results in hematopoietic malignancies and autoimmune diseases. Previously, elevated levels of miR-27 in T cells isolated from multiple sclerosis patients has been suggested to facilitate disease progression through inhibiting Th2 immunity and promoting pathogenic Th1 responses. Here we show while mice with T cell-specific overexpression of miR-27 harbor dysregulated Th1 responses and develop autoimmune pathology, these disease phenotypes are not driven by miR-27 in effector T cells in a cell autonomous manner but rather resulted from a perturbed regulatory T (Treg) cell compartment. Excessive miR-27 expression in T cells severely impairs thymic Treg cell development and TGFb-mediated conversion of peripheral Treg cells from conventional T (Tconv) cells. Moreover, Treg cells with exaggerated miR-27-mediated gene regulation exhibit diminished homeostatic potential and suppressor function in vivo. Mechanistically, miR-27 represses a subset of genes that play critical roles in controlling multiple aspects of Treg cell biology. Collectively, our data show miR-27 functions as a key regulator in Treg cell development and function and suggest that proper regulation of miR-27 is pivotal to safeguard Treg cell-mediated immunological tolerance.

Thymic B cells promote thymus-derived regulatory T cell development and proliferation

Thymic CD4+ FoxP3+ regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell–cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3+ Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFβ. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells.

Costimulation via the tumor-necrosis factor receptor superfamily couples TCR signal strength to the thymic differentiation of regulatory T cells.

Regulatory T (Treg) cells express tumor necrosis factor receptor superfamily (TNFRSF) members, but their role in thymic Treg development is undefined. We demonstrate that Treg progenitors highly express the TNFRSF members GITR, OX40, and TNFR2. Expression of these receptors correlates directly with T cell receptor (TCR) signal strength, and requires CD28 and the kinase TAK1. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF agonists enhanced Treg differentiation by augmenting IL-2R/STAT5 responsiveness. GITR-ligand costimulation elicited a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on Treg progenitors translates strong TCR signals into molecular parameters that specifically promote Treg differentiation and shape the Treg repertoire.

MARCHing for tolerance

Ubiquitylation of MHC class II molecules by DC-expressed MARCH1 is required for thymic TReg cell development.

Advances in basic and clinical immunology in 2012

Basic and clinical immunology articles published in the Journal in 2012 were mostly related to the expanding area of primary immunodeficiencies (PIDs). Novel forms of PID were identified by using whole-exome sequencing or after careful examination of flow cytometric data, as in the reports of lymphocyte-specific protein tyrosine kinase, CD27, and CD21 deficiencies. Absent IgG and IgA memory B cells were described in patients with hyper-IgE syndrome, which is consistent with defective antibody response and suggests a potential benefit of immunoglobulin replacement. Impaired production of antibodies to polysaccharide antigens by the human B-cell subset analog to murine B-1 cells was reported in a child with selective polysaccharide antibody deficiency. Increased production of inflammatory cytokines by monocyte-derived cells on Toll-like receptor activation was reported in patients with X-linked agammaglobulinemia, underscoring the important role of Bruton tyrosine kinase in modulation of inflammation. The mechanisms explaining susceptibility to yeast infections and development of chronic mucocutaneous candidiasis were extensively studied. Universal newborn screening for T-cell deficiencies is being implemented in several states, resulting in the diagnosis of a higher number of immunodeficient newborns than previously estimated. The use of laboratory testing to distinguish PIDs from HIV infection was clarified. In the management of PIDs, refinement of indication and strategies to hematopoietic stem cell transplantation resulted in improved outcomes. The use of anti-IL-6 mAbs showed promise as an alternative treatment in patients with Schnitzler syndrome.

