Lineage-defining Transcription Factor Research Articles

Premature expression of Foxp3 in double-negative thymocytes.

Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tTreg) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the Treg lineage defining transcription factor Foxp3 in developing tTreg 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 in vivo. 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-CD44highCD25low). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.

Wanda Blenska

<h3>ABSTRACT</h3> Lineage-defining transcription factors (LTFs) play key roles in tumor cell growth, making them highly attractive, but currently “undruggable”, small cell lung cancer (SCLC) vulnerabilities. Delineating LTF genomic binding sites...

The Transcription Factor KLF2 Restrains CD4+ T Follicular Helper Cell Differentiation

SummaryT follicular helper (Tfh) cells are essential for efficient B cell responses, yet the factors that regulate differentiation of this CD4+ T cell subset are incompletely understood. Here we found that the KLF2 transcription factor serves to restrain Tfh cell generation. Induced KLF2 deficiency in activated CD4+ T cells led to increased Tfh cell generation and B cell priming, while KLF2 overexpression prevented Tfh cell production. KLF2 promotes expression of the trafficking receptor S1PR1, and S1PR1 downregulation is essential for efficient Tfh cell production. However, KLF2 also induced expression of the transcription factor Blimp-1, which repressed transcription factor Bcl-6 and thereby impaired Tfh cell differentiation. Furthermore, KLF2 induced expression of the transcription factors T-bet and GATA3 and enhanced Th1 differentiation. Hence, our data indicate KLF2 is pivotal for coordinating CD4+ T cell differentiation through two distinct and complementary mechanisms: via control of T cell localization, and by regulation of lineage-defining transcription factors.

Insufficient interleukin-12 signalling favours differentiation of human CD4(+) and CD8(+) T cells into GATA-3(+) and GATA-3(+) T-bet(+) subsets in humanized mice.

Differentiation of CD4(+) T cells into type 1 or type 2 subsets is mediated by the expression of the opposing lineage defining transcription factors T-bet and GATA-3. However, the existence of GATA-3(+) T-bet(+) CD4(+) T cells in mice suggests functional plasticity of these subsets. Little is known about type 1 and type 2 plasticity of human T-cell subsets in vivo. Here, we show that in the xenogeneic environment of humanized mice, which lacks a functional immune-regulatory network, human CD4(+) and, notably, CD8(+) T cells preferentially differentiate into interleukin (IL)-4(+) GATA-3(+) and IL-4(+) interferon-γ(+) GATA-3(+) T-bet(+) subsets. Treatment with recombinant human IL-12 or expansion of IL-12-producing human dendritic cells in vivo reverted this phenotype and led to the down-regulation of GATA-3 expression. These changes also correlated with improved antiviral immune responses in humanized mice. In conclusion, our study shows the capacity of human CD4(+) and CD8(+) T cells for stable co-expression of GATA-3 and T-bet in humanized mice and reveals a critical role for IL-12 in regulating this phenotype.

Deciphering the epigenetic code of T lymphocytes.

The multiple lineages and differentiation states that constitute the T-cell compartment all derive from a common thymic precursor. These distinct transcriptional states are maintained both in time and after multiple rounds of cell division by the concerted actions of a small set of lineage-defining transcription factors that act in conjunction with a suite of chromatin-modifying enzymes to activate, repress, and fine-tune gene expression. These chromatin modifications collectively provide an epigenetic code that allows the stable and heritable maintenance of the T-cell phenotype. Recently, it has become apparent that the epigenetic code represents a therapeutic target for a variety of immune cell disorders, including lymphoma and acute and chronic inflammatory diseases. Here, we review the recent advances in epigenetic regulation of gene expression, particularly as it relates to the T-cell differentiation and function.

E and Id proteins influence invariant NKT cell sublineage differentiation and proliferation.

