Transcription Factors Eomes Research Articles

Ets2-dependent trophoblast signalling is required for gastrulation progression after primitive streak initiation

Although extraembryonic ectoderm trophoblast signals the embryo for primitive streak initiation, a prerequisite for gastrulation, it is unknown whether it also signals for the progression of gastrulation after primitive streak initiation. Here, using Ets2-/- mice, we show that trophoblast signalling is also required in vivo for primitive streak elongation, completion of intraembryonic mesoderm epithelial-mesenchymal transition and the development of anterior primitive streak derivatives such as the node. We show that Ets2-dependent trophoblast signalling is required for the maintenance of high levels of Nodal and Wnt3 expression in the epiblast and for the induction of Snail expression in the primitive streak, between embryonic day 6.3 and 6.7. Within extraembryonic ectoderm trophoblast, Ets2 maintains the expression of the transcription factors Elf5, Cdx2 and Eomes, and that of the signalling molecule Bmp4. We propose a model that provides a genetic explanation as to how Ets2 in trophoblast mediates the progression of gastrulation within the epiblast.

Natural killer cell maturation is blocked by mTOR inhibition in C57Bl/6 mice

Recent work established a pathway by which the energy sensitive kinase mammalian target of rapamycin (mTOR) regulates T cell biology, and this pathway is directly influenced by energy intake. Energy restriction (ER) is a dietary intervention shown to increase lifespan and reduce the incidence of tumors in rodents. The objective of this study was to examine the role of mTOR in NK cell metabolism and biology, based on the observation that ER results in fewer mature NK cells. NK cells from 40% ER mice exhibited mTOR inhibition directly ex vivo, and reduced expression of the transcription factors Eomes and T‐bet, which are required for NK cell maturation. Consequences of mTOR inhibition on NK cell biology was examined in vitro and revealed IL‐12 + IL‐18 induced NK cell maturation and function was impaired following rapamycin treatment. mTOR inhibition reduced upregulation of metabolic transporters, and repressed expression of NK cell maturation markers. Concurrently, mTOR inhibition in NK cells blocked upregulation of transcription factors Eomes and T‐bet following stimulation. Mature NK cells play a critical role in killing tumor cells and here we show NK cell maturation is linked to metabolic signaling through mTOR. Thus, manipulation of energy intake or energy sensitive pathways directly influences NK cell maturation, which has significant implications during the immune response to cancers or viral infections.Grant Funding Source: R01AG034949

Lineage relationships of human interleukin-22–producing CD56+ RORγt+ innate lymphoid cells and conventional natural killer cells

AbstractHuman interleukin (IL)-22–producing RORγt+ innate lymphoid cells (ILC22) and conventional natural killer (cNK) cells are present in secondary lymphoid tissues. Both have an immunophenotype corresponding to stage III NK progenitors (CD56+/−CD117highCD94−). Using an in vitro differentiation and primary human tissues, we investigated their developmental relationships. cNK cells showed a CD56+CD117+CD7+/−LFA-1high phenotype and expressed surface receptors, cytokines, and transcription factors found on mature cNK cells. In contrast, ILC22 cells were contained within the CD56+CD117highCD94−CD7−LFA-1− fraction and produced IL-22, IL-8, and granulocyte macrophage colony stimulating factor. Although ILC22 cells expressed NKp44 and CD161, they lacked most other NK receptors and NK-associated transcription factors (T-bet and Eomes) and were incapable of interferon-γ production or cytotoxic responses. Most purified CD56+CD117+CD7+/−LFA-1− remained as ILC22 cells and never became cNK cells. In the absence of IL-15, CD34+ cells showed a complete block in cNK differentiation and instead gave rise to a CD56+ population of ILC22 cells. Conversely, in the absence of IL-7 and stem cell factor, cNK cells were generated but ILC22 cells showed minimal differentiation. Although human ILC22 cells and cNK progenitors have a phenotype that overlaps with stage III NK progenitors, they have unique cytokine requirements and can be distinguished by LFA-1 expression.