Abrogation of CD40–CD154 Signaling Impedes the Homeostasis of Thymic Resident Regulatory T Cells by Altering the Levels of IL-2, but Does Not Affect Regulatory T Cell Development

Identification of costimulatory signals required for murine regulatory T (Treg) cell development relies on measuring the frequency of total thymic Treg cells. However, the thymus contains both resident and newly developed Treg cells; whether such signals target both populations is unknown. In this study, we show that CD40-CD154 blockade specifically targeted thymic resident Treg cells, but not, as was previously believed, newly developed Treg cells. Unlike CD28-CD80/CD86 signals, CD40-CD154 signals were not required for Treg cell precursor development. Instead we demonstrate that homeostatic proliferation of thymic resident Treg cells was dependent on CD40-CD154 signals maintaining IL-2 levels. Furthermore, in newborn mice, where all Treg cells are newly developed, blockade of CD40-CD154 signals had no effect on thymic Treg numbers or their proliferation. Our studies highlight the complexity in the study of thymic Treg cell development due to the heterogeneity of thymic Treg cells.

Vagaries of Fluorochrome Reporter Gene Expression in Foxp3+ Regulatory T Cells

CD4+CD25+ regulatory T (Treg) cell lineage commitment and expression of the transcription factor Foxp3 can be induced at the CD4+CD8+ double-positive (DP) and CD4+CD8? single-positive stages of thymic development, as well as in postthymic CD4+ T cells in peripheral lymphoid tissues. The availability of transgenic mice with Foxp3-dependent fluorochrome reporter gene expression has greatly facilitated studies on the intra- and extrathymic generation of murine Foxp3+ Treg cells. Here, we performed a comparative analysis of thymic Treg cell development and peripheral compartments of mature Treg cells in various transgenic strains with gene targeted and bacterial artificial chromosome (BAC)-driven Foxp3-fluorochrome expression. These studies revealed a relative deficiency of Foxp3+ DP thymocytes selectively in mice with targeted insertion of the fluorochrome reporter gene coding sequence into the endogenous Foxp3 gene. While Foxp3 BAC-driven fluorochrome expression in ex vivo CD4+ T cells was found to faithfully reflect Foxp3 protein expression, we provide evidence that Foxp3 BAC transgenesis can result in sizable populations of Foxp3+ Treg cells that lack fluorochrome reporter expression. This could be attributed to both timely delayed up-regulation of BAC expression in developing Treg cells and the accumulation of peripheral Foxp3+ Treg cells with continuous transcriptional inactivity of the Foxp3 BAC transgene.

Intraclonal competition limits the fate determination of regulatory T cells in the thymus

Because the deletion of self-reactive T cells is incomplete, thymic development of natural Foxp3+CD4+ regulatory T (Treg) cells is required for preventing autoimmunity. However, the role of T cell receptor (TCR) specificity in thymic Treg cell development remains controversial. To address this issue, we generated a transgenic line expressing a naturally occurring Treg cell-derived TCR. Surprisingly, efficient thymic Treg cell development occurred only when the antigen-specific Treg cell precursors were present at low clonal frequency (<1%) within a normal thymus. Using retroviral vectors and bone marrow chimeras, we observed similar behavior with two other Treg cell-derived TCRs. These data demonstrate that thymic Treg cell development is a TCR-instructive process involving a niche which can be saturable at much lower clonal frequencies than the niche for positive selection.

Characterization of Thymic Regulatory T cell development (89.6)

Abstract The development of Foxp3+ CD4+ regulatory T (Treg) cells in the thymus is important for the preservation of self-tolerance and the prevention of spontaneous autoimmunity. It has been suggested that the CD4+CD8+ double positive (DP) stage is the earliest that Foxp3+ expression can be observed. We hypothesized that the antigen specificity of Foxp3+ DP cells may differ from Foxp3+ CD4+CD8- (CD4SP) cells due to the different antigenic repertoires presented by cortical thymic epithelial cells versus medullary APCs. However, purification of Foxp3+ DP cells proved difficult as many of those events on flow cytometry represented doublets of Foxp3+ CD4SP cells and Foxp3- DP cells. Our data therefore suggest that the frequency of Foxp3+ cells that can be found at the DP stage is around 1%, which is much smaller than previously reported. Rather, our current data suggests that thymic Treg cell development occurs primarily at the HSAhi CD4SP stage involving a two-step process involving both TCR-instructive and TCR-independent steps.