Disease outcome is known to be influenced by defined subsets of invariant NKT (iNKT) cells residing in distinct locations within peripheral tissue. However, the factors governing the development of these unique iNKT sublineages during thymic development are unknown. In this study we explored the mechanism by which E protein transcription factors and their negative regulators, the Id proteins, control the development of iNKT sublineages after positive selection. We found that E proteins directly bound the promyelocytic leukemia zinc finger (PLZF) promoter and were required for expression of this lineage-defining transcription factor and for the maturation and expansion of thymic iNKT cells. Moreover, expression of the negative regulators of E proteins, Id2 and Id3, defined distinct iNKT cell sublineages. Id3 was expressed in PLZF(high) NKT2 cells and loss of Id3 allowed for increased thymic iNKT cell expansion and abundance of the PLZF(+) NKT2 sublineage. Id2 was expressed in T-BET(+) NKT1 cells, and both Id proteins were required for the formation of this sublineage. Thus, we provide insight into E and Id protein regulation of iNKT cell proliferation and differentiation to specific sublineages during development in the thymus.

A8.23 E and Id proteins influence invariant Natural Killer T cell sublineage differentiation and proliferation

Background and ObjectivesInvariant Natural Killer T (iNKT) cells are able to produce large amounts of cytokines within hours of activation. Disease outcome is known to be influenced depending on the...

Regulation of the NF-κB-Mediated Transcription of Inflammatory Genes

The NF-κB family of transcription factors plays a central role in the inducible expression of inflammatory genes during the immune response, and the proper regulation of these genes is a critical factor in the maintenance of immune homeostasis. The chromatin environment at stimulus-responsive NF-κB sites is a major determinant in transcription factor binding, and dynamic alteration of the chromatin state to facilitate transcription factor binding is a key regulatory mechanism. NF-κB is in turn able to influence the chromatin state through a variety of mechanisms, including the recruitment of chromatin modifying co-activator complexes such as p300, the competitive eviction of negative chromatin modifications, and the recruitment of components of the general transcriptional machinery. Frequently, the selective interaction with these co-activators is dependent on specific post-translational modification of NF-κB subunits. Finally, the mechanisms of inducible NF-κB activity in different immune cell types seem to be largely conserved. The diversity of cell-specific NF-κB-mediated transcriptional programs is established at the chromatin level during cell differentiation by lineage-defining transcription factors. These factors generate and maintain a cell-specific chromatin landscape that is accessible to NF-κB, thus restricting the inducible transcriptional response to a cell-appropriate output.

Intracellular staining for cytokines and transcription factors.

Within the past years immune cells in general and T cells in particular have been categorized into a vast variety of subsets with different functional properties. One of the key technologies fueling this emerging complexity is intracellular staining for effector cytokines and/or lineage-defining transcription factors. Here we discuss the critical steps for performing successful multicolor immunophenotyping of mouse T cells in combination with analysis of intracellular molecules after ex vivo isolation.

Foxp3+ Treg cells in humoral immunity

T(reg) cells are essential for the maintenance of immune homeostasis and prevention of autoimmunity. In humoral immune responses, loss of T(reg) cell function causes increased levels of serum autoantibodies, hyper-IgE, spontaneous generation of germinal centres, and enhanced numbers of specialised T follicular helper cells (T(fh) cells) controlled by the lineage-defining transcription factor BCL-6 (B-cell lymphoma 6). Recent studies have demonstrated that a subset of T(reg) cells [T follicular regulatory (T(freg)) cells] are able to co-opt the follicular T-cell program by gaining expression of BCL-6 and travelling to the follicle where they have an important role in the control of expansion of T(fh) cells and the germinal centre reaction. However, the mechanisms by which they exert this control are still under investigation. In this review, we discuss the effects of T(reg) cells on humoral immunity and the mechanisms by which they exert their regulatory function.