Technical Advance: Fluorescent reporter reveals insights into eomesodermin biology in cytotoxic lymphocytes

The T-box transcription factor Eomes is expressed in cytotoxic immune cells and plays an important role in development, maintenance, and function of these cell types. Identification and separation of cells with differential Eomes expression would allow for better understanding of the transcriptional program governing these cytotoxic lymphocytes. Here, we report the use of an Eomes(gfp)-targeted mouse allele that displays robust fidelity to Eomes protein expression in NK and T cells. Use of this reporter mouse revealed that Eomes expression in antiviral effector cells did not correlate with enhanced cytotoxicity but rather was associated with more efficient central memory differentiation. Weakening of reporter activity in Eomes-deficient CD8(+) T cells revealed a critical role for Eomes protein in maintaining central memory cells that have activated the Eomes locus. Characterization of reporter activity in NK lineage cells also permitted identification of a novel intermediate of NK cell maturation. Thus, the murine Eomes(gfp)-targeted allele provides a novel opportunity to explore Eomes biology in cytotoxic lymphocytes.

Roles of CDX2 and EOMES in human induced trophoblast progenitor cells

Abnormal trophoblast lineage proliferation and differentiation in early pregnancy have been associated with the pathogenesis of placenta diseases of pregnancy. However, there is still a gap in understanding the molecular mechanisms of early placental development due to the limited primary trophoblast cultures and fidelity of immortalized trophoblast lines. Trophoblasts stem (TS) cells, an in vitro model of trophectoderm that can differentiate into syncytiotrophoblasts and extravillous trophoblasts, can be an attractive tool for early pregnancy research. TS cells are well established in mouse but not in humans due to insufficient knowledge of which trophoblast lineage-specific transcription factors are involved in human trophectoderm (TE) proliferation and differentiation. Here, we applied induced pluripotent stem cell technique to investigate the human trophoblast lineage-specific transcription factors. We established human induced trophoblast progenitor (iTP) cells by direct reprogramming the fibroblasts with a pool of mouse trophoblast lineage-specific transcription factors consisting of CDX2, EOMES, and ELF5. The human iTP cells exhibit epithelial morphology and can be maintained in vitro for more than 2months. Gene expression profile of these cells was tightly clustered with human trophectoderm but not with human neuron progenitor cells, mesenchymal stem cells, or endoderm cells. These cells are capable of differentiating into cells with an invasive capacity, suggesting extravillous trophoblasts. They also form multi-nucleated cells which secrete human chorionic gonadotropin and estradiol, consistent with a syncytiotrophoblast phenotype. Our results provide the evidence that transcription factors CDX2 and EOMES may play critical roles in human iTP cell generation.

Histone Deacetylase 11 (HDAC11) Regulates Cytotoxic T-Cell Function and Memory Phenotype

Abstract 840T-cells are an essential component of immune mediated tumor rejection. Adoptive transfer of T-cells results in a durable anti-tumor response in some patients with hematological malignancies. To further improve the efficacy of T-cell adoptive transfers, a better understanding of the regulatory checkpoints of these cells is needed. Here we show that HDAC11 is a negative regulator of CD8+ T-cell function, thus representing a potential target in adoptive immunotherapy. HDACs are a group of enzymes initially known for their role in deacetylating histones, thereby condensing chromatin structure and repressing gene expression. The known roles of HDACs as epigenetic regulators have recently expanded to include more complex regulatory functions including interactions with non-histone targets. HDAC11 is the most recently identified member of the HDAC family, and is highly expressed in brain, testis and T-cells. Recently, our group reported HDAC11 as a regulator of IL-10 production in antigen presenting cells. To determine the role of HDAC11 in T-cell biology, T-cells from HDAC11 knock out (HDAC11KO) mice were compared to wild-type T-cells in number, function and phenotype. HDAC11KO T-cells had no differences in absolute number or percentages of CD4+ or CD8+ lymphocytes. However CD8+ T-cells were hyper-proliferative upon CD3/CD28 stimulation and produced significantly higher levels of the pro-inflammatory, Tc1 cytokines IL-2, INF-γ, and TNF-α. However, no significant increases in the production of the Tc2 cytokines IL-4, IL-6 or IL-10 were seen. Further investigation of phenotypic differences also revealed that HDAC11KO mice have a larger percentage of central memory CD8+ T-cells. Additionally, HDAC11KO CD8+ T-cells express higher levels of the transcription factor Eomes, a known contributor to central memory cell formation as well as a controller of granzyme B and perforin production in CD8+ T-cells. This Tc1 and central memory-like phenotype translated to delayed tumor progression and survival in vivo in C1498 AML bearing mice treated with adoptively transferred HDAC11KO T-cells, as compared with wild type T-cells. Collectively, we have demonstrated HDAC11 as a negative regulator of CD8+ T-cell function, and a novel potential target to augment the efficacy of adoptive T-cell tumor immunotherapy. Disclosures:No relevant conflicts of interest to declare.