The Tc17 Population Required For Sclerodermatous Chronic Gvhd Represents a Non-Cytolytic, Pluripotent Inflammatory Effector T Cell Lineage

IL-17 producing CD8+ T cells (Tc17) are a recently described effector T cell lineage. Donor T cell polarization is a critical factor influencing the severity and tissue distribution of graft-versus-host disease (GVHD) and the potency of graft-versus-leukemia (GVL) effects after bone marrow transplantation. However, CD8+ T cell lineage development during GVHD is not well understood. The recent shift to using G-CSF mobilized peripheral blood as a stem cell source for allogeneic stem cell transplantation (SCT) has increased the incidence of chronic GVHD (cGVHD) and we have recently reported that G-CSF promotes type-17 differentiation in both CD4 and CD8 T cells. Importantly, donor IL-17A, predominantly from CD8 T cells, mediates fibrotic skin pathology which manifests late after transplant as scleroderma (Hill GR, et al. Blood 2010, 116:819). To study the donor Tc17 population, we utilized the IL-17A-CrexR26ReYFP fate-mapping reporter mouse that permanently labels any cell that has expressed IL-17A. In time course experiments, CD8+YFP+ Tc17 cells were rapidly induced in all lymphoid tissues and GVHD target organs examined (skin, liver and lung). This was a GVHD phenomena since no CD8+YFP+ T cells developed in syngeneic recipients. Importantly, the use of these transgenic cells confirmed that the majority of the IL-17 signal seen following re-stimulation with PMA/Ionomycin is not within the reporter population (i.e. is artefact). At day 7 post transplant Tc17 cells were the dominant IL-17 producing population and following re-stimulation, over 35% of the CD8+YFP+ cells co-expressed IL-17 and IFNγ seven days after BMT, while the remaining CD8+YFP+ cells had switched off IL-17 but maintained IFNγ production (i.e. had a Tc1 phenotype). By D21 only 5% of the reporter cells continued to produce IL-17, whereas IFNγ production was maintained, demonstrating significant plasticity within the CD8+YFP+ population. Thus Tc17 differentiation is an early and transient phenomenon with most cells going on to a Tc1 phenotype. In order to understand the factors driving Tc17 differentiation during GVHD we utilized various cytokine neutralization strategies. The frequency of CD8+YFP+ Tc17 cells was significantly reduced in IL-6–/– recipients (1.8% vs 5.1%, p=0.0002, WT vs IL-6-/-) while IFNg neutralization (the dominant cytokine secreted early after BMT) resulted in a significant increase in frequency. CD8+YFP+ Tc17 development was dependent upon the presence of recipient DC as CD8+YFP+ frequencies were significantly reduced (5.2% vs 2.9%, p=0.0003) when host DC were depleted using diphtheria toxin (DT) treated CD11c-DTR recipients. CD8+YFP+ cells responded to stimulation with both mitogen and host-type DC by producing significantly increased levels of IL-17A (321 pg/mL ±11 pg/mL vs 3283 ± 147pg/mL, 24hrs p&lt;0.001), IL-13 (252 ±12 pg/mL vs 542 ± 10pg/mL, 24hrs p&lt;0.001), and TNF (1789 ±46 pg/mL vs 3140 ± 177pg/mL, 24hrs p&lt;0.001) relative to CD8+YFPneg cells, again suggestive of significant plasticity. These data demonstrate that recipient IL-6 production and recipient DC promote development of polyfunctional Tc17 cells. Examination of lineage defining transcription factors in these populations revealed the CD8+YFP+ T cells expressed high levels of both RORγt and T-bet, the transcription factors associated with Th17 and Th1 differentiation respectively. Although IL-17 cytokine production ceased over time, the transcription factor signature was highly stable. In contrast the CD8+YFPneg T cells expressed T-bet but not RORgT, suggesting these cells represent a different subset of cytotoxic Tc1 cells. This Tc1 phenotype was confirmed by their high level mRNA expression of the cytolytic proteins granzyme A and B which were minimally expressed in CD8+YFP+ Tc17 cells. The CD8+YFP+ Tc17 cells also failed to express granzyme B protein as determined by flow cytometry, in contrast to high expression by CD8+YFPneg cells. Finally, the CD8+YFP+ cells were poorly cytotoxic in chromium release CTL assays relative to Tc1 effectors when assessed after BMT. These data suggest that Tc17 differentiation is a very early and highly plastic differentiation program culminating in a non-cytolytic, pluripotent inflammatory donor T cell. Intervention to prevent this differentiation program, potentially via IL-6 or RORgt inhibition would be predicted to reduce cGVHD whilst maintaining therapeutic GVL effects. Disclosures: No relevant conflicts of interest to declare.

The Role of FOXP3 in Regulating Immune Responses

Regulatory T cells (Tregs) act in trans to control immune responses. The suppressive function of Tregs relies heavily on high and stable expression of the transcription factor FOXP3, which, together with other transcription factors, activates anti-inflammatory genes and represses proinflammatory genes. FOXP3 is required to shape the unique signaling mechanisms in Tregs, creating a positive-feedback pathway to further enhance its own expression. In addition, FOXP3 is thought to switch on a complex transcriptional network that leads to the stabilization of the Treg phenotype. Emerging data reveal that FOXP3 achieves this function in concert with several other transcription factors, many of which are associated with lineages of conventional T cells. In this review, we will discuss the structural features of FOXP3 and how it functions by interacting with other transcription factors. We will also summarize the role of FOXP3 in establishing the unique signaling cascades in Tregs. Finally, we will dissect the cooperative roles of FOXP3 and other T-cell lineage-defining transcription factors and discuss how these networks not only control the ability to Tregs to suppress different types of immune responses, but also enable Treg plasticity.

Master regulators or lineage-specifying? Changing views on CD4+ T cell transcription factors

There is an emerging body of research demonstrating that the co-expression of key lineage-specifying transcription factors, commonly referred to as 'master regulators', affects the functional capabilities and flexibility of CD4(+) T cell subsets. Here, we discuss how the natural co-expression of these lineage-specifying transcription factors has challenged the concept that the expression of a single 'master regulator' strictly establishes an absolute CD4(+) T cell phenotype. Instead, it is becoming clear that the interplay between the lineage-specifying (or lineage-defining) transcription factors, including T-bet, GATA3, RORγt, BCL-6 and FOXP3, contributes to the fate and flexibility of CD4(+) T cell subtypes. This in turn has led to the realization that CD4(+) T cell phenotypes are more diverse than previously recognized.

Decreased melanoma proliferation and cell survival: turn down your SOX10

Decreased melanoma proliferation and cell survival: turn down your <scp>SOX</scp>10

Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

Differential localization of T-bet and Eomes within distinct human CD8 T-cell memory populations (110.10)

Abstract In mice, two T-box transcription factors, T-bet and Eomes, drive the differentiation of CD8 T-cell lineages; however, little is known regarding their role in human CD8 T-cell differentiation. Here, we characterized T-bet and Eomes expression and localization within human CD8 memory T-cell populations. We find T-bet and Eomes are broadly expressed in human memory CD8 T cells, with increasing levels of T-bet and Eomes strongly correlating with differentiation from central memory to effector memory and effector subpopulations. Interestingly, T-bet levels directly correlate to subcellular localization, with a higher propensity for nuclear expression of T-bet within T-bethi cells and predominately cytoplasmic expression in T-betlo cells. In contrast, Eomes appears to be localized solely to the nucleus. In murine CD8 T cells, T-bet can be nuclear, cytoplasmic, or both; however, Eomes is detectable in both compartments suggesting differential regulation compared to humans. Additionally, in humans, Eomes tightly correlated with nuclear T-bet suggesting T-bet localization might impact Eomes expression. Together, our data provide a novel mechanism for CD8 T-cell differentiation in humans and mice based on subcellular localization of T-bet, and reveal the likely presence of previously unknown regulation mechanisms for these critical lineage defining transcription factors.