The Transcription Factors T-bet and Eomes Control Key Checkpoints of Natural Killer Cell Maturation

Natural killer (NK) cells play critical roles defending against tumors and pathogens. We show that mice lacking both transcription factors Eomesodermin (Eomes) and T-bet failed to develop NK cells. Developmental stability of immature NK cells constitutively expressing the death ligand TRAIL depended on T-bet. Conversely, maturation characterized by loss of constitutive TRAIL expression and induction of Ly49 receptor diversity and integrin CD49b (DX5(+)) required Eomes. Mature NK cells from which Eomes was deleted reverted to phenotypic immaturity if T-bet was present or downregulated NK lineage antigens if T-bet was absent, despite retaining expression of Ly49 receptors. Fetal and adult hepatic hematopoiesis restricted Eomes expression and limited NK development to the T-bet-dependent, immature stage, whereas medullary hematopoiesis permitted Eomes-dependent NK maturation in adult mice. These findings reveal two sequential, genetically separable checkpoints of NK cell maturation, the progression of which is metered largely by the anatomic localization of hematopoiesis.

Antigen cross-presentation in the liver generates protective memory CD8+ T cells in the absence of inflammation

The inflammatory context of antigen-presentation dictates the outcome of CD8+ T cell responses. Pathogen-induced inflammatory signals ensure the generation of an effector CD8+ T cell response and long-lasting T cell memory, whereas antigen-presentation in absence of inflammatory stimuli, e.g. of self-antigens, causes CD8+ T cell-deletion. The systemic distribution of antigens from certain pathogens without inflammatory stimuli therefore imposes the risk of deleting CD8+ T cells needed for anti-infectious defense. We hence wondered whether cross-presentation of exogenous antigens in the liver, the central organ for clearing soluble and gut-derived antigens, might allow for memory T cell generation even in the absence of inflammation. When restricting presentation of soluble ovalbumin to liver sinusoidal endothelial cells (LSEC), the most abundant population of liver-resident antigen-presenting cells, cross-priming by LSEC generated an antigen-experienced CD8+ T cell population that persisted long time and was insensitive to DC-mediated cross-tolerance. As LSEC-primed T cells re-expressed CD62L and relocated to secondary lymphoid organs (SLOs), a hallmark of central memory T cells (TCM), we directly compared the phenotype and in vivo distribution of both T cell populations. Clearly, LSEC-primed OT-I T cells resembled TCM induced by L. monocytogenes-OVA infection in their localization to SLOs and showed high expression of CD127, CD122 and the memory-specific transcription factor Eomes. In response to bacterial and viral infection, LSEC-primed T cells expanded and differentiated into effector T cells similar to TCM. Taken together, these data suggest that antigen-presentation under non-inflammatory conditions, if conducted by LSEC in the liver, allows for memory T cell generation. Those T cells may serve for defense against pathogens that in the first instance avoid causing inflammation.

Epitope Density Influences CD8+ Memory T Cell Differentiation

BackgroundThe generation of long-lived memory T cells is critical for successful vaccination but the factors controlling their differentiation are still poorly defined. We tested the hypothesis that the strength of T cell receptor (TCR) signaling contributed to memory CD8+ T cell generation.Methodology/Principal FindingsWe manipulated the density of antigenic epitope presented by dendritic cells to mouse naïve CD8+ T cells, without varying TCR affinity. Our results show that a two-fold decrease in antigen dose selectively affects memory CD8+ T cell generation without influencing T cell expansion and acquisition of effector functions. Moreover, we show that low antigen dose alters the duration of the interaction between T cells and dendritic cells and finely tunes the expression level of the transcription factors Eomes and Bcl6. Furthermore, we demonstrate that priming with higher epitope density results in a 2-fold decrease in the expression of Neuron-derived orphan nuclear receptor 1 (Nor-1) and this correlates with a lower level of conversion of Bcl-2 into a pro-apoptotic molecule and an increased number of memory T cells.ConclusionsOur results show that the amount of antigen encountered by naïve CD8+ T cells following immunization with dendritic cells does not influence the generation of functional effector CD8+ T cells but rather the number of CD8+ memory T cells that persist in the host. Our data support a model where antigenic epitope density sensed by CD8+ T cells at priming influences memory generation by modulating Bcl6, Eomes and Nor-1 expression.