Dominant Th2 Differentiation of Human Regulatory T Cells upon Loss of FOXP3 Expression

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) are pivotal for peripheral self-tolerance. They prevent immune responses to auto- and alloantigens and are thus under close scrutiny as cellular therapeutics for autoimmune diseases and the prevention or treatment of alloresponses after organ or stem cell transplantation. We previously showed that human Treg with a memory cell phenotype, but not those with a naive phenotype, rapidly downregulate expression of the lineage-defining transcription factor FOXP3 upon in vitro expansion. We now compared the transcriptomes of stable FOXP3(+) Treg and converted FOXP3(-) ex-Treg by applying a newly developed intranuclear staining protocol that permits the isolation of intact mRNA from fixed, permeabilized, and FACS-purified cell populations. Whole-genome microarray analysis revealed strong and selective upregulation of Th2 signature genes, including GATA-3, IL-4, IL-5, and IL-13, upon downregulation of FOXP3. Th2 differentiation of converted FOXP3(-) ex-Treg occurred even under nonpolarizing conditions and could not be prevented by IL-4 signaling blockade. Thus, our studies identify Th2 differentiation as the default developmental program of human Treg after downregulation of FOXP3.

The Krüppel-like zinc finger protein GLIS3 transactivates neurogenin 3 for proper fetal pancreatic islet differentiation in mice.

Mutations in GLIS3, which encodes a Krüppel-like zinc finger transcription factor, were found to underlie sporadic neonatal diabetes. Inactivation of Glis3 by gene targeting in mice was previously shown to lead to neonatal diabetes, but the underlying mechanism remains largely unknown. We aimed to elucidate the mechanism of action of GLIS family zinc finger 3 (GLIS3) in Glis3 ( -/- ) mice and to further decipher its action in in-vitro systems. We created Glis3 ( -/- ) mice and monitored the morphological and biochemical phenotype of their pancreatic islets at different stages of embryonic development. We combined these observations with experiments on Glis3 expressed in cultured cells, as well as in in vitro systems in the presence of other reconstituted components. In vivo and in vitro analyses placed Glis3 upstream of Neurog3, the endocrine pancreas lineage-defining transcription factor. We found that GLIS3 binds to specific GLIS3-response elements in the Neurog3 promoter, activating Neurog3 gene transcription both directly, and synergistically with hepatic nuclear factor 6 and forkhead box A2. These results indicate that GLIS3 controls fetal islet differentiation via direct transactivation of Neurog3, a perturbation that causes neonatal diabetes in mice.

The lineage-defining factors T-bet and Bcl-6 collaborate to regulate Th1 gene expression patterns

The T-box transcription factor T-bet is important for the differentiation of naive CD4(+) T helper cells (Th cells) into the Th1 phenotype. Much is known about T-bet's role as a transcriptional activator, but less is known about the mechanisms by which T-bet functionally represses alternative Th cell genetic programs. In this study, we first identify Socs1, Socs3, and Tcf7 (TCF-1) as gene targets that are negatively regulated by T-bet. Significantly, T-bet's role in the repression of these genes is through a direct interaction with their promoters. Consistent with this, we identified two T-bet DNA-binding elements in the Socs1 promoter that are functionally used to down-regulate transcription in primary Th1 cells. Importantly, T-bet's novel role in transcriptional repression is because of its ability to physically associate with, and functionally recruit, the transcriptional repressor Bcl-6 to a subset of promoters. Furthermore, T-bet functionally recruits Bcl-6 to the Ifng locus in late stages of Th1 differentiation to repress its activity, possibly to prevent the overproduction of IFN-γ, which could result in autoimmunity. Collectively, these data establish a novel mechanism for T-bet-mediated gene repression in which two lineage-defining transcription factors, one a classical activator and one a repressor, collaborate to promote and properly regulate Th1 development.