Human cytomegalovirus elicits fetal γδ T cell responses in utero

The fetus and infant are highly susceptible to viral infections. Several viruses, including human cytomegalovirus (CMV), cause more severe disease in early life compared with later life. It is generally accepted that this is a result of the immaturity of the immune system. γδ T cells are unconventional T cells that can react rapidly upon activation and show major histocompatibility complex–unrestricted activity. We show that upon CMV infection in utero, fetal γδ T cells expand and become differentiated. The expansion was restricted to Vγ9-negative γδ T cells, irrespective of their Vδ chain expression. Differentiated γδ T cells expressed high levels of IFN-γ, transcription factors T-bet and eomes, natural killer receptors, and cytotoxic mediators. CMV infection induced a striking enrichment of a public Vγ8Vδ1-TCR, containing the germline-encoded complementary-determining-region-3 (CDR3) δ1–CALGELGDDKLIF/CDR3γ8–CATWDTTGWFKIF. Public Vγ8Vδ1-TCR–expressing cell clones produced IFN-γ upon coincubation with CMV-infected target cells in a TCR/CD3-dependent manner and showed antiviral activity. Differentiated γδ T cells and public Vγ8Vδ1-TCR were detected as early as after 21 wk of gestation. Our results indicate that functional fetal γδ T cell responses can be generated during development in utero and suggest that this T cell subset could participate in antiviral defense in early life.

The Transcription Factor GATA3 Actively Represses RUNX3 Protein-Regulated Production of Interferon-γ

The transcription factor GATA3 is crucial for the differentiation of naive CD4(+) T cells into T helper 2 (Th2) cells. Here, we show that deletion of Gata3 allowed the appearance of interferon-gamma (IFN-gamma)-producing cells in the absence of interleukin-12 (IL-12) and IFN-gamma. Such IFN-gamma production was transcription factor T-bet independent. Another T-box-containing transcription factor Eomes, but not T-bet, was induced both in GATA3-deficient CD4(+) T cells differentiated under Th2 cell conditions and in Th2 cells with enforced Runx3 expression, contributing to IFN-gamma production. GATA3 overexpression blocked Runx3-mediated Eomes induction and IFN-gamma production, and GATA3 protein physically interacted with Runx3 protein. Furthermore, we found that Runx3 directly bound to multiple regulatory elements of the Ifng gene and that blocking Runx3 function in either Th1 or GATA3-deficient "Th2" cells results in diminished IFN-gamma production by these cells. Thus, the Runx3-mediated pathway, actively suppressed by GATA3, induces IFN-gamma production in a STAT4- and T-bet-independent manner.

IL-2 signals promote the differentiation of effector CD8+ T cells (132.26)

Abstract CD8+ T cells that do not receive IL-2 signals during the primary immune response to acute infection form stable memory populations but are unable to mount successful recall responses. In this study, we found a specific defect in the differentiation and persistence of effector and effector memory CD8+ T cells in the absence of IL-2 signals during the response to lymphocytic choriomeningitis virus (LCMV) infection. Although expression levels of the effector-associated transcription factors T-bet, Eomes, and Blimp-1 did not differ significantly, unbiased analysis of gene expression by WT or IL-2Rα-deficient CTLs revealed that optimal expression of CTL effector molecules, including Granzymes and Perforin, was IL-2-dependent. The end result of CD8+ T cell activation in the absence of IL-2 was therefore decreased effector CTL activity. The defect in effector differentiation was even more profound for secondary CTL responses and in models of localized tissue-specific infection. CD8+ T cells that homed to the lung following influenza infection failed to express Granzyme B. We conclude that IL-2 provides a differentiation signal to responding CD8+ T cells that enhances access to an effector gene expression program during the primary response to infection. Furthermore, our findings suggest that IL-2 permanently imparts a gene expression imprint to CD8+ memory T cells that enables them to re-access the CTL effector program upon subsequent encounter with antigen.

Mutation of the Variant α-Tubulin TUBA8 Results in Polymicrogyria with Optic Nerve Hypoplasia

The critical importance of cytoskeletal function for correct neuronal migration during development of the cerebral cortex has been underscored by the identities of germline mutations underlying a number of human neurodevelopmental disorders. The proteins affected include TUBA1A, a major alpha-tubulin isoform, and microtubule-associated components such as doublecortin, and LIS1. Mutations in these genes are associated with the anatomical abnormality lissencephaly, which is believed to reflect failure of neuronal migration. An important recent observation has been the dependence of cortical neuronal migration upon acetylation of alpha-tubulin at lysine 40 by the histone acetyltransferase Elongator complex. Here, we describe a recognizable autosomal recessive syndrome, characterized by generalized polymicrogyria in association with optic nerve hypoplasia (PMGOH). By autozygosity mapping, we show that the molecular basis for this condition is mutation of the TUBA8 gene, encoding a variant alpha-tubulin of unknown function that is not susceptible to the lysine 40 acetylation that regulates microtubule function during cortical neuron migration. Together with the unique expression pattern of TUBA8 within the developing cerebral cortex, these observations suggest a role for this atypical microtubule component in regulating mammalian brain development.

CTLA‐4 co‐receptor impacts on the function of Treg and CD8+ T‐cell subsets

CTLA-4 has potent regulatory effects on the threshold of T-cell signalling and, in the process, guards against the development of hyper-proliferation and autoimmunity. Despite this, the role of CTLA-4 on specific T-cell subsets has been unclear. Such studies could shed light on both the function of CTLA-4, and on the contribution of the subsets to the disease phenotype of the Ctla4(-/-) mouse. Recently, a role for this co-receptor in the function of Treg has been outlined and, in this issue of the European Journal of Immunology, the selective targeting of the T-box transcription factor Eomes by CTLA-4 in the regulation of CD8(+) cytolytic T-cell (CTL) effector function is shown. Together, these papers shed light on the role of CTLA-4 in different T-cell subsets.

113 CHARACTERIZATION OF THE BOVINE EMBRYO-DERIVED CT-1 CELL LINE FOR STUDYING REGULATION OF TRANSCRIPTION FACTORS EXPRESSED WITHIN THE BOVINE TROPHECTODERM

Cloned bovine embryos have been shown to display deviations in gene expression of trophectodermal (TE) lineage-specific transcription factors, including Cdx2, Errβ, Eomes, Mash2, and Hand1. Misregulation of these genes in the early TE has been implicated to play a causal role in abnormal placental formation and fetal loss. Because suboptimal in vitro culture conditions fail to support development beyond hatching, very little is known about regulation of peri-attachment bovine embryo development. Trophectodermal-derived bovine cell lines, such as the CT-1 cell line produced by Talbot and colleagues (Talbot et al. 2000 Biol. Reprod. 62, 235–247) could prove beneficial for understanding how regulatory factors govern early bovine TE development. The CT-1 cell line has been cultured continuously for over 2 years and appears morphologically similar to primary bovine TE outgrowths. Furthermore, these cells produce interferon τ, a cytokine unique to the early bovine TE. The objective of this study was to develop a gene expression profile of selected developmental genes that play a potential role in commitment to the TE fate, maintenance of multipotency, or differentiation into a binucleate phenotype. The CT-1 colonies were initially expanded on gamma-irradiated STO cells at 37°C and 5% CO2 and passaged weekly. Cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1 mm sodium pyruvate and were passaged whenever colony edges touched. Cultures designated for RNA extraction and RT-PCR were then passaged into collagen-coated flasks or Cellbind® flasks (Corning, Lowell, MA, USA) at an average dilution of 1:4 for at least 6 feeder-free passages. Feeder-free culture at 37°C and 8.5% CO2 was optimal for adherence and growth. There were no observable differences in morphology, adherence, or growth rate between CT-1 cells grown on collagen film or a Cellbind® surface. The RT-PCR results indicate that the transcription factor Cdx2, which is homologous to the mouse master TE regulator, is abundantly expressed, whereas pluripotency specific Oct4 is weakly expressed. However, pluripotency specific transcription factors Nanog and Sox2 were undetectable. Transcription factors Eomes, Ets2, Errβ, and Id2, which may be required for proper bovine trophoblast elongation and developmental potential, were also expressed. Hand1 mRNA, a basic helix-loop-helix transcription factor that directs mouse trophoblast cells to differentiate into a giant cell phenotype, was also present and may play a role in binucleate cell formation. Together the data indicate that the CT-1 cell line can be utilized as a permissive cell line for studying transcriptional control of key TE regulatory factors that define the bovine TE lineage and allow better interpretation of gene expression data collected from assisted reproductive technology-derived embryos.